title: Methyl antcinate A from Antrodia camphorata induces apoptosis in human liver cancer cells through oxidant-mediated cofilin- and Bax-triggered mitochondrial pathway.
abstract: We investigated the effects of antcin A, antcin C, and methyl antcinate A (MAA) isolated from Antrodia camphorata on the proliferation of human liver cancer cell lines Huh7, HepG2, and Hep3B and the normal cell rat hepatocytes. The three compounds selectively inhibit the proliferation of tumor cells rather than normal cells, with IC(50) values ranging from 30.2 to 286.4 microM. The compound MAA was a more potent cytotoxic agent than antcins A and C with IC(50) values of 52.2, 78.0, and 30.2 microM against HepG2, Hep3B, and Huh7 cells, respectively. To elucidate the molecular mechanism, treatment of Huh7 cells with 100 microM MAA induced an apoptotic cell death, which was characterized by the appearance of sub-G1 population, DNA fragmentation, TUNEL positive cells, and caspase activation. MAA triggered the mitochondrial apoptotic pathway, as indicated by an increase in the protein expression of Bax, Bak, and PUMA, as well as a decrease in Bcl-(XL) and Bcl-2 and disruption of mitochondrial membrane potential and promotion of mitochondrial cytochrome c release, as well as activation of caspases-2, -3, and -9. We also found that pretreatment with inhibitors of caspases-2, -3, and -9 noticeably blocked MAA-triggered apoptosis. Furthermore, intracellular reactive oxygen species (ROS) generation and NADPH oxidase activation were observed in MAA-stimulated Huh7 cells. Mechanistic studies showed that MAA induces mitochondrial translocation of cofilin. When Huh7 cells were treated with cyclosporine A and bongkrekic acid, an inhibitor of the mitochondria permeability transition pore, the levels of cell death induced by MAA were significantly attenuated. Additionally, pretreatment of Huh7 cells with antioxidants ascorbic acid and N-acetyl cysteine markedly attenuated the MAA-induced apoptosis by upregulation of Bax, Bak, and PUMA, mitochondrial translocation of cofilin, activation of caspase-3, and cell death. Taken together, our results provide the first evidence of the activation of the ROS-dependent cofilin- and Bax-triggered mitochondrial pathway as a critical mechanism of MAA-induced cell death in liver cancer cells.
authors.affiliation: Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan, Republic of China.
authors.forename: Yun-Chih
authors.initials: YC
authors.lastname: Hsieh
authors.forename: Yerra Koteswara
authors.initials: YK
authors.lastname: Rao
authors.forename: Chun-Chi
authors.initials: CC
authors.lastname: Wu
authors.forename: Chi-Ying F
authors.initials: CY
authors.lastname: Huang
authors.forename: Madamanchi
authors.initials: M
authors.lastname: Geethangili
authors.forename: Shih-Lan
authors.initials: SL
authors.lastname: Hsu
authors.forename: Yew-Min
authors.initials: YM
authors.lastname: Tzeng
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000627:therapeutic use / Q000633:toxicity*; D055491:Antrodia / Q000737:chemistry*; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D053143:Caspase 2 / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D051338:Cofilin 1 / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D014315:Triterpenes / Q000737:chemistry / Q000627:therapeutic use / Q000633:toxicity*; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; D051338:Cofilin 1; D016877:Oxidants; D011518:Proto-Oncogene Proteins; D017382:Reactive Oxygen Species; D014315:Triterpenes; D051028:bcl-2-Associated X Protein; C553655:methyl antcinate A; D045304:Cytochromes c; D019255:NADPH Oxidases; D053143:Caspase 2; D053148:Caspase 3; D053453:Caspase 9