study on biological characteristics and mechanism of paclitaxel...
TRANSCRIPT
Research ArticleStudy on Biological Characteristics and Mechanism of PaclitaxelInduced Drug Resistance in Endometrial Carcinoma Cells
Jie Ding1 Mengxiong Li2 Liuzhi Deng1 and Tian Li 2
1TheThird Affiliated Hospital Sun Yat-sen University Guangdong Province China2The Seventh Affiliated Hospital Sun Yat-sen University Guangdong Province China
Correspondence should be addressed to Tian Li litian2mailsysueducn
Received 20 February 2018 Accepted 29 April 2018 Published 5 August 2018
Academic Editor Ji-Fu Wei
Copyright copy 2018 Jie Ding et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Objective To study the biological characteristics of paclitaxel resistant endometrial carcinoma cells and its mechanism of drugresistanceMethodThe paclitaxel resistant cell lines were established by high-dose paclitaxel (TAX) injectionThe IC50 of paclitaxelwas determined by CCK-8 assay in Ishikawa and Ishikawa-TAXThe cell cycle and apoptosis rate were detected by flow cytometryWestern blot was used to detect the expression of p-AKT and p-p70S6K The expression of drug resistance-related genes Pgp andMDR1 was determined by RT-PCR Cell viability was determined by soft agarose assay and invasive ability in vitro by transwellassay Results Paclitaxel and NVP-BEZ235 cotreatment group can further inhibit the clonogenicity and invasion of Ishikawa andIshikawa-TAX cells compared with paclitaxel alone and NVP-BEZ235 treatment group Paclitaxel and NVP-BEZ235 cotreatedgroups increased the apoptosis rate of Ishikawa and increased G0G1 phase arrest in both cells Paclitaxel alone significantlyinhibited p-AK and p-p70 S6K protein expression in Ishikawa and Ishikawa-TAX cells and the inhibition was enhanced by NVP-BEZ235 when cotreated with paclitaxel Conclusion Paclitaxel can inhibit Ishikawa and Ishikawa-TAX cell via PI3KAktmTORsignaling pathway Paclitaxel and NVP-BEZ235 cotreatment can enhance the inhibitory effect
1 Introduction
Endometrial cancer is one of the most common gynecologicmalignancies The mortality is increasing because of thelack of no effective treatment for advanced and recurrentendometrial cancer which seriously affects the quality of lifeof women [1 2] At present chemotherapy is the primarymethod of treatment [3] The combination of paclitaxeltaxol(TAX) and platinum compounds is the first-line chemother-apy regime for endometrial cancer which shows a certaineffect on primary endometrial cancer [4] Paclitaxel is anantimicrotubule agent which binds to the h-tubulin subunitand stabilizes the microtubules resulting in disruption ofmicrotubule dynamics during cell division [5] Howeverwith the extension of dosage and duration the inducedpaclitaxel resistance often leads to the failure of chemotherapyin endometrial carcinoma [6] Therefore the study of drugresistance in endometrial cancer is of great significance forclinical treatment especially for the advanced and recurrentendometrial cancer
PI3KmTOR signaling pathway is frequently activated ina variety of human tumors such as breast cancer ovariancancer and bladder cancer [7ndash9] In recent years the studyshowed that the abnormal activation of phosphatidylinositol-3 kinaseprotein kinase Bmammalian target of rapamycin(PI3KAktmTOR) signaling pathway (eg gene mutation)can inhibit the apoptosis of endometrial cancer cells andpromote cancer cell proliferation invasion and angiogenesis[10 11] Therefore the biological characteristics of paclitaxelresistant endometrial cancer cells and their correlation withPI3KmTOR signaling were studied in this paper so as toprovide a theoretical basis for clinical treatment of paclitaxelresistant endometrial cancer
2 Materials and Methods
21 Cell Culture The endometrial carcinoma cell line(Ishikawa) was purchased from Shanghai cell bank of Chi-nese Academy of Sciences Ishikawa cells were cultured in
HindawiBioMed Research InternationalVolume 2018 Article ID 8372085 10 pageshttpsdoiorg10115520188372085
2 BioMed Research International
RPMI1640 culture medium containing 10 FBS 1 peni-cillin and 100 Uml streptomycin at 37∘C and 5 CO
2
22 Construction of Paclitaxel Resistant Cell Line (Ishikawa-TAX Cells) The MTT method was used to detect IC50 inIshikawa cells When the parent cell confluence reached80 the cells are treated with taxol (sigma USA) at theconcentration of 110 IC50 The concentration of taxol waskept in the culture medium by changing medium with taxolWhen cell growth was stable in the low concentration taxolmedium (110 IC50) the taxol concentration was raisedgradually until 5-10 times of IC50 The treated Ishikawa cellsgrew well with the high concentration of taxol and thereforethe paclitaxel resistant cell line (Ishikawa-TAX cells) wassuccessfully established
23 NVP-BEZ235 and Paclitaxel Treatment When cells grewto 80 density NVP-BEZ235 (MedChemExpress USA) wasadded to medium at concentration of 5 nM After 6 hoursTAXwas added to 001 ugml for 24 hours then the cells werecollected for follow-up experiments
24 MTT Assay Cells at logarithmic growth phase werecollected and suspended at the concentration of 104-105 ml100 ul of cell suspension was added to each pore of 96-wellplate and MTT assay was carried out on the correspondingday 20 ul MTT solution was added and the cells were incu-bated for 4 h The supernatant was then carefully dischargedand 150 ul DMSO was added to dissolve the crystallizationThe absorbance at OD492 nm was measured by MicroplateReader
25 Clone Formation Cell suspension was prepared bytrypsin-EDTAdigestion and cell densitywas adjusted accord-ing to the number of Trypan blue positive staining cells 100living cells were inoculated in each hole of 6-well plates andthen the cells were cultured for 15 days at normal conditionAt day 15 the culture medium was discharged and cells werefixed by paraformaldehyde After washing by PBS the cellswere stained by crystal violet for 10minThenumber of cloneswas counted All experiments were conducted in triplicate
26 Cell Apoptosis Detection Cells were harvested by trypsinand collected by centrifugation at 400timesg for 5 minutes Akit from BD company was used for cell apoptosis analy-sis Carefully decant all the supernatant and add 250 120583Lof Solution A (trypsin buffer) for 10 minutes and 200120583Lof Solution B (trypsin inhibitor and RNase buffer for) 10minutes Do not remove Solution A and Solution B Add200120583L of cold Solution C (propidium iodide stain solution)incubate for 10 minutes in the dark in the refrigerator (2∘to 8∘C) Cell apoptosis was analyzed using flow cytometryand Cell Quest software All experiments were conducted intriplicate
27 Cell Cycle Detection Cells were collected and washedwith cold PBS twice resuspended in 1timesbinding buffer at aconcentration of 105-106 cellsml and transferred 100120583l of cell
suspension to a 5ml flow cytometry tube 5 120583l of Annexin V-PE was added to the cell suspension and incubated for 15minin dark at room temperature Another 400 120583l of bindingbuffer was added and then analyzed by flow cytometry Allexperiments were conducted in triplicate
28 Transwell Assay Matrigel was dissolved at 4 C overnightand diluted with precooled serum-free medium at a volumeratio of 13 40 ul Matrigel was added to a precooled transwellchamber and incubated at 37 C for 2 h to solidify The excessliquid in the chamber was discharged counted the cellscarefully and resuspended 1lowast105 cells in 200 ul serum-freemedia Cell suspension was added to the upper chamber and1000 ul complete medium was added to the lower chamberAfter incubation for 24 hours at 37∘C and 5 CO
2 the
chamber was removed and the cells of the upper chamberwere wiped away with a cotton swab The cells in lowerchamber were fixed by 4polyformaldehyde 15min and thenwashed by PBS and stained by crystal violet All experimentswere conducted in triplicate
29 Western Blot Cells were harvested lysed and cen-trifuged at 15000 rpm for 5min at 4∘C Protein content in thesupernatants was determined by a BCA protein assay kit [12]Equal amounts of protein were separated by 10 SDS-PAGEtransferred onto PVDFmembranes and then incubated withp-AKT (1500 Santa Cruz USA) p-p70S6K (1500 SantaCruz USA) and 120573-actin (12000 Santa Cruz USA) antibodyovernight at 4∘C After incubation with secondary antibody(KPL Gaithersburg MD) membrane was treated with ECL-Western blot detecting reagent (Amersham Biosciences KKTokyo Japan) Protein bands detected were estimated usingQuantity One software (Bio-Rad Laboratories HerculesCA) The density measurement was correlated with proteinexpression and normalized to 120573-actin All experiments wereconducted in triplicate
210 Quantitative Real-Time PCR Total mRNA wasextracted with TRIzol reagent (Invitrogen) cDNA wasthen generated from 10 120583g of total RNA with avianmyeloblastosis virus reverse transcriptase (Takara OtsuJapan) Amplification was carried out with an ABI PRISM7000 (Applied Biosystems Tokyo Japan) using SYBR greenreagent for detection according to the protocol of SYBRPremix Ex Taq kit (Takara) [13] All experiments wereconducted in triplicate
Primer sequences for Pgp were 51015840GGAGAGATCCTC-ACCAAGCG-31015840(sense) and 51015840-CGAGCCTGGTAGTCA-ATGCT-31015840(antisense) Primer sequences for MDR1 were51015840-TTTGGAGCCTACTTGGTGGC-31015840(sense) and 51015840-GCTTTGGCATAGTCAGGAGC-31015840(antisense) Primer se-quences for Caspase-3 were 51015840-TGCATACTCCACAGCACC-TG-31015840(sense) and 51015840-TCAAGCTTGTCGGCATACTGT-31015840(antisense) Primer sequences for MMP-9 were 51015840-GTACTCGACCTGTACCAGCG-31015840(sense) and 51015840-AGA-AGCCCCACTTCTTGTCG-31015840(antisense) Primer sequen-ces for 120573-actin were 51015840-GCATGGGTCAGAAGGATTCCT-31015840(sense) and 51015840-TCGTCCCAGTTGGTGACGAT-31015840(anti-sense)
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Figure 1 Identification of Ishikawa-TAX resistant cell lines and their proliferation rate (a) IC50 test of Ishikawa and Ishikawa-TAX cells (b)Cell proliferation assay of Ishikawa and Ishikawa-TAX cells (c) Ishikawa and Ishikawa-TAX resistance-related gene test (P lt005)
211 Statistical Analysis All the experiments were repeated 3times independently Data were expressed as x + s and wereanalyzed by SPSS 17 softwareMultiple groupswere comparedby ANOVA and post hoc comparisons were carried out byLSDmethodThe difference was statistically significant whenP lt 005
3 Results
31 Identification of Ishikawa-TAX Resistant Cell Lines andTheir Proliferation Rate The results of CCK-8 showed thatIC50 of Ishikawa-TAX group was significantly higher thanthat of Ishikawa group (Figure 1(a)) The proliferation rateof Ishikawa-TAX group was lower than Ishikawa group(Figure 1(b)) QPCR results showed that the resistance-related genes of Pgp and MDR1 were significantly higher inIshikawa-TAX group than in Ishikawa group (Figure 1(c))
32 NVP-BEZ235 Cotreatment with Paclitaxel Can FurtherEnhance Paclitaxel Inhibition of Ishikawa and Ishikawa-TAXCloning Formation Clone formation results showed thattreatment with NVP-BEZ235 alone did not inhibit Ishikawaclonal formation whereas paclitaxel alone inhibited Ishikawaclonality and cotreatment with paclitaxel NVP-BEZ235further inhibited Ishikawa clonality (Figure 2) Howeverin Ishikawa-TAX NVP-BEZ235 treatment and paclitaxeltreatment inhibited Ishikawa clonogenicity whereas NVP-BEZ235 and paclitaxel further inhibited clonogenicity ofIshikawa-TAX (Figure 2)
33 NVP-BEZ235 Cotreatment with Paclitaxel Can FurtherInhibit the Invasion of Ishikawa and Ishikawa-TAX CellsInvasion results showed that NVP-BEZ235 alone and pacli-taxel alone inhibited Ishikawa invasion and NVP-BEZ235cotreatment with paclitaxel further inhibited Ishikawa inva-sion (Figure 3) In Ishikawa-TAX NVP-BEZ235 alone andpaclitaxel alone inhibited the invasion of Ishikawa whereasNVP-BEZ235 cotreated with paclitaxel further inhibitedIshikawa-TAX invasion (Figure 3)
34 NVP-BEZ235 Cotreatment with Paclitaxel Can Signif-icantly Lead to an Increase in Apoptosis Rate IshikawaApoptosis results showed that cotreatment of NVP-BEZ235with paclitaxel significantly increased apoptosis of Ishikawawhile cotreatment of NVP-BEZ235 with paclitaxel did notresult in significant apoptosis of Ishikawa-TAX (Figure 4)
35 NVP-BEZ235 Cotreatment with Paclitaxel IncreasedIshikawa and Ishikawa-TAX Cells in the Proportion of G1Phase The results of cell cycle showed that NVP-BEZ235and paclitaxel alone increased the proportion of Ishikawaand Ishikawa-TAX cells in G1 phase while NVP-BEZ235cotreatment with paclitaxel further increased the proportionof Ishikawa and Ishikawa-TAX cells in G1 phase (Figure 5)
36 NVP-BEZ235 Cotreatment with Paclitaxel IncreasedIshikawa and Ishikawa-TAX Cells p-AKT and p-p70 S6KProtein Expression Western blotting results showed thatNVP-BEZ235 and paclitaxel alone treatment reduced p-AKT
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Figure 2 Clonogenicity after treatment of Ishikawa and Ishikawa-TAX cells with NVP-BEZ235 and paclitaxel (a) Cell cloning followingtreatment of Ishikawa and Ishikawa-TAXcells withNVP-BEZ235 alone and in combinationwith paclitaxel (b)Clone formation quantification(lowastP lt005 lowastlowastP lt001)
and p-p70 S6K protein levels in Ishikawa and Ishikawa-TAXcells while NVP-BEZ235 cotreatment with paclitaxel couldfurther reduced p-AKT and p-p70 S6K protein expression(Figure 6)
37 LY294002 Cotreatment with Paclitaxel Can SignificantlyLead to the Increase in Apoptosis Rate and the Proportionof G1 Phase Apoptosis results showed that cotreatment ofLY294002 with paclitaxel significantly increased apoptosis ofIshikawa-TAX while the treatment of LY294002 or paclitaxeldid not result in significant apoptosis of Ishikawa-TAX(Figure 7) The results of cell cycle showed that cotreatmentof LY294002 and paclitaxel increased the proportion ofIshikawa-TAX cells in G1 phase (Figure 8)
38 LY294002 Cotreatment with Paclitaxel Decreased theExpression of p-AKT and p-p70 S6K Protein in Ishikawa-TAX Cells Western blotting results showed that LY294002and paclitaxel alone treatment reduced p-AKT and p-p70S6K protein levels in Ishikawa-TAX cells while LY294002
cotreatmentwith paclitaxel could further reduced p-AKTandp-p70 S6K protein expression (Figure 9)
4 Discussion
Endometrial cancer is a malignant tumor which originatesfrom endometrial glands with a high incidence It accountsfor 20-30 of the female reproductive system malignanttumors which seriously endangers womenrsquos health and life[14 15] Chemotherapy is the common treatment for endome-trial cancer and the sensitivity of tumor cells makes a big dif-ference in the prognosis Improving sensitivity and reducingof drug resistance will raise the survival rate of endometrialcancer and therefore improve the patientsrsquo life quality [16 17]Paclitaxel is one of the most commonly used chemotherapydrug which can be applied in endometrial cancer coloncancer and other malignancies [18ndash20] However in clinicemergence of paclitaxel resistance greatly affects its effect andrestricts its application as a chemotherapeutic drug
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In this study we used the taxol concentration gradientmethod to establish the paclitaxel resistant cell line (Ishikawa-TAX cells) by repeated induction of Ishikawa parent cellsThrough its IC50 resistance-related genes (Pgp and MDR1)were detected which confirmed that the constructed cellline was Ishikawa-TAX (Figure 1) The results showed thatPgp and MDR1 were increased in Ishikawa-TAX cells whichindicated that it was a promising therapy to focus on thegenes of Pgp and MDR1 in Ishikawa-TAX cells So far themechanism of Ishikawa cellsrsquo resistance to paclitaxel has notbeen clearly illustrated and need further study
The phosphatidylinositol-3-kinase (PI3K)AktmTORpathway is a commonly activated signaling pathway incancer which play an important role in drug resistance ofcancers including prostate cancer and lung cancer [21 22]while the relationships between Ishikawa and taxol is stillunclear In this study we used NVP-BEZ235 which is theinhibitor of PI3KAktmTOR pathway [23] NVP-BEZ235is also a promising chemotherapy drug in clinical trials formany cancers such as colon cancer [24 25]
Compared with paclitaxel alone the combined treat-ment of NVP-BEZ235 and paclitaxel further enhanced the
inhibitory effect on the clone formation and invasion ofIshikawa and Ishikawa-TAX cells (Figures 2 and 3) Thissuggests that the inhibitory effect of taxol on Ishikawa andIshikawa-TAX is mediated by the PI3KmTOR signalingpathway In apoptosis detection the combined treatment ofNVP-BEZ235 and paclitaxel showed similar result and raisedthe apoptosis induction effect of taxol on Ishikawa cells (Fig-ure 4) The combined treatment also enhanced the arrest ofG0G1 cells in Ishikawa and Ishikawa-TAX cellsThese resultscollectively showed that the cotreatment of NVP-BEZ235 andpaclitaxel could lead to a decrease in the proliferation activityof Ishikawa and Ishikawa-TAX cells (Figure 5) When welooked into the underlying mechanism we found that simplepaclitaxel treatment could significantly inhibit the expressionof p-AK and p-p70 S6K protein in Ishikawa and Ishikawa-TAX cells When combined with paclitaxel NVP-BEZ235can further inhibit the expression of p-AKT and p-p70 S6Kprotein in Ishikawa and Ishikawa-TAX cells indicating thatpaclitaxel inhibits Ishikawa and Ishikawa-TAX cells throughPI3KAktmTOR (Figure 6) LY294002 an inhibitor of PI3K-AKT pathway was used to further confirm the function ofAKT pathway Results showed that the apoptosis rate and
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Figure 5 Cell cycle changes after NVP-BEZ235 and paclitaxel treatment of Ishikawa and Ishikawa-TAX cells The cell cycle of Ishikawa andIshikawa-TAX cells changes of G0G1 S G2M cycles after NVP-BEZ235 and paclitaxel alone and in cotreatment (lowastlowastP lt001)
BioMed Research International 7
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8 BioMed Research International
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02+T
AX
lowastlowastlowastlowast lowastlowast
10
02
04
06
08
00
Figure 9 Changes of p-AKT and p-p70 S6K proteins after treatment with LY294002 and paclitaxel in Ishikawa-TAX cells (a) Changes ofp-AKT and p-p70 S6K proteins after treatment of Ishikawa-TAX cells with LY294002 and paclitaxel alone and in combination (b) Proteinrelative quantification (lowastlowastP lt001)
BioMed Research International 9
cell cycle were not different between Ishikawa group andIshikawa-TAX + TAX group which suggested that Ishikawa-TAX had the ability of resistance for paclitaxel (Figures 7 and8) What is more the results showed that paclitaxel inhibitsIshikawa-TAX cells through PI3KAktmTOR (Figures 7 8and 9)
Nowadays more and more clinical trials focus on thecombination of paxlitaxel with other PI3K inhibitors incancer treatment such as rapamycin and TORC12 inhibitorTAK-228 [26 27] It shows that PI3Kpathway plays an impor-tant role in cancer growth invasion and drug resistance
In summary taxol treatment can inhibit the Ishikawa cellsand Ishikawa-TAX cells through PI3KAktmTOR signalingpathway and therefore regulating PI3KAktmTOR pathwaymay been a promising therapeutic direction for the clinicaltreatment of drug-resistant endometrial carcinoma
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors have no conflicts of interest to declare
Authorsrsquo Contributions
Jie Ding and Mengxiong Li contributed equally to this work
Acknowledgments
This study was funded by Guangdong Medical ResearchFoundation (Grant A2014235) and Guangdong ProvincialScience and Technology Research (Grants 2013B0218000902013B022000044 and 2016A020215074)
References
[1] T Yamamoto T Mori M Sawada et al ldquoLoss of AF-6afadininduces cell invasion suppresses the formation of glandularstructures and might be a predictive marker of resistance tochemotherapy in endometrial cancerrdquo BMC Cancer vol 15 no1 article no 275 2015
[2] L Hoang S Chiang and C-H Lee ldquoEndometrial stromalsarcomas and related neoplasms New developments and diag-nostic considerationsrdquo Pathology 2017
[3] A Mihailovici M Rottenstreich S Kovel I Wassermann NSmorgick andZVaknin ldquoEndometriosis-associatedmalignanttransformation in abdominal surgical scarrdquo Medicine (UnitedStates) vol 96 no 49 Article ID e9136 2017
[4] VMakker A K Green RMWenham DMutch B Davidsonand D S Miller ldquoNew therapies for advanced recurrent andmetastatic endometrial cancersrdquoGynecologic Oncology Researchand Practice vol 4 no 1 2017
[5] N Dadi M Stanley S Shahda B H Orsquoneil and A SehdevldquoImpact of nab-paclitaxel-based second-line chemotherapy inmetastatic pancreatic cancerrdquoAnticancer Reseach vol 37 no 10pp 5533ndash5539 2017
[6] C M Bestvina and G F Fleming ldquoChemotherapy for endome-trial cancer in adjuvant and advanced disease settingsrdquo TheOncologist vol 21 no 10 pp 1250ndash1259 2016
[7] P Augereau A Patsouris E Bourbouloux et al ldquoHormonore-sistance in advanced breast cancer A new revolution inendocrine therapyrdquoTherapeutic Advances in Medical Oncologyvol 9 no 5 pp 335ndash346 2017
[8] D Xu R Li J Wu L Jiang and H A Zhong ldquoDrug designtargeting theCXCR4CXCR7CXCL12 pathwayrdquoCurrent Topicsin Medicinal Chemistry vol 16 no 13 pp 1441ndash1451 2016
[9] K Yuge E Kikuchi M Hagiwara et al ldquoNicotine inducestumor growth and chemoresistance through activation of thePI3KAktmTOR pathway in bladder cancerrdquoMolecular CancerTherapeutics vol 14 no 9 pp 2112ndash2120 2015
[10] V Makker F O Recio L Ma et al ldquoA multicenter single-arm open-label phase 2 study of apitolisib (GDC-0980) forthe treatment of recurrent or persistent endometrial carcinoma(MAGGIE study)rdquo Cancer vol 122 no 22 pp 3519ndash3528 2016
[11] K Inaba K Oda Y Ikeda et al ldquoAntitumor activity of acombination of dual PI3KmTOR inhibitor SAR245409 andselective MEK12 inhibitor pimasertib in endometrial carcino-masrdquo Gynecologic Oncology vol 138 no 2 pp 323ndash331 2015
[12] H K Kim S-H Hwang E Oh and S Abdi ldquoRolipram aselective phosphodiesterase 4 inhibitor ameliorates mechanicalhyperalgesia in a rat model of chemotherapy-induced neuro-pathic pain through inhibition of inflammatory cytokines in thedorsal root ganglionrdquo Frontiers in Pharmacology vol 8 2017
[13] X Chen X Sun J Guan et al ldquoRsf-1 Influences the Sensitivityof Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-120581B Pathway and Its Downstream Proteinsrdquo Cellular Physiologyand Biochemistry vol 44 no 6 pp 2322ndash2336 2018
[14] Y C Tsai Y H Chang Y Chang and C M Chuang ldquoSalvageintraperitoneal chemotherapy for relapsed type II endometrialcancer A pilot case-control studyrdquo Journal of the TurkishGerman Gynecological Association vol 17 no 4 pp 176ndash1812016
[15] L H Tan P H Sykes M M Alkaisi and J J Evans ldquoCell-likefeatures imprinted in the physical nano- andmicro-topographyof the environment modify the responses to anti-cancer drugsof endometrial cancer cellsrdquo Biofabrication vol 9 no 1 ArticleID 015017 2017
[16] K Ding Y Yuan Q-Y Chong et al ldquoAutocrine prolactinstimulates endometrial carcinoma growth and metastasis andreduces sensitivity to chemotherapyrdquo Endocrinology vol 158no 6 pp 1595ndash1611 2017
[17] L Kogan I Laskov Z Amajoud et al ldquoDose dense carboplatinpaclitaxel improves progression free survival in patients withendometrial cancerrdquo Gynecologic Oncology vol 147 no 1 pp30ndash35 2017
[18] H Koh H Park N Chandimali et al ldquoMicroRNA-128 sup-presses paclitaxel-resistant lung cancer by inhibiting MUC1-Cand BMI-1 in cancer stem cellsrdquo Oncotarget vol 8 no 66 pp110540ndash110551 2017
[19] S Tiwari B Tirosh and A Rubinstein ldquoIncreasing the affinityof cationized polyacrylamide-paclitaxel nanoparticles towardscolon cancer cells by a surface recognition peptiderdquo Interna-tional Journal of Pharmaceutics vol 531 no 1 pp 281ndash291 2017
[20] M Xiong Q Lei X You et al ldquoMannosylated liposomesimprove therapeutic effects of paclitaxel in colon cancer mod-elsrdquo Journal of Microencapsulation vol 34 no 6 pp 513ndash5212017
10 BioMed Research International
[21] S Xu J Ge Z Zhang and W Zhou ldquoMiR-129 inhibitscell proliferation and metastasis by targeting ETS1 viaPI3KAKTmTOR pathway in prostate cancerrdquo Biomedicine ampPharmacotherapy vol 96 pp 634ndash641 2017
[22] P-P Gan Y-Y Zhou M-Z Zhong Y Peng L Li and J-HLi ldquoEndoplasmic Reticulum Stress Promotes Autophagy andApoptosis and Reduces Chemotherapy Resistance in Mutantp53 Lung Cancer Cellsrdquo Cellular Physiology and Biochemistrypp 133ndash151 2017
[23] G Wu Z Sun Q Li et al ldquoNVP-BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3KAKT mTORphosphorylationrdquo OncoTargets andTherapy p 269
[24] J Chen R Shao F Li et al ldquoPI3KAktmTOR pathway dualinhibitor BEZ235 suppresses the stemness of colon cancer stemcellsrdquo Clinical and Experimental Pharmacology and Physiologyvol 42 no 12 pp 1317ndash1326 2015
[25] J Chen R Shao L Li Z P Xu and W Gu ldquoEffective inhi-bition of colon cancer cell growth with MgAl-layered doublehydroxide (LDH) loaded 5-FU and PI3KmTOR dual inhibitorBEZ-235 through apoptotic pathwaysrdquo International Journal ofNanomedicine vol 9 no 1 pp 3403ndash3411 2014
[26] D A Sabbah M G Brattain and H Zhong ldquoDual inhibitorsof PI3KmTOR or mTOR-selective inhibitors Which way shallwe gordquo Current Medicinal Chemistry vol 18 no 36 pp 5528ndash5544 2011
[27] H A Burris C D Kurkjian L Hart et al ldquoTAK-228 (formerlyMLN0128) an investigational dual TORC12 inhibitor pluspaclitaxel withwithout trastuzumab in patients with advancedsolid malignanciesrdquo Cancer Chemotherapy and Pharmacologyvol 80 no 2 pp 261ndash273 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
2 BioMed Research International
RPMI1640 culture medium containing 10 FBS 1 peni-cillin and 100 Uml streptomycin at 37∘C and 5 CO
2
22 Construction of Paclitaxel Resistant Cell Line (Ishikawa-TAX Cells) The MTT method was used to detect IC50 inIshikawa cells When the parent cell confluence reached80 the cells are treated with taxol (sigma USA) at theconcentration of 110 IC50 The concentration of taxol waskept in the culture medium by changing medium with taxolWhen cell growth was stable in the low concentration taxolmedium (110 IC50) the taxol concentration was raisedgradually until 5-10 times of IC50 The treated Ishikawa cellsgrew well with the high concentration of taxol and thereforethe paclitaxel resistant cell line (Ishikawa-TAX cells) wassuccessfully established
23 NVP-BEZ235 and Paclitaxel Treatment When cells grewto 80 density NVP-BEZ235 (MedChemExpress USA) wasadded to medium at concentration of 5 nM After 6 hoursTAXwas added to 001 ugml for 24 hours then the cells werecollected for follow-up experiments
24 MTT Assay Cells at logarithmic growth phase werecollected and suspended at the concentration of 104-105 ml100 ul of cell suspension was added to each pore of 96-wellplate and MTT assay was carried out on the correspondingday 20 ul MTT solution was added and the cells were incu-bated for 4 h The supernatant was then carefully dischargedand 150 ul DMSO was added to dissolve the crystallizationThe absorbance at OD492 nm was measured by MicroplateReader
25 Clone Formation Cell suspension was prepared bytrypsin-EDTAdigestion and cell densitywas adjusted accord-ing to the number of Trypan blue positive staining cells 100living cells were inoculated in each hole of 6-well plates andthen the cells were cultured for 15 days at normal conditionAt day 15 the culture medium was discharged and cells werefixed by paraformaldehyde After washing by PBS the cellswere stained by crystal violet for 10minThenumber of cloneswas counted All experiments were conducted in triplicate
26 Cell Apoptosis Detection Cells were harvested by trypsinand collected by centrifugation at 400timesg for 5 minutes Akit from BD company was used for cell apoptosis analy-sis Carefully decant all the supernatant and add 250 120583Lof Solution A (trypsin buffer) for 10 minutes and 200120583Lof Solution B (trypsin inhibitor and RNase buffer for) 10minutes Do not remove Solution A and Solution B Add200120583L of cold Solution C (propidium iodide stain solution)incubate for 10 minutes in the dark in the refrigerator (2∘to 8∘C) Cell apoptosis was analyzed using flow cytometryand Cell Quest software All experiments were conducted intriplicate
27 Cell Cycle Detection Cells were collected and washedwith cold PBS twice resuspended in 1timesbinding buffer at aconcentration of 105-106 cellsml and transferred 100120583l of cell
suspension to a 5ml flow cytometry tube 5 120583l of Annexin V-PE was added to the cell suspension and incubated for 15minin dark at room temperature Another 400 120583l of bindingbuffer was added and then analyzed by flow cytometry Allexperiments were conducted in triplicate
28 Transwell Assay Matrigel was dissolved at 4 C overnightand diluted with precooled serum-free medium at a volumeratio of 13 40 ul Matrigel was added to a precooled transwellchamber and incubated at 37 C for 2 h to solidify The excessliquid in the chamber was discharged counted the cellscarefully and resuspended 1lowast105 cells in 200 ul serum-freemedia Cell suspension was added to the upper chamber and1000 ul complete medium was added to the lower chamberAfter incubation for 24 hours at 37∘C and 5 CO
2 the
chamber was removed and the cells of the upper chamberwere wiped away with a cotton swab The cells in lowerchamber were fixed by 4polyformaldehyde 15min and thenwashed by PBS and stained by crystal violet All experimentswere conducted in triplicate
29 Western Blot Cells were harvested lysed and cen-trifuged at 15000 rpm for 5min at 4∘C Protein content in thesupernatants was determined by a BCA protein assay kit [12]Equal amounts of protein were separated by 10 SDS-PAGEtransferred onto PVDFmembranes and then incubated withp-AKT (1500 Santa Cruz USA) p-p70S6K (1500 SantaCruz USA) and 120573-actin (12000 Santa Cruz USA) antibodyovernight at 4∘C After incubation with secondary antibody(KPL Gaithersburg MD) membrane was treated with ECL-Western blot detecting reagent (Amersham Biosciences KKTokyo Japan) Protein bands detected were estimated usingQuantity One software (Bio-Rad Laboratories HerculesCA) The density measurement was correlated with proteinexpression and normalized to 120573-actin All experiments wereconducted in triplicate
210 Quantitative Real-Time PCR Total mRNA wasextracted with TRIzol reagent (Invitrogen) cDNA wasthen generated from 10 120583g of total RNA with avianmyeloblastosis virus reverse transcriptase (Takara OtsuJapan) Amplification was carried out with an ABI PRISM7000 (Applied Biosystems Tokyo Japan) using SYBR greenreagent for detection according to the protocol of SYBRPremix Ex Taq kit (Takara) [13] All experiments wereconducted in triplicate
Primer sequences for Pgp were 51015840GGAGAGATCCTC-ACCAAGCG-31015840(sense) and 51015840-CGAGCCTGGTAGTCA-ATGCT-31015840(antisense) Primer sequences for MDR1 were51015840-TTTGGAGCCTACTTGGTGGC-31015840(sense) and 51015840-GCTTTGGCATAGTCAGGAGC-31015840(antisense) Primer se-quences for Caspase-3 were 51015840-TGCATACTCCACAGCACC-TG-31015840(sense) and 51015840-TCAAGCTTGTCGGCATACTGT-31015840(antisense) Primer sequences for MMP-9 were 51015840-GTACTCGACCTGTACCAGCG-31015840(sense) and 51015840-AGA-AGCCCCACTTCTTGTCG-31015840(antisense) Primer sequen-ces for 120573-actin were 51015840-GCATGGGTCAGAAGGATTCCT-31015840(sense) and 51015840-TCGTCCCAGTTGGTGACGAT-31015840(anti-sense)
BioMed Research International 3
010
008
006
004
002
000
IC50
(ug
ml)
ishikawa ishikawa-TAX
lowastlowast
(a)
ishikawaishikawa-TAX
20
15
10
05
00
OD
(492
nm)
Days3210
lowastlowast
lowastlowast
lowastlowast
(b)
ishikawa ishikawa-TAX
30
20
10
0
Retiv
e mRN
A ex
pres
sion
Pgplowastlowast
(c)ishikawa ishikawa-TAX
40
30
20
10
0
Retiv
e mRN
A ex
pres
sion
MDR1
lowastlowast
(d)
Figure 1 Identification of Ishikawa-TAX resistant cell lines and their proliferation rate (a) IC50 test of Ishikawa and Ishikawa-TAX cells (b)Cell proliferation assay of Ishikawa and Ishikawa-TAX cells (c) Ishikawa and Ishikawa-TAX resistance-related gene test (P lt005)
211 Statistical Analysis All the experiments were repeated 3times independently Data were expressed as x + s and wereanalyzed by SPSS 17 softwareMultiple groupswere comparedby ANOVA and post hoc comparisons were carried out byLSDmethodThe difference was statistically significant whenP lt 005
3 Results
31 Identification of Ishikawa-TAX Resistant Cell Lines andTheir Proliferation Rate The results of CCK-8 showed thatIC50 of Ishikawa-TAX group was significantly higher thanthat of Ishikawa group (Figure 1(a)) The proliferation rateof Ishikawa-TAX group was lower than Ishikawa group(Figure 1(b)) QPCR results showed that the resistance-related genes of Pgp and MDR1 were significantly higher inIshikawa-TAX group than in Ishikawa group (Figure 1(c))
32 NVP-BEZ235 Cotreatment with Paclitaxel Can FurtherEnhance Paclitaxel Inhibition of Ishikawa and Ishikawa-TAXCloning Formation Clone formation results showed thattreatment with NVP-BEZ235 alone did not inhibit Ishikawaclonal formation whereas paclitaxel alone inhibited Ishikawaclonality and cotreatment with paclitaxel NVP-BEZ235further inhibited Ishikawa clonality (Figure 2) Howeverin Ishikawa-TAX NVP-BEZ235 treatment and paclitaxeltreatment inhibited Ishikawa clonogenicity whereas NVP-BEZ235 and paclitaxel further inhibited clonogenicity ofIshikawa-TAX (Figure 2)
33 NVP-BEZ235 Cotreatment with Paclitaxel Can FurtherInhibit the Invasion of Ishikawa and Ishikawa-TAX CellsInvasion results showed that NVP-BEZ235 alone and pacli-taxel alone inhibited Ishikawa invasion and NVP-BEZ235cotreatment with paclitaxel further inhibited Ishikawa inva-sion (Figure 3) In Ishikawa-TAX NVP-BEZ235 alone andpaclitaxel alone inhibited the invasion of Ishikawa whereasNVP-BEZ235 cotreated with paclitaxel further inhibitedIshikawa-TAX invasion (Figure 3)
34 NVP-BEZ235 Cotreatment with Paclitaxel Can Signif-icantly Lead to an Increase in Apoptosis Rate IshikawaApoptosis results showed that cotreatment of NVP-BEZ235with paclitaxel significantly increased apoptosis of Ishikawawhile cotreatment of NVP-BEZ235 with paclitaxel did notresult in significant apoptosis of Ishikawa-TAX (Figure 4)
35 NVP-BEZ235 Cotreatment with Paclitaxel IncreasedIshikawa and Ishikawa-TAX Cells in the Proportion of G1Phase The results of cell cycle showed that NVP-BEZ235and paclitaxel alone increased the proportion of Ishikawaand Ishikawa-TAX cells in G1 phase while NVP-BEZ235cotreatment with paclitaxel further increased the proportionof Ishikawa and Ishikawa-TAX cells in G1 phase (Figure 5)
36 NVP-BEZ235 Cotreatment with Paclitaxel IncreasedIshikawa and Ishikawa-TAX Cells p-AKT and p-p70 S6KProtein Expression Western blotting results showed thatNVP-BEZ235 and paclitaxel alone treatment reduced p-AKT
4 BioMed Research International
Ishikawa Ishikawa+TAXIshikawa+NVP-BEZ235Ishikawa+NVP-BEZ235
+TAX
Ishikawa-TAX Ishikawa-TAX +NVP-BEZ235
Ishikawa-TAX +TAX
Ishikawa-TAX +NVP-BEZ235
+TAX(a)
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
150
100
50
0Col
ony
num
ber p
er w
ell
lowastlowast
lowastlowastlowastlowast
lowastlowast
(b)
Figure 2 Clonogenicity after treatment of Ishikawa and Ishikawa-TAX cells with NVP-BEZ235 and paclitaxel (a) Cell cloning followingtreatment of Ishikawa and Ishikawa-TAXcells withNVP-BEZ235 alone and in combinationwith paclitaxel (b)Clone formation quantification(lowastP lt005 lowastlowastP lt001)
and p-p70 S6K protein levels in Ishikawa and Ishikawa-TAXcells while NVP-BEZ235 cotreatment with paclitaxel couldfurther reduced p-AKT and p-p70 S6K protein expression(Figure 6)
37 LY294002 Cotreatment with Paclitaxel Can SignificantlyLead to the Increase in Apoptosis Rate and the Proportionof G1 Phase Apoptosis results showed that cotreatment ofLY294002 with paclitaxel significantly increased apoptosis ofIshikawa-TAX while the treatment of LY294002 or paclitaxeldid not result in significant apoptosis of Ishikawa-TAX(Figure 7) The results of cell cycle showed that cotreatmentof LY294002 and paclitaxel increased the proportion ofIshikawa-TAX cells in G1 phase (Figure 8)
38 LY294002 Cotreatment with Paclitaxel Decreased theExpression of p-AKT and p-p70 S6K Protein in Ishikawa-TAX Cells Western blotting results showed that LY294002and paclitaxel alone treatment reduced p-AKT and p-p70S6K protein levels in Ishikawa-TAX cells while LY294002
cotreatmentwith paclitaxel could further reduced p-AKTandp-p70 S6K protein expression (Figure 9)
4 Discussion
Endometrial cancer is a malignant tumor which originatesfrom endometrial glands with a high incidence It accountsfor 20-30 of the female reproductive system malignanttumors which seriously endangers womenrsquos health and life[14 15] Chemotherapy is the common treatment for endome-trial cancer and the sensitivity of tumor cells makes a big dif-ference in the prognosis Improving sensitivity and reducingof drug resistance will raise the survival rate of endometrialcancer and therefore improve the patientsrsquo life quality [16 17]Paclitaxel is one of the most commonly used chemotherapydrug which can be applied in endometrial cancer coloncancer and other malignancies [18ndash20] However in clinicemergence of paclitaxel resistance greatly affects its effect andrestricts its application as a chemotherapeutic drug
BioMed Research International 5
Ishikawa Ishikawa+TAXIshikawa+NVP-BEZ235 Ishikawa+NVP-BEZ235+TAX
Ishikawa-TAXIshikawa-TAX +NVP-BEZ235
Ishikawa-TAX +TAX
Ishikawa-TAX+NVP -BEZ235 +TAX
(a)
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
150
200
250
100
50
0
Inva
ded
num
ber p
er w
ell
lowastlowast lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
(b)
Figure 3The cell invasion of Ishikawa and Ishikawa-TAX cells after NVP-BEZ235 treatment with paclitaxel (a)The cell invasion of Ishikawaand Ishikawa-TAX cells treated byNVP-BEZ235 alone and cotreated with paclitaxel treatment (b)The number of cell invasion was quantified(lowastlowastP lt001)
In this study we used the taxol concentration gradientmethod to establish the paclitaxel resistant cell line (Ishikawa-TAX cells) by repeated induction of Ishikawa parent cellsThrough its IC50 resistance-related genes (Pgp and MDR1)were detected which confirmed that the constructed cellline was Ishikawa-TAX (Figure 1) The results showed thatPgp and MDR1 were increased in Ishikawa-TAX cells whichindicated that it was a promising therapy to focus on thegenes of Pgp and MDR1 in Ishikawa-TAX cells So far themechanism of Ishikawa cellsrsquo resistance to paclitaxel has notbeen clearly illustrated and need further study
The phosphatidylinositol-3-kinase (PI3K)AktmTORpathway is a commonly activated signaling pathway incancer which play an important role in drug resistance ofcancers including prostate cancer and lung cancer [21 22]while the relationships between Ishikawa and taxol is stillunclear In this study we used NVP-BEZ235 which is theinhibitor of PI3KAktmTOR pathway [23] NVP-BEZ235is also a promising chemotherapy drug in clinical trials formany cancers such as colon cancer [24 25]
Compared with paclitaxel alone the combined treat-ment of NVP-BEZ235 and paclitaxel further enhanced the
inhibitory effect on the clone formation and invasion ofIshikawa and Ishikawa-TAX cells (Figures 2 and 3) Thissuggests that the inhibitory effect of taxol on Ishikawa andIshikawa-TAX is mediated by the PI3KmTOR signalingpathway In apoptosis detection the combined treatment ofNVP-BEZ235 and paclitaxel showed similar result and raisedthe apoptosis induction effect of taxol on Ishikawa cells (Fig-ure 4) The combined treatment also enhanced the arrest ofG0G1 cells in Ishikawa and Ishikawa-TAX cellsThese resultscollectively showed that the cotreatment of NVP-BEZ235 andpaclitaxel could lead to a decrease in the proliferation activityof Ishikawa and Ishikawa-TAX cells (Figure 5) When welooked into the underlying mechanism we found that simplepaclitaxel treatment could significantly inhibit the expressionof p-AK and p-p70 S6K protein in Ishikawa and Ishikawa-TAX cells When combined with paclitaxel NVP-BEZ235can further inhibit the expression of p-AKT and p-p70 S6Kprotein in Ishikawa and Ishikawa-TAX cells indicating thatpaclitaxel inhibits Ishikawa and Ishikawa-TAX cells throughPI3KAktmTOR (Figure 6) LY294002 an inhibitor of PI3K-AKT pathway was used to further confirm the function ofAKT pathway Results showed that the apoptosis rate and
6 BioMed Research International
Ishikawa Ishikawa+TAXIshikawa+NVP-BEZ235 Ishikawa+NVP-BEZ235+TAX
Ishikawa-TAX Ishikawa-TAX +NVP-BEZ235
Ishikawa-TAX +TAX
Ishikawa-TAX +NVP-BEZ235
+TAX
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
R2
9342 022
632 004Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9480 036
436 048Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9492 186
280 042Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9186 366
310 138
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
g
R2
9472 024
498 006Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9546 056
386 012Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9552 066
370 012Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9524 044
410 022
ANNEXINV-PEANNEXINV-PE ANNEXINV-PE
ANNEXINV-PE ANNEXINV-PE
ANNEXINV-PE
ANNEXINV-PEANNEXINV-PE
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
104103102101100 104103102101100 104103102101100 104103102101100
104103102101100 104103102101100 104103102101100 104103102101100
(a)
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
Apop
tosis
rate
() 15
10
5
0
lowastlowast
(b)
Figure 4 The apoptosis of Ishikawa and Ishikawa-TAX cells after treatment of NVP-BEZ235 and paclitaxel (a) NVP-BEZ235 alone andcotreated with paclitaxel Ishikawa and Ishikawa-TAX cells and the changes in apoptosis (b) Apoptosis quantification (lowastlowastP lt001)
Ratio
()
100
80
60
40
20
0
lowastlowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
01 S 2－
Cycle
Ishikawa
Ishikawa+TAX
Ishikawa+NVP-BEZ235
Ishikawa+NVP-BEZ235+TAX
Ishikawa-TAX
Ishikawa-TAX+NVP-BEZ235
Ishikawa-TAX+TAX
Ishikawa-TAX+NVP-BEZ235+TAX
Figure 5 Cell cycle changes after NVP-BEZ235 and paclitaxel treatment of Ishikawa and Ishikawa-TAX cells The cell cycle of Ishikawa andIshikawa-TAX cells changes of G0G1 S G2M cycles after NVP-BEZ235 and paclitaxel alone and in cotreatment (lowastlowastP lt001)
BioMed Research International 7
P-AKT
P-p70 S6K
-actin
(a)
Relat
ive e
xpre
ssio
nP-
AKT
-a
ctin lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
lowastlowast3
2
1
0
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
Relat
ive e
xpre
ssio
nP-
p70
S6K
-a
ctin
20
15
10
05
00
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
(b)Figure 6 Changes of p-AKT and p-p70 S6K proteins after treatment with NVP-BEZ235 and paclitaxel in Ishikawa and Ishikawa-TAX cells(a) Changes of p-AKT and p-p70 S6K proteins after treatment of Ishikawa and Ishikawa-TAX cells with NVP-BEZ235 and paclitaxel aloneand in combination (b) Protein relative quantification (lowastlowastP lt001)
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
Apop
tosis
rate
()
15
20
10
5
0
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
lowastlowast
Figure 7 The apoptosis of Ishikawa-TAX cells after treatment of LY294002 and paclitaxel (a) LY294002 alone and cotreated with paclitaxelIshikawa-TAX cells the changes in apoptosis (b) Apoptosis quantification (lowastlowastP lt001)
8 BioMed Research International
Ratio
()
lowastlowast
lowastlowast
lowastlowast
01 S 2－
Cycle
Ishikawa
Ishikawa-TAX+LY294002
Ishikawa-TAX+TAX
Ishikawa-TAX+LY294002+TAX
lowastlowast
lowastlowast
lowastlowast
100
80
60
40
20
0
lowastlowast
lowastlowast
lowastlowast
Figure 8 Cell cycle changes after LY294002 and paclitaxel treatment of Ishikawa-TAX cells The cell cycle of Ishikawa-TAX cells changes ofG0 G1 S G2 M cycles after LY294002 and paclitaxel alone and in cotreatment (lowastlowastP lt001)
P-AKT
P-p70S6K
-actin
lowastlowast
lowastlowast
lowastlowast
Relat
ive e
xpre
ssio
nP-
p70
S6K
-a
ctin
10
02
04
06
08
00
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
Relat
ive e
xpre
ssio
nP-
AKT
-a
ctin
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
lowastlowastlowastlowast lowastlowast
10
02
04
06
08
00
Figure 9 Changes of p-AKT and p-p70 S6K proteins after treatment with LY294002 and paclitaxel in Ishikawa-TAX cells (a) Changes ofp-AKT and p-p70 S6K proteins after treatment of Ishikawa-TAX cells with LY294002 and paclitaxel alone and in combination (b) Proteinrelative quantification (lowastlowastP lt001)
BioMed Research International 9
cell cycle were not different between Ishikawa group andIshikawa-TAX + TAX group which suggested that Ishikawa-TAX had the ability of resistance for paclitaxel (Figures 7 and8) What is more the results showed that paclitaxel inhibitsIshikawa-TAX cells through PI3KAktmTOR (Figures 7 8and 9)
Nowadays more and more clinical trials focus on thecombination of paxlitaxel with other PI3K inhibitors incancer treatment such as rapamycin and TORC12 inhibitorTAK-228 [26 27] It shows that PI3Kpathway plays an impor-tant role in cancer growth invasion and drug resistance
In summary taxol treatment can inhibit the Ishikawa cellsand Ishikawa-TAX cells through PI3KAktmTOR signalingpathway and therefore regulating PI3KAktmTOR pathwaymay been a promising therapeutic direction for the clinicaltreatment of drug-resistant endometrial carcinoma
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors have no conflicts of interest to declare
Authorsrsquo Contributions
Jie Ding and Mengxiong Li contributed equally to this work
Acknowledgments
This study was funded by Guangdong Medical ResearchFoundation (Grant A2014235) and Guangdong ProvincialScience and Technology Research (Grants 2013B0218000902013B022000044 and 2016A020215074)
References
[1] T Yamamoto T Mori M Sawada et al ldquoLoss of AF-6afadininduces cell invasion suppresses the formation of glandularstructures and might be a predictive marker of resistance tochemotherapy in endometrial cancerrdquo BMC Cancer vol 15 no1 article no 275 2015
[2] L Hoang S Chiang and C-H Lee ldquoEndometrial stromalsarcomas and related neoplasms New developments and diag-nostic considerationsrdquo Pathology 2017
[3] A Mihailovici M Rottenstreich S Kovel I Wassermann NSmorgick andZVaknin ldquoEndometriosis-associatedmalignanttransformation in abdominal surgical scarrdquo Medicine (UnitedStates) vol 96 no 49 Article ID e9136 2017
[4] VMakker A K Green RMWenham DMutch B Davidsonand D S Miller ldquoNew therapies for advanced recurrent andmetastatic endometrial cancersrdquoGynecologic Oncology Researchand Practice vol 4 no 1 2017
[5] N Dadi M Stanley S Shahda B H Orsquoneil and A SehdevldquoImpact of nab-paclitaxel-based second-line chemotherapy inmetastatic pancreatic cancerrdquoAnticancer Reseach vol 37 no 10pp 5533ndash5539 2017
[6] C M Bestvina and G F Fleming ldquoChemotherapy for endome-trial cancer in adjuvant and advanced disease settingsrdquo TheOncologist vol 21 no 10 pp 1250ndash1259 2016
[7] P Augereau A Patsouris E Bourbouloux et al ldquoHormonore-sistance in advanced breast cancer A new revolution inendocrine therapyrdquoTherapeutic Advances in Medical Oncologyvol 9 no 5 pp 335ndash346 2017
[8] D Xu R Li J Wu L Jiang and H A Zhong ldquoDrug designtargeting theCXCR4CXCR7CXCL12 pathwayrdquoCurrent Topicsin Medicinal Chemistry vol 16 no 13 pp 1441ndash1451 2016
[9] K Yuge E Kikuchi M Hagiwara et al ldquoNicotine inducestumor growth and chemoresistance through activation of thePI3KAktmTOR pathway in bladder cancerrdquoMolecular CancerTherapeutics vol 14 no 9 pp 2112ndash2120 2015
[10] V Makker F O Recio L Ma et al ldquoA multicenter single-arm open-label phase 2 study of apitolisib (GDC-0980) forthe treatment of recurrent or persistent endometrial carcinoma(MAGGIE study)rdquo Cancer vol 122 no 22 pp 3519ndash3528 2016
[11] K Inaba K Oda Y Ikeda et al ldquoAntitumor activity of acombination of dual PI3KmTOR inhibitor SAR245409 andselective MEK12 inhibitor pimasertib in endometrial carcino-masrdquo Gynecologic Oncology vol 138 no 2 pp 323ndash331 2015
[12] H K Kim S-H Hwang E Oh and S Abdi ldquoRolipram aselective phosphodiesterase 4 inhibitor ameliorates mechanicalhyperalgesia in a rat model of chemotherapy-induced neuro-pathic pain through inhibition of inflammatory cytokines in thedorsal root ganglionrdquo Frontiers in Pharmacology vol 8 2017
[13] X Chen X Sun J Guan et al ldquoRsf-1 Influences the Sensitivityof Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-120581B Pathway and Its Downstream Proteinsrdquo Cellular Physiologyand Biochemistry vol 44 no 6 pp 2322ndash2336 2018
[14] Y C Tsai Y H Chang Y Chang and C M Chuang ldquoSalvageintraperitoneal chemotherapy for relapsed type II endometrialcancer A pilot case-control studyrdquo Journal of the TurkishGerman Gynecological Association vol 17 no 4 pp 176ndash1812016
[15] L H Tan P H Sykes M M Alkaisi and J J Evans ldquoCell-likefeatures imprinted in the physical nano- andmicro-topographyof the environment modify the responses to anti-cancer drugsof endometrial cancer cellsrdquo Biofabrication vol 9 no 1 ArticleID 015017 2017
[16] K Ding Y Yuan Q-Y Chong et al ldquoAutocrine prolactinstimulates endometrial carcinoma growth and metastasis andreduces sensitivity to chemotherapyrdquo Endocrinology vol 158no 6 pp 1595ndash1611 2017
[17] L Kogan I Laskov Z Amajoud et al ldquoDose dense carboplatinpaclitaxel improves progression free survival in patients withendometrial cancerrdquo Gynecologic Oncology vol 147 no 1 pp30ndash35 2017
[18] H Koh H Park N Chandimali et al ldquoMicroRNA-128 sup-presses paclitaxel-resistant lung cancer by inhibiting MUC1-Cand BMI-1 in cancer stem cellsrdquo Oncotarget vol 8 no 66 pp110540ndash110551 2017
[19] S Tiwari B Tirosh and A Rubinstein ldquoIncreasing the affinityof cationized polyacrylamide-paclitaxel nanoparticles towardscolon cancer cells by a surface recognition peptiderdquo Interna-tional Journal of Pharmaceutics vol 531 no 1 pp 281ndash291 2017
[20] M Xiong Q Lei X You et al ldquoMannosylated liposomesimprove therapeutic effects of paclitaxel in colon cancer mod-elsrdquo Journal of Microencapsulation vol 34 no 6 pp 513ndash5212017
10 BioMed Research International
[21] S Xu J Ge Z Zhang and W Zhou ldquoMiR-129 inhibitscell proliferation and metastasis by targeting ETS1 viaPI3KAKTmTOR pathway in prostate cancerrdquo Biomedicine ampPharmacotherapy vol 96 pp 634ndash641 2017
[22] P-P Gan Y-Y Zhou M-Z Zhong Y Peng L Li and J-HLi ldquoEndoplasmic Reticulum Stress Promotes Autophagy andApoptosis and Reduces Chemotherapy Resistance in Mutantp53 Lung Cancer Cellsrdquo Cellular Physiology and Biochemistrypp 133ndash151 2017
[23] G Wu Z Sun Q Li et al ldquoNVP-BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3KAKT mTORphosphorylationrdquo OncoTargets andTherapy p 269
[24] J Chen R Shao F Li et al ldquoPI3KAktmTOR pathway dualinhibitor BEZ235 suppresses the stemness of colon cancer stemcellsrdquo Clinical and Experimental Pharmacology and Physiologyvol 42 no 12 pp 1317ndash1326 2015
[25] J Chen R Shao L Li Z P Xu and W Gu ldquoEffective inhi-bition of colon cancer cell growth with MgAl-layered doublehydroxide (LDH) loaded 5-FU and PI3KmTOR dual inhibitorBEZ-235 through apoptotic pathwaysrdquo International Journal ofNanomedicine vol 9 no 1 pp 3403ndash3411 2014
[26] D A Sabbah M G Brattain and H Zhong ldquoDual inhibitorsof PI3KmTOR or mTOR-selective inhibitors Which way shallwe gordquo Current Medicinal Chemistry vol 18 no 36 pp 5528ndash5544 2011
[27] H A Burris C D Kurkjian L Hart et al ldquoTAK-228 (formerlyMLN0128) an investigational dual TORC12 inhibitor pluspaclitaxel withwithout trastuzumab in patients with advancedsolid malignanciesrdquo Cancer Chemotherapy and Pharmacologyvol 80 no 2 pp 261ndash273 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
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Hindawiwwwhindawicom Volume 2018
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Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
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Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
BioMed Research International 3
010
008
006
004
002
000
IC50
(ug
ml)
ishikawa ishikawa-TAX
lowastlowast
(a)
ishikawaishikawa-TAX
20
15
10
05
00
OD
(492
nm)
Days3210
lowastlowast
lowastlowast
lowastlowast
(b)
ishikawa ishikawa-TAX
30
20
10
0
Retiv
e mRN
A ex
pres
sion
Pgplowastlowast
(c)ishikawa ishikawa-TAX
40
30
20
10
0
Retiv
e mRN
A ex
pres
sion
MDR1
lowastlowast
(d)
Figure 1 Identification of Ishikawa-TAX resistant cell lines and their proliferation rate (a) IC50 test of Ishikawa and Ishikawa-TAX cells (b)Cell proliferation assay of Ishikawa and Ishikawa-TAX cells (c) Ishikawa and Ishikawa-TAX resistance-related gene test (P lt005)
211 Statistical Analysis All the experiments were repeated 3times independently Data were expressed as x + s and wereanalyzed by SPSS 17 softwareMultiple groupswere comparedby ANOVA and post hoc comparisons were carried out byLSDmethodThe difference was statistically significant whenP lt 005
3 Results
31 Identification of Ishikawa-TAX Resistant Cell Lines andTheir Proliferation Rate The results of CCK-8 showed thatIC50 of Ishikawa-TAX group was significantly higher thanthat of Ishikawa group (Figure 1(a)) The proliferation rateof Ishikawa-TAX group was lower than Ishikawa group(Figure 1(b)) QPCR results showed that the resistance-related genes of Pgp and MDR1 were significantly higher inIshikawa-TAX group than in Ishikawa group (Figure 1(c))
32 NVP-BEZ235 Cotreatment with Paclitaxel Can FurtherEnhance Paclitaxel Inhibition of Ishikawa and Ishikawa-TAXCloning Formation Clone formation results showed thattreatment with NVP-BEZ235 alone did not inhibit Ishikawaclonal formation whereas paclitaxel alone inhibited Ishikawaclonality and cotreatment with paclitaxel NVP-BEZ235further inhibited Ishikawa clonality (Figure 2) Howeverin Ishikawa-TAX NVP-BEZ235 treatment and paclitaxeltreatment inhibited Ishikawa clonogenicity whereas NVP-BEZ235 and paclitaxel further inhibited clonogenicity ofIshikawa-TAX (Figure 2)
33 NVP-BEZ235 Cotreatment with Paclitaxel Can FurtherInhibit the Invasion of Ishikawa and Ishikawa-TAX CellsInvasion results showed that NVP-BEZ235 alone and pacli-taxel alone inhibited Ishikawa invasion and NVP-BEZ235cotreatment with paclitaxel further inhibited Ishikawa inva-sion (Figure 3) In Ishikawa-TAX NVP-BEZ235 alone andpaclitaxel alone inhibited the invasion of Ishikawa whereasNVP-BEZ235 cotreated with paclitaxel further inhibitedIshikawa-TAX invasion (Figure 3)
34 NVP-BEZ235 Cotreatment with Paclitaxel Can Signif-icantly Lead to an Increase in Apoptosis Rate IshikawaApoptosis results showed that cotreatment of NVP-BEZ235with paclitaxel significantly increased apoptosis of Ishikawawhile cotreatment of NVP-BEZ235 with paclitaxel did notresult in significant apoptosis of Ishikawa-TAX (Figure 4)
35 NVP-BEZ235 Cotreatment with Paclitaxel IncreasedIshikawa and Ishikawa-TAX Cells in the Proportion of G1Phase The results of cell cycle showed that NVP-BEZ235and paclitaxel alone increased the proportion of Ishikawaand Ishikawa-TAX cells in G1 phase while NVP-BEZ235cotreatment with paclitaxel further increased the proportionof Ishikawa and Ishikawa-TAX cells in G1 phase (Figure 5)
36 NVP-BEZ235 Cotreatment with Paclitaxel IncreasedIshikawa and Ishikawa-TAX Cells p-AKT and p-p70 S6KProtein Expression Western blotting results showed thatNVP-BEZ235 and paclitaxel alone treatment reduced p-AKT
4 BioMed Research International
Ishikawa Ishikawa+TAXIshikawa+NVP-BEZ235Ishikawa+NVP-BEZ235
+TAX
Ishikawa-TAX Ishikawa-TAX +NVP-BEZ235
Ishikawa-TAX +TAX
Ishikawa-TAX +NVP-BEZ235
+TAX(a)
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
150
100
50
0Col
ony
num
ber p
er w
ell
lowastlowast
lowastlowastlowastlowast
lowastlowast
(b)
Figure 2 Clonogenicity after treatment of Ishikawa and Ishikawa-TAX cells with NVP-BEZ235 and paclitaxel (a) Cell cloning followingtreatment of Ishikawa and Ishikawa-TAXcells withNVP-BEZ235 alone and in combinationwith paclitaxel (b)Clone formation quantification(lowastP lt005 lowastlowastP lt001)
and p-p70 S6K protein levels in Ishikawa and Ishikawa-TAXcells while NVP-BEZ235 cotreatment with paclitaxel couldfurther reduced p-AKT and p-p70 S6K protein expression(Figure 6)
37 LY294002 Cotreatment with Paclitaxel Can SignificantlyLead to the Increase in Apoptosis Rate and the Proportionof G1 Phase Apoptosis results showed that cotreatment ofLY294002 with paclitaxel significantly increased apoptosis ofIshikawa-TAX while the treatment of LY294002 or paclitaxeldid not result in significant apoptosis of Ishikawa-TAX(Figure 7) The results of cell cycle showed that cotreatmentof LY294002 and paclitaxel increased the proportion ofIshikawa-TAX cells in G1 phase (Figure 8)
38 LY294002 Cotreatment with Paclitaxel Decreased theExpression of p-AKT and p-p70 S6K Protein in Ishikawa-TAX Cells Western blotting results showed that LY294002and paclitaxel alone treatment reduced p-AKT and p-p70S6K protein levels in Ishikawa-TAX cells while LY294002
cotreatmentwith paclitaxel could further reduced p-AKTandp-p70 S6K protein expression (Figure 9)
4 Discussion
Endometrial cancer is a malignant tumor which originatesfrom endometrial glands with a high incidence It accountsfor 20-30 of the female reproductive system malignanttumors which seriously endangers womenrsquos health and life[14 15] Chemotherapy is the common treatment for endome-trial cancer and the sensitivity of tumor cells makes a big dif-ference in the prognosis Improving sensitivity and reducingof drug resistance will raise the survival rate of endometrialcancer and therefore improve the patientsrsquo life quality [16 17]Paclitaxel is one of the most commonly used chemotherapydrug which can be applied in endometrial cancer coloncancer and other malignancies [18ndash20] However in clinicemergence of paclitaxel resistance greatly affects its effect andrestricts its application as a chemotherapeutic drug
BioMed Research International 5
Ishikawa Ishikawa+TAXIshikawa+NVP-BEZ235 Ishikawa+NVP-BEZ235+TAX
Ishikawa-TAXIshikawa-TAX +NVP-BEZ235
Ishikawa-TAX +TAX
Ishikawa-TAX+NVP -BEZ235 +TAX
(a)
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
150
200
250
100
50
0
Inva
ded
num
ber p
er w
ell
lowastlowast lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
(b)
Figure 3The cell invasion of Ishikawa and Ishikawa-TAX cells after NVP-BEZ235 treatment with paclitaxel (a)The cell invasion of Ishikawaand Ishikawa-TAX cells treated byNVP-BEZ235 alone and cotreated with paclitaxel treatment (b)The number of cell invasion was quantified(lowastlowastP lt001)
In this study we used the taxol concentration gradientmethod to establish the paclitaxel resistant cell line (Ishikawa-TAX cells) by repeated induction of Ishikawa parent cellsThrough its IC50 resistance-related genes (Pgp and MDR1)were detected which confirmed that the constructed cellline was Ishikawa-TAX (Figure 1) The results showed thatPgp and MDR1 were increased in Ishikawa-TAX cells whichindicated that it was a promising therapy to focus on thegenes of Pgp and MDR1 in Ishikawa-TAX cells So far themechanism of Ishikawa cellsrsquo resistance to paclitaxel has notbeen clearly illustrated and need further study
The phosphatidylinositol-3-kinase (PI3K)AktmTORpathway is a commonly activated signaling pathway incancer which play an important role in drug resistance ofcancers including prostate cancer and lung cancer [21 22]while the relationships between Ishikawa and taxol is stillunclear In this study we used NVP-BEZ235 which is theinhibitor of PI3KAktmTOR pathway [23] NVP-BEZ235is also a promising chemotherapy drug in clinical trials formany cancers such as colon cancer [24 25]
Compared with paclitaxel alone the combined treat-ment of NVP-BEZ235 and paclitaxel further enhanced the
inhibitory effect on the clone formation and invasion ofIshikawa and Ishikawa-TAX cells (Figures 2 and 3) Thissuggests that the inhibitory effect of taxol on Ishikawa andIshikawa-TAX is mediated by the PI3KmTOR signalingpathway In apoptosis detection the combined treatment ofNVP-BEZ235 and paclitaxel showed similar result and raisedthe apoptosis induction effect of taxol on Ishikawa cells (Fig-ure 4) The combined treatment also enhanced the arrest ofG0G1 cells in Ishikawa and Ishikawa-TAX cellsThese resultscollectively showed that the cotreatment of NVP-BEZ235 andpaclitaxel could lead to a decrease in the proliferation activityof Ishikawa and Ishikawa-TAX cells (Figure 5) When welooked into the underlying mechanism we found that simplepaclitaxel treatment could significantly inhibit the expressionof p-AK and p-p70 S6K protein in Ishikawa and Ishikawa-TAX cells When combined with paclitaxel NVP-BEZ235can further inhibit the expression of p-AKT and p-p70 S6Kprotein in Ishikawa and Ishikawa-TAX cells indicating thatpaclitaxel inhibits Ishikawa and Ishikawa-TAX cells throughPI3KAktmTOR (Figure 6) LY294002 an inhibitor of PI3K-AKT pathway was used to further confirm the function ofAKT pathway Results showed that the apoptosis rate and
6 BioMed Research International
Ishikawa Ishikawa+TAXIshikawa+NVP-BEZ235 Ishikawa+NVP-BEZ235+TAX
Ishikawa-TAX Ishikawa-TAX +NVP-BEZ235
Ishikawa-TAX +TAX
Ishikawa-TAX +NVP-BEZ235
+TAX
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
R2
9342 022
632 004Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9480 036
436 048Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9492 186
280 042Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9186 366
310 138
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
g
R2
9472 024
498 006Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9546 056
386 012Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9552 066
370 012Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9524 044
410 022
ANNEXINV-PEANNEXINV-PE ANNEXINV-PE
ANNEXINV-PE ANNEXINV-PE
ANNEXINV-PE
ANNEXINV-PEANNEXINV-PE
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
104103102101100 104103102101100 104103102101100 104103102101100
104103102101100 104103102101100 104103102101100 104103102101100
(a)
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
Apop
tosis
rate
() 15
10
5
0
lowastlowast
(b)
Figure 4 The apoptosis of Ishikawa and Ishikawa-TAX cells after treatment of NVP-BEZ235 and paclitaxel (a) NVP-BEZ235 alone andcotreated with paclitaxel Ishikawa and Ishikawa-TAX cells and the changes in apoptosis (b) Apoptosis quantification (lowastlowastP lt001)
Ratio
()
100
80
60
40
20
0
lowastlowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
01 S 2－
Cycle
Ishikawa
Ishikawa+TAX
Ishikawa+NVP-BEZ235
Ishikawa+NVP-BEZ235+TAX
Ishikawa-TAX
Ishikawa-TAX+NVP-BEZ235
Ishikawa-TAX+TAX
Ishikawa-TAX+NVP-BEZ235+TAX
Figure 5 Cell cycle changes after NVP-BEZ235 and paclitaxel treatment of Ishikawa and Ishikawa-TAX cells The cell cycle of Ishikawa andIshikawa-TAX cells changes of G0G1 S G2M cycles after NVP-BEZ235 and paclitaxel alone and in cotreatment (lowastlowastP lt001)
BioMed Research International 7
P-AKT
P-p70 S6K
-actin
(a)
Relat
ive e
xpre
ssio
nP-
AKT
-a
ctin lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
lowastlowast3
2
1
0
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
Relat
ive e
xpre
ssio
nP-
p70
S6K
-a
ctin
20
15
10
05
00
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
(b)Figure 6 Changes of p-AKT and p-p70 S6K proteins after treatment with NVP-BEZ235 and paclitaxel in Ishikawa and Ishikawa-TAX cells(a) Changes of p-AKT and p-p70 S6K proteins after treatment of Ishikawa and Ishikawa-TAX cells with NVP-BEZ235 and paclitaxel aloneand in combination (b) Protein relative quantification (lowastlowastP lt001)
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
Apop
tosis
rate
()
15
20
10
5
0
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
lowastlowast
Figure 7 The apoptosis of Ishikawa-TAX cells after treatment of LY294002 and paclitaxel (a) LY294002 alone and cotreated with paclitaxelIshikawa-TAX cells the changes in apoptosis (b) Apoptosis quantification (lowastlowastP lt001)
8 BioMed Research International
Ratio
()
lowastlowast
lowastlowast
lowastlowast
01 S 2－
Cycle
Ishikawa
Ishikawa-TAX+LY294002
Ishikawa-TAX+TAX
Ishikawa-TAX+LY294002+TAX
lowastlowast
lowastlowast
lowastlowast
100
80
60
40
20
0
lowastlowast
lowastlowast
lowastlowast
Figure 8 Cell cycle changes after LY294002 and paclitaxel treatment of Ishikawa-TAX cells The cell cycle of Ishikawa-TAX cells changes ofG0 G1 S G2 M cycles after LY294002 and paclitaxel alone and in cotreatment (lowastlowastP lt001)
P-AKT
P-p70S6K
-actin
lowastlowast
lowastlowast
lowastlowast
Relat
ive e
xpre
ssio
nP-
p70
S6K
-a
ctin
10
02
04
06
08
00
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
Relat
ive e
xpre
ssio
nP-
AKT
-a
ctin
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
lowastlowastlowastlowast lowastlowast
10
02
04
06
08
00
Figure 9 Changes of p-AKT and p-p70 S6K proteins after treatment with LY294002 and paclitaxel in Ishikawa-TAX cells (a) Changes ofp-AKT and p-p70 S6K proteins after treatment of Ishikawa-TAX cells with LY294002 and paclitaxel alone and in combination (b) Proteinrelative quantification (lowastlowastP lt001)
BioMed Research International 9
cell cycle were not different between Ishikawa group andIshikawa-TAX + TAX group which suggested that Ishikawa-TAX had the ability of resistance for paclitaxel (Figures 7 and8) What is more the results showed that paclitaxel inhibitsIshikawa-TAX cells through PI3KAktmTOR (Figures 7 8and 9)
Nowadays more and more clinical trials focus on thecombination of paxlitaxel with other PI3K inhibitors incancer treatment such as rapamycin and TORC12 inhibitorTAK-228 [26 27] It shows that PI3Kpathway plays an impor-tant role in cancer growth invasion and drug resistance
In summary taxol treatment can inhibit the Ishikawa cellsand Ishikawa-TAX cells through PI3KAktmTOR signalingpathway and therefore regulating PI3KAktmTOR pathwaymay been a promising therapeutic direction for the clinicaltreatment of drug-resistant endometrial carcinoma
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors have no conflicts of interest to declare
Authorsrsquo Contributions
Jie Ding and Mengxiong Li contributed equally to this work
Acknowledgments
This study was funded by Guangdong Medical ResearchFoundation (Grant A2014235) and Guangdong ProvincialScience and Technology Research (Grants 2013B0218000902013B022000044 and 2016A020215074)
References
[1] T Yamamoto T Mori M Sawada et al ldquoLoss of AF-6afadininduces cell invasion suppresses the formation of glandularstructures and might be a predictive marker of resistance tochemotherapy in endometrial cancerrdquo BMC Cancer vol 15 no1 article no 275 2015
[2] L Hoang S Chiang and C-H Lee ldquoEndometrial stromalsarcomas and related neoplasms New developments and diag-nostic considerationsrdquo Pathology 2017
[3] A Mihailovici M Rottenstreich S Kovel I Wassermann NSmorgick andZVaknin ldquoEndometriosis-associatedmalignanttransformation in abdominal surgical scarrdquo Medicine (UnitedStates) vol 96 no 49 Article ID e9136 2017
[4] VMakker A K Green RMWenham DMutch B Davidsonand D S Miller ldquoNew therapies for advanced recurrent andmetastatic endometrial cancersrdquoGynecologic Oncology Researchand Practice vol 4 no 1 2017
[5] N Dadi M Stanley S Shahda B H Orsquoneil and A SehdevldquoImpact of nab-paclitaxel-based second-line chemotherapy inmetastatic pancreatic cancerrdquoAnticancer Reseach vol 37 no 10pp 5533ndash5539 2017
[6] C M Bestvina and G F Fleming ldquoChemotherapy for endome-trial cancer in adjuvant and advanced disease settingsrdquo TheOncologist vol 21 no 10 pp 1250ndash1259 2016
[7] P Augereau A Patsouris E Bourbouloux et al ldquoHormonore-sistance in advanced breast cancer A new revolution inendocrine therapyrdquoTherapeutic Advances in Medical Oncologyvol 9 no 5 pp 335ndash346 2017
[8] D Xu R Li J Wu L Jiang and H A Zhong ldquoDrug designtargeting theCXCR4CXCR7CXCL12 pathwayrdquoCurrent Topicsin Medicinal Chemistry vol 16 no 13 pp 1441ndash1451 2016
[9] K Yuge E Kikuchi M Hagiwara et al ldquoNicotine inducestumor growth and chemoresistance through activation of thePI3KAktmTOR pathway in bladder cancerrdquoMolecular CancerTherapeutics vol 14 no 9 pp 2112ndash2120 2015
[10] V Makker F O Recio L Ma et al ldquoA multicenter single-arm open-label phase 2 study of apitolisib (GDC-0980) forthe treatment of recurrent or persistent endometrial carcinoma(MAGGIE study)rdquo Cancer vol 122 no 22 pp 3519ndash3528 2016
[11] K Inaba K Oda Y Ikeda et al ldquoAntitumor activity of acombination of dual PI3KmTOR inhibitor SAR245409 andselective MEK12 inhibitor pimasertib in endometrial carcino-masrdquo Gynecologic Oncology vol 138 no 2 pp 323ndash331 2015
[12] H K Kim S-H Hwang E Oh and S Abdi ldquoRolipram aselective phosphodiesterase 4 inhibitor ameliorates mechanicalhyperalgesia in a rat model of chemotherapy-induced neuro-pathic pain through inhibition of inflammatory cytokines in thedorsal root ganglionrdquo Frontiers in Pharmacology vol 8 2017
[13] X Chen X Sun J Guan et al ldquoRsf-1 Influences the Sensitivityof Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-120581B Pathway and Its Downstream Proteinsrdquo Cellular Physiologyand Biochemistry vol 44 no 6 pp 2322ndash2336 2018
[14] Y C Tsai Y H Chang Y Chang and C M Chuang ldquoSalvageintraperitoneal chemotherapy for relapsed type II endometrialcancer A pilot case-control studyrdquo Journal of the TurkishGerman Gynecological Association vol 17 no 4 pp 176ndash1812016
[15] L H Tan P H Sykes M M Alkaisi and J J Evans ldquoCell-likefeatures imprinted in the physical nano- andmicro-topographyof the environment modify the responses to anti-cancer drugsof endometrial cancer cellsrdquo Biofabrication vol 9 no 1 ArticleID 015017 2017
[16] K Ding Y Yuan Q-Y Chong et al ldquoAutocrine prolactinstimulates endometrial carcinoma growth and metastasis andreduces sensitivity to chemotherapyrdquo Endocrinology vol 158no 6 pp 1595ndash1611 2017
[17] L Kogan I Laskov Z Amajoud et al ldquoDose dense carboplatinpaclitaxel improves progression free survival in patients withendometrial cancerrdquo Gynecologic Oncology vol 147 no 1 pp30ndash35 2017
[18] H Koh H Park N Chandimali et al ldquoMicroRNA-128 sup-presses paclitaxel-resistant lung cancer by inhibiting MUC1-Cand BMI-1 in cancer stem cellsrdquo Oncotarget vol 8 no 66 pp110540ndash110551 2017
[19] S Tiwari B Tirosh and A Rubinstein ldquoIncreasing the affinityof cationized polyacrylamide-paclitaxel nanoparticles towardscolon cancer cells by a surface recognition peptiderdquo Interna-tional Journal of Pharmaceutics vol 531 no 1 pp 281ndash291 2017
[20] M Xiong Q Lei X You et al ldquoMannosylated liposomesimprove therapeutic effects of paclitaxel in colon cancer mod-elsrdquo Journal of Microencapsulation vol 34 no 6 pp 513ndash5212017
10 BioMed Research International
[21] S Xu J Ge Z Zhang and W Zhou ldquoMiR-129 inhibitscell proliferation and metastasis by targeting ETS1 viaPI3KAKTmTOR pathway in prostate cancerrdquo Biomedicine ampPharmacotherapy vol 96 pp 634ndash641 2017
[22] P-P Gan Y-Y Zhou M-Z Zhong Y Peng L Li and J-HLi ldquoEndoplasmic Reticulum Stress Promotes Autophagy andApoptosis and Reduces Chemotherapy Resistance in Mutantp53 Lung Cancer Cellsrdquo Cellular Physiology and Biochemistrypp 133ndash151 2017
[23] G Wu Z Sun Q Li et al ldquoNVP-BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3KAKT mTORphosphorylationrdquo OncoTargets andTherapy p 269
[24] J Chen R Shao F Li et al ldquoPI3KAktmTOR pathway dualinhibitor BEZ235 suppresses the stemness of colon cancer stemcellsrdquo Clinical and Experimental Pharmacology and Physiologyvol 42 no 12 pp 1317ndash1326 2015
[25] J Chen R Shao L Li Z P Xu and W Gu ldquoEffective inhi-bition of colon cancer cell growth with MgAl-layered doublehydroxide (LDH) loaded 5-FU and PI3KmTOR dual inhibitorBEZ-235 through apoptotic pathwaysrdquo International Journal ofNanomedicine vol 9 no 1 pp 3403ndash3411 2014
[26] D A Sabbah M G Brattain and H Zhong ldquoDual inhibitorsof PI3KmTOR or mTOR-selective inhibitors Which way shallwe gordquo Current Medicinal Chemistry vol 18 no 36 pp 5528ndash5544 2011
[27] H A Burris C D Kurkjian L Hart et al ldquoTAK-228 (formerlyMLN0128) an investigational dual TORC12 inhibitor pluspaclitaxel withwithout trastuzumab in patients with advancedsolid malignanciesrdquo Cancer Chemotherapy and Pharmacologyvol 80 no 2 pp 261ndash273 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
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Volume 2018
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Hindawiwwwhindawicom Volume 2018
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Hindawiwwwhindawicom Volume 2018
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Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
4 BioMed Research International
Ishikawa Ishikawa+TAXIshikawa+NVP-BEZ235Ishikawa+NVP-BEZ235
+TAX
Ishikawa-TAX Ishikawa-TAX +NVP-BEZ235
Ishikawa-TAX +TAX
Ishikawa-TAX +NVP-BEZ235
+TAX(a)
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
150
100
50
0Col
ony
num
ber p
er w
ell
lowastlowast
lowastlowastlowastlowast
lowastlowast
(b)
Figure 2 Clonogenicity after treatment of Ishikawa and Ishikawa-TAX cells with NVP-BEZ235 and paclitaxel (a) Cell cloning followingtreatment of Ishikawa and Ishikawa-TAXcells withNVP-BEZ235 alone and in combinationwith paclitaxel (b)Clone formation quantification(lowastP lt005 lowastlowastP lt001)
and p-p70 S6K protein levels in Ishikawa and Ishikawa-TAXcells while NVP-BEZ235 cotreatment with paclitaxel couldfurther reduced p-AKT and p-p70 S6K protein expression(Figure 6)
37 LY294002 Cotreatment with Paclitaxel Can SignificantlyLead to the Increase in Apoptosis Rate and the Proportionof G1 Phase Apoptosis results showed that cotreatment ofLY294002 with paclitaxel significantly increased apoptosis ofIshikawa-TAX while the treatment of LY294002 or paclitaxeldid not result in significant apoptosis of Ishikawa-TAX(Figure 7) The results of cell cycle showed that cotreatmentof LY294002 and paclitaxel increased the proportion ofIshikawa-TAX cells in G1 phase (Figure 8)
38 LY294002 Cotreatment with Paclitaxel Decreased theExpression of p-AKT and p-p70 S6K Protein in Ishikawa-TAX Cells Western blotting results showed that LY294002and paclitaxel alone treatment reduced p-AKT and p-p70S6K protein levels in Ishikawa-TAX cells while LY294002
cotreatmentwith paclitaxel could further reduced p-AKTandp-p70 S6K protein expression (Figure 9)
4 Discussion
Endometrial cancer is a malignant tumor which originatesfrom endometrial glands with a high incidence It accountsfor 20-30 of the female reproductive system malignanttumors which seriously endangers womenrsquos health and life[14 15] Chemotherapy is the common treatment for endome-trial cancer and the sensitivity of tumor cells makes a big dif-ference in the prognosis Improving sensitivity and reducingof drug resistance will raise the survival rate of endometrialcancer and therefore improve the patientsrsquo life quality [16 17]Paclitaxel is one of the most commonly used chemotherapydrug which can be applied in endometrial cancer coloncancer and other malignancies [18ndash20] However in clinicemergence of paclitaxel resistance greatly affects its effect andrestricts its application as a chemotherapeutic drug
BioMed Research International 5
Ishikawa Ishikawa+TAXIshikawa+NVP-BEZ235 Ishikawa+NVP-BEZ235+TAX
Ishikawa-TAXIshikawa-TAX +NVP-BEZ235
Ishikawa-TAX +TAX
Ishikawa-TAX+NVP -BEZ235 +TAX
(a)
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
150
200
250
100
50
0
Inva
ded
num
ber p
er w
ell
lowastlowast lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
(b)
Figure 3The cell invasion of Ishikawa and Ishikawa-TAX cells after NVP-BEZ235 treatment with paclitaxel (a)The cell invasion of Ishikawaand Ishikawa-TAX cells treated byNVP-BEZ235 alone and cotreated with paclitaxel treatment (b)The number of cell invasion was quantified(lowastlowastP lt001)
In this study we used the taxol concentration gradientmethod to establish the paclitaxel resistant cell line (Ishikawa-TAX cells) by repeated induction of Ishikawa parent cellsThrough its IC50 resistance-related genes (Pgp and MDR1)were detected which confirmed that the constructed cellline was Ishikawa-TAX (Figure 1) The results showed thatPgp and MDR1 were increased in Ishikawa-TAX cells whichindicated that it was a promising therapy to focus on thegenes of Pgp and MDR1 in Ishikawa-TAX cells So far themechanism of Ishikawa cellsrsquo resistance to paclitaxel has notbeen clearly illustrated and need further study
The phosphatidylinositol-3-kinase (PI3K)AktmTORpathway is a commonly activated signaling pathway incancer which play an important role in drug resistance ofcancers including prostate cancer and lung cancer [21 22]while the relationships between Ishikawa and taxol is stillunclear In this study we used NVP-BEZ235 which is theinhibitor of PI3KAktmTOR pathway [23] NVP-BEZ235is also a promising chemotherapy drug in clinical trials formany cancers such as colon cancer [24 25]
Compared with paclitaxel alone the combined treat-ment of NVP-BEZ235 and paclitaxel further enhanced the
inhibitory effect on the clone formation and invasion ofIshikawa and Ishikawa-TAX cells (Figures 2 and 3) Thissuggests that the inhibitory effect of taxol on Ishikawa andIshikawa-TAX is mediated by the PI3KmTOR signalingpathway In apoptosis detection the combined treatment ofNVP-BEZ235 and paclitaxel showed similar result and raisedthe apoptosis induction effect of taxol on Ishikawa cells (Fig-ure 4) The combined treatment also enhanced the arrest ofG0G1 cells in Ishikawa and Ishikawa-TAX cellsThese resultscollectively showed that the cotreatment of NVP-BEZ235 andpaclitaxel could lead to a decrease in the proliferation activityof Ishikawa and Ishikawa-TAX cells (Figure 5) When welooked into the underlying mechanism we found that simplepaclitaxel treatment could significantly inhibit the expressionof p-AK and p-p70 S6K protein in Ishikawa and Ishikawa-TAX cells When combined with paclitaxel NVP-BEZ235can further inhibit the expression of p-AKT and p-p70 S6Kprotein in Ishikawa and Ishikawa-TAX cells indicating thatpaclitaxel inhibits Ishikawa and Ishikawa-TAX cells throughPI3KAktmTOR (Figure 6) LY294002 an inhibitor of PI3K-AKT pathway was used to further confirm the function ofAKT pathway Results showed that the apoptosis rate and
6 BioMed Research International
Ishikawa Ishikawa+TAXIshikawa+NVP-BEZ235 Ishikawa+NVP-BEZ235+TAX
Ishikawa-TAX Ishikawa-TAX +NVP-BEZ235
Ishikawa-TAX +TAX
Ishikawa-TAX +NVP-BEZ235
+TAX
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
R2
9342 022
632 004Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9480 036
436 048Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9492 186
280 042Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9186 366
310 138
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
g
R2
9472 024
498 006Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9546 056
386 012Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9552 066
370 012Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9524 044
410 022
ANNEXINV-PEANNEXINV-PE ANNEXINV-PE
ANNEXINV-PE ANNEXINV-PE
ANNEXINV-PE
ANNEXINV-PEANNEXINV-PE
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
104103102101100 104103102101100 104103102101100 104103102101100
104103102101100 104103102101100 104103102101100 104103102101100
(a)
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
Apop
tosis
rate
() 15
10
5
0
lowastlowast
(b)
Figure 4 The apoptosis of Ishikawa and Ishikawa-TAX cells after treatment of NVP-BEZ235 and paclitaxel (a) NVP-BEZ235 alone andcotreated with paclitaxel Ishikawa and Ishikawa-TAX cells and the changes in apoptosis (b) Apoptosis quantification (lowastlowastP lt001)
Ratio
()
100
80
60
40
20
0
lowastlowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
01 S 2－
Cycle
Ishikawa
Ishikawa+TAX
Ishikawa+NVP-BEZ235
Ishikawa+NVP-BEZ235+TAX
Ishikawa-TAX
Ishikawa-TAX+NVP-BEZ235
Ishikawa-TAX+TAX
Ishikawa-TAX+NVP-BEZ235+TAX
Figure 5 Cell cycle changes after NVP-BEZ235 and paclitaxel treatment of Ishikawa and Ishikawa-TAX cells The cell cycle of Ishikawa andIshikawa-TAX cells changes of G0G1 S G2M cycles after NVP-BEZ235 and paclitaxel alone and in cotreatment (lowastlowastP lt001)
BioMed Research International 7
P-AKT
P-p70 S6K
-actin
(a)
Relat
ive e
xpre
ssio
nP-
AKT
-a
ctin lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
lowastlowast3
2
1
0
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
Relat
ive e
xpre
ssio
nP-
p70
S6K
-a
ctin
20
15
10
05
00
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
(b)Figure 6 Changes of p-AKT and p-p70 S6K proteins after treatment with NVP-BEZ235 and paclitaxel in Ishikawa and Ishikawa-TAX cells(a) Changes of p-AKT and p-p70 S6K proteins after treatment of Ishikawa and Ishikawa-TAX cells with NVP-BEZ235 and paclitaxel aloneand in combination (b) Protein relative quantification (lowastlowastP lt001)
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
Apop
tosis
rate
()
15
20
10
5
0
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
lowastlowast
Figure 7 The apoptosis of Ishikawa-TAX cells after treatment of LY294002 and paclitaxel (a) LY294002 alone and cotreated with paclitaxelIshikawa-TAX cells the changes in apoptosis (b) Apoptosis quantification (lowastlowastP lt001)
8 BioMed Research International
Ratio
()
lowastlowast
lowastlowast
lowastlowast
01 S 2－
Cycle
Ishikawa
Ishikawa-TAX+LY294002
Ishikawa-TAX+TAX
Ishikawa-TAX+LY294002+TAX
lowastlowast
lowastlowast
lowastlowast
100
80
60
40
20
0
lowastlowast
lowastlowast
lowastlowast
Figure 8 Cell cycle changes after LY294002 and paclitaxel treatment of Ishikawa-TAX cells The cell cycle of Ishikawa-TAX cells changes ofG0 G1 S G2 M cycles after LY294002 and paclitaxel alone and in cotreatment (lowastlowastP lt001)
P-AKT
P-p70S6K
-actin
lowastlowast
lowastlowast
lowastlowast
Relat
ive e
xpre
ssio
nP-
p70
S6K
-a
ctin
10
02
04
06
08
00
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
Relat
ive e
xpre
ssio
nP-
AKT
-a
ctin
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
lowastlowastlowastlowast lowastlowast
10
02
04
06
08
00
Figure 9 Changes of p-AKT and p-p70 S6K proteins after treatment with LY294002 and paclitaxel in Ishikawa-TAX cells (a) Changes ofp-AKT and p-p70 S6K proteins after treatment of Ishikawa-TAX cells with LY294002 and paclitaxel alone and in combination (b) Proteinrelative quantification (lowastlowastP lt001)
BioMed Research International 9
cell cycle were not different between Ishikawa group andIshikawa-TAX + TAX group which suggested that Ishikawa-TAX had the ability of resistance for paclitaxel (Figures 7 and8) What is more the results showed that paclitaxel inhibitsIshikawa-TAX cells through PI3KAktmTOR (Figures 7 8and 9)
Nowadays more and more clinical trials focus on thecombination of paxlitaxel with other PI3K inhibitors incancer treatment such as rapamycin and TORC12 inhibitorTAK-228 [26 27] It shows that PI3Kpathway plays an impor-tant role in cancer growth invasion and drug resistance
In summary taxol treatment can inhibit the Ishikawa cellsand Ishikawa-TAX cells through PI3KAktmTOR signalingpathway and therefore regulating PI3KAktmTOR pathwaymay been a promising therapeutic direction for the clinicaltreatment of drug-resistant endometrial carcinoma
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors have no conflicts of interest to declare
Authorsrsquo Contributions
Jie Ding and Mengxiong Li contributed equally to this work
Acknowledgments
This study was funded by Guangdong Medical ResearchFoundation (Grant A2014235) and Guangdong ProvincialScience and Technology Research (Grants 2013B0218000902013B022000044 and 2016A020215074)
References
[1] T Yamamoto T Mori M Sawada et al ldquoLoss of AF-6afadininduces cell invasion suppresses the formation of glandularstructures and might be a predictive marker of resistance tochemotherapy in endometrial cancerrdquo BMC Cancer vol 15 no1 article no 275 2015
[2] L Hoang S Chiang and C-H Lee ldquoEndometrial stromalsarcomas and related neoplasms New developments and diag-nostic considerationsrdquo Pathology 2017
[3] A Mihailovici M Rottenstreich S Kovel I Wassermann NSmorgick andZVaknin ldquoEndometriosis-associatedmalignanttransformation in abdominal surgical scarrdquo Medicine (UnitedStates) vol 96 no 49 Article ID e9136 2017
[4] VMakker A K Green RMWenham DMutch B Davidsonand D S Miller ldquoNew therapies for advanced recurrent andmetastatic endometrial cancersrdquoGynecologic Oncology Researchand Practice vol 4 no 1 2017
[5] N Dadi M Stanley S Shahda B H Orsquoneil and A SehdevldquoImpact of nab-paclitaxel-based second-line chemotherapy inmetastatic pancreatic cancerrdquoAnticancer Reseach vol 37 no 10pp 5533ndash5539 2017
[6] C M Bestvina and G F Fleming ldquoChemotherapy for endome-trial cancer in adjuvant and advanced disease settingsrdquo TheOncologist vol 21 no 10 pp 1250ndash1259 2016
[7] P Augereau A Patsouris E Bourbouloux et al ldquoHormonore-sistance in advanced breast cancer A new revolution inendocrine therapyrdquoTherapeutic Advances in Medical Oncologyvol 9 no 5 pp 335ndash346 2017
[8] D Xu R Li J Wu L Jiang and H A Zhong ldquoDrug designtargeting theCXCR4CXCR7CXCL12 pathwayrdquoCurrent Topicsin Medicinal Chemistry vol 16 no 13 pp 1441ndash1451 2016
[9] K Yuge E Kikuchi M Hagiwara et al ldquoNicotine inducestumor growth and chemoresistance through activation of thePI3KAktmTOR pathway in bladder cancerrdquoMolecular CancerTherapeutics vol 14 no 9 pp 2112ndash2120 2015
[10] V Makker F O Recio L Ma et al ldquoA multicenter single-arm open-label phase 2 study of apitolisib (GDC-0980) forthe treatment of recurrent or persistent endometrial carcinoma(MAGGIE study)rdquo Cancer vol 122 no 22 pp 3519ndash3528 2016
[11] K Inaba K Oda Y Ikeda et al ldquoAntitumor activity of acombination of dual PI3KmTOR inhibitor SAR245409 andselective MEK12 inhibitor pimasertib in endometrial carcino-masrdquo Gynecologic Oncology vol 138 no 2 pp 323ndash331 2015
[12] H K Kim S-H Hwang E Oh and S Abdi ldquoRolipram aselective phosphodiesterase 4 inhibitor ameliorates mechanicalhyperalgesia in a rat model of chemotherapy-induced neuro-pathic pain through inhibition of inflammatory cytokines in thedorsal root ganglionrdquo Frontiers in Pharmacology vol 8 2017
[13] X Chen X Sun J Guan et al ldquoRsf-1 Influences the Sensitivityof Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-120581B Pathway and Its Downstream Proteinsrdquo Cellular Physiologyand Biochemistry vol 44 no 6 pp 2322ndash2336 2018
[14] Y C Tsai Y H Chang Y Chang and C M Chuang ldquoSalvageintraperitoneal chemotherapy for relapsed type II endometrialcancer A pilot case-control studyrdquo Journal of the TurkishGerman Gynecological Association vol 17 no 4 pp 176ndash1812016
[15] L H Tan P H Sykes M M Alkaisi and J J Evans ldquoCell-likefeatures imprinted in the physical nano- andmicro-topographyof the environment modify the responses to anti-cancer drugsof endometrial cancer cellsrdquo Biofabrication vol 9 no 1 ArticleID 015017 2017
[16] K Ding Y Yuan Q-Y Chong et al ldquoAutocrine prolactinstimulates endometrial carcinoma growth and metastasis andreduces sensitivity to chemotherapyrdquo Endocrinology vol 158no 6 pp 1595ndash1611 2017
[17] L Kogan I Laskov Z Amajoud et al ldquoDose dense carboplatinpaclitaxel improves progression free survival in patients withendometrial cancerrdquo Gynecologic Oncology vol 147 no 1 pp30ndash35 2017
[18] H Koh H Park N Chandimali et al ldquoMicroRNA-128 sup-presses paclitaxel-resistant lung cancer by inhibiting MUC1-Cand BMI-1 in cancer stem cellsrdquo Oncotarget vol 8 no 66 pp110540ndash110551 2017
[19] S Tiwari B Tirosh and A Rubinstein ldquoIncreasing the affinityof cationized polyacrylamide-paclitaxel nanoparticles towardscolon cancer cells by a surface recognition peptiderdquo Interna-tional Journal of Pharmaceutics vol 531 no 1 pp 281ndash291 2017
[20] M Xiong Q Lei X You et al ldquoMannosylated liposomesimprove therapeutic effects of paclitaxel in colon cancer mod-elsrdquo Journal of Microencapsulation vol 34 no 6 pp 513ndash5212017
10 BioMed Research International
[21] S Xu J Ge Z Zhang and W Zhou ldquoMiR-129 inhibitscell proliferation and metastasis by targeting ETS1 viaPI3KAKTmTOR pathway in prostate cancerrdquo Biomedicine ampPharmacotherapy vol 96 pp 634ndash641 2017
[22] P-P Gan Y-Y Zhou M-Z Zhong Y Peng L Li and J-HLi ldquoEndoplasmic Reticulum Stress Promotes Autophagy andApoptosis and Reduces Chemotherapy Resistance in Mutantp53 Lung Cancer Cellsrdquo Cellular Physiology and Biochemistrypp 133ndash151 2017
[23] G Wu Z Sun Q Li et al ldquoNVP-BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3KAKT mTORphosphorylationrdquo OncoTargets andTherapy p 269
[24] J Chen R Shao F Li et al ldquoPI3KAktmTOR pathway dualinhibitor BEZ235 suppresses the stemness of colon cancer stemcellsrdquo Clinical and Experimental Pharmacology and Physiologyvol 42 no 12 pp 1317ndash1326 2015
[25] J Chen R Shao L Li Z P Xu and W Gu ldquoEffective inhi-bition of colon cancer cell growth with MgAl-layered doublehydroxide (LDH) loaded 5-FU and PI3KmTOR dual inhibitorBEZ-235 through apoptotic pathwaysrdquo International Journal ofNanomedicine vol 9 no 1 pp 3403ndash3411 2014
[26] D A Sabbah M G Brattain and H Zhong ldquoDual inhibitorsof PI3KmTOR or mTOR-selective inhibitors Which way shallwe gordquo Current Medicinal Chemistry vol 18 no 36 pp 5528ndash5544 2011
[27] H A Burris C D Kurkjian L Hart et al ldquoTAK-228 (formerlyMLN0128) an investigational dual TORC12 inhibitor pluspaclitaxel withwithout trastuzumab in patients with advancedsolid malignanciesrdquo Cancer Chemotherapy and Pharmacologyvol 80 no 2 pp 261ndash273 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
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Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
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Hindawiwwwhindawicom Volume 2018
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Hindawiwwwhindawicom Volume 2018
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Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
BioMed Research International 5
Ishikawa Ishikawa+TAXIshikawa+NVP-BEZ235 Ishikawa+NVP-BEZ235+TAX
Ishikawa-TAXIshikawa-TAX +NVP-BEZ235
Ishikawa-TAX +TAX
Ishikawa-TAX+NVP -BEZ235 +TAX
(a)
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
150
200
250
100
50
0
Inva
ded
num
ber p
er w
ell
lowastlowast lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
(b)
Figure 3The cell invasion of Ishikawa and Ishikawa-TAX cells after NVP-BEZ235 treatment with paclitaxel (a)The cell invasion of Ishikawaand Ishikawa-TAX cells treated byNVP-BEZ235 alone and cotreated with paclitaxel treatment (b)The number of cell invasion was quantified(lowastlowastP lt001)
In this study we used the taxol concentration gradientmethod to establish the paclitaxel resistant cell line (Ishikawa-TAX cells) by repeated induction of Ishikawa parent cellsThrough its IC50 resistance-related genes (Pgp and MDR1)were detected which confirmed that the constructed cellline was Ishikawa-TAX (Figure 1) The results showed thatPgp and MDR1 were increased in Ishikawa-TAX cells whichindicated that it was a promising therapy to focus on thegenes of Pgp and MDR1 in Ishikawa-TAX cells So far themechanism of Ishikawa cellsrsquo resistance to paclitaxel has notbeen clearly illustrated and need further study
The phosphatidylinositol-3-kinase (PI3K)AktmTORpathway is a commonly activated signaling pathway incancer which play an important role in drug resistance ofcancers including prostate cancer and lung cancer [21 22]while the relationships between Ishikawa and taxol is stillunclear In this study we used NVP-BEZ235 which is theinhibitor of PI3KAktmTOR pathway [23] NVP-BEZ235is also a promising chemotherapy drug in clinical trials formany cancers such as colon cancer [24 25]
Compared with paclitaxel alone the combined treat-ment of NVP-BEZ235 and paclitaxel further enhanced the
inhibitory effect on the clone formation and invasion ofIshikawa and Ishikawa-TAX cells (Figures 2 and 3) Thissuggests that the inhibitory effect of taxol on Ishikawa andIshikawa-TAX is mediated by the PI3KmTOR signalingpathway In apoptosis detection the combined treatment ofNVP-BEZ235 and paclitaxel showed similar result and raisedthe apoptosis induction effect of taxol on Ishikawa cells (Fig-ure 4) The combined treatment also enhanced the arrest ofG0G1 cells in Ishikawa and Ishikawa-TAX cellsThese resultscollectively showed that the cotreatment of NVP-BEZ235 andpaclitaxel could lead to a decrease in the proliferation activityof Ishikawa and Ishikawa-TAX cells (Figure 5) When welooked into the underlying mechanism we found that simplepaclitaxel treatment could significantly inhibit the expressionof p-AK and p-p70 S6K protein in Ishikawa and Ishikawa-TAX cells When combined with paclitaxel NVP-BEZ235can further inhibit the expression of p-AKT and p-p70 S6Kprotein in Ishikawa and Ishikawa-TAX cells indicating thatpaclitaxel inhibits Ishikawa and Ishikawa-TAX cells throughPI3KAktmTOR (Figure 6) LY294002 an inhibitor of PI3K-AKT pathway was used to further confirm the function ofAKT pathway Results showed that the apoptosis rate and
6 BioMed Research International
Ishikawa Ishikawa+TAXIshikawa+NVP-BEZ235 Ishikawa+NVP-BEZ235+TAX
Ishikawa-TAX Ishikawa-TAX +NVP-BEZ235
Ishikawa-TAX +TAX
Ishikawa-TAX +NVP-BEZ235
+TAX
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
R2
9342 022
632 004Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9480 036
436 048Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9492 186
280 042Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9186 366
310 138
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
g
R2
9472 024
498 006Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9546 056
386 012Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9552 066
370 012Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9524 044
410 022
ANNEXINV-PEANNEXINV-PE ANNEXINV-PE
ANNEXINV-PE ANNEXINV-PE
ANNEXINV-PE
ANNEXINV-PEANNEXINV-PE
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
104103102101100 104103102101100 104103102101100 104103102101100
104103102101100 104103102101100 104103102101100 104103102101100
(a)
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
Apop
tosis
rate
() 15
10
5
0
lowastlowast
(b)
Figure 4 The apoptosis of Ishikawa and Ishikawa-TAX cells after treatment of NVP-BEZ235 and paclitaxel (a) NVP-BEZ235 alone andcotreated with paclitaxel Ishikawa and Ishikawa-TAX cells and the changes in apoptosis (b) Apoptosis quantification (lowastlowastP lt001)
Ratio
()
100
80
60
40
20
0
lowastlowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
01 S 2－
Cycle
Ishikawa
Ishikawa+TAX
Ishikawa+NVP-BEZ235
Ishikawa+NVP-BEZ235+TAX
Ishikawa-TAX
Ishikawa-TAX+NVP-BEZ235
Ishikawa-TAX+TAX
Ishikawa-TAX+NVP-BEZ235+TAX
Figure 5 Cell cycle changes after NVP-BEZ235 and paclitaxel treatment of Ishikawa and Ishikawa-TAX cells The cell cycle of Ishikawa andIshikawa-TAX cells changes of G0G1 S G2M cycles after NVP-BEZ235 and paclitaxel alone and in cotreatment (lowastlowastP lt001)
BioMed Research International 7
P-AKT
P-p70 S6K
-actin
(a)
Relat
ive e
xpre
ssio
nP-
AKT
-a
ctin lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
lowastlowast3
2
1
0
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
Relat
ive e
xpre
ssio
nP-
p70
S6K
-a
ctin
20
15
10
05
00
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
(b)Figure 6 Changes of p-AKT and p-p70 S6K proteins after treatment with NVP-BEZ235 and paclitaxel in Ishikawa and Ishikawa-TAX cells(a) Changes of p-AKT and p-p70 S6K proteins after treatment of Ishikawa and Ishikawa-TAX cells with NVP-BEZ235 and paclitaxel aloneand in combination (b) Protein relative quantification (lowastlowastP lt001)
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
Apop
tosis
rate
()
15
20
10
5
0
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
lowastlowast
Figure 7 The apoptosis of Ishikawa-TAX cells after treatment of LY294002 and paclitaxel (a) LY294002 alone and cotreated with paclitaxelIshikawa-TAX cells the changes in apoptosis (b) Apoptosis quantification (lowastlowastP lt001)
8 BioMed Research International
Ratio
()
lowastlowast
lowastlowast
lowastlowast
01 S 2－
Cycle
Ishikawa
Ishikawa-TAX+LY294002
Ishikawa-TAX+TAX
Ishikawa-TAX+LY294002+TAX
lowastlowast
lowastlowast
lowastlowast
100
80
60
40
20
0
lowastlowast
lowastlowast
lowastlowast
Figure 8 Cell cycle changes after LY294002 and paclitaxel treatment of Ishikawa-TAX cells The cell cycle of Ishikawa-TAX cells changes ofG0 G1 S G2 M cycles after LY294002 and paclitaxel alone and in cotreatment (lowastlowastP lt001)
P-AKT
P-p70S6K
-actin
lowastlowast
lowastlowast
lowastlowast
Relat
ive e
xpre
ssio
nP-
p70
S6K
-a
ctin
10
02
04
06
08
00
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
Relat
ive e
xpre
ssio
nP-
AKT
-a
ctin
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
lowastlowastlowastlowast lowastlowast
10
02
04
06
08
00
Figure 9 Changes of p-AKT and p-p70 S6K proteins after treatment with LY294002 and paclitaxel in Ishikawa-TAX cells (a) Changes ofp-AKT and p-p70 S6K proteins after treatment of Ishikawa-TAX cells with LY294002 and paclitaxel alone and in combination (b) Proteinrelative quantification (lowastlowastP lt001)
BioMed Research International 9
cell cycle were not different between Ishikawa group andIshikawa-TAX + TAX group which suggested that Ishikawa-TAX had the ability of resistance for paclitaxel (Figures 7 and8) What is more the results showed that paclitaxel inhibitsIshikawa-TAX cells through PI3KAktmTOR (Figures 7 8and 9)
Nowadays more and more clinical trials focus on thecombination of paxlitaxel with other PI3K inhibitors incancer treatment such as rapamycin and TORC12 inhibitorTAK-228 [26 27] It shows that PI3Kpathway plays an impor-tant role in cancer growth invasion and drug resistance
In summary taxol treatment can inhibit the Ishikawa cellsand Ishikawa-TAX cells through PI3KAktmTOR signalingpathway and therefore regulating PI3KAktmTOR pathwaymay been a promising therapeutic direction for the clinicaltreatment of drug-resistant endometrial carcinoma
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors have no conflicts of interest to declare
Authorsrsquo Contributions
Jie Ding and Mengxiong Li contributed equally to this work
Acknowledgments
This study was funded by Guangdong Medical ResearchFoundation (Grant A2014235) and Guangdong ProvincialScience and Technology Research (Grants 2013B0218000902013B022000044 and 2016A020215074)
References
[1] T Yamamoto T Mori M Sawada et al ldquoLoss of AF-6afadininduces cell invasion suppresses the formation of glandularstructures and might be a predictive marker of resistance tochemotherapy in endometrial cancerrdquo BMC Cancer vol 15 no1 article no 275 2015
[2] L Hoang S Chiang and C-H Lee ldquoEndometrial stromalsarcomas and related neoplasms New developments and diag-nostic considerationsrdquo Pathology 2017
[3] A Mihailovici M Rottenstreich S Kovel I Wassermann NSmorgick andZVaknin ldquoEndometriosis-associatedmalignanttransformation in abdominal surgical scarrdquo Medicine (UnitedStates) vol 96 no 49 Article ID e9136 2017
[4] VMakker A K Green RMWenham DMutch B Davidsonand D S Miller ldquoNew therapies for advanced recurrent andmetastatic endometrial cancersrdquoGynecologic Oncology Researchand Practice vol 4 no 1 2017
[5] N Dadi M Stanley S Shahda B H Orsquoneil and A SehdevldquoImpact of nab-paclitaxel-based second-line chemotherapy inmetastatic pancreatic cancerrdquoAnticancer Reseach vol 37 no 10pp 5533ndash5539 2017
[6] C M Bestvina and G F Fleming ldquoChemotherapy for endome-trial cancer in adjuvant and advanced disease settingsrdquo TheOncologist vol 21 no 10 pp 1250ndash1259 2016
[7] P Augereau A Patsouris E Bourbouloux et al ldquoHormonore-sistance in advanced breast cancer A new revolution inendocrine therapyrdquoTherapeutic Advances in Medical Oncologyvol 9 no 5 pp 335ndash346 2017
[8] D Xu R Li J Wu L Jiang and H A Zhong ldquoDrug designtargeting theCXCR4CXCR7CXCL12 pathwayrdquoCurrent Topicsin Medicinal Chemistry vol 16 no 13 pp 1441ndash1451 2016
[9] K Yuge E Kikuchi M Hagiwara et al ldquoNicotine inducestumor growth and chemoresistance through activation of thePI3KAktmTOR pathway in bladder cancerrdquoMolecular CancerTherapeutics vol 14 no 9 pp 2112ndash2120 2015
[10] V Makker F O Recio L Ma et al ldquoA multicenter single-arm open-label phase 2 study of apitolisib (GDC-0980) forthe treatment of recurrent or persistent endometrial carcinoma(MAGGIE study)rdquo Cancer vol 122 no 22 pp 3519ndash3528 2016
[11] K Inaba K Oda Y Ikeda et al ldquoAntitumor activity of acombination of dual PI3KmTOR inhibitor SAR245409 andselective MEK12 inhibitor pimasertib in endometrial carcino-masrdquo Gynecologic Oncology vol 138 no 2 pp 323ndash331 2015
[12] H K Kim S-H Hwang E Oh and S Abdi ldquoRolipram aselective phosphodiesterase 4 inhibitor ameliorates mechanicalhyperalgesia in a rat model of chemotherapy-induced neuro-pathic pain through inhibition of inflammatory cytokines in thedorsal root ganglionrdquo Frontiers in Pharmacology vol 8 2017
[13] X Chen X Sun J Guan et al ldquoRsf-1 Influences the Sensitivityof Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-120581B Pathway and Its Downstream Proteinsrdquo Cellular Physiologyand Biochemistry vol 44 no 6 pp 2322ndash2336 2018
[14] Y C Tsai Y H Chang Y Chang and C M Chuang ldquoSalvageintraperitoneal chemotherapy for relapsed type II endometrialcancer A pilot case-control studyrdquo Journal of the TurkishGerman Gynecological Association vol 17 no 4 pp 176ndash1812016
[15] L H Tan P H Sykes M M Alkaisi and J J Evans ldquoCell-likefeatures imprinted in the physical nano- andmicro-topographyof the environment modify the responses to anti-cancer drugsof endometrial cancer cellsrdquo Biofabrication vol 9 no 1 ArticleID 015017 2017
[16] K Ding Y Yuan Q-Y Chong et al ldquoAutocrine prolactinstimulates endometrial carcinoma growth and metastasis andreduces sensitivity to chemotherapyrdquo Endocrinology vol 158no 6 pp 1595ndash1611 2017
[17] L Kogan I Laskov Z Amajoud et al ldquoDose dense carboplatinpaclitaxel improves progression free survival in patients withendometrial cancerrdquo Gynecologic Oncology vol 147 no 1 pp30ndash35 2017
[18] H Koh H Park N Chandimali et al ldquoMicroRNA-128 sup-presses paclitaxel-resistant lung cancer by inhibiting MUC1-Cand BMI-1 in cancer stem cellsrdquo Oncotarget vol 8 no 66 pp110540ndash110551 2017
[19] S Tiwari B Tirosh and A Rubinstein ldquoIncreasing the affinityof cationized polyacrylamide-paclitaxel nanoparticles towardscolon cancer cells by a surface recognition peptiderdquo Interna-tional Journal of Pharmaceutics vol 531 no 1 pp 281ndash291 2017
[20] M Xiong Q Lei X You et al ldquoMannosylated liposomesimprove therapeutic effects of paclitaxel in colon cancer mod-elsrdquo Journal of Microencapsulation vol 34 no 6 pp 513ndash5212017
10 BioMed Research International
[21] S Xu J Ge Z Zhang and W Zhou ldquoMiR-129 inhibitscell proliferation and metastasis by targeting ETS1 viaPI3KAKTmTOR pathway in prostate cancerrdquo Biomedicine ampPharmacotherapy vol 96 pp 634ndash641 2017
[22] P-P Gan Y-Y Zhou M-Z Zhong Y Peng L Li and J-HLi ldquoEndoplasmic Reticulum Stress Promotes Autophagy andApoptosis and Reduces Chemotherapy Resistance in Mutantp53 Lung Cancer Cellsrdquo Cellular Physiology and Biochemistrypp 133ndash151 2017
[23] G Wu Z Sun Q Li et al ldquoNVP-BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3KAKT mTORphosphorylationrdquo OncoTargets andTherapy p 269
[24] J Chen R Shao F Li et al ldquoPI3KAktmTOR pathway dualinhibitor BEZ235 suppresses the stemness of colon cancer stemcellsrdquo Clinical and Experimental Pharmacology and Physiologyvol 42 no 12 pp 1317ndash1326 2015
[25] J Chen R Shao L Li Z P Xu and W Gu ldquoEffective inhi-bition of colon cancer cell growth with MgAl-layered doublehydroxide (LDH) loaded 5-FU and PI3KmTOR dual inhibitorBEZ-235 through apoptotic pathwaysrdquo International Journal ofNanomedicine vol 9 no 1 pp 3403ndash3411 2014
[26] D A Sabbah M G Brattain and H Zhong ldquoDual inhibitorsof PI3KmTOR or mTOR-selective inhibitors Which way shallwe gordquo Current Medicinal Chemistry vol 18 no 36 pp 5528ndash5544 2011
[27] H A Burris C D Kurkjian L Hart et al ldquoTAK-228 (formerlyMLN0128) an investigational dual TORC12 inhibitor pluspaclitaxel withwithout trastuzumab in patients with advancedsolid malignanciesrdquo Cancer Chemotherapy and Pharmacologyvol 80 no 2 pp 261ndash273 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
6 BioMed Research International
Ishikawa Ishikawa+TAXIshikawa+NVP-BEZ235 Ishikawa+NVP-BEZ235+TAX
Ishikawa-TAX Ishikawa-TAX +NVP-BEZ235
Ishikawa-TAX +TAX
Ishikawa-TAX +NVP-BEZ235
+TAX
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
R2
9342 022
632 004Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9480 036
436 048Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9492 186
280 042Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9186 366
310 138
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
g
R2
9472 024
498 006Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9546 056
386 012Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9552 066
370 012Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
Plot P02 gated on P01R1
7-A
AD
(RED
-B-H
Log)
R2
9524 044
410 022
ANNEXINV-PEANNEXINV-PE ANNEXINV-PE
ANNEXINV-PE ANNEXINV-PE
ANNEXINV-PE
ANNEXINV-PEANNEXINV-PE
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
104103102101100 104103102101100 104103102101100 104103102101100
104103102101100 104103102101100 104103102101100 104103102101100
(a)
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
Apop
tosis
rate
() 15
10
5
0
lowastlowast
(b)
Figure 4 The apoptosis of Ishikawa and Ishikawa-TAX cells after treatment of NVP-BEZ235 and paclitaxel (a) NVP-BEZ235 alone andcotreated with paclitaxel Ishikawa and Ishikawa-TAX cells and the changes in apoptosis (b) Apoptosis quantification (lowastlowastP lt001)
Ratio
()
100
80
60
40
20
0
lowastlowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
01 S 2－
Cycle
Ishikawa
Ishikawa+TAX
Ishikawa+NVP-BEZ235
Ishikawa+NVP-BEZ235+TAX
Ishikawa-TAX
Ishikawa-TAX+NVP-BEZ235
Ishikawa-TAX+TAX
Ishikawa-TAX+NVP-BEZ235+TAX
Figure 5 Cell cycle changes after NVP-BEZ235 and paclitaxel treatment of Ishikawa and Ishikawa-TAX cells The cell cycle of Ishikawa andIshikawa-TAX cells changes of G0G1 S G2M cycles after NVP-BEZ235 and paclitaxel alone and in cotreatment (lowastlowastP lt001)
BioMed Research International 7
P-AKT
P-p70 S6K
-actin
(a)
Relat
ive e
xpre
ssio
nP-
AKT
-a
ctin lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
lowastlowast3
2
1
0
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
Relat
ive e
xpre
ssio
nP-
p70
S6K
-a
ctin
20
15
10
05
00
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
(b)Figure 6 Changes of p-AKT and p-p70 S6K proteins after treatment with NVP-BEZ235 and paclitaxel in Ishikawa and Ishikawa-TAX cells(a) Changes of p-AKT and p-p70 S6K proteins after treatment of Ishikawa and Ishikawa-TAX cells with NVP-BEZ235 and paclitaxel aloneand in combination (b) Protein relative quantification (lowastlowastP lt001)
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
Apop
tosis
rate
()
15
20
10
5
0
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
lowastlowast
Figure 7 The apoptosis of Ishikawa-TAX cells after treatment of LY294002 and paclitaxel (a) LY294002 alone and cotreated with paclitaxelIshikawa-TAX cells the changes in apoptosis (b) Apoptosis quantification (lowastlowastP lt001)
8 BioMed Research International
Ratio
()
lowastlowast
lowastlowast
lowastlowast
01 S 2－
Cycle
Ishikawa
Ishikawa-TAX+LY294002
Ishikawa-TAX+TAX
Ishikawa-TAX+LY294002+TAX
lowastlowast
lowastlowast
lowastlowast
100
80
60
40
20
0
lowastlowast
lowastlowast
lowastlowast
Figure 8 Cell cycle changes after LY294002 and paclitaxel treatment of Ishikawa-TAX cells The cell cycle of Ishikawa-TAX cells changes ofG0 G1 S G2 M cycles after LY294002 and paclitaxel alone and in cotreatment (lowastlowastP lt001)
P-AKT
P-p70S6K
-actin
lowastlowast
lowastlowast
lowastlowast
Relat
ive e
xpre
ssio
nP-
p70
S6K
-a
ctin
10
02
04
06
08
00
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
Relat
ive e
xpre
ssio
nP-
AKT
-a
ctin
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
lowastlowastlowastlowast lowastlowast
10
02
04
06
08
00
Figure 9 Changes of p-AKT and p-p70 S6K proteins after treatment with LY294002 and paclitaxel in Ishikawa-TAX cells (a) Changes ofp-AKT and p-p70 S6K proteins after treatment of Ishikawa-TAX cells with LY294002 and paclitaxel alone and in combination (b) Proteinrelative quantification (lowastlowastP lt001)
BioMed Research International 9
cell cycle were not different between Ishikawa group andIshikawa-TAX + TAX group which suggested that Ishikawa-TAX had the ability of resistance for paclitaxel (Figures 7 and8) What is more the results showed that paclitaxel inhibitsIshikawa-TAX cells through PI3KAktmTOR (Figures 7 8and 9)
Nowadays more and more clinical trials focus on thecombination of paxlitaxel with other PI3K inhibitors incancer treatment such as rapamycin and TORC12 inhibitorTAK-228 [26 27] It shows that PI3Kpathway plays an impor-tant role in cancer growth invasion and drug resistance
In summary taxol treatment can inhibit the Ishikawa cellsand Ishikawa-TAX cells through PI3KAktmTOR signalingpathway and therefore regulating PI3KAktmTOR pathwaymay been a promising therapeutic direction for the clinicaltreatment of drug-resistant endometrial carcinoma
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors have no conflicts of interest to declare
Authorsrsquo Contributions
Jie Ding and Mengxiong Li contributed equally to this work
Acknowledgments
This study was funded by Guangdong Medical ResearchFoundation (Grant A2014235) and Guangdong ProvincialScience and Technology Research (Grants 2013B0218000902013B022000044 and 2016A020215074)
References
[1] T Yamamoto T Mori M Sawada et al ldquoLoss of AF-6afadininduces cell invasion suppresses the formation of glandularstructures and might be a predictive marker of resistance tochemotherapy in endometrial cancerrdquo BMC Cancer vol 15 no1 article no 275 2015
[2] L Hoang S Chiang and C-H Lee ldquoEndometrial stromalsarcomas and related neoplasms New developments and diag-nostic considerationsrdquo Pathology 2017
[3] A Mihailovici M Rottenstreich S Kovel I Wassermann NSmorgick andZVaknin ldquoEndometriosis-associatedmalignanttransformation in abdominal surgical scarrdquo Medicine (UnitedStates) vol 96 no 49 Article ID e9136 2017
[4] VMakker A K Green RMWenham DMutch B Davidsonand D S Miller ldquoNew therapies for advanced recurrent andmetastatic endometrial cancersrdquoGynecologic Oncology Researchand Practice vol 4 no 1 2017
[5] N Dadi M Stanley S Shahda B H Orsquoneil and A SehdevldquoImpact of nab-paclitaxel-based second-line chemotherapy inmetastatic pancreatic cancerrdquoAnticancer Reseach vol 37 no 10pp 5533ndash5539 2017
[6] C M Bestvina and G F Fleming ldquoChemotherapy for endome-trial cancer in adjuvant and advanced disease settingsrdquo TheOncologist vol 21 no 10 pp 1250ndash1259 2016
[7] P Augereau A Patsouris E Bourbouloux et al ldquoHormonore-sistance in advanced breast cancer A new revolution inendocrine therapyrdquoTherapeutic Advances in Medical Oncologyvol 9 no 5 pp 335ndash346 2017
[8] D Xu R Li J Wu L Jiang and H A Zhong ldquoDrug designtargeting theCXCR4CXCR7CXCL12 pathwayrdquoCurrent Topicsin Medicinal Chemistry vol 16 no 13 pp 1441ndash1451 2016
[9] K Yuge E Kikuchi M Hagiwara et al ldquoNicotine inducestumor growth and chemoresistance through activation of thePI3KAktmTOR pathway in bladder cancerrdquoMolecular CancerTherapeutics vol 14 no 9 pp 2112ndash2120 2015
[10] V Makker F O Recio L Ma et al ldquoA multicenter single-arm open-label phase 2 study of apitolisib (GDC-0980) forthe treatment of recurrent or persistent endometrial carcinoma(MAGGIE study)rdquo Cancer vol 122 no 22 pp 3519ndash3528 2016
[11] K Inaba K Oda Y Ikeda et al ldquoAntitumor activity of acombination of dual PI3KmTOR inhibitor SAR245409 andselective MEK12 inhibitor pimasertib in endometrial carcino-masrdquo Gynecologic Oncology vol 138 no 2 pp 323ndash331 2015
[12] H K Kim S-H Hwang E Oh and S Abdi ldquoRolipram aselective phosphodiesterase 4 inhibitor ameliorates mechanicalhyperalgesia in a rat model of chemotherapy-induced neuro-pathic pain through inhibition of inflammatory cytokines in thedorsal root ganglionrdquo Frontiers in Pharmacology vol 8 2017
[13] X Chen X Sun J Guan et al ldquoRsf-1 Influences the Sensitivityof Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-120581B Pathway and Its Downstream Proteinsrdquo Cellular Physiologyand Biochemistry vol 44 no 6 pp 2322ndash2336 2018
[14] Y C Tsai Y H Chang Y Chang and C M Chuang ldquoSalvageintraperitoneal chemotherapy for relapsed type II endometrialcancer A pilot case-control studyrdquo Journal of the TurkishGerman Gynecological Association vol 17 no 4 pp 176ndash1812016
[15] L H Tan P H Sykes M M Alkaisi and J J Evans ldquoCell-likefeatures imprinted in the physical nano- andmicro-topographyof the environment modify the responses to anti-cancer drugsof endometrial cancer cellsrdquo Biofabrication vol 9 no 1 ArticleID 015017 2017
[16] K Ding Y Yuan Q-Y Chong et al ldquoAutocrine prolactinstimulates endometrial carcinoma growth and metastasis andreduces sensitivity to chemotherapyrdquo Endocrinology vol 158no 6 pp 1595ndash1611 2017
[17] L Kogan I Laskov Z Amajoud et al ldquoDose dense carboplatinpaclitaxel improves progression free survival in patients withendometrial cancerrdquo Gynecologic Oncology vol 147 no 1 pp30ndash35 2017
[18] H Koh H Park N Chandimali et al ldquoMicroRNA-128 sup-presses paclitaxel-resistant lung cancer by inhibiting MUC1-Cand BMI-1 in cancer stem cellsrdquo Oncotarget vol 8 no 66 pp110540ndash110551 2017
[19] S Tiwari B Tirosh and A Rubinstein ldquoIncreasing the affinityof cationized polyacrylamide-paclitaxel nanoparticles towardscolon cancer cells by a surface recognition peptiderdquo Interna-tional Journal of Pharmaceutics vol 531 no 1 pp 281ndash291 2017
[20] M Xiong Q Lei X You et al ldquoMannosylated liposomesimprove therapeutic effects of paclitaxel in colon cancer mod-elsrdquo Journal of Microencapsulation vol 34 no 6 pp 513ndash5212017
10 BioMed Research International
[21] S Xu J Ge Z Zhang and W Zhou ldquoMiR-129 inhibitscell proliferation and metastasis by targeting ETS1 viaPI3KAKTmTOR pathway in prostate cancerrdquo Biomedicine ampPharmacotherapy vol 96 pp 634ndash641 2017
[22] P-P Gan Y-Y Zhou M-Z Zhong Y Peng L Li and J-HLi ldquoEndoplasmic Reticulum Stress Promotes Autophagy andApoptosis and Reduces Chemotherapy Resistance in Mutantp53 Lung Cancer Cellsrdquo Cellular Physiology and Biochemistrypp 133ndash151 2017
[23] G Wu Z Sun Q Li et al ldquoNVP-BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3KAKT mTORphosphorylationrdquo OncoTargets andTherapy p 269
[24] J Chen R Shao F Li et al ldquoPI3KAktmTOR pathway dualinhibitor BEZ235 suppresses the stemness of colon cancer stemcellsrdquo Clinical and Experimental Pharmacology and Physiologyvol 42 no 12 pp 1317ndash1326 2015
[25] J Chen R Shao L Li Z P Xu and W Gu ldquoEffective inhi-bition of colon cancer cell growth with MgAl-layered doublehydroxide (LDH) loaded 5-FU and PI3KmTOR dual inhibitorBEZ-235 through apoptotic pathwaysrdquo International Journal ofNanomedicine vol 9 no 1 pp 3403ndash3411 2014
[26] D A Sabbah M G Brattain and H Zhong ldquoDual inhibitorsof PI3KmTOR or mTOR-selective inhibitors Which way shallwe gordquo Current Medicinal Chemistry vol 18 no 36 pp 5528ndash5544 2011
[27] H A Burris C D Kurkjian L Hart et al ldquoTAK-228 (formerlyMLN0128) an investigational dual TORC12 inhibitor pluspaclitaxel withwithout trastuzumab in patients with advancedsolid malignanciesrdquo Cancer Chemotherapy and Pharmacologyvol 80 no 2 pp 261ndash273 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
BioMed Research International 7
P-AKT
P-p70 S6K
-actin
(a)
Relat
ive e
xpre
ssio
nP-
AKT
-a
ctin lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
lowastlowast3
2
1
0
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
Relat
ive e
xpre
ssio
nP-
p70
S6K
-a
ctin
20
15
10
05
00
Ishi
kaw
a
Ishi
kaw
a+TA
X
Ishi
kaw
a+N
VP-
BEZ2
35
Ishi
kaw
a+N
VP-
BEZ2
35+T
AX
Ishi
kaw
a-TA
X
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
Ishi
kaw
a-TA
X +T
AX
Ishi
kaw
a-TA
X +N
VP-
BEZ2
35
+TA
X
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
(b)Figure 6 Changes of p-AKT and p-p70 S6K proteins after treatment with NVP-BEZ235 and paclitaxel in Ishikawa and Ishikawa-TAX cells(a) Changes of p-AKT and p-p70 S6K proteins after treatment of Ishikawa and Ishikawa-TAX cells with NVP-BEZ235 and paclitaxel aloneand in combination (b) Protein relative quantification (lowastlowastP lt001)
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
7-A
AD
ANNEXINV-PE
101
102
103
104
100
104103102101100
Apop
tosis
rate
()
15
20
10
5
0
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
lowastlowast
Figure 7 The apoptosis of Ishikawa-TAX cells after treatment of LY294002 and paclitaxel (a) LY294002 alone and cotreated with paclitaxelIshikawa-TAX cells the changes in apoptosis (b) Apoptosis quantification (lowastlowastP lt001)
8 BioMed Research International
Ratio
()
lowastlowast
lowastlowast
lowastlowast
01 S 2－
Cycle
Ishikawa
Ishikawa-TAX+LY294002
Ishikawa-TAX+TAX
Ishikawa-TAX+LY294002+TAX
lowastlowast
lowastlowast
lowastlowast
100
80
60
40
20
0
lowastlowast
lowastlowast
lowastlowast
Figure 8 Cell cycle changes after LY294002 and paclitaxel treatment of Ishikawa-TAX cells The cell cycle of Ishikawa-TAX cells changes ofG0 G1 S G2 M cycles after LY294002 and paclitaxel alone and in cotreatment (lowastlowastP lt001)
P-AKT
P-p70S6K
-actin
lowastlowast
lowastlowast
lowastlowast
Relat
ive e
xpre
ssio
nP-
p70
S6K
-a
ctin
10
02
04
06
08
00
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
Relat
ive e
xpre
ssio
nP-
AKT
-a
ctin
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
lowastlowastlowastlowast lowastlowast
10
02
04
06
08
00
Figure 9 Changes of p-AKT and p-p70 S6K proteins after treatment with LY294002 and paclitaxel in Ishikawa-TAX cells (a) Changes ofp-AKT and p-p70 S6K proteins after treatment of Ishikawa-TAX cells with LY294002 and paclitaxel alone and in combination (b) Proteinrelative quantification (lowastlowastP lt001)
BioMed Research International 9
cell cycle were not different between Ishikawa group andIshikawa-TAX + TAX group which suggested that Ishikawa-TAX had the ability of resistance for paclitaxel (Figures 7 and8) What is more the results showed that paclitaxel inhibitsIshikawa-TAX cells through PI3KAktmTOR (Figures 7 8and 9)
Nowadays more and more clinical trials focus on thecombination of paxlitaxel with other PI3K inhibitors incancer treatment such as rapamycin and TORC12 inhibitorTAK-228 [26 27] It shows that PI3Kpathway plays an impor-tant role in cancer growth invasion and drug resistance
In summary taxol treatment can inhibit the Ishikawa cellsand Ishikawa-TAX cells through PI3KAktmTOR signalingpathway and therefore regulating PI3KAktmTOR pathwaymay been a promising therapeutic direction for the clinicaltreatment of drug-resistant endometrial carcinoma
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors have no conflicts of interest to declare
Authorsrsquo Contributions
Jie Ding and Mengxiong Li contributed equally to this work
Acknowledgments
This study was funded by Guangdong Medical ResearchFoundation (Grant A2014235) and Guangdong ProvincialScience and Technology Research (Grants 2013B0218000902013B022000044 and 2016A020215074)
References
[1] T Yamamoto T Mori M Sawada et al ldquoLoss of AF-6afadininduces cell invasion suppresses the formation of glandularstructures and might be a predictive marker of resistance tochemotherapy in endometrial cancerrdquo BMC Cancer vol 15 no1 article no 275 2015
[2] L Hoang S Chiang and C-H Lee ldquoEndometrial stromalsarcomas and related neoplasms New developments and diag-nostic considerationsrdquo Pathology 2017
[3] A Mihailovici M Rottenstreich S Kovel I Wassermann NSmorgick andZVaknin ldquoEndometriosis-associatedmalignanttransformation in abdominal surgical scarrdquo Medicine (UnitedStates) vol 96 no 49 Article ID e9136 2017
[4] VMakker A K Green RMWenham DMutch B Davidsonand D S Miller ldquoNew therapies for advanced recurrent andmetastatic endometrial cancersrdquoGynecologic Oncology Researchand Practice vol 4 no 1 2017
[5] N Dadi M Stanley S Shahda B H Orsquoneil and A SehdevldquoImpact of nab-paclitaxel-based second-line chemotherapy inmetastatic pancreatic cancerrdquoAnticancer Reseach vol 37 no 10pp 5533ndash5539 2017
[6] C M Bestvina and G F Fleming ldquoChemotherapy for endome-trial cancer in adjuvant and advanced disease settingsrdquo TheOncologist vol 21 no 10 pp 1250ndash1259 2016
[7] P Augereau A Patsouris E Bourbouloux et al ldquoHormonore-sistance in advanced breast cancer A new revolution inendocrine therapyrdquoTherapeutic Advances in Medical Oncologyvol 9 no 5 pp 335ndash346 2017
[8] D Xu R Li J Wu L Jiang and H A Zhong ldquoDrug designtargeting theCXCR4CXCR7CXCL12 pathwayrdquoCurrent Topicsin Medicinal Chemistry vol 16 no 13 pp 1441ndash1451 2016
[9] K Yuge E Kikuchi M Hagiwara et al ldquoNicotine inducestumor growth and chemoresistance through activation of thePI3KAktmTOR pathway in bladder cancerrdquoMolecular CancerTherapeutics vol 14 no 9 pp 2112ndash2120 2015
[10] V Makker F O Recio L Ma et al ldquoA multicenter single-arm open-label phase 2 study of apitolisib (GDC-0980) forthe treatment of recurrent or persistent endometrial carcinoma(MAGGIE study)rdquo Cancer vol 122 no 22 pp 3519ndash3528 2016
[11] K Inaba K Oda Y Ikeda et al ldquoAntitumor activity of acombination of dual PI3KmTOR inhibitor SAR245409 andselective MEK12 inhibitor pimasertib in endometrial carcino-masrdquo Gynecologic Oncology vol 138 no 2 pp 323ndash331 2015
[12] H K Kim S-H Hwang E Oh and S Abdi ldquoRolipram aselective phosphodiesterase 4 inhibitor ameliorates mechanicalhyperalgesia in a rat model of chemotherapy-induced neuro-pathic pain through inhibition of inflammatory cytokines in thedorsal root ganglionrdquo Frontiers in Pharmacology vol 8 2017
[13] X Chen X Sun J Guan et al ldquoRsf-1 Influences the Sensitivityof Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-120581B Pathway and Its Downstream Proteinsrdquo Cellular Physiologyand Biochemistry vol 44 no 6 pp 2322ndash2336 2018
[14] Y C Tsai Y H Chang Y Chang and C M Chuang ldquoSalvageintraperitoneal chemotherapy for relapsed type II endometrialcancer A pilot case-control studyrdquo Journal of the TurkishGerman Gynecological Association vol 17 no 4 pp 176ndash1812016
[15] L H Tan P H Sykes M M Alkaisi and J J Evans ldquoCell-likefeatures imprinted in the physical nano- andmicro-topographyof the environment modify the responses to anti-cancer drugsof endometrial cancer cellsrdquo Biofabrication vol 9 no 1 ArticleID 015017 2017
[16] K Ding Y Yuan Q-Y Chong et al ldquoAutocrine prolactinstimulates endometrial carcinoma growth and metastasis andreduces sensitivity to chemotherapyrdquo Endocrinology vol 158no 6 pp 1595ndash1611 2017
[17] L Kogan I Laskov Z Amajoud et al ldquoDose dense carboplatinpaclitaxel improves progression free survival in patients withendometrial cancerrdquo Gynecologic Oncology vol 147 no 1 pp30ndash35 2017
[18] H Koh H Park N Chandimali et al ldquoMicroRNA-128 sup-presses paclitaxel-resistant lung cancer by inhibiting MUC1-Cand BMI-1 in cancer stem cellsrdquo Oncotarget vol 8 no 66 pp110540ndash110551 2017
[19] S Tiwari B Tirosh and A Rubinstein ldquoIncreasing the affinityof cationized polyacrylamide-paclitaxel nanoparticles towardscolon cancer cells by a surface recognition peptiderdquo Interna-tional Journal of Pharmaceutics vol 531 no 1 pp 281ndash291 2017
[20] M Xiong Q Lei X You et al ldquoMannosylated liposomesimprove therapeutic effects of paclitaxel in colon cancer mod-elsrdquo Journal of Microencapsulation vol 34 no 6 pp 513ndash5212017
10 BioMed Research International
[21] S Xu J Ge Z Zhang and W Zhou ldquoMiR-129 inhibitscell proliferation and metastasis by targeting ETS1 viaPI3KAKTmTOR pathway in prostate cancerrdquo Biomedicine ampPharmacotherapy vol 96 pp 634ndash641 2017
[22] P-P Gan Y-Y Zhou M-Z Zhong Y Peng L Li and J-HLi ldquoEndoplasmic Reticulum Stress Promotes Autophagy andApoptosis and Reduces Chemotherapy Resistance in Mutantp53 Lung Cancer Cellsrdquo Cellular Physiology and Biochemistrypp 133ndash151 2017
[23] G Wu Z Sun Q Li et al ldquoNVP-BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3KAKT mTORphosphorylationrdquo OncoTargets andTherapy p 269
[24] J Chen R Shao F Li et al ldquoPI3KAktmTOR pathway dualinhibitor BEZ235 suppresses the stemness of colon cancer stemcellsrdquo Clinical and Experimental Pharmacology and Physiologyvol 42 no 12 pp 1317ndash1326 2015
[25] J Chen R Shao L Li Z P Xu and W Gu ldquoEffective inhi-bition of colon cancer cell growth with MgAl-layered doublehydroxide (LDH) loaded 5-FU and PI3KmTOR dual inhibitorBEZ-235 through apoptotic pathwaysrdquo International Journal ofNanomedicine vol 9 no 1 pp 3403ndash3411 2014
[26] D A Sabbah M G Brattain and H Zhong ldquoDual inhibitorsof PI3KmTOR or mTOR-selective inhibitors Which way shallwe gordquo Current Medicinal Chemistry vol 18 no 36 pp 5528ndash5544 2011
[27] H A Burris C D Kurkjian L Hart et al ldquoTAK-228 (formerlyMLN0128) an investigational dual TORC12 inhibitor pluspaclitaxel withwithout trastuzumab in patients with advancedsolid malignanciesrdquo Cancer Chemotherapy and Pharmacologyvol 80 no 2 pp 261ndash273 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
8 BioMed Research International
Ratio
()
lowastlowast
lowastlowast
lowastlowast
01 S 2－
Cycle
Ishikawa
Ishikawa-TAX+LY294002
Ishikawa-TAX+TAX
Ishikawa-TAX+LY294002+TAX
lowastlowast
lowastlowast
lowastlowast
100
80
60
40
20
0
lowastlowast
lowastlowast
lowastlowast
Figure 8 Cell cycle changes after LY294002 and paclitaxel treatment of Ishikawa-TAX cells The cell cycle of Ishikawa-TAX cells changes ofG0 G1 S G2 M cycles after LY294002 and paclitaxel alone and in cotreatment (lowastlowastP lt001)
P-AKT
P-p70S6K
-actin
lowastlowast
lowastlowast
lowastlowast
Relat
ive e
xpre
ssio
nP-
p70
S6K
-a
ctin
10
02
04
06
08
00
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
Relat
ive e
xpre
ssio
nP-
AKT
-a
ctin
Ishi
kaw
a
Ishi
kaw
a-TA
X+LY
2940
02
Ishi
kaw
a-TA
X+TA
X
Ishi
kaw
a-TA
X+LY
2940
02+T
AX
lowastlowastlowastlowast lowastlowast
10
02
04
06
08
00
Figure 9 Changes of p-AKT and p-p70 S6K proteins after treatment with LY294002 and paclitaxel in Ishikawa-TAX cells (a) Changes ofp-AKT and p-p70 S6K proteins after treatment of Ishikawa-TAX cells with LY294002 and paclitaxel alone and in combination (b) Proteinrelative quantification (lowastlowastP lt001)
BioMed Research International 9
cell cycle were not different between Ishikawa group andIshikawa-TAX + TAX group which suggested that Ishikawa-TAX had the ability of resistance for paclitaxel (Figures 7 and8) What is more the results showed that paclitaxel inhibitsIshikawa-TAX cells through PI3KAktmTOR (Figures 7 8and 9)
Nowadays more and more clinical trials focus on thecombination of paxlitaxel with other PI3K inhibitors incancer treatment such as rapamycin and TORC12 inhibitorTAK-228 [26 27] It shows that PI3Kpathway plays an impor-tant role in cancer growth invasion and drug resistance
In summary taxol treatment can inhibit the Ishikawa cellsand Ishikawa-TAX cells through PI3KAktmTOR signalingpathway and therefore regulating PI3KAktmTOR pathwaymay been a promising therapeutic direction for the clinicaltreatment of drug-resistant endometrial carcinoma
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors have no conflicts of interest to declare
Authorsrsquo Contributions
Jie Ding and Mengxiong Li contributed equally to this work
Acknowledgments
This study was funded by Guangdong Medical ResearchFoundation (Grant A2014235) and Guangdong ProvincialScience and Technology Research (Grants 2013B0218000902013B022000044 and 2016A020215074)
References
[1] T Yamamoto T Mori M Sawada et al ldquoLoss of AF-6afadininduces cell invasion suppresses the formation of glandularstructures and might be a predictive marker of resistance tochemotherapy in endometrial cancerrdquo BMC Cancer vol 15 no1 article no 275 2015
[2] L Hoang S Chiang and C-H Lee ldquoEndometrial stromalsarcomas and related neoplasms New developments and diag-nostic considerationsrdquo Pathology 2017
[3] A Mihailovici M Rottenstreich S Kovel I Wassermann NSmorgick andZVaknin ldquoEndometriosis-associatedmalignanttransformation in abdominal surgical scarrdquo Medicine (UnitedStates) vol 96 no 49 Article ID e9136 2017
[4] VMakker A K Green RMWenham DMutch B Davidsonand D S Miller ldquoNew therapies for advanced recurrent andmetastatic endometrial cancersrdquoGynecologic Oncology Researchand Practice vol 4 no 1 2017
[5] N Dadi M Stanley S Shahda B H Orsquoneil and A SehdevldquoImpact of nab-paclitaxel-based second-line chemotherapy inmetastatic pancreatic cancerrdquoAnticancer Reseach vol 37 no 10pp 5533ndash5539 2017
[6] C M Bestvina and G F Fleming ldquoChemotherapy for endome-trial cancer in adjuvant and advanced disease settingsrdquo TheOncologist vol 21 no 10 pp 1250ndash1259 2016
[7] P Augereau A Patsouris E Bourbouloux et al ldquoHormonore-sistance in advanced breast cancer A new revolution inendocrine therapyrdquoTherapeutic Advances in Medical Oncologyvol 9 no 5 pp 335ndash346 2017
[8] D Xu R Li J Wu L Jiang and H A Zhong ldquoDrug designtargeting theCXCR4CXCR7CXCL12 pathwayrdquoCurrent Topicsin Medicinal Chemistry vol 16 no 13 pp 1441ndash1451 2016
[9] K Yuge E Kikuchi M Hagiwara et al ldquoNicotine inducestumor growth and chemoresistance through activation of thePI3KAktmTOR pathway in bladder cancerrdquoMolecular CancerTherapeutics vol 14 no 9 pp 2112ndash2120 2015
[10] V Makker F O Recio L Ma et al ldquoA multicenter single-arm open-label phase 2 study of apitolisib (GDC-0980) forthe treatment of recurrent or persistent endometrial carcinoma(MAGGIE study)rdquo Cancer vol 122 no 22 pp 3519ndash3528 2016
[11] K Inaba K Oda Y Ikeda et al ldquoAntitumor activity of acombination of dual PI3KmTOR inhibitor SAR245409 andselective MEK12 inhibitor pimasertib in endometrial carcino-masrdquo Gynecologic Oncology vol 138 no 2 pp 323ndash331 2015
[12] H K Kim S-H Hwang E Oh and S Abdi ldquoRolipram aselective phosphodiesterase 4 inhibitor ameliorates mechanicalhyperalgesia in a rat model of chemotherapy-induced neuro-pathic pain through inhibition of inflammatory cytokines in thedorsal root ganglionrdquo Frontiers in Pharmacology vol 8 2017
[13] X Chen X Sun J Guan et al ldquoRsf-1 Influences the Sensitivityof Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-120581B Pathway and Its Downstream Proteinsrdquo Cellular Physiologyand Biochemistry vol 44 no 6 pp 2322ndash2336 2018
[14] Y C Tsai Y H Chang Y Chang and C M Chuang ldquoSalvageintraperitoneal chemotherapy for relapsed type II endometrialcancer A pilot case-control studyrdquo Journal of the TurkishGerman Gynecological Association vol 17 no 4 pp 176ndash1812016
[15] L H Tan P H Sykes M M Alkaisi and J J Evans ldquoCell-likefeatures imprinted in the physical nano- andmicro-topographyof the environment modify the responses to anti-cancer drugsof endometrial cancer cellsrdquo Biofabrication vol 9 no 1 ArticleID 015017 2017
[16] K Ding Y Yuan Q-Y Chong et al ldquoAutocrine prolactinstimulates endometrial carcinoma growth and metastasis andreduces sensitivity to chemotherapyrdquo Endocrinology vol 158no 6 pp 1595ndash1611 2017
[17] L Kogan I Laskov Z Amajoud et al ldquoDose dense carboplatinpaclitaxel improves progression free survival in patients withendometrial cancerrdquo Gynecologic Oncology vol 147 no 1 pp30ndash35 2017
[18] H Koh H Park N Chandimali et al ldquoMicroRNA-128 sup-presses paclitaxel-resistant lung cancer by inhibiting MUC1-Cand BMI-1 in cancer stem cellsrdquo Oncotarget vol 8 no 66 pp110540ndash110551 2017
[19] S Tiwari B Tirosh and A Rubinstein ldquoIncreasing the affinityof cationized polyacrylamide-paclitaxel nanoparticles towardscolon cancer cells by a surface recognition peptiderdquo Interna-tional Journal of Pharmaceutics vol 531 no 1 pp 281ndash291 2017
[20] M Xiong Q Lei X You et al ldquoMannosylated liposomesimprove therapeutic effects of paclitaxel in colon cancer mod-elsrdquo Journal of Microencapsulation vol 34 no 6 pp 513ndash5212017
10 BioMed Research International
[21] S Xu J Ge Z Zhang and W Zhou ldquoMiR-129 inhibitscell proliferation and metastasis by targeting ETS1 viaPI3KAKTmTOR pathway in prostate cancerrdquo Biomedicine ampPharmacotherapy vol 96 pp 634ndash641 2017
[22] P-P Gan Y-Y Zhou M-Z Zhong Y Peng L Li and J-HLi ldquoEndoplasmic Reticulum Stress Promotes Autophagy andApoptosis and Reduces Chemotherapy Resistance in Mutantp53 Lung Cancer Cellsrdquo Cellular Physiology and Biochemistrypp 133ndash151 2017
[23] G Wu Z Sun Q Li et al ldquoNVP-BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3KAKT mTORphosphorylationrdquo OncoTargets andTherapy p 269
[24] J Chen R Shao F Li et al ldquoPI3KAktmTOR pathway dualinhibitor BEZ235 suppresses the stemness of colon cancer stemcellsrdquo Clinical and Experimental Pharmacology and Physiologyvol 42 no 12 pp 1317ndash1326 2015
[25] J Chen R Shao L Li Z P Xu and W Gu ldquoEffective inhi-bition of colon cancer cell growth with MgAl-layered doublehydroxide (LDH) loaded 5-FU and PI3KmTOR dual inhibitorBEZ-235 through apoptotic pathwaysrdquo International Journal ofNanomedicine vol 9 no 1 pp 3403ndash3411 2014
[26] D A Sabbah M G Brattain and H Zhong ldquoDual inhibitorsof PI3KmTOR or mTOR-selective inhibitors Which way shallwe gordquo Current Medicinal Chemistry vol 18 no 36 pp 5528ndash5544 2011
[27] H A Burris C D Kurkjian L Hart et al ldquoTAK-228 (formerlyMLN0128) an investigational dual TORC12 inhibitor pluspaclitaxel withwithout trastuzumab in patients with advancedsolid malignanciesrdquo Cancer Chemotherapy and Pharmacologyvol 80 no 2 pp 261ndash273 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
BioMed Research International 9
cell cycle were not different between Ishikawa group andIshikawa-TAX + TAX group which suggested that Ishikawa-TAX had the ability of resistance for paclitaxel (Figures 7 and8) What is more the results showed that paclitaxel inhibitsIshikawa-TAX cells through PI3KAktmTOR (Figures 7 8and 9)
Nowadays more and more clinical trials focus on thecombination of paxlitaxel with other PI3K inhibitors incancer treatment such as rapamycin and TORC12 inhibitorTAK-228 [26 27] It shows that PI3Kpathway plays an impor-tant role in cancer growth invasion and drug resistance
In summary taxol treatment can inhibit the Ishikawa cellsand Ishikawa-TAX cells through PI3KAktmTOR signalingpathway and therefore regulating PI3KAktmTOR pathwaymay been a promising therapeutic direction for the clinicaltreatment of drug-resistant endometrial carcinoma
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors have no conflicts of interest to declare
Authorsrsquo Contributions
Jie Ding and Mengxiong Li contributed equally to this work
Acknowledgments
This study was funded by Guangdong Medical ResearchFoundation (Grant A2014235) and Guangdong ProvincialScience and Technology Research (Grants 2013B0218000902013B022000044 and 2016A020215074)
References
[1] T Yamamoto T Mori M Sawada et al ldquoLoss of AF-6afadininduces cell invasion suppresses the formation of glandularstructures and might be a predictive marker of resistance tochemotherapy in endometrial cancerrdquo BMC Cancer vol 15 no1 article no 275 2015
[2] L Hoang S Chiang and C-H Lee ldquoEndometrial stromalsarcomas and related neoplasms New developments and diag-nostic considerationsrdquo Pathology 2017
[3] A Mihailovici M Rottenstreich S Kovel I Wassermann NSmorgick andZVaknin ldquoEndometriosis-associatedmalignanttransformation in abdominal surgical scarrdquo Medicine (UnitedStates) vol 96 no 49 Article ID e9136 2017
[4] VMakker A K Green RMWenham DMutch B Davidsonand D S Miller ldquoNew therapies for advanced recurrent andmetastatic endometrial cancersrdquoGynecologic Oncology Researchand Practice vol 4 no 1 2017
[5] N Dadi M Stanley S Shahda B H Orsquoneil and A SehdevldquoImpact of nab-paclitaxel-based second-line chemotherapy inmetastatic pancreatic cancerrdquoAnticancer Reseach vol 37 no 10pp 5533ndash5539 2017
[6] C M Bestvina and G F Fleming ldquoChemotherapy for endome-trial cancer in adjuvant and advanced disease settingsrdquo TheOncologist vol 21 no 10 pp 1250ndash1259 2016
[7] P Augereau A Patsouris E Bourbouloux et al ldquoHormonore-sistance in advanced breast cancer A new revolution inendocrine therapyrdquoTherapeutic Advances in Medical Oncologyvol 9 no 5 pp 335ndash346 2017
[8] D Xu R Li J Wu L Jiang and H A Zhong ldquoDrug designtargeting theCXCR4CXCR7CXCL12 pathwayrdquoCurrent Topicsin Medicinal Chemistry vol 16 no 13 pp 1441ndash1451 2016
[9] K Yuge E Kikuchi M Hagiwara et al ldquoNicotine inducestumor growth and chemoresistance through activation of thePI3KAktmTOR pathway in bladder cancerrdquoMolecular CancerTherapeutics vol 14 no 9 pp 2112ndash2120 2015
[10] V Makker F O Recio L Ma et al ldquoA multicenter single-arm open-label phase 2 study of apitolisib (GDC-0980) forthe treatment of recurrent or persistent endometrial carcinoma(MAGGIE study)rdquo Cancer vol 122 no 22 pp 3519ndash3528 2016
[11] K Inaba K Oda Y Ikeda et al ldquoAntitumor activity of acombination of dual PI3KmTOR inhibitor SAR245409 andselective MEK12 inhibitor pimasertib in endometrial carcino-masrdquo Gynecologic Oncology vol 138 no 2 pp 323ndash331 2015
[12] H K Kim S-H Hwang E Oh and S Abdi ldquoRolipram aselective phosphodiesterase 4 inhibitor ameliorates mechanicalhyperalgesia in a rat model of chemotherapy-induced neuro-pathic pain through inhibition of inflammatory cytokines in thedorsal root ganglionrdquo Frontiers in Pharmacology vol 8 2017
[13] X Chen X Sun J Guan et al ldquoRsf-1 Influences the Sensitivityof Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-120581B Pathway and Its Downstream Proteinsrdquo Cellular Physiologyand Biochemistry vol 44 no 6 pp 2322ndash2336 2018
[14] Y C Tsai Y H Chang Y Chang and C M Chuang ldquoSalvageintraperitoneal chemotherapy for relapsed type II endometrialcancer A pilot case-control studyrdquo Journal of the TurkishGerman Gynecological Association vol 17 no 4 pp 176ndash1812016
[15] L H Tan P H Sykes M M Alkaisi and J J Evans ldquoCell-likefeatures imprinted in the physical nano- andmicro-topographyof the environment modify the responses to anti-cancer drugsof endometrial cancer cellsrdquo Biofabrication vol 9 no 1 ArticleID 015017 2017
[16] K Ding Y Yuan Q-Y Chong et al ldquoAutocrine prolactinstimulates endometrial carcinoma growth and metastasis andreduces sensitivity to chemotherapyrdquo Endocrinology vol 158no 6 pp 1595ndash1611 2017
[17] L Kogan I Laskov Z Amajoud et al ldquoDose dense carboplatinpaclitaxel improves progression free survival in patients withendometrial cancerrdquo Gynecologic Oncology vol 147 no 1 pp30ndash35 2017
[18] H Koh H Park N Chandimali et al ldquoMicroRNA-128 sup-presses paclitaxel-resistant lung cancer by inhibiting MUC1-Cand BMI-1 in cancer stem cellsrdquo Oncotarget vol 8 no 66 pp110540ndash110551 2017
[19] S Tiwari B Tirosh and A Rubinstein ldquoIncreasing the affinityof cationized polyacrylamide-paclitaxel nanoparticles towardscolon cancer cells by a surface recognition peptiderdquo Interna-tional Journal of Pharmaceutics vol 531 no 1 pp 281ndash291 2017
[20] M Xiong Q Lei X You et al ldquoMannosylated liposomesimprove therapeutic effects of paclitaxel in colon cancer mod-elsrdquo Journal of Microencapsulation vol 34 no 6 pp 513ndash5212017
10 BioMed Research International
[21] S Xu J Ge Z Zhang and W Zhou ldquoMiR-129 inhibitscell proliferation and metastasis by targeting ETS1 viaPI3KAKTmTOR pathway in prostate cancerrdquo Biomedicine ampPharmacotherapy vol 96 pp 634ndash641 2017
[22] P-P Gan Y-Y Zhou M-Z Zhong Y Peng L Li and J-HLi ldquoEndoplasmic Reticulum Stress Promotes Autophagy andApoptosis and Reduces Chemotherapy Resistance in Mutantp53 Lung Cancer Cellsrdquo Cellular Physiology and Biochemistrypp 133ndash151 2017
[23] G Wu Z Sun Q Li et al ldquoNVP-BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3KAKT mTORphosphorylationrdquo OncoTargets andTherapy p 269
[24] J Chen R Shao F Li et al ldquoPI3KAktmTOR pathway dualinhibitor BEZ235 suppresses the stemness of colon cancer stemcellsrdquo Clinical and Experimental Pharmacology and Physiologyvol 42 no 12 pp 1317ndash1326 2015
[25] J Chen R Shao L Li Z P Xu and W Gu ldquoEffective inhi-bition of colon cancer cell growth with MgAl-layered doublehydroxide (LDH) loaded 5-FU and PI3KmTOR dual inhibitorBEZ-235 through apoptotic pathwaysrdquo International Journal ofNanomedicine vol 9 no 1 pp 3403ndash3411 2014
[26] D A Sabbah M G Brattain and H Zhong ldquoDual inhibitorsof PI3KmTOR or mTOR-selective inhibitors Which way shallwe gordquo Current Medicinal Chemistry vol 18 no 36 pp 5528ndash5544 2011
[27] H A Burris C D Kurkjian L Hart et al ldquoTAK-228 (formerlyMLN0128) an investigational dual TORC12 inhibitor pluspaclitaxel withwithout trastuzumab in patients with advancedsolid malignanciesrdquo Cancer Chemotherapy and Pharmacologyvol 80 no 2 pp 261ndash273 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
10 BioMed Research International
[21] S Xu J Ge Z Zhang and W Zhou ldquoMiR-129 inhibitscell proliferation and metastasis by targeting ETS1 viaPI3KAKTmTOR pathway in prostate cancerrdquo Biomedicine ampPharmacotherapy vol 96 pp 634ndash641 2017
[22] P-P Gan Y-Y Zhou M-Z Zhong Y Peng L Li and J-HLi ldquoEndoplasmic Reticulum Stress Promotes Autophagy andApoptosis and Reduces Chemotherapy Resistance in Mutantp53 Lung Cancer Cellsrdquo Cellular Physiology and Biochemistrypp 133ndash151 2017
[23] G Wu Z Sun Q Li et al ldquoNVP-BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3KAKT mTORphosphorylationrdquo OncoTargets andTherapy p 269
[24] J Chen R Shao F Li et al ldquoPI3KAktmTOR pathway dualinhibitor BEZ235 suppresses the stemness of colon cancer stemcellsrdquo Clinical and Experimental Pharmacology and Physiologyvol 42 no 12 pp 1317ndash1326 2015
[25] J Chen R Shao L Li Z P Xu and W Gu ldquoEffective inhi-bition of colon cancer cell growth with MgAl-layered doublehydroxide (LDH) loaded 5-FU and PI3KmTOR dual inhibitorBEZ-235 through apoptotic pathwaysrdquo International Journal ofNanomedicine vol 9 no 1 pp 3403ndash3411 2014
[26] D A Sabbah M G Brattain and H Zhong ldquoDual inhibitorsof PI3KmTOR or mTOR-selective inhibitors Which way shallwe gordquo Current Medicinal Chemistry vol 18 no 36 pp 5528ndash5544 2011
[27] H A Burris C D Kurkjian L Hart et al ldquoTAK-228 (formerlyMLN0128) an investigational dual TORC12 inhibitor pluspaclitaxel withwithout trastuzumab in patients with advancedsolid malignanciesrdquo Cancer Chemotherapy and Pharmacologyvol 80 no 2 pp 261ndash273 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom