Bufalin Exerts Antitumor Effects By Triggering Apoptosis And Inducing Cell Cycle Arrest In Pancreatic Cancer Cells

As one of the most aggressive human malignancies, pancreatic cancer is a leading cause of cancer related deaths worldwide and only about4%of patients will live five years after diagnosis.80%～85%of patients are diagnosed with an un-resectable or metastatic disease, which is correlated with poor prognosis and low survival rate. For patients with locally advanced or metastatic pancreatic cancer, gemcitabine-based chemotherapy is the main mode of treatment. However, the development of resistance of pancreatic cancer cells against gemcitabine often leads to treatment failure. Therefore, it is tremendously significant to exploit novel chemicals to prevent and treat pancreatic cancer. Previous research and clinical studies have demonstrated that many natural products derived from traditional Chinese medicine(TCM)such as camptothecin derivatives and vinca alkaloids could be effective antitumor compounds, hinting that TCM is a promising source for developing new anti-cancer drugs. There have been researches demonstrating that bufalin, the major digoxin-like immuno-reactive component of Chan-Su extracts, could trigger apoptosis, cell cycle arrest and autophagy in many cancers including leukemia, gastric cancer, breast cancer, prostate cancer, melanoma and colon cancer cells. Other reports have proved that bufalin could increase the sensitivity of hepatic cancer and osteosarcoma cells to regular chemotherapeutics. However, the antitumor effect and especially the underlying molecular mechanisms have not been well understood yet in pancreatic cancer until now. In this study, we tested the effects of bufalin on PANC-1and CFPAC-1cells and further explored the potential mechanisms.Our work is presented in four parts as follows.Part oneThe inhibitory effects of bufalin on the proliferation of PANC-1and CFPAC-1cellsIn this study, we selected PANC-1and CFPAC-1cells to elucidate the inhibitory effects of bufalin on the proliferation of pancreatic cancer cells. PANC-1and CFPAC-1cells were treated with different concentrations(50nM,100nM,200nM,500nM,1000nM,2000nM) of bufalin and cells treated with0.1%DMSO served as control. After treatment for24h,48h and72h, CCK-8assay was used to test the proliferation of pancreatic cancer cells.The result showed that the proliferation of PANC-1and CFPAC-1cells was dramatically inhibited in a time-and dose-dependent manner.Part twoThe effects of bufalin on the apoptotic rates and the expression levels of apoptosis-related proteins of pancreatic cancer cellsInducing apoptosis is one of the most important mechanisms of action of many antitumor drugs. We know that the apoptotic process is regulated by both the proapoptotic proteins and the apoptosis inhibitory proteins. It has been proved that Bcl-2is an important apotosis inhibitory molecule, while p53acts as a supporter in DNA damage-induced apoptosis. Other apoptosis-related molecules include Bcr-abl, PML-RARa, Bax and so on. In recent years, there have been more and more reports demonstrating that heat shock proteins are important regulators of cell apoptosis, which can inhibit the apoptotic process by modulating the key molecules of the related signaling pathways. Hsp27, a member of the small heat shock protein family, could influence the extrinsic and intrinsic apoptotic signaling pathways at different levels to eventually suppress cell apoptosis and promote the proliferation of cancer cells. Therefore, exploring chemotherapeutics targeting at Hsp27is of great significance.Just as we mentioned before, bufalin could regulate different molecular signaling pathways to induce apoptosis in a variety of cancer cells. Interestingly, a recent study proved that bufalin was able to trigger apoptosis in osteosarcoma cells and the possible mechanism may be through targeting Hsp27. Herein, we wanted to study whether bufalin could affect the apoptotic rates of pancreatic cancer cells and its potential molecular mechanisms. In this part, we selected PANC-1and CFPAC-1cells as study models. After being treated with different concentrations of bufalin(0～200nM)for the indicated time(48h), pancreatic cancer cells were collected with appropriate methods and the fraction of apoptotic cells was analyzed by flow cytometry; western blotting analysis was used to detect the expression level of apoptosis-related proteins and the upstream modulating molecules.The results of flow cytometry indicated that the apoptotic rates of PANC-1and CFPAC-1cells increased significantly in a dose-dependent manner, which means that bufalin could actually induce apoptosis in pancreatic cancer cells. Meanwhile, we used western blotting analysis to further investigate the potential mechanisms of bufalin-induced apoptosis in pancreatic cacner cells. Our results showed us that after treatment with bufalin, the expression level of the anti-apoptotic protein Hsp27was down-regulated in a dose-dependent manner and the protein level of its downstream molecule p-Akt was also decreased. In addition, we found that pro-caspase-3and pro-caspase-9were activated, while Bax was up-regulated and Bcl-2was down-regulated in PANC-1and CFPAC-1cells. According to the above data, we speculated that bufalin might target Hsp27and thus regulate the key apoptosis-related proteins to eventually trigger apoptosis in pancreatic cancer cells. Part threeThe effects of bufalin on the cell cycle distribution and the expression levels of cell cycle-related proteins of pancreatic cancer cellsA variety of antitumor drugs such as dactinomycin D, cytosine arabinoside, fluorouracil, etoposide and vincristine could suppress the proliferation of cancer cells by inducing cell cycle arrest. There have been reports demonstrating that induction of cell cycle arrest is one of the most important mechanisms of bufalin in hepatic cancer, bladder cancer and endometrial cancer cells. What interested us is whether bufalin could trigger cell cycle arrest in pancreatic cancer cells. Therefore, we used different concentrations of bufalin(0-200nM)to treat PANC-1and CFPAC-1cells for48h, and flow cytometry was utilized to test the cell cycle distribution. The result showed that bufalin induced accumulation of G2/M phase in pancreatic cancer cells. Moreover, with western blotting analysis, we found that the protein level of CyclinBl and CDK1was down-regulated while the expression level of p21was increased after bufalin treatment.The data in this part suggested that bufalin could arrest pancreatic cancer cells at G2/M phase by down-regulating the expression level of CyclinB1and CDK1, which are the key modulators of the G2/M checkpoint. As our western blotting analysis also proved that bufalin could increase the expression level of p21, an inhibitor of the cyclinBl-CDK1complex, we suggested that bufalin might upregulate the expression of p21and down-regulate the expression and activity of CyclinB1and CDK1to activate G2/M cell cycle checkpoint in pancreatic cancer cells. But the specific molecular mechanisms of bufalin-induced cell cycle arrest in pancreatic cancer needs to be further investigated.Part fourBufalin affected the anti-cancer activity of gemcitabine in pancreatic cancerGemcitabine-based chemotherapy is the main treatment for the locally advanced or the recurrent and metastatic pancreatic cancer. Unfortunately, the resistance of pancreatic cancer cells against gemcitabine often results in treatment failure. Recent studies have proved that bufalin could enhance the chemosensitivity of hepatic cancer and osteosarcoma cells to regular chemotherapeutics. However,whether bufalin could affect the anti-cancer activity of gemcitabine in pancreatic cancer has not been reported. In this thesis, pancreatic cancer cells were treated with bufalin or gemcitabine alone or their combinations for48h, cells treated with0.1%DMSO served as control. After treatment, the results were analyzed with CCK-8assay.Our data showed that the combination treatment could suppress the proliferation of these two pancreatic cancer cell lines more than either bufalin or gemcitabine alone. This result suggested that bufalin could enhance the chemosensitivity of PANC-1and CFPAC-1cells and the possible mechanism should be further explored.