cytochrome-c-t and navitoclax

Topics: Aged; Aged, 80 and over; Aniline Compounds; Antineoplastic Agents; bcl-X Protein; Biological Assay; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cytochromes c; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Mitochondria; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Organ Specificity; Peptides; Plasma Cells; Proto-Oncogene Proteins c-bcl-2; Recurrence; Sulfonamides

Bcl-2 and Bcl-xL are critical regulators of apoptosis that are overexpressed in a variety of human cancers and pharmacological inhibition of Bcl-2 and Bcl-xL represents a promising strategy for cancer treatment. Using a structure-based design approach, we have designed BM-1197 as a potent and efficacious dual inhibitor of Bcl-2 and Bcl-xL. BM-1197 binds to Bcl-2 and Bcl-xL proteins with Ki values less than 1 nM and shows >1,000-fold selectivity over Mcl-1. Mechanistic studies performed in the Mcl-1 knockout mouse embryonic fibroblast (MEF) cells revealed that BM-1197 potently disassociates the heterodimeric interactions between anti-apoptotic and pro-apoptotic Bcl-2 family proteins, concomitant with conformational changes in Bax protein, loss of mitochondrial membrane potential and subsequent cytochrome c release to the cytosol, leading to activation of the caspase cascade and apoptosis. BM-1197 exerts potent growth-inhibitory activity in 7 of 12 small cell lung cancer cell lines tested and induces mechanism-based apoptotic cell death. When intravenously administered at daily or weekly in H146 and H1963 small-cell lung cancer xenograft models, it achieves complete and long-term tumor regression. Consistent with its targeting of Bcl-xL, BM-1197 causes transit platelet reduction in mice. Collectively, our data indicate that BM-1197 is a promising dual Bcl-2/Bcl-xL inhibitor which warrants further investigation as a new anticancer drug.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; bcl-X Protein; Cell Line; Cytochromes c; Humans; Lung Neoplasms; Membrane Proteins; Mice; Mice, SCID; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; Sulfonamides; Transplantation, Heterologous; Tumor Suppressor Proteins

Small-molecule Bcl-2/Bcl-xL inhibitor Navitoclax represents a promising cancer therapeutic since preclinical and clinical studies with Navitoclax have demonstrated strong anticancer activity in several types of cancers. However, because Navitoclax has a low binding affinity to Mcl-1, anticancer activity by Navitoclax is often attenuated by the elevated expression of Mcl-1 in hepatocellular carcinoma (HCC) and other cancers, posing a serious problem for its potential clinical utilities. Therefore, approaches that suppress the expression of Mcl-1 are urgently needed to overcome Navitoclax-resistance in these cancers. Here, we reported that aspirin markedly suppressed Mcl-1 expression, and significantly enhanced Navitoclax-mediated cell viability inhibition and apoptosis induction in HCC cells. We further showed that aspirin robustly enhanced Navitoclax-triggered cytosolic cytochrome c release, activation of initiator caspase-9 and effector caspase-3, and cleavage of PARP. Importantly, the cell death induction by the combination could be rescued by a cell-permeable caspase-9 inhibitor Z-LEHD-FMK, indicative of an indispensable role of mitochondrial apoptosis pathway during the combination effect. Taken together, our study suggests that aspirin can be used to enhance Navitoclax-mediated anticancer activity via suppression of Mcl-1. Since aspirin is one of the most commonly used medicines, our findings therefore have translational impacts on Navitoclax-based therapy for HCC.

Topics: Aniline Compounds; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Aspirin; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Hep G2 Cells; Humans; Liver Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Sulfonamides

The present study demonstrates the important structural features of ceramide required for proper regulation, binding and identification by both pro-apoptotic and anti-apoptotic Bcl-2 family proteins. The C-4=C-5 trans-double bond has little influence on the ability of Bax and Bcl-xL to identify and bind to these channels. The stereochemistry of the headgroup and access to the amide group of ceramide is indispensible for Bax binding, indicating that Bax may interact with the polar portion of the ceramide channel facing the bulk phase. In contrast, Bcl-xL binding to ceramide channels is tolerant of stereochemical changes in the headgroup. The present study also revealed that Bcl-xL has an optimal interaction with long-chain ceramides that are elevated early in apoptosis, whereas short-chain ceramides are not well regulated. Inhibitors specific for the hydrophobic groove of Bcl-xL, including 2-methoxyantimycin A3, ABT-737 and ABT-263 provide insights into the region of Bcl-xL involved in binding to ceramide channels. Molecular docking simulations of the lowest-energy binding poses of ceramides and Bcl-xL inhibitors to Bcl-xL were consistent with the results of our functional studies and propose potential binding modes.

Topics: Aniline Compounds; Animals; Apoptosis; bcl-2-Associated X Protein; Biphenyl Compounds; Caspases; Ceramides; Computer Simulation; Cytochromes c; Ion Channels; Male; Mitochondria, Liver; Models, Molecular; Nitrophenols; Oxidation-Reduction; Piperazines; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Sulfonamides

Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed "primed for death" and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O(2) consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O(2) consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers by "bioenergetics-based profiling" may eventually help identify the subset of patients with chemoresistant but primed tumors who can benefit from treatment that incorporates a BCL-2 antagonist.

Topics: Aniline Compounds; Animals; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Biphenyl Compounds; Cell Death; Cell Line; Cell Line, Tumor; Cell Respiration; Cytochromes c; Cytological Techniques; Energy Metabolism; Humans; Lymphoma, B-Cell; Mice; Mitochondria; Models, Biological; Nitrophenols; Oxygen Consumption; Phenotype; Piperazines; Proto-Oncogene Proteins c-bcl-2; Rats; Sulfonamides

As single agents, ABT-263 and ABT-737 (ABT), molecular antagonists of the Bcl-2 family, bind tightly to Bcl-2, Bcl-xL and Bcl-w, but not to Mcl-1, and induce apoptosis only in limited cell types. The compound 2-deoxyglucose (2DG), in contrast, partially blocks glycolysis, slowing cell growth but rarely causing cell death. Injected into an animal, 2DG accumulates predominantly in tumors but does not harm other tissues. However, when cells that were highly resistant to ABT were pre-treated with 2DG for 3 hours, ABT became a potent inducer of apoptosis, rapidly releasing cytochrome c from the mitochondria and activating caspases at submicromolar concentrations in a Bak/Bax-dependent manner. Bak is normally sequestered in complexes with Mcl-1 and Bcl-xL. 2DG primes cells by interfering with Bak-Mcl-1 association, making it easier for ABT to dissociate Bak from Bcl-xL, freeing Bak to induce apoptosis. A highly active glucose transporter and Bid, as an agent of the mitochondrial apoptotic signal amplification loop, are necessary for efficient apoptosis induction in this system. This combination treatment of cancer-bearing mice was very effective against tumor xenograft from hormone-independent highly metastasized chemo-resistant human prostate cancer cells, suggesting that the combination treatment may provide a safe and effective alternative to genotoxin-based cancer therapies.

Topics: Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Blotting, Western; Cell Line, Tumor; Cytochromes c; Deoxyglucose; Dose-Response Relationship, Drug; HeLa Cells; Hep G2 Cells; Humans; Male; Mice; Mice, Knockout; Mice, Nude; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasms; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Survival Analysis; Tumor Burden; Xenograft Model Antitumor Assays