BZG,a Candidate Compound Against Liver Cancer,combined With Sorafenib's Pharmacodynamic Evaluation And The Analysis Of Metabolic Profile And Target Affinity Of BZG

Background and Objectives Hepatocellular carcinoma(HCC)is the most common primary tumor of the liver and also one of the most deadly cancers in the world.Moreover,liver cancer is highly malignant and difficult to detect.Currently,sorafenib is the only targeted molecular drug for the first-line treatment of advanced hepatocellular carcinoma.Though it increases the hope of patients with advanced hepatocellular carcinoma,it's overall survival rate is still low and does have some severe side effects.Therefore,it is very critical to improve new therapeutic drugs for hepatocellular carcinoma.BZG is a multi-targeted enzyme inhibitor with a similar chemical structure to sorafenib,and our previous pharmacokinetic studies have shown that BZG can be rapidly and widely distributed to organs and tissues of animal models.Besides,BZG could inhibit a panel of human cancer cells' proliferation.Therefore,this research intends to explore the mechanism of BZG alone and also BZG with sorafenib on cell proliferation and cell apoptosis in vitro and in vivo.In addition,to study the process of drug metabolisms of BZG and analyze its active metabolites from the metabolic model in vivo and in vitro.Methods 1.CCK-8 assay and cell cloning formation were performed to evaluate the cell viabilities of hepatocarcinoma cells Hep3 B,SMMC-7721,Hep G2 and Huh-7 after treatment of BZG alone and BZG with sorafenib.Cell apoptosis were detected by Hoechst 33342 staining and flow cytometry.The effects of BZG and sorafenib with BZG's co-treatment were also done by flow cytometry.The expressions of AKT,p-AKT,ERK,p-ERK,PI3 K,m TOR,p-m TOR,RAF1 and VEGFR-3 were detected by western blot.2.Huh-7 cell xenografts were used to assess the effects of BZG and BZG with sorafenib's co-treatment in vivo.Cell apoptosis and tissue necrosis of the tumors were observed by HE staining and TUNEL separately.3.UPLC-QTOF MS was used to analyze metabolites of BZG through the metabolic model in vivo,and human liver microsomes and recombinant enzymes in vitro.4.Molecular docking software e Hi Ts was used to analyze the affinity of BZG and its metabolites to the molecular target VEGFR-2.Results 1.BZG can significantly reduce the viability rate of Hep3 B,SMMC-7721,Hep G2 and Huh-7 in a time and dose-dependent manner.Besides,the high concentration of BZG could dramatically inhibit the survival of Hep G2 cells compared to the other three kinds of HCC cells.BZG combined with sorafenib has significantly better efficacy than BZG or sorafenib alone.2.BZG can effectively promote the apoptosis of Hep3 B,SMMC-7721,Hep G2 and Huh-7 cells.However,BZG has a dose-dependent manner on the other three kinds of liver cancer cells except for SMMC-7721 cells,and moreover,BZG combined with sorafenib could more effectively promote the apoptosis of human liver cancer cells compared with BZG or sorafenib alone.3.BZG and sorafenib have different inhibitory effects on protein expression of Raf/ ERK and PI3K/AKT/m TOR pathways in four types of liver cancer cells.The combination of BZG and sorafenib could inhibit the expression of p-AKT more effectively and can inhibit the expression of PI3 K,p-m TOR,Raf1 and p-ERK to some extent.4.With the increasing concentration of BZG,the weights and volumes of the tumors of Huh-7 xenografts were significantly decreased.Besides,the anti-tumor outcomes of BZG and sorafenib's co-treatment were significantly better than that of BZG or sorafenib alone.Tissue necrosis and a large number of apoptotic cells were also observed in the tumors.5.BZG generated a total of 11 metabolites(M1-M11)through phase I and phases II metabolic reactions in vivo and in vitro.A screening of Cytochrome P450(CYP)enzymes confirmed that h CYP1A2,h CYP2B6,h CY2C19 and h CYP2C8 exhibited hydroxylation activity and h UGT1A9 exhibited glucuronidation activity.6.The affinities of BZG's metabolites M2,M3,M9,M10 and M11 to VEGFR-2 was higher than sorafenib.Conclusion 1.BZG can effectively inhibit the proliferation of human hepatocellular carcinoma cells such as Hep3 B,SMMC-7721,Hep G2 and Huh-7,and could promote the apoptosis of hepatocellular carcinoma cells.Besides,BZG can effectively enhance the anti-tumor effects of sorafenib.Through the AKT/m TOR signaling pathway,BZG and sorafenib's co-treatment promotes the apoptosis of hepatocellular carcinoma cells.2.Through the analysis of the BZG's metabolites of phase I and phase II,it is speculated that the major metabolic pathways of BZG may include hydroxylation,gluconate acidification,acetylation,sulfonation and degradation.And h CYP1A2,h CYP2B6,h CY2C19 and h CYP2C8 exhibited hydroxylation activity and h UGT1A9 exhibited glucuronidation activity.The metabolites M2,M3,M9,M10 and M11 are more effective in inhibiting VEGFR-2 than their parent BZG.