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Mode of Action of the Bioreductive Alkylating Agent, 2,3-Bis(chloromethyl)-1,4-naphthoquinone¹

Department of Pharmacology and Section of Developmental Therapeutics, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06510 [L. A. C., A. J. L., Y. M. H., K. M. H., A. C. S.], and Allie M. Lee Laboratory for Cancer Research, Division of Biochemistry, University of Nevada, Reno, Nevada 89507 [R. S. P., R. E. B., T. L. L.]

The bioreductive alkylating agent, 2,3-bis(chloromethyl)-1,4-naphthoquinone (CMNQ), has been shown to inhibit the growth of Sarcoma 180 ascites cells in vivo. Evidence for the reductive activation of this agent via the mitochondrial respiratory chain was provided by CMNQ-induced oxidation of reduced nicotinamide adenine dinucleotide; the interaction was shown to be on the substrate side of the site of rotenone inhibition. Consistent with the concept that reduction of CMNQ to a hydroquinone results in the generation of an alkylating species (i.e., a quinone methide) was the finding that radioactivity from [¹⁴C]CMNQ present in Sarcoma 180 ascites cells was associated with DNA, RNA, and protein for a period of up to 72 hr after exposure of tumorbearing animals to this agent. Inhibition of the incorporation of [³H]thymidine, [³H]uridine, and [¹⁴C]leucine into DNA, RNA, and protein, respectively, of Sarcoma 180 ascites cells was produced by this agent, with DNA biosynthesis being the most susceptible. The inhibitory effect of CMNQ on the formation of DNA was, at least in part, the result of a prevention of the conversion of thymidine to its nucleotide forms. This action was due to (a) a drug-induced decrease in intracellular levels of adenosine 5'-triphosphate, presumably resulting from uncoupling of oxidative phosphorylation by CMNQ; and (b) a partial loss of thymidine kinase activity in Sarcoma 180 cells, which did not appear to be due to direct inhibition of the enzyme by the drug. Although the primary event produced by CMNQ at the mitochondrial level appeared to be release of respiratory control, other effects on mitochondrial metabolism occurred. These included inhibition of reduced nicotinamide adenine dinucleotide and succinoxidase activities, as previously demonstrated, and mitochondrial swelling, which suggested interaction of CMNQ with the inner mitochondrial membrane. These findings indicate a variety of biochemical lesions are associated with the antineoplastic activity of CMNQ and demonstrate a relationship between the effects of this drug on mitochondrial respiratory control and DNA biosynthesis.

¹ This research was supported in part by USPHS Research Grants CA-02817 and CA-16359 from the National Cancer Institute.

Received 6/ 7/76. Accepted 7/29/76.