Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes

Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes.     

Bufalin Induces the Interplay between Apoptosis and Autophagy in Glioma Cells through Endoplasmic Reticulum Stress

Malignant gliomas are common primary tumors of the central nervous system. The prognosis of patients with malignant glioma is poor in spite of current intensive therapy and thus novel therapeutic modalities are necessary. Bufalin is the major component of Chan-Su (a traditional Chinese medicine) extracts from the venom of Bufo gargarizan. In this study, we evaluated the growth inhibitory effect of bufalin on glioma cells and explored the underlying molecular mechanisms. Our results showed that bufalin inhibited the growth of glioma cells significantly. Mechanistic studies demonstrated that bufalin induced apoptosis through mitochondrial apoptotic pathway. In addition, bufalin was also found to induce ER stress-mediated apoptosis, which was supported by the up- regulation of ER stress markers, CHOP and GRP78, and augmented phosphorylation of PERK and eIF2α as well as cleavage of caspase-4. Downregulation of CHOP using siCHOP RNA attenuated bufalin-induced apoptosis, further confirming the role of ER stress response in mediating bufalin-induced apoptosis. Evidence of bufalin-induced autophagy included formation of the acidic vesicular organelles, increase of autophagolysosomes and LC3-II accumulation. Further experiments showed that the mechanism of bufalin-induced autophagy associated with ATP deleption involved an increase in the active form of AMPK, decreased phosphorylation levels of mTOR and its downstream targets 4EBP1 and p70S6K1. Furthermore, TUDC and silencing of eIF2α or CHOP partially blocked bufalin-induced accumulation of LC3-II, which indicated that ER stress preceded bufalin-induced autophagy and PERK/eIF2α/CHOP signaling pathway played a major part in the process. Blockage of autophagy increased expression of ER stress associated proteins and the ratio of apoptosis, indicating that autophagy played a cytoprotective role in bufalin induced ER stress and cell death. In conclusion, bufalin inhibits glioma cell growth and induces interplay between apoptosis and autophagy through endoplasmic reticulum stress. It will provide molecular bases for developing bufalin into a drug candidate for the treatment of maglinant glioma.     

Endoplasmic Reticulum Stress Stimulates p53 Expression through NF-κB Activation

Background

Induction of apoptosis by endoplasmic reticulum (ER) stress is implicated as the major factor in the development of multiple diseases. ER stress also appears to be a potentially useful major response to many chemotherapeutic drugs and environmental chemical compounds. A previous study has indicated that one major apoptotic regulator, p53, is significantly increased in response to ER stress, and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood.

Principal Findings

In this report, we demonstrate that induction of p53 expression is mediated through NF-κB signaling pathways during ER stress in MCF-7 cells. Tunicamycin or brefeldin A, two ER stress inducers, increased p53 expression in MCF-7 and Hela cells. We found p53 nuclear localization, activity, and phosphorylation at serine 15 on p53 increased during ER stress. Nuclear translocation of NF-κB and activity of NF-κB were also observed during ER stress. ER stress-induced p53 expression was significantly inhibited by coincubation with the NF-κB inhibitor, Bay 11-7082 and downregulation of NF-κB p65 expression. The role of p53 in mediating Brefeldin A-induced apoptosis was also investigated. Induction of p53 expression by Brefeldin A was correlated to Brefeldin A-induced apoptosis. Furthermore, downregulation of p53 expression by p53 siRNA significantly reduced Brefeldin A-induced apoptosis in MCF-7 cells.

Significance

Taken together, NF-κB activation and induction of p53 expression is essential for ER stress-induced cell death which is important for therapeutic effects of clinical cancer drugs. Our results may provide insight into the mechanism of cancer chemotherapy efficacy that is associated with induction of ER stress.     

LncRNA SNHG15 modulates gastric cancer tumorigenesis by impairing miR-506-5p expression

The gastric cancer (GC) patients commonly have a poor prognosis due to its invasiveness and distant metastasis. Growing evidence proved that aberrant long non-coding RNAs (lncRNAs) expression contributes to tumor development and progression. LncRNA SNHG15 has been reported to be involved in many different kinds of cancer, while its role in GC remains unclear. In the present study, we found that SNHG15 was up-regulated in GC tissues and cell lines. Silencing SNHG15 suppressed proliferation migration, invasion and promoted apoptosis of AGS cells. More importantly, microRNA-506-5p (miR-506-5p) was predicted as a direct target of SNHG15 by binding its 3′-UTR and further verified using luciferase reporter assay. Meanwhile, the results of rescue experiments revealed that knockdown of miR-506-5p expression reversed the functional effects of SNHG15 silenced cell proliferation, migration, invasion and apoptosis. In conclusion, our findings revealed that SNHG15 executed oncogenic properties in GC progression through targeting miR-506-5p, which might provide a novel target for the GC treatment.     

Protective role of Gipie, a Girdin family protein, in endoplasmic reticulum stress responses in endothelial cells

Continued exposure of endothelial cells to mechanical/shear stress elicits the unfolded protein response (UPR), which enhances intracellular homeostasis and protect cells against the accumulation of improperly folded proteins. Cells commit to apoptosis when subjected to continuous and high endoplasmic reticulum (ER) stress unless homeostasis is maintained. It is unknown how endothelial cells differentially regulate the UPR. Here we show that a novel Girdin family protein, Gipie (78 kDa glucose-regulated protein [GRP78]-interacting protein induced by ER stress), is expressed in endothelial cells, where it interacts with GRP78, a master regulator of the UPR. Gipie stabilizes the interaction between GRP78 and the ER stress sensor inositol-requiring protein 1 (IRE1) at the ER, leading to the attenuation of IRE1-induced c-Jun N-terminal kinase (JNK) activation. Gipie expression is induced upon ER stress and suppresses the IRE1-JNK pathway and ER stress-induced apoptosis. Furthermore we found that Gipie expression is up-regulated in the neointima of carotid arteries after balloon injury in a rat model that is known to result in the induction of the UPR. Thus our data indicate that Gipie/GRP78 interaction controls the IRE1-JNK signaling pathway. That interaction appears to protect endothelial cells against ER stress-induced apoptosis in pathological contexts such as atherosclerosis and vascular endothelial dysfunction.     

p53 negatively regulates Pin1 expression under ER stress

Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a major role in the development of many diseases. A previous study indicated that the apoptotic regulator p53 is significantly increased in response to ER stress and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood. Here, we investigated whether p53 contributes to the impairment of Pin1 signaling under ER stress. We found that treatment with thapsigargin, a stimulator of p53 expression and an inducer of ER stress, decreased Pin1 expression in HCT116 cells. Also, we identified functional p53 response elements (p53REs) in the Pin1 promoter. Overexpression of p53 significantly decreased Pin1 expression in HCT116 cells while abolition of p53 gene expression induced Pin1 expression. Pin1 expression was significantly increased by treatment with the p53 inhibitor pifithrin-α or down-regulation of p53 expression. Taken together, ER stress decreased Pin1 expression through p53 activation, and this mechanism may be associated with ER stress-induced cell death. These data reported here support the importance of Pin1 as a potential target molecule mediating tumor development.     

Palmitic acid induces human osteoblast-like Saos-2 cell apoptosis via endoplasmic reticulum stress and autophagy

Palmitic acid (PA) is the most common saturated long-chain fatty acid in food that causes cell apoptosis. However, little is known about the molecular mechanisms of PA toxicity. In this study, we explore the effects of PA on proliferation and apoptosis in human osteoblast-like Saos-2 cells and uncover the signaling pathways involved in the process. Our study showed that endoplasmic reticulum (ER) stress and autophagy are involved in PA-induced Saos-2 cell apoptosis. We found that PA inhibited the viability of Saos-2 cells in a dose- and time-dependent manner. At the same time, PA induced the expression of ER stress marker genes (glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)), altered autophagy-related gene expression (microtubule-associated protein 1 light chain 3 (LC3), ATG5, p62, and Beclin), promoted apoptosis-related gene expression (Caspase 3 and BAX), and affected autophagic flux. Inhibiting ER stress with 4-PBA diminished the PA-induced cell apoptosis, activated autophagy, and increased the expression of Caspase 3 and BAX. Inhibiting autophagy with 3-MA attenuated the PA and ER stress-induced cell apoptosis and the apoptosis-related gene expression (Caspase 3 and BAX), but seemed to have no obvious effects on ER stress, although the CHOP expression was downregulated. Taken together, our results suggest that PA-induced Saos-2 cell apoptosis is activated via ER stress and autophagy, and the activation of autophagy depends on the ER stress during this process.     

Progranulin is required for proper ER stress response and inhibits ER stress-mediated apoptosis through TNFR2

Progranulin (PGRN) was reported to be a stress-response factor in response to hypoxia and acidosis. Here we present evidences demonstrating that PGRN is also an endoplasmic reticulum (ER) stress responsive factor: PGRN expression was induced and its activation of Erk1/2 and Akt signaling enhanced in response to ER stress; Normal ER stress response was lost in PGRN deficient cells and PGRN deficient cells became hypersusceptible to ER stress-induced apoptosis; additionally, recombinant PGRN could rescue the defects in ER-stress responses seen in PGRN deficient cells. Mechanistic studies indicated that PGRN/TNFR2 was critical for PGRN mediated regulation of ER stress response: similar to PGRN, the expression of TNFR2, but not TNFR1, was also induced in the course of ER stress; in addition, the association between PGRN and TNFR2 was markedly enhanced following ER stress; More importantly, PGRN protection of ER stress induced apoptosis was abolished when TNFR2 signaling was blocked. In addition, the 2nd and 3rd cysteine-rich domains (CRD) in the extracellular portion of TNFR2 (CRD2CRD3), known to directly bind to PGRN, disturbed the interaction of PGRN with TNFR2, and in turn abolished PGRN-mediated activation of Erk1/2 and Akt signaling and protection against apoptosis in response to ER-stress. Collectively, PGRN plays an important role in ER stress and regulates ER stress response through interacting with TNFR2. This study provides new insight into PGRN regulation of stress response and may also present PGRN as a potential molecular target for treating stress-associated disorders.     

长链非编码RNA SNHG1促进肺癌细胞增殖

近年来,大量证据表明长链非编码RNA (long non-coding RNAs, lncRNAs) 在肿瘤发生发展中发挥重要的作用。本研究通过生物信息学预测发现,核仁小分子RNA宿主基因1(small nucleolar RNA host gene 1,SNHG1)无蛋白质编码功能,且主要定位于细胞质。qRT-PCR结果显示,SNHG1在肺癌细胞中的表达量显著高于正常肺上皮细胞。在肺癌细胞NCI-H1299中,敲低SNHG1发现,该细胞增殖能力明显下降;在正常肺上皮细胞BEAS-2B中,过表达SNHG1可显著促进该细胞的增殖能力。深入研究发现,SNHG1可上调CDK4和下调p27kip1在蛋白质水平的表达,而对其mRNA水平表达量无影响。此外,在肺癌临床组织中也发现SNHG1高表达,且高表达的SNHG1与肺癌瘤体大小、TNM分期和远端转移正相关,而与患者年龄及吸烟史无关。综上所述,SNHG1在肺癌中高表达,通过上调CDK4和下调p27kip1推进肺癌进程。     

Scutellarin inhibits the glioma cell proliferation by downregulating BIRC5 to promote cell apoptosis

The expression changes of baculovirus inhibitor of apoptosis repeat-containing protein5 in brain glioma after administration of Scutellarin was detected. To explore the effort of scutellarin on anti-glioma by downregulating BIRC5.The effect of scutellarin on tumour growth and animal survival was detected by administering scutellarin to nude mice subcutaneous tumour formation and SD rats in situ tumour formation models. A significantly different gene BIRC5 was found by using the combination of TCGA databases and network pharmacology. And then qPCR was performed to detect the expression of BIRC5 in glioma tissues, cells and normal brain tissues and glial cells. CCK-8 was used to detect the IC50 of scutellarin on glioma cells. The wound healing assay, flow cytometry and MTT test were used to detect the effect of scutellarin on the apoptosis and proliferation of glioma cells. The expression of BIRC5 in glioma tissues was significantly higher than that in normal brain tissues. Scutellarin can significantly reduce tumour growth and improve animal's survival. After scutellarin was administered, the expression of BIRC5 in U251 cells was significantly reduced. And after same time, apoptosis increased and cell proliferation was inhibited. This original research showed that scutellarin can promote the apoptosis of glioma cells and inhibit the proliferation by downregulating the expression of BIRC5.     

The Orphan Nuclear Receptor NR4A1 Protects Pancreatic β-Cells from Endoplasmic Reticulum (ER) Stress-mediated Apoptosis

The role of NR4A1 in apoptosis is controversial. Pancreatic β-cells often face endoplasmic reticulum (ER) stress under adverse conditions such as high free fatty acid (FFA) concentrations and sustained hyperglycemia. Severe ER stress results in β-cell apoptosis. The aim of this study was to analyze the role of NR4A1 in ER stress-mediated β-cell apoptosis and to characterize the related mechanisms. We confirmed that upon treatment with the ER stress inducers thapsigargin (TG) or palmitic acid (PA), the mRNA and protein levels of NR4A1 rapidly increased in both MIN6 cells and mouse islets. NR4A1 overexpression in MIN6 cells conferred resistance to cell loss induced by TG or PA, as assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and TUNEL assays indicated that NR4A1 overexpression also protected against ER stress-induced apoptosis. This conclusion was further confirmed by experiments exploiting siRNA to knockdown NR4A1 expression in MIN6 cells or exploiting NR4A1 knock-out mice. NR4A1 overexpression in MIN6 cells reduced C/EBP homologous protein (CHOP) expression and Caspase3 activation induced by TG or PA. NR4A1 overexpression in MIN6 cells or mouse islets resulted in Survivin up-regulation. A critical regulatory element was identified in Survivin promoter (−1872 bp to −1866 bp) with a putative NR4A1 binding site; ChIP assays demonstrated that NR4A1 physically associates with the Survivin promoter. In conclusion, NR4A1 protects pancreatic β-cells against ER stress-mediated apoptosis by up-regulating Survivin expression and down-regulating CHOP expression, which we termed as “positive and negative regulation.”     

Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression

NF-kappaB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-kappaB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1alpha through the adapter protein TRAF2. ER stress-induced NF-kappaB activation is impaired in IRE1alpha knockdown cells and IRE1alpha(-/-) MEFs. We found, however, that inhibiting NF-kappaB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-alpha) was IRE1alpha and NF-kappaB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-alpha-induced activation of NF-kappaB and c-Jun N-terminal kinase and turns TNF-alpha from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-alpha induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.     

Long noncoding RNA SNHG12 promotes cell proliferation and activates Wnt/β-catenin signaling in prostate cancer through sponging microRNA-195

Long noncoding RNAs (lncRNAs) serve critical roles in multiple human malignant tumors, including prostate cancer (PCa). Currently, the biological role of oncogenic lncRNA SNHG12 in PCa remains largely unclear. In the present study, we found that SNHG12 was highly expressed in human PCa tissues and cell lines. In addition, gain-of-function and loss-of-function studies showed that overexpression of SNHG12 promoted, while downregulation suppressed the proliferation, invasion, and migration of PCa cells in vitro. Knockdown of SNHG12 also repressed PCa xenograft tumor growth in vivo. Further in-depth mechanistic studies showed that SNHG12 might serve as a competing endogenous RNA for miR-195 in PCa cells, and miR-195 expression level was negatively associated with the expression of SNHG12 in PCa tissues. Finally, we found that the activity of Wnt/β-catenin signaling is enhanced by SNHG12 overexpression and rescued by co-transfection with miR-195 mimics in PCa cells. Collectively, the present study indicated the oncogenic function of SNHG12 in PCa and our findings might provide a new target in the treatment of PCa.     

Aberrant expression of lncRNAs SNHG6, TRPM2-AS1, MIR4435-2HG,and hypomethylation of TRPM2-AS1 promoter in colorectal cancer

Accumulating evidence has indicated that deregulation of lncRNAs plays essential roles in colorectal cancer (CRC) carcinogenesis. The goal of this study was to analyze the expression of lncRNAs in colorectal cancer and their association with clinicopathological variables. Bioinformatics analysis of published CRC microarray data was performed to identify the important lncRNAs. The expression levels of candidate genes were assessed in the human colon cancer/normal cell lines, CRC, adenomatous colorectal polyps, and their marginal tissues by qRT-PCR. Moreover, the methylation status of the TRPM2-AS1 promoter was studied using qMSP assay. Furthermore, we investigated the molecular mechanisms of these lncRNAs in CRC progression using in silico analysis. Microarray analysis revealed that lncRNAs SNHG6, MIR4435-2HG, and TRPM2-AS1 were upregulated in CRC. These results were validated in colon cell lines. Moreover, qRT-PCR showed that the expression levels of SNHG6 and TRPM2-AS1 were upregulated in the colorectal tumor tissues compared with their paired tissues. Nonetheless, there was no significant increase in MIR4435-2HG expression in CRC samples. Furthermore, we observed a significant hypomethylation of TRPM2-AS1 promoter and its activation in CRC tissues. By in silico analysis, we found that the lncRNAs upregulation could promote proliferation and drug resistance of colorectal cancer cells via miRNAs sponging and modulation of their targets expression. In conclusion, based on our results upregulation of SNHG6 and TRPM2-AS1, and hypomethylation of TRPM2-AS1 promoter might be considered as potential diagnostic biomarkers for CRC initiation and development.