Activation of Neocarzinostatin by NADPH/Cytochrome P-450 Reductase: Proposal of an Ultimate Mode of Action in Cells Involving Oxygen Free Radicals

Abstract

Neocarzinostatin (NCS), a potent proteinaceous anticancer agent, is a single-chain polypeptide (MW, 11 700) with two disulfide bonds that contains a nonprotein chromophore (NPC) with a unique enediyne structure as described elsewhere in this volume. In this chapter, a new activation mechanism of NCS was explored by analyzing oxygen radical generation from NCS in a reduced nicotinamide adenine dinucleotide (NADPH)/cytochrome (cyt) P-450 reductase system. Electron spin resonance (ESR) spin-trapping was performed to identify oxygen radicals such as superoxide (O2·−) and hydroxyl radical (OH·) produced in the reaction mixture with NCS + NADPH/cyt P-450 reductase in the presence of NADPH. 5,5-Dimethyl-l-pyrroline N-oxide (DMPO) was used as a spin-trap for ESR study. Based on the Lineweaver—Burk plot of the DMPO-OOH, a spin adduct of O2·− generated in the reaction system against the concentration of NCS, mitomycin C, and adriamycin, K m values of cyt P-450 reductase for these antitumor agents were determined to be 2.89 × l0−5 M, 4.46 × 10−6 M, and 1.82 × 10−6 M, respectively. The K m value for NCS was comparable to those of mitomycin C and adriamycin. The most important observation in this study is that DNA cleavage of plasmid pBR322 occurred most effectively in the system with picomolar (10−12 M) concentrations of NCS in the presence of NADPH/cyt P-450 reductase system as revealed by agarose gel electrophoresis of the plasmid DNA, whereas at least 10−5~10−6 M of NCS is needed without NADPH/P450 reductase system. These results indicate the importance of the NADPH/cyt P-450 reductase system in activation of NCS, and the present mechanism may explain the potent cytotoxic action of NCS in cells because NADPH/cyt P-450 is a ubiquitous cellular component.