nitrophenols and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Renal cell carcinoma (RCC) is the most common malignancy in the kidney in the world, and the 5-year overall survival for patients remains poor due to the lack of effective treatment strategies. Although ABT-737, as a Bcl-2/Bcl-xL inhibitor, has recently emerged as a novel cancer therapeutic reagent, apoptosis induced by ABT-737 is often blocked in several types of cancer cells. This study investigated whether the combination of the small-molecule BH3 mimetic ABT-737 and the lysosome inhibitor chloroquine was an effective strategy for treating renal cancer cells. We found that the combination of ABT-737 and chloroquine synergistically decreased cell viability when compared to treatment with either single reagent. Cell apoptosis induced by a combined treatment was markedly inhibited by the caspase inhibitors z-DEVD-FMK and z-VAD-FMK. It was also inhibited by cathepsin inhibitor E-64 and CTSI (cathepsin inhibitor), which suggested that apoptosis was dependent on the cascade of caspase activation and cathepsins released from lysosomes. Furthermore, we found that ABT-737 could increase the cell level of ROS, which triggers cathepsin-mediated cell death and augments the role of chloroquine in cell death. So the combination of ABT-737 and chloroquine was an effective strategy for the treatment of renal cancer cells, and this combined strategy may widen the therapeutic window of ABT-737 and chloroquine as well as enhance the clinical efficacy of synergistic drug combinations.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Biphenyl Compounds; Carcinoma, Renal Cell; Cell Death; Cell Line, Tumor; Chloroquine; Drug Synergism; Humans; Kidney Neoplasms; Leucine; Nitrophenols; Oligopeptides; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Thiadiazoles

Primary chronic lymphocytic leukemia (CLL) cells are exquisitely sensitive to ABT-737, a small molecule BCL2-antagonist, which induces many of the classical biochemical and ultrastructural features of apoptosis, including BAX/BAK oligomerization, cytochrome c release, caspase activation and chromatin condensation. Surprisingly, ABT-737 also induces mitochondrial inner membrane permeabilization (MIMP) resulting in mitochondrial matrix swelling and rupture of the outer mitochondrial membrane (OMM), so permitting the rapid efflux of cytochrome c from mitochondrial cristae and facilitating rapid caspase activation and apoptosis. BAX and BAK appear to be involved in the OMM discontinuities as they localize to the OMM break points. Notably, ABT-737 induced mitochondrial matrix swelling and OMM discontinuities in other primary B-cell malignancies, including mantle cell, follicular and marginal zone lymphoma cells but not in several cell lines studied. Thus, we describe a new paradigm of apoptosis in primary B-cell malignancies, whereby targeting of BCL2 results in all the classical features of apoptosis together with OMM rupture independent of caspase activation. This mechanism may be far more prevalent than hitherto recognized due to the failure of most methods, used to measure apoptosis, to recognize such a mechanism.

Topics: Adult; Amino Acid Chloromethyl Ketones; Apoptosis; B-Lymphocytes; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Biphenyl Compounds; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Mitochondrial Membranes; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Previous suggestions of CpG-specific apoptotic commitment implied critical epigenetic modulation of housekeeping genes which have canonical CpG islands at 5' promoter regions. Differential housekeeping gene activity however has not been shown. Using a focussed microarray (genechip) of 22 housekeeping genes we show this in apoptosis induced in human Chang liver cells by DCNP (2,6-dichloro-4-nitrophenol), a non-genotoxic inhibitor of sulfate detoxification. 3-7 folds downregulation of 9 genes in glycolysis, tricarboxylic acid cycle and the respiratory electron transport chain suggested gene-directed energy depletion which was correlated with observed ATP depletion. 4 folds downregulation of the pyruvate dehydrogenease gene suggested gene-directed metabolic acidosis which was correlated with observed cell acidification. Other differential housekeeping gene activity, including 4 folds upregulation of microtubular alpha-tubulin gene, and 2 folds upregulation of ubiquitin, also had a bearing on apoptosis. Broadspectrum zVAD-fmk caspase inhibition abolished 200 bp DNA ladder fragmentations but not the CpG-specific megabase fragmentations and other hallmarks of cell destruction, suggesting a caspase-independent cell death. Death appeared committed at gene-level.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Cell Line; Cysteine Proteinase Inhibitors; DNA Fragmentation; Dose-Response Relationship, Drug; Energy Metabolism; Enzyme Activation; Flow Cytometry; Gene Expression Regulation; Hepatocytes; Humans; Hydrogen-Ion Concentration; Mitochondria; Nitrophenols; Oligonucleotide Array Sequence Analysis