Bax overexpression enhances cytochrome c release from mitochondria and sensitizes KATOIII gastric cancer cells to chemotherapeutic agent-induced apoptosis

To evaluate whether overexpression of Box, an apoptosis-promoting gene, sensitizes KATOIII gastric cancer cells to apoptosis induced by chemotherapeutic 'agents, three stable cell lines of KATOIII transfected with Box (KATOIII-Bax), Bcl-2 (KATOIII-Bcl-2), or control pCI-neo expression vector (KATOIII-pCI-neo) were established. The cells were treated with paclitaxel, 5-fluorouracil, or doxorubicin, and the apoptotic response was measured. Our results showed that the sensitivity of the KATOIII-Bax cells to chemotherapeutic agents was enhanced compared with that of the KATOIII-pCI-neo cells, and the KATOIII-Bcl-2 cells were more resistant to these agents. Western blotting revealed that cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was higher than that of the KATOIII-pCI-neo cells. Significant increase of cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was detected 24 h after exposure to chemotherapeutic agents, when apoptotic cells were less than 10%. The cytochrome c level in the cytosol fraction of the KATOIII-Bax cells was higher than that of the KATOIII-pCI-neo cells at all time points examined after exposure to chemotherapeutic agents. Marked activation of caspase-3 in the KATOIII-Bax cells was observed 48 h and 72 h after exposure to chemotherapeutic agents compared with that in the KATOIII-pCI-neo cells. Consistently, zVAn-fmk, a pancaspase inhibitor, repressed the paclitaxel-induced apoptosis. In addition, Bcl-2 overexpression strongly blocked KATOIII cell apoptosis by inhibiting the cytochrome c releasefrom mitochondria and caspase-3 activation. These findings suggest that cytochrome c release is a major mechanism of apoptotic response and Bax overexpression sensitizes KATOIII cells to chemotherapeutic agent-induced apoptosis through enhancing the release of cytochrome c from mitochondria.