Search

DT-Diaphorase catalyzes a two-electron reduction of mitomycin C (MC) and porfiromycin (POR) to reactive species. Many cell lines that overexpress DT-diaphorase and are sensitive to the mitomycins are protected from the aerobic cytotoxicity of these drugs by the DT-diaphorase inhibitor dicumarol. The cytoprotective properties of this relatively non-specific inhibitor, however, vanish under hypoxic conditions. To ascertain the role of DT-diaphorase in mitomycin bioactivation and cytotoxicity in living cells, a rat liver DT-diaphorase cDNA was transfected into Chinese hamster ovary cells. MC was equitoxic to the parental cells under oxygenated and hypoxic conditions. In contrast, POR was less toxic than MC to these cells under aerobic conditions, but significantly more toxic than MC under hypoxia. Two DT-diaphorase-transfected clones displayed increases in DT-diaphorase activity of 126- and 133-fold over parental cells. The activities of other oxidoreductases implicated in mitomycin bioreduction were unchanged. MC was more toxic to both DT-diaphorase-transfected lines than to parental cells; the toxicity of MC to the transfected lines was similar in air and hypoxia. POR was also more toxic to the DT-diaphorase-elevated clones than to parental cells under oxygenated conditions. Under hypoxia, however, the toxicity of POR to the transfected clones was unchanged from that of parental cells. The findings implicate DT-diaphorase in mitomycin bioactivation in living cells, but suggest that this enzyme does not contribute to the differential toxicity of MC or POR in air and hypoxia.

15 human tumour cell lines (lung, breast and colon) have been evaluated for their sensitivity to the quinone based anti-cancer drugs Mitomycin C, Porfiromycin, and E09 (3-hydroxymethyl-5-aziridinyl-l-methyl-2-(IH-indole-4,7-dione)prop-β-en-α-ol). Sensitivity has been compared with the intra-cellular levels of DT-diaphorase, an enzyme thought to be important in the reductive activation of these quinones. No correlation exists between levels of DT-diaphorase and sensitivity to Mitomycin C or Porfiromycin. However, for E09 those cell lines showing highest levels of DT-diaphorase activity tend to be the most sensitive.

Bioreductive alkylating agents require reductive activation prior to exerting their cytotoxic actions. This property results in preferential toxicity to hypoxic cells. Previous data have demonstrated that mitomycin C is activated by hypoxic tumor cells and is selectively cytotoxic to these oxygen-deficient cells. The biotransformation of mitomycin C was studied in liver microsomes and nuclei and in a reconstituted, partially purified cytochrome P-450 drug-metabolizing system to provide information on these reductive processes. Both the metabolism of mitomycin C, measured by disappearance of the quinone portion of the substrate, and the formation of an alkylating metabolite(s), determined by employing 4-(p-nitrobenzyl)pyridine as a trapping agent, required anaerobic conditions and an NADPH-generating system, and were inhibited by O2 and CO in both microsomes and nuclei. A reconstituted enzyme system consisting of NADPH, NADPH-cytochrome P-450 reductase, phospholipid and cytochrome P-450 converted mitomycin C to a reactive metabolite(s) under hypoxic conditions. Omission of N2 or any component of the system decreased the metabolic activation of mitomycin C. These findings support the concept that the cytochrome P-450 system is capable of activating mitomycin C under hypoxic conditions to the alkylating metabolite(s) that is responsible for antineoplastic activity.

Trypanosoma equiperdum , a member of the brucei-evansi group of African trypanosomes, is sensitive to the following tumor-inhibitory antibiotics: actinomycin D, mitomycin C, porfiromycin, phleomycin, and pactamycin. Only pactamycin inhibited trypanosomal carbohydrate metabolism in vitro , an action that might have contributed to the ability of this antibiotic to depress protein synthesis and various pathways of nucleic acid metabolism. The inhibitory effects of the other antibiotics were restricted to trypanosomal nucleic acid metabolism. Actinomycin D, 1.6 × 10 −8 M, inhibited the incorporation of 3 H-adenine and 3 H-uracil into RNA by approximately 50%, but had no effect at this concentration upon the incorporation of derivatives of these isotopes into DNA. Higher concentrations had no effect upon the incorporation of 3 H-adenine and 3 H-thymidine into DNA, but did inhibit the incorporation of derivatives of 3 H-uracil into DNA. The distribution of radioactivity in the nucleotide components of the acid-soluble fractions suggested that the predominant action of actinomycin D was to inhibit RNA polymerase. Mitomycin C and its methyl congener, porfiromycin, 2.2 × 10 −4 M and 5.7 × 10 −4 M, respectively, inhibited the incorporation of derivatives of 3 H-adenine and 3 H-uracil into RNA and DNA by approximately 50%. At 7.5 × 10 −6 M and 1.1 × 10 −5 M, mitomycin C and porfiromycin inhibited the incorporation of 3 H-thymidine into DNA by approximately 50%, and at 7.5 × 10 −5 M and 7.2 × 10 −5 M, the evolution of 14 CO 2 derived from orotic acid- 14 COOH. The ability of the mitosane antibiotics, at relatively low concentrations, to inhibit the incorporation of thymidine into DNA suggested that interference with DNA replication was a prominent feature of this group. The data also indicated that at higher concentrations, mitomycin C and porfiromycin inhibited RNA polymerase—whether by direct action or as a consequence of their effects upon DNA synthesis remains unsettled. Phleomycin, 1.3 × 10 −4 M, inhibited the incorporation of 3 H-adenine and 3 H-uracil into RNA by approximately 50%. At this concentration, phleomycin inhibited the incorporation of derivatives of 3 H-uracil into DNA to a greater extent than the derivatives of 3 H-adenine. At 3.2 × 10 −6 M, phleomycin inhibited the incorporation of 3 H-thymidine into DNA by approximately 50%. The action of phleomycin seemed to be qualitatively similar to that of the mitosane antibiotics, except for its inability to inhibit orotidylic acid decarboxylase.

Solid tumors have inadequate vascular networks; thus, significantly less blood flows through these tumor vascular beds than through those of the tissue of origin, and as neoplastic masses enlarge, the blood supply to these tumors further decreases [l-3]. This phenomenon is believed to be due in part to a slower growth rate for endothelial cells of blood vessels than for malignant cells; such a differential results in an inability to maintain an adequate vascular supply [4]. In addition, as tumor masses enlarge, changes in interstitial pressure are possible which can cause compression of tumor capillary beds and regional changes in tumor blood flow. The end result of these oxygen deficit producing effects is the formation of chronic and acute hypoxia in the neoplastic cell population (Fig. l), which may have significant impact on the curability of malignant lesions. There is evidence that hypoxic cell populations exist in both animal [5] and human (see ref. 6 for appropriate references) tumors. Characterization of hypoxic cell populations in transplanted animal tumors has shown that most tumors contain between 5 and 30% of the malignant cell population in an oxygen-deficient state [5]. Although tumor enlargement increases the quantity of hypoxic cells, it is clear that micrometastases also contain oxygendeficient cell populations. Oxygen-deficient malignant cells are significantly more resistant to ionizing radiation than their aerobic counterparts (see for example refs 7 and 8) and, in addition, hypoxia may limit the efficacy of chemotherapy. This latter phenomenon is due to the fact that hypoxic cell populations may be composed of cells which are either blocked or are slowly progressing through the cell cycle [g-11]. Thus, cell cycle active agents may have a relatively low efficacy against hypoxic tumor cell populations. Chemotherapeutic agents may also have difficulty in reaching hypoxic regions, since they must diffuse into the

When mice bearing ascitic L5178Y or L1210 lymphoblasts were treated with a combination of 1-β- d -arabinofuranosylcytosine (cytosine arabinoside) and porfiromycin, the median survival time and the number of survivors were increased. The combination is most effective when treatment is started within 24 hr after implanting the leukemie cells. The synergistic effect is noted only when the dosage level of the cytosine arabinoside component of the combination is optimal. The same synergistic effects were noted when the combination of mitomycin C and cytosine arabinoside was used.