Growth-inhibition Effects And Mechanisms Of Sorafenib In Combination With Tamoxifen On Cholangiocarcinoma

Objective: To investigate the in vitro antineoplastic effects of sorafenibcombined with tamoxifen in human Cholangiocarcinoma（CC） cells and toreveal the antineoplastic mechanisms of sorafenib alone and in combinationwith tamoxifen.Methods: Cell growth inhibition by sorafenib alone and in combination withtamoxifen was studied in vitro in3human CC cell lines（QBC939、SK-ChA-1and Mz-ChA-1） by MTT assay. Sorafenib-mediated changes of mRNAexpressions of Bcl-2、Bcl-xL、cFLIPL、Mcl-1and p21were analysised byreal-time RT-PCR in QBC939cell line. The antineoplastic mechanisms ofsorafenib alone and in combination with tamoxifen were assessed by Westernblotting for PARP、Bcl-2、Akt、p-Akt、Bcl-xL、Erk、p-Erk、c-FLIP_S/L and Mcl-1assay in human CC cell lines. In addition, the antineoplastic effect andmechanisms of Mek inhibitor PD98059were also assessed by Westernblotting for PARP、Erk、p-Erk、c-FLIPS/Land Mcl-1assay in QBC939cell line.Results: Sorafenib treatment alone inhibited the proliferation of human CCcells and this effect was enhanced by addition of7.5μM tamoxifen. Treatmentwith sorafenib combined with tamoxifen exerted synergistic antineoplasticeffects in human CC cells．The synergistic effects were most Significant inSK-ChA-1cell line but the effects could be observed only at high dosesorafenib combined with tamoxifen in Mz-ChA-1cell line. A dramatic increaseof mRNA levels of p21、cFLIPLand Mcl-1was observed in QBC939cell lineafter sorafenib treatment for24h. Sorafenib-induced apoptosis was observedin both QBC939and SK-ChA-1cell lines but not in Mz-ChA-1cell line aftersorafenib treatment for24h. Sorafenib reduced protein levels of p-Erk、cFLIPLand Mcl-1in both QBC939and SK-ChA-1cell lines but not in Mz-ChA-1cellline. In addition, sorafenib downregulated Bcl-2protein levels in QBC939cellline and p-Akt protein levels in SK-ChA-1cell line. PD98059significantlydownregulated p-Erk protein levels in QBC939cell line,but didn’t induceapoptosis and downregulate protein levels of both cFLIPLand Mcl-1inQBC939cell line after the treatment for24h. The addition of7.5μM tamoxifenenhanced the level of sorafenib-induced apoptosis in SK-ChA-1cell line. Thetreatment with sorafenib combined with tamoxifen resulted in a morepronounced decrease in cFLIPLprotein level.Conclusion: Sorafenib reduces apoptosis independent of its effects onMek/Erk signaling pathway in human CC cells. Sorafenib-induced apoptosis isassociated with downregulation of protein levels of both cFLIPLand Mcl-1.Both downregulation Bcl-2protein level and p-Akt protein level appear to alsoplay a key role in sorafenib-induced apoptosis, but the mechanism seem to becell type-specific. The treatment with sorafenib combined with tamoxifen results in synergistic antiproliferative effects in human CC cells. Tamoxifenresults in a pronounced increase in sorafenib-induced apoptosis, whichincrease is associated with the more pronounced decrease in cFLIPLproteinlevel.