NOR1is Oxidative Stress Reative Protein Regulating Tumor Cell Autophagy/apoptosis Molecular Switch

The previous research of the projectOxidored-nitro domain-containing protein1(NOR1) is a candidate TSG that is downregulated in nasopharyngeal carcinoma (NPC). Despite these findings, little is known regarding mechanisms underlying downregulation of the NOR1gene in NPC cell lines and biopsies. NOR1is an interaction partner of the mitochondrial adenosine triphosphate synthase subunit OSCP/ATP5O protein;Ectopic expression of NOR1in NPC HNE1cells inhibited tumor cell colony formation and viability, and NOR1gene may be a critical tumor suppressor involved in the development of NPC. Despite these findings, little is known regarding the influence and mechanism of NOR1to tumor cell homeostasis and tumor progression.Identification of a NOR1promoterSeveral bioinformatics tools such as PromoterScan, Promoter Inspector and EMBOSS CpG Plot were used to identify and characterize the potential promoter region of the NOR1gene. A753bp region spanning positions-517to+236bp relative to the transcription start site was identified as the potential promoter region of the NOR1gene. To assess whether this sequence could function to drive transcription, different regions of the sequence were cloned and linked to a luciferase-or eGFP-based reporter construct. The results using luciferase assay and visible eGFP showed that NOR1promoter located in a region spanning positions-258to+26bp.The NOR1promoter is hypermethylated and NOR1expression is downregulated in NPC tissue samples A CpG island was detected using the EMBOSS CpGplot program, with a GC content of58.8%. We investigated the methylation status of the NOR1promoter in primary NPC biopsies and non-cancerous nasopharyngeal tissue samples, several NPC cell lines and normal nasopharyngeal epithelial cell line by MSP and BSP.The results showed that methylated NOR1promoter could be detected in NPC biopsies and cell lines. The data using immunohistochemical staining and RT-PCR indicate that there might be a negative correlation between NOR1methylation and gene expression levels. Semi-quantitative RT-PCR and real-time RT-PCR analysis showed that NOR1expression levels were restored by5-aza-dC treatment. Thus, these data strongly suggest that hypermethylation of the NOR1promoter might be responsible for transcription silencing of the NOR1gene in human malignancies.Transcription factors HSF1and NRF1specifically bind to and activate the NOR1promoterSeveral bioinformatics tools MatInspector was used to characterize the potential promoter region of the NOR1gene. Within this promoter, one putative NRF1-binding site and ne putative HSF1-binding were identified at consecutive position. The results using EMAS and ChIP provide strong evidence demonstrating that transcription factors HSF1and NRF1specifically bind to and activate theNOR1promoter.Furthermore, cotransfection of the NOR1promoter constructs containing the HSF1-binding site with an HSF1-expressing vector showed that ecotopic expression of HSF1increased in the activity of the NOR1promoter. Western blot analyses showed that NOR1protein levels increased following exogenous expression of HSF1or NRF1. However, knockdown of endogenous HSF1or NRF1decreased NOR1mRNA levels. Taken together, our results provide strong evidence demonstrating that transcription factors HSF1and NRF1specifically bind to and activate the NOR1promoter.Hydrogen peroxide exposure upregulated NOR1expression in normal cells and cancer cells In previous study, NOR1gene promoter was showed to be regulated by HSF1and NRF1, both of which are critical mediators of oxidative stress response. Thus, in this study, we investigated expression of NOR1mRNA and protein in both cancer and non-cancerous cells after induced by hydrogen peroxide. Realtime PCR, FACS and immunofluorescence data showed that NOR1expression was induced by an acute oxidative stress in NP69normal human cells and MCF7tumor cells.NOR1suppress basal autophagy, mitochondrial oxidative phosphorylation and tumor cell viabilityIn this study, we investigated enforced expression of NOR1at physiologic levels in HNE1cells reduced HNE1tumor cell viability compared to vector-control cells. Overexpression of NOR1significantly suppressed an anchorage-independent growth of HNE1cells. In contrast, knockdown of NOR1expression using NOR1RNAi resulted in increase in cell viability.This study investigated effects of NOR1on autophagy under normal culture conditions. Western blot and transmission electron microscopy data showed overexpression of NOR1suppressed basal autophagy level in HNE1cells. In contrast, knockdown of NOR1expression using NOR1RNAi resulted in increase in autophagy level.Autophagy is required for tumor cell survival during starvation and tumorigenesis. We next investigated whether expression of NOR1reduced tumor cell glycolysis and mitochondrial oxidative phosphorylation. Our data showed that levels of lactate production and glucose consumption were decreased after NOR1expression in HNE1cells. Oxygen consumption was inhibited in NOR1expressing FNE1cells.Moreover, ROS generation was also decreased in NOR1expressing HNE1cells.Effects of NOR1-inhibited hydrogen peroxide-induced autophagy on tumor cell cytotoxicity; NOR1induced HNE1cell apoptosis through inhibited autophagy Here we determined the effects of NOR1-inhibited hydrogen peroxide-induced autophagy on regulation of tumor cell cytotoxicity. LDH assay and MTT data showed that LDH release is increased and tumor cell toxicity was significantly induced in NOR1expressing HNE1cells with H2O2. Furthermore, we found that restoration of NOR1expression down-regulated autophagy after treating with H2O2assessed by using an autophagy marker LC3conversion and transmission electron microscopy. Western blot, TUNEL and Hoechst33258staining identified that induction of apoptosis in NOR1expressing HNE1cells treated with H2O2. In contract, Western blot showed that an induced autophagy evident and a decreased level of cleaved PARP in NOR1siRNA-transfected cells treated with H2O2. These results confirmed that autophagy inhibition by NOR1might confer sensitivity to oxidative stress-induced apoptosis in cancer cells.Western blot resulted in an increased Bax/Bcl-xL ratio in NOR1expressing cells treated with H2O2and then facilitated the release of Smac/Diablo from the mitochondria into the cytosol. Consequently, cleavaged caspase-9, caspase-3, and PARP were clearly increased in NOR1expressing HNE1cells after hydrogen peroxide treatment.These results suggested that NOR1mediated tumor cells apoptosis in oxidative stress was associated with upregulation of the pro-apoptotic pathway through increase in the ratio of mitochondrial Bax/Bcl-xL ratio and corresponding release of mitochondrial Smac/Diablo proteins.NOR1expression sensitized tumor cells to cisplatin-induced apoptosisSince autophagy may be activated as a protective mechanism against chemotherapy agents. We assess whether NOR1sensitizes tumor cells to cisplatin-induced apoptosis as a novel strategy in future cancer therapy. We treated NPC5-8F cells or HNE1cells with a series of concentrations of cisplatin. The data showed that expression of NOR1significantly inhibited the cisplatin-induced autophagy, resulting in increased cisplatin cytotoxicity and apoptosis. Thus, promoter hypermethylation has been recognized as a commonmechanism leading to inactivation of TSGs. These data revealed NOR1may be a critical TSG in NPC and represents a potential attractive target for epigenetic therapy of NPC and revealed novel aspects of the interplay between autophagy and apoptosis in nasopharyngeal carcinoma cells that underlie the tumor suppression function of NOR1, possibly providing a novel insight into development of a combinatorial therapy for nasopharyngeal carcinoma.