Identification Of Genes Associated With Gemcitabine Chemoresistance And Potentiation Of The Effect Of Gemcitabine By Emodin In Pancreatic Cancer

1.Background and objective:Pancreatic cancer is a highly aggressive malignant tumor,which has a characteristics of late clinical presentation,advanced stage and extremely poor prognosis.The curative treatment for pancreatic cancer is surgery,but only 10-15% of patients can be diagnosed in surgically resectable stages.Postoperative patients often accompany with recurrence and metastasis of tumor.Thus,the prognosis of patients with pancreatic cancer is extremely poor,with a 5-year survival rate of＜5% and a total median survival of 6 months.Chemotherapy is an importantly adjunctive therapy to pancreatic cancer patients,which is helpful to prolonging the median survival and improving quality of life.Currently,gemcitabine remains the first line chemotherapeutic agent available for treatment of advanced pancreatic cancer. However,single gemcitabine treatment has a response rate of less than 20%and is associated with drug resistance.Therefore,it is desirable to find predictor of gemcitabine chemoresistance and to develop strategies for overcoming gemcitabine chemoresistance.Emodin(1,3,8-trihydroxy-6-methylanthraquinone) is a natural anthraquinone derivative isolated from natural plant.Pharmacological studies have demonstrated that emodin possesses variously biological function,such as anti-bacterial,antiinflammatory, anti-cancer and a potent inhibitor of the Casein KinaseⅡ.Gene expression of nontreated pancreatic cancer gemcitabine-resistant cell lines (Panc-1) was compared with nontreated gemcitanbine-sensitive cell lines(Bxpc-3) by using GeneChip Human Genome U133A.A number of pancreatic cancer gemcitabine chemoresistant genes which were identified in the current study.Further studies showed that emodin enhanced the apoptotic effects of gemcitabine in pancreatic cancer cells. The mechanism by which emodin enhances the antitumor activity of gemcitabine in pancreatic cancer cells was investigated.2.Materials and methods:2.1 Oligonucleotide array hybridizationGene expression of nontreated pancreatic cancer gemcitabine-resistant cell lines (Panc-1) was compared with nontreated gemcitanbine-sensitive cell lines(Bxpc-3) by using GeneChip HG U133A 2.0 arrays.Pancreatic cancer gemcitabine chemoresistant genes were identified.Gemcitabine chemoresistant genes selected in microarray data were confirmed by Real-time PCR analyses.2.2 Cell growth assayEffects of emodin(0-160μM) and gemcitabine(0-50μg/ml) on growth of pancreatic cancer cells cultured for 24h,48 h and 72h were examined using MTT assay. The concentration of drug in next test was determined.Subsequent researches were done to examine the effect of a combination of emodin(Bxpc-3 and Mia Paca-2,40μM; Panc-1,80μM) and gemcitabine(Panc-1 and Mia Paca-2,5μg/ml;Bxpc-3,0.5μg/ml) for 48h on cell viability by MTT assay. 2.3 ApoptosisPotentiation of gemcitabine-induced apoptosis by emodin was evaluated by using Annexin V/propidium iodide flow cytometry,caspase 3 activities and PARP cleavage.2.4 Tumor growth and treatments7×10~6 Bxpc-3 cells collected in 100μl serum-free RPMI-1640 media in log phase growth were injected subcutaneously in the backs of 20 g athymic nu/nu male mice 4-6 weeks old.Once rumor masses became established and palpable,animals were randomized to receive intraperitoneal(IP) injections of vehicle(0.9%sodium chloride), emodin(40 mg/kg) alone,gemcitabine(125 mg/kg) alone,or emodin and gemcitabine in combination twice per week for 2 weeks.Tumor volumes and body weight were measured twice per week to evaluate the potentiation of antitumor activity of gemcitabine by emodin in vivo.2.5 TUNEL and immunohistochemical analysisPotentiation of apoptotic activity and cell growth inhibition of gemcitabine by emodin in vivo was investigated by TUNEL and immunohlstochemical analysis.2.6 Western blotting,RT-PCR and qRT-PCREffects of emodin in combination with gemcitabine on the expression levels of genes associated with gemcitabine chemoresistance in pancreatic cancer cells were analyzed by western blotting,RT-PCR and qRT-PCR.The mechanism by which emodin enhances the antitumor activity of gemcitabine was probed.2.7 Statistical analysisData were expressed as mean values±SE and analyzed by a two-tailed t-test or ANOVA followed by the LSD's multiple comparison with P＜0.05 considered significant.Analyses were performed using SPSS 13.0 statistical software package. 3.Results:3.1 Identification of genes associated with gemcitabine chemoresistanceIn gene expression microarray analysis,the differential expression 2887 well-characterized human genes was identified between gemcitabine-resistant cell lines (Pane-1) and gemcitabine-sensitive cell lines(Bxpc-3).Compared with Bxpc-3 cells, 141 genes were identified that show significantly differential expression at least 5-fold in Pane-1 cells.Of these 141 genes,103 unique genes with high expression(≥5-fold) and 38 unique genes relatively lower expression(≥5-fold) were found.3.2 Validation of genes expression of gemcitabine ehemoresistance in pancreatic cancer cellTo validate the microarray results,expression patterns of 22 genes in Pane-1 and Bxpc-3 cell lines were subjected to quantitative real-time PCR(qRT-PCR).Among these results,expression patterns of 21 genes were consistent with those in the microarray data.3.3 Emodin potentiates growth inhibition induced by gemeitabine in pancreatic cancer cells in vitroTo investigate the effect of ernodinon cell viability,pancreatic cancer cell lines (Pane-1,Mia Paca-2 and Bxpc-3) were treated with increasing concentrations of emodin (0-160μM) for 24,48 and 72 hours.Cell viability was inhibited by emodin treatment in a dose-and time-dependent manner when compared with the control in tested cell lines. Subsequent researches were done to examine the effect of a combination of emodin and gemcitabine on cell viability by MTT assay.The results showed treatment with emodin (Bxpc-3 and Mia Paca-2 40μM,Pane-1 80μM) plus gemcitabine(Pane-1 and Mia Paca-2 5μg/ml,Bxpc-3 0.5μg/ml) for 48 hours resulted in significant cell growth inhibition compared with single agent alone in pancreatic cancer cells(P＜0.05).3.4 Emodin sensitizes pancreatic cancer cells to apoptosis induced by gemcitabine in vitroThe combination of gemcitabine(Panc-1 5μg/ml,Bxpc-3 0.5μg/ml) and emodin (Panc-1 80μM,Bxpc-3 40μM) resulted in a stronger apoptotic effect in comparison with either agent alone after 24 h treatment by using Annexin V/propidium iodide flow cytometry(P＜0.05).To further test the ability of the combination therapy to induce apoptosis,caspase-3 and PARP activation in both cell lines tested were evaluated. Co-treatment between emodin and gemcitabine caused an additional increase in caspase-3 activity(P＜0.05) and PARP cleavage in Panc-1 and Bxpc-3 cells by comparison with single agent alone after 24 h treatment.3.5 Potentiation of the effect of gemcitabine in vivo by emodinAt the end of four cycles treatment,the final tumor volumes showed significant decrease in the emodin+gemcitabine group compared with control(P＜0.05) or with gemcitabine alone(P＜0.05).More significantly,no apparent systemic toxicity occurred during the combination treatment,because neither mouse death nor damaged liver or kidney functions happened in these groups.TUNEL staining of tumor sections demonstrated a visibly higher level of apoptosis in tumors treated with combination therapy than controls or single treatment.Next study showed that emodin in combination with gemcitabine significantly downregulated the expression of Ki-67 in tumor tissues compared with the control group and single treatment group.3.6 The mechanism by which emodin potentiates the effect of gemcitabine in vitroOur results of western blotting,RT-PCR and qRT-PCR displayed the survivin expression was significantly up-regulated after gemcitabine treatment.Emodin enhances the effect of gemcitabine in pancreatic cancer by downregulating gemcitabine chemoresistant gene HS3ST1,AKRC13 and survivin.4.Conclusions:4.1 A differential gene expression pattern associated with gemcitabine chemoresistant phenotypes in pancreatic cancer cells was identified by using microarray.4.2 Emodin not only induces apoptosis and inhibits the growth of human pancreatic cancer cells but also increases the effect of gemcitabine in vitro.4.3 Emodin potentiates growth inhibition induced by gemcitabine in pancreatic cancer cells in vivo.4.4 Modulating several genes associated with gemcitabine chemoresistance may be the mechanisms by which emodin enhances the antitumor activity of gemcitabine in pancreatic cancer cells.