Cytotoxicity of cyclophosphamide, paclitaxel, and docetaxel for tumor cell lines in vitro: effects of concentration, time and cytochrome P450-catalyzed metabolism.

Abstract

Cytotoxic effects of cyclophosphamide (CPA), paclitaxel (PCT), and docetaxel (DTX) and their modulation by cytochrome P450 (CYP) metabolism were studied by incubating cell lines L929 and P388D1 with or without rat liver microsomes. The microsomes themselves were not cytotoxic. P388D1 cells were more sensitive to CPA, PCT, and DTX than L929 cells. CYP2B1-, CYP3A-, and CYP2E1-induced microsomes effectively oxidized the prodrug CPA to cytotoxic products in 2-h incubation periods. Cytotoxicity of DTX and PCT for P388D1 cells became apparent 24 h after a 2-h incubation period with the drugs, and their effects were enhanced by CYP2E1 microsomes, but markedly decreased by CYP3A-induced microsomes. DTX and PCT without microsomes caused a dose-related cytotoxicity in P388D1 and HeLa cells. P388D1 and HeLa cells did not grow after a 24-h exposure to 1-10 microM DTX, but about 0.1% of cells survived exposure to 1-10 microM PCT. After 4 weeks of multiplication, the surviving P388D1 cells displayed lower sensitivity to DTX and PCT, but cytotoxicity in HeLa cells was unchanged and their growth ability decreased. In P388D1 cells, PCT with DTX (0.1, 0.5. 1, 2.5, or 5 microM) showed only additive cytotoxicity, although they reportedly act in different phases of the cell cycle. In P388D1 cells treated with DTX or PCT, normal mononuclear cells disappeared and the cell diameter increased up to threefold. Mulberry-like nuclei developed, giving rise to multiple nuclei, which were hyper- or hypochromatic. Chromatin condensation in some multiple nuclei and cell shrinkage of some cells fit the definition of apoptosis, but enlargement of the surviving cells and numerous hypochromatic nuclei do not. In conclusion, L929 and P388D1 cells incubated with microsomes enabled the role of various CYP enzymes in the effect of anticancer drugs to be assessed. The delayed cytotoxicity of DTX and PCT compared to that of CPA was related to their different mode of action. Fluorescent microscopy revealed quantitatively different effects of PCT and DTX on the nuclei, indicating that their mode of action may not be completely identical.