Curcumin Induces Mitochondrial Apoptosis In Human Hepatoma Cells Through Bclaf1-mediated Modulation Of PI3K/AKT/GSK-3β Signaling

Background:Primary liver cancer has a high incidence and has been found to be associated with chemical carcinogenic stimulation,genetic factors,gene mutations,viral liver cancer,soil,water factors,and alcoholic cirrhosis.In recent years,Chinese traditional medicine has become a hot spot in the study of drugs for the treatment of liver cancer.Curcumin extracted from the root of turmeric has minimal toxicity and can inhibit cancer.However,the mechanism of curcumin’s anti-liver cancer action is not completely clear.Bcl-2 associated transcription factor 1(Bclaf1)is a multifunctional protein that plays an important role in gene transcription and post-transcription processing,Bclafl protein is highly expressed in liver cancer tissue proteomics analysis,which can regulate and inhibit the proliferation process of liver cancer cells,accelerate the apoptosis of liver cancer cells,and promote autophagy reaction.As a key pathway of apoptosis,the mitochondrial pathway is highly conserved and mainly regulated by apoptotic genes.Cell activity can be regulated and its growth and proliferation process can be changed.through the PI3K/AKT/GSK-3β pathway,When the pathway is overactivated,cells will be strongly stimulated and abnormal proliferative activities could be caused,that is tumorigenesis.Therefore,this study speculated that curcumin may induce apoptosis of human hepatoma cells through the Bclafl-mediated PI3K/AKT/GSK-3β pathway by the mitochondrial pathway.Objective:To investigate the mechanism of curcumin down-regulating Bclafl and inhibiting PI3K/AKT/GSK-3β pathway to induce apoptosis of human hepatoma cells by mitochondrial pathway in vivo and in vitro.Methods:In vitro experiment:The effects of curcumin(10,20,40,60 μM)and 5FU(10 μM)on the proliferation of HepG2 and SK-Hep-1 cells were determined by CCK-8 assay;the effect of curcumin on the cell cycle of HepG2 and SK-Hep-1 cells was detected by PI single staining;Hoechst33258 staining and Annexin/PI double staining were used to detect the apoptotic effect of curcumin on human hepatoma cells;the effect of curcumin on mitochondrial membrane potential of human hepatoma cells was determined by fluorescence probe method;to verify the effect of curcumin on PI3K/AKT/GSK-3β pathway after the addition of pathway inhibitor LY294002 in human hepatoma cells,Western blot was used to detect the expression levels of PI3K/AKT/GSK-3β pathway-related proteins,apoptosis-related proteins and Bclaf1;the Bclafl gene of HepG2 and SK-Hep-1 was knocked out by CRISPR/Cas9 technique;Annexin/PI double staining,PI single staining and fluorescence probe were used to detect the effect of curcumin on the mitochondrial pathway of Bclafl knockout human hepatoma cells;to verify the mechanism of Bclafl mediated the PI3K/AKT/GSK-3β pathway through mitochondrial pathway in human hepatoma cells,Western blot was used to detect the expression of curcumin on mitochondrial pathway proteins and PI3K/AKT/GSK-3β pathway proteins in Bclafl-knockout human hepatoma cells.In vivo experiment:HepG2 nude mouse transplanted tumor model was constructed and divided into negative control group,positive control group(5FU),curcumin(20 μM)group,curcumin(40 μM)group and curcumin(60 μM)group,the inhibitory effect of curcumin on transplanted tumor in nude mice was determined by calculating tumor volume and growth inhibition rate;H&E staining was used to observe the tissue structure of xenograft in nude mice;immunohistochemistry was used to detect the expression of Bcl-2,Bax and Bclafl in the transplanted tumor tissues of nude mice;the expressions of apoptosis proteins and PI3K/AKT/GSK-3β pathway-related proteins in the transplanted tumor tissues were detected by Western blot.Results:CCK-8 assay showed that curcumin could inhibit the proliferation of HepG2 and SK-Hep-1 cells,and the inhibitory effect was dependent on time and concentration;PI single staining showed that human hepatoma cells were blocked in G0/G1 phase;the results of Hoechst33258 staining and Annexin/PI double staining showed that curcumin promoted apoptosis of HCC cells,and the apoptosis rate increased gradually with the increase of curcumin concentration;the results showed that the mitochondrial membrane potential decreased after curcumin treatment;Western blot analysis revealed apoptosis-related proteins and mitochondrial pathway related proteins:Cleaved caspase-3,Cleaved Caspase-9,Cyt-c were up-regulated,Bcl-2/Bax were down-regulated,and curcumin inhibited the expression of pathway-related proteins PI3K,p-PI3K,AKT,p-AKT,GSK-3β,p-GSK-3β,the addition of pathway inhibitor LY294002 could enhance the inhibitory effect of curcumin on pathway and down-regulate Bcl-2/Bax;Western blot analysis showed that curcumin inhibited the expression of Bclafl protein;HepG2 and SK-Hep-1 cells with stable knockout of Bclafl gene were constructed,and the apoptosis rate of cells was significantly increased after curcumin treatment,the number of cells blocked in G0/G1 phase increased significantly,the expression of Bcl-2/Bax,PI3K/AKT/GSK-3β pathway related proteins was down-regulated,and the expression of Cyt-c was up-regulated;the xenograft tumor model of human hepatoma cell line HepG2 in nude mice was constructed.The experimental results showed that curcumin inhibited the growth of xenograft tumor in nude mice;H&E staining showed that curcumin destroyed the tissue structure of the transplanted tumor;immunohistochemistry and Western blot results showed that curcumin inhibited the expression of Bclafl,Bcl-2 and pathway-related proteins in the transplanted tumor tissues,the expression of Bax increased.Conclusion:Curcumin can induce apoptosis of human hepatoma cells through mitochondrial pathway,and its mechanism is related to inhibition of Bclafl protein expression and PI3K/AKT/GSK-3β pathway.