RESUMO
The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins can mediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted key residues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target.
RESUMO
Recent advances in melanoma therapy have significantly improved the prognosis of metastasized melanoma. However, large therapeutic gaps remain that need to be closed by new strategies. Antiapoptotic Bcl-2 proteins critically contribute to apoptosis deficiency and therapy resistance. They can be targeted by BH3 mimetics, small molecule antagonists that mimic the Bcl-2 homology domain 3 (BH3) of proapoptotic BH3-only proteins. By applying in vitro experiments, we aimed to obtain an overview of the possible suitability of BH3 mimetics for future melanoma therapy. Thus, we investigated the effects of ABT-737 and ABT-263, which target Bcl-2, Bcl-xL and Bcl-w as well as the Bcl-2-selective ABT-199 and the Mcl-1-selective S63845, in a panel of four BRAF-mutated and BRAF-WT melanoma cell lines. None of the inhibitors showed significant effectiveness when used alone; however, combination of S63845 with each one of the three ABTs almost completely abolished melanoma cell survival and induced apoptosis in up to 50-90% of the cells. Special emphasis was placed here on the understanding of the downstream pathways involved, which may allow improved applications of these strategies. Thus, cell death induction was correlated with caspase activation, loss of mitochondrial membrane potential, phosphorylation of histone H2AX, and ROS production. Caspase dependency was demonstrated by a caspase inhibitor, which blocked all effects. Upregulation of Mcl-1, induced by S63845 itself, as reported previously, was blocked by the combinations. Indeed, Mcl-1, as well as XIAP (X-linked inhibitor of apoptosis), were strongly downregulated by combination treatments. These findings demonstrate that melanoma cells can be efficiently targeted by BH3 mimetics, but the right combinations have to be selected. The observed pronounced activation of apoptosis pathways demonstrates the decisive role of apoptosis in the loss of cell viability by BH3 mimetics.
Assuntos
Antineoplásicos , Melanoma , Pirimidinas , Tiofenos , Humanos , Melanoma/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Proteína bcl-X/metabolismo , Apoptose , Caspases/metabolismo , Linhagem Celular Tumoral , Antineoplásicos/farmacologiaRESUMO
Targeting of MAP kinase pathways by BRAF inhibitors has evolved as a key therapy for BRAF-mutated melanoma. However, it cannot be applied for BRAF-WT melanoma, and also, in BRAF-mutated melanoma, tumor relapse often follows after an initial phase of tumor regression. Inhibition of MAP kinase pathways downstream at ERK1/2, or inhibitors of antiapoptotic Bcl-2 proteins, such as Mcl-1, may serve as alternative strategies. As shown here, the BRAF inhibitor vemurafenib and the ERK inhibitor SCH772984 showed only limited efficacy in melanoma cell lines, when applied alone. However, in combination with the Mcl-1 inhibitor S63845, the effects of vemurafenib were strongly enhanced in BRAF-mutated cell lines, and the effects of SCH772984 were enhanced in both BRAF-mutated and BRAF-WT cells. This resulted in up to 90% loss of cell viability and cell proliferation, as well as in induction of apoptosis in up to 60% of cells. The combination of SCH772984/S63845 resulted in caspase activation, processing of poly (ADP-ribose) polymerase (PARP), phosphorylation of histone H2AX, loss of mitochondrial membrane potential, and cytochrome c release. Proving the critical role of caspases, a pan-caspase inhibitor suppressed apoptosis induction, as well as loss of cell viability. As concerning Bcl-2 family proteins, SCH772984 enhanced expression of the proapoptotic Bim and Puma, as well as decreased phosphorylation of Bad. The combination finally resulted in downregulation of antiapoptotic Bcl-2 and enhanced expression of the proapoptotic Noxa. In conclusion, combined inhibition of ERK and Mcl-1 revealed an impressive efficacy both in BRAF-mutated and WT melanoma cells, and may thus represent a new strategy for overcoming drug resistance.
Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Humanos , Vemurafenib/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Regulação para Cima , Sobrevivência Celular , Potencial da Membrana Mitocondrial , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Melanoma/metabolismo , Caspases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Linhagem Celular TumoralRESUMO
BACKGROUND: Photodynamic therapy with a photosensitizer such as protoporphyrin-IX, a light sensitive metabolite of heme synthesis, is a highly selective treatment for various carcinomas. In previous studies, we found a significant down regulation of the relevant enzyme ferrochelatase in gastrointestinal carcinomas leading to an accumulation of protoporphyrin-IX within the tumor cells. Recent studies showed that a novel anti-cancer drug, Alectinib, an orally available, highly selective, potent second-generation inhibitor of anaplastic lymphoma tyrosinkinase binds to ferrochelatase. Therefore, we were interested to see whether Alectinib treatment might lead to an accumulation of protoporphyrin IX. METHODS: Tumor cells of different origin were cultured, treated with LED-light and Alectinib. Results were gained by flow cytometry, immunohistochemistry and western blotting. Apoptosis was determined by flow cytometric analysis of Annexin V-FITC stained cells. In addition, cells were counterstained with propidium iodide to distinguish early apoptotic cells and late apoptotic/necrotic cells. RESULTS: Here, we report that photodynamic treatment of tumor cell lines of different origin in combination with Alectinib increased protoporphyrin-IX specific fluorescence and concomitantly cell death. CONCLUSIONS: The usage of Alectinib could be another step for enhancing the effectiveness of photodynamic therapy. Further experiments will show whether photodynamic therapy in combination with Alectinib could be a new strategy for the treatment of e.g. peritoneal disseminated carcinomas.
Assuntos
Ácido Aminolevulínico/farmacologia , Carbazóis/farmacologia , Luz , Neoplasias/tratamento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Piperidinas/farmacologia , Protoporfirinas/metabolismo , Fluorescência , Humanos , Neoplasias/patologia , Células Tumorais CultivadasRESUMO
Renal cell carcinoma (RCC) is polyresistant to chemo- and radiotherapy and biologicals, including TNF-related apoptosis-inducing ligand (TRAIL). Sorafenib, a multikinase inhibitor approved for the treatment of RCC, has been shown to sensitize cancer cells to TRAIL-induced apoptosis, in particular by down-regulation of the Bak-inhibitory Bcl-2 family protein Mcl-1. Here we demonstrate that sorafenib overcomes TRAIL resistance in RCC by a mechanism that does not rely on Mcl-1 down-regulation. Instead, sorafenib induces rapid dissipation of the mitochondrial membrane potential (ΔΨm) that is accompanied by the accumulation of reactive oxygen species (ROS). Loss of ΔΨm and ROS production induced by sorafenib are independent of caspase activities and do not depend on the presence of the proapoptotic Bcl-2 family proteins Bax or Bak, indicating that both events are functionally upstream of the mitochondrial apoptosis signaling cascade. More intriguingly, we find that it is sorafenib-induced ROS accumulation that enables TRAIL to activate caspase-8 in RCC. This leads to apoptosis that involves activation of an amplification loop via the mitochondrial apoptosis pathway. Thus, our mechanistic data indicate that sorafenib bypasses central resistance mechanisms through a direct induction of ΔΨm breakdown and ROS production. Activation of this pathway might represent a useful strategy to overcome the cell-inherent resistance to cancer therapeutics, including TRAIL, in multiresistant cancers such as RCC.
Assuntos
Carcinoma de Células Renais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Renais/metabolismo , Mitocôndrias/metabolismo , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Apoptose , Carcinoma de Células Renais/tratamento farmacológico , Caspase 8/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Ativação Enzimática , Citometria de Fluxo , Humanos , Neoplasias Renais/tratamento farmacológico , Potencial da Membrana Mitocondrial , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Niacinamida/farmacologia , Conformação Proteica , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sorafenibe , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
The pro-apoptotic multidomain Bcl-2 proteins Bax and Bak (also known as BAK1) are considered the gatekeepers of the intrinsic pathway of apoptosis by triggering the mitochondrial release of cytochrome c The role of the third Bax- and Bak-homologous multidomain protein Bok, however, is still unresolved. As cells doubly deficient for Bax and Bak are largely resistant to various apoptotic stimuli, it has been proposed that Bok is either dispensable for apoptosis or that its role is dependent on Bax and Bak. Here, we demonstrate, in several cell systems, that Bok efficiently induces cytochrome c release and apoptosis even in the complete absence of both Bak and Bax. Moreover, modulation of endogenous Bok levels affects the apoptosis response. By RNA interference and targeted deletion of the Bok gene, we demonstrate that Bok can significantly influence the apoptotic response to chemotherapeutic drugs in ovarian carcinoma cells. Hence, our results not only establish Bok as a Bak- and Bax-independent apoptosis inducer, but also suggest a potential impact of Bok expression in ovarian cancer therapy.
Assuntos
Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose/efeitos dos fármacos , Citocromos c/metabolismo , Citostáticos/farmacologia , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Células HCT116 , Humanos , Células MCF-7 , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismoRESUMO
Oral candidiasis is the most frequent fungal infection of the oral cavity. Clinical diagnoses require mycological confirmation, which is time-consuming in case of culture testing. The aim of the study was to identify signature volatiles to develop a chairside breath test to diagnose oral candidiasis. Headspaces above Candida albicans, glabrata, tropicalis, krusei cultures, and growth media as control were analysed after eight and 24 h using offline gas chromatography and mass spectrometry. The identification of signature volatiles was assisted using various microbial databases. Retrieved volatile patterns enabled Candida species discrimination in vitro. For C. albicans 3-methyl-2-butanone and styrene and for C. krusei a combination of p-xylene, 2-octanone, 2-heptanone and n-butyl acetate were found to be specific. 1-hexanol was found in C. tropicalis, but is emitted by a variety of other microorganisms. C. glabrata was characterised through the absence of these volatiles. The development of a breath test is a promising approach in confirming suspicions of oral candidiasis. To confirm the retrieved results in vivo, breath tests in affected and healthy subjects have to be performed.
Assuntos
Candida/classificação , Candida/isolamento & purificação , Candidíase Bucal/diagnóstico , Compostos Orgânicos Voláteis/análise , Acetatos/análise , Adulto , Testes Respiratórios , Candida/química , Candida albicans/classificação , Candida albicans/isolamento & purificação , Candida glabrata/classificação , Candida glabrata/isolamento & purificação , Candida tropicalis/classificação , Candida tropicalis/isolamento & purificação , Candidíase Bucal/microbiologia , Cromatografia Gasosa/métodos , Diagnóstico Diferencial , Hexanóis/análise , Humanos , Cetonas/análise , Masculino , Espectrometria de Massas/métodos , Pentanonas/análise , Estireno/análise , Xilenos/análiseRESUMO
Enhanced expression of anti-apoptotic B-cell lymphoma 2 (BCL-2) protein is frequent in cancer. Targeting of BCL-2 with the specific inhibitor ABT-199 (Venetoclax) has significant clinical activity in malignant diseases such as chronic lymphocytic leukemia and multiple myeloma. The small molecule drug ABT-199 mimics the pro-apoptotic BCL-2 homology domain 3 of BH3-only proteins and blocks the hydrophobic BC-groove in BCL-2. We have previously shown that ABT-199 synergizes with the proteasome inhibitor (PI) bortezomib in soft tissue sarcoma derived cells and cell lines to induce apoptosis. Synergistic apoptosis induction relies on the pore-forming effector BAX and expression of the pro-apoptotic BH3-only protein NOXA. Bortezomib augments expression of NOXA by blocking its proteasomal degradation. Interestingly, shown here for the first time, expression of NOXA is strongly enhanced by ABT-199 induced integrated stress response (ISR). ISR transcription factors ATF3 & ATF4 mediate transactivation of the BH3-only protein NOXA which specifically inhibits the anti-apoptotic MCL-1. Thus, NOXA potentiates the efficacy of the BCL-2 inhibitor ABT-199 by simultaneous inhibition of MCL-1. Hence, ABT-199 has a double impact by directly blocking anti-apoptotic BCL-2 and inhibiting MCL-1 via transactivated NOXA. By preventing degradation of NOXA PIs synergize with ABT-199. Synergism of ABT-199 and PIs therefore occurs on several, previously unexpected levels. This finding should prompt clinical evaluation of combinatorial regimens in further malignancies.
RESUMO
Circular RNAs (circRNAs) are dynamically regulated during differentiation and show cell type-specific expression, which is altered in cancer and can have a direct impact on its various hallmarks. We hypothesized that circRNA expression is deregulated in acute myeloid leukemia (AML) and that circRNA candidates might contribute to the pathogenesis of the disease. To identify leukemia-associated and differentiation-independent changes in circRNA expression, we determined the circular RNAome of 61 AML patients and 16 healthy hematopoietic stem and progenitor cell (HSPC) samples using ribosomal RNA-depleted RNA sequencing. We found hundreds of circRNAs that were differentially expressed between AML and healthy HSPCs. Gene set analysis found that many of these circRNAs were transcribed from genes implicated in leukemia biology. We discovered a circRNA derived from the T-cell transcription factor gene B cell CLL/lymphoma 11B, circBCL11B, which was exclusively expressed in AML patients, but not detected in healthy HSPCs, and associated with a T-cell-like gene expression signature. We were able to validate this finding in an independent cohort of 332 AML patients. Knockdown of circBCL11B had a negative effect on leukemic cell proliferation and resulted in increased cell death of leukemic cells, thereby suggesting circBCL11B as a novel functionally relevant candidate in AML pathogenesis. In summary, our study enables comprehensive insights into circRNA expression changes upon leukemic transformation and provides valuable information on the biology of leukemic cells and potential novel pathway dependencies that are relevant for AML therapy.
Assuntos
Leucemia Mieloide Aguda , MicroRNAs , Proliferação de Células , Humanos , Leucemia Mieloide Aguda/genética , RNA Circular , TranscriptomaRESUMO
BACKGROUND: Graft-versus-host disease (GvHD) presents a major cause for morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Rabbit-derived antithymocyte globulin (rATG) treatment reduces the incidence of GvHD after allogeneic hematopoietic stem cell transplantation. However, delayed immune reconstitution following rATG treatment, partly caused by hampered thymic function, is being discussed. The present study aims at elucidating possible cytotoxic effects of 2 commonly used rATG preparations on cultured human thymic stroma, especially thymic epithelial cells (TECs). METHODS: A primary TEC culture was established and the binding and cytotoxicity of 2 rATG preparations to the aforementioned cells were assessed by flow cytometry and immunofluorescence analyses. The release of several cytokines by cultured thymic stroma cells in response to rATG was analyzed via multiplex enzyme-linked immunosorbent assays. RESULTS: Both preparations showed a comparable dose-dependent binding to TECs and exerted a similar complement-independent, dose-dependent cytotoxicity. rATG exposure further resulted in hampered secretion of interleukin (IL)-7, IL-15, and IL-6, cytokines being involved in thymic T cell development and proliferation. Pretreatment with keratinocyte growth factor diminished rATG-induced cytotoxicity of TECs and restored their IL-7 and IL-15 secretion. CONCLUSIONS: Cytotoxic effects on TECs link the rATG-induced thymic damage to the delayed T cell reconstitution, witnessed after rATG treatment. Our data support a combination treatment of rATG and thymus-protective strategies such as keratinocyte growth factor to simultaneously offer sufficient GvHD prophylaxis and overcome delayed T cell reconstitution caused by thymic damage.
Assuntos
Soro Antilinfocitário/imunologia , Células Epiteliais/imunologia , Doença Enxerto-Hospedeiro/imunologia , Timo/citologia , Animais , Técnicas de Cultura de Células , Proliferação de Células , Pré-Escolar , Proteínas do Sistema Complemento/imunologia , Ensaio de Imunoadsorção Enzimática , Fator 7 de Crescimento de Fibroblastos/farmacologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Lactente , Recém-Nascido , Interleucina-15/imunologia , Interleucina-6/imunologia , Interleucina-7/imunologia , Células Jurkat , Coelhos , Linfócitos T/imunologia , Timócitos/citologia , Transplante Homólogo/efeitos adversosRESUMO
Central to intrinsic apoptosis signaling is the release of cytochrome c from mitochondria, which depends on the pro-apoptotic effector proteins Bax, Bak or Bok. These pore-forming effector proteins share four Bcl-2 homology (BH) domains, a functionally essential and conserved sequence of hydrophobic amino acids in their BH3-domain and a C-terminal transmembrane-domain whose specific function remains rather unknown. To elucidate the molecular basis of Bok-mediated apoptosis we analyzed apoptosis induction by transmembrane-domain deficient BokΔTM compared to the respective Bax and Bak proteins and proteins in which the first leucine in the BH3-stretch was mutated to glutamic acid. We show that deletion of the C-terminal transmembrane-domain reduces the pro-apoptotic function of each protein. Mutation of the first leucine in the BH3-domain (L78E) blocks activity of Bak, while mutation of the homologue residues in Bax or Bok (L63E and L70E respectively) does not affect apoptosis induction. Unexpectedly, combined mutation of the BH3-domain and deletion of the transmembrane-domain enhances the pro-apoptotic activity of Bok(L70E)ΔTM by abolishing the interaction with anti-apoptotic proteins, especially the primary Bok-inhibitory protein Mcl-1. These results therefore suggest a specific contribution of the transmembrane-domain to the pro-apoptotic function and interaction of Bok.
Assuntos
Domínios Proteicos/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Humanos , Células MCF-7 , Proteínas de Membrana/metabolismo , Camundongos Knockout , Mitocôndrias/metabolismoRESUMO
MACC1 was identified as a novel player in cancer progression and metastasis, but its role in death receptor-mediated apoptosis is still unexplored. We show that MACC1 knockdown sensitizes cancer cells to death receptor-mediated apoptosis. For the first time, we provide evidence for STAT signaling as a MACC1 target. MACC1 knockdown drastically reduced STAT1/3 activating phosphorylation, thereby regulating the expression of its apoptosis targets Mcl-1 and Fas. STAT signaling inhibition by the JAK1/2 inhibitor ruxolitinib mimicked MACC1 knockdown-mediated molecular signatures and apoptosis sensitization to Fas activation. Despite the increased Fas expression, the reduced Mcl-1 expression was instrumental in apoptosis sensitization. This reduced Mcl-1-mediated apoptosis sensitization was Bax and Bak dependent. MACC1 knockdown also increased TRAIL-induced apoptosis. MACC1 overexpression enhanced STAT1/3 phosphorylation and increased Mcl-1 expression, which was abrogated by ruxolitinib. The central role of Mcl-1 was strengthened by the resistance of Mcl-1 overexpressing cells to apoptosis induction. The clinical relevance of Mcl-1 regulation by MACC1 was supported by their positive expression correlation in patient-derived tumors. Altogether, we reveal a novel death receptor-mediated apoptosis regulatory mechanism by MACC1 in solid cancers through modulation of the STAT1/3-Mcl-1 axis.
Assuntos
Apoptose , Neoplasias Colorretais/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Receptor fas/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Janus Quinases/antagonistas & inibidores , Janus Quinases/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Nitrilas , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Fatores de Tempo , Transativadores , Fatores de Transcrição/genética , Transfecção , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
The proapoptotic BH3-only protein natural born killer / Bcl-2 interacting killer (Nbk/Bik) has been described to inhibit Bcl-2 and Bcl-xL, thereby supporting the death promoting ability of Bax. In order to evaluate its function in melanoma, we investigated the response after Nbk/Bik overexpression in cultured human melanoma cells and in a melanoma mouse model. Untransfected melanoma cell lines expressed Nbk/Bik only weakly at the mRNA and protein level. Conditional expression of Nbk/Bik by applying the inducible tetracycline-responsive expression system triggered apoptosis and enhanced sensitivity to proapoptotic stimuli as to agonistic CD95 activation and to chemotherapeutics etoposide, doxorubicin and pamidronate. For investigating the effects of Nbk/Bik in vivo, stably transfected melanoma cells were subcutaneously injected into nude mice. Significantly delayed tumor growth was the result when mice received doxycycline for induction of Nbk/Bik expression. By investigating the mechanism of Nbk/Bik-induced cell death, typical hallmarks of apoptosis such as DNA fragmentation and chromatin condensation were seen after induction. Interestingly, no indications for cytochrome c release and caspase processing were found, and selective caspase inhibition remained without effect. These data indicate the high potential of Nbk/Bik in regulating apoptosis in melanoma by a caspase-independent pathway and may corroborate the potency of novel antimelanoma strategies based on activation of BH3-only proteins such as Nbk/Bik.
Assuntos
Apoptose , Caspases/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Cromatina/metabolismo , Citocromos c/metabolismo , Difosfonatos/farmacologia , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Feminino , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Injeções Subcutâneas , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pamidronato , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pele/metabolismo , Tetraciclina/farmacologia , Células Tumorais Cultivadas , Proteína X Associada a bcl-2/metabolismo , Receptor fas/metabolismoRESUMO
We previously demonstrated that the forced expression of pro-caspase-3 can revert acquired chemoresistance in MT1-Adr breast cancer cells which show a defective activation of the mitochondrial pathway of apoptosis. We now asked whether the manipulation of mitochondrial apoptosis signaling can revert different types of drug resistance, i.e. the resistance due to impaired mitochondrial activation in the MT1-Adr cells and the resistance in MT3-Adr cells which is caused by increased expression of the Mdr-1/p-glycoprotein ABC transporter. Here we show that Bcl-2 overexpression is the underlying cause for the resistant phenotype in the MT1-Adr cells. Overexpression of the apoptosis-promoting Bax homologue Bak or the BH3 only protein Nbk/Bik reverts, as expected, acquired drug resistance in the MT1-Adr cells as recently demonstrated for pro-caspase-3. Moreover, we show that both apoptosis-promoters, Nbk/Bik and Bak, antagonize acquired chemoresistance for epirubicin-mediated apoptosis in MT3-Adr breast cancer cells. Neither drug uptake nor drug efflux were influenced by Bak or Nbk/Bik. Thus, our data show that manipulation of the downstream apoptosis signaling cascade by Bak and Nbk/Bik can overcome not only drug resistance due to mitochondrial apoptosis deficiency (in the MT1-Adr cells) but also classical, i.e. efflux-mediated, resistance for drug-induced cell death in the MT3-Adr cell line. Nbk/Bik and Bak could therefore be target genes to increase chemosensitivity and overcome different types of drug resistance.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Neoplasias da Mama/genética , Proteínas de Membrana/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Apoptose/genética , Proteínas Reguladoras de Apoptose , Neoplasias da Mama/patologia , Morte Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos , Epirubicina/toxicidade , Feminino , Humanos , Cinética , Proteínas de Membrana/genética , Proteínas Mitocondriais , Reação em Cadeia da Polimerase , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais Cultivadas , Proteína Killer-Antagonista Homóloga a bcl-2RESUMO
The mitochondrial apoptosis pathway mediates cell death through the release of various pro-apoptotic factors including cytochrome c and Smac, the second mitochondrial activator of caspases, into the cytosol. Smac was shown previously to inhibit IAP proteins and to facilitate initiation of the caspase cascade upon cytochrome c release. To investigate Smac function during apoptosis and to explore Smac as an experimental cancer therapeutic, we constructed an expression system based on a single adenoviral vector containing Smac under control of the Tet-off system supplied in cis. Conditional expression of Smac induced apoptosis in human HCT116 and DU145 carcinoma cells regardless of the loss of Bax or overexpression of Bcl-x(L). Nevertheless, apoptosis induced by Smac was associated with cytochrome c release and breakdown of the mitochondrial membrane potential. This indicates that Smac acts independently of Bax and Bcl-x(L) during initiation of apoptosis and triggers a positive feedback loop that results in Bax/Bcl-x(L)-independent activation of mitochondria. In caspase-proficient cells, Smac-induced apoptosis could be inhibited partially by cell-permeable LEHD (caspase-9 inhibitor) and DEVD (caspase-3 inhibitor) peptides. Furthermore, loss of caspase-3 expression in MCF-7 cells carrying a caspase-3 null mutation completely abrogated the sensitivity for Smac-induced apoptotic or nonapoptotic, necrosis-like cell death, while re-expression of caspase-3 conferred sensitivity. Altogether, caspase-3 but not caspase-9 activation was necessary for execution of Smac-induced cell death. Notably, Smac did not induce caspase-9 processing in the absence of caspase-3. Thus, caspase-9 processing occurs secondary to caspase-3 activation during Smac-induced apoptosis. Altogether, Smac is capable of circumventing defects in mitochondrial apoptosis signaling such as loss of Bax or overexpression of Bcl-x(L) that are frequently observed in tumor cells resistant to anticancer therapy. Consequently, Smac appears to be a promising therapeutic target in anticancer treatment.
Assuntos
Carcinoma/metabolismo , Proteínas de Transporte/metabolismo , Caspases/metabolismo , Citocromos c/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Adenoviridae/genética , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Neoplasias da Mama/metabolismo , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Proteínas de Transporte/genética , Caspase 3 , Caspase 9 , Inibidores de Caspase , Caspases/genética , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Mitocondriais/genética , Mutação , Oligopeptídeos/farmacologia , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Células Tumorais Cultivadas , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF), which are frequently inactivated in human cancer. Whereas p16(INK4a) acts through engagement of the Rb-cdk4/6-cyclin D pathway, both the pro-apoptotic and cell cycle-regulatory functions of p14(ARF) were shown to be primarily dependent on the presence of functional p53. Recent reports have also implicated p14(ARF) in p53-independent mechanisms of cell cycle regulation and apoptosis induction, respectively. To further explore the pro-apoptotic function of p14(ARF) in relation to functional cellular p53, we constructed a replication-deficient adenoviral vector for overexpression of p14(ARF) (Ad-p14(ARF)). As expected, Ad-p14(ARF) efficiently induced apoptosis in p53/Rb wild-type U-2OS osteosarcoma cells at low multiplicities of infection. Interestingly, Ad-p14(ARF) also induced apoptosis in both p53-deleted SAOS-2 osteosarcoma cells and HCT116 colon cancer cells with a bi-allelic knock-out of p53 (HCT116-p53(-/-)). Similarly, adenovirus-mediated overexpression of p14(ARF) induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells as well as in HCT116 cells devoid of functional Bax (HCT116-Bax(-/-)). Restoration of Bax expression by retroviral gene transfer in DU145 cells did not further enhance p14(ARF)-triggered cell death. Infection with Ad-p14(ARF) induced activation of mitochondrial permeability shift transition, caspase activation and apoptotic DNA fragmentation irrespective of the presence or absence of either Bax or functional cellular p53. Nevertheless, overexpression of the anti-apoptotic Bcl-2 homolog Bcl-x(L) markedly inhibited p14(ARF)-induced apoptosis. This may indicate that p14(ARF) triggers a so far unknown activator of mitochondrial apoptosis which can be inhibited by Bcl-2 but which acts either independently or downstream of Bax. Taken together, this report demonstrates the participation of signaling pathways apart from the p53/Mdm-2 rheostat and Bax in p14(ARF)-mediated apoptosis.
Assuntos
Apoptose/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteína Supressora de Tumor p14ARF/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Adenocarcinoma/patologia , Adenoviridae/genética , Apoptose/genética , Neoplasias Ósseas/patologia , Regulação Viral da Expressão Gênica , Vetores Genéticos/genética , Humanos , Masculino , Mitocôndrias/fisiologia , Modelos Biológicos , Proteínas de Neoplasias/fisiologia , Osteossarcoma/patologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p53/deficiência , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
The death ligand TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation. Here, we show that the synergism of TRAIL and 5-fluorouracil (5-FU) and cross-sensitization between TRAIL and 5-FU for induction of apoptosis, entirely depend on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to release cytochrome c and to activate caspase-3 and -9 when exposed to TRAIL and 5-FU. In contrast, TRAIL sensitized for 5-FU-induced apoptosis and vice versa upon reconstitution of Bax expression. Isobolographic analyses of ED50 doses for 5-FU at increasing TRAIL concentrations showed a clear synergism of TRAIL and 5-FU in Bax-expressing cells. In contrast, the effect was merely additive in DU145 cells lacking Bax. Notably, both DU145 and HCT116 Bax-deficient cells still express Bak. This indicates that Bak is not sufficient to mediate cross-sensitization and synergism between 5-FU and TRAIL. Stable overexpression of Bak in DU145 sensitized for epirubicin-induced apoptosis but failed to confer synergy between TRAIL and 5-FU. Moreover, we show by the use of EGFP-tagged Bax and Bak that TRAIL and 5-FU synergistically trigger oligomerization and clustering of Bax but not Bak. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade and delineate a higher degree of specificity in signaling for cell death by multidomain Bcl-2 homologs.
Assuntos
Apoptose/fisiologia , Fluoruracila/farmacologia , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Reguladoras de Apoptose , Western Blotting , Caspases/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Ativação Enzimática , Humanos , Masculino , Ligante Indutor de Apoptose Relacionado a TNF , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2RESUMO
UNLABELLED: Resistance to cell death is the major cause of chemotherapy failure in most kinds of cancers, including Burkitt lymphoma (BL). When analyzing therapy resistance in Burkitt lymphoma (BL), we discovered a link between apoptosis resistance and ploidy control. We therefore studied systematically a panel of 15 BL lines for apoptosis induction upon treatment with microtubule inhibitors and compared three types of microtubule toxins, i.e., paclitaxel, nocodazole and vincristine. We found an inverse relationship between apoptosis sensitivity and ploidy control. Thus, cells resistant to paclitaxel- or nocodazole-induced apoptosis underwent mitotic catastrophe and developed polyploidy (>4N). Mechanistically, apoptosis resistance was linked to failure of caspase activation, which was most pronounced in cells lacking the pro-apoptotic multidomain Bcl-2 homologs Bax and Bak. Pharmacological caspase inhibition promoted polyploidy upon exposure to paclitaxel and nocodazole supporting the relationship between resistance to apoptosis and polyploidization. Of note, vincristine induced persistent mitotic arrest but no loss of ploidy control. Considering targets to facilitate Bax/Bak-independent cell death and to avoid drug-induced mitotic catastrophe and consecutive mitotic catastrophe should be of great importance to overcome therapy resistance and therapy-related events that result in ploidy changes and tumor progression. KEY MESSAGE: Inverse relation of apoptosis and polyploidy induction by paclitaxel or nocodazole in BL. Resistant cells undergo mitotic catastrophe and develop polyploidy. Lack of Bax/Bak confers resistance and leads to induction of polyploidy in BL. Intact apoptosis response protects from polyploidy as a result of mitotic catastrophe.
Assuntos
Apoptose/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Mitose/genética , Ploidias , Animais , Apoptose/efeitos dos fármacos , Inibidores de Caspase/farmacologia , Caspases/metabolismo , Linhagem Celular , Fragmentação do DNA , Citometria de Fluxo , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Knockout , Mitose/efeitos dos fármacos , Nocodazol/farmacologia , Paclitaxel/farmacologia , Poliploidia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Moduladores de Tubulina/farmacologia , Vincristina/farmacologiaRESUMO
Melanoma cells are characterized by apoptosis deficiency coinciding with reduced expression of the proapoptotic Bcl-2 protein Bim. An adenoviral vector was constructed with the BimL cDNA controlled by an inducible promoter. Highly efficient apoptosis induction and abrogated cell proliferation was seen in melanoma cells upon BimL overexpression. Loss of mitochondrial membrane potential, release of mitochondrial apoptogenic factors and caspase-9 processing indicated the activation of mitochondrial apoptosis pathways. BimL activated both Bax and Bak, as shown by siRNA knockdown and activation-specific antibodies. Of note, BimL overrode the apoptosis blockade by Bcl-2 overexpression or by Bax/Bak single knockdown. The high efficacy correlated to BimL interaction with all antiapoptotic Bcl-2 family members in melanoma cells, shown by co-immunoprecipitation analyses for Bcl-2, Bcl-xL, Mcl-1 and Bcl-w. Thus, BimL reveals an outstanding proapoptotic potential in melanoma cells, and strategies for its re-expression appear of interest. These have been reported for B-Raf inhibitors, and their efficacy may be partly attributed to BimL.