Anti-cancer Effects Of Garcinol On Human Hepatocellular Carcinoma Cells:the Role Of Circadian Clock Genes BMAL1 And Per2

Objective: Garcinol(Garc)is extracted from the shell of the mangosteen plant in the genus Garcinia in the garciniaceae family.but its exact mechanism of anti-cancer action remains unclear.Therefore,in our study,we explored the function of Garc on inhibiting the proliferation,migration,invasion and apoptosis of liver cancer cells and the relationship between the proliferation,migration of tumors and biological rhythm genes.Providing therapeutic targets for the future of novel antitumor agents.Methods: 1.Cell proliferation was measured by CCK-8 assay,clonogenic formation assay and Edu assay.Cell migration and invasion ability was assessed by cell scratch assay,Transwell cell migration and Matrigel Transwell cell migration assay,Huvec angiogenesis assay to detect angiogenesis ability,Cell apoptosis was examined by Mitochondrial membrane potential detection experiment(JC-1),cleaved-PARP was assayed by western blot 2.We detected biological rhythm genes Bmal1 and Per through western blotting,cellular immunofluorescence experiments and RT-PCR 3.Using lentiviral vectors that overexpress and interfere with the biological rhythm genes Bmal1 and Per2,explore the effects of the biological rhythm genes Bmal1 and Per2 on the proliferation and migration of liver cancer cells.Results: 1.Bioinformatics analysis showed that the Bmal1 and Per2 proteins are closely related to DNA transcription,apoptosis,hypoxic stress response and tumorigenesis.2.Garc reduced hepatocarcinoma cell viability significantly(P<0.05).The colongenic formation assay along with Edu assay were inhibited by Garc significantly(P<0.05).The JC-1 assay showed the level of early apoptosis increased in a concentration-dependent manner.Garc increased the level of cleaved-PARP(Y34)and cleaved-PAR(E51)proteins in HCC(P<0.05).3.Garc reduced cell migration and proliferation ability and angiogenesis ability when comparing with the control group(P<0.05).4.Garc reduced the level of biological rhythm gene Bmal1 and Per2 protein expression,and cell immunofluorescence suggested that rhythm gene Per2 had entered the nucleus,while the mRNA change was contrary to the protein trend.5.Overexpression of Bmal1 and Per2 genes can significantly antagonize Garc’s ability to inhibit proliferation and migration of HepG2 cells and huh7 cells,suggesting that Garc may play an anti-tumor effect by targeting the biological rhythm genes Bmal1 and Per2.Conclusion: Garc has inhibitory effects on proliferation,migration,invasion and angiogenesis,apoptosis induction of hepatocellular carcinoma cells,showing a concentration-effect and time-effect relationship,and the anti-hepatoma effect is related to the changes of biological rhythm genes Bmal1 and Per2,suggesting that Garc may pass targeting the biological rhythm genes Bmal1 and Per2 to play an anti-liver cancer effect.