2'-epi-2'-O-Acetylthevetin B extracted from seeds of Cerbera manghas L. induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.
2'-epi-2'-O-Acetylthevetin B (GHSC-74) is a cardiac glycoside isolated from the seeds of Cerbera manghas L. We have demonstrated that GHSC-74 reduced the viability of HepG2 cells in a time- and dose-dependent manner. The present study was designed to explore cellular mechanisms whereby GHSC-74 led to cell cycle arrest and apoptosis in HepG2 cells. Cell cycle flow cytometry demonstrated that HepG2 cells treated with GHSC-74 (4microM) resulted in S and G2 phase arrest in a time-dependent manner, as confirmed by mitotic index analysis. G2 phase arrest was accompanied with down-regulation of CDC2 and Cyclin B1 protein. Furthermore, GHSC-74-induced apoptotic killing, as demonstrated by DNA fragmentation, DAPI staining, and flow cytometric detection of sub-G1 DNA content in HepG2 cells. GHSC-74 treatment resulted in a significant increase in reactive oxygen species, activation of caspase-9, dissipation of mitochondrial membrane potential, and translocation of apoptosis-inducing factor (AIF) from the mitochondrion to the nucleus in HepG2 cells. Nevertheless, after GHSC-74 exposure, no significant Fas and FasL up-regulation was observed in HepG2 cells by flow cytometry. In addition, treatment with antioxidant N-acetyl-l-cysteine (NAC) and broad-spectrum caspase inhibitor z-VAD-fmk partially prevented apoptosis but did not abrogate GHSC-74-induced nuclear translocation of AIF. In conclusion, we have demonstrated that GHSC-74 inhibited growth of HepG2 cells by inducing S and G2 phase arrest of the cell cycle and by triggering apoptosis via mitochondrial disruption including both caspase-dependent and -independent pathways, and ROS generation.
Feng
Bo
B
Department of Biochemistry and Molecular Biology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, PR China.
Guo
Yue-Wei
YW
Huang
Cai-Guo
CG
Li
Liang
L
Chen
Ruo-Hua
RH
Jiao
Bing-Hua
BH
D000111:Acetylcysteine / Q000494:pharmacology; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D029062:Apocynaceae / Q000737:chemistry*; D017209:Apoptosis; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy*; D002301:Cardiac Glycosides / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D053453:Caspase 9 / Q000378:metabolism; D056744:Cyclin B1 / Q000378:metabolism; D016195:G2 Phase; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D016196:S Phase; D012639:Seeds / Q000737:chemistry
C545239:2'-epi-2'-O-acetylthevetin B; D000590:Amino Acid Chloromethyl Ketones; D000970:Antineoplastic Agents; D051033:Apoptosis Inducing Factor; D002301:Cardiac Glycosides; D056744:Cyclin B1; D017382:Reactive Oxygen Species; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D016203:CDC2 Protein Kinase; D053453:Caspase 9; D000111:Acetylcysteine