)joebxj1vcmjtijoh$psqpsbujpo …downloads.hindawi.com/journals/bmri/2016/4567580.pdf · pogostemon...

Research ArticleMolecular Role of EGFR-MAPK Pathway in PatchouliAlcohol-Induced Apoptosis and Cell Cycle Arrest on A549 CellsIn Vitro and In Vivo

XinGang Lu1 Liu Yang2 ChengHua Lu3 ZhenYu Xu4 HongFu Qiu1 JiaJia Wu5

JingWen Wang6 JiaFeng Tong1 Yin Zhu1 and Jie Shen7

1Department of Traditional Chinese Medicine Huadong Hospital Fudan University Shanghai 200040 China2Department of Oncology Baoshan Hospital of Integrated Traditional Chinese and Western MedicineShanghai University of Traditional Chinese Medicine Shanghai 201999 China3Department of Respiratory Longhua Hospital Shanghai University of Traditional Chinese Medicine Shanghai 200030 China4Department of Traditional Chinese Medicine Wujing Hospital Shanghai University of Traditional Chinese MedicineShanghai 200241 China5Department of Rehabilitation The Second People Huaian Hospital Xuzhou Medical College Huaian 223002 China6Department of Oncology Huadong Hospital Fudan University Shanghai 200040 China7Department of Pharmacy Huadong Hospital Fudan University Shanghai 200040 China

Correspondence should be addressed to Jie Shen shj421126com

Received 5 May 2016 Accepted 20 July 2016

Academic Editor Pratheeshkumar Poyil

Copyright copy 2016 XinGang Lu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Nowadays chemotherapy is still the main effective treatment for cancer Herb prescriptions containing Pogostemon cablin Benth(also known as ldquoGuang-Huo-Xiangrdquo) have been widely used in Chinese medicine today In our research we found that patchoulialcohol a compound isolated from the oil of Pogostemon cablin Benth exerted antitumor ability against human lung cancer A549cells ability both in vitro and in vivo MTT assay was used to assess cell viability Hoechst 33342 staining and TUNEL cover glassstaining provided the visual evidence of apoptosis Caspase activity measurement showed that patchouli alcohol activated caspase9 and caspase 3 of mitochondria-mediated apoptosis Consistently patchouli alcohol inhibited the xenograft tumor in vivo Furtherinvestigation of the underlying molecular mechanism showed that MAPK and EGFR pathway might contribute to the antitumoreffect of patchouli alcohol Our study proved that patchouli alcohol might be able to serve as a novel antitumor compound in theclinical treatment of lung cancer

1 Introduction

Pogostemon cablin Benth also known as Guang-Huo-Xiangin China is a medicinal plant widely planted in PhilippinesMalaysia India and China [1] In the clinical practice oftraditionally Chinese medicine Guang-Huo-Xiang has beenwidely used in numerous prescriptions including Lianhua-Qingwen capsule [2] Additionally the patchouli oil a natureperfume from Guang-Huo-Xiang has been widely used toresolve damp improve appetite arrest vomiting and dispelsummer-heat as well as summer-damp [3ndash5] for a long timein history since ancient times in China

It has been found that patchouli alcohol (PA) is the maineffective component of Guang-Huo-Xiang which accountsfor 20ndash26 weight of the patchouli oil [6] The chemicalstructure of PA is shown in Figure 1(a) PA is used for thequality control objective of patchouli oil in pharmaceuticalindustry (The Pharmacopoeia Commission of PRC 2010)Accumulating reports have demonstrated that PA had mul-tiple effects such as anti-inflammatory [7 8] antivirus [9ndash12] antimicrobial [13ndash17] and insecticidal [18 19] protectiveeffect on fluidity of intestinal epithelial cells [20] protectiveeffect on acute lung injury [21] radical-scavenging activity

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 4567580 12 pageshttpdxdoiorg10115520164567580

2 BioMed Research International

SS

S

H

H RR

OHMe Me

MeMe

(a)

4824120

20

40

60

80

100

120

0

Time (h)

Cel

l via

bilit

y

Control50 gmL

75 gmL100 gmL

( o

f con

trol) lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

(b)

300250200150

70

80

90

100

10060

Concentration (gmL)

HEK293

L02HFL-1

Cel

l via

bilit

y (

of c

ontro

l)

(c)

104103102101100

101

102

103

104

100

FL1-H

FL2

-H

0

FL2

-H

PI

104103102101100

101

102

103

104

100

FL1-H Annexin V FITC

50

104103102101100

101

102

103

104

100


FL2

-H

PI

75

104103102101100

101

102

103

104

100

FL1-H

FL2

-H

100

104103102101100

101

102

103

104

100


FL2

-H

PI

75 + EGF

75 + EGF1007550Control

5

10

15

20

25

30

35

40

45

50

0

Concentration

Early apoptosisLate apoptosis

lowastlowastlowast


lowastlowastlowast

(gmL)

A

nnex

in V

-pos

itive

(d)Figure 1 Continued

BioMed Research International 3

10075500 (gmL)

BAX

Bcl-2

c-Myc

PARPp-PARP

BAX

c-M

yc

PARP

p-PR

RP

Actin

15

20

25

30

05

10

00

o

f con

trol

50 gmL0 gmL

lowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast




lowastlowast

lowastlowast


75gmL100gmL

Cleaved-

Cleaved-

cas9

cas3

Clea

ved-

cas9

Clea

ved-

cas3

Bcl-2

Cyclin E

(e)

Figure 1 Growth inhibition and apoptosis effect of PA (a)The chemical structure of PA (b) Inhibition effect of PA on A549 cells usingMTTassay (c) Effect of PA on HEK293 L02 and HFL-1 cells for 48 h using MTT assay (d) The increasing tendency of apoptotic ratio inducedby increasing PA concentrations of 0 50 75 and 100120583gmL and 75 120583gmL combined with EGF (e) Mechanism of PA induced apoptosis onA549 cells A549 cells were exposed for 48 h with increasing PA Proteins levels were probed by western blotting assay Statistical differenceswere compared between PA treatment group and control group and considered significant at the levels of lowastlowast119875 lt 001 or lowastlowastlowast119875 lt 0001

[22] inhibitory activity on platelet-activating factor (PAF)activation [23] and antiemetic activity [24]

Recently several studies have revealed the anticanceractivity of PA and the possible underlying mechanisms PAsuppressed cell proliferation and induced apoptosis in aconcentration-dependent manner in two human colorectalcancer cell lines (HCT116 SW480) Moreover PA suppressedcell growth in MCF7 BxPC3 PC3 and HUVEC cells PAtreatment to HCT116 and SW480 cells triggered p21 expres-sion and inhibited the expressions of Cyclin D1 and Cyclin-dependent kinase 4 (CDK4) Meanwhile PA attenuated theexpressions of histone deacetylases (HDACs) and c-myc intwo human colorectal cancer cells PA treatment also resultedin the transcriptional activity of NF-120581B via an increase ofp65 nuclear translocation [25] Additionally PA inhibited theproliferation of HeLa cells in vitro [26]

However the anticancer ability on human lung cancerof PA has not been investigated In this study we foundthat PA was able to suppress the growth of A549 cells bothin vitro and in vivo Treatment of PA induced apoptosis inand arrested the cell cycle progression of A549 cells Furtherinvestigation on the molecular mechanism showed that PAinhibited the phosphorylation of EGFR and thereby blockedthe downstream signaling which may be responsible for theanticancer activity of PA Our study proved that PA mightserve as a potential compound for the treatment of non-small-cell lung cancer in the clinical practice and providedmore insights for developing novel anticancer agents frommedicinal plants

2 Materials and Methods

21 Materials Pancreatin penicillin RPMI-1640 mediumFetal Bovine Serum (FBS) Phosphate Buffered Saline (PBS)and streptomycin were supplied from Gibco (Gibco Carls-bad USA) A549 L02 HEK293 and HFL-1 cells weregained from the Cell Bank of Chinese Academy of Sciences(Shanghai China) Terminal Deoxynucleotidyl Transferase-(TdT-) mediated dUTP nick end labeling (TUNEL) Apo-Green Detection Kit was supplied by Roche (Roche BaselSwitzerland) while Annexin VampPI Kit was purchased byBiotool (Selleck Chemicals Houston Texas USA) Proteinextraction kit RNA extraction kit BCA Protein Assay Kitcaspases 3 and 9 activity kit Propidium Iodide (PI) dimethyl-sulfoxide (DMSO) 410158406-diamidino-2-phenylindole (DAPI)and Hoechst 33243 were purchased from Beyotime Instituteof Biotechnology (Beyotime Haimen China) PA stand-ard preparation (purity gt 98) was purchased fromNationalInstitute for the Control of Pharmaceutical and Biologi-cal Products (Beijing China) JC-10 Mitochondrial Mem-brane Potential Assay Kit was obtained by AAT Bio-quest (AAT Bioquest CA USA) ECL Advanced Detec-tion Kit was provided by Thermo Fisher (Thermo FisherWaltham USA) 3-(45-Dimethylthiazol-2-yl)-25-diphenyl-2H-tetrazolium bromide (MTT) and albumin from bovineserum (ABS) were purchased from Sigma-Aldrich (St LouisUSA) Desired primary antibodies HRP-labeled secondaryanti-mouseanti-rabbit antibodies were provided by CellSignaling Technology (CST Beverly USA) All other unmen-tioned chemical reagents were of analytic grade


22 Cell Culture and PA Treatment A549 carcinoma celllines and three types of normal cell lines (HEK293 L02and HFL-1) were cultured in a humidified atmosphere of5 CO

2with RPMI-1640 medium containing 10 fetal calf

serum and antibiotics (100 IUmL penicillin and 100 IUmLstreptomycin) PA standard was dissolved in DMSO forfurther experiment A549 cells were treated with increasingconcentrations of PA for desired hours in PA exposuregroups The control group was conducted with the samevolumes of DMSO under the same conditions

23 Cytotoxicity and Cell Viability Analysis Cytotoxicity ofPA on carcinoma and normal cells was analyzed using theMTT assay The A549 HEK293 L02 and HFL-1 cells wereseeded in culture plates and exposed to PA by increasingconcentrations Concisely 5 times 103 cells were cultured with200120583L culture medium and 20120583L MTT (5mgmL) for 4hoursThe absorbance at 570 nmwas assessed using a micro-plate reader (Perkin-Elmer IncWaltham USA) after remov-ing the supernatant and dissolving the formazan in DMSOCell viability of PA-treated cells and control cells was esti-mated as (absorbance of PA-treated groupabsorbance ofcontrol) times 100

24 Hoechst 33342 Staining TheNSCLCA549 cells were cul-tured and treated by PA as described aboveTheNSCLCA549cells were stained with Hoechst 33342 (5120583gmL) at roomtemperature for 5 minutes in the dark Stained cells wereobserved by an inverted immunofluorescence microscopy(D5100 Nikon Tokyo Japan)

25 TUNEL Staining DNA fragmentation in apoptotic cellsinduced by anticarcinoma drugs can be detected by TUNELstaining assay according to the manufacturerrsquos instructionThe NSCLC A549 cells after PA treatment and control werefixed by TUNEL and DAPI We analyzed the PA-treatedgroup and control cells immediately using immunofluo-rescence microscopy described above to view the green(TUNEL) at 520 nm and the blue (DAPI) at 460 nm

26 Flow Cytometry for Cell Cycle and Apoptosis Measure-ment To detect the cell cycle and apoptotic subpopulationin PA-treated A549 cells the A549 cells were cultured andinduced by PA as mentioned PA-treated A549 cells wereharvested stained with Annexin-FITCPI and measuredusing a FACS Calibur cytometer (BD Biosciences San JoseCA USA) according to the manufacturerrsquos instruction Asfor the investigation of cell cycle progression PA-treated cellswere harvested fixed and stained with PI according to themanufacturersquos instruction and then measured by the FACSCalibur cytometer

27 Mitochondrial Membrane Potential (MMP) AAT JC-10assay kit was used to measure the loss in mitochondrialmembrane potential of PA-treated A549 cells PA-treatedA549 cells were stained according to the manual and thenthe fluorescence was measured using a microplate reader(Perkin-Elmer IncWalthamUSA)TheMMPwas indicated

by the ratio of the green fluorescence absorbance at 525 nm tothe red fluorescence absorbance at 590 nm

28 Caspases 3 and 9 Activity Assay Caspases 3 and 9 activ-ities were determined by using caspases 3 and 9 activitykit according to the kit instruction manual Release of p-nitroanilide (pNA) was determined by absorbance at 405 nmusing a microplate reader (Perkin-Elmer Inc WalthamUSA) The caspase activity was demonstrated as the ratio ofPA-treated cellscontrol A549 cells

29 In Vivo Study We conducted all the animal experimentsaccording to the Declaration of Helsinki and the Guide forthe Care and the Use of Laboratory Animals as adoptedand promulgated by the United States National Institutes ofHealth The animal study was licensed by the InstitutionalAnimal Care and Use Committee of Fudan University The6-week-old BALBC nude mice (Shanghai Slac LaboratoryAnimal) were injected with A549 cells intraperitoneally with1 times 107 cells per mouse There were 4 randomly dividedgroups (6 mice each group) control group (vehicle) andPA treatment group with low medium and high PA dosePA standard crystal was dissolved in saline containing 5DMSO When the volume of the tumor reached 100mm3200120583L 5 DMSOsaline solutions including increasing doseof PA were injected intraperitoneally into the right flank ofnude mice in PA treatment group The disinfecting saline insame volumes containing 5 DMSO was administrated intothe vehicle group PA was injected into all the PA treatmentgroups every 3 days After the last treatment the nude micewere put to death in 24 h Tumor xenografts removed frommice were measured and fixed for next experiment Tumorsizes were evaluated every 3 days using micrometer calipersand tumor weights were determined at last

210 Immunohistochemistry The A549 xenografted tumorsamples were stained using cleaved-caspase 3 (1 100) andKi67 (1 150) antibodies separately for immunohistochem-istry Images were recorded with a fluorescence microscope(D5100 Nikon Tokyo Japan)

211 Western Blotting The levels of apoptotic proteins wereanalyzed by western blotting analysis Proteins of PA-treatedcells and control were extracted by the extraction kit proteinconcentrations were measured using BCA protein assaykit The proteins were subjected to SDS-PAGE and elec-trophoretically transferred to PDVF membrane (MilliporeCorp Bedford MA USA) The membranes were incubatedwith desired primary antibodies (1 1000) and incubated withsecondary antibody (1 5000) conjugated with peroxidasesubsequentlyThese signals were then detected by a detectionsystem (BD Biosciences San Jose CA USA) The 120573-actinantibody was employed as control

212 Statistical Analysis Triplicate experiments were con-ducted with independent samplesThe data were representedas mean plusmn SD Statistical intergroup differences were evalu-ated using one-way ANOVA followed by post hoc Bonferroni


Table 1 The inhibitory effect of PA on A549 implantation tumor growth in BALBC-nu mice

Groups (mgkg) Number of animalssurvived

Body weights (g) Tumor weights (g) Inhibition rate ()Start End

Control 6 2038 plusmn 066 2478 plusmn 093 135 plusmn 0185 6 207 plusmn 044 2448 plusmn 068 078 plusmn 011lowast 423510 6 2067 plusmn 042 2448 plusmn 143 051 plusmn 023lowastlowastlowast 625015 6 2067 plusmn 041 2338 plusmn 057 027 plusmn 019lowastlowastlowast 7970lowast119875 lt 005 versus the vehicle controllowastlowastlowast119875 lt 0001 versus the vehicle control

test and were represented as lowast119875 lt 005 lowastlowast119875 lt 001 or lowastlowastlowast119875 lt0001 level All statistical analyses were performed using SPSS190 (Chicago IL USA)

3 Results

31 Effect of PA Antigrowth Capability in Four Cells We firstinvestigated whether PA treatment could inhibit the growthof non-small-cell lung cancer cells The A549 is most com-mon cell model used for multiple anti-lung cancer research[27] In our results it was shown that PA suppressed thegrowth of A549 cells in a concentration- and time-dependentmanner (Figure 1(b)) Lower viability was observed in A549cells treated with PA for 48 h The IC

50value of PA on A549

cells was 7980 plusmn 409 120583gmL Further we determined thecytotoxicity of PA in human normal cells L02 HEK293 andHFL-1 However more than 60 of cells were still viablewhen treated with PA of the concentration up to 300120583gmL(Figure 1(c)) Taken together these data suggested that PAmight serve as a potent and safe anticancer agent against non-small-cell lung cancer

32 Effects of PA on Cell Apoptosis In Vitro The best per-formances based on MTT results indicate that 48 h wasbest experiment time PA exposure at 50ndash100120583gmL at 48 htreated cells showed amarked increase byAnnexinVPI assayshown in Figure 1(d)The apoptotic population in PA-treatedA549 cells increased in a dose-dependent manner

We further hypothesized that PA might induce apoptosisvia the mitochondrial pathway Firstly western blottingassays showed prompted cleavage of PARP and caspases 3and 9 in PA-treated cells Additionally PA inhibited theexpression of Bcl-2 but had little effect on the level of Baxwhich led to decreased ratio of Bcl-2Bax (Figure 1(e))Consistent with above result JC-10 assays determine themitochondrial outer membrane permeabilization inducedby PA treatment Most anticancer agents induce collapse ofMMP mainly in the mitochondria apoptotic early phase Inour present study 24 h was conducted as PA treatment timeto view the loss of MMP It was found that PA treatmentincreased the ratio of greenred fluorescence of stained cellswhich indicated a loss of MMP (Figure 2(a)) Activity assaysrevealed caspases 3 and 9 activation in the PA induced A549cells (Figures 2(b) and 2(c)) These results suggested thatinduction of mitochondrial apoptosis was at least partlyresponsible for the cytotoxicity of PA in A549 cells

Additionally the apoptosis elicited by PA exposure wasobserved visually as well Hoechst staining exhibited imageresults with shrinkage nuclear fragmentation and chromatincompaction of PA-treated cells indicating apoptosis induc-tion as well (Figure 2(d)) Consistently TUNEL stainingdemonstrated that PA at the concentration of 50ndash100120583gmLled to significantly increased population of apoptotic cells(green Figure 2(e))

Western blotting of the PA treatment A549 versus controlshowed the apoptosis-related signal pathway In presentsurvey we found that the phosphorylation of EGFR a criticaltyrosine kinase in the genesis and development of numeroushuman cancers was suppressed by PA treatment The EGFRphosphorylation level containing RASRAFMEKERK isblocked with the increasing concentration of PA as well TheMAPK signaling pathway activities (JNK ERK and P38)were also evaluated after PA treatment As shown in Fig-ure 2(f) western blotting indicated that phosphorylation ofERK (which lies downstream of EGFR) was downregulatedHowever PA caused the increasing of p-JNK while showingno impact on p-P38 The phosphorylation activation ofJNK signaling pathway and downphosphorylation of EGFRpathway induced the MMP loss These caused the unbalanceof Bcl-2Bax which elicits the following mitochondrial apop-tosis pathways releasing of c-Myc and activation of caspase 9and caspase 3 subsequently

33 Effects of PA on the Cell Cycle Arrest In Vitro We con-ducted cell cycle assay to evaluate the PArsquos impact on A549using PI Meanwhile we performed western blotting toreveal the underlying mechanism Figure 3(a) manifestedan accumulating cell cycle progression in G

1S phase in a

dose-dependent manner in PA-treated A549 cells Then wedetermined the PArsquos impact on the expression of P53 P21Cyclin E and Cyclin-dependent kinase 2 (CDK2) Westernblotting demonstrated that the expression level of P53 wasactivated in a dose-dependent manner and an appreciabledownregulation of Cyclin E and CDK2 subsequently (Fig-ure 3(b))

34 Antiproliferation Effect of PA on A549 Nude XenograftTumor We performed a study in vivo to investigate the anti-proliferation effect of PA on A549 nude xenograft tumorThere were few changes in mice body weight in the experi-ment as shown in Figure 4(a)This implied the relative safetyof the injection of PA to the nude mice After 21 days tumorstreatedwith PA grew smaller than control as shown in Table 1


Concentration (gmL)1007550Control

200

300

400

500

100

0

Ratio

(525

590

) o

f con

trol

lowastlowastlowast

lowastlowastlowast

lowastlowast

(a)


0

6

8

4

2

lowastlowastlowast

lowastlowastlowast

lowastlowast

Rela

tive c

aspa

se9

activ

ity

(b)


0

6

8

4

2

10

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Rela

tive c

aspa

se3

activ

ity

(c)

Concentration (gmL)

1007550

80

100

40

60

20

0

Hoe

chst

posit

ive c

ells

()

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0(gmL) 50 75 100

0

(d)

Figure 2 Continued


0

50

75

100

MergeDAPI(gmL)

Concentration (gmL)1007550

0

0

lowastlowastlowast

lowastlowast30

40

20

10

50

TUNEL

TUN

EL p

ositi

ve ce

ll (

)

(e)

P38

p-P38

JNK

p-JNK

ERK

p-ERK

EGFR

p-EGFR

MEK

p-MEK

10075500 10075500(gmL) (gmL)

Actin

p-M

EK

MEK

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38

75 gmL100gmL

o

f con

trol lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast lowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowast lowast

-Actin

0gmL50gmL

10

15

20

25

30

05

00

(f)

Figure 2 The apoptosis effect and involved signaling pathway activity of PA on A549 cells The A549 cells were exposed for 48 h withincreasing PA (a) PA induced MMP collapse for 48 h treatment (b) PA exposure induced caspase 9 activation in A549 cells for 48 h (c) PAexposure induced caspase 3 activation in A549 cells for 48 h (d) The Hoechst staining immunofluorescence microscopy image of A549 cellsfor Hoechst in vitro Scale Bar 10 120583m (e)The TUNEL staining immunofluorescencemicroscopy image of A549 cells for DAPI (blue) TUNELFITC (green) and their merge in vitro Scale Bar 50120583m (f) Effect of PA on EGFR and MAPK signaling pathways in A549 cells Statisticaldifferences were compared between PA treatment group and control group and considered significant at the levels of lowast119875 lt 005 lowastlowast119875 lt 001or lowastlowastlowast119875 lt 0001

and Figures 4(b) and 4(c) The significantly decreasing levelsof Ki67-positive cells and the increasing levels of cleaved-caspase 3 induced by the PA treatment were found in thestudy in vivo as shown in Figure 4(d) The study in vivodemonstrated the effects of apoptosis and antiproliferationeffect of PA as well

4 Discussion

Pogostemon cablin Benth possessed multiple pharmacologyactivities in previous reports [5] PA is the main biologicalactive constituent of Pogostemon cablin Benth Publishedstudies had shown that PA inhibited the proliferation ofseveral carcinoma cells and normal cells such as HUVEC[25] However higher IC

50in our study demonstrated tissue

specific effect of PA on HEK293 (normal kidney cell) L02

(normal liver cell) and HFL-1 Exposure to PA caused apop-tosis and cell cycle arrest in A549 in a time- and dose-dependent manner Therefore PA may be a candidate anti-tumor agent although the administration or the structure ofPA may call for new efforts in future

Scientists paid attention to the drugrsquos antiproliferationeffect and intrinsic mechanism during the past decades [28ndash30] Antiproliferation mechanisms are commonly associatedwith apoptosis and cell cycle arrest [31] Firstly we observedthat the antiproliferation is activated by apoptosis throughTUNEL andHoechst staining Using flow cytometry analysisthe apoptosis induced by PA was validated by Annexin VPIassay Apoptosis by caspase activations has two main typescaspase 8 activation is linked to extrinsic apoptotic pathwaydownstream death receptor like TNF-120572 receptor while cas-pase 9 activation is involved in the intrinsic mitochondrial


200

300

400

100

0250200150100502502001501005025020015010050

25020015010050 25020015010050

75

200

300

400

100

0

500

50

200

300

400

100

0

0

200

300

400

100

0

500100

200

300

400

100

0

75 + EGF

DNA content DNA content

Cel

l cou

nt

Cel

l cou

nt

SG1

G2M


lowastlowastlowast

40

60

80

100

20

0

Cell

cycle

dist

ribut

ion

()

lowastlowastlowastlowastlowast

lowast

(G1)

Concentration (gmL)

(a)

(gmL)10075500

P21

P53

CDK2

ActinCDK2P53P21

10

15

20

25

30

35

40

05

00

o

f con

trol

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast lowastlowastlowast

lowastlowastlowastlowastlowast lowast

50 gmL0 gmL 75gmL

100gmL

Cyclin E

Cyclin E

(b)

Figure 3 Typical G1S cell cycle arrest on A549 cells induced by increasing PA exposure of 0 50 75 and 100120583gmL (a) PA induced G

1S cell

cycle arrest (b) The involved proteins activities Statistical differences were compared between PA treatment group and control group andconsidered significant at the levels of lowast119875 lt 005 lowastlowast119875 lt 001 or lowastlowastlowast119875 lt 0001

apoptotic pathways [32] Anticancer drugs induce caspase9 activation in most apoptosis cases [33] As demonstratedby caspase activation assay and western blotting caspases 9and 3 activations in PA exposure were detected The Bcl-2family proteins play important roles in apoptosis [34] Twomain proteins of Bcl-2 family are the antiapoptotic Bax andthe proapoptotic Bcl [35] The unbalance of Bcl-2Bax ratiocontributes to the loss ofMMP [36] PA caused the unbalanceof Bcl-2Bax collapse of MMP and thus cascade of caspasepathway MMP is an important converge point for manysignaling intracellular apoptotic pathways [37] Transcrip-tional factor NF-120581B plays an important role in the regulationof mitochondrial apoptotic pathway [38] Many antitumoragents exert their ability through NF-120581B signaling pathway

The antiproliferation effect of PA to A549 cell was weakerthan SW480 cell but stronger thanHCT116 cell in vitro How-ever apparent antitumor effect of PA to A549 xenograft nudemodel was witnessed in our study in vivo As previous reportmentioned PA induced two types of human colon cancercell lines apoptosis via activating NF-120581B signaling pathwayand downregulating c-myc [25] Our result is associated withthe published reports in mitochondrial apoptotic pathwayFurthermore we conducted the cell cycle arrest assay inPA-treated A549 cell In published report PA treatment inHCT116 and SW480 cells activated P21 expression and sup-pressed the expressions of Cyclin D1 and Cyclin-dependentkinase 4 (CDK4) in a dose-dependent manner [25] Similarto previous result PA induced accumulating G

1S cell cycle


21181512963

22

24

26

20

18

Body

wei

ghts

(g)

Control5mgkg

10mgkg15mgkg

Days after first administration

(a)

21181512963

Control5mgkg

10mgkg15mgkg

lowast

lowastlowast


lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

400

600

800

1000

1200

200

0

Tum

or si

ze (m

m3)


(b)

0mgkg

5mgkg

10mgkg

15mgkg

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


0

20

40

60

Ki67

posit

ive c

ells

()


5

10

15

20

0

(mgkg)151050

Ki67

Cleaved-cas3

Clea

ved-

cas3

posit

ive c

ells

()

(d)

Figure 4 The antiproliferation effect of PA on A549 xenograft model (a) The body weights of nude A549 models in vivo (b) The tumorsizes of A549 nude models in vivo (c) The tumor of each group (d) Effect of PA on the expression levels of cleaved-caspase 3 and Ki67 inA549 nude model Scale Bar 100 120583m Statistical differences were compared between PA treatment group and control group and consideredsignificant at the levels of lowast119875 lt 005 lowastlowast119875 lt 001 or lowastlowastlowast119875 lt 0001


EGFR

p-EGFR

ERK

p-ERK

P38

p-P38

JNK

p-JNK

(gmL)75 + EGF750

Actin

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38

0 gmL75gmL75gmL + EGF

40

35

30

25

20

15

10

05

00

o

f con

trol



-Actin

(a)

(b)

Figure 5 A549 cells were exposed for 48 h combined with EGF (a) Mechanism certification of PA induced apoptosis and cell cycle arreston A549 cells (b) The graphical abstract of PArsquos action on A549 cells Statistical differences were compared between PA treatment group andcontrol group and considered significant at the levels of lowastlowastlowast119875 lt 0001

arrest through activation in P53 and P21 and suppression inCDK2 and Cyclin E

EGFR plays important roles in cell proliferation differen-tiation oncogenesis and development [39] The expressionlevel of EGFR was commonly upregulated in many cancersfor instance colon [40] ovarian [41] breast [42] and lungespecially [43] We conducted western blotting assay tocertify apoptotic mechanism We used the EGF (100 ngmL)in activation of EGFR signal pathway [44] The apoptosis

in PA combined with EGF group was decreased comparedwith PA while cell cycle distribution was altered as shownin Figures 1(d) and 3(a) The EGFR phosphorylation levelswere reupregulated in the combination group as shownin Figure 5(a) Our results show that PA treatment sig-nificantly blocked the phosphorylation of EGFR and itsdownstream signaling phosphorylation of MEK and ERKproteins Importantly exotic addition of EGF reactivated thedownstream pathway of EGFR and reversed the apoptosis as


well as cell cycle arrest induced by PA This indicated thatblocking the phosphorylation of EGFR is necessary for thecytotoxicity of PA

In summary we reported that PA induced apoptosis andcell cycle arrest in A549 cancer cells in vitro and in vivothrough blocking phosphorylation of EGFR pathways andactivating JNK pathways for the first time to our knowledgePA also arrests G

1S cycle distribution through impact on

CDK2Cyclin E complex The graphical mechanism of PArsquosaction on A549 cells was demonstrated in Figure 5(b) Ourfindings suggested that PA may be a promising candidate forantitumor agent

Competing Interests

No potential competing interests were disclosed by all au-thors

Authorsrsquo Contributions

XinGang Lu Liu Yang and ChengHua Lu contributed to thiswork equally

Acknowledgments

This study was financially supported by ldquoShanghai XinLinNew Star Planrdquo (ZY3-RCPY-2-2081) and State Administra-tion of Traditional Chinese Medicine ldquoTwelfth Five-YearPlanrdquo Key Specialty (Chinese Medicine Geriatrics) Technol-ogy supports were obtained from Shanghai Key Laboratoryof Clinical Geriatric Medicine

References

[1] J-B Liao Y-Z Liang Y-L Chen et al ldquoNovel patchouli alcoholternary solid dispersion pellets prepared by poloxamersrdquo Ira-nian Journal of Pharmaceutical Research vol 14 no 1 pp 15ndash262015

[2] W Jia C Wang Y Wang et al ldquoQualitative and quantitativeanalysis of the major constituents in Chinese medical prepa-ration Lianhua-Qingwen capsule by UPLC-DAD-QTOF-MSrdquoThe Scientific World Journal vol 2015 Article ID 731765 19pages 2015

[3] L F Hu S P Li H Cao et al ldquoGC-MS fingerprint of Pogos-temon cablin in Chinardquo Journal of Pharmaceutical and Biomed-ical Analysis vol 42 no 2 pp 200ndash206 2006

[4] M K Swamy and U R Sinniah ldquoA comprehensive review onthe phytochemical constituents and pharmacological activitiesof Pogostemon cablin Benth an aromatic medicinal plant ofindustrial importancerdquoMolecules vol 20 no 5 pp 8521ndash85472015

[5] S P Bhatia C S Letizia and A M Api ldquoFragrance materialreviewonpatchouli alcoholrdquoFood andChemical Toxicology vol46 supplement 11 pp S255ndashS256 2008

[6] Z Zhao J Lu K Leung C L Chan and Z-H Jiang ldquoDeter-mination of patchoulic alcohol in Herba Pogostemonis by GC-MS-MSrdquo Chemical amp Pharmaceutical Bulletin vol 53 no 7 pp856ndash860 2005

[7] Y-F Xian Y-C Li S-P Ip Z-X Lin X-P Lai and Z-R SuldquoAnti-inflammatory effect of patchouli alcohol isolated from

Pogostemonis Herba in LPS-stimulated RAW2647 macro-phagesrdquo Experimental and Therapeutic Medicine vol 2 no 3pp 545ndash550 2011

[8] J B Jeong Y K Shin and S-H Lee ldquoAnti-inflammatoryactivity of patchouli alcohol in RAW2647 and HT-29 cellsrdquoFood and Chemical Toxicology vol 55 pp 229ndash233 2013

[9] H Kiyohara C Ichino Y Kawamura T Nagai N Sato and HYamada ldquoPatchouli alcohol in vitro direct anti-influenza virussesquiterpene in Pogostemon cablin Benthrdquo Journal of NaturalMedicines vol 66 no 1 pp 55ndash61 2012

[10] H Wu B Li X Wang M Jin and G Wang ldquoInhibitory effectand possible mechanism of action of patchouli alcohol againstinfluenza A (H2N2) virusrdquo Molecules vol 16 no 8 pp 6489ndash6501 2011

[11] Y-C Li S-Z Peng H-M Chen et al ldquoOral administration ofpatchouli alcohol isolated from Pogostemonis Herba augmentsprotection against influenza viral infection in micerdquo Interna-tional Immunopharmacology vol 12 no 1 pp 294ndash301 2012

[12] X-L Wu D-H Ju J Chen et al ldquoImmunologic mechanism ofpatchouli alcohol anti-H1N1 influenza virus may through regu-lation of the RLH signal pathway in vitrordquoCurrentMicrobiologyvol 67 no 4 pp 431ndash436 2013

[13] X Yang X Zhang S-P Yang and X-D Yu ldquoPreliminary anti-bacterial evaluation of the chemical compositions in Herbapogostemonis oilrdquo Pakistan Journal of Pharmaceutical Sciencesvol 26 no 6 pp 1173ndash1179 2013

[14] X-D Yu J-H Xie Y-H Wang et al ldquoSelective antibacterialactivity of patchouli alcohol against Helicobacter pylori basedon inhibition of ureaserdquo Phytotherapy Research vol 29 no 1pp 67ndash72 2015

[15] S Pattnaik V R Subramanyam and C Kole ldquoAntibacterial andantifungal activity of ten essential oils in vitrordquo Microbios vol86 no 349 pp 237ndash246 1996

[16] X Yang X Zhang S-P Yang and W-Q Liu ldquoEvaluation ofthe antibacterial activity of patchouli oilrdquo Iranian Journal ofPharmaceutical Research vol 12 no 3 pp 307ndash316 2013

[17] D Vazquez-Sanchez M L Cabo and J J Rodrıguez-HerreraldquoAntimicrobial activity of essential oils against Staphylococcusaureus biofilmsrdquo Food Science and Technology International vol21 no 8 pp 559ndash570 2015

[18] R Pavela ldquoInsecticidal properties of several essential oils on thehouse fly (Musca domestica L)rdquo Phytotherapy Research vol 22no 2 pp 274ndash278 2008

[19] L Tan J Su D Wu et al ldquoKinetics and mechanism study ofcompetitive inhibition of Jack-Bean urease by baicalinrdquo TheScientific World Journal vol 2013 Article ID 879501 9 pages2013

[20] Y-C Xie and F Tang ldquoProtective effect of Pogostemon cablinon membrane fluidity of intestinal epithelia cell in ischemiareperfusion rats after ischemiareperfusionrdquo Zhongguo ZhongXi Yi Jie He Za Zhi vol 29 no 7 pp 639ndash641 2009

[21] J-L Yu X-S Zhang X Xue and R-M Wang ldquoPatchoulialcohol protects against lipopolysaccharide-induced acute lunginjury in micerdquoThe Journal of Surgical Research vol 194 no 2pp 537ndash543 2015

[22] HW Kim S J Cho B-Y Kim S I Cho and Y K Kim ldquoPogos-temon cablin as ROS scavenger in oxidant-induced cell death ofhuman neuroglioma cellsrdquo Evidence-Based Complementary andAlternative Medicine vol 7 no 2 pp 239ndash247 2010

[23] Y-C Tsai H-C Hsu W-C Yang W-J Tsai C-C Chen andT Watanabe ldquo120572-Bulnesene a PAF inhibitor isolated from the


essential oil of Pogostemon cablinrdquo Fitoterapia vol 78 no 1 pp7ndash11 2007

[24] Y Yang K Kinoshita K Koyama et al ldquoAnti-emetic principlesof Pogostemon cablin (Blanco) Benthrdquo Phytomedicine vol 6 no2 pp 89ndash93 1999

[25] J B Jeong J Choi Z Lou X Jiang and S-H Lee ldquoPatchoulialcohol an essential oil of Pogostemon cablin exhibits anti-tumorigenic activity in human colorectal cancer cellsrdquo Interna-tional Immunopharmacology vol 16 no 2 pp 184ndash190 2013

[26] J Yu Y Qi G Luo H-Q Duan and J Zhou ldquoExtraction andanalysis of the essential oil in Pogostemon cablin by enzymatichydrolysis and inhibitory activity against Hela cell prolifera-tionrdquo Zhong Yao Cai vol 35 no 5 pp 796ndash799 2012

[27] D J Giard S A Aaronson G J Todaro et al ldquoIn vitro cul-tivation of human tumors establishment of cell lines derivedfrom a series of solid tumorsrdquo Journal of the National CancerInstitute vol 51 no 5 pp 1417ndash1423 1973

[28] A Spira B Halmos and C A Powell ldquoUpdate in lung can-cer 2014rdquo American Journal of Respiratory and Critical CareMedicine vol 192 no 3 pp 283ndash294 2015

[29] G P Kalemkerian ldquoAdvances in pharmacotherapy of small celllung cancerrdquo Expert Opinion on Pharmacotherapy vol 15 no16 pp 2385ndash2396 2014

[30] R Sundar R Soong B-C Cho J R Brahmer and R A SooldquoImmunotherapy in the treatment of non-small cell lung can-cerrdquo Lung Cancer vol 85 no 2 pp 101ndash109 2014

[31] H Shi B Tan G Ji et al ldquoZedoary oil (Ezhu You) inhibitsproliferation of AGS cellsrdquoChineseMedicine vol 8 no 1 article13 2013

[32] P Li D Nijhawan I Budihardjo et al ldquoCytochrome c anddATP-dependent formation of Apaf-1caspase-9 complex initi-ates an apoptotic protease cascaderdquo Cell vol 91 no 4 pp 479ndash489 1997

[33] E Ondrouskova and B Vojtesek ldquoProgrammed cell death incancer cellsrdquo Klinicka Onkologie vol 27 supplement 1 pp S7ndashS14 2014

[34] S Thomas B A Quinn S K Das et al ldquoTargeting the Bcl-2 family for cancer therapyrdquo Expert Opinion on TherapeuticTargets vol 17 no 1 pp 61ndash75 2013

[35] A Shamas-Din J Kale B Leber and D W Andrews ldquoMech-anisms of action of Bcl-2 family proteinsrdquo Cold Spring HarborPerspectives in Biology vol 5 no 4 Article ID a008714 2013

[36] M Degli Esposti and C Dive ldquoMitochondrial membrane per-meabilisation by BaxBakrdquo Biochemical and Biophysical Re-search Communications vol 304 no 3 pp 455ndash461 2003

[37] J Estaquier F Vallette J-L Vayssiere and B Mignotte ldquoThemitochondrial pathways of apoptosisrdquo inAdvances inMitochon-drial Medicine R Scatena P Bottoni and B Giardina Eds vol942 of Advances in Experimental Medicine and Biology pp 157ndash183 2012

[38] J D Ly D R Grubb and A Lawen ldquoThe mitochondrial mem-brane potential (998779Ψm) in apoptosis an updaterdquo Apoptosis vol8 no 2 pp 115ndash128 2003

[39] M P Cunningham S EssapenHThomas et al ldquoCoexpressionprognostic significance and predictive value of EGFR EGFRvIIIand phosphorylated EGFR in colorectal cancerrdquo InternationalJournal of Oncology vol 27 no 2 pp 317ndash325 2005

[40] M Shimizu A Deguchi J T E Lim H Moriwaki L Kope-lovich and I B Weinstein ldquo(minus)-Epigallocatechin gallate andpolyphenon E inhibit growth and activation of the epidermalgrowth factor receptor and human epidermal growth factor

receptor-2 signaling pathways in human colon cancer cellsrdquoClinical Cancer Research vol 11 no 7 pp 2735ndash2746 2005

[41] P K Kandala S E Wright and S K Srivastava ldquoBlock-ing epidermal growth factor receptor activation by 331015840-diin-dolylmethane suppresses ovarian tumor growth in vitro and invivordquoThe Journal of Pharmacology and ExperimentalTherapeu-tics vol 341 no 1 pp 24ndash32 2012

[42] M R Maiello A DrsquoAlessio S Bevilacqua M Gallo N Nor-manno and A De Luca ldquoEGFR and MEK blockade in triplenegative breast cancer cellsrdquo Journal of Cellular Biochemistryvol 116 no 12 pp 2778ndash2785 2015

[43] S M Gadgeel S Ali P A Philip F Ahmed A Wozniak andF H Sarkar ldquoResponse to dual blockade of epidermal growthfactor receptor (EGFR) and cycloxygenase-2 in nonsmall celllung cancer may be dependent on the EGFR mutational statusof the tumorrdquo Cancer vol 110 no 12 pp 2775ndash2784 2007

[44] L Li Y Gao L Zhang J Zeng D He and Y Sun ldquoSilibinininhibits cell growth and induces apoptosis by caspase activationdown-regulating survivin and blocking EGFR-ERK activationin renal cell carcinomardquo Cancer Letters vol 272 no 1 pp 61ndash69 2008

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


SS

S

H

H RR

OHMe Me

MeMe

(a)

4824120

20

40

60

80

100

120

0

Time (h)

Cel

l via

bilit

y

Control50 gmL

75 gmL100 gmL

( o

f con

trol) lowastlowast


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

(b)

300250200150

70

80

90

100

10060

Concentration (gmL)

HEK293

L02HFL-1

Cel

l via

bilit

y (

of c

ontro

l)

(c)

104103102101100

101

102

103

104

100

FL1-H

FL2

-H

0

FL2

-H

PI

104103102101100

101

102

103

104

100


50

104103102101100

101

102

103

104

100


FL2

-H

PI

75

104103102101100

101

102

103

104

100

FL1-H

FL2

-H

100

104103102101100

101

102

103

104

100


FL2

-H

PI

75 + EGF


5

10

15

20

25

30

35

40

45

50

0

Concentration

Early apoptosisLate apoptosis

lowastlowastlowast


lowastlowastlowast

(gmL)

A

nnex

in V

-pos

itive

(d)Figure 1 Continued


10075500 (gmL)

BAX

Bcl-2

c-Myc

PARPp-PARP

BAX

c-M

yc

PARP

p-PR

RP

Actin

15

20

25

30

05

10

00

o

f con

trol

50 gmL0 gmL

lowastlowast


lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast




lowastlowast

lowastlowast


75gmL100gmL

Cleaved-

Cleaved-

cas9

cas3

Clea

ved-

cas9

Clea

ved-

cas3

Bcl-2

Cyclin E

(e)




























3 Results









1S phase in a





200

300

400

500

100

0

Ratio

(525

590

) o

f con

trol

lowastlowastlowast

lowastlowastlowast

lowastlowast

(a)


0

6

8

4

2

lowastlowastlowast

lowastlowastlowast

lowastlowast

Rela

tive c

aspa

se9

activ

ity

(b)


0

6

8

4

2

10

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Rela

tive c

aspa

se3

activ

ity

(c)

Concentration (gmL)

1007550

80

100

40

60

20

0

Hoe

chst

posit

ive c

ells

()

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0(gmL) 50 75 100

0

(d)

Figure 2 Continued


0

50

75

100

MergeDAPI(gmL)


0

0

lowastlowastlowast

lowastlowast30

40

20

10

50

TUNEL

TUN

EL p

ositi

ve ce

ll (

)

(e)

P38

p-P38

JNK

p-JNK

ERK

p-ERK

EGFR

p-EGFR

MEK

p-MEK

10075500 10075500(gmL) (gmL)

Actin

p-M

EK

MEK

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38

75 gmL100gmL

o

f con


lowastlowastlowast

lowastlowastlowast


-Actin

0gmL50gmL

10

15

20

25

30

05

00

(f)



4 Discussion







200

300

400

100

0250200150100502502001501005025020015010050

25020015010050 25020015010050

75

200

300

400

100

0

500

50

200

300

400

100

0

0

200

300

400

100

0

500100

200

300

400

100

0

75 + EGF


Cel

l cou

nt

Cel

l cou

nt

SG1

G2M


lowastlowastlowast

40

60

80

100

20

0

Cell

cycle

dist

ribut

ion

()


lowast

(G1)

Concentration (gmL)

(a)

(gmL)10075500

P21

P53

CDK2

ActinCDK2P53P21

10

15

20

25

30

35

40

05

00

o

f con

trol

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




50 gmL0 gmL 75gmL

100gmL

Cyclin E

Cyclin E

(b)


1S cell




1S cell cycle


21181512963

22

24

26

20

18

Body

wei

ghts

(g)

Control5mgkg

10mgkg15mgkg


(a)

21181512963

Control5mgkg

10mgkg15mgkg

lowast

lowastlowast



lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

400

600

800

1000

1200

200

0

Tum

or si

ze (m

m3)


(b)

0mgkg

5mgkg

10mgkg

15mgkg

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


0

20

40

60

Ki67

posit

ive c

ells

()


5

10

15

20

0

(mgkg)151050

Ki67

Cleaved-cas3

Clea

ved-

cas3

posit

ive c

ells

()

(d)



EGFR

p-EGFR

ERK

p-ERK

P38

p-P38

JNK

p-JNK

(gmL)75 + EGF750

Actin

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38


40

35

30

25

20

15

10

05

00

o

f con

trol



-Actin

(a)

(b)










Competing Interests




Acknowledgments


References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


10075500 (gmL)

BAX

Bcl-2

c-Myc

PARPp-PARP

BAX

c-M

yc

PARP

p-PR

RP

Actin

15

20

25

30

05

10

00

o

f con

trol

50 gmL0 gmL

lowastlowast


lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast




lowastlowast

lowastlowast


75gmL100gmL

Cleaved-

Cleaved-

cas9

cas3

Clea

ved-

cas9

Clea

ved-

cas3

Bcl-2

Cyclin E

(e)




























3 Results









1S phase in a





200

300

400

500

100

0

Ratio

(525

590

) o

f con

trol

lowastlowastlowast

lowastlowastlowast

lowastlowast

(a)


0

6

8

4

2

lowastlowastlowast

lowastlowastlowast

lowastlowast

Rela

tive c

aspa

se9

activ

ity

(b)


0

6

8

4

2

10

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Rela

tive c

aspa

se3

activ

ity

(c)

Concentration (gmL)

1007550

80

100

40

60

20

0

Hoe

chst

posit

ive c

ells

()

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0(gmL) 50 75 100

0

(d)

Figure 2 Continued


0

50

75

100

MergeDAPI(gmL)


0

0

lowastlowastlowast

lowastlowast30

40

20

10

50

TUNEL

TUN

EL p

ositi

ve ce

ll (

)

(e)

P38

p-P38

JNK

p-JNK

ERK

p-ERK

EGFR

p-EGFR

MEK

p-MEK

10075500 10075500(gmL) (gmL)

Actin

p-M

EK

MEK

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38

75 gmL100gmL

o

f con


lowastlowastlowast

lowastlowastlowast


-Actin

0gmL50gmL

10

15

20

25

30

05

00

(f)



4 Discussion







200

300

400

100

0250200150100502502001501005025020015010050

25020015010050 25020015010050

75

200

300

400

100

0

500

50

200

300

400

100

0

0

200

300

400

100

0

500100

200

300

400

100

0

75 + EGF


Cel

l cou

nt

Cel

l cou

nt

SG1

G2M


lowastlowastlowast

40

60

80

100

20

0

Cell

cycle

dist

ribut

ion

()


lowast

(G1)

Concentration (gmL)

(a)

(gmL)10075500

P21

P53

CDK2

ActinCDK2P53P21

10

15

20

25

30

35

40

05

00

o

f con

trol

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




50 gmL0 gmL 75gmL

100gmL

Cyclin E

Cyclin E

(b)


1S cell




1S cell cycle


21181512963

22

24

26

20

18

Body

wei

ghts

(g)

Control5mgkg

10mgkg15mgkg


(a)

21181512963

Control5mgkg

10mgkg15mgkg

lowast

lowastlowast



lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

400

600

800

1000

1200

200

0

Tum

or si

ze (m

m3)


(b)

0mgkg

5mgkg

10mgkg

15mgkg

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


0

20

40

60

Ki67

posit

ive c

ells

()


5

10

15

20

0

(mgkg)151050

Ki67

Cleaved-cas3

Clea

ved-

cas3

posit

ive c

ells

()

(d)



EGFR

p-EGFR

ERK

p-ERK

P38

p-P38

JNK

p-JNK

(gmL)75 + EGF750

Actin

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38


40

35

30

25

20

15

10

05

00

o

f con

trol



-Actin

(a)

(b)










Competing Interests




Acknowledgments


References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






















3 Results









1S phase in a





200

300

400

500

100

0

Ratio

(525

590

) o

f con

trol

lowastlowastlowast

lowastlowastlowast

lowastlowast

(a)


0

6

8

4

2

lowastlowastlowast

lowastlowastlowast

lowastlowast

Rela

tive c

aspa

se9

activ

ity

(b)


0

6

8

4

2

10

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Rela

tive c

aspa

se3

activ

ity

(c)

Concentration (gmL)

1007550

80

100

40

60

20

0

Hoe

chst

posit

ive c

ells

()

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0(gmL) 50 75 100

0

(d)

Figure 2 Continued


0

50

75

100

MergeDAPI(gmL)


0

0

lowastlowastlowast

lowastlowast30

40

20

10

50

TUNEL

TUN

EL p

ositi

ve ce

ll (

)

(e)

P38

p-P38

JNK

p-JNK

ERK

p-ERK

EGFR

p-EGFR

MEK

p-MEK

10075500 10075500(gmL) (gmL)

Actin

p-M

EK

MEK

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38

75 gmL100gmL

o

f con


lowastlowastlowast

lowastlowastlowast


-Actin

0gmL50gmL

10

15

20

25

30

05

00

(f)



4 Discussion







200

300

400

100

0250200150100502502001501005025020015010050

25020015010050 25020015010050

75

200

300

400

100

0

500

50

200

300

400

100

0

0

200

300

400

100

0

500100

200

300

400

100

0

75 + EGF


Cel

l cou

nt

Cel

l cou

nt

SG1

G2M


lowastlowastlowast

40

60

80

100

20

0

Cell

cycle

dist

ribut

ion

()


lowast

(G1)

Concentration (gmL)

(a)

(gmL)10075500

P21

P53

CDK2

ActinCDK2P53P21

10

15

20

25

30

35

40

05

00

o

f con

trol

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




50 gmL0 gmL 75gmL

100gmL

Cyclin E

Cyclin E

(b)


1S cell




1S cell cycle


21181512963

22

24

26

20

18

Body

wei

ghts

(g)

Control5mgkg

10mgkg15mgkg


(a)

21181512963

Control5mgkg

10mgkg15mgkg

lowast

lowastlowast



lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

400

600

800

1000

1200

200

0

Tum

or si

ze (m

m3)


(b)

0mgkg

5mgkg

10mgkg

15mgkg

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


0

20

40

60

Ki67

posit

ive c

ells

()


5

10

15

20

0

(mgkg)151050

Ki67

Cleaved-cas3

Clea

ved-

cas3

posit

ive c

ells

()

(d)



EGFR

p-EGFR

ERK

p-ERK

P38

p-P38

JNK

p-JNK

(gmL)75 + EGF750

Actin

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38


40

35

30

25

20

15

10

05

00

o

f con

trol



-Actin

(a)

(b)










Competing Interests




Acknowledgments


References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



200

300

400

500

100

0

Ratio

(525

590

) o

f con

trol

lowastlowastlowast

lowastlowastlowast

lowastlowast

(a)


0

6

8

4

2

lowastlowastlowast

lowastlowastlowast

lowastlowast

Rela

tive c

aspa

se9

activ

ity

(b)


0

6

8

4

2

10

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Rela

tive c

aspa

se3

activ

ity

(c)

Concentration (gmL)

1007550

80

100

40

60

20

0

Hoe

chst

posit

ive c

ells

()

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0(gmL) 50 75 100

0

(d)

Figure 2 Continued


0

50

75

100

MergeDAPI(gmL)


0

0

lowastlowastlowast

lowastlowast30

40

20

10

50

TUNEL

TUN

EL p

ositi

ve ce

ll (

)

(e)

P38

p-P38

JNK

p-JNK

ERK

p-ERK

EGFR

p-EGFR

MEK

p-MEK

10075500 10075500(gmL) (gmL)

Actin

p-M

EK

MEK

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38

75 gmL100gmL

o

f con


lowastlowastlowast

lowastlowastlowast


-Actin

0gmL50gmL

10

15

20

25

30

05

00

(f)



4 Discussion







200

300

400

100

0250200150100502502001501005025020015010050

25020015010050 25020015010050

75

200

300

400

100

0

500

50

200

300

400

100

0

0

200

300

400

100

0

500100

200

300

400

100

0

75 + EGF


Cel

l cou

nt

Cel

l cou

nt

SG1

G2M


lowastlowastlowast

40

60

80

100

20

0

Cell

cycle

dist

ribut

ion

()


lowast

(G1)

Concentration (gmL)

(a)

(gmL)10075500

P21

P53

CDK2

ActinCDK2P53P21

10

15

20

25

30

35

40

05

00

o

f con

trol

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




50 gmL0 gmL 75gmL

100gmL

Cyclin E

Cyclin E

(b)


1S cell




1S cell cycle


21181512963

22

24

26

20

18

Body

wei

ghts

(g)

Control5mgkg

10mgkg15mgkg


(a)

21181512963

Control5mgkg

10mgkg15mgkg

lowast

lowastlowast



lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

400

600

800

1000

1200

200

0

Tum

or si

ze (m

m3)


(b)

0mgkg

5mgkg

10mgkg

15mgkg

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


0

20

40

60

Ki67

posit

ive c

ells

()


5

10

15

20

0

(mgkg)151050

Ki67

Cleaved-cas3

Clea

ved-

cas3

posit

ive c

ells

()

(d)



EGFR

p-EGFR

ERK

p-ERK

P38

p-P38

JNK

p-JNK

(gmL)75 + EGF750

Actin

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38


40

35

30

25

20

15

10

05

00

o

f con

trol



-Actin

(a)

(b)










Competing Interests




Acknowledgments


References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

50

75

100

MergeDAPI(gmL)


0

0

lowastlowastlowast

lowastlowast30

40

20

10

50

TUNEL

TUN

EL p

ositi

ve ce

ll (

)

(e)

P38

p-P38

JNK

p-JNK

ERK

p-ERK

EGFR

p-EGFR

MEK

p-MEK

10075500 10075500(gmL) (gmL)

Actin

p-M

EK

MEK

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38

75 gmL100gmL

o

f con


lowastlowastlowast

lowastlowastlowast


-Actin

0gmL50gmL

10

15

20

25

30

05

00

(f)



4 Discussion







200

300

400

100

0250200150100502502001501005025020015010050

25020015010050 25020015010050

75

200

300

400

100

0

500

50

200

300

400

100

0

0

200

300

400

100

0

500100

200

300

400

100

0

75 + EGF


Cel

l cou

nt

Cel

l cou

nt

SG1

G2M


lowastlowastlowast

40

60

80

100

20

0

Cell

cycle

dist

ribut

ion

()


lowast

(G1)

Concentration (gmL)

(a)

(gmL)10075500

P21

P53

CDK2

ActinCDK2P53P21

10

15

20

25

30

35

40

05

00

o

f con

trol

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




50 gmL0 gmL 75gmL

100gmL

Cyclin E

Cyclin E

(b)


1S cell




1S cell cycle


21181512963

22

24

26

20

18

Body

wei

ghts

(g)

Control5mgkg

10mgkg15mgkg


(a)

21181512963

Control5mgkg

10mgkg15mgkg

lowast

lowastlowast



lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

400

600

800

1000

1200

200

0

Tum

or si

ze (m

m3)


(b)

0mgkg

5mgkg

10mgkg

15mgkg

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


0

20

40

60

Ki67

posit

ive c

ells

()


5

10

15

20

0

(mgkg)151050

Ki67

Cleaved-cas3

Clea

ved-

cas3

posit

ive c

ells

()

(d)



EGFR

p-EGFR

ERK

p-ERK

P38

p-P38

JNK

p-JNK

(gmL)75 + EGF750

Actin

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38


40

35

30

25

20

15

10

05

00

o

f con

trol



-Actin

(a)

(b)










Competing Interests




Acknowledgments


References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


200

300

400

100

0250200150100502502001501005025020015010050

25020015010050 25020015010050

75

200

300

400

100

0

500

50

200

300

400

100

0

0

200

300

400

100

0

500100

200

300

400

100

0

75 + EGF


Cel

l cou

nt

Cel

l cou

nt

SG1

G2M


lowastlowastlowast

40

60

80

100

20

0

Cell

cycle

dist

ribut

ion

()


lowast

(G1)

Concentration (gmL)

(a)

(gmL)10075500

P21

P53

CDK2

ActinCDK2P53P21

10

15

20

25

30

35

40

05

00

o

f con

trol

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




50 gmL0 gmL 75gmL

100gmL

Cyclin E

Cyclin E

(b)


1S cell




1S cell cycle


21181512963

22

24

26

20

18

Body

wei

ghts

(g)

Control5mgkg

10mgkg15mgkg


(a)

21181512963

Control5mgkg

10mgkg15mgkg

lowast

lowastlowast



lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

400

600

800

1000

1200

200

0

Tum

or si

ze (m

m3)


(b)

0mgkg

5mgkg

10mgkg

15mgkg

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


0

20

40

60

Ki67

posit

ive c

ells

()


5

10

15

20

0

(mgkg)151050

Ki67

Cleaved-cas3

Clea

ved-

cas3

posit

ive c

ells

()

(d)



EGFR

p-EGFR

ERK

p-ERK

P38

p-P38

JNK

p-JNK

(gmL)75 + EGF750

Actin

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38


40

35

30

25

20

15

10

05

00

o

f con

trol



-Actin

(a)

(b)










Competing Interests




Acknowledgments


References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


21181512963

22

24

26

20

18

Body

wei

ghts

(g)

Control5mgkg

10mgkg15mgkg


(a)

21181512963

Control5mgkg

10mgkg15mgkg

lowast

lowastlowast



lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

400

600

800

1000

1200

200

0

Tum

or si

ze (m

m3)


(b)

0mgkg

5mgkg

10mgkg

15mgkg

(c)

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


0

20

40

60

Ki67

posit

ive c

ells

()


5

10

15

20

0

(mgkg)151050

Ki67

Cleaved-cas3

Clea

ved-

cas3

posit

ive c

ells

()

(d)



EGFR

p-EGFR

ERK

p-ERK

P38

p-P38

JNK

p-JNK

(gmL)75 + EGF750

Actin

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38


40

35

30

25

20

15

10

05

00

o

f con

trol



-Actin

(a)

(b)










Competing Interests




Acknowledgments


References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


EGFR

p-EGFR

ERK

p-ERK

P38

p-P38

JNK

p-JNK

(gmL)75 + EGF750

Actin

p-EG

FR

EGFR

p-ER

K

ERK

p-JN

K

JNK

p-P3

8

P38


40

35

30

25

20

15

10

05

00

o

f con

trol



-Actin

(a)

(b)










Competing Interests




Acknowledgments


References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






Competing Interests




Acknowledgments


References





















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

)joebxj1vcmjtijoh$psqpsbujpo …downloads.hindawi.com/journals/bmri/2016/4567580.pdf · pogostemon...

Documents