Cellular effects of BMS: cell cycle inhibition and induction of the mitochondrial caspase activation pathway.To characterize the mechanisms through which BMS kills H cells, we determined its transcriptional effects at a preapoptotic stage, measured at h or at h. At h, BMS caused the upregulation of f genes and the downregulation of f genes by a factor, with a Thymine profile that was markedly different from that induced by CDDP. Hence, part of the changes in the transcriptome are specific for BMS and do not reflect a general property of Amiodarone hydrochloride apoptosis in H cells.In the same conditions, BMS led to a G cycle arrest. In contrast, BMS mediates cytotoxicantiproliferative effects even after pretreatment with CDDP, as shown in experiments in which CDDP and BMS were added in different orders to H cells. BMS influenced several genes that are involved in the mitochondrial cell death pathway.In this context, it may be noteworthy that HSP promotes the stability of mutant EGFR and that the use of HSP inhibitors. BMS induced signs of the mitochondrial apoptosis pathway.Thus, H cells treated with BMS manifested the release of cytochromecand subsequent caspase activation, as determined by twocolor immunofluorescence staining, immunoblotting, and cytofluorimetric quantification. Blockade of caspase activation by the broadspectrum inhibitor A.Antiproliferative and proapoptotic effects of BMS on NSCLC cell lines.A, H, H, H, and A cells were treated with the indicated concentrations of BMS or erlotinib for h, before the colorimetric assessment of cell proliferation.White columns, percentage of cells that have a low mitochondrial membrane potential but are still viable. Light gray columns, percentage of cells with disrupted plasma membrane. Columns, means of two independent experiments; bars, SE.To this aim, transfected cells were treated with BMS for h, before the assessment of proliferation by a tetrazolium salt reduction assay.The effect of each siRNA was evaluated by comparing the residual proliferation, as measured upon the treatment with BMS, to the level observed in untreated. Results were then compared with the negative control provided by an irrelevant, unrelated siRNA.Microarray analysis of the transcriptome of H cells treated with BMS or CDDP.Total RNA was then retrotranscribed into doublestranded cDNA followed by T RNA polymerasemediated linear amplification, labeling, and hybridization to a human whole genome k oligonucleotide array.A, hierarchical cluster analysis of genes, the transcription of which in treated cells compared with untreated controls.Each row represents the combination of two dyeswap is modified in a statistically significant fashion reports some examples of the differential transcriptional effects promoted by BMS and CDDP.Thereafter, cell proliferation was assessed by an assay based on the reduction of the tetrazolium salt WST.The switch between the two treatments was done either at h only.For each panel, the proliferation of treated cultures was normalized to that of control cells subjected to the exchange of the culture medium at the same time points.Novel EGFRTKI overcoming drug resistance might represent important weapons in the fight against cancer.As shown here, BMS was able to arrest proliferation andor to induce apoptosis in all tested NSCLC cell lines, although at different levels of potency.

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