Combined Inhibitory Effect Of Cucurbitacin B And Gemcitabine In Pancreatic Cancer

IntroductionPancreatic cancer is a common malignant tumor of gastrointestinal tract.Even though pancreatic cancer accounts for only 2%of all cancer diagnoses in the world,it is the fourth-leading cause of cancer death and one of the most difficult malignancies to manage.Because of the usually late onset of symptoms,only 15%-20%or less of patients present with resectable disease.Despite a lack of consistent evidence from previous clinical trials,surgical resection provides the best chance for cure or long-term survival,and chemotherapy is the most commonly used approach in treating locally advanced pancreatic cancer.Novel trends to prevent resistance and enhance efficacy incorporate biologically targeted agents.The signal transducer and activator of transcription(STAT) proteins are so named due to the role they play in relaying signals received by specific cell surface receptors to induce transcriptional changes within the cells.So far,seven distinct STAT molecules have been identified.These include STAT 1,2,3,4,5a,5b and 6.STATs were originally discovered in the context of cytokine signalling in 1992.STAT3 plays important roles at all levels of tumorigenesis and has been identified as an oncogene. Constitutively activated STAT3 has been demonstrated in many human carcinomas. Recent in vitro and in vivo studies have demonstrated that several strategies to target STAT3 signalling have been proposed as cancer therapies and tested in preclinical studies.In addition,chemical compound screens have identified several molecules, such as cucurbitacins,that appear to specifically alter the STAT3 pathway.Cucurbitacins are compounds isolated from various plant families which have been used as folk medicines for centuries in countries such as India and China because of their wide spectrum of pharmacological activities such as cytotoxic,anti-inflammatory, and anticancer effects.Of these compounds,cucurbitacin B is one of the most widely used for in vivo and in vitro studies on tumor inhibition.Accumulated evidences have shown that cucurbitacins such as B,D,E,I and Q can inhibit the growth of numerous human cancer cell lines and tumor xenografts.Cucurbitacin B suppresses the activation of STAT3,regulates STAT3 downstream genes such as cell cycle regulating genes and apoptosis-related genes,consequently inhibits tumor growth and induces cell apoptosis.Gemcitabine is the first-line drug for patients with advanced pancreatic cancer,but there's not so much effect on the patients' life survival using gemcitabine alone.Now drug combinations are often used on the clinical treatment,such as gemcitabine in combination with 5-fluorouracil,cisplatin,taxotere and the molecular tagarted drug like erlotinib.Compared with the use of single drug,drug combinations chemotherapy can improve overall survival,objective response rate and progression-free survival period, and achieved statistical significance.Gemcitabine combined with the molecular targeted drugs are currently hot topics.To date,however,no study has been conducted in the evaluation of the effect of cucurbitacin B or in combination with gemcitabine on pancreatic cancer cells.In the present study,the anti-tumor effect and mechanism of cucurbitacin B alone or in combination with gemcitabine on human pancreatic carcinoma cells were investigated in vitro,including the effects on cell growth,cell cycle distribution,apoptosis,the release of ROS,and the expression of proteins relevant to the regulation of cell cycle and apoptosis pathway.Materials and methodsPANC-1 cells were treated with cucurbitacin B or in combination with gemcitabine.The cell morphologic changes,cell proliferation,cell cycle distribution, and cell apoptosis were evaluated using Hoechst 33258 staining,fluorescent microscopy,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and flow cytometry.The expression of p-STAT3,Bcl-2,p53 and p21 was evaluated by Western blot analysis.The results was analysed by the software SPSS 16.0.Results1,Cucurbitacin B inhibited cellular proliferation in a dose and time dependent manner.One way ANOVA followed by LSD-t test showed that P＜0.05 or P＜0.01.2,FCM analysis showed that cucurbitacin B caused an enrichment of cells in G₂/M phase in a dose and time dependent manner(P＜0.05,or 0.01 vs.control), accompanied by a reduction in G₀/G₁ phase cells.3,Treating PANC-1 cells with different concentrations of cucurbitacin B for certain time,marked morphological changes of apoptosis such as condensation of chromatin,nuclear fragmentations and apoptotic bodies were found clearly using Hoechst 33258 staining.4,Annexin V/PI double staining analysis showed that cucurbitacin B increased the fraction of apoptotic cells(P＜0.05,or 0.01 vs.control).5,Cucurbitacin B increased the generation of ROS in a dose dependent manner (P＜0.01 vs.control).6,Westen blot analysis showed that cucurbitacin B suppressed STAT3 activation in a dose and time dependent manner,subsequently decreased the level of Bcl-2 proteins and increased the level of p53 and p21.7,Our results showed that,in comparison with single agent treatment,the combination of cucurbitacin B and gemcitabine produced greater efficacy in growth inhibition,cell cycle arrest,and apoptosis induction(P＜0.05 or P＜0.01).Measuring the modulation of regulators in the cell cycle,apoptosis and signal transductions by Western blot analysis showed that the combination effect of cucurbitacin B and gemcitabine was due to suppress the expression of p-STAT3 and increase the level of p53. Conclusion1,Cucurbitacin B inhibits the growth of pancreatic cancer PANC-1 in vitro. Cucurbitacin B in combination with gemcitabine had addictive or synergistic antitumor activity against pancreatic cancer in vitro.2,The antitumor effect of cucurbitacin B on PANC-1 cells was due to the induction of cell cycle arrest as well as apoptosis.The anti-tumor efficacy mediated via the combination of cucurbitacin B and gemcitabine was mainly due to the induction of cell cycle arrest as well as apoptosis.3,The possible mechanisms underlying the action of cucurbitacin B might be attributed to the suppression of STAT3 activation,Subsequently cucurbitacin B increased the level of p53 and p21 and decreased the level of Bcl-2 proteins.The possible mechanisms underlying the antitumor activity of cucurbitacin B in combination with gemcitabine might be attributed to the suppression of STAT3 activation,concomitantly increasing the expression level of p53 protein.The detail mechanism by which cucurbitacin B in combination with gemcitabine enhances the antitumor effects of gemcitabine on pancreatic cancer needs futher investigation.