Selective inhibition of cyclooxygenase-2 enhances mitomycin-C-induced apoptosis.
OBJECTIVE
Cyclooxygenase-2 (COX-2) is involved in antiapoptosis signaling, and its induction may require activation of protein kinase C (PKC). Safingol (SAF), a PKC inhibitor, has been shown to enhance apoptosis induced by mitomycin-C (MMC) in human gastric cancer MKN-74 cells. The aim of this study was to identify the role of COX-2 in MMC-induced apoptosis in MKN-74 cells.


METHODS
Protein expression of COX-2 and Bcl-2 and activation of PKCalpha were examined by Western blot analysis. Apoptosis induction was examined by staining with bisbenzimide trihydrochloride (Hoechst-33258) of condensed chromatin, which characterizes the cells undergoing apoptosis. COX-2 mRNA levels were examined by Northern blot analysis.


RESULTS
After exposure for 1-2 h to 1 microg/ml MMC, upregulation of COX-2 and Bcl-2 protein expression was noted. The activation of PKCalpha occurred within 1 h of MMC exposure, and temporally preceded the induction of COX-2. Similar results were observed in cells exposed to the PKC activator, 3-phorbol 12-myristate 13-acetate. Cotreatment with SAF and MMC abolished the induction of COX-2 by MMC. Furthermore, NS-398, a selective COX-2 inhibitor, significantly enhanced MMC-induced apoptosis by fivefold from 4 +/- 2% (MMC alone) to 20 +/- 2% (MMC plus NS-398). There was no discernible change in COX-2 mRNA levels after a 2-h exposure to MMC but a twofold increase after a 24-h exposure.


CONCLUSIONS
MMC upregulates COX-2 expression, which appears to be an antiapoptotic signal downstream of PKC. Selective inhibition of COX-2 can therefore provide a novel way to enhance MMC-induced apoptosis independent of inhibiting PKC.
Hsueh
C T
CT
Department of Internal Medicine, China Medical College Hospital, Taichung, Taiwan. d7198@hpd.cmch.org.tw
Chiu
C F
CF
Kelsen
D P
DP
Schwartz
G K
GK
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D004357:Drug Synergism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000502:physiology*; D008565:Membrane Proteins; D016685:Mitomycin / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000903:Antibiotics, Antineoplastic; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D016685:Mitomycin; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate