GRP78 Protein Expression Research Articles

Concomitant high expression of ERα36, GRP78 and GRP94 is associated with aggressive papillary thyroid cancer behavior.

Papillary thyroid cancer (PTC) is more common in women than in men. It has been suggested that estrogen may be involved in its development, as has previously been shown for breast, endometrial and ovarian cancer. The purpose of this study was to assess correlations between the expression of the estrogen receptor alpha36 (ERα36) and the glucose regulated proteins GRP78 and GRP94 (chaperones involved in glycoprotein folding) and various PTC clinicopathological features, as well as to evaluate the potential usefulness of these three potential oncogenic proteins in the prediction of aggressive PTC behavior. ERα36, GRP78 and GRP94 protein expression in 218 primary PTC tissues and PTC-derived BCPAP cells was examined using immunohistochemistry, Western blotting and immunocytochemistry. The proliferative, invasive and migrative capacities ofBCPAP cells in which the respective genes were either exogenously over-expressed or silenced were assessed using BrdU incorporation and Transwell assays, respectively. We found that ERα36, GRP78 and GRP94 protein expression was upregulated in the primary PTC tissues tested. We also found that ERα36, GRP78 and GRP94 expression modulation affected the proliferation, invasion and migration of PTC-derived BCPAP cells. A positive correlation and a positive feedback loop were noted between ERα36, GRP78 and GRP94 protein expression in the primary PTC tissues and in BCPAP cells, respectively. High ERα36 expression in combination with a high GRP78/ GRP94 expression was found to have a stronger correlation with extrathyroid extension (ETE), lymph node metastasis (LNM), distant metastasis (DM) and high TNM stage than high ERα36 expression in combination with either high GRP78 or high GRP94 expression (p = 0.028 for ETE, p = 0.002 for DM and p ≤ 0.001 for LNM and high TNM stage) or high ERα36 expression alone (p < 0.001 for ETE, LNM, DM and high TNM stage). From our data we conclude that a concomitant high expression of ERα36, GRP78 and GRP94 is strongly associated with aggressive PTC behavior and may be used as a predictor for ETE, LNM, DM and high TNM stage.

Hydroxytyrosol and olive leaf extract exert cardioprotective effects by inhibiting GRP78 and CHOP expression.

Myocardial infarction triggers massive biochemical changes, even cardiac cell death. Endoplasmic reticulum stress is involved in the pathology of myocardial infarction-mediated apoptosis. In the present study, myocardial cell line H9c2 cells were treated with cobalt chloride (CoCl2) to induce hypoxia. Isoproterenol was used for two successive days to induce myocardial infarction in SD rats. The cardioprotective effect of olive leaf extract (OLE) and its main constituent hydroxytyrosol and the underlying mechanisms were evaluated. The results showed that hydroxytyrosol markedly protected H9c2 cells against CoCl2-induced apoptosis. Hydroxytyrosol could reduce the mRNA and protein expression of GRP78 and CHOP induced by CoCl2 in vitro. In vivo, the decreased ejection fraction and fractional shortening, increased heart weight/body ratio, the formation of infarction, disordered cardiac muscle fibers and infiltration of inflammatory cells induced by isoproterenol could be significantly ameliorated by pretreatment with OLE for a month. Similarly, OLE could also reverse the increase of GRP78 and CHOP expression induced by isoproterenol. Therefore, OLE and hydroxytyrosol exert a cardioprotective effect through endoplasmic reticulum stress, which could be a new target for the prevention and treatment of cardiovascular diseases.

Abstract B11: Prostate apoptosis response-4 (PAR4) secretion in breast tumor and normal cell lines cultured in hypoxic conditions

Abstract The prostate apoptosis response-4 (PAR4) gene encodes a 38kDa proapoptotic protein with tumor cell selective action and/or cell sensitization to an additional proapoptotic insult. This selective apoptotic ability is associated with PAR4 nuclear translocation and phosphorylation state. PAR-4 acts through intrinsic and extrinsic apoptotic pathways. PAR-4 can also be secreted by normal/immortalized and cancer cells via the classical ER stress pathway mediated by GRP78. The previous study from our group provided evidence that MCF10A cells cultured in three-dimensional conditions display an increased expression of PAR4 in the center of the acini, where the cells are dying to lumen formation and where the O2 levels are expected to be lower. In the present study, we sought to evaluate the effects of hypoxia on PAR4 secretion in mammary epithelial cells and breast cancer cells. MCF10A (immortalized mammary epithelial cells) and the breast cancer cells MCF7, MDA-MB-231, and SKBR3 were cultured in normoxic and hypoxic (1% O2) conditions. The conditioned medium (CM) was concentrated using Amicon Ultra 10kDA filters and resolved by SDS-PAGE gel electrophoresis in parallel to cell lysate analysis. Cells maintained under hypoxic conditions for 6 h, 24 h, and 48 h showed no alteration in intracellular PAR4 protein levels in relation to cells maintained in normoxia. After 72 h we observed a decrease in intracellular PAR4 protein expression. The GRP78 protein expression did not change in the same time periods. However, Caspase-7 activation could be seen in 24 h, 48 h, and 72 h. Furthermore, we observed an increase (2.7 fold) in PAR4 protein expression in the CM after 72 h under hypoxia. Our results demonstrated for the first time that PAR4 secretion is modulated by hypoxia in normal and tumor breast cells. Further experimental and clinical studies are needed to determine the mechanism and role played by PAR4 secretion in cells' sensitivity to standard chemotherapeutics agents. Supported by FAPESP and CNPq. Citation Format: Simone Aparecida de Bessa Garcia, Irina Gueroldovna Brobovnitchaia, Flavia Rotea Mangone, Maria Aparecida Nagai. Prostate apoptosis response-4 (PAR4) secretion in breast tumor and normal cell lines cultured in hypoxic conditions [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B11.

PGK1 and GRP78 overexpression correlates with clinical significance and poor prognosis in Chinese endometrial cancer patients.

The aim of this study was to measure the expression patterns of PGK1 and GRP78 in normal endometrial tissues and endometrial carcinoma, and associations between their combined effects and the pathological features of endometrial carcinoma. We used 30 normal endometrial tissue samples and 130 endometrial carcinoma samples, and separately evaluated PGK1 and GRP78 protein expression by immunohistochemistry. Scores ranging from 0 to 9 were obtained by multiplying the percentage of positive cells by the staining intensity (0–3). Immunohistochemical analysis revealed increased PGK1 and GRP78 expression in the cytoplasm of endometrial carcinoma cells compared with that in normal endometrial tissues. High PGK1 expression positively correlated with the FIGO stage (P < 0.001), histological grade (P = 0.002), and lymph node status (P < 0.001). High GRP78 expression positively correlated with the pathological type (P = 0.0125), FIGO stage (P < 0.001), and lymph node status (P < 0.001). In addition, PGK1 overexpression was positively correlated with GRP78 overexpression in endometrial carcinoma patients (P < 0.001), and the concurrent expression of both oncogenes in endometrial carcinoma patients correlated significantly with the lymph node status (P < 0.001) and FIGO stage (P < 0.001). Patients with high PGK1 and GRP78 expression levels had poorer overall survival rates than those with low expression levels of both proteins (P < 0.001). Our results suggested that the co-occurrence of PGK1 and GRP78 expression is potentially an unfavorable factor for endometrial carcinoma progression.

Liraglutide attenuates atherosclerosis via inhibiting ER-induced macrophage derived microvesicles production in T2DM rats

BackgroundWe investigated the effects of liraglutide on the formation and progression of atherosclerosis in type 2 diabetes mellitus (T2DM) rats.MethodsSprague–Dawley rats were divided into control group, diabetes group and liraglutide treated group. The T2DM rats model with atherosclerosis were induced by high fat diet followed small dosage streptozotocin injection. Body weight and blood glucose levels were monitored once a week for 3 months and then the rats were sacrificed.Peripheral blood and aorta tissues were collected for further biochemical and pathological estimation respectively. Moreover, immunohistochemistry staining was used to detect the infiltration of macrophages and cell apoptosis in tissue samples. The amount of microvesicles of atherosclerotic plaques was determined by ELISA. Western blot was applied to detect the protein expressions of CHOP, GRP78 and caspase-3 in tissue samples. The mRNA expressions of SREBP-1c and FAS were detected by RT-PCR.ResultsThe rat model of diabetic atherosclerosis was established successfully. Compared with the control group, glucose, triglycerides, total cholesterol, AST, ALT, BUN, fasting insulin and homeostatic model assessment insulin resistance levels in peripheral blood were significantly increased in the diabetes group. While, these indicators in the liraglutide group were significantly lower than that in the diabetes group. Moreover, the atherosclerotic plaques were observed in the rats of diabetes group but not remarkable in the liraglutide group. The ratio between aorta intima and media thickness was significantly greater in the diabetes group than that in the liraglutide group. Compared with the diabetes group, the infiltration and apoptosis of macrophages were milder in the liraglutide group. The expressions of CD68, caspase-3, CHOP and GRP78 in aorta tissue samples were significantly downregulated in the liraglutide group than that in the diabetes group. Furthermore, the microvesicles of aorta tissues in the liraglutide group were significantly decreased than that in the diabetes group. The mRNA expressions of SREBP-1c and FAS were lower in the liraglutide group than that in the diabetes group.ConclusionLiraglutide attenuates diabetic atherosclerosis by inhibition of ER stress and subsequent macrophage apoptosis and microvesicles production in T2DM rats.

Mechanism of Astragalus polysaccharides in attenuating insulin resistance in Rats with type 2 diabetes mellitus via the regulation of liver microRNA‑203a‑3p.

Insulin resistance (IR) is a common feature of type 2 diabetes mellitus (T2DM). Astragalus polysaccharides (APS) is a natural medicine that is used to treat T2DM. However, the mechanism by which APS regulates micro (mi)RNA in the treatment of IR has not been investigated. The purpose of the present study was to investigate differential miRNA expression between normal, T2DM model and APS treatment rats, as well as changes in miRNA and its downstream gene expression levels after APS treatment in T2DM Goto Kakizaki (GK) rats. Results suggested that miRNA (miR)-203a-3p expression level was significantly decreased in the liver of T2DM GK rats. Furthermore, it was identified that glucose-regulated protein (GRP)78 was the target gene of miR-203a-3p. GRP78 mRNA and protein expression levels of GRP78, CAAT-enhancer-binding protein homologous protein (CHOP), phosphorylated-c-Jun N-terminal kinase (pJNK)1, and caspase-12 were significantly increased in the liver of T2DM GK rats. Furthermore, miR-203a-3p was upregulated following APS treatment, and the protein expression levels of GRP78, CHOP, pJNK1 and caspase-12 were significantly decreased. In addition, miR-203a-3p overexpression in IR cells decreased the protein expression levels of these factors and anti-miR-203a-3p produced the opposite result. These findings provided evidence that miR-203a-3p may have a functional role in endoplasmic reticulum stress (ERS) signaling in the liver of T2DM GK rats. In addition, APS attenuated IR in T2DM, likely through upregulating or maintaining the miR-203a-3p expression levels, decreasing GRP78 mRNA and protein expression levels and regulating the protein expression of the ERS signaling pathway.

Steroids from Helleborus caucasicus reduce cancer cell viability inducing apoptosis and GRP78 down-regulation

Helleborus caucasicus (Ranunculaceae) is an endemic plant of the Caucasian flora, widely distributed in West Georgia. Biological activities for the extracts of some Helleborus species including H. caucasicus have been reported. In this work we found that butanolic extract of the underground parts of H. caucasicus and isolated compounds decreased cell viability in vitro on cancer cell line of lung origin (Calu-1) in a concentration-dependent manner, compared to the normal cell line. In particular, we identified that furostanol derivative (25S)-22α,25-epoxyfurost-5-ene-3β,11β,26-triol 26-O-β-d-glucopyranoside (5), 20-hydroxyecdysone (6), and 3β,5β,14β-trihydroxy-19-oxo-bufa-20,22-dienolide 3-O-α-l-rhamnopyranoside, known as deglucohellebrin (7) exerted a strong cytotoxic effect on the same cells and on other cancer cell lines (HepG2 and Caco-2) reducing the S-phase entry (compound 6) and inducing cell apoptosis associated with activation of caspase-3 (compound 7). Moreover we demonstrated that 6 and 7 significantly decreased protein expression of GRP78, a general ER-stress marker, suggesting pro-apoptotic functions. These findings indicated that selected compounds from H. caucasicus are potential interesting agents in anti-cancer therapy.

Prostaglandin E1 protects hepatocytes against endoplasmic reticulum stress-induced apoptosis via protein kinase A-dependent induction of glucose-regulated protein 78 expression.

AIMTo investigate the protective effect of prostaglandin E1 (PGE1) against endoplasmic reticulum (ER) stress-induced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODSThapsigargin (TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinoma-derived cell line HepG2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RT-PCR. Apoptotic index and cell viability of L02 cells and HepG2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay.RESULTSPretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and HepG2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein (CHOP), glucose-regulated protein (GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and mRNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phospho-eukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A (PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSIONOur study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathway-dependent induction of GRP78 expression.

Effects of HMGB1 inhibition on endoplasmic reticulum stress after myocardial ischemia/reperfusion injury in rats

To investigate the effect of high mobility group protein B1 (HMGB1) inhibition on endoplasmic reticulum stress (ERS) after myocardial ischemia/reperfusion (I/R) in rats. Forty male Sprague-Dawley (SD) rats were randomly divided into four groups (n = 10): sham operation group, I/R model group, Gene silencing (HMGB1-siRNA) group, and empty vector (Scrambled-siRNA) group. Coronary blood flow of the rats were ligated for 30 minutes, relaxed the ligament line for 2 hours, to establish I/R injury model; not ligation with the sham operation group. Each group was injected 1 mL phosphate buffer (PBS), HMGB1-siRNA mixture or Scrambled-siRNA mixture preoperative by tail vein 0 hour, 12 hours, and 24 hours before surgery. After 2 hours reperfusion, the levels of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-8) in the serum were detected by enzyme linked immunosorbent assay (ELISA); the expression of HMGB1 protein in myocardium was detected by immunohistochemistry; the protein and mRNA expressions of HMGB1, GRP78, CHOP and caspase-12 in myocardium were detected by Western Blot and real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR). Compared with sham operation group, the levels of serum inflammatory factor, HMGB1 positive cells, and the protein and mRNA expressions of GRP78, CHOP, caspase-12 were significantly increased in I/R model group. The levels of serum inflammatory factor in HMGB1-siRNA group were significantly lower than those in the I/R model group [TNF-α (ng/L): 783.4±203.4 vs. 963.9±214.1, IL-6 (ng/L): 358.8±94.8 vs. 452.3±103.7, IL-8 (ng/L): 180.5±73.6 vs. 347.3±90.3, all P < 0.05], HMGB1 positive cells, and the protein and mRNA expressions of GRP78, CHOP, caspase-12 in HMGB1-siRNA group were significantly lower than I/R model group (HMGB1 protein: 1.59±0.26 vs. 3.21±0.40, GRP78 protein: 2.59±0.28 vs. 4.21±0.42, CHOP protein: 2.01±0.23 vs. 3.21±0.43, caspase-12 protein: 1.48±0.22 vs. 3.01±0.48; HMGB1 mRNA: 2.35±0.26 vs. 4.67±0.45, GRP78 mRNA: 6.59±0.26 vs. 11.21±0.40, CHOP mRNA: 2.01±0.43 vs. 5.21±0.63, caspase-12 mRNA: 4.48±0.32 vs. 8.41±0.52, all P < 0.05). There was no significant difference between the Scrambled-siRNA group and the I/R model group. HMGB1 may be involved in the activation of ERS in myocardial I/R injury and increase the damage of myocardial cells.

Targeting oral cancer stemness and chemoresistance by isoliquiritigenin-mediated GRP78 regulation.

Cancer stem cells (CSCs) are cells that drive tumorigenesis, contributing to metastasis and cancer recurrence as well as resistance to chemotherapy of oral squamous cell carcinomas (OSCC). Therefore, approaches to target CSCs become the subject of intense research for cancer therapy. In this study, we demonstrated that isoliquiritigenin, a chalcone-type flavonoid isolated from licorice root, exhibited more toxicity in oral cancer stem cells (OSCC-CSCs) compared to normal cells. Treatment of isoliquiritigenin not only inhibited the self-renewal ability but also reduced the expression of CSC markers, including the ALDH1 and CD44. In addition, the capacities of OSCC-CSCs to invade, metastasize and grow into a colony were suppressed by isoliquiritigenin. Most importantly, we showed that isoliquiritigenin potentiated chemotherapy along with downregulated expression of an ABC transporter that is associated with drug resistance, ABCG2. Moreover, a combination of isoliquiritigenin and Cisplatin significantly repressed the invasion and colony formation abilities of OSCC-CSCs. Our results suggested that administration of isoliquiritigenin reduced the protein expression of mRNA and membrane GRP78, a critical mediator of tumor biology. Overexpression of GRP78 reversed the inhibitory effect of isoliquiritigenin on OSCC-CSCs. Furthermore, isoliquiritigenin retarded the tumor growth in nude mice bearing OSCC xenografts. Taken together, these findings showed that isoliquiritigenin is an effective natural compound that can serve as an adjunct to chemotherapy for OSCC.

Necrostatin-1 Mitigates Endoplasmic Reticulum Stress After Spinal Cord Injury

Necrostatin-1 (Nec-1) has been shown to inhibit necroptosis and convey a significant protective effect after spinal cord injury (SCI). This small molecule inhibitor may reduce tissue damage and restore neurological function by lessening mitochondrial injury after SCI and preserving energy homeostasis. However, the effects of Nec-1 on endoplasmic reticulum stress (ERS)-an important pathological consequence of SCI-are still not clear. The present study investigates the relationship between necroptosis and ERS in a rat model of SCI. Electron microscopy was employed to observe ultra-structural changes in the endoplasmic reticulum and mitochondria after lesioning. Real-time quantitative PCR was used to measure the mRNA levels of ERS-related pro-apoptotic molecules such as C/EBP homologous protein (CHOP), immunoglobulin-binding protein (BiP/GRP78) and X box-binding protein-1 (XBP-1). Western blot and immunofluorescence were conducted to analyze CHOP, GRP78 and XBP-1 protein expression after lesioning. Results demonstrated that applying Nec-1 in SCI reduces ultra-structural damage to the endoplasmic reticulum and mitochondria and inhibits expression of ERS-related genes and proteins after lesioning. Immunofluorescence also shows ERS-related proteins mainly expressed in the cytoplasm of nerve cells. Taken together, these results demonstrate that Nec-1 has protective effect on the endoplasmic reticulum and mitochondria and alleviates ERS after SCI.

Questiomycin A stimulates sorafenib-induced cell death via suppression of glucose-regulated protein 78

Hepatocellular carcinoma (HCC) is one of the most difficult cancers to treat owing to the lack of effective chemotherapeutic methods. Sorafenib, the first-line and only available treatment for HCC, extends patient overall survival by several months, with a response rate below 10%. Thus, the identification of an agent that enhances the anticancer effect of sorafenib is critical for the development of therapeutic options for HCC. Endoplasmic reticulum (ER) stress response is one of the methods of sorafenib-induced cell death. Here we report that questiomycin A suppresses expression of GRP78, a cell-protective ER chaperone protein. Analysis of the molecular mechanisms of questiomycin A revealed that this compound stimulated GRP78 protein degradation in an ER stress response-independent manner. Cotreatment with sorafenib and questiomycin A suppressed GRP78 protein expression, which is essential for the stimulation of sorafenib-induced cell death. Moreover, our in vivo study demonstrated that the coadministration of sorafenib and questiomycin A suppressed tumor formation in HCC-induced xenograft models. These results suggest that cotreatment with sorafenib and questiomycin A is a novel therapeutic strategy for HCC by enhancing sorafenib-dependent ER stress-induced cell death, and downregulation of GRP78 is a new target for the stimulation of the therapeutic effects of sorafenib in HCC.

Metformin induced apoptosis of papillary thyroid carcinoma BCPAP cells

Objective:To investigate the role of metformin in inducing apoptosis of papillary thyroid carcinoma BCPAP cells. Method:Using MTT methods to detect effects of metformin on cell proliferation of BCPAP in different concentrations (0 mmol/L, 1 mmol/L, 5 mmol/L, 10 mmol/L, 20 mmol/L) and time course (0 h, 4 h, 8 h, 16 h, 24 h, 48 h). The experiment was divided into four groups: Con, Met, Met+Sal and Met+DM, flow cytometry to detect the rate of apoptosis of BCPAP. Then detect the protein expressions of CHOP, GRP78 and Caspase-12 of 4 groups by Western blot. Result:Compared with the experimental control group, the percentage of cell proliferation index significantly decreased in metformin (0-20 mmol/L and 0-48 h) treatment group. Compared with the experimental control group, the percentage of apoptosis cells significantly increased in metformin treatment group. Compared with the control group, the protein expressions of GRP78, CHOP and Caspase-12 were significantly increased in Met group; While compared with the Met group, the protein expressions of GRP78, CHOP and Caspase-12 were significantly inhibited in the Met+Sal group. Conclusion:Metformin can induced availably BCPAP cell apoptosis by activating endoplasmic reticulum stress mechanism.

P 119 - ER stress related lipid accumulation and apoptotic cell death in nonalcoholic fatty liver diesease

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease that ranges from benign steatosis to nonalcoholic steatohepatitis (NASH) . The role of cholesterol alterations in NAFLD progression have been shown in various studies. Additionally, activation of ER stress, induces inflammation, insulin resistance and apoptotic cell death which results in NAFLD/NASH transition. UPR is mediated by the activation of three ER proteins: IRE1, PERK and ATF-6 . These proteins are inactivated by binding to the ER chaperone GRP78 under normal conditions. Following disassociation of GRP78, each of these sensor proteins (PERK, ATF6 and IRE1) use a unique mechanism to induce transcription factors. If the stress is too severe and excess the capacity of defense mechanisms, cells switch to apoptotic cell death pathways which is mainly driven by CHOP. The aim of this study is to identify the effect of ER stress on the molecular mechanisms of apoptosis in hypercholesterolemia induced NAFLD. In this direction, lipid accumulation and protein expressions of GRP78, GRP94, IRE1, PERK, Bax, CHOP, Caspase 3 has been investigated in the liver tissues of NAFLD rabbit model. Our model demostrated that high cholesterol diet increase ER stress, that triggers apoptosis in the progresion of NAFLD.

Oxidative and endoplasmic reticulum stress is impaired in leukocytes from metabolically unhealthy vs healthy obese individuals.

Oxidative stress and inflammation are related to obesity, but the influence of metabolic disturbances on these parameters and their relationship with endoplasmic reticulum (ER) stress is unknown. Therefore, this study was performed to evaluate whether metabolic profile influences ER and oxidative stress in an obese population with/without comorbidities. A total of 113 obese patients were enrolled in the study; 29 were metabolically healthy (MHO), 53 were metabolically abnormal (MAO) and 31 had type 2 diabetes (MADO). We assessed metabolic parameters, proinflammatory cytokines (TNFα and IL-6), mitochondrial and total reactive oxygen species (ROS) production, glutathione levels, antioxidant enzymes activity, total antioxidant status, mitochondrial membrane potential and ER stress marker expression levels (glucose-regulated protein (GRP78), spliced X-box binding protein 1 (XBP1), P-subunit 1 alpha (P-eIF2α) and activating transcription factor 6 (ATF6). The MAO and MADO groups showed higher blood pressure, atherogenic dyslipidemia, insulin resistance and inflammatory profile than that of MHO subjects. Total and mitochondrial ROS production was enhanced in MAO and MADO patients, and mitochondrial membrane potential and catalase activity differed significantly between the MADO and MHO groups. In addition, decreases in glutathione levels and superoxide dismutase activity were observed in the MADO vs MAO and MHO groups. GRP78 and CHOP protein and gene expression were higher in the MAO and MADO groups with respect to MHO subjects, and sXBP1 gene expression was associated with the presence of diabetes. Furthermore, MAO patients exhibited higher levels of ATF6 than their MHO counterparts. Waist circumference was positively correlated with ATF6 and GRP78, and A1c was positively correlated with P-Eif2α. Interestingly, CHOP was positively correlated with TNFα and total ROS production and GRP78 was negatively correlated with glutathione levels. Our findings support the hypothesis that both inflammation and oxidative stress are involved in the induction of ER stress signaling pathways in the leukocytes of metabolically unhealthy obese vs healthy obese subjects.

The Novel Mechanisms Concerning the Inhibitions of Palmitate-Induced Proinflammatory Factor Releases and Endogenous Cellular Stress with Astaxanthin on MIN6 β-Cells.

Astaxanthin, an antioxidant agent, can protect pancreatic β-cells of db/db mice from glucotoxicity and resolve chronic inflammation in adipose tissue. Nonetheless, the effects of astaxanthin on free-fatty-acid-induced inflammation and cellular stress in β-cells remain to be demonstrated. Meanwhile, palmitate enhances the secretion of pro-inflammatory adipokines monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF120). We therefore investigated the influence of astaxanthin on palmitate-stimulated MCP-1 and VEGF120 secretion in mouse insulinoma (MIN6) pancreatic β-cells. Furthermore, whether astaxanthin prevents cellular stress in MIN6 cells was also assessed. Pre-treatment with astaxanthin or with N-acetyl-cysteine (NAC) which is an antioxidant drug, significantly attenuated the palmitate-induced MCP-1 release through downregulation of phosphorylated c-Jun NH2-terminal protein kinase (JNK) pathways, and suppressed VEGF120 through the PI3K/Akt pathways relative to the cells stimulated with palmitate alone. In addition, palmitate significantly upregulated homologous protein (CHOP) and anti-glucose-regulated protein (GRP78), which are endoplasmic reticulum (ER) stress markers, in MIN6 cells. On the other hand, astaxanthin attenuated the increased CHOP content, but further up-regulated palmitate-stimulated GRP78 protein expression. By contrast, NAC had no effects on either CHOP or GRP78 enhancement induced by palmitate in MIN6 cells. In conclusion, astaxanthin diminishes the palmitate-stimulated increase in MCP-1 secretion via the downregulation of JNK pathways in MIN6 cells, and affects VEGF120 secretion through PI3K/Akt pathways. Moreover, astaxanthin can prevent not only oxidative stress caused endogenously by palmitate but also ER stress, which NAC fails to attenuate, via upregulation of GRP78, an ER chaperon.

Effects of vitamin D and resveratrol on metabolic associated markers in liver and adipose tissue from SAMP8 mice

SAMP8 mice exhibit multiple metabolic characteristics associated with age, and it is a suitable candidate for researching aging associated metabolic dysfunction. ObjectivesWe aimed to 1) explore how key metabolic markers will be altered in both liver and adipose tissue with aging in SAMP8 mice; and 2) how the combination of vitamin D (VD) with resveratrol (RSV) will affect aging associated metabolic impairment in liver and adipose tissue from SAMP8 mice. MethodsSAMP8 mice and their control SAMR1 mice were divided into 5 groups, i.e. SAMR1, SAMP8, SAMP8 mice supplemented with VD, RSV and VD combined with RSV group, respectively. At the end of the intervention, glucose and insulin tolerance, p-AMP-activated protein kinase (AMPK) and amyloid precursor protein (APP), and endoplasmic reticulum (ER) stress markers in liver and adipose tissue, adiponectin secretion, p-NF-κBp65 and TNF-α protein expression in adipose tissue were determined. ResultsCompared to SAMR1 control, SAMP8 mice demonstrate impaired glucose tolerance and reduction in circulating adiponectin level; in the liver, SAMP8 mice have reduction in p-Aktser473, elevation in PTP1B and APP, p-eIF2α, GRP78 and p-JNK protein expression. In epididymal (EPI) fat, SAMP8 mice also have elevated p-Aktser473 and PTP1B compared to SAMR1 mice. In both epididymal (EPI) and subcutaneous (SC) fat, there were elevated ER stress markers, reduced p-AMPK and elevated APP, as well as elevated p-NF-κBp65 and TNF-α protein expression from SAMP8 compared to SAMR1 mice. In liver, the combined intervention significantly restored p-Aktser473, p-eIF2α and p-JNK protein expression. In both EPI and SC fat, the combined intervention is effective for reducing p-NF-κB p65 and TNF-α in both fat depot, while only partially reduced ER stress markers in SC fat. As for adiponectin, their combination is unable to reverse reduction in adiponectin level. Adiponectin secretion in SC fat from VD, RSV and VDRSV group were also significantly reduced compared to SAMR1. ConclusionThe combined intervention might exert greater beneficial effects for reversing aging associated metabolic dysfunction in liver and adipose tissue from SAMP8 mice.

Metabolic syndrome enhances endoplasmic reticulum, oxidative stress and leukocyte–endothelium interactions in PCOS

ObjectivePolycystic ovary syndrome (PCOS) is associated with insulin resistance, which can lead to metabolic syndrome (MetS). Oxidative stress and leukocyte–endothelium interactions are related to PCOS. Our aim was to evaluate whether the presence of MetS in PCOS patients can influence endoplasmic reticulum (ER) and oxidative stress and leukocyte–endothelium interactions. Material and MethodsThis was a prospective controlled study conducted in an academic medical center. The study population consisted of 148 PCOS women (116 without/32 with MetS) and 112 control subjects (87 without / 25 with MetS). Metabolic parameters, reactive oxygen species (ROS) production, ER stress markers (GRP78, sXBP1, ATF6), leukocyte–endothelium interactions, adhesion molecules (VCAM-1, ICAM-1, E-Selectin), TNF-α and IL-6 were determined. ResultsTotal ROS, inflammatory parameters and adhesion molecules were enhanced in the presence of MetS (p<0.05), and the PCOS+MetS group showed higher levels of IL-6 and ICAM-1 than controls (p<0.05). Increased adhesion and leukocyte rolling flux were observed in PCOS and PCOS+MetS groups vs their respective controls (p<0.05). GRP78 protein expression was higher in the PCOS groups (p<0.05 vs controls) and sXBP1 was associated with the presence of MetS (p<0.05 vs controls without MetS). Furthermore, PCOS+MetS patients exhibited higher GRP78 and ATF6 levels than controls and PCOS patients without MetS (p<0.05). In PCOS women, HOMA-IR was positively correlated with ICAM-1 (r=0.501; p<0.01), ROS (r=0.604; p<0.01), rolling flux (r=0.455;p<0.05) and GRP78 (r=0.574; p<0.001). ConclusionOur findings support the hypothesis of an association between altered metabolic status, increased ROS production, ER stress and leukocyte–endothelium interactions in PCOS, all of which are related to vascular complications.

Effects of 4-phenylbutyrate on the ischemia-reperfusion injury of rat skin flaps by inhibiting endoplasmic reticulum stress

Objective To observe the effects of 4-phenylbutyrate (4-PBA) on the ischemia-reperfusion injury of rat skin flaps by inhibiting endoplasmic reticulum stress. Methods Seventy-five Wistar rats were used to create the abdominal island skin flaps. They were then randomly divided into three groups: control group, 4-PBA group and saline group (NC group). In NC and 4-PBA groups, a skin flap tissue sample was removed at the time the skin flap was created 0, 1, 6, 12 and 24 hours following ischemia reperfusion. In the control group, a skin flap tissue sample was removed at the time the skin flap was created 0, 1, 6, 12 and 24 hours following the surgical procedure. Pathological observation, detection of apoptosis by TUNEL method, RT-PCR detection of the expression level of GRP78 and CHOP mRNA, Western-blot and immunofluorescence staining for expression level of GRP78 and CHOP proteins were carried out. The correlation was analyzed. Results Histology showed that the tissue swelling degree, necrotic cells and inflammatory cells in NC group was more severe than 4-PBA group following 6 hours of ischemia and reperfusion. The number of TUNEL positive cells in 4-PBA group, compared with NC group, were reduced by 19.4% and 26.1%, respectively, following 6 and 24 hours of ischemia and reperfusion with significant difference (P<0.05). The levels of both mRNA and protein expression of GRP78 and CHOP were lower in 4-PBA group than NC group following 6, 12 and 24 hours of ischemia and reperfusion with significant difference (P<0.05). Conclusion 4-PBA reduces cell apoptosis in rat skin flaps following ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress-mediated apoptosis mechanism. It plays an important role in protecting rat skin flaps against ischemia reperfusion injury and apoptosis. Key words: Surgical flaps; Animal experimentation; Ischemia reperfusion injury; Endoplasmic reticulum stress; 4-phenylbutyrate

Screening and Characterization of Drugs That Protect Corneal Endothelial Cells Against Unfolded Protein Response and Oxidative Stress.

PurposeTo screen for and characterize compounds that protect corneal endothelial cells against unfolded protein response (UPR) and oxidative stress.MethodsBovine corneal endothelial cells (BCECs) were treated for 48 hours with 640 compounds from a Food and Drug Administration (FDA)-approved drug library and then challenged with thapsigargin or H2O2 to induce UPR or oxidative stress, respectively. Cell viability was measured using the CellTiter-Glo survival assay. Selected “hits” were subjected to further dose-response testing, and their ability to modulate expression of UPR and oxidative stress markers was assessed by RT-PCR, Western blot, and measurement of protein carbonyl and 8-hydroxydeoxyguanosine (8-OHdG) adducts in immortalized human corneal endothelial cells (iHCECs).ResultsForty-one drugs at 20 μM and 55 drugs at 100 μM increased survival of H2O2-challenged cells, and 8 drugs at 20 μM and 2 drugs at 100 μM increased survival of thapsigargin-challenged cells, compared with untreated control cells. Nicergoline, ergothioneine, nimesulide, oxotremorine, and mefenamic acid increased survival of both H2O2- and thapsigargin-challenged cells. Oxotremorine altered DNA damage inducible 3 (CHOP) gene expression, glucose-regulated protein 78 kDa (GRP78) and activating transcription factor 4 (ATF4) protein expression, and protein carbonyl and 8-OHdG levels. Mefenamic acid altered GRP78 protein expression and protein carbonyl and 8-OHdG levels.ConclusionsOxotremorine and mefenamic acid are potential survival factors for corneal endothelial cells under UPR and oxidative stress. The described assay can be further expanded to screen additional drugs for potential therapeutic effect in corneal endothelial diseases such as Fuchs' endothelial corneal dystrophy.