Pilot Study On The Anti-cancer Effects Of Celastrol On Human Cholangiocarcinoma Cell Line TFK-1

Part1The Effect of Celastrol on Cell Proliferation and Cell Cycle of Cholangiocarcinoma CellObjective:To investigate the modulation of Celastrol on cell proliferation and cell cycle of human cholangiocarcinoma cell line TFK-1.Methods:TFK-1cells were seeded in96-well plates and exposed to different concentrations of Celastrol. After incubation for24h,48h, and72h, cell viability was measured by CCK-8assay followed by the calculation of IC50values. Cell cycle was analyzed by flow cytometry (FCM) with propidium iodide (PI) labeling method.Results:1. Celastrol suppressed the proliferation of TFK-1cells, and the inhibitory effect showed a time-and dose-dependent manner.2. The FCM results demonstrated that Celastrol can cause arrest of cell cycle. G2/M phase delay was observed significantly.Conclusions:1. Celastrol can inhibit the growth of cholangiocarcinoma cell with a time-and dose-dependent manner in vivo.2. Celastrol can initiate cell cycle arrest at the G2/M phase. Part2The Effect of Celastrol on Cell Apoptosis and Apoptosis-related Proteins of Cholangiocarcinoma CellObjective:To observe the modulation of Celastrol on TFK-1cell apoptosis, meanwhile, to determine the expression of apoptosis-related proteins after drug intervention, followed by a preliminary discussion on its possible mechanism of action.Methods:TFK-1cells were exposed to Celastrol (2μg/ml), and incubated for24h,48h, and72h respectively. Then, cells were double-stained with Annexin V-FITC and propidium iodide, and analyzed by FCM. After exposure to tripterine for48h, the apoptosis-related proteins extracted from TFK-1cells were examined by Western Blot analysis.Results:1. Annexin V-FITC/PI double staining results indicated that Celastrol induced apoptosis in TFK-1cells in a time-dependent manner. Apoptotic rate can reach to56.01%after incubation for72h.2. Western blotting results demonstrated that Celastrol can downregulated the expression of IAPs such as Survivin and Bcl-2, and upregulated the level of pro-apoptosis protein Bax and Caspase-3.Conclusions:1. Celastrol can induce apoptosis in TFK-1cells in a time-dependent manner.2. TFK-1cell apoptosis induced by Celastrol through a mitochondrial-dependent caspase pathway is correlated well with its ability to modulate the expression of apoptosis-related proteins. Part3Anti-cancer Activity and Mechanism of Celastrol on Human Cholangiocarcinoma Xenograft Model in Nude MiceObjective:To evaluate the anti-cancer activity of Celastrol on human cholangiocarcinoma xenograft nude mice tumor model via measuring the tumor growth inhibition in vivo. Then the anti-tumor mechanism was analyzed by determination of the expression of apoptosis-related proteins.Methods:Human cholangiocarcinoma xenograft model was established via injecting s.c. TFK-1cells into the backs area of the nude mice. Celastrol was administrated i.p. at the doses of1mg/kg/day, and2mg/kg/day, then tumor size was determined every three days. After administration, the mice were sacrificed, and the expression of target proteins from tumor homogenates was determined using Western blotting.Results:1. Both treatment groups showed inhibitory effects of Celastrol on tumor growth, while the higer dose group (2mg/kg/day) was more significant than the other (p<0.01), with the tumor growth inhibition rate of49.71%.2. It was observed that no mice died in either the control group or the experiment group, though there was weight loss at the2mg/kg dose.3. The expression of apoptosis-related proteins in the Celastrol-treated xenograft tumors was in accordance with that detected in vitro.Conclusions:1. Celastrol can suppress xenograft tumor growth significantly, presenting a dose-dependent manner. However, higher doses can lead to certain side effects.2. Celastrol can play an anti-tumor role via induction of apoptosis both in vitro and in vivo, which might be involved in the inhibition of NF-κB pathway. Whereas, further studies are needed to completely undersand the mechanism(s) of action of Celastrol and its therapeutic potential for the treatment of cholangiocarcinoma.