RESUMO
Drug resistance is a multifactorial process involving a variety of mechanisms and genes. Taxane drug class like Docetaxel is not effective for all types' breast cancers and presents a huge clinical challenge. To improve cancer treatment outcome, it is important to distinguish which proteins can kill the cancer cells and whether the expression levels of these proteins affect treatment. Cancer cells are wildly known to be protected from apoptosis, due to low level of apoptotic protease activating factor-1 (Apaf-1) compared with normal cells. Apaf-1 is an essential protein that defines whether cytochrome c released form mitochondria remains stable or degrades. According to this hypothesis, increasing of Apaf-1 expression in MCF7 breast cancer cells was performed and Docetaxel efficacy examined. The immunoassay techniques were used to investigate Apaf-1 and cytochrome c levels, and different apoptosis assay methods applied to better understand the effect of Apaf-1 expression levels in cellular response to apoptotic stimuli by Docetaxel. Our results determined that cytoplasmic cytochrome c level elevated along with increasing Apaf-1 and MCF7 cells were sensitised to Docetaxel, suggesting that loss of Apaf-1 may cause Docetaxel-resistance in breast cancer cells through less apoptosome formation. ROS level increased in cells transfected with Apaf-1 and induced mitochondrial permeability for cytochrome c release, which subsequently promoted apoptosome formation, intrinsic apoptosis and ATP depletion.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Docetaxel/farmacologia , Taxoides/farmacologia , Regulação para Cima/efeitos dos fármacos , Apoptose/fisiologia , Relação Dose-Resposta a Droga , Humanos , Células MCF-7 , Regulação para Cima/fisiologiaRESUMO
Background Gonadotrophins exert their functions by binding follicle-stimulating hormone receptor (FSHR) or luteinizing hormone and human chorionic gonadotropin receptor (LHCGR) present on endometrium. Within ovaries, FSH induces autophagy and apoptosis of granulosa cells leading to atresia of non-growing follicles, whereas hCG and LH have anti-apoptotic functions. Endometrial cells express functioning gonadotrophin receptors. The objective of this study was to analyze the effect of gonadotrophins on physiology and endometrial cells survival. Materials and methods Collected endometria were incubated for 48 or 72 h with 100 ng/mL of recombinant human FSH (rhFSH), recombinant human LH (rhLH) or highly purified hCG (HPhCG) alone or combined. Controls omitted gonadotrophins. The effect of gonadotrophins on cytochrome P450 family 11 subfamily A polypeptide 1 (CYP11A1), hypoxia inducible factor 1α (HIF1A), and cell-death-related genes expression was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Immunohistochemistry for microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) and apoptotic protease activating factor 1 (APAF-1) was performed. Results Gonadotrophins are able to modulate the endometrial cells survival. FSH induced autophagy and apoptosis by increasing the relative expression of MAP1LC3B and FAS receptor. In FSH-treated samples, expression of apoptosis marker APAF-1 was detected and co-localized on autophagic cells. hCG and LH does not modulate the expression of cell-death-related genes while the up-regulation of pro-proliferative epiregulin gene was observed. When combined with FSH, hCG and LH prevent autophagy and apoptosis FSH-induced. Conclusions Different gonadotrophins specifically affect endometrial cells viability differently: FSH promotes autophagy and apoptosis while LH and hCG alone or combined with rhFSH does not.
Assuntos
Gonadotropina Coriônica/farmacologia , Endométrio/efeitos dos fármacos , Hormônio Foliculoestimulante/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio Luteinizante/farmacologia , Adulto , Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Fator Apoptótico 1 Ativador de Proteases/genética , Autofagia/efeitos dos fármacos , Enzima de Clivagem da Cadeia Lateral do Colesterol/biossíntese , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Gonadotropina Coriônica/fisiologia , Endométrio/metabolismo , Epirregulina/biossíntese , Epirregulina/genética , Feminino , Hormônio Foliculoestimulante/fisiologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hormônio Luteinizante/fisiologia , Ciclo Menstrual , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Proteínas Recombinantes/farmacologia , Receptor fas/biossíntese , Receptor fas/genéticaRESUMO
The orchestrated events required during brain development, as well as the maintenance of adult neuronal plasticity, highly depend on the accurate responses of neuronal cells to various cellular stress or environmental stimuli. Recent studies have defined a previously unrecognized, broad class of multidomain proteins, designated as signal transduction ATPases with numerous domains (STAND), which comprises a large number of proteins, including the apoptotic peptidase activating factor 1 (Apaf1) and nucleotide-binding oligomerization domain-like receptors (NLRs), central players in cell death and innate immune responses, respectively. Although the involvement of STANDs in the central nervous system (CNS) has been postulated in terms of neuronal development and function, it remains largely unclear. Here, we identified Nwd1 (NACHT and WD repeat domain-containing protein 1), as a novel STAND protein, expressed in neural stem/progenitor cells (NSPCs). Structurally, Nwd1 was most analogous to the apoptosis regulator Apaf1, also involved in mitosis and axonal outgrowth regulation in the CNS. Using a specific antibody, we show that, during the embryonic and postnatal period, Nwd1 is expressed in nestin-positive NSPCs in vivo and in vitro, while postnatally it is found in terminally differentiated neurons and blood vessels. At the subcellular level, we demonstrate that Nwd1 is preferentially located in the cytosolic compartment of cultured NSPCs, partially overlapping with cytochrome c. These observations imply that Nwd1 might be involved in the neuronal lineage as a new STAND gene, including having a pro-apoptotic or nonapoptotic role, similar to Apaf1.
Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Animais Recém-Nascidos , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Fator Apoptótico 1 Ativador de Proteases/genética , Sistema Nervoso Central/embriologia , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mitose/genética , Células-Tronco Neurais/metabolismo , Distribuição TecidualRESUMO
Porphyromonas gingivalis is able to invade and modulate host-immune response to promote its survival. This bacterium modulates the cell cycle and programed cell death, contributing to periodontal lesion worsening. Several molecular pathways have been identified as key triggers of apoptosis, including apoptosome apoptotic peptidase activating factor 1 (APAF-1). Apaf-1 and X-linked inhibitor of apoptosis protein (Xiap) mRNA were differentially expressed between gingival samples harvested from human healthy and chronic periodontitis tissues (Apaf-1, 19.2-fold; caspase-9, 14.5-fold; caspase-3, 6.8-fold; Xiap: 2.5-fold in chronic periodontitis) (P < 0.05), highlighting their potential role in periodontitis. An increased proteic expression of APAF-1 was also observed in a murine experimental periodontitis model induced by P. gingivalis-soaked ligatures. In vitro, it was observed that P. gingivalis targets APAF-1, XIAP, caspase-3, and caspase-9, to inhibit epithelial cell death at both mRNA and protein levels. Opposite effect was observed in fibroblasts in which P. gingivalis increased cell death and apoptosis. To assess if the observed effects were associated to APAF-1, epithelial cells and fibroblasts were transfected with siRNA targeting Apaf-1. Herein, we confirmed that APAF-1 is targeted by P. gingivalis in both cell types. This study identified APAF-1 apoptosome and XIAP as intracellular targets of P. gingivalis, contributing to the deterioration of periodontal lesion through an increased persistence of the bacteria within tissues and the subversion of host-immune response.
Assuntos
Apoptossomas/metabolismo , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Infecções por Bacteroidaceae/metabolismo , Periodontite Crônica/microbiologia , Porphyromonas gingivalis/patogenicidade , Idoso , Animais , Fator Apoptótico 1 Ativador de Proteases/genética , Infecções por Bacteroidaceae/patologia , Células Cultivadas , Periodontite Crônica/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica/fisiologia , Gengiva/metabolismo , Gengiva/microbiologia , Gengiva/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , RNA Mensageiro/genéticaRESUMO
BACKGROUND: Despite the improvement in the diagnosis and the management of laryngeal squamous cell carcinoma (LSCC), many patients with advanced-stage have poor prognosis in the form of recurrence, metastasis or death. So, recognition of new molecular markers would facilitate the development of targeted therapies. OBJECTIVES: To investigate miR-221 expression in LSCC and its possible correlation to apoptotic protease activating factor-1 (Apaf-1). Also, we aimed to investigate the association between miR-221 and Apaf-1 expressions and the clinicopathological features of LSCC. METHODS: We investigated the expression of miR-221 (by qRT-PCR) and Apaf-1 (by qRT-PCR and immunohistochemistry) in primary LSCC and adjacent normal tissues. RESULTS: We found significant up-regulation of miR-221 and significant down-regulation of Apaf-1 expression in LSCC tissues compared to normal nearby laryngeal tissues. In addition, significant associations between up-regulated miR-221 and down-regulated Apaf-1 expressions and clinical stage and lymph node (LN) metastasis (P< 0.001 for each) were found. Furthermore, there was a negative correlation between miR-221 gene expression and Apaf-1 gene expression (r=-0.73, P< 0.001). CONCLUSION: miR-221 can be considered as a diagnostic marker in LSCC and Apaf-1 may be considered as a possible target of miR-221.
Assuntos
Fator Apoptótico 1 Ativador de Proteases/biossíntese , Carcinoma de Células Escamosas/diagnóstico , Neoplasias Laríngeas/diagnóstico , MicroRNAs/metabolismo , Adulto , Idoso , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Biomarcadores Tumorais , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Neoplasias Laríngeas/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Regulação para CimaRESUMO
Natural product lingenol, a purified diterpenoid compound derived from the root of Euphorbia kansui, exerts serious hepatotoxicity; however, the molecular mechanisms remain to be defined. In the present study, cell counting Kit-8 (CCK-8), inverted phase contrast microscope and flow cytometry were used to demonstrate that lingenol significantly inhibited L-O2 cells proliferation, and induced cell cycle arrest and apoptosis. Moreover, the results investigated that lingenol markedly disrupted mitochondrial functions by high content screening (HCS). In addition, the up-regulation of cytochrome c, AIF and Apaf-1 and activation of caspases were found in L-O2 cells detected by Western blotting and ELISA assay, which was required for lingenol activation of cytochrome c-mediated caspase cascades and AIF-mediated DNA damage. Mechanistic investigations revealed that lingenol significantly down-regulated the Bcl-2/Bax ratio and enhanced the reactive oxygen species (ROS) in L-O2 cells. These data collectively indicated that lingenol modulation of ROS and Bcl-2/Bax ratio led to cell cycle arrest and mitochondrial-mediated apoptosis in L-O2 cells in vitro. All of these results will be helpful to reveal the hepatotoxicity mechanism of Euphorbia kansui and to effectively guide safer and better clinical application of this herb.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Diterpenos/efeitos adversos , Extratos Vegetais/administração & dosagem , Fator de Indução de Apoptose/biossíntese , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Citocromos c/biossíntese , Diterpenos/administração & dosagem , Euphorbia/química , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Extratos Vegetais/efeitos adversos , Extratos Vegetais/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
PURPOSE: To investigate predictive value of APAF-1 and COX-2 expression in pathologic complete response (pCR) for patients with rectal adenocarcinoma (RAC) who were treated with neoadjuvant chemoradiotherapy (neo-CRT) followed by total mesorectal excision (TME). MATERIALS AND METHODS: Immunohistochemistry assay was used to detect expression of APAF-1 and COX-2 in paraffin-wax embedded tissues obtained before neo-CRT for patients with RAC. A 5-point tumor-regression grade (TRG) based on the ratio of residual tumor to fibrosis according to Dworak's scoring system was used to assess neo-CRT response. The relationship between expression of APAF-1 and COX-2 genes and pCR was explored. RESULTS: pCR (TRG4) was observed in 23 patients (28.0%). pCR were more likely to be achieved for those with APAF-1 over-expression or lower expression of COX-2. pCR rate in patients with combination of high APAF-1 and low COX-2 expression was 56.0%, significantly higher than those with other combination of APAF1 and COX-2 expression. Multivariate analysis showed that over-expression of APAF-1 and suppressed expression of COX-2 were independent predictive factors for pCR. CONCLUSION: Immunohistochemical evaluation of APAF-1 and COX-2 expression on pretreatment specimen may be used to predict pCR to neo-CRT in patients with RAC. The potential of the markers in monitoring pCR patient merits further investigation.
Assuntos
Adenocarcinoma/patologia , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Biomarcadores Tumorais/análise , Ciclo-Oxigenase 2/biossíntese , Neoplasias Retais/patologia , Adenocarcinoma/mortalidade , Adenocarcinoma/terapia , Adolescente , Adulto , Idoso , Fator Apoptótico 1 Ativador de Proteases/análise , Quimioterapia Adjuvante , Ciclo-Oxigenase 2/análise , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Valor Preditivo dos Testes , Neoplasias Retais/mortalidade , Neoplasias Retais/terapia , Adulto JovemRESUMO
The p53 tumor suppressor controls cell growth, metabolism, and death by regulating the transcription of various target genes. The target-specific transcriptional activity of p53 is highly regulated. Here we demonstrate that acetylation of p53 at Lys-120 up-regulates its transcriptional activity toward Apaf-1, a core component in the mitochondrial apoptotic pathway, and thus sensitizes caspase activation and apoptosis. We found that histone deacetylase (HDAC) inhibitors, including butyrate, augment Lys-120 acetylation of p53 and thus Apaf-1 expression by inhibiting HDAC1. In p53-null cells, transfection of wild-type but not K120R mutant p53 can restore the p53-dependent sensitivity to butyrate. Strikingly, transfection of acetylation-mimicking K120Q mutant p53 is sufficient to up-regulates Apaf-1 in a manner independent of butyrate treatment. Therefore, HDAC inhibitors can induce p53 acetylation at lysine 120, which in turn enhances mitochondrion-mediated apoptosis through transcriptional up-regulation of Apaf-1.
Assuntos
Apoptose , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Mitocôndrias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Acetilação/efeitos dos fármacos , Substituição de Aminoácidos , Fator Apoptótico 1 Ativador de Proteases/genética , Células HeLa , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Lisina/genética , Lisina/metabolismo , Mitocôndrias/genética , Mutação de Sentido Incorreto , Proteína Supressora de Tumor p53/genéticaRESUMO
Laryngeal cancer is one of the most commonly occurring malignant cancers of the head and neck region. In the present study, we investigated the roles of miR-221 in laryngeal squamous cell carcinoma cell line, Hep-2. We examined the function and mechanism of miR-221 in Hep-2 cells using techniques of cell biology and molecular pathology, such as western blotting, quantitative PCR, immunohistochemical staining and flow cytometry. Using a luciferase assay, the apoptotic protease activating factor-1 (Apaf-1) mRNA 3'-UTR was shown to have complementary binding sites using bioinformatics prediction software including TargetScan, PicTar and miRanda. In conclusion, our results showed that miR-221 inhibition caused elevated expression levels of the Apaf-1 apoptotic pathway proteins caspase-3, -8 and -9. miR-221 may therefore be used as a novel therapeutic target for laryngeal cancer.
Assuntos
Apoptose/genética , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Neoplasias Laríngeas/genética , MicroRNAs/genética , Animais , Fator Apoptótico 1 Ativador de Proteases/genética , Sítios de Ligação/genética , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Laríngeas/patologia , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transplante HeterólogoRESUMO
Fluorinated organic compounds, such as perfluorooctane sulfonate, are stable chemicals with a wide range of industrial applications. The potential toxicity of perfluorooctane sulfonate is not well characterized, and even less known are the mechanisms underlying its toxic effects. Perfluorooctane sulfonate change of inner mitochondrial membrane permeability has been implicated as a potential mechanism of toxicity. In this study, we research that perfluorooctane sulfonate effects the expression of Apaf1 and Caspase3 genes in the amnion and fetal lung cell line that initiate the cells to undergo apoptosis. The expression of Caspase3 and Apaf1 was determined by using quantitative RT-PCR. In the study there is significant increase in expression of Caspase3 and Apaf1 in amnion and fetal lung cell line exposed to high dose (p < 0.001, p = 0.004). Also there is significant increase in cell lines exposed for a long period of time to perfluorooctane sulfonate (p = 0.001). But no significant increase was seen in the low doses and exposed for a short period of time. In conclusion, apoptotic gene expression is increase in cells exposed perfluorooctane sulfonate by dose dependent manner was determined. So this work is the first study examines the apoptotic effects of perfluorooctane sulfonate in human embryonic cells it will lead the way to the other topical studies.
Assuntos
Ácidos Alcanossulfônicos/administração & dosagem , Apoptose/efeitos dos fármacos , Fluorocarbonos/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Ácidos Alcanossulfônicos/toxicidade , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Caspase 3/biossíntese , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Poluentes Ambientais/administração & dosagem , Poluentes Ambientais/toxicidade , Fluorocarbonos/toxicidade , Humanos , Técnicas In Vitro , Membranas Mitocondriais/efeitos dos fármacosRESUMO
Pseudolaric acid B (PAB) is a diterpene acid isolated from the root and trunk bark of Pseudolarix kaempferi Gordon (Pinaceae). Recent studies have reported that PAB exhibits cytotoxic effects in several cancer cell lines. In the present study, we assessed its antitumor activity and molecular mechanisms in HO-8910 and A2780 ovarian cancer cells in vitro. We found that PAB reduced cell viability and induced apoptosis in a dose- and time-dependent manner in HO-8910 and A2780 human ovarian cancer cells. The induction of apoptosis was also accompanied by the regulation of Bcl-2 and XIAP family proteins, cytochrome c and Apaf-1. Moreover, we observed that PAB treatment resulted in the activation of caspase-3 and -9, which may partly explain the anticancer activity of PAB. Collectively, the present study for the first time suggests that PAB enhances apoptosis of HO-8910 and A2780 cells through regulation of Bcl-2 and IAP family proteins. Moreover, the triggering of caspase-3 and -9 activation mediated apoptotic induction. Our results suggest that PAB may be a new therapeutic option for the treatment of ovarian cancers.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/metabolismo , Diterpenos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Benzoquinonas/farmacologia , Inibidores de Caspase/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/biossíntese , Citocromos c/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossínteseRESUMO
Melatonin exhibits anti-inflammatory and anticancer effects and could be a chemopreventive and chemotherapeutic agent against cancers, but the precise mechanisms involved remain largely unresolved. In this study, we evaluated the mechanism of action of melatonin in human MDA-MB-361 breast cancer cells. Melatonin at pharmacological concentrations (10(-3) m) significantly suppressed cell proliferation and induced apoptosis in a dose-dependent manner. The observed suppression of proliferation was accompanied by the melatonin-mediated inhibition of COX-2, p300, and NF-κB signaling. Melatonin significantly inhibited COX-2 expression and prostaglandin E(2) (PGE2) production, abrogated p300 histone acetyltransferase activity and p300-mediated NF-κB acetylation, thereby blocking NF-κB binding and p300 recruitment to COX-2 promoter. Pretreatment with a COX-2- or p300-selective inhibitor abrogated the melatonin-induced inhibition of cell proliferation, whereas PGE2 treatment or COX-2 transfection reversed the inhibition by melatonin. Moreover, melatonin markedly inhibited phosphorylation of PI3K, Akt, PRAS40, and GSK-3 proteins, thereby inactivating the PI3K/Akt signaling pathway. Pretreatment with a PI3K- or an Akt-selective inhibitor or an Akt-specific siRNA blocked the melatonin-mediated inhibition of cell proliferation. Conversely, gene delivery of a constitutively active Akt effectively reversed the inhibition by melatonin. Furthermore, melatonin induced Apaf-1 expression, triggered cytochrome C release, and stimulated caspase-3 and caspase-9 activities and cleavage, leading to an activation of the Apaf-1-dependent apoptotic pathway. Pretreatment with an Apaf-1-specific siRNA effectively attenuated the melatonin-induced apoptosis. These results therefore indicate that melatonin inhibits cell proliferation and induces apoptosis in MDA-MB-361 breast cancer cells in vitro by simultaneously suppressing the COX-2/PGE2, p300/NF-κB, and PI3K/Akt/signaling and activating the Apaf-1/caspase-dependent apoptotic pathway.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Neoplasias da Mama/metabolismo , Ciclo-Oxigenase 2/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melatonina/farmacologia , Proteínas Proto-Oncogênicas c-akt/biossíntese , Fatores de Transcrição de p300-CBP/biossíntese , Fator Apoptótico 1 Ativador de Proteases/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Dinoprostona/biossíntese , Feminino , Humanos , Proteínas Proto-Oncogênicas c-akt/genética , Fatores de Transcrição de p300-CBP/genéticaRESUMO
Mice homozygous for the targeted disruption of the growth hormone (GH) receptor (Ghr) gene (GH receptor knockout; GHRKO; KO) are hypoinsulinemic, highly insulin sensitive, normoglycemic, and long-lived. Visceral fat removal (VFR) is a surgical intervention which improves insulin signaling in normal (N) mice and rats and extends longevity in rats. We have previously demonstrated decreased expression level of certain pro-apoptotic genes in skeletal muscles and suggested that this may contribute to the regulation of longevity in GHRKO mice. Alterations in apoptosis-related genes expression in the kidneys also may potentially lead to lifespan extension. In this context, we decided to examine the renal expression of the following genes: caspase-3, caspase-9, caspase-8, bax, bad, bcl-2, Smac/DIABLO, Apaf-1, p53, and cytochrome c1 (cyc1) in male GHRKO and N mice subjected to VFR or sham surgery, at approximately 6 months of age. The kidneys were collected 2 months after VFR. As a result, caspase-3, caspase-9, and bax expressions were decreased in KO mice as compared to N animals. Expressions of Smac/DIABLO, caspase-8, bcl-2, bad, and p53 did not differ between KOs and N mice. VFR did not change the expression of the examined genes in KO or N mice. In conclusion, endocrine abnormalities in GHRKO mice result in decreased expression of pro-apoptotic genes and VFR did not alter the examined genes expression in N and KO mice. These data are consistent with a model in which alterations of GH signaling and/or insulin sensitivity lead to increased lifespan mediated by decreased renal expression of pro-apoptotic genes.
Assuntos
Envelhecimento/genética , Fator Apoptótico 1 Ativador de Proteases/genética , Expressão Gênica , Gordura Intra-Abdominal/cirurgia , Longevidade/genética , RNA/genética , Receptores da Somatotropina/genética , Envelhecimento/metabolismo , Animais , Apoptose/genética , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Restrição Calórica , Feminino , Deleção de Genes , Gordura Intra-Abdominal/metabolismo , Rim/citologia , Rim/metabolismo , Masculino , Camundongos , Camundongos Knockout , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptores da Somatotropina/biossíntese , Transdução de SinaisRESUMO
Di (2-ethylhexyl) phthalate (DEHP) exposure is thought to lead to hepatocellular hypertrophy and hyperplasia in rodents mediated via peroxisome proliferator-activated receptor alpha (PPAR alpha). A recent study revealed that long-term exposure to relatively low-dose DEHP (0.05%) caused liver tumors including hepatocellular carcinomas, hepatocellular adenomas, and chologiocellular carcinomas at a higher incidence in Ppar alpha-null mice (25.8%) than in wild-type mice (10.0%). Using tissues with hepatocellular adenoma, microarray (Affymetrix MOE430A) as well as, in part, real-time quantitative PCR analysis was conducted to elucidate the mechanisms of the adenoma formation resulting from DEHP exposure in both genotyped mice. The microarray profiles showed that the up- or down-regulated genes were quite different between hepatocellular adenoma tissues of wild-type and Ppar alpha-null mice exposed to DEHP. The gene expressions of apoptotic peptidase activating factor 1 (Apaf1) and DNA-damage-inducible 45 alpha (Gadd45a) were increased in the hepatocellular adenoma tissues of wild-type mice exposed to DEHP, whereas they were unchanged in corresponding tissues of Ppar alpha-null mice. On the other hand, the expressions of cyclin B2 and myeloid cell leukemia sequence 1 were increased only in the hepatocellular adenoma tissues of Ppar alpha-null mice. Taken together, DEHP may induce hepatocellular adenomas, in part, via suppression of G2/M arrest regulated by Gadd45a and caspase 3-dependent apoptosis in Ppar alpha-null mice, but these genes may not be involved in tumorigenesis in the wild-type mice. In contrast, the expression level of Met was notably increased in the liver adenoma tissue of wild-type mice, which may suggest the involvement of Met in DEHP-induced tumorigenesis in wild-type mice.
Assuntos
Dietilexilftalato/toxicidade , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Fígado/efeitos dos fármacos , PPAR alfa/deficiência , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Caspase 3/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/biossíntese , Divisão Celular/genética , Dietilexilftalato/administração & dosagem , Fase G2/genética , Perfilação da Expressão Gênica , Hepatócitos/efeitos dos fármacos , Hiperplasia , Fígado/patologia , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Knockout , Análise em Microsséries , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/biossíntese , PPAR alfa/genética , PPAR alfa/metabolismo , Plastificantes/toxicidade , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
FUS1 is a novel tumor suppressor gene located in the human chromosome 3p21.3 region. We previously showed that restoration of FUS1 function in 3p21.3-deficient human non-small-cell lung cancer (NSCLC) cells significantly inhibited tumor cell growth in vitro and in vivo. In this study, we evaluated the combined effects of the tumor suppressor FUS1 and the chemotherapeutic drug cisplatin on tumor cell growth and apoptosis induction in NSCLC cells, and explored the molecular mechanism of their mutual action. Exogenous expression of FUS1 by nanoparticle-mediated gene transfer sensitized the response of NSCLC cells to cisplatin, resulting in a 4- to 6-fold increase in tumor-suppressing activity. A systemic treatment with a combination of FUS1-nanoparticles and cisplatin in a human H322 lung cancer orthotopic xenograft mouse model dramatically enhanced the therapeutic efficacy of cisplatin. We also found that the FUS1-enhanced chemosensitivity is associated with the downregulation of MDM2, accumulation of p53 and activation of the Apaf-1-dependent apoptosis pathway. Our results demonstrated an important role of FUS1 in modulating chemosensitivity of lung cancer cells, and suggested that a proper combination of molecular therapeutics such as the proapoptotic tumor suppressor FUS1 and the conventional chemotherapeutic drugs such as cisplatin may be an efficient treatment strategy for human lung cancer.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Cisplatino/farmacologia , Terapia Genética , Neoplasias Pulmonares/terapia , Proteínas Supressoras de Tumor/biossíntese , Animais , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Cromossomos Humanos Par 3/genética , Cromossomos Humanos Par 3/metabolismo , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Transferência de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Nanopartículas , Proteínas Proto-Oncogênicas c-mdm2/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
E2F-6 is a dominant-negative transcriptional repressor against other members of the E2F family. In this study, we investigated the expression and function of E2F-6 in human hematopoietic progenitor cells to clarify its role in hematopoiesis. We found that among E2F subunits, E2F-1, E2F-2, E2F-4, and E2F-6 were expressed in CD34(+) human hematopoietic progenitor cells. The expression of E2F-6 increased along with proliferation and decreased during differentiation of hematopoietic progenitors, whereas the other three species were upregulated in CD34(-) bone marrow mononuclear cells. Overexpression of E2F-6 did not affect the growth of immature hematopoietic cell line K562 but suppressed E2F-1-induced apoptosis, whereas it failed to inhibit apoptosis induced by differentiation inducers and anticancer drugs. Among E2F-1-dependent apoptosis-related molecules, E2F-6 specifically inhibited upregulation of Apaf-1 by competing with E2F-1 for promoter binding. E2F-6 similarly suppressed apoptosis and Apaf-1 upregulation in primary hematopoietic progenitor cells during cytokine-induced proliferation but had no effect when they were differentiated. As a result, E2F-6 enhanced the clonogenic growth of colony-forming unit-granulocyte, erythroid, macrophage, and megakaryocyte. These results suggest that E2F-6 provides a failsafe mechanism against loss of hematopoietic progenitor cells during proliferation. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Apoptose/efeitos dos fármacos , Fator de Transcrição E2F6/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Fator Apoptótico 1 Ativador de Proteases/genética , Ligação Competitiva , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Meios de Cultura Livres de Soro/farmacologia , Citocinas/farmacologia , Fatores de Transcrição E2F/biossíntese , Fatores de Transcrição E2F/genética , Fator de Transcrição E2F1/antagonistas & inibidores , Fator de Transcrição E2F1/farmacologia , Células-Tronco Hematopoéticas/citologia , Humanos , Células K562/citologia , Células K562/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Recombinantes de Fusão/fisiologia , Transdução Genética , Regulação para CimaRESUMO
Caspases play an essential role in the initiation/regulation of apoptosis. Aberrant apoptotic regulation has been associated with carcinogenesis and therapeutic resistance. To explore the possible involvement of altered caspase expression in breast cancer, we have systematically examined the expression of both protein and mRNA levels of 7 caspases in a panel of 18 breast cancer cell lines. We found that variation of caspase expression can occur at both protein and RNA levels. Down-regulation of these caspases, especially caspase-8 and -10, was frequently observed. Functional screening of these selected cell lines using TNF-alpha, doxorubicin and radiation induced cell injury showed that a lack of functional caspase-8 resulted in resistance to TNF-alpha-induced apoptosis. Array style examination of caspase expression profiles in breast cancer cell lines yields massive information that is valuable in establishing cell line models to study the role of caspase down-regulation/deficiency in breast cancer development and therapeutic resistance.
Assuntos
Neoplasias da Mama/enzimologia , Caspases/biossíntese , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Fator Apoptótico 1 Ativador de Proteases/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Caspases/genética , Linhagem Celular Tumoral , Regulação para Baixo , Doxorrubicina/farmacologia , Humanos , Isoenzimas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Chronic myelogenous leukemia (CML) is a stem cell disorder that eventually progresses to a blast crisis phase (BC) characterized by distorted apoptotic pathways. The exact mechanism leading to failure in apoptotic pathways during CML progression is unclear. In view of the central role of p53 and apaf1 in the apoptotic machinery we examined six human paired chronic and BC phases samples for their expression. Real-time PCR (RQ-PCR) experiments showed an elevation of p53 mRNA in all patients during transition to BC. However, elevation of apaf1 during BC was observed in five patients only. In contrast, one patient displayed a significant 11.5-fold reduction of apaf1 expression during the transition to BC. No apaf1 promoter methylation was observed. The reduced apaf1 expression was accompanied by a trans-dominant point mutation (H179R) in one p53 allele and the loss of the other. This mutant p53, when tested using functionality assays, was unable to activate apaf1, consequently explaining the reduced expression observed in this patient. Furthermore, the same mutant failed to activate either genes involved in apoptotic or cell cycle arrest pathways, and can be considered as a complete loss of function mutation. This specific mutation was reported in several types of cancer, but was not implicated in CML. To conclude, in this study we have demonstrated mRNA elevation of p53 and apaf1 during CML blast crisis, indicating that genes and proteins involved in cellular apoptosis might be involved in disease progression/response to therapy. Moreover, the mutated p53 discovered in the patient exhibiting lowered apaf1 expression provides, in a clinical case, the first correlation between p53 and apaf1 transcription regulation in humans.
Assuntos
Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Fator Apoptótico 1 Ativador de Proteases/genética , Crise Blástica/genética , Regulação Leucêmica da Expressão Gênica/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação Puntual , Proteína Supressora de Tumor p53/genética , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Arginina/genética , Crise Blástica/metabolismo , Histidina/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismoRESUMO
PURPOSE: Clinical observations suggest that intratumoral hypoxia increases the aggressiveness of tumors through clonal selection of cancer cells that have lost their apoptotic potential. The aim of this study, therefore, was to investigate the expression of the proapoptotic protein apoptotic protease activating factor-1 (Apaf-1) in cervical cancers and to analyze its relation to intratumoral hypoxia and apoptosis. Furthermore, the effect of hypoxia and apoptosis on survival was examined. EXPERIMENTAL DESIGN: In 56 patients, intratumoral oxygenation measurements and subsequent needle biopsies were done. The obtained tissue was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays and by immunohistochemistry with an Apaf-1 antibody. RESULTS: Apaf-1 was expressed in 86% of cancers. The median apoptosis rate was 1.0%. There was no correlation between Apaf-1 expression and intratumoral hypoxia. However, Apaf-1 expression was negative in 37.5% of hypoxic cervical cancers (pO(2) Assuntos
Apoptose/fisiologia
, Fator Apoptótico 1 Ativador de Proteases/biossíntese
, Neoplasias do Colo do Útero/metabolismo
, Adulto
, Idoso
, Fator Apoptótico 1 Ativador de Proteases/deficiência
, Fator Apoptótico 1 Ativador de Proteases/metabolismo
, Hipóxia Celular/fisiologia
, Feminino
, Humanos
, Pessoa de Meia-Idade
, Estadiamento de Neoplasias
, Oxigênio/metabolismo
, Pressão Parcial
, Estudos Prospectivos
, Neoplasias do Colo do Útero/patologia
RESUMO
Matrine is a major component of Sophora Flavescens and has been reported to stimulate differentiation of erythroleukemia cells. Here we show that matrine inhibits cell proliferation or induces apoptosis in a cell type-specific manner. The latter effect was investigated in more detail in the p53 deficient erythroleukemia cell line, K562. Matrine exposure induced apoptosis in a time- and dose-dependent manner in these cells. Interestingly, co-treatment with etoposide potentiated apoptosis. Further analysis of matrine-induced apoptotic changes revealed that E2F-1 and Apaf-1 were upregulated, whereas Rb was downregulated after 24 h of exposure. This was followed by Bax translocation, cytochrome c release, and caspase-9 and -3 activation. These results demonstrate that matrine triggers apoptosis of K562 cells primarily through the mitochondrial pathway and that matrine is a potential anti-tumor drug.