Insights into cellular and molecular mechanisms of apoptosis induced by the anticancer drug cisplatin.

摘要

Cisplatin (cis-diamminedichloroplatinum (II)) is widely used for the treatment of ovarian, testicular, and colorectal cancers. The drug arrests the G2 phase of the cell cycle, inducing apoptosis or programmed cell death. In order to understand the molecular mechanism of apoptosis, phosphorus and proton NMR experiments were conducted in Chinese hamster ovarian cells in the presence and absence of cisplatin at different time intervals. The NMR experiments revealed that apoptosis was initiated after six hours as measured by the increase in phosphocholine and glycerophosphocholine metabolites due to membrane disintegration. These NMR experiments also provided an insight into cellular energetics, based on variation of phosphocreatine and nucleoside triphosphate concentrations.; To unveil the roles of zinc finger transcription factors, the structures of a model zinc finger motif, Cys-X4-Cys-X13-Cys-X 4-Cys and its platinum analog, were evaluated by two-dimensional NMR, circular dichroism and fluorescence spectroscopy. Zinc-peptide structures revealed a distorted alpha-helix followed by an antiparallel beta-sheet, which are connected by a type II' beta-turn. These structures were consistent with CD spectra in which features for alpha-helical and beta-sheet secondary structures were observed. Fluorescence data yielded the primary zinc binding constant, 2.5x106M-1 along with a much smaller secondary binding constant, 2.0x103M-1.; The reaction of cisplatin with the zinc finger peptide revealed severe structural perturbations. These perturbations were clearly seen with the appearance of an intense negative peak at 215 nm in the CD spectra, with the concomitant disappearance of the helical features at 208 and 222 nm. HPLC separations indicate the existence of two products, which were supported by the two-dimensional NMR experiments. Based on chromatographic and spectroscopic data, it was concluded that two cysteines at both N- and C- termini were bound to the platinum atom yielding two different adducts. Platinum binding was accompanied by formation of two intermediates, one of which is due to a conformational change of the first intermediate. The rate constant for the formation of the first intermediate was evaluated to be 16.7+/-1.3M-1.s-1, followed by a slow unfolding process with a rate of 2.9+/-0.4x10 -4s-1, and subsequent conversion to products with a first-order reaction (8.5+/-0.5x10-5s -1).