RESUMO
UNLABELLED: Viruses have coevolved with their host to ensure efficient replication and transmission without inducing excessive pathogenicity that would indirectly impair their persistence. This is exemplified by the bovine leukemia virus (BLV) system in which lymphoproliferative disorders develop in ruminants after latency periods of several years. In principle, the equilibrium reached between the virus and its host could be disrupted by emergence of more pathogenic strains. Intriguingly but fortunately, such a hyperpathogenic BLV strain was never observed in the field or designed in vitro. In this study, we sought to understand the role of envelope N-linked glycosylation with the hypothesis that this posttranslational modification could either favor BLV infection by allowing viral entry or allow immune escape by using glycans as a shield. Using reverse genetics of an infectious molecular provirus, we identified a N-linked envelope glycosylation site (N230) that limits viral replication and pathogenicity. Indeed, mutation N230E unexpectedly leads to enhanced fusogenicity and protein stability. IMPORTANCE: Infection by retroviruses requires the interaction of the viral envelope protein (SU) with a membrane-associated receptor allowing fusion and release of the viral genomic RNA into the cell. We show that N-linked glycosylation of the bovine leukemia virus (BLV) SU protein is, as expected, essential for cell infection in vitro. Consistently, mutation of all glycosylation sites of a BLV provirus destroys infectivity in vivo. However, single mutations do not significantly modify replication in vivo. Instead, a particular mutation at SU codon 230 increases replication and accelerates pathogenesis. This unexpected observation has important consequences in terms of disease control and managing.
Assuntos
Vírus da Leucemia Bovina/genética , Vírus da Leucemia Bovina/patogenicidade , Proteínas do Envelope Viral/genética , Replicação Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Gatos , Fusão Celular , Chlorocebus aethiops , Glicosilação , Células HEK293 , Células HeLa , Humanos , Vírus da Leucemia Bovina/metabolismo , Fusão de Membrana/genética , Mutação , Estabilidade Proteica , Alinhamento de Sequência , Análise de Sequência de RNA , Ovinos , Proteínas do Envelope Viral/metabolismo , Carga ViralRESUMO
BACKGROUND: it is now well established that hypoxia renders tumor cells resistant to radio- but also chemotherapy. However, few elements are currently available as for the mechanisms underlying this protection. RESULTS: in this study, physiological hypoxia was shown to inhibit apoptosis induced in HepG2 cells by etoposide. Indeed, hypoxia reduced DNA fragmentation, caspase activation and PARP cleavage. The DNA binding activity of 10 transcription factors was followed while the actual transcriptional activity was measured using specific reporter plasmids. Of note is the inhibition of the etoposide-induced activation of p53 under hypoxia. In parallel, data from low density DNA microarrays indicate that the expression of several pro- and anti-apoptotic genes was modified, among which are Bax and Bak whose expression profile paralleled p53 activity. Cluster analysis of data unravels several possible pathways involved in the hypoxia-induced protection against etoposide-induced apoptosis: one of them could be the inhibition of p53 activity under hypoxia since caspase 3 activity parallels Bax and Bak expression profile. Moreover, specific downregulation of HIF-1alpha by RNA interference significantly enhanced apoptosis under hypoxia possibly by preventing the hypoxia mediated decrease in Bak expression without altering Bax expression. CONCLUSION: these results are a clear demonstration that hypoxia has a direct protective effect on apoptotic cell death. Moreover, molecular profiling points to putative pathways responsible for tumor growth in challenging environmental conditions and cancer cell resistance to chemotherapeutic agents.
Assuntos
Apoptose/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Etoposídeo/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Análise por Conglomerados , DNA de Neoplasias/metabolismo , Inativação Gênica , Genes Reporter , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
BACKGROUND: It is more and more recognized that hypoxia plays a role in the resistance of cancer cells to chemotherapy. However, the mechanisms underlying this resistance still need deeper understanding. The aim of this study was to investigate the effect of hypoxia on this process since hypoxia is one of the hallmarks of tumor environment. RESULTS: The effect of hypoxia on the apoptosis induced by etoposide, one drug commonly used in chemotherapy, was investigated using three different cancer cell lines. Gene expression changes were also studied in order to delineate the mechanisms responsible for the hypoxia-induced chemoresistance. We observed that hypoxia differentially influenced etoposide-induced cell death according to the cancer cell type. While hypoxia inhibited apoptosis in hepatoma HepG2 cells, it had no influence in lung carcinoma A549 cells and further enhanced it in breast cancer MCF-7 cells. Etoposide increased p53 activity in all cell lines while hypoxia alone decreased it only in HepG2 cells. Hypoxia had no influence on the etoposide-induced p53 activity in A549, increased p53 abundance in MCF-7 cells but markedly decreased p53 activity in HepG2 cells. Using low density DNA arrays to detect the expression of genes involved in the regulation of apoptosis, etoposide and hypoxia were shown to each influence the expression of numerous genes, many of the ones influenced by etoposide being p53 target genes. Again, the influence of hypoxia on the etoposide-induced changes was different according to the cell type. CONCLUSION: These results evidenced that there was a striking parallelism between the effect of hypoxia on the etoposide-induced p53 stabilization as well as p53 target gene expression and its effect on the etoposide-induced apoptosis according to the cell type. They are very interesting not only because they provide one possible mechanism for the induction of chemoresistance under hypoxic conditions in cells like HepG2 but also because they indicate that not all cell types respond the same way. This knowledge is of importance in designing adequate treatment according to the type of tumors.
Assuntos
Apoptose/efeitos dos fármacos , Etoposídeo/farmacologia , Neoplasias/metabolismo , Neoplasias/patologia , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , DNA/genética , Dano ao DNA/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Supressora de Tumor p53/metabolismoRESUMO
With 5-year survival rates below 5%, small cell lung carcinoma (SCLC) has very poor prognosis and requires improved therapies. Despite an excellent overall response to first-line therapy, relapses are frequent and further treatments are disappointing. The goal of the study was to improve second-line therapy of SCLC. The effect of chemotherapeutic agents was evaluated in cell lines (apoptosis, reactive oxygen species, and RNA and protein expression) and in mouse models (tumour development). We demonstrate here that valproic acid, a histone deacetylase inhibitor, improves the efficacy of a second-line regimen (vindesine, doxorubicin and cyclophosphamide) in SCLC cells and in mouse models. Transcriptomic profiling integrating microRNA and mRNA data identifies key signalling pathways in the response of SCLC cells to valproic acid, opening new prospects for improved therapies.
RESUMO
Malignant pleural mesothelioma (MPM) is an almost invariably fatal cancer of the pleura due to asbestos exposure. Increasing evidence indicates that unresponsiveness to chemotherapy is due to epigenetic errors leading to inadequate gene expression in tumor cells. The availability of compounds that modulate epigenetic modifications, such as histone acetylation or DNA methylation, offers new prospects for treatment of MPM. Here, we review latest findings on epigenetics in mesothelioma and present novel strategies for promising epigenetic therapies.
Assuntos
Epigênese Genética , Neoplasias Pulmonares/genética , Mesotelioma/genética , Neoplasias Pleurais/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transformação Celular Neoplásica/genética , Ensaios Clínicos como Assunto , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Epigênese Genética/efeitos dos fármacos , Epigenômica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Mesotelioma/tratamento farmacológico , Mesotelioma MalignoRESUMO
Tumor hypoxia is a common characteristic of most solid tumors and is correlated with poor prognosis for patients partly because hypoxia promotes resistance to cancer therapy. Hypoxia selects cancer cells that are resistant to apoptosis and allows the onset of mechanisms that promote cancer cells survival including autophagy. Previously, we showed that human hepatoma HepG2 cells were protected under hypoxia against the etoposide-induced apoptosis. In this study, respective putative contribution of autophagy and BNIP3 in the protection conferred by hypoxia against the etoposide-induced apoptosis was investigated. We report that autophagy is induced by etoposide, a process that is not affected by hypoxic conditions. Using Atg5 siRNA, we show that etoposide-induced autophagy promotes apoptotic cell death under normoxia but not under hypoxia. Then, we investigated whether the hypoxia-induced protein BNIP3 could explain the different effect of autophagy on cell death under hypoxia or normoxia. We show that the silencing of BNIP3 does not affect autophagy whatever the pO(2) but participates in the protective effect of hypoxia against etoposide-induced apoptosis. Together, these results suggest that autophagy might be involved in etoposide-induced cell death only under normoxia and that BNIP3 is a major effector of the protective mechanism conferred by hypoxia to protect cancer cells against etoposide-induced apoptotic cell death.
Assuntos
Antineoplásicos/farmacologia , Autofagia/fisiologia , Hipóxia Celular/fisiologia , Etoposídeo/farmacologia , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Consumo de Oxigênio , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , VacúolosRESUMO
Tumor hypoxia is one of the features of tumor microenvironment that contributes to chemoresistance in particular by cellular adaptations that modulate the apoptotic process. However, the mechanisms involved in this resistance still need deeper understanding. In this study, we investigated the involvement of four transcription factors, c-Myc, nuclear factor kappaB (NF-kappaB), p53, and c-jun/activator protein 1 (AP-1) in the hypoxia-induced resistance to etoposide in HepG2 cells. Whereas the profile of c-Myc and NF-kappaB activity did not fit the effect of hypoxia on caspase 3 activity, hypoxia decreased basal p53 abundance and DNA binding activity as well as p53 etoposide-induced activation. Short interfering RNA (siRNA) silencing evidenced that p53 was required for etoposide-induced apoptosis under normoxia. An inhibition of its activity under hypoxia could thus be responsible at least in part for the protection observed under hypoxic conditions. Moreover, p53 was found to induce the expression of Bak1. We showed that Bak1 was involved in the etoposide-induced apoptosis because Bak1 siRNA decreased it. Conversely, hypoxia increased c-jun DNA binding activity in the presence of etoposide. siRNA-mediated silencing of c-jun increased the responsiveness of cells to etoposide under hypoxia, as shown by an increase in caspase 3 activity and lactate dehydrogenase release. These effects occurred in a p53-independent manner. These data evidenced that hypoxia decreased the responsiveness of HepG2 cells to etoposide at least by two independent pathways involving p53 inhibition and c-jun activation.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Etoposídeo/farmacologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Hipóxia Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/genéticaRESUMO
A solid tumour forms an organ-like structure that is comprised of cancer cells as well as stroma cells (fibroblasts, inflammatory cells) that are embedded in an extracellular matrix and are nourished by vascular network. However, tumoral microenvironment is heterogeneous due to the abnormal vasculature network and high proliferation rate of cancer cells. Because of these features, some regions are starved from oxygen, a phenomenon called hypoxia. Transient hypoxia is associated with inadequate blood flow while chronic hypoxia is the consequence of the increased oxygen diffusion distance due to tumour expansion. Both types of hypoxia are correlated with poor outcome for patients. Moreover, hypoxia also enhances chemoresistance of cancer cells. Firstly, the delivery of drugs in hypoxic area and cellular uptake of it are affected by hypoxia or associated acidity. Secondly, some chemotherapeutic drugs require oxygen to generate free radicals that contribute to cytotoxicity. Last, hypoxia induces cellular adaptations that compromise the effectiveness of chemotherapy. In response to nutrient deprivation due to hypoxia, the rate of proliferation of cancer cells decreases but chemotherapeutic drugs are more effective against proliferating cells. On the other hand, hypoxia induces adaptation by post-translational and transcriptional changes that promote cell survival and resistance to chemotherapy. Through these changes, hypoxia promotes angiogenesis, shift to glycolytic metabolism, expression of ABC transporters, cell survival by inducing the expression of genes encoding growth factors and the modulation of apoptotic process. The aim of this review is to provide a description of known hypoxia-induced mechanisms of chemoresistance at a cellular level.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Hipóxia Celular , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Invasividade Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Tumor hypoxia has been described to increase the resistance of cancer cells to radiation therapy and chemotherapy. It also supports the invasiveness and metastatic potential of the tumor. However, few data are available on the transduction pathway set up under hypoxia and leading to this resistance against anti-cancer therapies. HIF-1, the main transcription factor activated by hypoxia, has been recently shown to participate to this process although its role as an anti- or a pro-apoptotic protein is still controversy. In this study, we showed that hypoxia protected HepG2 cells against etoposide-induced apoptosis. The effect of hypoxia on cell death was assayed by measuring different parameters of the apoptotic pathway, like DNA fragmentation, caspase activity and PARP-1 cleavage. The possible implication of HIF-1 in the anti-apoptotic role of hypoxia was investigated using HIF-1alpha siRNA. Our results indicated that HIF-1 is not involved in the hypoxia-induced anti-apoptotic pathway. Another transcription factor, AP-1, was studied for its potential role in the hypoxia-induced protection against apoptosis. Specific inhibition of AP-1 decreased the protection effect of hypoxia against etoposide-induced apoptosis. Together, all these data underline that hypoxia could mediate its anti-apoptotic role via different transcription factors depending on the cellular context and pro-apoptotic stimuli.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose , Etoposídeo/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator de Transcrição AP-1/metabolismo , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Caspase 3 , Caspases/metabolismo , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Cobalto/metabolismo , Fragmentação do DNA , Imunofluorescência , Humanos , Neoplasias Hepáticas/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transfecção , Células Tumorais CultivadasRESUMO
Increased levels of Mcl-1 (myeloid cell factor-1) have been reported in several cancers, suggesting an important role played by Mcl-1 in cancer cell survival. Mcl-1 is an anti-apoptotic protein shown to delay or block apoptosis. In this work, using semiquantitative immunofluorescence, real-time PCR, and RNase protection assay, an increase in Mcl-1 expression was detected in hepatoma HepG2 cells incubated under hypoxia or in the presence of cobalt chloride. Through analysis of the Mcl-1 promoter sequence, a putative HIF-1 (hypoxiainducible factor-1) binding site was identified. A Mcl-1 promoter fragment containing this hypoxia-responsive element was able to bind HIF-1 in vitro. It also induced hypoxia-dependent transcription of a luciferase reporter gene, which was suppressed by anti-HIF-1alpha short interfering RNA. Finally, overexpression of Mcl-1 protected HepG2 cells against apoptosis induced by tert-butyl hydroperoxide as shown by inhibition of caspase-3 activation and DNA fragmentation. All these data suggest a potential anti-apoptotic role of HIF-1 that could protect cells against apoptosis under hypoxia by overexpression of the Mcl-1 protein.