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Molecular Pharmacology Dec 2012Previous studies showed that lapatinib and obatoclax interact in a greater-than-additive fashion to cause cell death and do so through a toxic form of autophagy. The...
Previous studies showed that lapatinib and obatoclax interact in a greater-than-additive fashion to cause cell death and do so through a toxic form of autophagy. The present studies sought to extend our analyses. Lapatinib and obatoclax killed multiple tumor cell types, and cells lacking phosphatase and tensin homolog (PTEN) function were relatively resistant to drug combination lethality; expression of PTEN in PTEN-null breast cancer cells restored drug sensitivity. Coadministration of lapatinib with obatoclax elicited autophagic cell death that was attributable to the actions of mitochondrial reactive oxygen species. Wild-type cells but not mitochondria-deficient rho-zero cells were radiosensitized by lapatinib and obatoclax treatment. Activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase 1/2 (JNK1/2) by the drug combination was enhanced by radiation, and signaling by p38 MAPK and JNK1/2 promoted cell killing. In immunohistochemical analyses, the autophagosome protein p62 was determined to be associated with protein kinase-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1, as well as with binding immunoglobulin protein/78-kDa glucose-regulated protein, in drug combination-treated cells. Knockdown of PERK suppressed drug-induced autophagy and protected tumor cells from the drug combination. Knockdown of PERK suppressed the reduction in Mcl-1 expression after drug combination exposure, and overexpression of Mcl-1 protected cells. Our data indicate that mitochondrial function plays an essential role in cell killing by lapatinib and obatoclax, as well as radiosensitization by this drug combination.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Autophagy; Breast Neoplasms; Cell Line, Tumor; Endoplasmic Reticulum Stress; Female; HSP70 Heat-Shock Proteins; Humans; Indoles; Lapatinib; Membrane Proteins; Mice; Mice, Nude; Mitochondria; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinase 9; Pyrroles; Quinazolines; Reactive Oxygen Species; eIF-2 Kinase; p38 Mitogen-Activated Protein Kinases
PubMed: 22989520
DOI: 10.1124/mol.112.081539 -
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 -
Neuro-oncology Advances 2023IDH-wildtype glioblastoma (GBM) is a highly malignant primary brain tumor with a median survival of 15 months after standard of care, which highlights the need for...
BACKGROUND
IDH-wildtype glioblastoma (GBM) is a highly malignant primary brain tumor with a median survival of 15 months after standard of care, which highlights the need for improved therapy. Personalized combination therapy has shown to be successful in many other tumor types and could be beneficial for GBM patients.
METHODS
We performed the largest drug combination screen to date in GBM, using a high-throughput effort where we selected 90 drug combinations for their activity onto 25 patient-derived GBM cultures. 43 drug combinations were selected for interaction analysis based on their monotherapy efficacy and were tested in a short-term (3 days) as well as long-term (18 days) assay. Synergy was assessed using dose-equivalence and multiplicative survival metrics.
RESULTS
We observed a consistent synergistic interaction for 15 out of 43 drug combinations on patient-derived GBM cultures. From these combinations, 11 out of 15 drug combinations showed a longitudinal synergistic effect on GBM cultures. The highest synergies were observed in the drug combinations Lapatinib with Thapsigargin and Lapatinib with Obatoclax Mesylate, both targeting epidermal growth factor receptor and affecting the apoptosis pathway. To further elaborate on the apoptosis cascade, we investigated other, more clinically relevant, apoptosis inducers and observed a strong synergistic effect while combining Venetoclax (BCL targeting) and AZD5991 (MCL1 targeting).
CONCLUSIONS
Overall, we have identified via a high-throughput drug screening several new treatment strategies for GBM. Moreover, an exceptionally strong synergistic interaction was discovered between kinase targeting and apoptosis induction which is suitable for further clinical evaluation as multi-targeted combination therapy.
PubMed: 37455945
DOI: 10.1093/noajnl/vdad073 -
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 -
Oncotarget Mar 2016Obatoclax, a pan-inhibitor of anti-apoptotic Bcl-2 proteins, exhibits cytotoxic effect on cancer cells through both apoptosis-dependent and -independent pathways. Here...
Obatoclax, a pan-inhibitor of anti-apoptotic Bcl-2 proteins, exhibits cytotoxic effect on cancer cells through both apoptosis-dependent and -independent pathways. Here we show that obatoclax caused cytotoxicity in both cisplatin-sensitive and -resistant esophageal cancer cells. Although obatoclax showed differential apoptogenic effects in these cells, it consistently blocked autophagic flux, which was evidenced by concomitant accumulation of LC3-II and p62. Obatoclax was trapped in lysosomes and induced lysosome clustering. Obatoclax also substantially reduced the expression of lysosomal cathepsins B, D and L. Moreover, cathepsin knockdown was sufficient to induce cytotoxicity, connecting lysosomal function to cell viability. Consistent with the known function of autophagy, obatoclax caused the accumulation of polyubiquitinated proteins and showed synergy with proteasome inhibition. Taken together, our studies unveiled impaired lysosomal function as a novel mechanism whereby obatoclax mediates its cytotoxic effect in esophageal cancer cells.
Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Cathepsin B; Cell Proliferation; Cisplatin; Drug Resistance, Neoplasm; Enzyme Inhibitors; Esophageal Neoplasms; Humans; Indoles; Lysosomes; Pyrroles; Tumor Cells, Cultured
PubMed: 26910910
DOI: 10.18632/oncotarget.7492 -
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 -
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 -
Oral Oncology Feb 2014More than half of head and neck squamous cell carcinoma (HNSCC) patients are initially treated with curative intent, but will relapse over the course of their disease...
OBJECTIVES
More than half of head and neck squamous cell carcinoma (HNSCC) patients are initially treated with curative intent, but will relapse over the course of their disease and have poor prognosis with a median survival of approximately 6months. Novel therapeutic approaches are in desperate need for this patient population. The anti-apoptotic BCL-2 family proteins such as BCL-2, BCL-XL, and MCL-1 are involved in oncogenesis and chemoresistance and are overexpressed in HNSCC. Obatoclax is a small-molecule antagonist of the BH3-binding groove of anti-apoptotic BCL-2 family. We evaluated the activity of obatoclax against 4 HNSCC cell lines (UMSCC-1, Cal33, 1483, UMSCC-22A).
METHODS
Cell viability was determined by MTT assay, cell cycle status by propidium iodide staining, and apoptosis by Annexin-V staining and immunoblotting. Autophagy was assessed by immunofluorescence and immunoblotting.
RESULTS
All four HNSCC cell lines were highly sensitive to single-agent obatoclax with IC50's ranging from 46 to 177nM. Obatoclax induced apoptosis in all four HNSCC cell lines as evidenced by increases in sub-G1 DNA content, Annexin-V staining, and PARP cleavage. In addition, obatoclax induced autophagy in all 4 cell lines, and the addition of the autophagy inhibitor chloroquine enhanced obatoclax cytotoxicity.
CONCLUSION
Our findings demonstrate potent monotherapeutic activity of obatoclax against HNSCC cells, and enhancement of this activity in the presence of chloroquine. This preclinical study suggests that obatoclax might have therapeutic value in the treatment of HNSCC, either alone or in combination with inhibitors of autophagy.
Topics: Apoptosis; Autophagy; Carcinoma, Squamous Cell; Cell Survival; Head and Neck Neoplasms; Humans; Indoles; Inhibitory Concentration 50; Proto-Oncogene Proteins c-bcl-2; Pyrroles
PubMed: 24216166
DOI: 10.1016/j.oraloncology.2013.10.013 -
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