RESUMO
BACKGROUND: Next-generation sequencing is used in cancer research to identify somatic and germline mutations, which can predict sensitivity or resistance to therapies, and may be a useful tool to reveal drug repurposing opportunities between tumour types. Multigene panels are used in clinical practice for detecting targetable mutations. However, the value of clinical whole-exome sequencing (WES) and whole-genome sequencing (WGS) for cancer care is less defined, specifically as the majority of variants found using these technologies are of uncertain significance. PATIENTS AND METHODS: We used the Cancer Genome Interpreter and WGS in 726 tumours spanning 10 cancer types to identify drug repurposing opportunities. We compare the ability of WGS to detect actionable variants, tumour mutation burden (TMB) and microsatellite instability (MSI) by using in silico down-sampled data to mimic WES, a comprehensive sequencing panel and a hotspot mutation panel. RESULTS: We reveal drug repurposing opportunities as numerous biomarkers are shared across many solid tumour types. Comprehensive panels identify the majority of approved actionable mutations, with WGS detecting more candidate actionable mutations for biomarkers currently in clinical trials. Moreover, estimated values for TMB and MSI vary when calculated from WGS, WES and panel data, and are dependent on whether all mutations or only non-synonymous mutations were used. Our results suggest that TMB and MSI thresholds should not only be tumour-dependent, but also be sequencing platform-dependent. CONCLUSIONS: There is a large opportunity to repurpose cancer drugs, and these data suggest that comprehensive sequencing is an invaluable source of information to guide clinical decisions by facilitating precision medicine and may provide a wealth of information for future studies. Furthermore, the sequencing and analysis approach used to estimate TMB may have clinical implications if a hard threshold is used to indicate which patients may respond to immunotherapy.
Assuntos
Exoma , Neoplasias , Biomarcadores Tumorais , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Instabilidade de Microssatélites , Mutação , Sequenciamento do ExomaRESUMO
INTRODUCTION: Mesothelioma remains a lethal cancer. To date, systemic therapy with pemetrexed and a platinum drug remains the only licensed standard of care. As the median survival for patients with mesothelioma is 12.1 months, surgery is an important consideration to improve survival and/or quality of life. Currently, only two surgical trials have been performed which found that neither extensive (extra-pleural pneumonectomy) or limited (partial pleurectomy) surgery improved survival (although there was some evidence of improved quality of life). Therefore, clinicians are now looking to evaluate pleurectomy decortication, the only radical treatment option left. METHODS AND ANALYSIS: The MARS 2 study is a UK multicentre open parallel group randomised controlled trial comparing the effectiveness and cost-effectiveness of surgery-(extended) pleurectomy decortication-versus no surgery for the treatment of pleural mesothelioma. The study will test the hypothesis that surgery and chemotherapy is superior to chemotherapy alone with respect to overall survival. Secondary outcomes include health-related quality of life, progression-free survival, measures of safety (adverse events) and resource use to 2 years. The QuinteT Recruitment Intervention is integrated into the trial to optimise recruitment. ETHICS AND DISSEMINATION: Research ethics approval was granted by London - Camberwell St. Giles Research Ethics Committee (reference 13/LO/1481) on 7 November 2013. We will submit the results for publication in a peer-reviewed journal. TRIAL REGISTRATION NUMBERS: ISRCTN-ISRCTN44351742 and ClinicalTrials.gov-NCT02040272.
Assuntos
Neoplasias Pulmonares , Mesotelioma Maligno , Mesotelioma , Neoplasias Pleurais , Humanos , Londres , Neoplasias Pulmonares/cirurgia , Mesotelioma/cirurgia , Estudos Multicêntricos como Assunto , Neoplasias Pleurais/cirurgia , Qualidade de Vida , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do TratamentoRESUMO
The treatment of advanced non-small cell lung cancer (NSCLC) may be changing, but the cisplatin-based doublet remains the foundation of treatment for the majority of patients with advanced NSCLC. In this respect, changes in practice to various aspects of cisplatin use, such as administration schedules and the choice of methods and frequency of monitoring for toxicities, have contributed to an incremental improvement in patient management and experience. Chemoresistance, however, limits the clinical utility of this drug in patients with advanced NSCLC. Better understanding of the molecular mechanisms of cisplatin resistance, identification of predictive markers and the development of newer, more effective and less toxic platinum agents is required. In addition to maximising potential benefits from advances in molecular biology and associated therapeutics, modification of existing cisplatin-based treatments can still lead to improvements in patient outcomes and experiences.
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma de Células Escamosas/tratamento farmacológico , Cisplatino/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Adenocarcinoma/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/patologia , Cisplatino/história , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Docetaxel , Descoberta de Drogas/história , História do Século XIX , História do Século XX , História do Século XXI , Humanos , Neoplasias Pulmonares/patologia , Paclitaxel/administração & dosagem , Taxoides/administração & dosagem , GencitabinaRESUMO
Inhibition of the chaperone heat-shock protein 90 (HSP90) induces apoptosis, and it is a promising anti-cancer strategy. The mechanisms underpinning apoptosis activation following HSP90 inhibition and how they are modified during acquired drug resistance are unknown. We show for the first time that, to induce apoptosis, HSP90 inhibition requires the cooperation of multi BH3-only proteins (BID, BIK, PUMA) and the reciprocal suppression of the pro-survival BCL-2 family member MCL1, which occurs via inhibition of STAT5A. A subset of tumour cell lines exhibit dependence on MCL1 expression for survival and this dependence is also associated with tumour response to HSP90 inhibition. In the acquired resistance setting, MCL1 suppression in response to HSP90 inhibitors is maintained; however, a switch in MCL1 dependence occurs. This can be exploited by the BH3 peptidomimetic ABT737, through non-BCL-2-dependent synthetic lethality.
Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Linhagem Celular Tumoral , Humanos , Peptidomiméticos , Fator de Transcrição STAT5/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1-3 selective inhibitors such as entinostat to exploit high procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Caspase 8/metabolismo , Western Blotting , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Caspase 8/genética , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Citometria de Fluxo , Humanos , Técnicas In Vitro , Estudos RetrospectivosRESUMO
Estrogen receptor (ER)-ß has been shown to possess a tumor suppressive effect, and is a potential target for cancer therapy. Using gene-expression meta-analysis of human malignant pleural mesothelioma, we identified an ESR2 (ERß coding gene) signature. High ESR2 expression was strongly associated with low succinate dehydrogenase B (SDHB) (which encodes a mitochondrial respiratory chain complex II subunit) expression. We demonstrate that SDHB loss induced ESR2 expression, and that activated ERß, by over-expression or by selective agonist stimulation, negatively affected oxidative phosphorylation compromising mitochondrial complex II and IV activity. This resulted in reduced mitochondrial ATP production, increased glycolysis dependence and impaired cell proliferation. The observed in vitro effects were phenocopied in vivo using a selective ERß agonist in a mesothelioma mouse model. On the whole, our data highlight an unforeseen interaction between ERß-mediated tumor suppression and energy metabolism that may be exploited to improve on the therapy for clinical management of malignant mesothelioma.
RESUMO
Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFα) signaling for their activity. As circulating TNFα expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFα. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFα promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA.
Assuntos
Antineoplásicos/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Ativação Enzimática , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Mesotelioma , Mimetismo Molecular , Neoplasias Pleurais , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , VorinostatRESUMO
Failure to efficiently induce apoptosis contributes to cisplatin resistance in non-small-cell lung cancer (NSCLC). Although BCL-2-associated X protein (BAX) and BCL-2 antagonist killer (BAK) are critical regulators of the mitochondrial apoptosis pathway, their requirement has not been robustly established in relation to cisplatin. Here, we show that cisplatin can efficiently bypass mitochondrial apoptosis block caused by loss of BAX and BAK, via activation of the extrinsic death receptor pathway in some model cell lines. Apoptosis resistance following cisplatin can only be observed when both extrinsic and intrinsic pathways are blocked, consistent with redundancy between mitochondrial and death receptor pathways in cisplatin-induced apoptosis. In H460 NSCLC cells, caspase-8 cleavage was shown to be induced by cisplatin and is dependent on death receptor 4, death receptor 5, Fas-associated protein with death domain, acid sphingomyelinase and ceramide synthesis. In contrast, cisplatin-resistant cells fail to activate caspase-8 via this pathway despite conserving sensitivity to death ligand-driven activation. Accordingly, caspase-8 activation block acquired during cisplatin resistance, can be bypassed by death receptor agonism.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Caspase 8/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/enzimologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Caspase 8/genética , Linhagem Celular Tumoral , Cisplatino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacosRESUMO
BACKGROUND: We have previously demonstrated that Tcf-4 regulates osteopontin (OPN) in rat breast epithelial cells, Rama37. In this report, we have examined the importance of this regulation in human breast cancer. METHODS: The regulatory roles of Tcf-4 on cell invasion and OPN expression were investigated. The mRNA expression of Tcf-4 and OPN, and survival of breast cancer patients were correlated. RESULTS: Tcf-4 enhanced cell invasion in both MCF10AT and MDA MB 231 breast cancer cells by transcriptionally activating OPN expression. Osteopontin was activated by Wnt signalling in MDA MB 231 cells. Paradoxical results on Tcf-4-regulated OPN expression in MCF10AT (activation) and Rama37 (repression) cells were shown to be a result of differential Wnt signalling competency in MCF10AT and Rama37 cells. High levels of OPN and Tcf-4 mRNA expression were significantly associated with survival in breast cancer patients. Most importantly, Tcf-4-positive patients had a poorer prognosis when OPN was overexpressed, while OPN-negative patients had a better prognosis when Tcf-4 was overexpressed. CONCLUSION: Our results suggest that Tcf-4 can act as a repressor or activator of breast cancer progression by regulating OPN expression in a Wnt-dependent manner and that Tcf-4 and OPN together may be a novel prognostic indicator for breast cancer progression.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Osteopontina/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Western Blotting , Imunoprecipitação da Cromatina , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Reação em Cadeia da Polimerase , Prognóstico , Análise Serial de Proteínas , Transdução de Sinais , Fator de Transcrição 4 , Regulação para CimaRESUMO
Three-dimensional (3D) cultures are a valuable platform to study acquired multicellular apoptotic resistance of cancer. We used spheroids of cell lines and actual tumor to study resistance to the proteasome inhibitor bortezomib in mesothelioma, a highly chemoresistant tumor. Spheroids from mesothelioma cell lines acquired resistance to bortezomib by failing to upregulate Noxa, a pro-apoptotic sensitizer BH3-only protein that acts by displacing Bim, a pro-apoptotic Bax/Bak-activator protein. Surprisingly, despite their resistance, spheroids also upregulated Bim and thereby acquired sensitivity to ABT-737, an inhibitor of anti-apoptotic Bcl-2 molecules. Analysis using BH3 profiling confirmed that spheroids acquired a dependence on anti-apoptotic Bcl-2 proteins and were 'primed for death'. We then studied spheroids grown from actual mesothelioma. ABT-737 was active in spheroids grown from those tumors (5/7, â¼70%) with elevated levels of Bim. Using immunocytochemistry of tissue microarrays of 48 mesotheliomas, we found that most (33, 69%) expressed elevated Bim. In conclusion, mesothelioma cells in 3D alter the expression of Bcl-2 molecules, thereby acquiring both apoptotic resistance and sensitivity to Bcl-2 blockade. Mesothelioma tumors ex vivo also show sensitivity to Bcl-2 blockade that may depend on Bim, which is frequently elevated in mesothelioma. Therefore, mesothelioma, a highly resistant tumor, may have an intrinsic sensitivity to Bcl-2 blockade that can be exploited therapeutically.
Assuntos
Apoptose , Resistencia a Medicamentos Antineoplásicos , Mesotelioma/metabolismo , Mesotelioma/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Humanos , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Esferoides Celulares/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfonamidas/farmacologia , Células Tumorais CultivadasRESUMO
Non-small-cell lung cancer (NSCLC) remains by far the major cause of cancer-related death in the Western world in both men and women. The majority of patients will be diagnosed with metastatic disease, and chemotherapy doublets remain the cornerstone of treatment for these patients. However, chemotherapy has a minimal impact on long-term survival and prognosis remains poor for these patients. Further improvement in treatment is likely to require incorporation of novel targeted therapies. Among these agents, inhibitors of the epidermal growth factor receptor (EGFR) have demonstrated significant activity in the first-, second- or third-line treatment of NSCLC. The purpose of current paper is to present the evidence for using several proposed molecular biomarkers as a tool for selection of NSCLC patients for anti-EGFR treatment. According to current data, EGFR mutation status appears to be the strongest predictor for the selection of NSCLC patients to first-line treatment with EGFR tyrosine kinase inhibitors vs chemotherapy. Use of other biomarkers remains investigational.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/genética , Humanos , Neoplasias Pulmonares/genética , Prognóstico , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Direct pharmacological targeting of the anti-apoptotic B-cell lymphoma-2 (BCL-2) family is an attractive therapeutic strategy for treating cancer. Obatoclax is a pan-BCL-2 family inhibitor currently in clinical development. Here we show that, although obatoclax can induce mitochondrial apoptosis dependent on BCL-2 associated x protein/BCL-2 antagonist killer (BAX/BAK) consistent with its on-target pharmacodynamics, simultaneous silencing of both BAX and BAK did not abolish acute toxicity or loss of clonogenicity. This is despite complete inhibition of apoptosis. Obatoclax dramatically reduced viability without inducing loss of plasma membrane integrity. This was associated with rapid processing of light chain-3 (LC3) and reduction of S6 kinase phosphorylation, consistent with autophagy. Dramatic ultrastructural vacuolation, not typical of autophagy, was also induced. Silencing of beclin-1 failed to prevent LC3 processing, whereas knockout of autophagy-related (Atg)7 abolished LC3 processing but failed to prevent obatoclax-induced loss of clonogenicity or ultrastructural changes. siRNA silencing of Atg7 in BAX/BAK knockout mouse embryonic fibroblasts did not prevent obatoclax-induced loss of viability. Cells selected for obatoclax resistance evaded apoptosis independent of changes in BCL-2 family expression and displayed reduced LC3 processing. In summary, obatoclax exhibits BAX- and BAK-dependent and -independent mechanisms of toxicity and activation of autophagy. Mechanisms other than autophagy and apoptosis are blocked in obatoclax resistant cells and contribute significantly to obatoclax's anticancer efficacy.
Assuntos
Autofagia/efeitos dos fármacos , Pirróis/farmacologia , Enzimas Ativadoras de Ubiquitina/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Proteína X Associada a bcl-2/fisiologia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/fisiologia , Proteína 7 Relacionada à Autofagia , Proteína Beclina-1 , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Indóis , Proteínas de Membrana/fisiologia , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , 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
We found that procaspase 8 was overexpressed in non-small-cell lung cancers (NSCLCs) compared with matched normal tissues. The caspase 8 inhibitor FLICE-inhibitory protein (FLIP) was also overexpressed in the majority of NSCLCs. Silencing FLIP induced caspase 8 activation and apoptosis in NSCLC cell lines, but not in normal lung cell lines. Apoptosis induced by FLIP silencing was mediated by the TRAIL death receptors DR4 and DR5, but was not dependent on ligation of the receptors by TRAIL. Furthermore, the apoptosis induced by FLIP silencing was dependent on the overexpression of procaspase 8 in NSCLC cells. Moreover, in NSCLC cells, but not in normal cells, FLIP silencing induced co-localization of DR5 and ceramide, and disruption of this co-localization abrogated apoptosis. FLIP silencing supra-additively increased TRAIL-induced apoptosis of NSCLC cells; however, normal lung cells were resistant to TRAIL, even when FLIP was silenced. Importantly, FLIP silencing sensitized NSCLC cells but not normal cells to chemotherapy in vitro, and silencing FLIP in vivo retarded NSCLC xenograft growth and enhanced the anti-tumour effects of cisplatin. Collectively, our results suggest that due to frequent procaspase 8 overexpression, NSCLCs may be particularly sensitive to FLIP-targeted therapies.
Assuntos
Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Caspase 8/metabolismo , Neoplasias Pulmonares/enzimologia , Animais , Antineoplásicos/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Caspase 8/genética , Caspase 8/fisiologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Precursores Enzimáticos/metabolismo , Feminino , Citometria de Fluxo , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Transplante HeterólogoRESUMO
Death receptors can directly (type I cells) or indirectly induce apoptosis by activating mitochondrial-regulated apoptosis (type II cells). The level of caspase 8 activation is thought to determine whether a cell is type I or II, with type II cells less efficient at activating this caspase following death receptor activation. FLICE-inhibitory protein (FLIP) blocks death receptor-mediated apoptosis by inhibiting caspase 8 activation; therefore, we assessed whether silencing FLIP could convert type II cells into type I. FLIP silencing-induced caspase 8 activation in Bax wild-type and null HCT116 colorectal cancer cells; however, complete caspase 3 processing and apoptosis were only observed in Bax wild-type cells. Bax-null cells were also more resistant to chemotherapy and tumor necrosis factor-related apoptosis inducing ligand and, unlike the Bax wild-type cells, were not sensitized to these agents by FLIP silencing. Further analyses indicated that release of second mitochondrial activator of caspases from mitochondria and subsequent inhibition of X-linked inhibitor of apoptosis protein (XIAP) was required to induce full caspase 3 processing and apoptosis following FLIP silencing. These results indicate that silencing FLIP does not necessarily bypass the requirement for mitochondrial involvement in type II cells. Furthermore, targeting FLIP and XIAP may represent a therapeutic strategy for the treatment of colorectal tumors with defects in mitochondrial-regulated apoptosis.
Assuntos
Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/antagonistas & inibidores , Neoplasias Colorretais/patologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Inativação Gênica , Humanos , Mitocôndrias/enzimologia , Estadiamento de Neoplasias , RNA Interferente Pequeno/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Bortezomib (Velcade, PS341) was licensed in 2003 as a first-in-class 20S proteasome inhibitor indicated for treatment of multiple myeloma, and is currently being evaluated clinically in a range of solid tumours. The mechanisms underlying its cancer cell toxicity are complex. A growing body of evidence suggests proteasome inhibition-dependent regulation of the BCL-2 family is a critical requirement. In particular, the stabilization of BH3-only proteins BIK, NOXA and BIM, appear to be essential for effecting BAX- and BAK-dependent cell death. These mechanisms are reviewed and the implications for favourable novel drug interactions are highlighted.
Assuntos
Ácidos Borônicos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Família Multigênica/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Proteínas Proto-Oncogênicas c-bcl-2/genética , Pirazinas/farmacologia , Animais , Bortezomib , Humanos , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/fisiologiaRESUMO
Malignant pleural mesothelioma (MPM) is a highly chemoresistant cancer with a poor prognosis. Hypoxia is a specific property of solid tumours, contributes to low apoptotic potential, and can be selectively targeted by bioreductive drugs. Hypoxia-inducible factor 1alpha (HIF-1alpha) is a subunit of a heterodimeric transcription complex that regulates several genes associated with tumour progression and anti-apoptosis. In this study, we measured for the first time the expression of HIF-1alpha in MPM. Our results show that HIF-1alpha is commonly expressed in MPM but not in normal mesothelium, consistent with the presence of hypoxia. HIF-1alpha does not appear to predict survival; however, this study suggests that bioreductive drugs should be investigated in clinical trials of MPM.
Assuntos
Biomarcadores Tumorais/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Mesotelioma/metabolismo , Neoplasias Pleurais/metabolismo , Apoptose , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Progressão da Doença , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Mesotelioma/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Neoplasias Pleurais/patologia , PrognósticoRESUMO
Robust quantitative estimation of average whole cell mitochondrial dysfunction is a useful tool for assessing sensitivity to apoptotic stimuli induced either by novel agents, or following manipulation of apoptotic threshold by pharmacological or functional genomics approaches. We have mathematically modelled the kinetics of whole cell mitochondrial membrane potential depolarisation within a population of cells as a Bernouli transition. An exponential distribution enables the median latency preceding mitochondrial membrane potential dissipation to be derived. The kinetic model can be fitted to in vitro single cell resolution data derived from kinetic flow cytometric studies by non-linear regression. We propose that kinetic determination of cumulative frequency distributions provides a useful approach for estimating apoptosis sensitivity across cell populations over short time-frames.
Assuntos
Apoptose , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Cinética , Potenciais da Membrana , Mitocôndrias/patologia , Modelos Estatísticos , Modelos Teóricos , Processos Estocásticos , Fatores de TempoRESUMO
BACKGROUND: Cholangiocarcinoma cells express high levels of the antiapoptotic proteins Bcl-X(L) and Mcl-1 and are markedly chemo- and radioresistant. Mitochondria have emerged as central players in apoptosis. Antiapoptotic members of the Bcl-2 protein family localise to the outer mitochondrial membrane and regulate mitochondrial release of apoptogenic proteins. Mitochondrial benzodiazepine receptor (mBzR) ligands have been shown to reverse Bcl-2 action and facilitate apoptosis. AIM: We evaluated the ability of the mBzR antagonist Pk11195 to overcome preapoptotic mitochondrial dysfunction in Egi-1 and Tfk-1, two human cholangiocarcinoma cell lines expressing high levels of Bcl-X(L) and Mcl-1. MATERIALS AND METHODS: Cells growing in culture were used to perform in vitro experiments over 48-96 hours following treatment. The cytotoxic agents used were 5 fluorouracil 10 microM and etoposide (Vp16) 10 microM, together with ultraviolet and 0.5-1 Gy x ray irradiation with or without 75 microM Pk11195. Apoptosis and mitochondrial dysfunction were measured at single cell resolution by flow cytometry using the mitochondrial fluorochrome DiOC6(3). Severe combined immunodeficient non-obese diabetic (SCID-NOD) mice with subcutaneous xenografts using the Egi-1 and Tfk-1 cell lines were treated with etoposide with or without addition of Pk11195 over a 72 hour period during which time the xenograft growth patterns were monitored. RESULTS: In vitro, the effect of Pk11195 on induction of apoptosis in cholangiocarcinoma cells following stimulation by chemotherapy or radiotherapy was found to be both time and dose dependent, with Pk11195 increasing rates of apoptosis by 50-95%. Intraperitoneal administration of Pk11195 in combination with Vp16 was found to increase the growth inhibiting effects of Vp16 on xenografts during the treatment phase. PK11195 75 microM on its own had no intrinsic cytotoxic efficacy. CONCLUSION: This is the first study to demonstrate that functional antagonism of coexpressed Bcl-X(L) and Mcl-1 proteins using the mBzR antagonist Pk11195 can facilitate apoptosis in cholangiocarcinoma following chemotherapy and radiotherapy.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias dos Ductos Biliares/tratamento farmacológico , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/tratamento farmacológico , Isoquinolinas/farmacologia , Neoplasias dos Ductos Biliares/radioterapia , Colangiocarcinoma/radioterapia , Citometria de Fluxo , Imunofluorescência , Antagonistas de Receptores de GABA-A , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células Tumorais Cultivadas , Proteína bcl-XRESUMO
Resistance to apoptosis is a major obstacle preventing effective therapy for malignancy. Mitochondria localized anti-death proteins of the Bcl-2 family play a central role in inhibiting apoptosis and therefore present valid targets for novel therapy. The peripheral benzodiazepine receptor (PBR) shares a close physical association with the permeability transition pore complex (PTPC), a pivotal regulator of cell death located at mitochondrial contact sites. In this study we investigated the cytotoxicity of the PBR ligand, PK11195, in the micromolar concentration range. PK11195 induced antioxidant inhibitable collapse of the inner mitochondrial membrane potential (DeltaPsi(m)) and mitochondrial swelling in HL60 human leukaemia cells, but not in SUDHL4 lymphoma cells (which exhibited a higher level of reduced glutathione and relative tolerance to chemotherapy or pro-oxidant induced DeltaPsi(m)dissipation). PK11195 induced the production of hydrogen peroxide that was not inhibited by Bcl-2 transfection, nor depletion of mitochondrial DNA. ROS production was however blocked by protonophore, implicating a requirement for DeltaPsi(m). Our findings suggest that PK11195-induced cytotoxicity relies upon Bcl-2 resistant generation of oxidative stress; a process only observed at concentrations several orders of magnitude higher that required to saturate its receptor.
Assuntos
Antineoplásicos/toxicidade , Genes bcl-2 , Isoquinolinas/toxicidade , Mitocôndrias/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/fisiologia , Receptores de GABA-A/fisiologia , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HL-60 , Humanos , Peróxido de Hidrogênio/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Células K562 , Linfoma , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptores de GABA-A/efeitos dos fármacos , Transfecção , Células Tumorais CultivadasRESUMO
Bcl-XL, a member of the Bcl-2-related anti-apoptosis protein family, antagonizes a diverse range of apoptosis-inducing stimuli by preventing mitochondrial permeability transition, release of apoptogenic factors including cytochrome C, and caspase activation. We have tested the hypothesis that the susceptibility of Bcl-XL-expressing leukaemic cells to apoptosis induced by VP16 (etoposide) can be enhanced by pharmacological downregulation of Bcl-XL in vivo. Two subcutaneous xenograft models of B-cell leukaemia-employing SEMK-2 and BV173 cell lines were established in severe combined immunodeficient/non-obese diabetic mice followed by 14 d of continuous subcutaneous administration of Bcl-XL-specific second generation oligonucleotides ISIS 16009 or ISIS 15999. Tumours were disaggregated, enabling investigation of Bcl-XL expression and apoptosis susceptibility at single-cell resolution using cytofluorimetry. Marked sequence-specific reduction of Bcl-XL was associated with sequence-specific enhancement of VP16-induced mitochondrial permeability transition, caspase-3 activation and loss of membrane asymmetry. A negative correlation between Bcl-XL expression and apoptosis susceptibility was observed, together with a positive correlation with respect to a reduced redox state. Bcl-XL downregulation reduces the threshold for VP16-induced apoptosis by potentiating mitochondrial dysfunction and its sequelae, and therefore presents a novel therapeutic strategy for reversing chemoresistance.