RESUMO
Necroptosis is a regulated cell death mechanism. However, it is unknown whether necroptosis is involved in the death of tumor necrosis factor-α (TNF-α)-treated osteoblasts. Therefore, we conducted the study with TNF-α, Nec-1 (a specific inhibitor of necroptosis), and Z-IETD-FMK (a specific inhibitor of apoptosis) to determine whether necroptosis plays a role in the death of TNF-α-treated osteoblast cell line MC3T3-E1. Cell viability, cell death, and lactate dehydrogenase (LDH) release were assayed to evaluate cytotoxicity. Specific marker proteins receptor interacting protein kinase (RIPK3) and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) for necroptosis, and cleaved caspase 3 for apoptosis were detected by western blot, and mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). We found that TNF-α inhibited cell proliferation in a dose- and time-dependent manner. Nec-1 plus Z-IETD-FMK restored cell viability and significantly decreased LDH release. In addition, TNF-α alone increased the cell population of AV+PI−, while Z-IETD-FMK caused a shift in the cell population from AV+PI− to AV+PI+. Furthermore, TNF-α significantly increased protein cleaved caspase 3. TNF-α plus Z-IETD-FMK significantly increased the proteins RIPK3 and MLKL phosphorylation in MC3T3-E1 cells, while the changes in mRNA levels of RIPK3, MLKL, and caspase 3 were not consistent with the changes in the corresponding protein expression levels. In conclusion, TNF-α induced preferentially apoptosis in osteoblast cell line and necroptosis played a decisive role when TNF-α-induced death was inhibited by the inhibitor of apoptosis. Combined treatment with Nec-1 and Z-IETD-FMK protected mouse osteoblasts from death induced by TNF-α.
Assuntos
Animais , Coelhos , Osteoblastos/patologia , Fator de Necrose Tumoral alfa/farmacologia , Caspase 8/efeitos dos fármacos , Inibidores de Caspase/farmacologia , Necrose/patologia , Oligopeptídeos/farmacologia , Osteoblastos/efeitos dos fármacos , Fosforilação , Sobrevivência Celular/efeitos dos fármacos , Imidazóis/farmacologia , Indóis/farmacologia , L-Lactato Desidrogenase/farmacologiaRESUMO
Necroptosis is a regulated cell death mechanism. However, it is unknown whether necroptosis is involved in the death of tumor necrosis factor-α (TNF-α)-treated osteoblasts. Therefore, we conducted the study with TNF-α, Nec-1 (a specific inhibitor of necroptosis), and Z-IETD-FMK (a specific inhibitor of apoptosis) to determine whether necroptosis plays a role in the death of TNF-α-treated osteoblast cell line MC3T3-E1. Cell viability, cell death, and lactate dehydrogenase (LDH) release were assayed to evaluate cytotoxicity. Specific marker proteins receptor interacting protein kinase (RIPK3) and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) for necroptosis, and cleaved caspase 3 for apoptosis were detected by western blot, and mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). We found that TNF-α inhibited cell proliferation in a dose- and time-dependent manner. Nec-1 plus Z-IETD-FMK restored cell viability and significantly decreased LDH release. In addition, TNF-α alone increased the cell population of AV+PI-, while Z-IETD-FMK caused a shift in the cell population from AV+PI- to AV+PI+. Furthermore, TNF-α significantly increased protein cleaved caspase 3. TNF-α plus Z-IETD-FMK significantly increased the proteins RIPK3 and MLKL phosphorylation in MC3T3-E1 cells, while the changes in mRNA levels of RIPK3, MLKL, and caspase 3 were not consistent with the changes in the corresponding protein expression levels. In conclusion, TNF-α induced preferentially apoptosis in osteoblast cell line and necroptosis played a decisive role when TNF-α-induced death was inhibited by the inhibitor of apoptosis. Combined treatment with Nec-1 and Z-IETD-FMK protected mouse osteoblasts from death induced by TNF-α.
Assuntos
Caspase 8/efeitos dos fármacos , Inibidores de Caspase/farmacologia , Necrose/patologia , Osteoblastos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Imidazóis/farmacologia , Indóis/farmacologia , L-Lactato Desidrogenase/farmacologia , Camundongos , Oligopeptídeos/farmacologia , Osteoblastos/patologia , FosforilaçãoRESUMO
PURPOSE: To assess the cytotoxicity and genotoxicity of intravitreal adalimumab treatment in an animal experimental model using cytological and molecular techniques. METHODS: Eighteen rabbits were randomly assigned to three groups: control, adalimumab treatment, and placebo. Cytotoxicity on retinal cells was evaluated using flow cytometry assays to determine the level of apoptosis and necrosis. Genotoxicity was evaluated by comet assays to assess DNA damage, and quantitative real-time polymerase chain reaction (qPCR) was used to evaluate expression of apoptosis-inducing caspases (8 and 3). RESULTS: No cytotoxicity or genotoxicity was observed in any of the two treatment groups (adalimumab and placebo) following intravitreal administration compared with the control group. Flow cytometry analysis revealed that more than 90% of the cells were viable, and only a low proportion of retinal cells presented apoptotic (~10%) or necrotic (<1%) activity across all groups. Molecular damage was also low with a maximum of 6.4% DNA degradation observed in the comet assays. In addition, no increase in gene expression of apoptosis-inducing caspases was observed on retinal cells by qPCR in both the adalimumab and placebo groups compared with the control group. CONCLUSION: The use of adalimumab resulted in no detectable cytotoxicity or genotoxicity on retinal cells for up to 60 days upon administration. These results therefore indicate that adalimumab may be a safe option for intravitreal application to treat ocular inflammatory diseases in which TNF-α is involved.
Assuntos
Anti-Inflamatórios/toxicidade , Anticorpos Monoclonais Humanizados/toxicidade , Injeções Intravítreas/métodos , Retina/efeitos dos fármacos , Adalimumab , Animais , Apoptose/efeitos dos fármacos , Caspase 3/análise , Caspase 3/efeitos dos fármacos , Caspase 8/análise , Caspase 8/efeitos dos fármacos , Sobrevivência Celular , Ensaio Cometa , Dano ao DNA , Citometria de Fluxo , Masculino , Modelos Animais , Necrose , Coelhos , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70â µM) for 24, 48, and 72â h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48â h of treatment with 50â µM KA, and 31% for cells submitted to 48â h of treatment with 70â µM KA. Similarly, in U87 cells treated with KA for 48â h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.
Assuntos
Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diterpenos/farmacologia , Glioblastoma/tratamento farmacológico , Mikania/química , Caspase 3/efeitos dos fármacos , Caspase 8/efeitos dos fármacos , Linhagem Celular Tumoral , Diterpenos/isolamento & purificação , Proteína Ligante Fas , Citometria de Fluxo , Glioblastoma/enzimologia , Glioblastoma/patologia , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Fatores de TempoRESUMO
Treatment of P388D1, a macrophage-like cell line, with staurosporine triggered apoptosis through the activation of caspase-9 and caspase-3. Unexpected effects of staurosporine on the induction of apoptosis were the activation of caspase-8, and an increase of the levels of TNF-alpha. The increased TNF-alpha levels led to activation of caspase-8 by an autocrine effect via the TNF receptor expressed by the P388D1 macrophages. In contrast, P388D1 macrophages that either had been exposed to UV light or treated with dexamethasone did not undergo apoptosis.