Since the cure of solid
tumors is limited by the presence of cells with low
oxygen contents, we have approached the development of treatment regimens and of new drugs for these
tumors by investigating agents which are preferentially bioactivated under
hypoxia. Major emphasis has been directed at studying the mode of action of the
mitomycin antibiotics, as bioreductive
alkylating agents. Using primarily the EMT6 mouse mammary
carcinoma as a solid
tumor model, we have found that
mitomycin C and
porfiromycin are preferentially toxic to cells with low
oxygen contents. The
mitomycin analog
BMY-25282 is more toxic to hypoxic cells than are
mitomycin C and
porfiromycin; however, unlike these
antibiotics,
BMY-25282 is preferentially toxic to well-oxygenated cells. With these three
mitomycins, we have observed a correlation between cytotoxicity to hypoxic cells, the rate of generation of reactive products, and the redox potentials of the drugs. Investigations of the
enzymes in EMT6 cells that could possibly activate
mitomycin C have revealed that
cytochrome P-450 and
xanthine oxidase are not present in measurable quantities and therefore are not responsible for activation of
mitomycin C. Activities representative of
NADPH-cytochrome c reductase and
DT-diaphorase are present in these neoplastic cells. Comparison of these enzymatic activities in EMT6, CHO, and V79 cells with the rate of generation of reactive products under
hypoxia shows a direct correlation between these two parameters, but there is no quantitative correlation between these two parameters and the amount of cytotoxicity. Use of purified
NADPH-cytochrome c reductase and inhibitors of this
enzyme demonstrated that
NADPH-cytochrome c reductase can activate
mitomycin C, but that it is probably not the only
enzyme participating in this bioactivation in EMT6 cells. The
DT-diaphorase inhibitor
dicoumarol was employed to show that this
enzyme is not involved in the activation of
mitomycin C to a
cytotoxic agent. Instead,
DT-diaphorase appears to metabolize
mitomycin C to a nontoxic product. This property has been exploited to develop a new treatment regimen for solid
tumors. Using X-rays to eliminate well oxygenated cells of a solid
tumor implant of the EMT6
carcinoma, we have found that the combination of
dicoumarol plus
mitomycin C is more toxic to hypoxic
tumor cells in vivo than
mitomycin C alone. Furthermore, knowledge of the biochemical mechanism of
mitomycin C activation permits a prediction of which
tumors can best be treated with this combination of drugs by measuring enzymatic activities in biopsy specimens.