2-Methoxyestradiol-induced caspase-3 activation and apoptosis occurs through G(2)/M arrest dependent and independent pathways in gastric carcinoma cells.
BACKGROUND
2-Methoxyestradiol (2-Me), one of the estrogen metabolites, has recently been found to possess anti-angiogenesis activity in vivo. Many chemotherapeutic agents, such as taxol, docetaxel, and vinblastine, interact with microtubules and then induce apoptosis. It has been suggested that 2-Me acts on microtubules and results in G(2)/M-cycle arrest of tumor cells. Whether 2-Me induces apoptosis in gastric carcinoma cell lines is not known. Moreover, reactive oxygen species (ROS) produced by 2-Me may be involved in cytotoxicity of tumor cells. Thus, another objective of this study was to evaluate the relation between cell cycle arrest, ROS formation, and caspase activity levels after 2-Me treatment in gastric carcinoma cells.


METHODS
It was determined whether 2-Me directly induced apoptosis in two gastric carcinoma cell lines (SC-M1 and NUGC-3) through caspase-3 and caspase-8 activation and, eventually, induced DNA fragmentation. To clarify the effect of 2-Me-induced G(2)/M arrest in apoptosis, calcium ionophore, A23187, and thapsigargin were used to modulate 2-Me-induced cell cycle responses. Moreover, the role of 2-Me-induced ROS formation in the cell cycle responses also was evaluated.


RESULTS
It was found that 2-Me treatment resulted in G(2)/M-cycle arrest, caspase-8 and caspase-3 activation, and DNA fragmentation. In addition, the 2-Me induced, concomitant increases of peroxide and superoxide anions were correlated with G(2)/M-cycle arrest. Treatment with calcium ionophore A23187 and thapsigargin partially reversed the 2-Me-induced G(2)/M-cycle arrest, with a concomitant decrease in both peroxide and superoxide levels. Moreover, A23187 blocked the 2-Me-induced caspase-3 activation, whereas thapsigargin had no effect. Treatment with calcium channel blockers did not affect 2-Me-induced cell cycle arrest or caspase-3 activation.


CONCLUSIONS
These results suggest that the 2-Me-induced apoptosis of gastric carcinoma cells by DNA fragmentation accompanied caspase activation. Elevation of free radicals was associated with G(2)/M-cycle arrest. The induction of G(2)/M-cycle arrest is not a prerequisite for caspase activation.
Lin
H L
HL
Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan, Republic of China.
Liu
T Y
TY
Wu
C W
CW
Chi
C W
CW
D000077584:2-Methoxyestradiol; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D053938:DNA Fragmentation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology*; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D008938:Mitosis / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013274:Stomach Neoplasms / Q000473:pathology*; D014407:Tumor Cells, Cultured
D017382:Reactive Oxygen Species; D004958:Estradiol; D000077584:2-Methoxyestradiol; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases