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Oncotarget Jun 2016Poorly differentiated and anaplastic thyroid carcinomas are very aggressive, almost invariably lethal neoplasms for which no effective treatment exists. These tumors are...
Poorly differentiated and anaplastic thyroid carcinomas are very aggressive, almost invariably lethal neoplasms for which no effective treatment exists. These tumors are intrinsically resistant to cell death, even when their driver oncogenic signaling pathways are inhibited.We have undertaken a detailed analysis, in mouse and human thyroid cancer cells, of the mechanism through which Obatoclax, a pan-inhibitor of the anti-apoptotic proteins of the BCL2 family, effectively reduces tumor growth in vitro and in vivo.We demonstrate that Obatoclax does not induce apoptosis, but rather necrosis of thyroid cancer cells, and that non-transformed thyroid cells are significantly less affected by this compound. Surprisingly, we show that Obatoclax rapidly localizes to the lysosomes and induces loss of acidification, block of lysosomal fusion with autophagic vacuoles, and subsequent lysosomal permeabilization. Notably, prior lysosome neutralization using different V-ATPase inhibitors partially protects cancer cells from the toxic effects of Obatoclax. Although inhibition of autophagy does not affect Obatoclax-induced cell death, selective down-regulation of ATG7, but not of ATG5, partially impairs Obatoclax effects, suggesting the existence of autophagy-independent functions for ATG7. Strikingly, Obatoclax killing activity depends only on its accumulation in the lysosomes, and not on its interaction with BCL2 family members.Finally, we show that also other lysosome-targeting compounds, Mefloquine and LLOMe, readily induce necrosis in thyroid cancer cells, and that Mefloquine significantly impairs tumor growth in vivo, highlighting a clear vulnerability of these aggressive, apoptosis-resistant tumors that can be therapeutically exploited.
Topics: Animals; Antineoplastic Agents; Apoptosis; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Cell Proliferation; Enzyme Inhibitors; Humans; Indoles; Lysosomes; Mefloquine; Mice; Mice, Knockout; Necrosis; Proto-Oncogene Proteins c-bcl-2; Pyrroles; RNA Interference; RNA, Small Interfering; Spheroids, Cellular; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Tumor Cells, Cultured
PubMed: 27144341
DOI: 10.18632/oncotarget.9121 -
International Journal of Molecular... Mar 2019Several studies by our group and others have determined that expression levels of Bcl-2 and/or Bcl-xL, pro-survival molecules which are associated with chemoresistance,...
Obatoclax, a BH3 Mimetic, Enhances Cisplatin-Induced Apoptosis and Decreases the Clonogenicity of Muscle Invasive Bladder Cancer Cells via Mechanisms That Involve the Inhibition of Pro-Survival Molecules as Well as Cell Cycle Regulators.
Several studies by our group and others have determined that expression levels of Bcl-2 and/or Bcl-xL, pro-survival molecules which are associated with chemoresistance, are elevated in patients with muscle invasive bladder cancer (MI-BC). The goal of this study was to determine whether combining Obatoclax, a BH3 mimetic which inhibits pro-survival Bcl-2 family members, can improve responses to cisplatin chemotherapy, the standard of care treatment for MI-BC. Three MI-BC cell lines (T24, TCCSuP, 5637) were treated with Obatoclax alone or in combination with cisplatin and/or pre-miR-34a, a molecule which we have previously shown to inhibit MI-BC cell proliferation via decreasing Cdk6 expression. Proliferation, clonogenic, and apoptosis assays confirmed that Obatoclax can decrease cell proliferation and promote apoptosis in a dose-dependent manner. Combination treatment experiments identified Obatoclax + cisplatin as the most effective treatment. Immunoprecipitation and Western analyses indicate that, in addition to being able to inhibit Bcl-2 and Bcl-xL, Obatoclax can also decrease cyclin D1 and Cdk4/6 expression levels. This has not previously been reported. The combined data demonstrate that Obatoclax can inhibit cell proliferation, promote apoptosis, and significantly enhance the effectiveness of cisplatin in MI-BC cells via mechanisms that likely involve the inhibition of both pro-survival molecules and cell cycle regulators.
Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Indoles; Neoplasm Invasiveness; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Urinary Bladder; Urinary Bladder Neoplasms; bcl-X Protein
PubMed: 30875757
DOI: 10.3390/ijms20061285 -
Clinical Cancer Research : An Official... Jan 2009Prosurvival Bcl-2 proteins inhibit the mitochondrial and death receptor-mediated apoptotic pathways. Obatoclax is a small-molecule antagonist of the BH3-binding groove...
PURPOSE
Prosurvival Bcl-2 proteins inhibit the mitochondrial and death receptor-mediated apoptotic pathways. Obatoclax is a small-molecule antagonist of the BH3-binding groove of Bcl-2 proteins that may enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity and efficacy.
EXPERIMENTAL DESIGN
Human pancreatic cancer cell lines were incubated with obatoclax and/or TRAIL and cell viability, Annexin V labeling, caspase cleavage, and cytochrome c release were measured. In drug-treated cell lines, protein-protein interactions were studied by immunoprecipitation. Bax/Bak activation was analyzed using conformation-specific antibodies. Lentiviral short hairpin RNA was used to knockdown Bim, Bid, and apoptosis-inducing factor (AIF) expression.
RESULTS
Obatoclax reduced the viability of PANC-1 and BxPC-3 cell lines and synergistically enhanced TRAIL-mediated cytotoxicity. Obatoclax enhanced TRAIL-mediated apoptosis, as shown by Annexin V labeling, which was accompanied by caspase activation (caspase-8, -9, and -3) and cleavage of Bid. Obatoclax potentiated TRAIL-mediated Bax/Bak activation and the release of mitochondrial cytochrome c, Smac, and AIF. Mechanisms underlying the apoptotic effect of obatoclax include displacement of Bak from its sequestration by Bcl-x(L) or Mcl-1 and release of Bim from Bcl-2 or Mcl-1. Bid knockdown by short hairpin RNA attenuated caspase cleavage and cytotoxicity of obatoclax plus TRAIL. Bim knockdown failed to inhibit the cytotoxic effect of obatoclax alone or combined with TRAIL yet attenuated TRAIL-mediated cytotoxicity. AIF knockdown attenuated cytotoxicity of the drug combination.
CONCLUSIONS
Obatoclax potentiates TRAIL-mediated apoptosis by unsequestering Bak and Bim from Bcl-2/Bcl-x(L) or Mcl-1 proteins. This drug combination enhances Bid-mediated cross-talk between the mitochondrial and death receptor-mediated apoptotic pathways and may represent a novel therapeutic strategy against pancreatic cancer.
Topics: Apoptosis; BH3 Interacting Domain Death Agonist Protein; Caspases; Cell Line, Tumor; Cytochromes c; Humans; Indoles; Myeloid Cell Leukemia Sequence 1 Protein; Pancreatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Pyrroles; TNF-Related Apoptosis-Inducing Ligand; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 19118042
DOI: 10.1158/1078-0432.CCR-08-1575 -
The FEBS Journal Dec 2017The ERK1/2 signalling pathway is best known for its role in connecting activated growth factor receptors to changes in gene expression due to activated ERK1/2 entering... (Review)
Review
The ERK1/2 signalling pathway is best known for its role in connecting activated growth factor receptors to changes in gene expression due to activated ERK1/2 entering the nucleus and phosphorylating transcription factors. However, active ERK1/2 also translocate to a variety of other organelles including the endoplasmic reticulum, endosomes, golgi and mitochondria to access specific substrates and influence cell physiology. In this article, we review two aspects of ERK1/2 signalling at the mitochondria that are involved in regulating cell fate decisions. First, we describe the prominent role of ERK1/2 in controlling the BCL2-regulated, cell-intrinsic apoptotic pathway. In most cases ERK1/2 signalling promotes cell survival by activating prosurvival BCL2 proteins (BCL2, BCL-x and MCL1) and repressing prodeath proteins (BAD, BIM, BMF and PUMA). This prosurvival signalling is co-opted by oncogenes to confer cancer cell-specific survival advantages and we describe how this information has been used to develop new drug combinations. However, ERK1/2 can also drive the expression of the prodeath protein NOXA to control 'autophagy or apoptosis' decisions during nutrient starvation. We also describe recent studies demonstrating a link between ERK1/2 signalling, DRP1 and the mitochondrial fission machinery and how this may influence metabolic reprogramming during tumorigenesis and stem cell reprogramming. With advances in subcellular proteomics it is likely that new roles for ERK1/2, and new substrates, remain to be discovered at the mitochondria and other organelles.
Topics: Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Bridged Bicyclo Compounds, Heterocyclic; Drug Screening Assays, Antitumor; Humans; Indoles; MAP Kinase Signaling System; Mitochondrial Dynamics; Molecular Targeted Therapy; Neoplasm Proteins; Neoplasms; Oncogene Addiction; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides
PubMed: 28548464
DOI: 10.1111/febs.14122 -
Molecular Cancer Therapeutics Aug 2008Human cholangiocarcinomas evade apoptosis by overexpression of Mcl-1. The drug obatoclax (GX15-070) inhibits antiapoptotic members of the Bcl-2 family including Mcl-1....
Human cholangiocarcinomas evade apoptosis by overexpression of Mcl-1. The drug obatoclax (GX15-070) inhibits antiapoptotic members of the Bcl-2 family including Mcl-1. The purpose of this study is to determine if obatoclax sensitizes human cholangiocarcinoma cells to apoptosis. The human cholangiocarcinoma cell lines, KMCH, KMBC, and TFK, were employed for these studies. Protein expression was assessed by immunoblot and protein-protein interactions detected by coprecipitation of the polypeptide of interest with S-tagged Mcl-1. Activation of Bak and Bax was observed by immunocytochemistry with conformation-specific antisera. Obatoclax induced minimal apoptosis alone; however, it increased apoptosis 3- to 13-fold in all three cancer cell lines when combined with Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Obatoclax did not alter cellular expression of Bid, Bim, Puma, Noxa, Bak, Bax, Mcl-1, or cFLIP. Mcl-1 binding to Bak was readily identified in untreated cells, and this association was disrupted by treating the cells with obatoclax. Additionally, Bim binding to Mcl-1 was markedly decreased by obatoclax treatment. We also identified alterations in Bak and Bax conformation following treatment with obatoclax plus Apo2L/TRAIL but not with either Apo2L/TRAIL or obatoclax alone. In conclusion, obatoclax releases Bak and Bim from Mcl-1 and sensitizes human cholangiocarcinoma cells to Apo2L/TRAIL-induced apoptosis. Obatoclax is a potentially promising adjunctive agent for the treatment of this cancer.
Topics: Apoptosis; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cell Line, Tumor; Cholangiocarcinoma; Fluorescent Antibody Technique; Humans; Indoles; Molecular Mimicry; Pyrroles; TNF-Related Apoptosis-Inducing Ligand
PubMed: 18723481
DOI: 10.1158/1535-7163.MCT-08-0285 -
IScience Oct 2023We identified cytoprotective small molecules (CSMs) by a cell-based high-throughput screening of Bax inhibitors. Through a medicinal chemistry program, M109S was...
We identified cytoprotective small molecules (CSMs) by a cell-based high-throughput screening of Bax inhibitors. Through a medicinal chemistry program, M109S was developed, which is orally bioactive and penetrates the blood-brain/retina barriers. M109S protected retinal cells in ocular disease mouse models. M109S directly interacted with Bax and inhibited the conformational change and mitochondrial translocation of Bax. M109S inhibited ABT-737-induced apoptosis both in Bax-only and Bak-only mouse embryonic fibroblasts. M109S also inhibited apoptosis induced by staurosporine, etoposide, and obatoclax. M109S decreased maximal mitochondrial oxygen consumption rate and reactive oxygen species production, whereas it increased glycolysis. These effects on cellular metabolism may contribute to the cytoprotective activity of M109S. M109S is a novel small molecule protecting cells from mitochondria-dependent apoptosis both and . M109S has the potential to become a research tool for studying cell death mechanisms and to develop therapeutics targeting mitochondria-dependent cell death pathway.
PubMed: 37841588
DOI: 10.1016/j.isci.2023.107916 -
Current Hematologic Malignancy Reports Feb 2017The BCL-2 family of proteins integrates pro- and anti-apoptotic signals within the cell and is responsible for initiation of caspase-dependent apoptosis. Chronic... (Review)
Review
The BCL-2 family of proteins integrates pro- and anti-apoptotic signals within the cell and is responsible for initiation of caspase-dependent apoptosis. Chronic lymphocytic leukemia (CLL) cells are particularly dependent on the anti-apoptotic protein BCL-2 for their survival, making this an attractive therapeutic target in CLL. Several early efforts to create inhibitors of the anti-apoptotic family members faced significant challenges, but eventually, the BCL-2 specific inhibitor venetoclax moved forward in CLL. Overall and complete response rates to venetoclax monotherapy in relapsed, refractory CLL are approximately 80 and 20%, respectively, even in patients with high-risk 17p deletion. Toxicities have been manageable and include neutropenia, diarrhea, and nausea. The risk of tumor lysis syndrome (TLS), seen in early experience with the drug, has been mitigated by the use of appropriate TLS risk assessment, prophylaxis, and management. Future studies of venetoclax will focus on combination approaches, predictive biomarker discovery, and mechanisms of resistance.
Topics: Aniline Compounds; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Gossypol; Humans; Indoles; Leukemia, Lymphocytic, Chronic, B-Cell; Neutropenia; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides; Thionucleotides
PubMed: 28116634
DOI: 10.1007/s11899-017-0359-0 -
PloS One 2013Anti-apoptotic Bcl-2 family proteins, in particular, Mcl-1, are known to play a critical role in resistance of human melanoma cells to induction of apoptosis by...
Anti-apoptotic Bcl-2 family proteins, in particular, Mcl-1, are known to play a critical role in resistance of human melanoma cells to induction of apoptosis by endoplasmic reticulum stress and other agents. The present study examined whether the BH3 mimetics, Obatoclax and ABT-737, which inhibit multiple anti-apoptotic Bcl-2 family proteins, would overcome resistance to apoptosis. We report that both agents induced a strong unfolded protein response (UPR) and that RNAi knockdown of UPR signalling proteins ATF6, IRE1α and XBP-1 inhibited Mcl-1 upregulation and increased sensitivity to the agents. These results demonstrate that inhibition of anti-apoptotic Bcl-2 proteins by Obatoclax and ABT-737 appears to elicit a protective feedback response in melanoma cells, by upregulation of Mcl-1 via induction of the UPR. We also report that Obatoclax, but not ABT-737, strongly induces autophagy, which appears to play a role in determining melanoma sensitivity to the agents.
Topics: Antineoplastic Agents; Apoptosis; Autophagy; Biphenyl Compounds; Calcium; Cell Line, Tumor; Cytosol; Drug Resistance, Neoplasm; Endoplasmic Reticulum Stress; Humans; Indoles; Melanoma; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides; Unfolded Protein Response; Up-Regulation
PubMed: 24367627
DOI: 10.1371/journal.pone.0084073 -
Blood Apr 2013Survival in infants younger than 1 year who have acute lymphoblastic leukemia (ALL) is inferior whether MLL is rearranged (R) or germline (G). MLL translocations confer...
Survival in infants younger than 1 year who have acute lymphoblastic leukemia (ALL) is inferior whether MLL is rearranged (R) or germline (G). MLL translocations confer chemotherapy resistance, and infants experience excess complications. We characterized in vitro sensitivity to the pan-antiapoptotic BCL-2 family inhibitor obatoclax mesylate in diagnostic leukemia cells from 54 infants with ALL/bilineal acute leukemia because of the role of prosurvival BCL-2 proteins in resistance, their imbalanced expression in infant ALL, and evidence of obatoclax activity with a favorable toxicity profile in early adult leukemia trials. Overall, half maximal effective concentrations (EC50s) were lower than 176 nM (the maximal plasma concentration [Cmax] with recommended adult dose) in 76% of samples, whether in MLL-AF4, MLL-ENL, or other MLL-R or MLL-G subsets, and regardless of patients' poor prognostic features. However, MLL status and partner genes correlated with EC50. Combined approaches including flow cytometry, Western blot, obatoclax treatment with death pathway inhibition, microarray analyses, and/or electron microscopy indicated a unique killing mechanism involving apoptosis, necroptosis, and autophagy in MLL-AF4 ALL cell lines and primary MLL-R and MLL-G infant ALL cells. This in vitro obatoclax activity and its multiple killing mechanisms across molecular cytogenetic subsets provide a rationale to incorporate a similarly acting compound into combination strategies to combat infant ALL.
Topics: Antineoplastic Agents; Apoptosis; Autophagy; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; Histone-Lysine N-Methyltransferase; Humans; Indoles; Infant; Infant, Newborn; Myeloid-Lymphoid Leukemia Protein; Necrosis; Oncogene Proteins, Fusion; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; Pyrroles
PubMed: 23393050
DOI: 10.1182/blood-2012-04-425033 -
Core Evidence 2013Obatoclax mesylate is an intravenously-administered drug under investigation in Phase I and II clinical trials as a novel anticancer therapeutic for hematological...
Obatoclax mesylate is an intravenously-administered drug under investigation in Phase I and II clinical trials as a novel anticancer therapeutic for hematological malignancies and solid tumors. Obatoclax was developed as a pan-inhibitor of antiapoptotic members of the B cell chronic lymphocytic leukemia/lymphoma 2 (BCL-2) family of proteins, which control the intrinsic or mitochondrial pathway of apoptosis. Resistance to apoptosis through dysregulation of BCL-2 family members is commonly observed in hematological malignancies, and can be linked to therapeutic resistance and poor clinical outcomes. By inhibiting pro-survival BCL-2 family proteins, including MCL-1, obatoclax is proposed to (1) trigger cell death as a single agent, and (2) potentiate the anticancer effects of other therapeutics. Preclinical investigations have supported these proposals and have provided evidence suggestive of a promising therapeutic index for this drug. Phase I trials of obatoclax mesylate in leukemia and lymphoma have defined well-tolerated regimens and have identified transient neurotoxicity as the most common adverse effect of this drug. In these studies, a limited number of objective responses were observed, along with hematological improvement in a larger proportion of treated patients. Published Phase II evaluations in lymphoma and myelofibrosis, however, have not reported robust single-agent activity. Emerging evidence from ongoing preclinical and clinical investigations suggests that the full potential of obatoclax mesylate as a novel anticancer agent may be realized (1) in rational combination treatments, and (2) when guided by molecular predictors of therapeutic response. By understanding the molecular underpinnings of obatoclax response, along with optimal therapeutic regimens and indications, the potential of obatoclax mesylate for the treatment of hematological malignancies may be further clarified.
PubMed: 23515850
DOI: 10.2147/CE.S42568