The Anti-tumor Effect And Mechanism Of Epidithiodiketopiperazine Derivative Chaetocochin J On Hepatocellular Carcinoma

Background and purpose: Hepatocellular carcinoma(HCC),as one of the common primary liver cancers,has the characteristics of complex pathogenesis,strong metastasis,high degree of malignancy and poor prognosis,resulting in very limited clinical therapeutic effects.Natural products are an important resource library for clinical small molecule drugs.Epipolythiodioxopiperazine(ETP)alkaloids are a class of natural products with various biological activities,which have strong antitumor,antibacterial,antiviral and immunosuppressive pharmacological effects.Chaetocochin J(CJ)belongs to ETP alkaloid derivative,and has good antitumor activity.In this study,CJ was isolated from Chaetomium sp.,and we explored its anti-HCC mechanism under normoxic and hypoxic conditions.Methods: CCK-8 assay was used to detect the viability of Hep G2 and Hep3 B cells with different concentrations of CJ;Colony formation assay was evaluated the proliferation ability of CJ on HCC cells;Cell cycle,apoptosis and glucose uptake rates were detected by flow cytometry;JC-1 and DAPI staining were observed by fluorescence microscope;Wound healing assay and transwell assay were evaluated the migration and invasion ability of CJ on HCC cells;Co-immunoprecipitation assay was measured the binding ability of HIF-1α to p300;The m RNA levels of Glut1,HK2,VEGF,e NOS,LDHA,MMP9 and COX-2 were detected by q RT-PCR;Western blot was used to detect proteins related to cell cycle,apoptosis,migration and metabolism;Western blot and immunofluorescence were used to detect HIF-1α protein;We constructed a Hep G2 xenograft tumor model.Tumor tissue was analyzed by H&E staining,and HIF-1α and cleaved-caspase3 protein levels were analyzed by western blot and immunohistochemistry;Results: CJ inhibited proliferation of Hep G2 and Hep3 B cells in a dose-dependent manner and induced G2/M phase arrest.Moreover,CJ induced apoptosis through the caspase dependent pathway both under normoxic and hypoxic conditions.In addition,CJ inhibited the expression of HIF-1α through the PI3K/Akt/m TOR/p70S6K/4EBP1 pathway,inhibited the binding of HIF-1α/p300,and inhibited the glycolysis before inducing apoptosis in Hep G2 and Hep3 B cells,further inhibited migration and invasion in HCC cells.We demonstrated that CJ inhibited tumor growth without significant toxicity in the Hep G2 nude xenograft model.Although sorafenib can effectively inhibit tumor proliferation,nude mice died.Western blot and immunohistochemistry results showed that the expressions of HIF-1α and PCNA were down-regulated,while the expression of cleaved-caspase3 was up-regulated after intraperitoneal injection of CJ.Conclusion: we showed the anti-HCC effect of CJ in vitro and in vivo,and demonstrated that the key to its function is mainly associate with the inhibition of PI3K/Akt/m TOR/p70S6K/4EBP1 pathway independent of hypoxic,and it also could downregulate the expression of HIF-1α as well as disrupt the binding of HIF-1α/p300 under hypoxic condition,leading to the disorder of metabolism,inhibits cell migration and invasion,and induces apoptosis in HCC cells.