Cytotoxic Mechanisms of FK317, a New Class of Bioreductive Agent with Potent Antitumor Activity

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Abstract
FK317 is a member of a new class of bioreductive agents that exhibit strong cytotoxicity against various human cancer cells. The effect of FK317 was found to be stronger than that of mitomycin C (MMC), adriamycin (ADR) or cisplatin (CDDP). Alkaline elution analysis indicated that FK317 formed interstrand DNA‐DNA and DNA‐protein cross‐links in cells. On the other hand, no DNA single‐strand breaks were observed in the cells treated with FK317. In a cell‐free system the deacetylated metabolites produced cross‐linked DNA under reductive conditions, though FK317 itself did not form DNA‐DNA cross‐links. In order to elucidate the metabolic activation mechanisms, we established an FK317‐resistant subline from human non‐small cell lung cancer cells (Lu99) by stepwise and brief exposure (1 h) to FK317. The resistant subline (Lu99/317) showed cross‐resistance to MMC and carboquone (CQ), but not to ADR or CDDP. DT‐diaphorase, which is one of the activation enzymes of MMC and CQ, was deficient in Lu99/317 cells as determined by enzyme activity assay. However, the levels of NADPH:cytochrome P450 reductase, which is another activation enzyme for MMC and CQ, were comparable in resistant and parent cell lines. Treatment of the cells with dicumarol, an inhibitor of DT‐diaphorase, reduced the cytotoxicity of FK317 to Lu99 cells, but not to Lu99/317 cells. These results indicate that deacetylation of FK317 is necessary for its reductive activation, and deacetylated FK317 is reduced by DT‐diaphorase to form an active metabolite, which produces DNA‐DNA interstrand and DNA‐protein cross‐links that lead to cell death.