Regulation And Role Of Aquaporin-3 In The Sensitivity Of Hepatocellular Carcinoma Cells To Sorafenib

BackgroundExtensive identification of hepatocellular carcinoma(HCC)progression drivers and their regulation during treatment is vital not only to understanding the mechanisms of resistance,but also to sensitizing cells to the drugs.Previous studies have extensively revealed the role of aquaporins,particularly aquaporin-3(AQP3),in promoting tumor progression in multiple tumors,including HCC.However,the regulation and role of AQP3 in the treatment of HCC with sorafenib needs to be further studied.This dissertation mainly investigated the effect of sorafenib on the expression of AQP3 in HCC cells,and the expression change and role of AQP3 in hypoxia-induced insensitivity of HCC cells to sorafenib.Methods and materialsReal-time quantitative polymerase chain reaction(qPCR)and Western blotting were used to detect the mRNA and protein levels of AQP3 in HCC cells exposed to sorafenib versus unexposed to drug or exposed to hypoxic condition versus normoxic condition,respectively.UO126(Mek1/2 inhibitor)and LY294002-PI3 K inhibitor were used to interrogate the signaling pathway involved in the regulation of AQP3 in sorafenib or hypoxic-treated HCC cells.The proliferation and cytotoxicity of the transfected cells were detected by CCK-8,and cell cycle and apoptosis were detected by flow cytometry.In addition,proteins involved in the regulation of cell proliferation,cell cycle progression,and apoptosis were determined via western blotting.ResultsIn sorafenib treated cells,AQP3 mRNA and protein levels were significantly reduced,while in hypoxic cells,AQP3 levels were significantly increased.The inhibition of Raf/Mek/Erk signaling pathway by sorafenib led to the down-regulation of AQP3,while the activation of PI3K/Akt signaling pathway by hypoxia led to the up-regulation of AQP3.In addition,the results showed that the upregulation of AQP3 in hypoxic HCC cells activated the PI3K/Akt signaling pathway through positive feedback,resulting in reduced sensitivity of the cells to sorafenib.In hypoxic cells with down-regulated AQP3 expression,cell proliferation was inhibited,cell cycle arrest was induced,cell apoptosis was increased,and sensitivity to sorafenib was restored.In combination with LY294002,the sensitivity of AQP3 down-regulated hypoxic cells to sorafenib increased even more.ConclusionThis study revealed the effect of sorafenib on the expression of AQP3 in HCC cells and the role of AQP3 in hypoxia-induced HCC cells’ insensitivity to sorafenib.The findings may indicate that AQP3 is a potential therapeutic target for increasing the sensitivity of HCC cells to sorafenib.Further in vivo studies are needed to confirm the findings.