title: Bax overexpression enhances cytochrome c release from mitochondria and sensitizes KATOIII gastric cancer cells to chemotherapeutic agent-induced apoptosis.
abstract: To evaluate whether overexpression of Bax, an apoptosis-promoting gene, sensitizes KATOIII gastric cancer cells to apoptosis induced by chemotherapeutic agents, three stable cell lines of KATOIII transfected with Bax (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, zVAD-fmk, a pancaspase inhibitor, repressed the paclitaxel-induced apoptosis. In addition, Bcl-2 overexpression strongly blocked KATOIII cell apoptosis by inhibiting the cytochrome c release from 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.
authors.affiliation: Second Department of Internal Medicine, Mie University School of Medicine, Tsu 514-8507, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Sawa
authors.forename: T
authors.initials: T
authors.lastname: Kobayashi
authors.forename: K
authors.initials: K
authors.lastname: Mukai
authors.forename: W
authors.initials: W
authors.lastname: Zhang
authors.forename: H
authors.initials: H
authors.lastname: Shiku
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D003574:Cytochrome c Group / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000970:Antineoplastic Agents; C494826:BAX protein, human; D003574:Cytochrome c Group; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone