RESUMEN
The present study aims to investigate if Cimicifuga racemosa (L.) Nutt extract (CIMI) reduces deleterious effects of dexamethasone (DEXA) in ovaries cultured in vitro. Mouse ovaries were collected and cultured in DMEM+ only or supplemented with 5 ng/mL of CIMI, or 4 ng/mL DEXA, or both CIMI and DEXA. The ovaries were cultured at 37.5°C in 5% CO2 for 6 days. Ovarian morphology, follicular ultrastructure, and the levels of mRNA for Bax, Bcl-2, and Caspase-3 were evaluated. The results showed that DEXA reduced the percentage of morphologically normal follicles, while CIMI prevented the deleterious effects caused by DEXA. In addition, DEXA negatively affected the stromal cellular density, while CIMI prevented these adverse effects. Ovaries cultured with DEXA and CIMI showed similar levels of mRNA for Bax, Bcl-2, and Caspase-3 compared to those cultured in control medium, while ovaries cultured with DEXA had increased expression of the above genes. Additionally, the ultrastructure of the ovaries cultured with CIMI was well preserved. Thus, the extract of CIMI was able to prevent the deleterious effects caused by DEXA on cultured mouse ovaries.
Asunto(s)
Cimicifuga , Femenino , Animales , Ratones , Caspasa 3 , Proteína X Asociada a bcl-2/metabolismo , Proteína X Asociada a bcl-2/farmacología , Cimicifuga/genética , Cimicifuga/metabolismo , Folículo Ovárico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , ARN Mensajero/metabolismo , Dexametasona/toxicidadRESUMEN
OBJECTIVES: This study aims to explore the effect of silencing Beclin-1 gene on autophagy and apoptosis of Benign Prostatic Hyperplasia (BPH) (BPH-1) cells under the condition of Androgen Deprivation (AD) and Autophagy Inhibition (AI). METHODS: Control group (BPH-1 group), empty carrier group (sh-RNA-BPH-1 group) and Beclin-1 silenced group (sh-Beclin1-BPH-1 group) were set. The Beclin-1 gene silencing efficiency was detected by RT-PCR and Western blot. Autophagic flux was monitored by GFP-LC3 cleavage assay and cell apoptosis was analyzed by flow cytometry. The protein expression levels of LC3, Caspase-3, PARP-1, Bcl-2, and Bax were detected by Western blot. RESULTS: The transfection of sh-Beclin-1 obviously down-regulated the expression of Beclin-1 at both mRNA and protein levels. Under the conditions of AD and AI, silencing of Beclin-1 restrained the autophagy of BPH-1 cells, as evidenced by a decreased number of autophagosomes and down-regulation of LC3-II protein (p < 0.001). The results of flow cytometry showed that the apoptotic rate of sh-Beclin-1 group was elevated significantly compared to the other two groups (p < 0.01). Western blot results showed that silencing of Beclin-1 promoted 89 kd fragmentation of PARP-1 (p < 0.001) and Caspase-3 activation (p < 0.01). Moreover, silencing of Beclin-1 resulted in declined Bcl-2 and augmented Bax protein expression in BPH-1 cells (p < 0.01), which ultimately led to a decreased Bcl-2/Bax ratio. CONCLUSIONS: The results indicated that the silencing of Beclin-1 gene hampered autophagy while activating apoptosis in BPH-1 cells. Thus, Beclin-1 may participate in an antagonistic relationship between autophagy and apoptosis in BPH.
Asunto(s)
Hiperplasia Prostática , Neoplasias de la Próstata , Antagonistas de Andrógenos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/farmacología , Autofagia , Beclina-1/genética , Beclina-1/metabolismo , Beclina-1/farmacología , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 3/farmacología , Células Epiteliales/metabolismo , Silenciador del Gen , Humanos , Masculino , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Hiperplasia Prostática/genética , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Proteína X Asociada a bcl-2/farmacologíaRESUMEN
The intracellular parasite Leishmania mexicana inhibits camptothecin (CPT)-induced apoptosis of monocyte-derived dendritic cells (moDC) through the down-regulation of p38 and JNK phosphorylation, while the kinase Akt is maintained active for 24 h. In addition, the infection of moDC with L. mexicana promastigotes increases the protein presence of the antiapoptotic protein Bcl-xL. In the present work, we aimed to investigate the role of Akt in the inhibition of apoptosis of moDC by L. mexicana and in the modulation of the expression of the antiapoptotic proteins Bcl-2, Mcl-1 and Bcl-xL. moDC were infected with L. mexicana metacyclic promastigotes and treated with CPT, an Akt inhibitor, or both and the mitochondrial outer membrane permeabilization (MOMP) and protein presence of active caspase 3, Bcl-2, Mcl-1 and Bcl-xL were evaluated. Our results show that the specific inhibition of Akt reverts the apoptosis protective effect exerted by L. mexicana on moDC reflected by a reduction in MOMP, caspase 3 activation, and upregulation of Bcl-xL. Interestingly, we also found that the infection of moDC with L. mexicana promastigotes induces a decrease in Bcl-2 along with an isoform change of Mcl-1, this independently to Akt activity. We demonstrated that Akt is deeply involved in the inhibition of apoptosis of moDC by L. mexicana.
Asunto(s)
Leishmania mexicana , Apoptosis , Proteínas Reguladoras de la Apoptosis , Camptotecina/farmacología , Caspasa 3 , Células Dendríticas/parasitología , Leishmania mexicana/fisiología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , Proteína bcl-X/metabolismoRESUMEN
Mitochondrial permeability transition (MPT) is a nonselective inner membrane permeabilization that contributes to neuronal cell death under circumstances such as brain trauma, ischemia, and hypoglycemia. Here we study the participation of MPT and the Bcl-2-sensitive apoptotic cell death pathway in glutamate receptor-mediated excitotoxicity. Intrastriatal infusions of the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid caused massive striatal neurodegeneration in both rats and mice. Interestingly, transgenic mice overexpressing human Bcl-2 and rats systemically treated with cyclosporin A did not exhibit reduced sensitivity to quinolinic acid-induced striatal toxicity. Both Bcl-2 and cyclosporin A are inhibitors of MPT; in addition Bcl-2 also inhibits apoptotic stimuli-mediated release of mitochondrial apoptogenic factors. Isolated brain mitochondria from cyclosporin A-treated rats showed resistance to Ca(2+)-induced dissipation of the membrane potential, indicating protection against MPT. We conclude that quinolinic acid-mediated striatal excitotoxicity is not dependent on MPT and Bcl-2-sensitive apoptotic cell death pathways.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Ciclosporina/farmacología , Neurotoxinas/toxicidad , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Ácido Quinolínico/toxicidad , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Animales , Apoptosis/efectos de los fármacos , Química Encefálica , Calcio/farmacología , Cuerpo Estriado/citología , Inhibidores Enzimáticos/farmacología , Femenino , Expresión Génica , Humanos , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Transgénicos , Mitocondrias/química , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , Ratas , Ratas WistarRESUMEN
Redox imbalance due to oxidative stress or excessive antioxidant levels can alter apoptotic responses. Recently, antioxidants like N-acetylcysteine (NAC) were reported to inhibit H(2)O(2)-mediated necrotic cell death, although they were inactive against apoptosis induced by other agents like etoposide. NAC was also found to kill preferentially tumor cells compared to normal fibroblasts at 20-50mM, but these concentrations are lethal to normal splenocytes. We now demonstrate that 10mM NAC, a non-toxic concentration, can enhance the UV radiation-mediated apoptosis of human C8161 melanoma cells. Compared to treatment with UV radiation alone, combination treatment with NAC doubled the ratio of activated caspase-3 to pro-caspase-3 and produced greater fragmentation of the retinoblastoma protein and the E2F-4 transcription factor without affecting the E2F-1 protein. These effects of joint NAC-UV radiation treatment were counteracted by the overexpression of the bcl-2 gene. To our knowledge, this report is the first to: (i) demonstrate a synergy between DNA-damaging agents, like UV radiation, and antioxidants, like NAC, and (ii) show that a Bcl-2-inhibitable E2F-4 fragmentation occurs concurrently with caspase-3 activation and apoptosis.