small interfering rna against the apurinic or apyrimidinic endonuclease enhances the sensitivity of...

Small interfering RNA against the apurinic or apyrimidinicendonuclease enhances the sensitivity of human pancreatic

cancer cells to gemcitabine in vitro

Guang Su XIONG, Hui Ling SUN,1 Shu Ming WU & Jian Zhong MO

Department of Gastroenterology, Renji Hospital, Shanghai Jiaotong University School of Medicine, ShanghaiInstitute of Digestive Disease, Shanghai, China

OBJECTIVE: To investigate whether the down-regulation of human apurinic or apyrimidinicendonuclease/redox factor-1 gene (APE1/Ref-1)expression by ribonucleic acid interference (RNAi)would increase the sensitivity of SW1990 cells togemcitabine.

METHODS: Chemically synthesized small interfer-ing RNA (siRNA) directed against human APE1/Ref-1(si-APE1) was transfected into SW1990 cells throughtransfection reagents. The mRNA expression of APE1/Ref-1 was detected by semi-quantitative RT-PCR andthe protein expression of APE1/Ref-1 was detected byWestern blot; cell proliferation and apoptosis werestudied by a Cell Counting Kit 8 (CCK-8) and flowcytometry (FCM) and fluorescence microscopy.

RESULTS: After transfecting the SW1990 cells withsiRNA directed against human APE1/Ref-1, the mRNA

expression of APE1/Ref-1 of these cells was reduced,and its protein expression was reduced by 55.41 �3.58%. The CCK-8 assay showed that the absorbanceand the inhibition of cell growth transfected withsi-APE1 were significantly different from the blank(cultured with Dulbecco’s modified Eagle’s medium)and negative control (given 50 nmol/L scrambledcontrol siRNA). The inhibition rates of cell growth ofthe si-APE1 group at 24, 48, 72 h were 41.69 � 2.78%,24.83 � 3.70% and 21.27 � 9.82%, respectively. AFCM analysis and cell morphology study showed thatthe apoptotic rate of SW1990 cells transfected withsi-APE1 combined with gemcitabine treatment wassignificantly different from the blank control andothers.

CONCLUSION: To knock down APE1/Ref-1 geneexpression may significantly sensitize the SW1990cells to gemcitabine and enhance cell apoptosis.

KEY WORDS: APE1/Ref-1, gemcitabine, pancreatic cancer, siRNA.

INTRODUCTIONcdd_442 224..230

Pancreatic cancer is a highly vascular and extremelydestructive malignancy and the survival of patientswith pancreatic cancer has not improved significantlyin recent years. With a 5-year survival rate of only 3%and a median survival of less than 6 months, itremains the fourth cause of death from cancer afterlung, prostate (breast in women) and colorectalcancers since the 1970s in USA in spite of tremendousclinical and experimental efforts.1,2 Gemcitabine

Correspondence to: Shu Ming WU, Department of Gastroenterology,Renji Hospital, Shanghai Jiaotong University School of Medicine,Shanghai Institute of Digestive Disease, Shanghai 200001, China.Email: [email protected] address: Liaocheng People’s Hospital, Liaocheng, ShandongProvince, China.

The first two authors contributed equally to this work.

© 2010 The AuthorsJournal compilation © 2010 Chinese Medical AssociationShanghai Branch, Chinese Society of Gastroenterology, RenjiHospital Affiliated to Shanghai Jiaotong University School ofMedicine and Blackwell Publishing Asia Pty Ltd.

Journal of Digestive Diseases 2010; 11; 224–230 doi: 10.1111/j.1751-2980.2010.00442.x

224

(2′-deoxy-2′,2′-difluorocytidine monohydrochloride)is a strong and specific deoxycytidine analogue withtherapeutic activity in a variety of solid tumors includ-ing non-small cell lung cancer, pancreatic cancer,bladder and ovarian cancer, as well as breast tumors.3

Single-agent chemotherapy with gemcitabine for pan-creatic cancer has shown significantly increased sur-vival rates and a better quality of life, and it is currentlythe recommended first-line treatment for advanced,unresectable pancreatic cancer.1,4,5 Unfortunately,there have also been many data showing that, despiteits promising activity against cancer cells, responserates to gemcitabine in pancreatic cancer patientsremain low, with a 23.8% objective response inclinical cases, and the median overall survival ofgemcitabine-treated patients is only 5.65 months andthe 1- year survival rate is only 18%.5,6 As it is thusimportant to develop new methods or drugs to treatpancreatic cancers, understanding the molecularmechanism of pancreatic cancer is one of the mostimportant issues for treatment.

DNA repair systems, as the molecular basis of defend-ing against environmental damage to cellular DNA,play an important role in protecting genomic stabili-zation and integrity.7 However, an elevated DNArepair capacity in tumor cells leads to drug or radia-tion resistance and severely limits the efficacy of theseagents. A new and emerging concept designed to sen-sitize cancer cells to DNA-damaging agents, that is,chemotherapy or radiation, is the inhibition ofvarious proteins in the DNA repair pathways.8–10

Human apurinic or apyrimidinic endonuclease(APE1) is an essential enzyme in the base excisionrepair pathway, which is responsible for the repair ofoxidative and alkylating DNA damage, thus protectscells against toxic effects of endogenous and exog-enous agents including chemotherapeutic agents.APE1 is a multifunctional protein that not only playsa role in DNA repair but also functions as a reduction-oxidation factor, known as redox factor-1 (Ref-1) inthe literature. It may increase the DNA binding abilityof several transcription factors involved in differentgrowth signaling pathways.11 Because of the criticalrole of APE1 in DNA repair and transcription factorregulation and its altered levels of expression andfunction in a variety of cancers such as prostate,ovarian, cervical and germ cell tumors and osteosar-comas, we focused on this key enzyme in pancreaticcancer.11–16

Previous studies by Lau17 and ourselves18 have shownan elevated level of APE1 in pancreatic cancer and a

gemcitabine dose-dependent increase in APE1 expres-sion. Thus, the goal of this study was to investigate theimpact of APE1 gene silencing on pancreatic tumorgrowth. Additionally, we examined whether the genetherapy with small interfering RNA (siRNA) directedagainst human APE1/Ref-1 combined with gemcitab-ine has therapeutic potential for pancreatic cancer.

MATERIALS AND METHODS

Cell culture and reagents

The human pancreatic cancer cell line used in thisstudy, SW1990, was maintained in Dulbecco’s modi-fied Eagle’s medium (DMEM) supplemented with10% fetal bovine serum, 100 units/mL penicillin, and100 units/mL streptomycin (Invitrogen, Carlsbad,CA). The SW1990 cells were cultured at 37°C in ahumidified incubator containing 5% CO2. Cell cul-tures were passaged routinely once a week andre-established from frozen stock every 2 months.

The monoclonal antibody against human APE1 waspurchased from Novus Biologicals (Novus, Littleton,CA). Anti-b-actin was purchased from Kangchen Bio-logical (Kangchen Biological, Shanghai, China). AllAPE1 siRNA and scrambled control siRNA were com-mercially obtained from Genepharm (ShanghaiGenepharm, Shanghai, China), and this 21-basesequence was subjected to a comprehensive search inthe National Center for Biotechnology Informationdatabase of expressed sequence tag libraries to ensurethat only one gene was targeted. The sense strand ofRNA oligonucleotides corresponding to APE1 (5′-GTCTGGTAAGACTGGAATACC-3′) was chemicallysynthesized by means of using standard methods andhigh-performance liquid chromatography purifica-tion. Gemcitabine was purchased from HansenPharmaceutical (Jiangsu Province, China). TheDharmaFECT 1 siRNA Transfection Reagent waspurchased from Dharmacon (Lafayette, CO, USA).

Transient transfection of APE1 siRNA

SW1990 cells were seeded on 6-well culture plates anddivided into the following groups: (i) a blank controlgroup of cells cultured in DMEM medium; (ii) a nega-tive control group of cells in culture with the additionof 50 nmol/L scrambled control siRNA; and (iii) asi-APE1 group of cells in culture with 50 nmol/LsiRNA targeted to the APE1 gene. According to Wanget al.’s study,13 sequences of the double-strandedsiRNA are antisense (5′-GUCUGGUACGACUGGAGU

Journal of Digestive Diseases 2010; 11; 224–230 siRNA against APE1 of pancreatic cancer 225

© 2010 The AuthorsJournal compilation © 2010 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to ShanghaiJiaotong University School of Medicine and Blackwell Publishing Asia Pty Ltd.

ACC-3′, 5′-UACUCCAGUCGUACCAGACCU-3′) andnonsense (5′-CCAUGAGGUCAGCAUGGUCUG-3′,5′-GACCAUGCUGACCUCAUGGAA-3′). The cellswere transfected with siRNA (50 nmol/L) using Dhar-maFECT 1 siRNA Transfection Reagent according tothe manufacturer’s instructions. The selective silencingof APE1 was confirmed by semi-quantitative RT-PCRand Western blot analysis.

Semi-quantitative RT-PCR

Total RNA extractions were performed using TRIzolreagent (Invitrogen, Carlsbad, CA) and treated withRNase-free Dnase I. The total RNA was reverse tran-scribed and amplified by PCR. The correspondingAPE1 primers were, forwards: 5′-ACTTCAGGAGCTGCCTGGACT-3′, reverse: 5′-AATGCAGGTAACAGAGTGGGA-3′, and the predicted product was 564 bp.b-actin was used as an internal control and its primersare forwards: 5′-CACCCACACTGTGCCCATC-3′ andreverse: 5′-CCACAGGACTCCATGCCC-3′. The pre-dicted product was 342 bp. PCR for APE1 gene werecarried out as follows: 3 min at 94°C, followed by 35cycles (94°C for 45 s, then 56°C for 1 min, and 72°Cfor 1 min) and a final extension step (72°C for10 min). For b-actin, PCR was incubated as followed:3 min at 94°C, 45 s at 94°C, then 1 min at 56°C, and1 min at 72°C, repeated 28 times, and finally 10 minat 72°C. The experiments were repeated thrice foreach assay. The size and quality of the PCR productswere verified by electrophoresis on 1.5% agarose gelstaining with ethidium bromide. Densitometric analy-sis was performed using ImageJ software (NationalInstitutes of Health, USA).

Western blot and antibodies

A Western blot analysis was performed using standardtechniques, as described previously.19 Briefly, equalprotein aliquots in each sample were resolved in 12%sodium dodecyl sulfate polyacrylamide gel electro-phoresis and the proteins transferred onto nitrocellu-lose membranes. After blocking with 5% skimmeddried milk, the membranes were incubated with a1:2000 dilution of primary mouse monoclonal anti-bodies against human APE1, b-actin. The membraneswere then incubated with a horseradish peroxidase-conjugated secondary antibody (1:2000; Pierce,Thermo Fisher Scientific Inc., IL, USA). The proteinswere detected by an enhanced chemiluminescencedetection system (SuperSignal West Femto Substrate;Pierce), and light emission was captured on KodakX-ray films.

Cell viability assay

Cell viability was assessed by a tetrazolium salt (WST-8)–based colorimetric assay in the Cell Counting Kit 8(CCK-8; Dojindo, Kumamoto, Japan). Briefly, controland treated SW1990 cells were seeded onto 96-wellplates at an initial density of 5 ¥ 103 cells/well. Atspecified time points, 10 mL of CCK-8 solution wasadded to each well of the plate, then the plate wasincubated for 1 hour. Cell viability was determined byscanning the absorbance with a microplate reader at450 nm. Data were expressed as the percentage ofviable cells as follows:

Relative viabilitytreated blank contro

%( )= [ ] − [ ]( )A A A450 450 450 ll

blank[ ](

− [ ]) ×A450 100%.

Chemotherapy and apoptosis detected by flowcytometry and Hoechst 33258 stain

SW1990 cells were seeded in 6-well culture plates anddivided into the following groups: (i) the blankcontrol group of cells cultured in DMEM medium;(ii) the chemotherapy group of cells in culture withthe addition of 20 nmol/L gemcitabine; (iii) thesi-APE1 group of cells cultured with 50 nmol/L siRNAtargeted to APE1 gene; and (iv) a combined group ofcells cultured with gemcitabine and si-APE1. Apopto-sis was determined by two methods. For nuclear mor-phology, cells were fixed and stained with Hoechst33258 according to the manufacturer’s instructions(Beyotime, Beijing, China). Then the stained nucleiwere immediately observed under a fluorescencemicroscope (Nikon, Tokyo, Japan). For flow cytom-etry analysis, an annexin-V fluorescein isothiocyanate/propidium iodide (PI) double-stain assay wasperformed in accordance with the manufacturer’s pro-tocol (BioVision, Mountain View, CA, USA). Briefly,after treatment, both floating and trypsinized adher-ent cells (5 ¥ 105) were collected and resuspended in500 mL of binding buffer containing 5 mL of annexin-Vfluorescein isothiocyanate and 5 mL of PI, and thenincubated for 5 min in the dark at room temperature.Analysis was immediately performed using a flowcytometer.

Statistical analysis

Results were expressed as mean � SD. Differencesbetween groups were examined for statistical signifi-cance with ANOVA or the Student Newman–Keuls test

Journal of Digestive Diseases 2010; 11; 224–230226 GS Xiong et al.

© 2010 The AuthorsJournal compilation © 2010 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai

Jiaotong University School of Medicine and Blackwell Publishing Asia Pty Ltd.

using SPSS 11.0 software (SPSS, Chicago, IL). P < 0.05indicated a statistically significant difference.

RESULTS

Transient suppression of APE1 expression byribonucleic acid interference (RNAi)

A densitometric analysis was performed using Image Jsoftware. Our data showed that compared with thoseof the blank and the negative control group, theexpression levels of APE1 mRNA and of protein in thesiRNA APE1 group both declined. Gray band intensi-ties of mRNA expressions in the blank control, nega-tive control and si-APE1 group were 1.2593 � 0.0512,1.0626 � 0.0719 and 0.6596 � 0.0261, respectively(Fig. 1) (P < 0.05). Meanwhile, the inhibitory degree

of APE1 protein expression in the si-APE1 group was55.41 � 3.58% (Fig. 2) (P < 0.05). However, there wasno obvious difference in APE1 mRNA and proteinexpression levels between the negative control and theblank control group (P > 0.05). So these data indi-cated that chemically synthesized siRNA could effec-tively suppress APE1 expression both in mRNA andprotein levels in pancreatic cancer cells.

Inhibition of cell growth by RNAi

Cell viability was determined by scanning the absor-bance at 450 nm (Table 1). As detected by the CCk-8assay, data showed that both the negative and blankcontrol group and the si-APE1 group in which siRNAwas directed against APE1 decreased the growth ofpancreatic cancer cells in a time-dependent manner(Fig. 3), but the si-APE1 group cells were decreasedmore significantly (P < 0.05). After treatment for 24 h,48 h and 72 h, the inhibitory rate of cell growth in thesi-APE1 group was 41.69 � 2.78%, 24.83 � 3.70%and 21.27 � 9.82%, respectively. The highest

Figure 1. Downregulation of apurinic or apyrimidinicendonuclease/redox factor-1(APE1) mRNA expressiondetected by semi-quantitive RT-PCR showing (1) SW1990with APE1 siRNA, (2) negative control group, (3) SW1990with blank group, and (4) marker.

Figure 2. Downregulation of apurinic or apyrimidinicendonuclease/redox factor-1 (APE1/Ref-1) protein expres-sion detected by Western blot analysis showing (1) theblank control group, (2) the negative control group, and(3) the si-APE1 (siRNA directed against APE1/Ref-1) group.

Table 1. The effects of small interfering RNA silencing the apurinic or apyrimidinic endonuclease gene on the growth ofSW1990 cells

Group 24 h (x̄ � s) 48 h (x̄ � s) 72 h (x̄ � s)

Blank control 0.4451 � 0.1057 0.9551 � 0.0495 1.4873 � 0.0667Negative control 0.4127 � 0.0625 0.9047 � 0.0423 1.4394 � 0.0621Si-APE1 0.2615 � 0.0680 0.7167 � 0.0061 1.1670 � 0.1683

(The absorbance at 450 nm by a cell counting kit 8 assay).

Inhib

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Figure 3. Inhibition of cell growth. Cell growth assay dem-onstrating a significant decrease in the si-APE1 group, com-pared with the negative control group and blank controlgroup in a time-dependent manner. The highest inhibitoryrate was 41.69 � 2.78%. ( ) negative blank group,( ) si-APE1 (siRNA directed against APE1/Ref-1) group.



inhibitory rate was 41.69 � 2.78% at the 24th hourafter treatment.

siRNA silencing APE1 gene enhances thesensitivity of SW1990 cells to gemcitabine

We have determined that siRNA targeted to the APE1gene could effectively inhibit APE1 expression inSW1990 cells and decrease cell growth. So, to explorethe effect of APE1 siRNA on apoptosis induction bygemcitabine in vitro, we measured the apoptotic cell byHochest 33258 staining and flow cytometry. To evalu-ate whether the decrease in cell viability might haveoccurred due to apoptotic cell death, we first examinedthe nuclear morphology by staining the cells withHoechst 33258 after treatment with gemcitabine andsiRNA. SW1990 cells stained with Hoechst 33258showed typical morphological features of apoptosis,including nuclear condensation and fragmentation 48hours after treatment with a final concentration20 mmol/L gemcitabine plus 50 nmol/L APE1 siRNA(Fig. 4). To quantify the apoptotic cell death we per-formed a flow cytometry analysis (Fig. 5). As shown inour result, 48 h after chemotherapy the apoptosis ratesin the blank control, gemcitabine group, si-APE1 andcombined group were 2.73 � 1.41%, 7.7 � 1.14%,8.37 � 0.98% and 19.8 � 3.47%, respectively. Thismeans that APE1 siRNA could significantly increasecell apoptosis induction by gemcitabine (P < 0.05).Taken together, our data demonstrate that treatmentwith APE1 siRNA not only inhibits cell growth butalso enhances gemcitabine-induced apoptosis inSW1990 cells, and this means that suppressing the

expression of APE1 gene markedly enhanced thechemosensitivity of pancreatic cancer. These findingssuggest the possibility of a better therapeutic resultfrom a combination therapy with siRNA against APE1and chemotherapeutic agents.

DISCUSSION

Mammalian APE1 is a ubiquitous and remarkablymultifunctional protein. Previous studies showed thatAPE1 is important in regulating cellular apoptosis andits overexpression, as detected in many tumors, suchas lung cancer, ovarian cancer, colorectal cancer andso on.7,11,12 In this study our data demonstrated thatdownregulating the APE1 level by chemically syn-thesized APE1 siRNA not only inhibited cell growthbut also enhanced the sensitivity of SW1990 cells togemcitabine.

As a rate-limiting enzyme, APE1 plays a central role inbase excision repair initiated by various DNA glycosy-lases. APE1 has 3′, 5′-AP endonuclease activity and3′-phosphatase activity. Specifically knocking downor inhibiting APE1 by using RNA interference andantisense oligonucleotide technology hypersensitizesmammalian cancer cells to several laboratory andclinical DNA damaging agents such as photodynamic

Figure 4. Hochest 33258 staining (¥ 400) on (a) blankcontrol group, (b) gemcitabine group, (c) si-APE1 group,and (d) combined group.

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105104103102 105104103102

FITC-A FITC-A

Q1 Q2

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Figure 5. SW1990 cell apoptosis detected by flow cytom-etry showing (a) blank control group, (b) gemcitabinegroup, (c) si-APE1(siRNA directed against APE1/Ref-1)group, and (d) combined group. FITC-A, fluoresceinisothiocyanate.




therapy, radio therapy, methyl methane sulfonate,hydrogen peroxide, bleomycin, temozolomide andgemcitabine.13,17,19–24

Importantly, APE1 also functions as a redox factormaintaining transcription factors in an active reducedstate. It stimulates the DNA binding activity of numer-ous transcription factors that are involved in cancerpromotion and progression such as activator protein1, nuclear factor-kappa B (NF-kB), hypoxia-induciblefactor-1a (HIF-1a), cAMP-responsive element bindingprotein, p53 and others.7,11,25,26 These transcriptionfactors have all been implicated in important aspectsof cancer including cell survival, angiogenesis, tumorpromotion, progression and prognosis. There arearticles reporting that, NF-kB and HIF-1a have beenlinked to chemoresistance in pancreatic cancer cells,and the inhibition of NF-kB enhances the sensitivityof human pancreatic cancers to chemotherapeuticdrugs.27–29

Some studies have showed that polymorphisms ofAPE1 also participate in the risk of tumors. Lo et al.30

reported that single-nucleotide polymorphism-656T>G located in the promoter region of APE1 wassignificantly associated with the risk of lung cancer.Jiao et al.31 reported that XRCC1(194) and MGMT(84)polymorphisms of genes involved in the repair ofalkylating DNA adduct and DNA base damage mayplay a role in modulating the risk of pancreatic cancer.

Although the accumulating weight of evidencestrongly implicates APE1 as a cancer potentiating mol-ecule with diverse functions, there is scant informa-tion at present vis-à-vis the relative importance of theDNA-binding versus redox domains of APE1 in sus-taining the growth of cancer cells. One study revealedthat resveratrol inhibited, in a dose-dependentmanner, Ref-1 activated activator protein-1 DNA-binding activities as well as Ref-1 endonuclease activi-ties and rendered melanoma cells more sensitive todacarbazine treatment.12 Many studies also indicatethat E3330 is a specific inhibitor of the APE1 redoxfunction without interfering with its DNA repair role,and Zou et al.’s data show that E3330 directly inhibitsthe growth and migration of human pancreatic cancercells.26,32,33

In conclusion, ours and other laboratory studies haveall shown that APE1 plays a critical role in the devel-opment, metastasis and prognosis of pancreaticcancer. Future studies should work out an optimal

regime for the selective inhibition of APE1’s activity,which is a promising avenue for developing novelcancer therapeutics.

ACKNOWLEDGMENTS

Supported by Shanghai Leading Academic DisciplineProject, Project Number Y0205 and the 2009 DoctoralFund of the Ministry of Education of China, ProjectNumber 20090073120092.

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