The Anti-tumor Effects And Mechanisms Of Diterpenoid GH02 In Hepatocullar Carcinoma

Objective:Hepatocellular carcinoma(HCC)is a most common type of primary liver cancer and has high insistance to conventional chemotherapy.It has been found that diterpenes can inhibit the expression and activity of signaling pathway-related proteins and induce apoptosis,therefore playing an anti-tumor role.In this study,we aim to explore the anti-tumor effects and mechanisms of diterpenoid compound GH02 in hepatocellular caicinoma.Methods:(1)In vitro inhibitions of GH02 on the proliferation of various liver cell lines were determined by CCK-8 assay;(2)Inverted fluorescence microscope,flow cytometry,and Western blotting were used to analysize apoptosis by charactreristic morphological changes,quantification,and protein expression;(3)Extending these in vitro results,HepG2 tumors and Hepal-6 tumors in a xenograft model and a murine orthotopic tumor model of hepatocellular carcinoma were established to observe the anti-tumor effects in vivo;(4)The proteomics assay was used to determined the protein expression in HepG2 cells following treated with GH02.(5)The mitochondrial changes were observed by JC-1 staining and Inverted fluorescence microscope;(6)Western blotting analysis for apoptotic and signaling pathway protien expression was determined in HCC cell lines exposed to GH02 for indicated time.Results:The significant inhibitions of hepatucullar carcinoma cell clines(HepG2,Hep3B,and SK-HEP-1)and normal liver cells(QSG7701 and L02)exposed to GH02 for 48 h in vitro were observed by CCK-8 assay,and the half maximal inhibitory concentrations(IC50)were 9.315±1.92 ?mol/L,7.107±0.61 ?mol/L,5.93±0.89?mol/L,33.5±3.34?mol/L,and 31.04±3.79?mol/L;GH02 can block cell cycle distrubution in G1/S,and induce apoptosis in HCC cell lines by upregulating cleaved caspase-9 but not cleaved caspase-3;(3)We also found that GH02 administration inhibited the growth of HepG2 tumors and Hepal-6 tumors in a xenograft model and a murine orthotopic tumor model of hepatocellular carcinoma;(4)The expressions of apoptotic proteins(caspase-7 and CTSL),cell cycle-raleted proteins(CDKN2A,ANAPC5,CDK7,and ORC6),and various signaling pathway proteins were regulated in HepG2 cells following treatment with GH02 for 24 h.(5)GH02 can change mitochondrial membrane permeability,down-regulate Bcl-2,Mcl-1 and Cyclin D proteins,inhibit phosphorylation levels of Src and STAT3 to induce apoptosis and inhibit the proliferation of HCC cells.Conclusion:GHO2 exhibits anti-tumor effects in HCC by inducing apoptosis via downregulating the expression of Bcl-2 and Mcl-1,and inhibiting the Src/STAT3 signaling pathway.Taken together,GH02 may be a potential precursor compound for the development of antitumor drugs against HCC.