Formation of a novel topotecan metabolite in the hormone-independent human prostate carcinoma cell lines DU-145 and PC-3.

To investigate the implications of drug metabolism on topotecan (TPT) resistance in prostate cancer cells, we measured the time-dependent uptake, metabolismrand efflux of TPT in the prostate cancer-derived cell lines DU-145 and PC-3 by HPLC. Exposure of DU-145 to 10 microM TPT resulted in a maximal intracellular concentration of TPT of 12.6 +/- 0.53 pmol/10(6) cells (t = 10 min) with a decrease to 4.4 +/- 0.25 pmol/10(6) cells after 2 hours. Incubation of PC-3 cells, however, revealed a more than 2-fold higher level of cytoplasmatic TPT (25.3 +/- 4.8 pmol/10(6) cells). In both cell lines, an intracellular metabolite was detectable after 30 minutes. Its concentration continuously increased reaching saturation after 6 hours (0.015 +/- 0.003 pmol/10(6) cells in DU-145 and 0.0059 +/- 0.0020 pmol/10(6) cells in PC-3 cells). Analysis of the culture supernatant of DU-145 and PC-3 cells revealed that this metabolite is secreted into the medium at increasing concentrations (0.220 +/- 0.025 and 0.079 +/- 0.008 pmol/10(6) cells, respectively). In accordance with the elevated formation of the TPT-metabolite in DU-145 cells, the expression of cytochrome P450 (CYP) isoenzymes CYP3A, CYP2B, CYP2D and CYP2E as measured by Western blot analysis was also higher in this cancer cell line. In conclusion, we found that TPT is rapidly taken up by the two prostate cancer cell lines and metabolized to a minor biotransformation product dependent on their content of cytochrome P450 isoenzymes. The structural identification of this TPT metabolite and the CYP isoenzyme(s) responsible for its formation remain to be elucidated.