Cell cycle disturbances and apoptosis induced by topotecan and gemcitabine on human lung cancer cell lines

Abstract

Apoptosis is a major mode of cell death in response to cytotoxic drug treatment. A correlation between induction of apoptosis and chemosensitivity has been documented in some preclinical models. Topotecan (a topoisomerase I inhibitor) and gemcitabine (a deoxycytidine analogue), two active new drugs for the treatment of lung cancer, were evaluated for their growth inhibitory effect on human lung cancer cell lines and their effect on cell cycle perturbation, apoptosis and apoptosis-related genes. The cytotoxicities of topotecan and gemcitabine on the human lung cancer cell lines H460 (wild-type- p53) and H322 (mutant p53) were determined after 72 h drug exposure employing the MTT assay. The apoptotic index (AI) was assessed by three methods: analysis of morphological changes using May–Grünwald–Giemsa (MGG) staining, the TUNEL assay and FACS analysis. Cell cycle disturbances were studied by FACS and the number of cells expressing p53 and p21 were determined by immunohistochemistry. Both gemcitabine and topotecan had potent growth inhibitory effects in human lung cancer cell lines; combination treatment with these two drugs showed some additivity but no synergism. Induction of apoptosis after treatment was concentration- and time-dependent with both drugs and ic 80 concentrations induced the highest values. The DNA histograms at 4, 24, 48 and 72 h indicate that topotecan at ic 80 concentrations causes accumulation of cells in S and G2/M phases, whereas gemcitabine at ic 80 concentrations causes, accumulation of cells in G1 phase. Both compounds induced p53 and p21 expression in the H460 cell line but not in the H322 cell line; the percentage of cells expressing p53 was highest at ic 80 values, whereas the highest percentage of p21 positive cells could be induced with ic 50 values. This could suggest that p53 induces cell cycle arrest at low drug concentrations, whereas p53 induces apoptosis at higher concentrations. In conclusion, p53-dependent and independent pathways of apoptosis exist in lung cancer cell lines. Activation of the p53 pathway depends on the induced cellular damage. Understanding the cell cycle disturbances induced by these drugs may help in the design of more rational treatment schedules.