Molecular Mechanisms Of Parthenolide And Arsenic Trioxide Synergizing To Trigger Autophagy And Apoptosis In Hepatoma Cells

Background purpose Hepatocellular carcinoma is a common malignant tumor with higher morbidity and mortality that endangers human health in China.Arsenic trioxide(ATO)possesses high killing activity against leukemia cells and solid tumor cells,and has achieved good efficacy in the treatment of clinic leukemia and solid tumors,but the toxic and side effects limit its clinical application.Parthenolide(PTL)is the first natural sesquiterpene lactone,an active compound from feverfew,which targeted-kills cancer stem cells and has little toxicity to normal cells.PTL is able to sensitize chemotherapeutics and specifically eliminate cancer stem cells.Whether combined application of PTL can enhance killing effect of ATO to hepatocellular carcinoma cells(HCC)and the possible cellular and molecular mechanisms remain unclear.In this study,the hepatocellular carcinoma cell models,with different malignancy degree and metastatic potential,and animal model were used to comparatively study the cytotoxic effect,as well as the abilities inducing apoptosis and autophagy,of ATO alone or combined with PTL in hepatocellular carcinoma cells,and the cellular and molecular mechanisms of the combined action of PTL and ATO were explored.These may provide experimental basis for the combination of PTL and ATO in the clinical treatment of liver cancer.Methods Hep G2 cells,MHCC 97 H cells and Huh7 cells were used as human hepatocellular carcinoma cell model,and immortalized hepatic L02 cells as the normal hepatic cell model.Mouse hepatocellular carcinoma H22 cells were employed to construct transplanted liver cancer animal model.Cell proliferation activity was detected by MTT colorimetry,and apoptosis was assessed by Annexin V/PI double staining.Intracellular mitochondrial membrane potential and reactive oxygen species were detected by fluorescence probe labeling assay.The protein expression,protein modification and protein-protein interactions in apoptosis-and autophagy-related signaling pathways were examined by western blotting and co-immunoprecipitation(Co-IP),and CRISPR-Cas9 technology was performed to construct gene-knockout cell model.A mouse model of subcutaneous liver cancer transplantation was established to study the antitumor effect in vivo.Results PTL promoted the inhibitory effect of ATO on the proliferation of Hep G2 cells,MHCC 97 H cells and Huh7 cells,and accelerated the apoptosis of these cancer cells.After co-treatment of PTL and ATO,the cell-cycle distribution was arrested at the G2/M phase,the CD133 expression,stemness and renewal ability of the hepatocellular carcinoma cells were inhibited.In PTL-ATO-co-treated HCC,the activity of deubiquitinating enzyme USP7 was inhibited and its substrate,E3 ubiquitin ligase HUWE1,was significantly reduced,the degradation of P53,the substrate of HUWE1,was inhibited and its content increased,and USP7 and HUWE1 interaction weakened.The above results suggested that the USP7-HUWE1-P53 axis might mediate regulation of PTL enhancing the sensitivity of HCC to ATO.The combined action of PTL and ATO initiated autophagy activity of HCC cells and inhibited PI3K/AKT/m TOR signaling pathway.Autophagy inhibitor chloroquine(CQ)and knock-out of the autophagy key gene Atg5 significantly increased the content of activated caspase3 and the sensitivity to ATO and PTL in PTL/ATO-treated HCC cells,which led to the augment of HCC apoptosis.These suggested that combination of PTL and ATO could induce autophagy of HCC cells in the early stage,and PTL cooperated with ATO to regulate autophagy and apoptosis of HCC cells through PI3K/AKT/m TOR signaling pathway.In vivo animal experiments showed that PTL and ATO alone or in combination could significantly reduce the growth of H22 liver cancer in mice,the weight and volume of tumor mass obviously decreased,and autophagy inhibitor CQ could enhance the anti-hepatoma effect of PTL and ATO in vivo.Conclusions1 Parthenolide combined with arsenic trioxide significantly inhibits the proliferation activity,reduces self-renewal ability and promotes apoptosis in Hep G2,MHCC 97 H,Huh7 and H22 hepatoma cells,and parthenolide enhances the sensitivity of the hepatocellular carcinoma cells to arsenic trioxide through the USP7-HUWE1-P53 axis.2 Parthenolide combined with arsenic trioxide can evoke the autophagy response of hepatocellular carcinoma cells by inhibiting the PI3K/AKT/m TOR pathway,and inhibition of autophagy activity can significantly promote the cytotoxic effect of arsenic trioxide to hepatocellular carcinoma cells.3 In vivo parthenolide and arsenic trioxide have significant anti-hepatoma effects,and inhibition of autophagy can strengthen the combined anti-hepatoma effect of parthenolide and arsenic trioxide.