| Objective: To study the inhibitory effect of Cinobufagin(CINO)and its molecular mechanism on human hepatocellular carcinoma cell line.Methods: Human hepatocellular carcinoma cell line SK-Hep1 was cultured and treated with CINO.(1)Cell proliferation was detected by CCK8 and cell clonal formationassay.(2)Flow cytometry was used to analyze the cell cycle changes after CINO treatment.(3)Western blot was used to analyze the expression of the Cyclin d1,P27,c-PARP?c-Caspase3,p-AKT,p-P70S6 k,,p-S6,p-e IF2?,ATF4,?H2AX after CINO treatment.Using TUN as a positive control,the activation shearing of XBP1 was detected by PCR.(4)After transfection with e IF2?-S51 A plasmid and si-e IF2? RNA,it was combined with CINO to analyze the expression of c-PARP and e IF2? pathway related proteins by Western blot.(5)Through AKT/m TOR inhibitor LY294002,transfection of AKT activated plasmid with CINO,Western blot was used to analyze the expression of c-PARP and AKT/m TOR pathway related proteins.Results:(1)CINO inhibited the proliferation of human hepatocellular carcinoma cell line SK-Hep1: After treated with CINO,CCK8 and cell clone formation assay showed that the proliferation of cells treated with CINO was inhibited.(2)CINO blocks the cell cycle of SK-Hep1: Flow cytometry results showed that the cell cycle of hepatocellular carcinoma cell was blocked at G0/G1 phase after CINO treatment.(3)CINO affects cell cycle by down-regulating Cyclin d1 and up-regulating P27 protein expression in SK-Hep1: Western Blot assay was performed to detect the expressions of cell cycle-associated protein Cyclin d1 and P27.Compared with the control group,the Cyclin d1 protein was down-regulated and the P27 protein was up-regulated after CINO treatment.(4)CINO induced apoptosis of SK-Hep1: Western Blot assay was performed to detect the expression of c-PARP and c-Caspase3.Compared with the control group,the abundance of c-PARP and c-Caspase3 were increased after CINO treatment.(5)CINO activates e IF2? pathway in SK-Hep1: Western Blot assay was performed to detects e IF2? pathway related protein expression,compared with the control group,the abundance of p-e IF2?and ATF4 increased after CINO treatment.The m RNA level of XBP1 was detected by PCR,and compared to the TUN group,the CINO treatment group did not show the activation band of XBP1.(6)Inhibiting the phosphorylation of e IF2? attenuates the apoptosis of human hepatocellular carcinoma cell SK-Hep1 induced by CINO: Transfection of e IF2?-S51 A plasmid and si-e IF2?RNA in combination with CINO,Western blot analysis showed that the expression of c-PARP protein was down-regulated in the transfection + CINO group compared with the single treatment group.(7)CINO inhibits the AKT/m TOR pathway in SK-Hep1: The expression of AKT/m TOR pathway-related proteins(p-AKT,p-P70S6 K,p-S6)was detected by Western Blot.Compared with the control group,CINO treatment down-regulated the expression level of AKT/m TOR pathway-related proteins.(8)Inhibition of AKT/m TOR pathway enhances apoptosis of SK-Hep1 induced by CINO: After LY294002 inhibited the AKT/m TOR pathway,Western Blot analysis showed that the expression of PARP was significantly increased in the combined group compared with the single treatment group.(9)Activation of AKT/m TOR pathway attenuated CINO-induced apoptosis in SK-Hep1: Western Blot analysis showed that the abundance of PARP in AKT+CINO combination group was significantly decreased than that in the single treatment group.(10)CINO induced DNA damage of cell SK-Hep1: Western blot assay detected the abundance of DNA damage bio-marker ?H2AX,compared with the control group,was increased after CINO treatment.Conclusion: CINO blocks the cell cycle of human hepatocellular carcinoma cell line SK-Hep1 in G0/G1 phase by regulating the expression of Cyclin d1 and P27 proteins,thereby inhibiting the proliferation of SK-Hep1;CINO induces the apoptosis of human hepatocellular carcinoma cell SK-Hep1 by activating e IF2?/ATF4 pathway,inhibiting AKT/m TOR pathway,and inducing DNA damage. |
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