title: Supercritical carbon dioxide extraction of aromatic turmerone from Curcuma longa Linn. induces apoptosis through reactive oxygen species-triggered intrinsic and extrinsic pathways in human hepatocellular carcinoma HepG2 cells.
abstract: The mechanisms underlying the antiproliferative and antitumor activities of aromatic turmerone (ar-turmerone), a volatile turmeric oil isolated from  Curcuma longa  Linn., have been largely unknown. In this study, 86% pure ar-turmerone was extracted by supercritical carbon dioxide and liquid-solid chromatography and its potential effects and molecular mechanisms on cell proliferation studied in human hepatocellular carcinoma cell lines. Ar-turmerone exhibited significant antiproliferative activity, with 50% inhibitory concentrations of 64.8 ± 7.1, 102.5 ± 11.5, and 122.2 ± 7.6 μg/mL against HepG2, Huh-7, and Hep3B cells, respectively. Ar-turmerone-induced apoptosis, confirmed by increased annexin V binding and DNA fragmentation, was accompanied by reactive oxygen species (ROS) production, mitochondrial membrane potential dissipation, increased Bax and p53 up-regulated modulator of apoptosis (PUMA) levels, Bax mitochondrial translocation, cytochrome c release, Fas and death receptor 4 (DR4) augmentation, and caspase-3, -8, and -9 activation. Exposure to caspase inhibitors, Fas-antagonistic antibody, DR4 antagonist, and furosemide (a blocker of Bax translocation) effectively abolished ar-turmerone-triggered apoptosis. Moreover, ar-turmerone stimulated c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) phosphorylation and activation; treatment with JNK and ERK inhibitors markedly reduced PUMA, Bax, Fas, and DR4 levels and reduced apoptosis but not ROS generation. Furthermore, antioxidants attenuated ar-turmerone-mediated ROS production; mitochondrial dysfunction; JNK and ERK activation; PUMA, Bax, Fas, and DR4 expression; and apoptosis. Taken together, these results suggest that ar-turmerone-induced apoptosis in HepG2 cells is through ROS-mediated activation of ERK and JNK kinases and triggers both intrinsic and extrinsic caspase activation, leading to apoptosis. On the basis of these observations, ar-turmerone deserves further investigation as a natural anticancer and cancer-preventive agent.
authors.affiliation: Division of General Surgery, Department of Surgery,  Taichung Veterans General Hospital , Taiwan.
authors.forename: Shao-Bin
authors.initials: SB
authors.lastname: Cheng
authors.forename: Li-Chen
authors.initials: LC
authors.lastname: Wu
authors.forename: Yun-Chih
authors.initials: YC
authors.lastname: Hsieh
authors.forename: Chi-Hao
authors.initials: CH
authors.lastname: Wu
authors.forename: Yu-Ju
authors.initials: YJ
authors.lastname: Chan
authors.forename: Li-Hsun
authors.initials: LH
authors.lastname: Chang
authors.forename: Chieh-Ming J
authors.initials: CM
authors.lastname: Chang
authors.forename: Shih-Lan
authors.initials: SL
authors.lastname: Hsu
authors.forename: Chieh-Lin
authors.initials: CL
authors.lastname: Teng
authors.forename: Chun-Chi
authors.initials: CC
authors.lastname: Wu
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D002245:Carbon Dioxide / Q000737:chemistry; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D025924:Chromatography, Supercritical Fluid / Q000379:methods*; D030024:Curcuma / Q000737:chemistry*; D045304:Cytochromes c / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D053222:Fas Ligand Protein / Q000378:metabolism; D056945:Hep G2 Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007659:Ketones / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012717:Sesquiterpenes / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C438378:BBC3 protein, human; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D007659:Ketones; D011518:Proto-Oncogene Proteins; D017382:Reactive Oxygen Species; D012717:Sesquiterpenes; D051028:bcl-2-Associated X Protein; D002245:Carbon Dioxide; C078098:ar-turmerone; D045304:Cytochromes c; D048049:Extracellular Signal-Regulated MAP Kinases; D048670:MAP Kinase Kinase 4; D020169:Caspases