Effect And Mechanism Of Tetrandrine Against Liver Cancer Cells Through Hippo Signaling Path Way

For a long time,humans have regarded cancer as the"first killer"that endangers the health of the body.Cancer is a highly concealed disease.Because the body does not show obvious symptoms in the early stage,it is easy to miss the best time for treatment.According to statistics,due to the high resilience of tumor cells,the cancer recurrence rate of cancer patients in China within one year after operation is as high as 60%,which leads to the second injury of the body due to the infinite spread of tumor cells.Therefore,in order to help patients alleviate symptoms and prolong patient survival,we urgently need to find other new treatments to improve this dilemma.In recent years,with the growing international development of the Chinese medicine industry,more and more people have begun to study the mechanism of treating liver cancer through Chinese medicine treatment methods.Tetrandrine（TET）is extracted from the root tuber of Stephania tetrandra S.Moore.Studies have shown that tetrandrine,as a bisbenzyl isoquinoline alkaloid,has strong anti-cancer activity,it can prevent the proliferation of tumor cells and inhibit the growth,invasion and migration of tumor cells.The discovery of Hippo signaling pathway comes from Drosophila,which can mainly regulate cell size by regulating cell proliferation and apoptosis.According to reports,the Hippo-YAP signaling pathway is an important pathway to regulate the formation of liver cancer in many signaling pathways,so the intensity of YAP activation needs to be precisely regulated.In the early stage of this research group,tetrandrine was extracted from the roots of tetrandrine plant powder,and it was found through preliminary experiments that tetrandrine was sensitive to the antitumor activity of human liver cancer HepG-2.Taking the pre-experiment results as the starting point,the anti-tumor activity of tetrandrine and the mechanism of inducing apoptosis of tumor cells through the Hippo pathway were preliminarily studied through in vitro experiments.The completion of this paper provides a new theoretical basis for the in-depth study of the antitumor effect of tetrandrine through the Hippo pathway,which is conducive to promoting the development and clinical application of tetrandrine antitumor drugs.In this experiment,the inhibitory effect of tetrandrine on human liver cancer HepG-2 cells was measured by CCK-8.The experimental results show that the growth inhibition rates of human tetracrine HepG-2 cells treated with tetrandrine at concentrations of 2,4,8,16,and32μmol·L^-1for 72 h were 18.21%,39.19%,57.89%,68.36%,and 68.41%.The inhibitory effect of tetrandrine on proliferation is within a certain range.With the increase of its concentration,the growth inhibition rate of cells is gradually increased,showing a dose-dependent relationship.According to the inhibition rate of each administration group,the IC50 of tetrandrine-treated human liver cancer HepG-2 cells after 72 h was 7.76μmol·L^-1.The reference IC50 value was 3.75,7.5,and 15μmol·L^-1of tetrandrine as the low,medium,and high doses of the subsequent experimental drug group.Then use an inverted microscope and a fluorescence microscope to observe the cell morphology of tetrandrine on human liver cancer HepG-2 cells after 48 h.The experimental results show that after using tetrandrine at a concentration of 3.75μmol·L^-1,7.5μmol·L^-1,and 15μmol·L^-1to treat human liver cancer HepG-2 cells for 48 hours,The majority of human liver cancer HepG-2 cells gradually changed from adherent spindle or polygonal shape to round shape,suspended cells appeared,and cell fragmentation occurred in the high-dose group.Cells in the three dose groups all formed apoptotic bodies,and the nuclei showed strong fluorescence,and the phenomenon became more pronounced with the increase in the concentration of Lycoline.After further detecting the effect of tetrandrine on apoptosis of human liver cancer HepG-2 cells by flow cytometry,the experimental results show that within a certain range,as the concentration of tetrandrine increases,the apoptosis rate will also follow Rising and dose-dependent.The apoptotic rates of the blank group,positive drug group,low,medium,and high dose groups were（1.579±0.197,21.197±1.459,5.125±0.797,19.698±1.860,36.882±1.511）%,respectively.Finally,Western Blot was used to analyze the expression of major protein factors（MST1,LATS1,P-LATS1,YAP1,P-YAP1,TAZ）on the Hippo pathway in human liver cancer HepG-2 cells.The expression levels of MST1 in low,medium and high doses were（49.672±2.089）%,（75.140±1.625）%,and（82.731±3.035）%,compared with the blank control group（42.672±2.308）%.The expression rises significantly.The expression levels of LATS1 in low,medium and high doses were（50.084±1.664）%,（63.758±3.080）%and（73.823±1.275）%,respectively.Compared with the blank control group（43.022±2.679）%,the protein expression The expression rises significantly.The expression levels of P-LATS1 in low,medium and high doses were（65.258±1.447）%,（79.307±1.859）%,and（83.115±3.363）%,compared with（50.255±0.877）%of the blank control group.,Protein expression increased significantly.The expression levels of P-YAP1 in low,medium and high doses were（63.664±3.958）%,（86.411±3.390）%,and（96.549±1.281）%,compared with the blank control group（37.885±1.844）%.,Protein expression increased significantly.With the increase of the concentration of the administration group,the expression of MST1,LATS1,P-LATS1,and P-YAP1 protein was up-regulated.The expression levels of YAP1 in low,middle and high doses were（81.256±1.386）%,（39.622±2.137）%,and（32.420±2.129）%,respectively.Compared with the blank control group（89.852±1.776）%,the protein expression The expression is significantly reduced.The expression levels of low,medium and high TAZ histones were（53.539±0.503）%,（31.110±1.643）%,and（20.387±1.201）%,respectively.Compared with the blank control group（55.947±1.006）%,the protein expression The expression level was significantly reduced.With the increase of drug concentration,the expression of YAP1 and TAZ protein was down-regulated.In summary,tetrandrine has an inhibitory effect on the growth of liver cancer cells.After tetrandrine activates the MST1 protein factor in the Hippo pathway,the activated MST1 can phosphorylate LATS1,resulting in enhanced LATS1 activation ability.The activated LATS1inactivates the downstream targets YAP1 and TAZ after phosphorylation,and YAP1 fails to bind to the transcription factor TEAD,resulting in the termination of oncogene transcription and ultimately inhibiting tumor cell proliferation.