Effect of Beclin-1 gene silencing on autophagy and apoptosis of the prostatic hyperplasia epithelial cells.

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.

Late autophagy inhibitor chloroquine improves efficacy of the histone deacetylase inhibitor SAHA and temozolomide in gliomas.

Glioblastoma multiforme is the most aggressive type of primary brain tumor associated with few therapeutic opportunities and poor prognosis. In this study, we evaluated the efficacy of combining temozolomide (TMZ) with suberoylanilide hydroxamic acid (SAHA) - a specific histone deacetylases inhibitor - in glioma models in vitro and in vivo. In glioma cell lines, combined TMZ/SAHA promoted more cytotoxicity, G2/M arrest and apoptosis than either drugs alone. G2/M arrest was detected as soon as 24 h post drug exposure and preceded apoptosis, which occurred from 72 h treatment. TMZ and SAHA, alone or combined, also stimulated autophagy as evaluated by means of acridine orange staining and immunodetection of LC3I-II conversion and p62/SQSTM1 degradation. Time-course of autophagy accompanied G2/M arrest and preceded apoptosis, and blockage of late steps of autophagy with chloroquine (CQ) augmented SAHA/TMZ toxicity leading to apoptosis. In orthotopic gliomas in vivo, combined SAHA/TMZ showed better antitumor efficacy than either drugs alone, and adding CQ to the regimen improved antiglioma effects of SAHA and TMZ monotherapies without further benefit on combined SAHA/TMZ. In summary, the herein presented data suggest that autophagy acts as a protective response that impairs efficacy of SAHA and TMZ. Inhibiting autophagy termination with CQ may offer means to improve antitumor effects of SAHA and TMZ in gliomas and possibly other cancers.