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Cell Stem Cell Mar 2013Most forms of chemotherapy employ mechanisms involving induction of oxidative stress, a strategy that can be effective due to the elevated oxidative state commonly...
Most forms of chemotherapy employ mechanisms involving induction of oxidative stress, a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However, recent studies have shown that relative redox levels in primary tumors can be heterogeneous, suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies, we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First, the majority of functionally defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed "ROS-low"). Second, ROS-low LSCs aberrantly overexpress BCL-2. Third, BCL-2 inhibition reduced oxidative phosphorylation and selectively eradicated quiescent LSCs. Based on these findings, we propose a model wherein the unique physiology of ROS-low LSCs provides an opportunity for selective targeting via disruption of BCL-2-dependent oxidative phosphorylation.
Topics: Apoptosis; Humans; Indoles; Leukemia, Myeloid, Acute; Neoplastic Stem Cells; Oxidative Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Reactive Oxygen Species; Tumor Cells, Cultured
PubMed: 23333149
DOI: 10.1016/j.stem.2012.12.013 -
Cancers Dec 2018Paclitaxel is a treatment option for advanced or metastatic bladder cancer after the failure of first-line cisplatin and gemcitabine, although resistance limits its...
Paclitaxel is a treatment option for advanced or metastatic bladder cancer after the failure of first-line cisplatin and gemcitabine, although resistance limits its clinical benefits. Mcl-1 is an anti-apoptotic protein that promotes resistance to paclitaxel in different tumors. Obatoclax, a BH3 mimetic of the Bcl-2 family of proteins, antagonizes Mcl-1 and hence may reverse paclitaxel resistance in Mcl-1-overexpressing tumors. In this study, paclitaxel-sensitive 5637 and -resistant HT1197 bladder cancer cells were treated with paclitaxel, obatoclax, or combinations of both. Apoptosis, cell cycle, and autophagy were measured by Western blot, flow cytometry, and fluorescence microscopy. Moreover, Mcl-1 expression was analyzed by immunohistochemistry in bladder carcinoma tissues. Our results confirmed that paclitaxel alone induced Mcl-1 downregulation and apoptosis in 5637, but not in HT1197 cells; however, combinations of obatoclax and paclitaxel sensitized HT1197 cells to the treatment. In obatoclax-treated 5637 and obatoclax + paclitaxel-treated HT1197 cells, the blockade of the autophagic flux correlated with apoptosis and was associated with caspase-dependent cleavage of beclin-1. Obatoclax alone delayed the cell cycle in 5637, but not in HT1197 cells, whereas combinations of both retarded the cell cycle and reduced mitotic slippage. In conclusion, obatoclax sensitizes HT1197 cells to paclitaxel-induced apoptosis through the blockade of the autophagic flux and effects on the cell cycle. Furthermore, Mcl-1 is overexpressed in many invasive bladder carcinomas, and it is related to tumor progression, so Mcl-1 expression may be of predictive value in bladder cancer.
PubMed: 30563080
DOI: 10.3390/cancers10120490 -
Pharmaceutics Dec 2022Metastasis is the primary cause of death in cancer patients. Many current chemotherapeutic agents only show cytotoxic, but not antimetastatic properties. This leads to a...
Metastasis is the primary cause of death in cancer patients. Many current chemotherapeutic agents only show cytotoxic, but not antimetastatic properties. This leads to a reduction in tumor size, but allows cancer cells to disseminate, which ultimately causes patient death. Therefore, novel anticancer compounds with both effects need to be developed. In this work, we analyze the antimetastatic properties of prodigiosin and obatoclax (GX15-070), anticancer drugs of the Prodiginines (PGs) family. We studied PGs' effects on cellular adhesion and morphology in the human primary and metastatic melanoma cell lines, SK-MEL-28 and SK-MEL-5, and in the murine melanoma cell line, B16F10A. Cell adhesion sharply decreased in the treated cells, and this was accompanied by a reduction in filopodia protrusions and a significant decrease in the number of focal-adhesion structures. Moreover, cell migration was assessed through the wound-healing assay and cell motility was severely inhibited after 24 h of treatment. To elucidate the molecular mechanisms involved, changes in metastasis-related genes were analyzed through a gene-expression array. Key genes related to cellular invasion, migration and chemoresistance were significantly down-regulated. Finally, an in vivo model of melanoma-induced lung metastasis was established and significant differences in lung tumors were observed in the obatoclax-treated mice. Altogether, these results describe, in depth, PGs' cellular antimetastatic effects and identify in vivo antimetastatic properties of Obatoclax.
PubMed: 36678726
DOI: 10.3390/pharmaceutics15010097 -
Cells Sep 2023Aging is associated with the disruption of protein homeostasis and causally contributes to multiple diseases, including amyotrophic lateral sclerosis (ALS). One strategy...
Aging is associated with the disruption of protein homeostasis and causally contributes to multiple diseases, including amyotrophic lateral sclerosis (ALS). One strategy for restoring protein homeostasis and protecting neurons against age-dependent diseases such as ALS is to de-repress autophagy. BECN1 is a master regulator of autophagy; however, is repressed by BCL2 via a BH3 domain-mediated interaction. We used an induced pluripotent stem cell model of ALS caused by mutant FUS to identify a small molecule BH3 mimetic that disrupts the BECN1-BCL2 interaction. We identified obatoclax as a brain-penetrant drug candidate that rescued neurons at nanomolar concentrations by reducing cytoplasmic FUS levels, restoring protein homeostasis, and reducing degeneration. Proteomics data suggest that obatoclax protects neurons via multiple mechanisms. Thus, obatoclax is a candidate for repurposing as a possible ALS therapeutic and, potentially, for other age-associated disorders linked to defects in protein homeostasis.
Topics: Humans; Amyotrophic Lateral Sclerosis; Motor Neurons; Induced Pluripotent Stem Cells; Mutation; Autophagy; Phenotype; Proto-Oncogene Proteins c-bcl-2; RNA-Binding Protein FUS
PubMed: 37759469
DOI: 10.3390/cells12182247 -
Emerging Microbes & Infections Dec 2022(COVID-19) caused by the emerging (SARS-CoV-2) has set off a global pandemic. There is an urgent unmet need for safe, affordable, and effective therapeutics against...
(COVID-19) caused by the emerging (SARS-CoV-2) has set off a global pandemic. There is an urgent unmet need for safe, affordable, and effective therapeutics against COVID-19. In this regard, drug repurposing is considered as a promising approach. We assessed the compounds that affect the endosomal acidic environment by applying human angiotensin-converting enzyme 2 (hACE2)- expressing cells infected with a SARS-CoV-2 spike (S) protein-pseudotyped HIV reporter virus and identified that obatoclax resulted in the strongest inhibition of S protein-mediated virus entry. The potent antiviral activity of obatoclax at nanomolar concentrations was confirmed in different human lung and intestinal cells infected with the SARS-CoV-2 pseudotype system as well as clinical virus isolates. Furthermore, we uncovered that obatoclax executes a double-strike against SARS-CoV-2. It prevented SARS-CoV-2 entry by blocking endocytosis of virions through diminished endosomal acidification and the corresponding inhibition of the enzymatic activity of the endosomal cysteine protease cathepsin L. Additionally, obatoclax impaired the SARS-CoV-2 S-mediated membrane fusion by targeting the MCL-1 protein and reducing furin protease activity. In accordance with these overarching mechanisms, obatoclax blocked the virus entry mediated by different S proteins derived from several SARS-CoV-2 variants of concern such as, Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2). Taken together, our results identified obatoclax as a novel effective antiviral compound that keeps SARS-CoV-2 at bay by blocking both endocytosis and membrane fusion. Our data suggested that obatoclax should be further explored as a clinical drug for the treatment of COVID-19.
Topics: COVID-19; Cathepsins; Furin; Humans; Hydrogen-Ion Concentration; Indoles; Pyrroles; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Virus Internalization
PubMed: 34989664
DOI: 10.1080/22221751.2022.2026739 -
PloS One 2016Obatoclax belongs to a class of compounds known as BH3 mimetics which function as antagonists of Bcl-2 family apoptosis regulators. It has undergone extensive...
Obatoclax belongs to a class of compounds known as BH3 mimetics which function as antagonists of Bcl-2 family apoptosis regulators. It has undergone extensive preclinical and clinical evaluation as a cancer therapeutic. Despite this, it is clear that obatoclax has additional pharmacological effects that contribute to its cytotoxic activity. It has been claimed that obatoclax, either alone or in combination with other molecularly targeted therapeutics, induces an autophagic form of cell death. In addition, obatoclax has been shown to inhibit lysosomal function, but the mechanism of this has not been elucidated. We have evaluated the mechanism of action of obatoclax in eight ovarian cancer cell lines. Consistent with its function as a BH3 mimetic, obatoclax induced apoptosis in three cell lines. However, in the remaining cell lines another form of cell death was evident because caspase activation and PARP cleavage were not observed. Obatoclax also failed to show synergy with carboplatin and paclitaxel, chemotherapeutic agents which we have previously shown to be synergistic with authentic Bcl-2 family antagonists. Obatoclax induced a profound accumulation of LC-3 but knockdown of Atg-5 or beclin had only minor effects on the activity of obatoclax in cell growth assays suggesting that the inhibition of lysosomal function rather than stimulation of autophagy may play a more prominent role in these cells. To evaluate how obatoclax inhibits lysosomal function, confocal microscopy studies were conducted which demonstrated that obatoclax, which contains two basic pyrrole groups, accumulates in lysosomes. Studies using pH sensitive dyes demonstrated that obatoclax induced lysosomal alkalinization. Furthermore, obatoclax was synergistic in cell growth/survival assays with bafilomycin and chloroquine, two other drugs which cause lysosomal alkalinization. These studies explain, for the first time, how obatoclax inhibits lysosomal function and suggest that lysosomal alkalinization contributes to the cytotoxic activity of obatoclax.
Topics: Biological Transport; Biomimetic Materials; Cathepsins; Cell Death; Cell Line, Tumor; Humans; Hydrogen-Ion Concentration; Indoles; Lysosomes; Proto-Oncogene Proteins c-bcl-2; Pyrroles
PubMed: 26950068
DOI: 10.1371/journal.pone.0150696 -
PLoS Neglected Tropical Diseases Mar 2020Parasitic infections are a major source of human suffering, mortality, and economic loss, but drug development for these diseases has been stymied by the significant...
Parasitic infections are a major source of human suffering, mortality, and economic loss, but drug development for these diseases has been stymied by the significant expense involved in bringing a drug though clinical trials and to market. Identification of single compounds active against multiple parasitic pathogens could improve the economic incentives for drug development as well as simplifying treatment regimens. We recently performed a screen of repurposed compounds against the protozoan parasite Entamoeba histolytica, causative agent of amebic dysentery, and identified four compounds (anisomycin, prodigiosin, obatoclax and nithiamide) with low micromolar potency and drug-like properties. Here, we extend our investigation of these drugs. We assayed the speed of killing of E. histolytica trophozoites and found that all four have more rapid action than the current drug of choice, metronidazole. We further established a multi-institute collaboration to determine whether these compounds may have efficacy against other parasites and opportunistic pathogens. We found that anisomycin, prodigiosin and obatoclax all have broad-spectrum antiparasitic activity in vitro, including activity against schistosomes, T. brucei, and apicomplexan parasites. In several cases, the drugs were found to have significant improvements over existing drugs. For instance, both obatoclax and prodigiosin were more efficacious at inhibiting the juvenile form of Schistosoma than the current standard of care, praziquantel. Additionally, low micromolar potencies were observed against pathogenic free-living amebae (Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba castellanii), which cause CNS infection and for which there are currently no reliable treatments. These results, combined with the previous human use of three of these drugs (obatoclax, anisomycin and nithiamide), support the idea that these compounds could serve as the basis for the development of broad-spectrum anti-parasitic drugs.
Topics: Animals; Anisomycin; Antiparasitic Agents; Cell Line; Cell Survival; Drug Repositioning; Fibroblasts; Humans; Indoles; Mice; Parasites; Parasitic Sensitivity Tests; Prodigiosin; Pyrroles; Rats
PubMed: 32196500
DOI: 10.1371/journal.pntd.0008150 -
International Journal of Molecular... Mar 2020Colorectal cancer (CRC) is a highly prevailing cancer and the fourth leading cause of cancer mortality worldwide. Aberrant expression of antiapoptotic BCL-2 family...
Colorectal cancer (CRC) is a highly prevailing cancer and the fourth leading cause of cancer mortality worldwide. Aberrant expression of antiapoptotic BCL-2 family proteins is closely linked to neoplastic progression and chemoresistance. Obatoclax is a clinically developed drug, which binds antiapoptotic BCL-2, BCL-xL, and MCL-1 for inhibition to elicit apoptosis. Survivin is an antiapoptotic protein, whose upregulation correlates with pathogenesis, therapeutic resistance, and poor prognosis in CRC. Herein, we provide the first evidence delineating the functional linkage between Obatoclax and survivin in the context of human CRC cells. In detail, Obatoclax was found to markedly downregulate survivin. This downregulation was mainly achieved via transcriptional repression, as Obatoclax lowered the levels of both mRNA and promoter activity, while blocking proteasomal degradation failed to prevent survivin from downregulation by Obatoclax. Notably, ectopic survivin expression curtailed Obatoclax-induced apoptosis and cytotoxicity, confirming an essential role of survivin downregulation in Obatoclax-elicited anti-CRC effect. Moreover, Obatoclax was found to repress hyperactive WNT/β-catenin signaling activity commonly present in human CRC cells, and, markedly, ectopic expression of dominant-active β-catenin mutant rescued the levels of survivin along with elevated cell viability. We further revealed that, depending on the cell context, Obatoclax suppresses WNT/β-catenin signaling in HCT 116 cells likely via inducing β-catenin destabilization, or by downregulating LEF1 in DLD-1 cells. Collectively, we for the first time define survivin downregulation as a novel, pro-apoptotic mechanism of Obatoclax as a consequence of Obatocalx acting as an antagonist to WNT/β-catenin signaling.
Topics: Apoptosis; Biomarkers, Tumor; Cell Proliferation; Colorectal Neoplasms; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Indoles; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Survivin; Tumor Cells, Cultured; Wnt Proteins; beta Catenin
PubMed: 32150830
DOI: 10.3390/ijms21051773 -
BMC Cancer Aug 2015Obatoclax is a clinical stage drug candidate that has been proposed to target and inhibit prosurvival members of the Bcl-2 family, and thereby contribute to cancer cell...
BACKGROUND
Obatoclax is a clinical stage drug candidate that has been proposed to target and inhibit prosurvival members of the Bcl-2 family, and thereby contribute to cancer cell lethality. The insolubility of this compound, however, has precluded the use of many classical drug-target interaction assays for its study. Thus, a direct demonstration of the proposed mechanism of action, and preferences for individual Bcl-2 family members, remain to be established.
METHODS
Employing modified proteins and lipids, we recapitulated the constitutive association and topology of mitochondrial outer membrane Mcl-1 and Bak in synthetic large unilamellar liposomes, and measured bakdependent bilayer permeability. Additionally, cellular and tumor models, dependent on Mcl-1 for survival, were employed.
RESULTS
We show that regulation of bilayer permeabilization by the tBid - Mcl-1 - Bak axis closely resemblesthe tBid - Bcl-XL - Bax model. Obatoclax rapidly and completely partitioned into liposomal lipid but also rapidly exchanged between liposome particles. In this system, obatoclax was found to be a direct and potent antagonist of liposome-bound Mcl-1 but not of liposome-bound Bcl-XL, and did not directly influence Bak. A 2.5 molar excess of obatoclax relative to Mcl-1 overcame Mcl-1-mediated inhibition of tBid-Bak activation. Similar results were found for induction of Bak oligomers by Bim. Obatoclax exhibited potent lethality in a cellmodel dependent on Mcl-1 for viability but not in cells dependent on Bcl-XL. Molecular modeling predicts that the 3-methoxy moiety of obatoclax penetrates into the P2 pocket of the BH3 binding site of Mcl-1. A desmethoxy derivative of obatoclax failed to inhibit Mcl-1 in proteoliposomes and did not kill cells whose survival depends on Mcl-1. Systemic treatment of mice bearing Tsc2(+) (/) (-) Em-myc lymphomas (whose cells depend on Mcl-1 for survival) with obatoclax conferred a survival advantage compared to vehicle alone (median 31 days vs 22 days, respectively; p=0.003). In an Akt-lymphoma mouse model, the anti-tumor effects of obatoclax synergized with doxorubicin. Finally, treatment of the multiple myeloma KMS11 cell model (dependent on Mcl-1 for survival) with dexamethasone induced Bim and Bim-dependent lethality. As predicted for an Mcl-1 antagonist, obatoclax and dexamethasone were synergistic in this model.
CONCLUSIONS
Taken together, these findings indicate that obatoclax is a potent antagonist of membranerestricted Mcl-1. Obatoclax represents an attractive chemical series to generate second generation Mcl-1 inhibitors.
Topics: Animals; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Cell Line, Tumor; Disease Models, Animal; Doxorubicin; Drug Synergism; Humans; Indoles; Lymphoma; Membrane Proteins; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins; Pyrroles; Xenograft Model Antitumor Assays
PubMed: 26231047
DOI: 10.1186/s12885-015-1582-5 -
British Journal of Haematology Jun 2011Bcl-2 proteins represent a rheostat that controls cellular viability. Obatoclax, a BH3-mimetic, has been designed to specifically target and counteract anti-apoptotic...
Bcl-2 proteins represent a rheostat that controls cellular viability. Obatoclax, a BH3-mimetic, has been designed to specifically target and counteract anti-apoptotic Bcl-2 proteins. We evaluated the biological effects of obatoclax on the anti-tumour activity of rituximab and chemotherapy agents. Obatoclax induced cell death of rituximab/chemotherapy-sensitive (RSCL), -resistant cell lines (RRCL) and primary tumour-cells derived from patients with B-cell lymphomas (N=39). Obatoclax also enhanced the activity of rituximab and had synergistic activity when combined with chemotherapy agents. The ability of Obatoclax to induce PARP cleavage varied between patient samples and was not observed in some RRCL. Inhibition of caspase activity did not affect obatoclax activity, suggesting the existence of caspase-independent death pathways. Autophagy was detected by LC3 conversion and/or electron microscopy in RRCL and in patient-derived tumour cells. Moreover, obatoclax activity was inhibited by Beclin-1 knockdown. In summary, obatoclax is an active Bcl-2 inhibitor that potentiates the activity of chemotherapy agents and, to a lesser degree, rituximab. Defining the molecular events triggered by obatoclax is necessary to further its clinical development and identify potential biomarkers that are predictive of response.
Topics: Antibodies, Monoclonal, Murine-Derived; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Combined Chemotherapy Protocols; Apoptosis Regulatory Proteins; Autophagy; Caspases; Cell Death; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Drug Synergism; Humans; Indoles; Lymphoma, B-Cell; Lymphoma, Follicular; Lymphoma, Large B-Cell, Diffuse; Neoplasm Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Rituximab; Tumor Cells, Cultured; Up-Regulation; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein
PubMed: 21492126
DOI: 10.1111/j.1365-2141.2011.08669.x