Anti-Hepatocellular Carcinoma Activity Study Of Tylophora Ovata Alkaloids(HTBPI) Through Inhibiting Akt

Objectives: Phenanthroindolizidine alkaloid(13a R,14R)-9,11,12,13,13 a,14-hexahydro-3,6,7-trimeth-oxydibenzo [f,h]pyrrolo[1,2-b]isoquinolin-14-ol(HTBPI)is derived from Tylophora ovata(Lindl.)Hook.ex Steud of traditional Chinese medicinal herb.HTBPI has good anti-tumor biological activity,but its anti-tumor mechanism research is not yet clear.This subject aims to study anti-hepatocellular carcinoma(HCC)of HTBPI,exploring its mechanism and providing a theoretical basis for the clinical application of HTBPI.Methods:(1)Cell counting kit-8(CCK8)and plate clone formation experiments were used to detect the effects of HTBPI on HCC cell viability and single cell proliferation ability;(2)4’,6-diamidino-2-phenylindole(DAPI)staining,Western blot analysis and flow cytometry were used to detect the effects of HTBPI on HCC cell apoptosis;(3)Western blot analysis,transmission Electron microscopy(TEM)and immunofluorescence(IF)staining to detect the effect of HTBPI on cell autophagy;(4)When autophagy inhibitor Bafilomycin A1(BA1)inhibiting autophagy,Western blot analysis and flow cytometry to detect the effect of HTBPI on apoptosis;(5)Cell thermal transformation analysis experiments(CETSA)and drug affinity response target stability assay(DARTS)experiments were used to analyze the binding relationship between HTBPI and protein kinase B(Akt);Molecular docking was used to simulate the interaction site between HTBPI and Akt,and Western blot experiments were performed on it;(6)Overexpression of Akt in two HCC cell lines Hep G2 and Hep3 B,and the effects of HTBPI on apoptosis and autophagy of HCC cells were analyzed by Western blot and immunofluorescence staining experiment;(7)A mouse model of liver cancer PDX was established using hematoxylin-Eosin(HE)staining and immunohistochemical(IHC)tests to detect the pathological effects of HTBPI on mouse organs and related proteins of apoptosis and autophagy;(8)TCGA database further evaluates Akt The relationship between the level of acidification and lifetime cancer patients.Results:(1)The results of CCK8 and plate clone formation experiments showed that HTBPI significantly reduced the viability of HCC cells and reduced the ability of single cell proliferation;(2)Western blot experiments showed that HTBPI could significantly change the expression levels of apoptosis-related such as Poly-ADP-ribose polymerase(PARP),Cleaved-caspase3,B-Cell CLL/Lymphoma 2(Bcl-2)and Bcl-2 Associated X Protein(Bax).After DAPI staining,the nucleosomes were obviously broken;The detection of HTBPI by flow cytometry can significantly induce the apoptosis of HCC cells;(3)HTBPI significantly changed the expression levels of autophagy-related proteins such as Sequestosome1(p62),Microtubule Associated Protein Light Chain 3(LC-3)and the fluorescence intensity of p62 and autolysosome;(4)After inhibiting autophagy,under the effect of HTBPI,the viability of HCC cells was significantly reduced,and the single cell proliferation ability was further reduced.The results of Western blot experiments and apoptosis kit tests showed that apoptosis was significantly increased;(5)HTBPI could slow the streptomycin or the rate of degradation of Akt protein by temperature,HTBPI can directly bind to the C-terminal domain of Akt,and can significantly inhibit the phosphorylation of Thr308(T308);(6)After overexpression of Akt,HTBPI inhibits the cell viability to a certain extent Recovery,apoptosis-related proteins and autophagy-related proteins were all reversed(7)To a certain extent,once every other day,after 21 consecutive days PDX tumor volume in a mouse model than the control group significantly reduced,and the heart,HE staining of heart,liver,spleen,lung,kidney abnormalities were not found;(8)Clinical data Analysis of the survival time of tumor patients was negatively correlated with the high expression of phosphorylation of Akt(T308).Conclusions: HTBPI can induce apoptosis and autophagy in HCC cells;inhibiting autophagy,HTBPI induced apoptosis of HCC cells is significantly enhanced;HTBPI binding in vivo and in vitro inhibits phosphorylation of Akt(T308),inducing apoptosis and autophagy;clinical data Analysis of the survival time of tumor patients was negatively correlated with the high expression of phosphorylation of Akt(T308).