The natural diterpenoid ovatodiolide induces cell cycle arrest and apoptosis in human oral squamous cell carcinoma Ca9-22 cells.
OBJECTIVE
Oral squamous cell carcinoma (OSCC) is a common worldwide malignancy and there has been little improvement in survival rates in recent decades. Ovatodiolide, a diterpenoid from a Chinese herb, has been reported to exhibit cytotoxicity against several human cancer cell lines. In the present study, the mechanism of action of ovatodiolide was further investigated in the p53 mutant OSCC cell line Ca9-22.


METHODS
The effect of ovatodiolide on cell viability was examined by MTT assay. Cell cycle analysis, DNA fragmentation, and reactive oxygen species (ROS) were investigated by flow cytometry. Caspases and other regulatory molecules were studied by Western blotting.


RESULTS
Treatment of Ca9-22 cells with ovatodiolide led to cell cycle arrest at G2/M phase. Ovatodiolide treatment also induced apoptosis, as indicated by caspase activation, DNA fragmentation, and poly (ADP-ribose) polymerase (PARP) cleavage. By using specific inhibitors of caspase-9 and -8, we demonstrated that ovatodiolide-induced apoptosis is dependent on both intrinsic and extrinsic pathways. The action of ovatodiolide was correlated with a rapid and sustained increase in ROS production and down-regulation of FLICE inhibitory protein (FLIP), which is an endogenous caspase-8 inhibitor and is sensitive to intracellular redox status. Pretreatment of Ca9-22 cells with N-acetylcysteine, a thiol antioxidant, abolished all of ovatodiolide-induced effects, including ROS generation, down-regulation of FLIP, caspase activation, apoptosis as well as cell cycle arrest.


CONCLUSIONS
Our results suggest that ovatodiolide causes cell cycle arrest and apoptosis in Ca9-22 cells through disturbance of intracellular redox balance. Furthermore, ovatodiolide may serve as a lead compound for developing new anticancer drugs.
Hou
Yu-Yi
YY
Department of Otorhinolaryngology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.
Wu
Mu-Ling
ML
Hwang
Yu-Chun
YC
Chang
Fang-Rong
FR
Wu
Yang-Chang
YC
Wu
Chin-Chung
CC
D000972:Antineoplastic Agents, Phytogenic; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004224:Diterpenes / Q000633:toxicity*; D004789:Enzyme Activation / Q000187:drug effects; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D009062:Mouth Neoplasms / Q000188:drug therapy* / Q000473:pathology; D018384:Oxidative Stress / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism; D013778:Tetrazolium Salts; D013844:Thiazoles
D000972:Antineoplastic Agents, Phytogenic; D004224:Diterpenes; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D013844:Thiazoles; C432447:ovatodiolide; D020169:Caspases; C022616:thiazolyl blue