Establishment and characterization of non-small cell lung cancer cell lines resistant to mitomycin C under aerobic conditions.

Abstract

To elucidate the mechanisms of acquired resistance to mitomycin C (MMC) in non‐small cell lung cancer (NSCLC), we established two MMC‐resistant NSCLC sublines by continuous exposure to MMC, using PC‐9 as a parent cell line. The sublines, PC‐9/MC2 and PC‐9/MC4, were 6.4‐ and 10‐fold more resistant to MMC than their parent cell line, respectively, at the IC50 value as determined by MTT assay. They exhibited cross‐resistance to EO9, but were not resistant to cisplatin, vindesine, etoposide, carboquone, or KW‐2149, a novel MMC derivative. They were collaterally sensitive to adriamycin and menadione. Accumulation of the drug was decreased in the resistant sublines to about 60% of that in the parent cells. Cytosolic DT‐diaphorase (DTD) activities were decreased to 13.5±3.2 in PC9/MC2 and 1.3±0.6 in PC‐9/MC4 from 261.5±92.7 nmol/min/mg protein in the parent PC‐9. NADH:cytochrome b5 reductase activities in both of the resistant cell lines were significantly decreased as compared to that in the parent cell line. Addition of dicumarol resulted in a two‐fold increase in IC50 value in PC‐9, whereas the IC50 value showed no change in PC‐9/MC4. Moreover, dicumarol did not affect the sensitivities to KW‐2149 but decreased the sensitivities to EO9 in both the parent and the resistant cell lines. Formation of an alkylating metabolite was significantly decreased in the resistant cells, in parallel to the degree of resistance. We concluded that deficient drug activation due to decreased DTD activity was important as a mechanism of resistance to MMC in PC‐9, a relatively DTD‐rich NSCLC cell line.