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International Journal of Molecular... Dec 2016Colorectal cancer is the third most common cancer worldwide. Aberrant overexpression of antiapoptotic BCL-2 (B-cell lymphoma 2) family proteins is closely linked to...
Colorectal cancer is the third most common cancer worldwide. Aberrant overexpression of antiapoptotic BCL-2 (B-cell lymphoma 2) family proteins is closely linked to tumorigenesis and poor prognosis in colorectal cancer. Obatoclax is an inhibitor targeting all antiapoptotic BCL-2 proteins. A previous study has described the antiproliferative action of obatoclax in one human colorectal cancer cell line without elucidating the underlying mechanisms. We herein reported that, in a panel of human colorectal cancer cell lines, obatoclax inhibits cell proliferation, suppresses clonogenicity, and induces G₁-phase cell cycle arrest, along with cyclin D1 downregulation. Notably, ectopic cyclin D1 overexpression abrogated clonogenicity suppression but also G₁-phase arrest elicited by obatoclax. Mechanistically, pre-treatment with the proteasome inhibitor MG-132 restored cyclin D1 levels in all obatoclax-treated cell lines. Cycloheximide chase analyses further revealed an evident reduction in the half-life of cyclin D1 protein by obatoclax, confirming that obatoclax downregulates cyclin D1 through induction of cyclin D1 proteasomal degradation. Lastly, threonine 286 phosphorylation of cyclin D1, which is essential for initiating cyclin D1 proteasomal degradation, was induced by obatoclax in one cell line but not others. Collectively, we reveal a novel anticancer mechanism of obatoclax by validating that obatoclax targets cyclin D1 for proteasomal degradation to downregulate cyclin D1 for inducing antiproliferation.
Topics: Carcinoma; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Down-Regulation; G1 Phase Cell Cycle Checkpoints; HCT116 Cells; HT29 Cells; Humans; Indoles; Proteolysis; Proto-Oncogene Proteins c-bcl-2; Pyrroles
PubMed: 28035994
DOI: 10.3390/ijms18010044 -
Antimicrobial Agents and Chemotherapy Mar 2017As new pathogenic viruses continue to emerge, it is paramount to have intervention strategies that target a common denominator in these pathogens. The fusion of viral...
As new pathogenic viruses continue to emerge, it is paramount to have intervention strategies that target a common denominator in these pathogens. The fusion of viral and cellular membranes during viral entry is one such process that is used by many pathogenic viruses, including chikungunya virus, West Nile virus, and influenza virus. Obatoclax, a small-molecule antagonist of the Bcl-2 family of proteins, was previously determined to have activity against influenza A virus and also Sindbis virus. Here, we report it to be active against alphaviruses, like chikungunya virus (50% effective concentration [EC] = 0.03 μM) and Semliki Forest virus (SFV; EC = 0.11 μM). Obatoclax inhibited viral entry processes in an SFV temperature-sensitive mutant entry assay. A neutral red retention assay revealed that obatoclax induces the rapid neutralization of the acidic environment of endolysosomal vesicles and thereby most likely inhibits viral fusion. Characterization of escape mutants revealed that the L369I mutation in the SFV E1 fusion protein was sufficient to confer partial resistance against obatoclax. Other inhibitors that target the Bcl-2 family of antiapoptotic proteins inhibited neither viral entry nor endolysosomal acidification, suggesting that the antiviral mechanism of obatoclax does not depend on its anticancer targets. Obatoclax inhibited the growth of flaviviruses, like Zika virus, West Nile virus, and yellow fever virus, which require low pH for fusion, but not that of pH-independent picornaviruses, like coxsackievirus A9, echovirus 6, and echovirus 7. In conclusion, obatoclax is a novel inhibitor of endosomal acidification that prevents viral fusion and that could be pursued as a potential broad-spectrum antiviral candidate.
Topics: Animals; Antiviral Agents; Cell Line; Cell Membrane; Chikungunya virus; Cricetinae; Drug Resistance, Viral; Endosomes; Epithelial Cells; Gene Expression; Hepatocytes; Humans; Hydrogen-Ion Concentration; Indoles; Lysosomes; Membrane Fusion; Membrane Glycoproteins; Mutation; Neutral Red; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Semliki forest virus; Viral Envelope Proteins; Virus Internalization; Virus Replication; West Nile virus; Yellow fever virus; Zika Virus
PubMed: 27993855
DOI: 10.1128/AAC.02227-16 -
The FEBS Journal Jan 2017Influenza A viruses (IAVs) remain serious threats to public health because of the shortage of effective means of control. Developing more effective virus control...
Influenza A viruses (IAVs) remain serious threats to public health because of the shortage of effective means of control. Developing more effective virus control modalities requires better understanding of virus-host interactions. It has previously been shown that IAV induces the production of kynurenine, which suppresses T-cell responses, enhances pain hypersensitivity and disturbs behaviour in infected animals. However, the regulation of kynurenine biosynthesis during IAV infection remains elusive. Here we showed that IAV infection induced expression of interferons (IFNs), which upregulated production of indoleamine-2,3-dioxygenase (IDO1), which catalysed the kynurenine biosynthesis. Furthermore, IAV attenuated the IDO1 expression and the production of kynurenine through its NS1 protein. Interestingly, inhibition of viral replication prior to IFN induction limited IDO1 expression, while inhibition after did not. Finally, we showed that kynurenine biosynthesis was activated in macrophages in response to other stimuli, such as influenza B virus, herpes simplex virus 1 and 2 as well as bacterial lipopolysaccharides. Thus, the tight regulation of the kynurenine biosynthesis by host cell and, perhaps, pathogen might be a basic signature of a wide range of host-pathogen interactions, which should be taken into account during development of novel antiviral and antibacterial drugs.
Topics: Animals; Antiviral Agents; Female; Gene Expression Regulation; Host-Pathogen Interactions; Humans; Immunologic Factors; Indoleamine-Pyrrole 2,3,-Dioxygenase; Indoles; Influenza A Virus, H1N1 Subtype; Interferons; Kynurenine; Lung; Macrophages; Metabolic Networks and Pathways; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Oxazoles; Oximes; Primary Cell Culture; Pyrroles; Sulfonamides; Thiazoles; Transcriptome; Tryptophan; Viral Nonstructural Proteins; Virus Replication
PubMed: 27860276
DOI: 10.1111/febs.13966 -
Oncotarget Oct 2016Multiple myeloma (MM) is a malignancy characterized by monoclonal paraproteinemia and tissue plasmocytosis. In advanced MM cytopenia and osteopathy may occur. Although...
Multiple myeloma (MM) is a malignancy characterized by monoclonal paraproteinemia and tissue plasmocytosis. In advanced MM cytopenia and osteopathy may occur. Although several effective treatment strategies have been developed in recent years, there is still a need to identify new drug targets and to develop more effective therapies for patients with advanced MM. We examined the effects of 15 targeted drugs on growth and survival of primary MM cells and 5 MM cell lines (MM.1S, NCI-H929, OPM-2, RPMI-8226, U-266). The PI3-kinase blocker BEZ235, the pan-BCL-2 inhibitor obatoclax, the Hsp90-targeting drug 17AAG, and the Polo-like kinase-1 inhibitor BI2536, were found to exert major growth-inhibitory effects in all 5 MM cell lines tested. Moreover, these drugs suppressed the in vitro proliferation of primary bone marrow-derived MM cells and induced apoptosis at pharmacologic drug concentrations. Apoptosis-inducing effects were not only seen in the bulk of MM cells but also in MM stem cell-containing CD138-/CD20+/CD27+ memory B-cell fractions. Synergistic growth-inhibitory effects were observed in MM cell lines using various drug combinations, including 17AAG+BI2536 in MM.1S, OPM-2, RPMI-8226, and U-266 cells, 17AAG+BEZ235 in MM.1S, OPM-2, RPMI-8226, and U-266 cells, 17AAG+obatoclax in MM.1S, NCI-H929, OPM-2, and RPMI-8226 cells, BI2536+BEZ235 in MM.1S, NCI-H929, OPM-2, and RPMI-8226 cells, BI2536+obatoclax in MM.1S, OPM-2 and RPMI-8226 cells, and BEZ235+obatoclax in MM.1S and RPMI-8226 cells. Together, our data show that various targeted drugs induce profound and often synergistic anti-neoplastic effects in MM cells which may have clinical implications and may contribute to the development of novel treatment strategies in advanced MM.
Topics: Aged; Aged, 80 and over; Antineoplastic Agents; Apoptosis; Cell Proliferation; Female; Humans; In Vitro Techniques; Male; Middle Aged; Multiple Myeloma; Neoplastic Stem Cells; Plasma Cells; Prognosis; Tumor Cells, Cultured
PubMed: 27582537
DOI: 10.18632/oncotarget.11593 -
Oncotarget Aug 2016Bcl2 and IAP families are anti-apoptotic proteins deregulated in multiple myeloma (MM) cells. Pharmacological inhibition of each of these families has shown significant...
Bcl2 and IAP families are anti-apoptotic proteins deregulated in multiple myeloma (MM) cells. Pharmacological inhibition of each of these families has shown significant activity only in subgroups of MM patients. Here, we have examined a broad-spectrum Bcl2 family inhibitor Obatoclax (OBX) in combination with a Smac mimetic LCL161 in MM cell lines and patient cells. LCL161/OBX combination induced synergistic cytotoxicity and anti-proliferative effects on a broad range of human MM cell lines. The cytotoxicity was mediated through inhibition of the IAPs, activation of caspases and up regulation of the pro-apoptotic proteins Bid, Bim, Puma and Noxa by the drug combination. In addition, we observed that OBX caused ER stress and activated the Unfolded Protein Response (UPR) leading to drug resistance. LCL161, however inhibited spliced Xbp-1, a pro-survival factor. In addition, we observed that OBX increased GRP78 localization to the cell surface, which then induced PI3K dependent Akt activation and resistance to cell death. LCL161 was able to block OBX induced Akt activation contributing to synergistic cell death. Our results support clinical evaluation of this combination strategy in relapsed refractory MM patients.
Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Humans; Indoles; Intracellular Signaling Peptides and Proteins; Mitochondrial Proteins; Multiple Myeloma; Neoplasm Recurrence, Local; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Thiazoles; Unfolded Protein Response; Up-Regulation; X-Box Binding Protein 1
PubMed: 27494845
DOI: 10.18632/oncotarget.11028 -
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 -
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 -
Cell Communication and Signaling : CCS Mar 2016Protein-protein interactions (PPI) are involved in virtually every cellular process and thus represent an attractive target for therapeutic interventions. A significant... (Review)
Review
Protein-protein interactions (PPI) are involved in virtually every cellular process and thus represent an attractive target for therapeutic interventions. A significant number of protein interactions are frequently formed between globular domains and short linear peptide motifs (DMI). Targeting these DMIs has proven challenging and classical approaches to inhibiting such interactions with small molecules have had limited success. However, recent new approaches have led to the discovery of potent inhibitors, some of them, such as Obatoclax, ABT-199, AEG-40826 and SAH-p53-8 are likely to become approved drugs. These novel inhibitors belong to a wide range of different molecule classes, ranging from small molecules to peptidomimetics and biologicals. This article reviews the main reasons for limited success in targeting PPIs, discusses how successful approaches overcome these obstacles to discovery promising inhibitors for human protein double minute 2 (HDM2), B-cell lymphoma 2 (Bcl-2), X-linked inhibitor of apoptosis protein (XIAP), and provides a summary of the promising approaches currently in development that indicate the future potential of PPI inhibitors in drug discovery.
Topics: Animals; Drug Discovery; Humans; Macrocyclic Compounds; Models, Molecular; Peptidomimetics; Protein Interaction Domains and Motifs; Protein Interaction Maps; Small Molecule Libraries
PubMed: 26936767
DOI: 10.1186/s12964-016-0131-4 -
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 -
Tumour Biology : the Journal of the... Aug 2016Obatoclax and ABT-737 belong to a new class of anticancer agents known as BH3-mimetics. These agents antagonize the anti-apoptotic members of Bcl-2 family. The Bcl-2... (Comparative Study)
Comparative Study
Obatoclax and ABT-737 belong to a new class of anticancer agents known as BH3-mimetics. These agents antagonize the anti-apoptotic members of Bcl-2 family. The Bcl-2 proteins modulate sensitivity of many types of cancer cells to chemotherapy. Therefore, the objective of the present study was to examine and compare the antileukemic activity of obatoclax and ABT-737 applied alone, and in combination with anticancer agent, mafosfamide and daunorubicin. The in vitro cytotoxic effects of the tested agents on human leukemia cells were determined using the spectrophotometric MTT test, Coulter electrical impedance method, flow cytometry annexin V-fluorescein/propidium iodide assay, and light microscopy technique. The combination index analysis was used to quantify the extent of agent interactions. BH3 mimetics significantly decreased the leukemia cell viability and synergistically enhanced the cytotoxic effects induced by mafosfamide and daunorubicin. Obatoclax affected the cell viability to a greater degree than did ABT-737. In addition, various patterns of temporary changes in the cell volume and count, and in the frequency of leukemia cells undergoing apoptosis, were found 24 and 48 h after the tested agent application. ABT-737 combined with anticancer agents induced apoptosis more effectively than obatoclax when given in the same combination regimen. The results of the present study point to the different antileukemic activities of obatoclax and ABT-737, when applied alone, and in combination with anticancer agents. A better understanding of the exact mechanisms of BH3 mimetic action is of key importance for their optional use in cancer therapy.
Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Cell Survival; Flow Cytometry; Humans; Indoles; Leukemia; Nitrophenols; Piperazines; Pyrroles; Sulfonamides
PubMed: 26880588
DOI: 10.1007/s13277-016-4943-z