Study On The Extracts From Phellinus Igniarius Inducing Apoptosis And Antitumor Activity Of Hepatoma SMMC-7721 Cells

Objective: This study aimed to investigate the antitumor effect of extracts from Phellinus igniarius(EPI)on human hepatocellular carcinoma SMMC-7721 cells and to investigate the molecular mechanism of antitumor activity.Methods: MTT assay was used to detect the total extracts content of P.igniarius in SMMC-7721,U87-MG,SKOV3,MDA-MB-231 and A375 cells at different concentrations of 10,20,40,80,160,320 and 640 ?g / mL inhibition of five tumor cells.Observed under a microscope,photographed and combined with Hoechst staining observed under a fluorescence microscope and photographed to analyze the different concentrations of P.igniarius hepatoma SMMC-7721 cells after 48 h effect on cell morphology and nuclear morphology.Flow cytometry was used to detect the changes of apoptosis rate of hepatoma SMMC-7721 cells by using Annexin-V/PI double staining method,including live cells,early apoptotic cells,the relative proportion of late apoptotic cells and necrotic cells.The expressions of Bcl-2,Bax,Caspase-3 and cytochrome C in hepatocellular carcinoma SMMC-7721 cells treated with different concentrations of P.igniarius extracts were detected by Western blot.The expression of AMPK,phosphorylated AMPK,mTOR,phosphorylated mTOR,ULK1,phosphorylated ULK1 and Beclin-1 in the autophagy markers LC3 and P62 and the autophagy pathway AMPK / mTOR / ULK1 were detected by Western blot.Cell scratch assaywas used to detect the changes of the number of migrated cells within the scratch after treated with the extracts of P.igniarius in SMMC-7721 cells for 24 h.Results: The results of MTT assay showed that the extract of P.igniarius had the best inhibitory effect on SMMC-7721 hepatocellular carcinoma cells and SK-OV-3 human ovarian cancer cells,followed by human malignant glioma cells U87-MG and human Melanoma A375 is less effective on human breast cancer cells MDA-MB-231.Morphological observation revealed that hepatoma SMMC-7721 cells changed from regular shuttle to irregular shape,round,cell shrinkage and fragmentation.Hoechst staining shows that the cytoplasm of the cell shrinks,the nuclei become irregular,fragmented,and the refraction of the fluorescence is enhanced.As the concentration of the extracts of P.igniarius increases,the blue fluorescence becomes stronger and stronger.The results of Western blot showed that the expression of Bax,Cyt C and Caspace-3 in SMMC-7721 cells increased with the increase of the extracts of P.igniarius,while the expression of Bcl-2 The amount of expression is reduced.The results of Western blot showed that the change of LC3-I / LC3-II,which is the downstream marker of autophagy,led to a decrease in the expression of LC3-I in LC3-II and inhibited the autophagic expression of P62 protein decreased in the upstream of AMPK / mTOR / ULK1 pathway protein,mTOR phosphorylation of autophagy regulator decreased,and to promote autophagy protein such as phosphorylated AMPK,phosphorylated ULK1,Beclin-1 expression levels have rising.In the cell scratch assay,the migration of SMMC-7721 cells in the control group was more obvious,with more cells in the center of the scratch.With the increase of the concentration of the extracts of P.igniarius,the number of migrated cells in the middle of the scratch compared with the control group was significantly reduced.Conclusion: The extracts of P.igniarius can significantly inhibit human hepatocellular carcinoma SMMC-7721 cells.The molecular mechanism of its anti-tumor effect may be related to a variety of pathways,including inhibiting the proliferation and promoting the apoptosis of hepatocellular carcinoma SMMC-7721 cells autophagic death,inhibition of its migration activity to achieve control of cancercell proliferation and so on.