Isoobtusilactone A-induced apoptosis in human hepatoma Hep G2 cells is mediated via increased NADPH oxidase-derived reactive oxygen species (ROS) production and the mitochondria-associated apoptotic mechanisms.
Chemoprevention by the use of naturally occurring substances is becoming a promising strategy to prevent cancer. In this study, the effects of isoobtusilactone A, a novel constituent isolated from the leaves of Cinnamomum kotoense, on the proliferation of human hepatoma Hep G2 cells were studied. Under our experimental conditions, isoobtusilactone A was found to elicit a concentration-dependent growth impediment (IC(50)=37.5 microM). The demise of these cells induced by isoobtusilactone A was apoptotic in nature, exhibiting a concentration-dependent increase in sub-G(1) fraction and DNA fragmentation. Subcellular fractionation analysis further revealed that Bax translocation to mitochondria resulted in a rapid release of cytochrome c, followed by activation of caspase 3 and PARP cleavage, and finally cell death. Isoobtusilactone A-treated cells also displayed transient increase of ROS during the earlier stage of the experiment, followed by the disruption of mitochondrial transmembrane potential (DeltaPsi(m)). The presence of a ROS scavenger (N-acetyl-L-cysteine) and an inhibitor of NADPH oxidase (diphenyleneiodonium chloride) blocked ROS production and the subsequent apoptotic cell death. In addition, in order to investigate the acute toxicity of isoobtusilactone A, groups of 5-6-week old Sprague-Dawley rats were subjected to oral administration of 350, or 700 mg/kg bw isoobtusilactone A four times each week for two weeks. There was no significant difference between control animals and treated animals with respect to the body weight gain, the body weight ratio of liver, spleen and kidney, haematological and clinical chemistry parameters. Taken together, our data suggest that ROS generated through the activation of NADPH oxidase plays an essential role in apoptosis induced by isoobtusilactone A, and the dosages of isoobtusilactone A tested in this study did not cause animal toxicity.
Chen
Chung-Yi
CY
Basic Medical Science Education Center, Fooyin University, Ta-Liao, Kaohsiung Hsien, Taiwan, ROC. xx377@mail.fy.edu.tw
Liu
Tsan-Zon
TZ
Chen
Ching-Hsein
CH
Wu
Chih-Chung
CC
Cheng
Jiin-Tsuey
JT
Yiin
Shuenn-Jiun
SJ
Shih
Ming-Kuei
MK
Wu
Mei-Jem
MJ
Chern
Chi-Liang
CL
D000284:Administration, Oral; D000473:Alkanes / Q000494:pharmacology* / Q000627:therapeutic use; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology* / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D027424:Cinnamomum / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D007783:Lactones / Q000494:pharmacology* / Q000627:therapeutic use; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D008564:Membrane Potentials / Q000187:drug effects; D008930:Mitochondria, Liver / Q000473:pathology*; D019255:NADPH Oxidases / Q000378:metabolism*; D010936:Plant Extracts / Q000494:pharmacology / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*
D000473:Alkanes; D000972:Antineoplastic Agents, Phytogenic; D007783:Lactones; D010936:Plant Extracts; D017382:Reactive Oxygen Species; C521340:isoobtusilactone A; D019255:NADPH Oxidases