Tyrosine-nitration of caspase 3 and cytochrome c does not suppress apoptosis induction in squamous cell carcinoma cells.
The influence of tyrosine nitration of cytochrome c and caspase 3 on apoptosis induction was investigated in an established squamous carcinoma cell line, OSC-4. The intracellular NO and O2(-) levels were increased up to about 110-120% and 140-180% of the control levels, respectively, after the treatment of OSC-4 cells with 5-FU (100 microg/ml), PLM (10 microg/ml), CDDP (10 microg/ml), or gamma-rays (20 Gy). The treatment of OSC-4 cells with ONOO(-) (1 mM) and the above anticancer agents induced tyrosine nitration of 14, 32 kDa protein among others and nitration of tyrosine residues of cytochrome c and caspase 3 was identified by the Western blotting of immunoprecipitates obtained by antibodies to these proapoptotic proteins. When cytochrome c and procaspase 3 were treated with ONOO(-), tyrosine nitration was increased in a ONOO(-)-dose dependent manner. Tyrosine nitration of cleaved (17 kDa) caspase 3, however, was not induced by ONOO(-). Procaspase 3 in the cytosol of HeLa cells was activated by the addition of ONOO(-)-treated as well as ONOO(-)-untreated cytochrome c. In addition, cleavage of ICAD and PARP were not suppressed in OSC-4 cells by pretreatment with ONOO(-). Activity of cleaved caspase 3 was not suppressed at low concentrations or by treatment with ONOO(-) or NO donors, SIN-1 and SNP. Furthermore, apoptosis of OSC-4 cells by the anticancer agents was not suppressed by ONOO(-). In conclusion, these results suggest that nitration of tyrosine residues of cytochrome c and procaspase 3 is induced by chemoradiotherapy but their nitration does not suppress cancer cell apoptosis.
Ueta
Eisaku
E
Department of Oral Surgery, Kochi Medical School, Kohasu, Oko-cho, Nankoku-city, Kochi, Japan. uetaei@kochi-ms.ac.jp
Kamatani
Takaaki
T
Yamamoto
Tetsuya
T
Osaki
Tokio
T
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000196:embryology / Q000473:pathology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D003574:Cytochrome c Group / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D006367:HeLa Cells / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D009062:Mouth Neoplasms / Q000201:enzymology / Q000473:pathology*; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D015538:Nitrosation; D030421:Peroxynitrous Acid / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011233:Precipitin Tests; D013481:Superoxides / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000473:pathology / Q000528:radiation effects; D014443:Tyrosine / Q000378:metabolism*
D000970:Antineoplastic Agents; D003574:Cytochrome c Group; D004792:Enzyme Precursors; D020030:Nitric Oxide Donors; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D014443:Tyrosine; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases