Research On The Anti-hepatocellular Carcinoma Activity And Related Mechanism Of Brevilin A

Hepatocellular carcinoma(HCC)is one of the common malignant tumors.China has the highest incidence rate and mortality of HCC in the world.Exploring the diagnosis and treatment of HCC is conducive to improving the health level of our people and reducing the disease burden caused by HCC.The previous research of our research group found that Brevelin A(BA),derived from the anti-tumor Chinese medicine Centipeda minima L.,has a good inhibitory activity against a variety of tumors,including triple negative breast cancer,nasopharyngeal cancer,melanoma,colon cancer,etc.However,there are still no research reports on the application of Brevelin A in the treatment of HCC.The purpose of this article is to study the in vitro and in vivo activities of Brevelin A on HCC cells,and to explore its mechanism of action.In this essay,MTT method was employed to measure the inhibitory effects of BA on the viability of HCC cells,including HepG2,Huh7,Hep3B,and PLC/PRF/5 cells.Colony formation method was used to measure the inhibitory effects of BA on the proliferation of HCC cells,including HepG2,Huh7,and PLC/PRF/5 cells.Transwell migration and invasion methods were applied to measure the inhibitory effects of BA on the migration and invasion of HepG2 and Huh7 cells.Flow cytometry was employed to measure the cell cycle arrest effects of BA on HepG2 and Huh7 cells.Western Blot method was used to measure the effects of BA on the expression of cell cycle related proteins in HepG2 and Huh7 cells.Flow cytometry was employed to measure the induction effects of BA on HepG2 and Huh7 cells.Nude mice subcutaneously inoculated with Huh7 cells were applied to measure the inhibitory effects of BA on the proliferation of human HCC cell line Huh7 in vivo.H&E staining method was used to measure the effects of BA on tumor tissue and main organs.RNA-sequence and Western Blot methods were employed to investigate the mechanism of BA against HCC.In vitro,BA inhibited proliferation viability of various HCC cells.Specifically,BA inhibited the growth of HepG2,Huh7,Hep3B,and PLC/PRF/5 cells,inhibited the colony formation of HepG2,Huh7,and PLC/PRF/5 cells,inhibited the migration and invasion ability of HepG2 and Huh7,blocked the cycle of HepG2 and Huh7 cells in the G2/M phase,and induced apoptosis of HepG2 and Huh7 cells.In vivo,in the nude mice subcutaneously inoculated with human HCC cell line Huh7,BA inhibited the growth of HCC cells,and had no significant impact on the body weight of nude mice.The results of H&E staining showed that the tumor tissue of the nude mice in the treatment group had significant intercellular spaces,lighter color of nuclei,decreased volume of nuclei,and increased necrotic area.Compared with the negative control group,the heart,liver,spleen,lungs,and kidneys of the nude mice in the treatment group showed no significant tissue damage and pathological changes,indicating that BA could inhibit the growth of the tumors in vivo and had good biological safety.Studies on the mechanism suggested that the induction of cell cycle by BA is related to the downregulation of cycle related proteins cdc2 and CDK6.The results of RNA-seq and Western Blot experiments showed that BA could promote the expression of MAPK signaling pathway and inhibit the expression of mTOR and STAT-3 signaling pathway.In summary,in vitro,BA inhibited growth of various HCC cells,blocked cell cycle,induced apoptosis,and inhibited migration and invasion.In vivo,BA inhibited significantly the tumor growth in the nude mice bearing tumor without significant toxicity.Preliminary studies on the mechanism of action have found that the anti-hepatocellular carcinoma activity of BA is related to the upregulation of MAPK signaling pathway and the downregulation of mTOR and STAT-3 signaling pathways.This completion of this paper provides a theoretical basis for the future application of BA in prevention and treatment of HCC.