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1.
PLoS One ; 8(6): e65489, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23762382

RESUMO

Based on promising preclinical efficacy associated with the 20S proteasome inhibitor bortezomib in malignant pleural mesothelioma (MPM), two phase II clinical trials have been initiated (EORTC 08052 and ICORG 05-10). However, the potential mechanisms underlying resistance to this targeted drug in MPM are still unknown. Functional genetic analyses were conducted to determine the key mitochondrial apoptotic regulators required for bortezomib sensitivity and to establish how their dysregulation may confer resistance. The multidomain proapoptotic protein BAK, but not its orthologue BAX, was found to be essential for bortezomib-induced apoptosis in MPM cell lines. Immunohistochemistry was performed on tissues from the ICORG-05 phase II trial and a TMA of archived mesotheliomas. Loss of BAK was found in 39% of specimens and loss of both BAX/BAK in 37% of samples. However, MPM tissues from patients who failed to respond to bortezomib and MPM cell lines selected for resistance to bortezomib conserved BAK expression. In contrast, c-Myc dependent transactivation of NOXA was abrogated in the resistant cell lines. In summary, the block of mitochondrial apoptosis is a limiting factor for achieving efficacy of bortezomib in MPM, and the observed loss of BAK expression or NOXA transactivation may be relevant mechanisms of resistance in the clinic.


Assuntos
Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Mesotelioma/metabolismo , Mesotelioma/patologia , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazinas/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Animais , Bortezomib , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Embrião de Mamíferos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Mesotelioma/genética , Camundongos , Mitocôndrias/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo
2.
Cell Oncol (Dordr) ; 35(2): 85-93, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22278362

RESUMO

BACKGROUND: The evolutionarily conserved septin family of genes encode GTP binding proteins involved in a variety of cellular functions including cytokinesis, apoptosis, membrane dynamics and vesicle trafficking. Septin proteins can form hetero-oligomeric complexes and interact with other proteins including actin and tubulin. The human SEPT9 gene on chromosome 17q25.3 has a complex genomic architecture with 18 different transcripts that can encode 15 distinct polypeptides. Two distinct transcripts with unique 5' ends (SEPT9_v4 and SEPT9_v4*) encode the same protein. In tumours the ratio of these transcripts changes with elevated levels of SEPT9_v4* mRNA, a transcript that is translated with enhanced efficiency leading to increased SEPT9_i4 protein. METHODS: We have examined the effect of over-expression of SEPT9_i4 on the dynamics of microtubule polymer mass in cultured cells. RESULTS: We show that the microtubule network in SEPT9_i4 over-expressing cells resists disruption by paclitaxel or cold incubation but also repolymerises tubulin more slowly after microtubule depolymerisation. Finally we show that SEPT9_i4 over-expressing cells have enhanced survival in the presence of clinically relevant microtubule acting drugs but not after treatment with DNAinteracting agents. CONCLUSIONS: Given that SEPT9 over-expression is seen in diverse tumours and in particular ovarian and breast cancer, such data indicate that SEPT9_v4 expression may be clinically relevant and contribute to some forms of drug resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Paclitaxel/farmacologia , Septinas/metabolismo , Vimblastina/análogos & derivados , Linhagem Celular Tumoral , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Citometria de Fluxo , Humanos , Gelo , Polimerização/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Vimblastina/farmacologia , Vinorelbina
3.
Apoptosis ; 16(3): 311-20, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21107700

RESUMO

Resistance to cisplatin chemotherapy remains a major hurdle preventing effective treatment of many solid cancers. BAX and BAK are pivotal regulators of the mitochondrial apoptosis pathway, however little is known regarding their regulation in cisplatin resistant cells. Cisplatin induces DNA damage in both sensitive and resistant cells, however the latter exhibits a failure to initiate N-terminal exposure of mitochondrial BAK or mitochondrial SMAC release. Both phenotypes are highly sensitive to mitochondrial permeabilisation induced by exogenous BH3 domain peptides derived from BID, BIM, NOXA (which targets MCL-1 and A1), and there is no significant change in their prosurvival BCL2 protein expression profiles. Obatoclax, a small molecule inhibitor of pro-survival BCL-2 family proteins including MCL-1, decreases cell viability irrespective of platinum resistance status across a panel of cell lines selected for oxaliplatin resistance. In summary, selection for platinum resistance is associated with a block of mitochondrial death signalling upstream of BAX/BAK activation. Conservation of sensitivity to BH3 domain induced apoptosis can be exploited by agents such as obatoclax, which directly target the mitochondria and BCL-2 family.


Assuntos
Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias/patologia , Peptídeos/farmacologia , Platina/farmacologia , Pirróis/farmacologia , Trifosfato de Adenosina/metabolismo , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Dano ao DNA , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indóis , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Peptídeos/química , Platina/uso terapêutico , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteína Killer-Antagonista Homóloga a bcl-2/antagonistas & inibidores , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
4.
BMC Cancer ; 10: 380, 2010 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-20646307

RESUMO

BACKGROUND: Activation of the extrinsic apoptosis pathway by tumour necrosis factor related apoptosis inducing ligand (TRAIL) is a novel therapeutic strategy for treating cancer that is currently under clinical evaluation. Identification of molecular biomarkers of resistance is likely to play an important role in predicting clinical anti tumour activity. The involvement of the mitochondrial type 1 voltage dependent anion channel (VDAC1) in regulating apoptosis has been highly debated. To date, a functional role in regulating the extrinsic apoptosis pathway has not been formally excluded. METHODS: We carried out stable and transient RNAi knockdowns of VDAC1 in non-small cell lung cancer cells, and stimulated the extrinsic apoptotic pathway principally by incubating cells with the death ligand TRAIL. We used in-vitro apoptotic and cell viability assays, as well as western blot for markers of apoptosis, to demonstrate that TRAIL-induced toxicity is VDAC1 dependant. Confocal microscopy and mitochondrial fractionation were used to determine the importance of mitochondria for caspase-8 activation. RESULTS: Here we show that either stable or transient knockdown of VDAC1 is sufficient to antagonize TRAIL mediated apoptosis in non-small cell lung cancer (NSCLC) cells. Specifically, VDAC1 is required for processing of procaspase-8 to its fully active p18 form at the mitochondria. Loss of VDAC1 does not alter mitochondrial sensitivity to exogenous caspase-8-cleaved BID induced mitochondrial depolarization, even though VDAC1 expression is essential for TRAIL dependent activation of the intrinsic apoptosis pathway. Furthermore, expression of exogenous VDAC1 restores the apoptotic response to TRAIL in cells in which endogenous VDAC1 has been selectively silenced. CONCLUSIONS: Expression of VDAC1 is required for full processing and activation of caspase-8 and supports a role for mitochondria in regulating apoptosis signaling via the death receptor pathway.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Caspase 8/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Apoptose , Western Blotting , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/antagonistas & inibidores , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Citometria de Fluxo , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Receptores de Morte Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Células Tumorais Cultivadas , Receptor fas/antagonistas & inibidores , Receptor fas/genética , Receptor fas/metabolismo
5.
Apoptosis ; 13(11): 1386-93, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18814032

RESUMO

We have developed a versatile and rapid method for the quantitative estimation of cell death kinetics, following direct single-shot activation of the mitochondrial death pathway by a cell permeable BH3 activator peptide (D-R(8)BH3(BID)). This approach employs timelapse epifluorescent imaging of live cells and a machine- vision based feature extraction algorithm, to measure unidirectional stochastic transitions associated with mitochondrial inner membrane potential depolarization and/or permeability transition, at single cell resolution. This data is transformed to enable construction of a right step-wise survival function using the product limit estimator, and estimation of a median latency parameter (lambda), defined for the entire imaged cell population. Estimates of lambda computed for cells exhibiting two-colour fluorescence can be compared statistically using the Mantel-Hansel test. This general method has been applied to measure the kinetics and temporal ordering of BH3 domain induced mitochondrial depolarization and inner membrane permeabilization in cancer cells, and demonstrates the robustness of this technique in resolving temporally distinct intracellular events within individual cells.


Assuntos
Mitocôndrias/metabolismo , Algoritmos , Apoptose , Biofísica/métodos , Dicroísmo Circular , Citocromos c/metabolismo , Humanos , Cinética , Potenciais da Membrana , Microscopia de Fluorescência/métodos , Modelos Biológicos , Conformação Proteica , Estrutura Terciária de Proteína , Processos Estocásticos , Fatores de Tempo
6.
J Pathol ; 206(4): 458-65, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15902694

RESUMO

Several lines of evidence indicate that altered expression of SEPT9 is seen in human neoplasia. In particular there is evidence of altered expression of the SEPT9_v4 isoform. The functional consequences of this remain unclear. We have studied the expression of wild-type- and GTP-binding mutants (G144V and S148N) of the SEPT9_v4 isoform in the MCF7 cell line as a model for its deregulation in neoplasia. We find that SEPT9_v4 expression induces dramatic actin cytoskeletal reorganization with the formation of processes around the cell periphery. Expression of the SEPT9_v4 isoform and a G144V mutant cause delocalization of endogenous SEPT9 from filamentous structures but the S148N mutant does not have this effect. In addition SEPT9_v4 isoform expression enhances cell motility and is associated with perturbation of directional movement. Expression of SEPT9_v4 GTP binding mutants also has potent effects on morphology and motility and causes loss of normal polarity, as judged by Golgi reorientation assays. The phenotypes induced by expression of the SEPT9_v4 isoform and the GTP mutants provide an insight into possible mechanisms of SEPT9_v4 function and suggest that the GTPase functions have both ras- and rab-like features. We propose a model in which overexpression of the SEPT9_v4 isoform in neoplasia is associated with perturbation of SEPT9 complexes, leading to phenotypes associated with neoplasia.


Assuntos
GTP Fosfo-Hidrolases/genética , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Actinas/análise , Linhagem Celular Tumoral , Movimento Celular/genética , Polaridade Celular/genética , Citoesqueleto/genética , GTP Fosfo-Hidrolases/metabolismo , Complexo de Golgi/genética , Humanos , Mutação/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fenótipo , Isoformas de Proteínas/genética , Septinas , Transgenes/genética
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