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.