title: Ethanol extract of Kalopanax septemlobus leaf induces caspase-dependent apoptosis associated with activation of AMPK in human hepatocellular carcinoma cells.
abstract: The Kalopanax septemlobus leaf (Thunb.) Koidz. has been used as a traditional medicine herb for the treatment of various human diseases for hundreds of years. In this study, we investigated the mechanism underlying the inhibitory effects of an ethanol extract of K. septemlobus leaf (EEKS) on proliferation of HepG2 hepatocellular carcinoma cells. For this study, cell viability and apoptosis were evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, DAPI (4,6-diamidino-2-phenylindole) staining, agarose gel electrophoresis, and flow cytometry. Measurements of the mitochondrial membrane potential (MMP), caspase activity assays and western blots were conducted to determine whether HepG2 cell death occurred by apoptosis. Treatment of HepG2 cells with EEKS concentration-dependently reduced cell survival while significantly increasing the ratio of apoptotic cells. EEKS treatment increased the levels of the death receptors (DRs), DR4 and DR5, and activated caspases, as well as promoting proteolytic degradation of poly(ADP-ribose)-polymerase associated with the downregulation of protein expression of members of the inhibitor of apoptosis protein family. Treatment with EEKS also caused truncation of Bid, translocation of pro-apoptotic Bax to the mitochondria, and loss of mitochondrial membrane permeabilization, thereby inducing the release of cytochrome c into the cytosol. However, treatment of HepG2 cells with a pan-caspase inhibitor reversed EEKS-induced apoptosis and growth suppression, indicating that EEKS appears to induce apoptosis though a caspase-dependent mechanism involving both intrinsic and extrinsic apoptotic pathways. In addition, the phosphorylation level of AMP-activated protein kinase (AMPK) was elevated when cells were exposed to EEKS. A specific inhibitor for AMPK attenuated the EEKS-induced activation of caspases, and consequently prevented the EEKS-induced apoptosis and reduction in cell viability. Overall, our findings suggest that EEKS inhibits the growth of HepG2 cells by inducing AMPK-mediated caspase-dependent apoptosis, suggesting the potential therapeutic application of EEKS in the treatment or prevention of cancers.
authors.affiliation: Department of Molecular Biology, College of Natural Sciences and Human Ecology, Dongeui University, Busan 614-714, Republic of Korea.
authors.forename: Cheol
authors.initials: C
authors.lastname: Park
authors.affiliation: Anti-Aging Research Center, Dongeui University, Busan 614-714, Republic of Korea.
authors.forename: Ji-Suk
authors.initials: JS
authors.lastname: Jeong
authors.affiliation: Gurye Wild Flower Institute, Gurye 504-805, Republic of Korea.
authors.forename: Jin-Woo
authors.initials: JW
authors.lastname: Jeong
authors.affiliation: Gurye Wild Flower Institute, Gurye 504-805, Republic of Korea.
authors.forename: Yong-Joo
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Gurye-gun Agricultural Center, Gurye 504-805, Republic of Korea.
authors.forename: Yeon-Kwon
authors.initials: YK
authors.lastname: Jung
authors.affiliation: Gurye-gun Agricultural Center, Gurye 504-805, Republic of Korea.
authors.forename: Geun-Bae
authors.initials: GB
authors.lastname: Go
authors.affiliation: Department of Food Science and Biotechnology, College of Engineering, Kyungsung University, Busan 608-736, Republic of Korea.
authors.forename: Sung Ok
authors.initials: SO
authors.lastname: Kim
authors.affiliation: Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea.
authors.forename: Gi-Young
authors.initials: GY
authors.lastname: Kim
authors.affiliation: Department of Biochemistry, Dongeui University College of Korean Medicine, Busan 614-714, Republic of Korea.
authors.forename: Su-Hyun
authors.initials: SH
authors.lastname: Hong
authors.affiliation: Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan 602-714, Republic of Korea.
authors.forename: Young Hyun
authors.initials: YH
authors.lastname: Yoo
authors.affiliation: Anti-Aging Research Center, Dongeui University, Busan 614-714, Republic of Korea.
authors.forename: Yung Hyun
authors.initials: YH
authors.lastname: Choi
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000096:biosynthesis* / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D061945:Caspase Inhibitors / Q000008:administration & dosage; D020169:Caspases / Q000096:biosynthesis; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D036882:Kalopanax / Q000737:chemistry; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D010936:Plant Extracts / Q000008:administration & dosage* / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry; D051028:bcl-2-Associated X Protein
chemical_list: D061945:Caspase Inhibitors; D010936:Plant Extracts; D051028:bcl-2-Associated X Protein; D055372:AMP-Activated Protein Kinases; D020169:Caspases