Linifanib Activate Autophagy As A Cytoprotective Response In Human Hepatocelluar Carcinoma

Multikinase inhibitor linifanib (ABT-869) has shown a promising prospect against hepatocellular carcinoma (HCC) in pre-clinical trials and phase II clinical trials. However, intrinsic or acquired resistance is common and inevitably follows susceptibility. As a novel cytoprotective mechanism for tumor cell to survive under unfavorable conditions, autophagy has been proposed to play a role in drug resistance of tumor cells. Whether linifanib can activate autophagy to impair the sensitivity of HCC cells remains unknown.Objective:To explore whether multikinase inhibitor linifanib can activate autophagy to impair the sensitivity of linifanib in hepatocellular carcinoma.Methods:HCC cell lines Bel-7404and HepG2were cultured. Cells were treated with different concentrations of linifanib and then cell proliferation and autophagy were measured.To investigate whether PDGFR-β, Akt/mTOR and Mek/Erk signaling pathway involved in autophagy activation by linifanib, the expression of PDGFR-β, Akt/mTOR and Mek/Erk were detected by Western blot.To identify whether the function of linifanib-induced autophagy is a cell survival or death mechanism, we evaluated the effect of autophagy on the growth of HCC by specific autophagy inhibitors chloroquine (CQ),3-methyladenine (3-MA) and small interference RNA targeting Atg5or Atg7in vitro. Further, we also examined the effect of autophagy inhibitor HCQ plus linifanib on the growth of HCC xenograft models in vivo. Results:First, cells were treated with linifanib (0.31,0.625,1.25,2.5,5uM) for24h,48h and72h. The proliferation of HCC cells were minor inhibited by linifanib treatment. Meantime, a high level of autophagy was induced by linifanib in a dose-dependent manner.Second, linifanib treatment suppressed the PDGFR-β, Akt/mTOR and Mek/Erk phosphorylation levels in a dose-dependent manner. PDGFR-β siRNA transfection reduced the expression of PDGFR-P and promoted the transition of LC3-1to LC3-II.The last, we observed the effect of blocking autophagy in HCC in vitro and in vivo. Our results showed that co-treatment with linifanib and CQ or3-MA significantly suppressed the cell proliferation and enhanced apoptosis, compared with linifanib treatment alone in vitro. Further studies also showed that siRNA Atg5or Atg7significantly reduced the expression of Atg5or Atg7and promoted the anti-proliferative effect of linifanib. Meantime, apoptotic population was enhanced in siRNA Atg5or Atg7combined linifanib group than linifanib treated alone. In vivo, linifanib plus HCQ administration significantly suppressed the tumor growth, compared with linifanib alone (p<0.05). In the xenograft tumors tissue, LC3and cleaved caspase-3were increased markedly in linifanib plus HCQ group compared with linifanib treatment only. Further studies showed that linifanib suppressed the PDGFR-β, Akt/mTOR and Mek/Erk phosphorylation levels in tumor tissues.Conclusions:Linifanib could activate autophagy in HCC cell lines, promote expression of LC3and formation of autophagosome and suppress the PDGFR-β, Akt/mTOR and Mek/Erk phosphorylation levels. Induction of autophagy was associated with inhibition of the PDGFR-β and downstream signal Akt/mTOR and Mek/Erk pathways. Autophagy activation plays a cytoprotective role in linifanib treated HCC. The disruption of autophagy process facilitates the anti-proliferative effect of linifanib and enhances apoptotic cell death in HCC in vitro and in vivo.