A new mechanism of action for the anticancer drug mitomycin C: mechanism-based inhibition of thioredoxin reductase.
Mitomycin C (MMC) is a chemotherapeutic drug that requires an enzymatic bioreduction to exert its biological effects. Upon reduction, MMC is converted into a highly reactive bis-electrophilic intermediate that alkylates cellular nucleophiles. Alkylation of DNA is the most favored mechanism of action for MMC, but other modes of action, such as redox cycling and inhibition of rRNA, may also contribute to the biological action of the drug. In this work, we show that thioredoxin reductase (TrxR) is also a cellular target for MMC. We show that MMC inhibits TrxR in vitro, using purified enzyme, and in vivo, using cancer cell cultures. The inactivation presents distinctive parameters of mechanism-based inhibitors: it is time- and concentration-dependent and irreversible. Additionally, spectroscopic experiments (UV, circular dichroism) show that the inactivated enzyme contains a mitosene chromophore. On the basis of kinetic and spectroscopic data, we propose a chemical mechanism for the inactivation of the enzyme that starts with a reduction of the quinone ring of MMC by the selenolthiol active site of TrxR and a subsequent alkylation of the active site by the activated drug. We also report that MMC inactivates TrxR in cancer cell cultures and that this inhibition correlates directly with the cytotoxicity of the drug, indicating that inhibition of TrxR may play a major role in the biological mode of action of the drug.
Department of Medical Biochemistry and Biophysics, Division of Biochemistry, Karolinska Institutet, SE-17177 Stockholm, Sweden. manuel.paz@usc.es
Manuel M
MM
Paz
Xu
X
Zhang
Jun
J
Lu
Arne
A
Holmgren
D000477:Alkylating Agents / Q000737:chemistry / Q000378:metabolism*; D000478:Alkylation; D000818:Animals; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016685:Mitomycin / Q000737:chemistry / Q000378:metabolism*; D009249:NADP / Q000737:chemistry; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000477:Alkylating Agents; D011994:Recombinant Proteins; D016685:Mitomycin; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase