All known mechanisms of apoptosis induced by resveratrol act through cell cycle arrest and changes in mitochondrial membrane potential. It is currently unknown whether resveratrol-induced apoptosis is associated with other physiological processes, such as autophagy. Apoptosis-related markers involved in the intrinsic and extrinsic apoptotic pathways, and autophagic markers were detected by using western blotting and immunofluorescence. Mitochondrial membrane potential was assayed by flow cytometry. Pharmaceutical or genetic inhibition of autophagy involved were carried by 3- methyladenine or knockdown of autophagy-related (Atg) genes by siRNA. Differences between two values were tested by Student’s unpaired t test. We show that resveratrol-induced apoptosis occurs through both the intrinsic and extrinsic apoptotic pathways. Mitochondrial membrane potential and apoptosis-related markers, such as an increased Bax/Bcl-2 ratio, and cleaved forms of caspase-8 and caspase-3, arise following resveratrol addition. Moreover, we find that resveratrol increases both the levels of microtubule-associated protein 1 light chain 3-II and the number of autophagosomes, and further demonstrate that resveratrol-induced autophagy depends on the LKB1-AMPK-mTOR pathway. We next reveal that some apoptosis-related markers induced by resveratrol are further attenuated by the inhibition of autophagy with 3-methyladenine or knockdown of autophagy-related (Atg) genes by siRNA. These results suggest that resveratrol induced apoptotic cell death of HL-60 cells depends on the autophagy activated through both the LKB1-AMPK and PI3K/AKT-regulated mTOR signaling pathways.

P62 regulates resveratrol-mediated Fas/Cav-1 complex formation and transition from autophagy to apoptosis P62 Regulates resveratrol-mediated Fas/Cav-1 complex formation and transition from autophagy to apoptosis

NAD(P)H:quinone oxidoreductase 1 (NQO1), a phase II flavoenzyme that catalyzes reduction reactions to protect cells against electrophiles and oxidants, is involved in tumorigenesis. Altered methylation of the NQO1 gene has been observed and is speculated to result in aberrant NQO1 expression in rat cells undergoing chemical carcinogenesis, although this has not been proven experimentally. In this study, we first investigated the potential epigenetic mechanisms underlying the phenomenon of NQO1 differential expression in individual subclones of rat arsenic-transformed lung epithelial cells (TLECs). NQO1 expression of TLEC subclones with or without 5-aza-2′-deoxycytidine (5-Aza-CdR) treatment was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), western blot analysis, and real-time PCR. Methylation status of the NQO1 promoter in TLEC subclones was analyzed by bisulfite sequencing. Transcriptional activity of NQO1 promoter in vitro methylated was determined by luciferase assay using a CpG-free luciferase reporter driven by the NQO1 promoter region (−435 to +229). We found that non-CpG island (non-CpGI) within the NQO1 promoter was hyper- or hypo-methylated in TLEC subclones and corresponded to low and high gene expressions, respectively. Following the treatment with 5-Aza-CdR, transcription of the NQO1 gene in the hypermethylated subclones was restored, accompanied by demethylation of the NQO1 promoter. In vitro promoter methylation almost completely silenced reporter activity in TLECs. These results indicate that DNA methylation of the non-CpGI promoter contributes to epigenetic silencing of NQO1 in rat TLECs.

[This corrects the article DOI: 10.18632/oncotarget.2733.].