RESUMO
Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as "apoptosis-necrosis continuum." To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death.
Assuntos
Antineoplásicos/farmacologia , Caspases/metabolismo , Cromatina/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Necrose/enzimologia , Clorometilcetonas de Aminoácidos/farmacologia , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzofenantridinas/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspases/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromatina/ultraestrutura , Colchicina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Necrose/induzido quimicamente , Necrose/genética , Neurônios , Nocodazol/farmacologia , Peptidomiméticos/farmacologia , Quinolinas/farmacologia , Rotenona/farmacologia , Transdução de Sinais , Estaurosporina/farmacologia , Tapsigargina/farmacologiaRESUMO
BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that regulate a broad array of cellular and disease processes. Several miRNAs are differentially expressed in cancer and many are being considered as biomarkers for predicting clinical outcomes. Here we quantified the expression of three miRNAs, miR-21, miR-141, and miR-221, from prostate cancer surgical specimens and evaluated their association with disease recurrence after primary therapy. METHODS: A pilot nested case-control study was designed from a large cohort of men who underwent radical prostatectomy between 1993 and 2001. Total RNA was extracted from malignant prostate tissue of 59 cases (recurrence) and 59 controls. Cases and controls were matched on age, race, pathologic stage, and grade. The relative expression of each miRNA was then determined for each sample by quantitative real-time RT-PCR. Conditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) of recurrence for tertiles of miRNA expression. We noted block storage time effects and thus, used separate tertile cutpoints based on the controls by calendar year of prostatectomy. RESULTS: Lower miR-221 expression was associated with a higher risk of recurrence; the ORs were 3.21 for the lowest tertile and 2.63 for the middle tertile compared with the highest tertile of expression (P-trend = 0.02). This pattern was unchanged after multivariable adjustment (P-trend = 0.05). No statistically significant trends were observed for miR-21 or miR-141 after multivariable adjustment. CONCLUSIONS: Based on this small pilot study, men with localized prostate cancers with lower miR-221 expression may have a greater risk for recurrence after surgery.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , MicroRNAs/metabolismo , Recidiva Local de Neoplasia/epidemiologia , Prostatectomia , Neoplasias da Próstata/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Idoso , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Regulação Neoplásica da Expressão Gênica , Humanos , Incidência , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Projetos Piloto , Prognóstico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Fatores de Risco , Resultado do TratamentoRESUMO
miR-21 is the most commonly over-expressed microRNA (miRNA) in cancer and a proven oncogene. Hsa-miR-21 is located on chromosome 17q23.2, immediately downstream of the vacuole membrane protein-1 (VMP1) gene, also known as TMEM49. VMP1 transcripts initiate â¼ 130 kb upstream of miR-21, are spliced, and polyadenylated only a few hundred base pairs upstream of the miR-21 hairpin. On the other hand, primary miR-21 transcripts (pri-miR-21) originate within the last introns of VMP1, but bypass VMP1 polyadenylation signals to include the miR-21 hairpin. Here, we report that VMP1 transcripts can also bypass these polyadenylation signals to include miR-21, thus providing a novel and independently regulated source of miR-21, termed VMP1-miR-21. Northern blotting, gene-specific RT-PCR, RNA pull-down and DNA branching assays support that VMP1-miR-21 is expressed at significant levels in a number of cancer cell lines and that it is processed by the Microprocessor complex to produce mature miR-21. VMP1 and pri-miR-21 are induced by common stimuli, such as phorbol-12-myristate-13-acetate (PMA) and androgens, but show differential responses to some stimuli such as epigenetic modifying agents. Collectively, these results indicate that miR-21 is a unique miRNA capable of being regulated by alternative polyadenylation and two independent gene promoters.
Assuntos
Proteínas de Membrana/genética , MicroRNAs/genética , Poliadenilação , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Ribonuclease III/metabolismo , Transcrição GênicaRESUMO
Chemical inhibitors of cyclin-dependent kinase (CDK), like roscovitine, are promising drugs in the context of new cancer therapies. Roscovitine and related compounds, like seliciclib and olomoucine, are effective inducers of apoptosis in many proliferating cells in culture. These compounds are known to activate the intrinsic or mitochondrial pathway of apoptosis. In order to better characterize this intrinsic pathway, a transcriptional analysis was performed using the reverse transcriptase-multiplex ligation-dependent probe amplification procedure (RT-MLPA). In five cell lines, we detected an early and marked reduction of most transcripts, which is consistent with the disruption of transcription that results from the inhibition of CDK7 and CDK9. However, the mRNA of p53-upregulated modulator of apoptosis (PUMA) gene escaped from this transcription inhibition in neuroblastoma cells with a functional p53 protein. The increase of PUMA mRNA was not found in roscovitine-treated cell lines defective in p53, which underwent apoptosis like their p53 proficient counterparts. In addition, in SH-SY5Y cells, sublethal and lethal concentrations of roscovitine produced equivalent increases of PUMA mRNA and protein. In conclusion, the increased expression of PUMA was not associated with apoptosis induction. On the contrary, mRNA and protein depletion of MCL-1 gene correlated the best with cell demise. Moreover, NOXA protein suffered a far minor decrease than MCL-1. Because of the selective neutralization of NOXA by MCL-1, we hypothesize that the disruption of this balance is a critical event in apoptosis induction by roscovitine and related compounds.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Purinas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Roscovitina , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo , Proteína bcl-X/metabolismoRESUMO
Glioblastoma (GBM) is a highly aggressive brain tumor and almost all patients die because of relapses. GBM-derived cells undergo cell death without nuclear fragmentation upon treatment with different apoptotic agents. Nuclear dismantling determines the point-of-no-return in the apoptotic process. DFF40/CAD is the main endonuclease implicated in apoptotic nuclear disassembly. To be properly activated, DFF40/CAD should reside in the cytosol. However, the endonuclease is poorly expressed in the cytosol and remains cumulated in the nucleus of GBM cells. Here, by employing commercial and non-commercial patient-derived GBM cells, we demonstrate that the natural terpenoid aldehyde gossypol prompts DFF40/CAD-dependent nuclear fragmentation. A comparative analysis between gossypol- and staurosporine-treated cells evidenced that levels of neither caspase activation nor DNA damage were correlated with the ability of each compound to induce nuclear fragmentation. Deconvoluted confocal images revealed that DFF40/CAD was almost completely excluded from the nucleus early after the staurosporine challenge. However, gossypol-treated cells maintained DFF40/CAD in the nucleus for longer times, shaping a ribbon-like structure piercing the nuclear fragments and building a network of bridged masses of compacted chromatin. Therefore, GBM cells can fragment their nuclei if treated with the adequate insult, making the cell death process irreversible.
RESUMO
SPP1 was found to be significantly upregulated in many kinds of malignant tumors, including gliomas. Considering that gene polymorphisms have been implicated in the development of gliomas, we performed an association study between SPP1 functional promoter region polymorphisms and glioma risk in a Chinese population. We found significant evidence of an association between SPP1 promoter polymorphisms and glioma risk. For the -155_156insG variant, the -155_156GG allele was found to be significantly associated with an increased risk of glioma (P=0.020, odds ratio (OR)=1.202, 95% confidence interval (CI): 1.028-1.408). Individuals with the genotype containing the GG allele had a 1.372-fold increased risk (P=0.006, OR=1.372, 95% CI: 1.095-1.719). Further stratified analyses suggested that a significant association existed in adult glioma patients, male subjects and in cases without a family history of cancer. Alternatively, the study of single-nucleotide polymorphism -443C/T in a recessive model revealed that the genotype CC+CT significantly decreased the risk of glioma when compared with TT (P=0.023, OR=0.774, 95% CI: 0.621-0.966). After the analysis of haplotypes, the haplotype -155_156GG/-443T was represented at a significantly higher frequency in cases (P=0.029, OR=1.192, 95% CI: 1.018-1.395). Cellular assay indicated that the transcriptional activity of the SPP1 promoter containing the -155_156GG allele significantly increased in glioma cells. Thus, variants of the SPP1 promoter might influence the risk of glioma by regulating promoter activity. Further analyses are necessary to validate our observation in larger samples or in other ethnic groups.
Assuntos
Povo Asiático/genética , Etnicidade/genética , Predisposição Genética para Doença , Glioma/genética , Osteopontina/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Adolescente , Adulto , Estudos de Casos e Controles , China , Feminino , Haplótipos/genética , Humanos , Masculino , Fatores de Risco , Transcrição GênicaRESUMO
Macroautophagy (hereafter autophagy) is a multistep intracellular catabolic process with pleiotropic implications in cell fate. Attending to its activation, autophagy can be classified into inducible or constitutive. Constitutive, or basal autophagy, unfolds under nutrient-replete conditions to maintain the cellular homeostasis. Autophagy inhibitory drugs are powerful tools to interrogate the role of autophagy and its consequences on cell fate. However, 3-methyladenine and various of these compounds present an intrinsic capacity to trigger cell death, for instance the broadly-employed 3-methyladenine. To elucidate whether the inhibition of basal autophagy is causative of cell demise, we have employed several representative compounds acting at different phases of the autophagic process: initiation (SBI0206965 and MHY1485), nucleation (3-methyladenine, SAR405, Spautin-1 and Cpd18), and completion (Bafilomycin A1 and Chloroquine). These compounds inhibited the basal autophagy of MEF cultures in growing conditions. Among them, 3-methyladenine, SBI-0206965, Chloroquine, and Bafilomycin A1 triggered BAX- and/or BAK-dependent cytotoxicity and caspase activation. 3-methyladenine was the only compound to induce a consistent and abrupt decrease in cell viability across a series of ontologically unrelated human cell lines. 3-methyladenine-induced cytotoxicity was not driven by the inhibition of the AKT/mTOR axis. Autophagy-deficient Fip200-/- MEFs displayed an increased sensitivity to activate caspases and to undergo cell death in response to 3-methyladenine. The cytotoxicity induced by 3-methyladenine correlated with a massive DNA damage, as shown by γ-H2A.X. This genotoxicity was observed at 10 mM 3-methyladenine, the usual concentration to inhibit autophagy and was maximized in Fip200-/- MEFs. In sum, our results suggest that, in growing conditions, autophagy acts as a protective mechanism to diminish the intrinsic cytotoxicity of 3-methyladenine. However, when the cellular stress exerted by 3-methyladenine surpasses the protective effect of basal autophagy, caspase activation and DNA damage compromise the cell viability.
RESUMO
In previous reports, we have shown in SH-SY5 cells that olomoucine and roscovitine, two inhibitory drugs of cyclin-dependent kinases, caused apoptosis independent of the extrinsic pathway. In this experimental paradigm, apoptosis was refractory to the protective effects of either Bcl-2 or Bcl-XL overexpression. We are now reporting that the failure of Bcl-XL to prevent dell death was consistent with no effect on the kinetics of caspase activation and cytochrome c release. To further characterize this issue, we have discarded a direct effect of either olomoucine or roscovitine on mitochondrial permeability transition. Moreover, we have evidence that an intrinsic pathway took place in SH-SY5Y cells by showing the mitochondrial translocation of a GFP-Bax construct on transfection and treatment with cyclin-dependent kinase inhibitory drugs. Finally, we tested the effect of olomoucine and roscovitine on wild-type, bax-/-, bak-/-, and double bax-/-bak-/- mouse embryonic fibroblasts (MEF). In wild-type MEFs, both drugs induced cell death by apoptosis in a dose-dependent manner. In bax-/-, bak-/-, and, particularly, double bax-/-bak-/- MEFs, we observed the inhibition of apoptosis. In conclusion, olomoucine and roscovitine caused apoptosis through an intrinsic pathway, with Bax and Bak proteins being involved.
Assuntos
Apoptose , Quinases Ciclina-Dependentes/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Proteína X Associada a bcl-2/fisiologia , Animais , Linhagem Celular Tumoral , Citocromos c/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Olomoucine and Roscovitine are pharmacological inhibitors of cyclin-dependent kinases (CDK) displaying a promising profile as anticancer agents. Both compounds are effective inductors of apoptosis in a human neuroblastoma cell line, SH-SY5Y. The characterization of this process had suggested the involvement of an extrinsic pathway [Ribas, J., Boix, J., 2004. Cell differentiation, Caspase inhibition, and macromolecular synthesis blockage, but not Bcl-2 or Bcl-XL proteins, protect SH-SY5Y cells from apoptosis triggered by two CDK inhibitory drugs. Exp. Cell Res. 295 9-24.], which depends on either Caspase 8 or Caspase 10 activation. However, neither Caspase 8 nor Caspase 10 is expressed in SH-SY5Y cells because of gene silencing. Upon Olomoucine or Roscovitine treatment, no re-expression of Caspase 8 or Caspase 10 was found. Therefore, in SH-SY5Y cells, this type of drugs is not triggering a canonical, Caspase 8/10-mediated, extrinsic apoptotic pathway.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Caspase 10 , Caspase 8 , Caspase 9 , Inibidores de Caspase , Linhagem Celular Tumoral , Humanos , Células Jurkat , Cinetina/farmacologia , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Purinas/farmacologia , Roscovitina , Fatores de TempoRESUMO
Aerobic metabolism of mammalian cells leads to the generation of reactive oxygen species (ROS). To cope with this toxicity, evolution provided cells with effective antioxidant systems like glutathione. Current anticancer therapies focus on the cancer dependence on oncogenes and non-oncogenes. Tumors trigger mechanisms to circumvent the oncogenic stress and to escape cell death. In this context we have studied 2-phenylethinesulfoxamine (PES), which disables the cell protective mechanisms to confront the proteotoxicity of damaged and unfolded proteins. Proteotoxic stress is increased in tumor cells, thus providing an explanation for the anticancer selectivity of PES. In addition, we have found that PES induces a severe oxidative stress and the activation of p53. The reduction of the cell content in glutathione by means of L-buthionine-sulfoximine (BSO) synergizes with PES. In conclusion, we have found that ROS constitutes a central element in a series of positive feed-back loops in the cell. ROS, p53, proteotoxicity, autophagy and mitochondrial dynamics are interconnected with the mechanisms leading to cell death, either apoptotic or necrotic. This network of interactions provides multiple targets for drug discovery and development in cancer.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/farmacologia , Butionina Sulfoximina/farmacologia , Butionina Sulfoximina/uso terapêutico , Descoberta de Drogas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Dinâmica Mitocondrial/efeitos dos fármacos , Neoplasias/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Proteína Supressora de Tumor p53/metabolismoRESUMO
Autophagy is generally regarded as a mechanism to promote cell survival. However, autophagy can occasionally be the mechanism responsible of cell demise. We have found that a concomitant depletion of glucose, nutrients and growth factors provoked cell death in a variety of cell lines. This death process was contingent upon caspase activation and was mediated by BAX/BAK proteins, thus indicating its apoptotic nature and the engagement of an intrinsic pathway. In order to abrogate autophagy, 3-methyladenine (3-MA), BECLIN-1 siRNA and Atg5 knock-out (Tet-Off type) approaches were alternatively employed. Irrespective of the procedure, at short times of starvation, we found that the ongoing autophagy was sensitizing cells to the permeabilization of the mitochondrial outer membrane (MOMP), caspase activation and, therefore, apoptosis. On the contrary, at longer times of starvation, autophagy displayed its characteristic pro-survival effect on cells. As far as we know, we provide the first experimental paradigm where time is the only variable determining the final outcome of autophagy. In other words, we have circumscribed in time the shift transforming autophagy from a cell death to a protection mechanism. Moreover, at short times, starvation-driven autophagy exacerbated the apoptotic cell death caused by several antitumor agents. In agreement with this fact, their apoptotic effects were greatly diminished by autophagy inhibition. The implications of these facts in tumor biology will be discussed.
Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Caspases/metabolismo , Animais , Morte Celular/fisiologia , Linhagem Celular Transformada , Sobrevivência Celular/fisiologia , Células HeLa , Humanos , Células MCF-7 , Camundongos , Camundongos Knockout , Fatores de TempoRESUMO
2-Phenylethynesulfonamide (PES) or pifithrin-µ is a promising anticancer agent with preferential toxicity for cancer cells. The type of cell death and the molecular cascades activated by this compound are controversial. Here, we demonstrate PES elicits a caspase- and BAX/BAK-independent non-necroptotic necrotic cell death, since it is not inhibited by necrostatin-1. This process is characterized by an early generation of reactive oxygen species (ROS) resulting in p53 up-regulation. Accordingly, thiolic antioxidants protect cells from PES-induced death. Furthermore, inhibiting the natural sources of glutathione with l-buthionine-sulfoximine (BSO) strongly cooperates with PES in triggering cytotoxicity. Genetically modified p53-null or p53 knocked-down cells show resistance to PES-driven necrosis. The predominant localization of p53 in chromatin-enriched fractions added to the up-regulation of the p53-responsive gene p21, strongly suggest the involvement of a transcription-dependent p53 program. On the other hand, we report an augmented production of ROS in p53-positive cells that, added to the increased p53 content in response to PES-elicited ROS, suggests that p53 and ROS are mutually regulated in response to PES. In sum, p53 up-regulation by ROS triggers a positive feedback loop responsible of further increasing ROS production and reinforcing PES-driven non-necroptotic necrosis.
Assuntos
Antineoplásicos/farmacologia , Genes p53 , Estresse Oxidativo/efeitos dos fármacos , Sulfonamidas/farmacologia , Butionina Sulfoximina/farmacologia , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Cromatina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células HCT116/efeitos dos fármacos , Humanos , Necrose/induzido quimicamente , Espécies Reativas de Oxigênio/metabolismoRESUMO
PES (2-phenylethynesulfonamide) was initially identified as an inhibitor of p53 translocation to mitochondria and named Pifithrin-µ. Further studies showed that PES selectively killed tumour cells and was thus a promising anticancer agent. PES-induced cell death was characterised by a non-apoptotic, autophagosome-rich phenotype. We observed this phenotype via electron microscopy in wild type (wt) and double Bax-/- Bak-/- (DKO) mouse embryonic fibroblasts (MEFs) treated with PES. We excluded the involvement of effector caspases, BAX and BAK, in causing PES-triggered cell death. Therefore, apoptosis was ruled out as the lethal mode of action of PES. Surprisingly, MEFs containing BAX were significantly protected from PES treatments. BAX overexpression in Bax-/- MEFs confirmed this pro-survival effect. Moreover, this protective effect required the ability of BAX to localise to mitochondrial membranes. Conversely, mitochondrial fusion induced by treatment with Mdivi-1 conferred increased resistance to MEFs subjected to PES treatment. The involvement of BAX in the regulation of mitochondrial dynamics has been reported. We propose the promotion of mitochondrial fusion by BAX to be the pro-survival function attributed to BAX.
Assuntos
Sulfonamidas/química , Proteína X Associada a bcl-2/metabolismo , Animais , Antineoplásicos/química , Apoptose , Caspases/metabolismo , Morte Celular , Sobrevivência Celular , Fibroblastos/citologia , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Mutação , Fenótipo , Quinazolinonas/química , Proteína Supressora de Tumor p53/metabolismoRESUMO
Cervical cancer is a major health concern among women in Latin America due to its high incidence and mortality. Therefore, the discovery of molecular markers for cervical cancer screening and triage is imperative. The aim of this study was to use a genome wide DNA methylation approach to identify novel methylation biomarkers in cervical cancer. DNA from normal cervical mucosa and cervical cancer tissue samples from Chile was enriched with Methylated DNA Immunoprecipitation (MeDIP), hybridized to oligonucleotide methylation microarrays and analyzed with a stringent bioinformatics pipeline to identify differentially methylated regions (DMRs) as candidate biomarkers. Quantitative Methylation Specific PCR (qMSP) was used to study promoter methylation of candidate DMRs in clinical samples from two independent cohorts. HPV detection and genotyping were performed by Reverse Line Blot analysis. Bioinformatics analysis revealed GGTLA4, FKBP6, ZNF516, SAP130, and INTS1 to be differentially methylated in cancer and normal tissues in the Discovery cohort. In the Validation cohort FKBP6 promoter methylation had 73% sensitivity and 80% specificity (AUC = 0.80). ZNF516 promoter methylation was the best biomarker, with both sensitivity and specificity of 90% (AUC = 0.92), results subsequently corroborated in a Prevalence cohort. Together, ZNF516 and FKBP6 exhibited a sensitivity of 84% and specificity of 81%, when considering both cohorts. Our genome wide DNA methylation assessment approach (MeDIP-chip) successfully identified novel biomarkers that differentiate between cervical cancer and normal samples, after adjusting for age and HPV status. These biomarkers need to be further explored in case-control and prospective cohorts to validate them as cervical cancer biomarkers.
Assuntos
Metilação de DNA , Papillomavirus Humano 18 , Regiões Promotoras Genéticas , Proteínas de Ligação a Tacrolimo/genética , Neoplasias do Colo do Útero/genética , Dedos de Zinco , Adulto , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Chile , Feminino , Genoma Humano , Humanos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Ligação a Tacrolimo/metabolismo , Neoplasias do Colo do Útero/etnologia , Neoplasias do Colo do Útero/virologiaRESUMO
Dose-escalated radiation therapy for localized prostate cancer (PCa) has a clear therapeutic benefit; however, escalated doses may also increase injury to noncancerous tissues. Radiosensitizing agents can improve ionizing radiation (IR) potency, but without targeted delivery, these agents will also sensitize surrounding normal tissues. Here we describe the development of prostate-targeted RNAi agents that selectively sensitized prostate-specific membrane antigen-positive (PSMA-positive) cells to IR. siRNA library screens identified DNA-activated protein kinase, catalytic polypeptide (DNAPK) as an ideal radiosensitization target. DNAPK shRNAs, delivered by PSMA-targeting RNA aptamers, selectively reduced DNAPK in PCa cells, xenografts, and human prostate tissues. Aptamer-targeted DNAPK shRNAs, combined with IR, dramatically and specifically enhanced PSMA-positive tumor response to IR. These findings support aptamer-shRNA chimeras as selective sensitizing agents for the improved treatment of high-risk localized PCa.
Assuntos
Adenocarcinoma/radioterapia , Antígenos de Superfície/análise , Aptâmeros de Nucleotídeos/administração & dosagem , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Glutamato Carboxipeptidase II/análise , Proteínas Nucleares/antagonistas & inibidores , Neoplasias da Próstata/radioterapia , RNA Interferente Pequeno/administração & dosagem , Radiossensibilizantes/uso terapêutico , Adenocarcinoma/patologia , Animais , Linhagem Celular , Humanos , Masculino , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Neoplasias da Próstata/patologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Tolerância a Radiação , Radiossensibilizantes/administração & dosagem , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MicroRNAs (miRNAs) are a natural part of the most recently discovered and global regulatory pathway known as RNA interference. Functional studies have shown how specific miRNAs can function as tumor suppressors or oncogenes and, correspondingly, deregulated miRNA profiles have been observed in prostate and other cancers. However, the upstream pathways which regulate miRNA expression are only currently being uncovered. The Androgen Receptor (AR) is a nuclear hormone receptor and transcription factor which plays a paramount role in prostate cancer (PCa) pathobiology. We performed high throughput miRNA microarray analysis on two AR-responsive cell lines to identified 16 candidate AR-regulated miRNAs.(1) One of the most androgen-induced candidates was a known oncogenic miRNA, miR-21. In a small study of early grade PCa samples we found that miR-21 levels were frequently elevated in comparison to adjacent normal tissue. This observation was supported in the literature(2,3) and suggests clinical relevance. We found that the activated AR directly interacts with miR-21 regulatory regions, indicating direct transcriptional induction. Furthermore, we provide new reporter studies supporting AR-regulation. Importantly, in functional studies, we found that a modest overexpression of miR-21 enhanced tumor xenograft growth and was sufficient to support androgen-independent proliferation following surgical castration. Thus, our studies suggest a model where miR-21 contributes to androgen-dependent and androgen-independent PCa growth. However, the AR is only one of many reported transcriptional regulators of miR-21. Here we review our recent discoveries and further analyze the reported miR-21 regulatory regions, inhibitory and stimulatory signaling pathways, and primary transcripts.
Assuntos
MicroRNAs/metabolismo , Neoplasias da Próstata/genética , Linhagem Celular Tumoral , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Transcrição GênicaRESUMO
MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in regulation of eukaryotic gene expression. Aberrant expression and structural alternation of miRNAs are considered to participate in tumorigenesis and cancer development. Recently, different genotypes of miR-196a polymorphisms (SNP, rs11614913) were found to be associated with the survival of patients with lung cancer and increased risk of breast cancer. To further investigate whether this polymorphism may influence glioma risk or not, we examined the SNP allele frequency in Chinese population. Our data shows the genotype CC of miR-196a (rs11614913) polymorphism is associated with decreased risk of glioma in the Chinese population (OR = 0.74, 95% CI:0.56-0.98). Furthermore, a significant association was observed between this genotype and glioma risk in the subgroups of adult glioma (OR = 0.73, 95% CI:0.55-0.98), male glioma (OR = 0.69, 95% CI:0.48-0.99) and patients with glioblastoma (OR = 0.58, 95% CI:0.37-0.91). This was the first study investigating the association between the miR-196a rs11614913 and glioma risk. Compared with the results from previous studies in lung cancer and breast cancer, our data suggest a different genotype association in glioma. This may be related to the diversity on the tissue origin, tumor type, tumorigenesis, and developing process.
Assuntos
Neoplasias do Sistema Nervoso Central/genética , Glioma/genética , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Alelos , Povo Asiático/genética , Estudos de Casos e Controles , Neoplasias do Sistema Nervoso Central/etnologia , Distribuição de Qui-Quadrado , Criança , China , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Glioma/etnologia , Humanos , Masculino , Fatores de Risco , Adulto JovemRESUMO
Androgen receptor (AR)-mediated oncogenic pathways have not been fully elucidated. In this study, we used high-throughput microarray analysis on two AR-positive prostate cancer (CaP) cell lines to identify 16 AR-responsive microRNAs (miRNA). We focused on miR-21 because of its previously reported oncogenic activity in other cancers. We show androgen-induced AR binding to the defined miR-21 promoter, miPPR-21, suggesting direct transcriptional regulation. Inhibition of miR-21 diminished androgen-induced CaP cell proliferation, providing new evidence that miRNAs can contribute to androgen-driven cell growth. Elevated expression of miR-21 enhanced CaP tumor growth in vivo and, surprisingly, was sufficient for androgen-dependent tumors to overcome castration-mediated growth arrest. Thus, elevated miR-21 expression alone is sufficient to impart castration resistance. Moreover, quantitative reverse transcription-PCR analysis revealed elevated miR-21 expression in CaP when compared with adjacent normal tissue. These results suggest that miR-21 may contribute to CaP pathogenesis.
Assuntos
MicroRNAs/genética , Neoplasias Hormônio-Dependentes/genética , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Neoplasias Hormônio-Dependentes/metabolismo , Orquiectomia , Regiões Promotoras Genéticas , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Transfecção , Transplante HeterólogoRESUMO
The new 7-bromoindirubin-3'-oxime (7BIO) compound induces caspase-independent cell death in all cell lines tested to date. Irrespective of the cell line, a 25 microM treatment for 24 h is lethal for the entire cell population. In SH-SY5Y and Jurkat cells, 7BIO (25 microM) was found to collapse the mitochondrial transmembrane potential (DeltaPsi m) at only 2-3 h of treatment. Concomitantly mitochondria swelled, cristae disrupted and, after 9 h, external cell membranes ruptured. In addition, endoplasmic reticulum dilated and, unexpectedly, the acute cytoplasmic destruction yielded isolated nuclei with preserved morphology and DNA integrity. Furthermore, the process was independent of both Bax and Bak, since cell viability and DeltaPsi m decayed indistinguishably in double Bax-/-Bak-/- mouse embryonic fibroblasts (MEFs) and their wild type counterparts. Pharmacological inhibition of the mitochondrial permeability transition pore (MPTP) did not prevent 7BIO-induced DeltaPsi m loss in none of the aforementioned cell lines. Caspase-independent inducers of cell death like AIF (Apoptosis Inducing Factor), cathepsins and calpains were not involved. Only the chemical inhibitors of serine proteases and, particularly, AEBSF afforded a significant protection thus suggesting a process regulated by this type of enzymes. As far as we know, these features are quite unique once taken together. Therefore, we propose 7BIO is triggering a specific type of necrotic cell death. Finally, the cytotoxicity of 7BIO on apoptosis-resistant cells like double Bax-/-Bak-/- MEFs seems of great interest envisaging cancer therapy.
Assuntos
Apoptose/efeitos dos fármacos , Indóis/farmacologia , Oximas/farmacologia , Serina Endopeptidases/metabolismo , Animais , Western Blotting , Linhagem Celular , DNA/metabolismo , Citometria de Fluxo , Humanos , Camundongos , Microscopia Eletrônica , NecroseRESUMO
In this study, we have analyzed the consequences, on several neuroblastoma cell lines, of combined treatments with (R)-roscovitine (CYC202, Seliciclib), a CDK inhibitory drug, and nutlin-3, a p53 activating drug. Both compounds were found to synergize, causing significant levels of apoptosis in cultured cells when combined at sublethal concentrations. In SH-SY5Y cells, Bcl-XL protein overexpression protected from apoptosis induced by either nutlin-3 alone or the (R)-roscovitine plus nutlin-3 association but failed to prevent apoptosis triggered by (R)-roscovitine alone. Moreover, Western blot studies showed that (R)-roscovitine increased nutlin-3-mediated p53 stabilization. Therefore, we conclude the contribution of (R)-roscovitine to the synergism is basically the sensitization of SH-SY5Y cells to the action of nutlin-3 on p53. The relevance of this pharmacological synergism with respect to the treatment of neuroblastoma is discussed.