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1.
Cell Death Dis ; 13(5): 436, 2022 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-35508606

RESUMO

The recruitment of DRP1 to mitochondrial membranes prior to fission is facilitated by the wrapping of endoplasmic reticulum (ER) membranes around the mitochondria. To investigate the complex interplay between the ER membranes and DRP1 in the context of mitochondrial structure and function, we downregulate two key ER shaping proteins, RTN4 and CLIMP-63, and demonstrate pronounced mitochondrial hyperfusion and reduced ER-mitochondria contacts, despite their differential regulation of ER architecture. Although mitochondrial recruitment of DRP1 is unaltered in cells lacking RTN4 or CLIMP-63, several aspects of mitochondrial function, such as mtDNA-encoded translation, respiratory capacity and apoptosis are significantly hampered. Further mechanistic studies reveal that CLIMP-63 is required for cristae remodeling (OPA1 proteolysis) and DRP1-mediated mitochondrial fission, whereas both RTN4 and CLIMP-63 regulate the recruitment of BAX to ER and mitochondrial membranes to enable cytochrome c release and apoptosis, thereby performing novel and distinct roles in the regulation of mitochondrial structure and function.


Assuntos
Dinaminas , Mitocôndrias , Apoptose/genética , Dinaminas/metabolismo , Metabolismo Energético , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo
2.
Cell Death Dis ; 10(12): 912, 2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31801952

RESUMO

Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide, with overall survival of less than 50%. Current therapeutic strategies involving a combination of surgery, radiation, and/or chemotherapy are associated with debilitating side effects, highlighting the need for more specific and efficacious therapies. Inhibitors of BCL-2 family proteins (BH3 mimetics) are under investigation or in clinical practice for several hematological malignancies and show promise in solid tumors. In order to explore the therapeutic potential of BH3 mimetics in the treatment of SCCHN, we assessed the expression levels of BCL-2, BCL-XL, and MCL-1 via Western blots and immunohistochemistry, in cell lines, primary cells derived from SCCHN patients and in tissue microarrays containing tumor tissue from a cohort of 191 SCCHN patients. All preclinical models exhibited moderate to high levels of BCL-XL and MCL-1, with little or no BCL-2. Although expression levels of BCL-XL and MCL-1 did not correlate with patient outcome, a combination of BH3 mimetics to target these proteins resulted in decreased clonogenic potential and enhanced apoptosis in all preclinical models, including tumor tissue resected from patients, as well as a reduction of tumor volume in a zebrafish xenograft model of SCCHN. Our results show that SCCHN is dependent on both BCL-XL and MCL-1 for apoptosis evasion and combination therapy targeting both proteins may offer significant therapeutic benefits in this disease.


Assuntos
Fragmentos de Peptídeos/química , Proteínas Proto-Oncogênicas/química , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
3.
Cell Death Dis ; 10(7): 521, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285422

RESUMO

The endoplasmic reticulum (ER) with its elaborate network of highly curved tubules and flat sheets interacts with several other organelles, including mitochondria, peroxisomes and endosomes, to play vital roles in their membrane dynamics and functions. Previously, we identified structurally diverse chemicals from different pharmacological classes, which induce a reversible reorganisation of ER membranes. Using apogossypol as a prototypic tool compound, we now show that ER membrane reorganisation occurs at the level of ER tubules but does not involve ER sheets. Reorganisation of ER membranes prevents DRP-1-mediated mitochondrial fission, thereby antagonising the functions of several mitochondrial fission-inducing agents. Previous reports have suggested that ER membranes mark the constriction sites of mitochondria by localising DRP-1, as well as BAX on mitochondrial membranes to facilitate both mitochondrial fission and outer membrane permeabilisation. Following ER membrane reorganisation and subsequent exposure to an apoptotic stimulus (BH3 mimetics), DRP-1 still colocalises with the reorganised ER membranes but BAX translocation and activation, cytochrome c release and phosphatidylserine externalisation are all inhibited, thereby diminishing the ability of BH3 mimetics to induce the intrinsic apoptotic pathway. Strikingly, both ER membrane reorganisation and its resulting inhibition of apoptosis could be reversed by inhibitors of dihydroorotate dehydrogenase (DHODH), namely teriflunomide and its active metabolite, leflunomide. However, neither genetic inhibition of DHODH using RNA interference nor metabolic supplementation with orotate or uridine to circumvent the consequences of a loss of DHODH activity rescued the effects of DHODH inhibitors, suggesting that the effects of these inhibitors in preventing ER membrane reorganisation is most likely independent of their ability to antagonise DHODH activity. Our results strengthen the hypothesis that ER is fundamental for key mitochondrial functions, such as fusion-fission dynamics and apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Gossipol/análogos & derivados , Dinâmica Mitocondrial/efeitos dos fármacos , Crotonatos/farmacologia , Citocromos c/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Gossipol/farmacologia , Células HeLa , Humanos , Hidroxibutiratos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Leflunomida/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Nitrilas , Transporte Proteico/efeitos dos fármacos , Toluidinas/farmacologia , Proteína X Associada a bcl-2/metabolismo
4.
Cell Death Discov ; 5: 117, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31341643

RESUMO

Maintenance of mitochondrial integrity is critical for normal cellular homoeostasis. Most cells respond to stress stimuli and undergo apoptosis by perturbing mitochondrial structure and function to release proteins, such as cytochrome c, which are essential for the execution of the intrinsic apoptotic cascade. Cancer cells evade these events by overexpressing the anti-apoptotic BCL-2 family of proteins on mitochondrial membranes. Inhibitors of the anti-apoptotic BCL-2 family proteins, also known as BH3 mimetics, antagonise the pro-survival functions of these proteins and result in rapid apoptosis. Although the precise mechanism by which BH3 mimetics induce apoptosis has been well characterised, not much is known in terms of the structural changes that occur in mitochondria during apoptosis. Using a panel of highly selective BH3 mimetics and a wide range of cell lines, we demonstrate that BH3 mimetics induce extensive mitochondrial fission, accompanied by swelling of the mitochondrial matrix and rupture of the outer mitochondrial membrane. These changes occur in a BAX/ BAK-dependent manner. Although a major mitochondrial fission GTPase, DRP-1, has been implicated in mitochondrial apoptosis, our data demonstrate that DRP-1 might function independently/downstream of BH3 mimetic-mediated mitochondrial fission to facilitate the release of cytochrome c and apoptosis. Moreover, downregulation of DRP-1 prevented cytochrome c release and apoptosis even when OPA1, a protein mediating mitochondrial fusion, was silenced. Although BH3 mimetic-mediated displacement of BAK and other BH3-only proteins from BCL-XL and MCL-1 was unaffected by DRP-1 downregulation, it prevented BAK activation significantly, thus placing DRP-1 as one of the most critical players, along with BAX and BAK, that governs BH3 mimetic-mediated cytochrome c release and apoptosis.

5.
Biol Chem ; 400(2): 181-185, 2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-29924730

RESUMO

Induction of apoptosis by selective BH3-mimetics is currently investigated as a novel strategy for cancer treatment. Here, we report that selective BH3-mimetics induce apoptosis in a variety of hematological malignancies. Apoptosis is accompanied by severe mitochondrial toxicities upstream of caspase activation. Specifically, the selective BH3-mimetics ABT-199, A-1331852 and S63845, which target BCL-2, BCL-XL and MCL-1, respectively, induce comparable ultrastructural changes including mitochondrial swelling, a decrease of mitochondrial matrix density and severe loss of cristae structure. These shared effects on mitochondrial morphology indicate a similar function of these anti-apoptotic BCL-2 proteins in maintaining mitochondrial integrity and function.


Assuntos
Mitocôndrias/efeitos dos fármacos , Mimetismo Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteína bcl-X/efeitos dos fármacos , Apoptose , Caspases/metabolismo , Ativação Enzimática , Humanos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismo
6.
Cell Death Differ ; 26(6): 1037-1047, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30185825

RESUMO

The impressive selectivity and efficacy of BH3 mimetics for treating cancer has largely been limited to BCL-2 dependent hematological malignancies. Most solid tumors depend on other anti-apoptotic proteins, including MCL-1, for survival. The recent description of S63845 as the first specific and potent MCL-1 inhibitor represents an important therapeutic advance, since MCL-1 is not targeted by the currently available BH3 mimetics, Navitoclax or Venetoclax, and is commonly associated with chemoresistance. In this study, we confirm a high binding affinity and selectivity of S63845 to induce apoptosis in MCL-1-dependent cancer cell lines. Furthermore, S63845 synergizes with other BH3 mimetics to induce apoptosis in cell lines derived from both hematological and solid tumors. Although the anti-apoptotic BCL-2 family members in these cell lines interact with a spectrum of pro-apoptotic BH3-only proteins to regulate apoptosis, these interactions alone do not explain the relative sensitivities of these cell lines to BH3 mimetic-induced apoptosis. These findings necessitated further investigation into the requirement of BH3-only proteins in BH3 mimetic-mediated apoptosis. Concurrent inhibition of BCL-XL and MCL-1 by BH3 mimetics in colorectal HCT116 cells induced apoptosis in a BAX- but not BAK-dependent manner. Remarkably this apoptosis was independent of all known BH3-only proteins. Although BH3-only proteins were required for apoptosis induced as a result of BCL-XL inhibition, this requirement was overcome when both BCL-XL and MCL-1 were inhibited, implicating distinct mechanisms by which different anti-apoptotic BCL-2 family members may regulate apoptosis in cancer.


Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Pirimidinas/farmacologia , Tiofenos/farmacologia , Proteína bcl-X/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Relação Estrutura-Atividade , Proteína bcl-X/metabolismo
7.
Haematologica ; 104(5): 1016-1025, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30467206

RESUMO

BH3 mimetics are novel targeted drugs with remarkable specificity, potency and enormous potential to improve cancer therapy. However, acquired resistance is an emerging problem. We report the rapid development of resistance in chronic lymphocytic leukemia cells isolated from patients exposed to increasing doses of navitoclax (ABT-263), a BH3 mimetic. To mimic such rapid development of chemoresistance, we developed simple resistance models to three different BH3 mimetics, targeting BCL-2 (ABT-199), BCL-XL (A-1331852) or MCL-1 (A-1210477), in relevant hematologic cancer cell lines. In these models, resistance could not be attributed to either consistent changes in expression levels of the anti-apoptotic proteins or interactions among different pro- and anti-apoptotic BCL-2 family members. Using genetic silencing, pharmacological inhibition and metabolic supplementation, we found that targeting glutamine uptake and its downstream signaling pathways, namely glutaminolysis, reductive carboxylation, lipogenesis, cholesterogenesis and mammalian target of rapamycin signaling resulted in marked sensitization of the chemoresistant cells to BH3 mimetic-mediated apoptosis. Furthermore, our findings highlight the possibility of repurposing widely used drugs, such as statins, to target intermediary metabolism and improve the efficacy of BH3 mimetic therapy.


Assuntos
Antineoplásicos/farmacologia , Biomimética , Resistencia a Medicamentos Antineoplásicos , Glutamina/metabolismo , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Fragmentos de Peptídeos/química , Proteínas Proto-Oncogênicas/química , Benzotiazóis/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Colesterol/biossíntese , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Humanos , Indóis/farmacologia , Isoquinolinas/farmacologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Lipogênese/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Células Tumorais Cultivadas , Proteína bcl-X/antagonistas & inibidores
8.
Oncotarget ; 9(28): 20075-20088, 2018 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-29732004

RESUMO

Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, well-tolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.

9.
Cell Death Differ ; 25(3): 486-541, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29362479

RESUMO

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.


Assuntos
Morte Celular , Animais , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Necrose/metabolismo , Necrose/patologia
10.
Cell Death Dis ; 8(1): e2552, 2017 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-28079887

RESUMO

The concept of using BH3 mimetics as anticancer agents has been substantiated by the efficacy of selective drugs, such as Navitoclax and Venetoclax, in treating BCL-2-dependent haematological malignancies. However, most solid tumours depend on MCL-1 for survival, which is highly amplified in multiple cancers and a major factor determining chemoresistance. Most MCL-1 inhibitors that have been generated so far, while demonstrating early promise in vitro, fail to exhibit specificity and potency in a cellular context. To address the lack of standardised assays for benchmarking the in vitro binding of putative inhibitors before analysis of their cellular effects, we developed a rapid differential scanning fluorimetry (DSF)-based assay, and used it to screen a panel of BH3 mimetics. We next contrasted their binding signatures with their ability to induce apoptosis in a MCL-1 dependent cell line. Of all the MCL-1 inhibitors tested, only A-1210477 induced rapid, concentration-dependent apoptosis, which strongly correlated with a thermal protective effect on MCL-1 in the DSF assay. In cells that depend on both MCL-1 and BCL-XL, A-1210477 exhibited marked synergy with A-1331852, a BCL-XL specific inhibitor, to induce cell death. Despite this selectivity and potency, A-1210477 induced profound structural changes in the mitochondrial network in several cell lines that were not phenocopied following MCL-1 RNA interference or transcriptional repression, suggesting that A-1210477 induces mitochondrial fragmentation in an MCL-1-independent manner. However, A-1210477-induced mitochondrial fragmentation was dependent upon DRP-1, and silencing expression levels of DRP-1 diminished not just mitochondrial fragmentation but also BH3 mimetic-mediated apoptosis. These findings provide new insights into MCL-1 ligands, and the interplay between DRP-1 and the anti-apoptotic BCL-2 family members in the regulation of apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Proteínas Quinases Associadas com Morte Celular/genética , Neoplasias Hematológicas/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Benzotiazóis/administração & dosagem , Benzotiazóis/química , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/química , Linhagem Celular Tumoral , Proteínas Quinases Associadas com Morte Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Indóis/administração & dosagem , Indóis/química , Isoquinolinas/administração & dosagem , Isoquinolinas/química , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/química , Proteínas Proto-Oncogênicas/administração & dosagem , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/administração & dosagem , Sulfonamidas/química
11.
Br J Cancer ; 114(6): 638-41, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26954718

RESUMO

BACKGROUND: Anti-apoptotic BCL-2 family members antagonise apoptosis by sequestering their pro-apoptotic counterparts. The balance between the different BCL-2 family members forms the basis of BH3 profiling, a peptide-based technique used to predict chemosensitivity of cancer cells. Recent identification of cell-permeable, selective inhibitors of BCL-2, BCL-XL and MCL-1, further facilitates the determination of the BCL-2 family dependency of cancer cells. METHODS: We use BH3 profiling in combination with cell death analyses using a chemical inhibitor toolkit to assess chemosensitivity of cancer cells. RESULTS: Both BH3 profiling and the inhibitor toolkit effectively predict chemosensitivity of cells addicted to a single anti-apoptotic protein but a combination of both techniques is more instructive when cell survival depends on more than one anti-apoptotic protein. CONCLUSIONS: The inhibitor toolkit provides a rapid, inexpensive and simple means to assess the chemosensitivity of tumour cells and in conjunction with BH3 profiling offers much potential in personalising cancer therapy.


Assuntos
Materiais Biomiméticos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fragmentos de Peptídeos/análise , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas/análise , Sequência de Aminoácidos , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzotiazóis/farmacologia , Materiais Biomiméticos/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Humanos , Isoquinolinas/farmacologia , Dados de Sequência Molecular , Neoplasias/patologia , Fragmentos de Peptídeos/química , Proteínas Proto-Oncogênicas/química , Sulfonamidas/farmacologia
12.
Oncotarget ; 6(14): 12668-81, 2015 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-26059440

RESUMO

The anti-apoptotic BCL-2 family proteins are important targets for cancer chemotherapy. Specific and potent inhibitors of the BCL-2 family, such as ABT-263 (navitoclax) and ABT-199, are only effective against some members of the BCL-2 family but do not target MCL-1, which is commonly amplified in tumors and associated with chemoresistance. In this report, the selectivity and potency of two putative MCL-1 inhibitors, dinaciclib and maritoclax, were assessed. Although both compounds induced Bax/Bak- and caspase-9-dependent apoptosis, dinaciclib was more potent than maritoclax in downregulating MCL-1 and also in inducing apoptosis. However, the compounds induced apoptosis, even in cells lacking MCL-1, suggesting multiple mechanisms of cell death. Furthermore, maritoclax induced extensive mitochondrial fragmentation, and a Bax/Bak- but MCL-1-independent accumulation of mitochondrial reactive oxygen species (ROS), with an accompanying loss of complexes I and III of the electron transport chain. ROS scavengers, such as MitoQ, could not salvage maritoclax-mediated effects on mitochondrial structure and function. Taken together, our data demonstrate that neither dinaciclib nor maritoclax exclusively target MCL-1. Although dinaciclib is clearly not a specific MCL-1 inhibitor, its ability to rapidly downregulate MCL-1 may be beneficial in many clinical settings, where it may reverse chemoresistance or sensitize to other chemotherapeutic agents.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Compostos de Piridínio/farmacologia , Pirróis/farmacologia , Western Blotting , Linhagem Celular Tumoral , Óxidos N-Cíclicos , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Humanos , Indolizinas
13.
Cell Rep ; 5(2): 409-20, 2013 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-24120870

RESUMO

BAK activation represents a key step during apoptosis, but how it converts into a mitochondria-permeabilizing pore remains unclear. By further delineating the structural rearrangements involved, we reveal that BAK activation progresses through a series of independent steps: BH3-domain exposure, N-terminal change, oligomerization, and membrane insertion. Employing a "BCL-XL-addiction" model, we show that neutralization of BCL-XL by the BH3 mimetic ABT-737 resulted in death only when cells were reconstituted with BCL-XL:BAK, but not BCL-2/ BCL-XL:BIM complexes. Although this resembles the indirect model, release of BAK from BCL-XL did not result in spontaneous adoption of the pore conformation. Commitment to apoptosis required association of the direct activator BIM with oligomeric BAK promoting its conversion to a membrane-inserted pore. The sequential nature of this cascade provides multiple opportunities for other BCL-2 proteins to interfere with or promote BAK activation and unites aspects of the indirect and direct activation models.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2 , Compostos de Bifenilo/toxicidade , Células HEK293 , Humanos , Células Jurkat , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nitrofenóis/toxicidade , Piperazinas/toxicidade , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Sulfonamidas/toxicidade , Proteína Killer-Antagonista Homóloga a bcl-2/antagonistas & inibidores , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo
15.
Neoplasia ; 15(5): 568-78, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23633928

RESUMO

Owing to the high levels of antiapoptotic B-cell lymphoma 2 (BCL-2) family members observed in several cancers, there has been a major effort to develop inhibitors of the BCL2-family as chemotherapeutic agents. Of the different members in the BCL-2 family, myeloid cell leukemia sequence 1 (MCL-1) is commonly amplified in human tumors and is associated with their relapse and chemoresistance. As a result, specific inhibitors of MCL-1 are being designed to treat resistant tumors. However, there is increasing evidence for other nonapoptotic roles of the BCL-2 family, ranging from ionic homeostasis and autophagy to the regulation of fission-fusion dynamics in subcellular organelles, including the endoplasmic reticulum and mitochondria. In this study, we characterize the specificity of two novel putative MCL-1 inhibitors, BI97C1 (Sabutoclax) and BI112D1, in inducing apoptosis in a BAX/BAK-dependent manner and in an MCL-1-dependent system. In addition to their being proapoptotic, these inhibitors also cause enhanced mitochondrial fragmentation that accompanies a time-dependent loss of optic atrophy 1 (OPA1), suggesting an impairment of mitochondrial fusion. This mitochondrial fragmentation occurs independently of dynamin-related protein 1 (DRP1)-mediated fission activity and, unlike most apoptotic stimuli, occurs upstream of and/or independent of BAX, BAK, and other BH3-only proteins. Furthermore, this mitochondrial fragmentation occurred rapidly and preceded other hallmarks of apoptosis, including the loss in mitochondrial membrane potential and the release of cytochrome c. Although such mitochondrial fragmentation did not deplete total cellular adenosine triphosphate (ATP) or alter other mitochondrial complexes, there was significant accumulation of reactive oxygen species.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Gossipol/análogos & derivados , Mitocôndrias/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Compostos de Anilina/farmacologia , Animais , Linhagem Celular , Dinaminas , GTP Fosfo-Hidrolases/metabolismo , Gossipol/farmacologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sulfonamidas/farmacologia
16.
PLoS One ; 8(2): e56603, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23457590

RESUMO

Recently we described a new, evolutionarily conserved cellular stress response characterized by a reversible reorganization of endoplasmic reticulum (ER) membranes that is distinct from canonical ER stress and the unfolded protein response (UPR). Apogossypol, a putative broad spectrum BCL-2 family antagonist, was the prototype compound used to induce this ER membrane reorganization. Following microarray analysis of cells treated with apogossypol, we used connectivity mapping to identify a wide range of structurally diverse chemicals from different pharmacological classes and established their ability to induce ER membrane reorganization. Such structural diversity suggests that the mechanisms initiating ER membrane reorganization are also diverse and a major objective of the present study was to identify potentially common features of these mechanisms. In order to explore this, we used hierarchical clustering of transcription profiles for a number of chemicals that induce membrane reorganization and discovered two distinct clusters. One cluster contained chemicals with known effects on Ca(2+) homeostasis. Support for this was provided by the findings that ER membrane reorganization was induced by agents that either deplete ER Ca(2+) (thapsigargin) or cause an alteration in cellular Ca(2+) handling (calmodulin antagonists). Furthermore, overexpression of the ER luminal Ca(2+) sensor, STIM1, also evoked ER membrane reorganization. Although perturbation of Ca(2+) homeostasis was clearly one mechanism by which some agents induced ER membrane reorganization, influx of extracellular Na(+) but not Ca(2+) was required for ER membrane reorganization induced by apogossypol and the related BCL-2 family antagonist, TW37, in both human and yeast cells. Not only is this novel, non-canonical ER stress response evolutionary conserved but so also are aspects of the mechanism of formation of ER membrane aggregates. Thus perturbation of ionic homeostasis is important in the regulation of ER membrane reorganization.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Homeostase , Membranas Intracelulares/metabolismo , Calmodulina/antagonistas & inibidores , Análise por Conglomerados , Retículo Endoplasmático/efeitos dos fármacos , Gossipol/análogos & derivados , Gossipol/farmacologia , Células HeLa , Homeostase/efeitos dos fármacos , Humanos , Membranas Intracelulares/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Sódio/metabolismo , Molécula 1 de Interação Estromal
17.
Blood ; 117(26): 7145-54, 2011 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-21562047

RESUMO

Apoptosis in megakaryocytes results in the formation of platelets. The role of apoptotic pathways in platelet turnover and in the apoptotic-like changes seen after platelet activation is poorly understood. ABT-263 (Navitoclax), a specific inhibitor of antiapoptotic BCL2 proteins, which is currently being evaluated in clinical trials for the treatment of leukemia and other malignancies, induces a dose-limiting thrombocytopenia. In this study, the relationship between BCL2/BCL-X(L) inhibition, apoptosis, and platelet activation was investigated. Exposure to ABT-263 induced apoptosis but repressed platelet activation by physiologic agonists. Notably, ABT-263 induced an immediate calcium response in platelets and the depletion of intracellular calcium stores, indicating that on BCL2/BCL-X(L) inhibition platelet activation is abrogated because of a diminished calcium signaling. By comparing the effects of ABT-263 and its analog ABT-737 on platelets and leukemia cells from the same donor, we show, for the first time, that these BCL2/BCL-X(L) inhibitors do not offer any selective toxicity but induce apoptosis at similar concentrations in leukemia cells and platelets. However, reticulated platelets are less sensitive to apoptosis, supporting the hypothesis that treatment with ABT-263 induces a selective loss of older platelets and providing an explanation for the transient thrombocytopenia observed on ABT-263 treatment.


Assuntos
Compostos de Anilina/farmacologia , Apoptose/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Sulfonamidas/farmacologia , Proteína bcl-X/antagonistas & inibidores , Compostos de Anilina/efeitos adversos , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/metabolismo , Compostos de Bifenilo/efeitos adversos , Compostos de Bifenilo/farmacologia , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Expressão Gênica , Homeostase/efeitos dos fármacos , Humanos , Cinética , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Terapia de Alvo Molecular , Nitrofenóis/efeitos adversos , Nitrofenóis/farmacologia , Piperazinas/efeitos adversos , Piperazinas/farmacologia , Inibidores da Agregação Plaquetária/efeitos adversos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/efeitos adversos , Trombocitopenia/induzido quimicamente , Trombopoese , Proteína bcl-X/metabolismo
18.
Biochem Biophys Res Commun ; 408(2): 344-9, 2011 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-21514278

RESUMO

Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using (3)H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Compostos de Anilina/metabolismo , Compostos de Bifenilo/metabolismo , Nitrofenóis/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/metabolismo , Compostos de Anilina/farmacologia , Animais , Compostos de Bifenilo/farmacologia , Linhagem Celular , Cães , Nitrofenóis/farmacologia , Piperazinas/metabolismo , Piperazinas/farmacologia , Especificidade por Substrato , Sulfonamidas/farmacologia
19.
Autophagy ; 6(8): 1042-56, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21121091

RESUMO

It is becoming increasingly clear that there is crosstalk between the apoptotic and autophagic pathways, with autophagy helping to contribute to cell death by providing energy to allow the energy-requiring programmed cell death process to complete, as well as degrading cellular material in its own right. Recent evidence has suggested that Atg proteins can themselves be targets of caspases, providing potential regulation of autophagy as well as uncovering novel functions for fragments derived from Atg proteins. However, to date there has not been a detailed examination of which Atg proteins may be the targets of which death proteases. We show that the majority of human Atg (hAtg) proteins can be cleaved by calpain 1, which is activated in some apoptotic paradigms, as well as other forms of death. We also show that hAtg3 is cleaved by caspases-3, -6 and -8, hAtg6 (Beclin 1) is cleaved by caspase-3 and -6, while hAtg9, hAtg7 and the hAtg4 homologues can be cleaved by caspase-3. Cleavage of Beclin 1 was also seen in apoptosis of HeLa cells induced by staurosporine and TRAIL, along with cleavage of Atg3 and Atg4C. There were subtle effects of caspase inhibition on GFP-LC3 lipidation but more marked effects on the formation of GFP-LC3 puncta (a marker of autophagosome formation) and p62 degradation, indicating that caspase cleavage of autophagy-related proteins can affect the autophagic process. Notably we show that p62 is a target for caspase-6 and -8 cleavage.


Assuntos
Calpaína/metabolismo , Caspases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Sequência de Aminoácidos , Morte Celular/efeitos dos fármacos , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , Endocitose/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/química , Dados de Sequência Molecular , Plasmídeos/genética , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Estaurosporina/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Transcrição Gênica/efeitos dos fármacos , Ubiquitina/metabolismo
20.
Clin Cancer Res ; 16(16): 4217-25, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20601444

RESUMO

PURPOSE: Inhibition of the antiapoptotic BCL2 family is one of the most promising areas of anticancer drug development. However, ABT-737, a specific BCL2 inhibitor, is neither orally bioavailable nor metabolically stable. To overcome these problems, the structurally related molecule ABT-263 was synthesized and recently entered clinical trials in hematologic malignancies, including chronic lymphocytic leukemia (CLL). Almost all laboratory studies have been carried out with ABT-737 rather than ABT-263, the drug being used in clinical trials. Currently there are no published data on the comparative effects of these inhibitors. To gain insight into the potential value or limitations of ABT-263 in the clinic, we assessed its ability to induce apoptosis in clinically relevant cellular models of CLL. EXPERIMENTAL DESIGN: The susceptibility of freshly isolated primary CLL cells to these inhibitors was compared in standard culture conditions and in conditions that more closely mimic in vivo conditions in a whole blood assay system. RESULTS: ABT-737 was more potent than ABT-263 at inducing apoptosis in CLL cells. In whole blood, approximately 100-fold higher concentrations of both drugs were required to induce apoptosis. We found that ABT-263 was highly bound by albumin and that an increased albumin binding of ABT-263 as compared with ABT-737 accounted for the differential sensitivity of CLL cells. CONCLUSIONS: Our data indicate that the exquisite in vitro sensitivity of CLL cells to BCL2 inhibitors may be lost in vivo due to high cell densities and the albumin binding of ABT-263. Modification of ABT-263 may yield a BCL2 inhibitor with greater bioavailability and more favorable pharmacokinetics.


Assuntos
Albuminas/metabolismo , Compostos de Anilina/farmacocinética , Antineoplásicos/farmacocinética , Compostos de Bifenilo/farmacocinética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Nitrofenóis/farmacocinética , Sulfonamidas/farmacocinética , Compostos de Anilina/metabolismo , Compostos de Anilina/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Disponibilidade Biológica , Compostos de Bifenilo/metabolismo , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Humanos , Imunoprecipitação , Leucemia Linfocítica Crônica de Células B/metabolismo , Nitrofenóis/metabolismo , Nitrofenóis/farmacologia , Piperazinas/metabolismo , Piperazinas/farmacocinética , Piperazinas/farmacologia , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia
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