Toxicity against gastric cancer cells by combined treatment with 5-fluorouracil and mitomycin c: implication in oxidative stress.
OBJECTIVE
Combined chemotherapy of 5-fluorouracil (5FU) and mitomycin c (MMC) is clinically used for gastric cancer, but the precise conditions and molecular mechanism of these agents when used together remain unclear. We examined the administration sequence of combining 5FU with MMC to maximize toxicity against a human gastric cancer cell line, and then investigated the possible molecular mechanisms underlying the observed toxic effects.


METHODS
Human gastric cancer MKN45 cells were treated with a combination of 5FU and MMC, and the changes in cell viability and apoptosis-related proteins were determined by a tetrazolium dye-based cytotoxicity assay and Western blot analysis, respectively. The intracellular levels of reactive oxygen species (ROS) were monitored using a fluorescent probe or by a cytochrome c reduction assay.


RESULTS
Pretreatment for 24 h with 5FU augmented the toxic effect of MMC in MKN45 cells. The synergic effect was mediated mainly via ROS formation and the p53-dependent apoptotic pathway, leading to mitochondrial dysfunction and caspase activation. In vitro experiments using extracts of the treated cells showed superoxide anion generation in a redox cycle of MMC, involving alterations in superoxide dismutase.


CONCLUSIONS
Pretreatment with 5FU enhanced the MMC-induced toxicity against gastric cancer cells via alterations in antioxidant enzymes with resulting ROS generation. This observation will need confirmation in the clinical setting.
Matsunaga
Toshiyuki
T
Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu, 502-8585, Japan. matsunagat@gifu-pu.ac.jp
Tsuji
Yoshitaka
Y
Kaai
Kairyu
K
Kohno
Saki
S
Hirayama
Renzo
R
Alpers
David H
DH
Komoda
Tsugikazu
T
Hara
Akira
A
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D004357:Drug Synergism; D005472:Fluorouracil / Q000008:administration & dosage; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D016685:Mitomycin / Q000008:administration & dosage; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000473:pathology; D013482:Superoxide Dismutase / Q000187:drug effects / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D013481:Superoxides; D016685:Mitomycin; D045304:Cytochromes c; D013482:Superoxide Dismutase; D020169:Caspases; D005472:Fluorouracil