Upregulation Of Glucose-regulated Protein Research Articles

Pan-Inhibition of Protein Disulfide Isomerase Caused Cell Death through Disrupting Cellular Proteostasis in Pancreatic Ductal Adenocarcinoma Cells.

The protein disulfide isomerase (PDI) family is a group of thioredoxin endoplasmic reticulum (ER)-resident enzymes and molecular chaperones that play crucial roles in the correct folding of proteins. PDIs are upregulated in multiple cancer types and are considered a novel target for cancer therapy. In this study, we found that a potent pan-PDI inhibitor, E64FC26, significantly decreased the proliferation of pancreatic ductal adenocarcinoma (PDAC) cells. As expected, E64FC26 treatment increased ER stress and the unfolded protein response (UPR), as evidenced by upregulation of glucose-regulated protein, 78-kDa (GRP78), phosphorylated (p)-PKR-like ER kinase (PERK), and p-eukaryotic initiation factor 2α (eIF2α). Persistent ER stress was found to lead to apoptosis, ferroptosis, and autophagy, all of which are dependent on lysosomal functions. First, there was little cleaved caspase-3 in E64FC26-treated cells according to Western blotting, but a higher dose of E64FC26 was needed to induce caspase activity. Then, E64FC26-induced cell death could be reversed by adding the iron chelator, deferoxamine, and the reactive oxygen species scavengers, ferrostatin-1 and N-acetylcysteine. Furthermore, the autophagosome-specific marker, light chain 3B (LC3B)-II, increased, but the autolysosome marker, sequestosome 1 (SQSTM1)/p62, was not degraded in E64FC26-treated cells. Using the FUW mCherry-LC3 plasmid and acridine orange staining, we also discovered a lower number of acidic vesicles, such as autolysosomes and mature lysosomes, in E64FC26-treated cells. Finally, E64FC26 treatment increased the cathepsin L precursor (pre-CTSL) but decreased mature CTSL expression according to Western blotting, indicating a defective lysosome. These results suggested that the PDI inhibitor, E64FC26, might initially impede proper folding of proteins, and then induce ER stress and disrupt proteostasis, subsequently leading to lysosomal defects. Due to defective lysosomes, the extents of apoptosis and ferroptosis were limited, and fusion with autophagosomes was blocked in E64FC26-treated cells. Blockade of autolysosomal formation further led to the autophagic cell death of PDAC cells.

Protective Effects of Astragalus Polysaccharide on Sepsis-Induced Acute Kidney Injury.

Objective To explore the protective roles of Astragalus polysaccharide (APS) on acute renal injury (AKI) induced by sepsis. Methods Firstly, an animal model of sepsis-induced AKI was established by injecting lipopolysaccharide (LPS) into mice. The mice were pretreated with an intraperitoneal injection of 1, 3, and 5 mg/(kg·d) APS for 3 consecutive days. The severity of kidney injury was then scored by histopathological analysis, and the concentrations of serum urea nitrogen (BUN) and serum creatinine (SCr) and the levels of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) were determined as well. In in vitro experiments, lipopolysaccharide (LPS) was used to induce HK-2 cell injury to establish a sepsis-induced AKI cell model, and the cell counting kit-8 (CCK-8) method was performed to determine the cytotoxicity and appropriate experimental concentration of APS. Then, cells were divided into the control, LPS, and APS+LPS groups. Cell apoptosis and inflammation-related TNF-α, IL-1β, IL-6, and IL-8 were determined by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. The microscope was used to observe the morphological changes of cells, and the cell migration ability was measured by wound healing assay. RT-qPCR and Western blot assay were used to determine the mRNA and protein levels of apoptosis-related factors including caspase-3, caspase-9, Bax, and Bcl-2; endoplasmic reticulum stress- (ERS-) related biomarkers including C/EBP homologous protein (CHOP) and glucose-regulated protein78 (GRP78); and epithelial-mesenchymal transition- (EMT-) related biomarkers including E-cadherin, Snail, α-smooth muscle actin (α-SMΑ), and Vimentin. Results In vivo experiments in mice showed that APS can reverse LPS-induced kidney damage in a concentration-dependent manner (P < 0.05); the concentrations of BUN and Scr were increased (all P < 0.05); similarly, the levels of TNF-α and IL-1β were increased as well (all P < 0.05). In in vitro experiments, the results showed that LPS can significantly cause HK-2 cell damage and induce apoptosis, inflammation, ERS, and EMT. When APS concentration was in the range of 0-200 μg/mL, it had no cytotoxicity in HK-2 cells, and 100 μg/mL APS pretreatment could significantly mitigate the decrease of cell activity induced by LPS (P < 0.05). Compared with the LPS group, APS pretreatment could inhibit the expression of inflammatory factors including TNF-α, IL-1 β, IL-6, and IL-8 (all P < 0.05), reducing the number of apoptotic cells (P < 0.05), suppressing the expression of caspase-3, caspase-9, and Bax, but upregulating the expression levels of Bcl-2. In ERS, APS pretreatment inhibited LPS-induced upregulation of CHOP and GRP78. Moreover, in EMT, APS pretreatment could inhibit the morphological changes of cells, downregulate the migration, decrease the expression of EMT biomarkers, and inhibit the process of EMT. Conclusion APS could alleviate sepsis-induced AKI by regulating inflammation, apoptosis, ERS, and EMT.

TRPV4 contributes to ER stress: Relation to apoptosis in the MPP+-induced cell model of Parkinson's disease

AimsParkinson's disease (PD) is a multifactorial neurodegenerative disorder. Its molecular mechanism is still unclear. Endoplasmic reticulum (ER) stress has been highlighted in PD. Transient receptor potential vanilloid 4 (TRPV4) is a kind of nonselective calcium cation channel. A defined role for TRPV4 in PD has not been reported. The purpose of the present research was to investigate the molecular mechanisms by which TRPV4 regulates ER stress induced by the 1-methyl-4-phenylpyridinium ion (MPP+) in PC12 cells. Main methodsPC12 cells were pretreated with the TRPV4-specific antagonist HC067047 or transfected with TRPV4 siRNA followed by treatment with MPP+. Cell viability was measured by the CCK-8 Assay. The expression of TRPV4, sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2), glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94), C/EBP homologous protein (CHOP), procaspase-12, and tyrosine hydroxylase (TH) was detected by western blot and RT-PCR. Key findingsThe expression of TRPV4 was upregulated, while cell viability was decreased by MPP+, which was reversed by HC067047. The ER stress common molecular signature SERCA2 was depressed by MPP+. Moreover, MPP+ induced upregulation of GRP78, GRP94, CHOP, and decrease in procaspase-12 and TH. HC067047 and TRPV4 siRNA reversed MPP+-induced ER stress and restored TH production. SignificanceTRPV4 functions upstream of ER stress induced by MPP+ and holds promise as a prospective pharmacotherapy target for PD.

Nifedipine Upregulates ATF6-α, Caspases -12, -3, and -7 Implicating Lipotoxicity-Associated Renal ER Stress.

Nifedipine (NF) is reported to have many beneficial effects in antihypertensive therapy. Recently, we found that NF induced lipid accumulation in renal tubular cells. Palmitic acid-induced renal lipotoxicity was found to be partially mediated by endoplasmic reticular (ER) stress, while it can also be elicited by NF in kidney cells; we examined the induction of suspected pathways in both in vitro and in vivo models. NRK52E cells cultured in high-glucose medium were treated with NF (30 µM) for 24–48 h. ER stress-induced lipotoxicity was explored by staining with thioflavin T and Nile red, transmission electron microscopy, terminal uridine nick-end labeling, and Western blotting. ER stress was also investigated in rats with induced chronic kidney disease (CKD) fed NF for four weeks. NF induced the production of unfolded protein aggregates, resulting in ER stress, as evidenced by the upregulation of glucose-regulated protein, 78 kDa (GRP78), activating transcription factor 6α (ATF6α), C/EBP-homologous protein (CHOP), and caspases-12, -3, and -7. In vitro early apoptosis was more predominant than late apoptosis. Most importantly, ATF6α was confirmed to play a unique role in NF-induced ER stress in both models. CKD patients with hypertension should not undergo NF therapy. In cases where it is required, alleviation of ER stress should be considered to avoid further damaging the kidneys.

Prosurvival roles mediated by the PERK signaling pathway effectively prevent excessive endoplasmic reticulum stress-induced skeletal muscle loss during high-stress conditions of hibernation.

Stress conditions like hypoxia, ischemia, and ischemia/reperfusion can trigger excessive endoplasmic reticulum stress (ERS), which can lead to cell apoptosis-induced skeletal muscle atrophy in non-hibernators. However, although hibernators experience multiple stress conditions during hibernation, their skeletal muscles appear to be well protected. We hypothesize that hibernators effectively avoid cell apoptosis, at least partially, by controlling ERS level. Here, we focused on the potential occurrence of ERS and how hibernators cope with it during different hibernation states. Results indicated that the protein expression levels of glucose-regulated protein 78 (GRP78), phosphorylated PKR-like ER protein kinase, phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α), and activating transcription factor 4 were significantly increased during hibernation, but primarily recovered in posthibernation. In the torpor-arousal cycle, the expression levels of the above indicators were lower during inter-bout arousal (IBA) than that during late torpor (LT). However, there was no change in C/EBP homologous protein expression and no apoptosis in skeletal muscles during the different hibernation states. In conclusion, the upregulation of p-eIF2α and GRP78 were identified as two crucial mechanisms mediated by the PERK signaling pathway to alleviate elevated ERS. The downregulation of ERS during IBA may be a unique countermeasure for hibernating squirrels to prevent excessive ERS. Thus, these special anti-excessive ERS abilities of ground squirrels contribute to the prevention of skeletal muscle cell apoptosis during hibernation.

Glucose-Regulated Protein 78 Signaling Regulates Hypoxia-Induced Epithelial-Mesenchymal Transition in A549 Cells.

Objective: Metastasis and therapeutic resistance are the major determinants of lung cancer progression and high mortality. Epithelial–mesenchymal transition (EMT) plays a key role in the metastasis and therapeutic resistance. Highly expressed glucose-regulated protein 78 (GRP78) is a poor prognostic factor in lung cancer and possibly correlated with EMT. This study aims to examine whether the up-regulation of GRP78 is involved in EMT in lung adenocarcinoma and explore the underlying downstream molecular pathways.Study Design: EMT was assessed by analysis of cell morphology and expression of EMT protein markers in A549 cells under normoxia, hypoxia and silencing GRP78 conditions. The expression levels of Smad2/3, Src, and MAPK (p38, ERK, and JNK) proteins were examined by Western blot analysis under hypoxia and treatments with phosphorylation inhibitors.Results: Under hypoxic conditions, the EMT morphology significantly changed and the GRP78 expression was significantly up-regulated in A549 cells compared with those in normoxia control. The expression and phosphorylation levels of smad2/3, Src, p38, ERK, and JNK were also upregulated. When GRP78 was silenced, EMT was inhibited, and the levels of phospho-smad2/3, phospho-Src, phospho-p38, phospho-ERK, and phospho-JNK were suppressed. When the activation of Smad2/3, Src, p38, ERK, and JNK was inhibited, EMT was also inhibited. The inhibition effect on EMT by these phosphorylation inhibitors was found to be weaker than that of GRP78 knockdown.Conclusions: Hypoxia-induced EMT in A549 cells is regulated by GRP78 signaling pathways. GRP78 promotes EMT by activating Smad2/3 and Src/MAPK pathways. Hence, GRP78 might be a potential target for treatment of lung adenocarcinoma.

Fucosterol from an Edible Brown Alga Ecklonia stolonifera Prevents Soluble Amyloid Beta-Induced Cognitive Dysfunction in Aging Rats.

Fucosterol from edible brown seaweeds has various biological activities, including anti-inflammatory, anti-adipogenic, antiphotoaging, anti-acetylcholinesterase, and anti-beta-secretase 1 activities. However, little is known about its effects on soluble amyloid beta peptide (sAβ)-induced endoplasmic reticulum (ER) stress and cognitive impairment. Fucosterol was isolated from the edible brown seaweed Ecklonia stolonifera, and its neuroprotective effects were analyzed in primary hippocampal neurons and in aging rats. Fucosterol attenuated sAβ1-42-induced decrease in the viability of hippocampal neurons and downregulated sAβ1-42-induced increase in glucose-regulated protein 78 (GRP78) expression in hippocampal neurons via activation of tyrosine receptor kinase B-mediated ERK1/2 signaling. Fucosterol co-infusion attenuated sAβ1-42-induced cognitive impairment in aging rats via downregulation of GRP78 expression and upregulation of mature brain-derived neurotrophic factor expression in the dentate gyrus. Fucosterol might be beneficial for the management of cognitive dysfunction via suppression of aging-induced ER stress.

Glucose-regulated protein 78 in lipid rafts elevates vascular smooth muscle cell proliferation of spontaneously hypertensive rats by controlling platelet-derived growth factor receptor signaling.

The multifunctional glucose-regulated protein 78 (GRP78) is known to be differentially expressed in the lipid rafts of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) and normal Wistar-Kyoto (WKY) rats. However, its role in VSMCs from SHRs remains to be elucidated. This work was conducted to investigate the contribution made by GRP78 in VSMCs. GRP78 expression in VSMC lipid rafts decreased in WKY rats with age, but not in SHRs. Transfection with GRP78-siRNA attenuated not only platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation and aortic sprout outgrowth but also the phosphorylation of PDGF receptor (PDGFR)-β, Akt, and extracellular signal-regulated kinase (Erk) 1/2 in VSMCs in response to PDGF-BB. Moreover, GRP78 knockdown also reduced the PDGF-BB-induced dimerization of PDGFR-β and GRP78 in SHR VSMCs. The phosphorylation of GRP78 and PDGFR-β was elevated in VSMCs treated with PDGF-BB and was completely abolished by AG1296 (a PDGFR inhibitor). Moreover, the binding of PDGFR-β to GRP78 and the co-localization of GRP78 to PDGFR-β in VSMCs were stronger in SHRs than in WKY rat controls. This study demonstrates that the PDGF-BB-induced proliferation of SHR VSMCs is mediated by the expressional upregulation of GRP78 on VSMC lipid rafts in SHRs, probably via the regulation of PDGFR-β-GRP78 binding and their cross-activation. These observations indicate that GRP78 may play important roles in the pathological progression of SHR VSMCs.

Apelin‑13 attenuates ER stress‑associated apoptosis induced by MPP+ in SH‑SY5Y cells.

Apelin‑13, a neuropeptide that acts as a ligand for a putative receptor related to the angiotensin II type receptor, elicits neuroprotective effects in numerous neurological conditions, such as Huntington's disease and cerebral ischemia. Parkinson's disease (PD), one of the most prevalent neurodegenerative diseases, is caused by damage to neurons in the brain; however, the underlying mechanism remains unclear. The present study explored the effects of apelin‑13 on SH‑SY5Y human neuroblastoma cells treated with 1‑methyl‑4‑phenylpyridine (MPP+). Cell growth, cell viability, and apoptosis were measured by real‑time cell analysis, the Cell Counting Kit‑8 assay, and flow cytometry, respectively. In addition, the expression levels of extracellular signal‑regulated kinase (ERK) 1/2, p38 mitogen‑activated protein kinase (MAPK), glucose‑regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and cleaved caspase‑12 were assessed by western blotting. MPP+ treatment decreased the viability of SH‑SY5Y cells and increased their apoptosis; however, these changes were attenuated by pretreatment with apelin‑13. Treatment with MPP+ for 24h significantly increased the expression levels of phospho‑ERK1/2, phospho‑p38, GRP78, CHOP, and cleaved caspase‑12 in SH‑SY5Y cells. Pretreatment with apelin‑13 significantly attenuated the upregulation of GRP78, CHOP and cleaved caspase‑12 in MPP+‑treated SH‑SY5Y cells, and significantly enhanced the expression levels of phospho‑ERK1/2. Taken together, the present results support a model in which apelin‑13 inhibits MPP+‑induced apoptosis of SH‑SY5Y cells by decreasing the expression of GRP78, CHOP, and cleaved caspase‑12, and by increasing the expression of phospho‑ERK1/2. The present findings suggest that apelin‑13 may be useful for the treatment of PD.

GRP78 Promotes Hepatocellular Carcinoma proliferation by increasing FAT10 expression through the NF-κB pathway

Glucose-regulated protein 78(GRP78) and the ubiquitin-like protein FAT10 each promote proliferation in hepatocellular carcinoma(HCC). However, the relationship of GRP78 and FAT10 in HCC proliferation are still not known. In this study, we found that GRP78 and FAT10 were significantly overexpressed in HCC tissues compare with adjacent non-cancerous tissues, and a positive correlation was found between their expression and associated proliferation characteristics. High expression of GRP78 and FAT10 were positively correlated with tumor proliferation and poor prognosis in HCC. Moreover, GRP78 knockdown reduced FAT10 expression and suppressed HCC proliferation in vitro and in vivo. The effects of GRP78 knockdown were rescued by FAT10 up-regulation, whereas FAT10 knockdown reduced HCC proliferation enhanced by GRP78 up-regulation. Furthermore, GRP78 modulated FAT10 expression by regulating the NF-κB pathway, direct activation of the NF-κB pathway increased the expression of FAT10, a gene counteracting the tumor suppressor p53. Taken together, these results suggest that this newly identified GRP78–NF-κB–FAT10 axis will provide novel insight into the understanding of the regulatory mechanisms of proliferation in human HCC.

Chrysophanol inhibits endoplasmic reticulum stress in cerebral ischemia and reperfusion mice

Endoplasmic reticulum (ER) stress plays a critical role in mediating ischemia/reperfusion (I/R) damage in the brain. Our previous study showed that Chrysophanol (CHR) alleviated cerebral ischemic injury in mice and nuclear factor-κB (NF-κB) involved in its neuroprotective effect, but the precise mechanism remains not fully understood. The present study investigated the effect of CHR treatment on I/R-induced ER stress. Mice were subjected to middle cerebral artery occlusion (MCAO) for 45min and received either vehicle or CHR (0.1mg/kg) for 14 days after reperfusion. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) was used to detect apoptotic cells in penumbral tissue. The expression of ER stress-related factors including glucose-regulated protein 78 (GRP78), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), CCAAT-enhancer-binding protein homologous protein (CHOP), and caspase-12 as well as inhibitory κB-α (IκB-α), the inhibitor of NF-κB, was assessed. Our results demonstrated that CHR treatment reduced MCAO-induced upregulation of GRP78, p-eIF2α, CHOP, and caspase-12 in the ischemic brain. Moreover, the TUNEL-positive neuronal cells, which were colocalized with CHOP and caspase-12, decreased in response to CHR treatment, indicating that CHR protects against I/R injury by inhibiting ER stress-associated neuronal apoptosis. In addition, CHR reversed the decrease in IκB-α level induced by MCAO, which was attributed at least in part to the attenuation of translational inhibition induced by eIF2α phosphorylation, indicating that CHR exerts anti-inflammatory effects following I/R by inhibiting ER stress response. These results suggest that attenuation of ER stress may be involved in the mechanisms of neuroprotective effects of CHR.

Glucose-regulated protein 78 demonstrates antiviral effects but is more suitable for hepatocellular carcinoma prevention in hepatitis B

BackgroundHepatitis B virus (HBV) is the leading cause of liver cirrhosis and hepatocellular carcinoma in Asia and Africa. Existing antivirals cannot cure HBV or eliminate risk of hepatocellular carcinoma. Glucose-regulated protein 78 (GRP78) can inhibit HBV replication, but promote virion secretion and hepatocellular cancer cell invasion. For these reasons, the overall effect of GRP78 on HBV production and whether to utilize the HBV replication-inhibitory effect of GRP78 up-regulation or the hepatocellular cancer cell invasion-inhibitory effect of its down-regulation were further investigated in order to improve the efficacy of current antiviral therapy.MethodsGRP78 regulations in HepG2.2.15 cells were conducted by transfections of expressing vector and small interfering RNA, respectively. The changes in HBV replication, hepatitis B e antigen (HBeAg) synthesis and hepatoma cell motility were monitored.ResultsGRP78 overall decreased HBV production due to its HBV replication-inhibitory effect time-dependently overwhelming virion secretion-promoting effect in HepG2.2.15 cells. Unlike the parental cells (HepG2), HepG2.2.15 cells demonstrated decreased expressions of the major genes in the interferon-β1-dependent pathway. Moreover, the expressions of these genes were not affected by GRP78 regulations. However, GRP78 was found to inhibit HBeAg secretion and to increase the retro-transportation of capsid assembly-interfering HBeAg precursor from the endoplasmic reticulum into the cytosol where new viral nucleocapsids formed. Furthermore, GRP78 overexpression promoted wound healing process (the motility) of HepG2.2.15 cells. In contrast, GRP78 knockdown enhanced HBV replication and HBeAg secretion, but they were abolished by entecavir and furin inhibitor, respectively.ConclusionsGRP78 mainly demonstrates anti-HBV effects, reducing HBV production and HBeAg secretion. With due regard to the hepatocellular cancer invasion risk of the overexpression and the rectifiability of the unpleasant effects of the knockdown, GRP78 down-regulation may be more suitable to serve as an additive strategy to cover the hepatocellular cancer prevention shortage of current antiviral therapy in the future.

Negative regulation of ERK1/2 by PI3K is required for the protective effects of Pyropia yezoensis peptide against perfluorooctane sulfonate-induced endoplasmic reticulum stress.

Perfluorooctane sulfonate (PFOS) is a stable fluorosurfactant, which causes adverse effects in various organisms. The present study was designed to investigate the effects of Pyropia yezoensis peptide (PYP), a peptide comprised of 11 residues (ALEGGKSSGGG), on PFOS-induced endoplasmic reticulum (ER) stress in Chang cells. PFOS exposure (400 µM) for 24 h significantly decreased cell viability, which was upregulated by 250–1,000 pg/ml PYP treatment. Exposure to PFOS also significantly increased expression of the ER stress response protein, glucose-regulated protein 78 (GRP78), and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). These elevations were significantly decreased by PYP (250 pg/ml), and, in particular, the PFOS-induced GRP78 upregulation was decreased following treatment with 10 µM SL327, an ERK-kinase inhibitor. However, PYP-induced decreases in GRP78 expression and ERK1/2 phosphorylation were upregulated following treatment with LY294002 (20 µM), a phosphatidylinositol-3 kinase (PI3K) inhibitor. PFOS-induced apoptosis was also significantly attenuated by PYP (250 pg/ml) treatment, and the PYP-induced reduction in apoptosis was abolished by inhibition of PI3K. These findings indicate that negative regulation of ERK1/2 by PI3K is essential for the protective effects of PYP against PFOS-induced cell death, suggesting that PYP may be a candidate for therapeutic use.

Renin-angiotensin system activation accelerates atherosclerosis in experimental renal failure by promoting endoplasmic reticulum stress-related inflammation.

In this study, we investigated the association between the renin-angiotensin system (RAS), endoplasmic reticulum (ER) stress and atherosclerosis (AS) in uremic apolipo-protein E knockout (apoE−/−) mice. Mild uremia was induced by a 5/6 nephrectomy (5/6 Nx) in 10-week-old apoE−/− mice. Four weeks after nephrectomy, the mice received losartan or no treatment for 16 weeks. Sham-operated mice served as the controls. We found that uremia accelerated AS at the aortic root. The activation of ER stress and the significant upregulation of pro-inflammatory cytokines and chemokines were observed in the uremic mice. Phosphorylated inositol-requiring 1α (p-IRE1α), an ER stress marker protein, was mainly expressed in macrophages in the atherosclerotic lesions. Treatment with losartan significantly attenuated aortic AS, inhibited ER stress and reduced aortic inflammation. In in vitro experiments, angiotensin II (Ang II) increased the levels of the common ER stress maker, glucose-regulated protein 78 (GRP78) and the phosphorylation of IRE1α in RAW264.7 macrophages. Treatment with losartan inhibited the activation of ER stress and the upregulation of GRP78, and enhanced the expression of nuclear factor-κB (NF-κB) inhibitor (IκB) in Ang II-stimulated RAW264.7 macrophages. IRE1α-siRNA suppressed inflammation and downregulated IκB expression and IκB kinase (IKK) phosphorylation, which inhibited IκB degradation and NF-κB p65 nuclear translocation in Ang II-treated RAW264.7 macrophages. These findings suggest that RAS activation accelerates AS by promoting ER stress-related inflammation in uremic mice.

Upregulation of Glucose-Regulated Protein 78 in Metastatic Cancer Cells Is Necessary for Lung Metastasis Progression

Metastasis is the cause of more than 90% of all cancer deaths. Despite this fact, most anticancer therapeutics currently in clinical use have limited efficacy in treating established metastases. Here, we identify the endoplasmic reticulum chaperone protein, glucose-regulated protein 78 (GRP78), as a metastatic dependency in several highly metastatic cancer cell models. We find that GRP78 is consistently upregulated when highly metastatic cancer cells colonize the lung microenvironment and that mitigation of GRP78 upregulation via short hairpin RNA or treatment with the small molecule IT-139, which is currently under clinical investigation for the treatment of primary tumors, inhibits metastatic growth in the lung microenvironment. Inhibition of GRP78 upregulation and an associated reduction in metastatic potential have been shown in four highly metastatic cell line models: three human osteosarcomas and one murine mammary adenocarcinoma. Lastly, we show that downmodulation of GRP78 in highly metastatic cancer cells significantly increases median survival times in our in vivo animal model of experimental metastasis. Collectively, our data indicate that GRP78 is an attractive target for the development of antimetastatic therapies.

V8 induces apoptosis and the endoplasmic reticulum stress response in human multiple myeloma RPMI 8226 cells via the PERK-eIF2α-ATF4 signaling pathway.

Multiple myeloma (MM) is a fatal hematological cancer characterized by clonal plasma cell proliferation in the bone marrow. MM has an increasing global incidence and a poor prognosis. There are limited treatment options available for MM, and this is further compounded by the development of drug resistance. The present study demonstrated that 7-{4-[Bis-(2-hydroxyethyl)-amino]-butoxy}-5-hydroxy-8-methoxy-2-phenylchromen-4-one (V8), a novel synthetic flavonoid, induced apoptosis in human MM RPMI 8226 cells in a dose- and time-dependent manner, using cell viability assays and flow cytometry. Subsequently, V8-induced apoptosis in RPMI 8226 cells was revealed to occur via mitochondria-mediated pathways. The activity of caspase-3, -8 and -9, and the mRNA level of B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large were greatly increased, while the expression of Bcl-2-like protein 4 and BH3 interacting domain death agonist was significantly decreased in RPMI 8226 cells following V8 treatment, as observed using quantitative polymerase chain reaction (qPCR). In addition, western blotting revealed that the release of mitochondrial cytochrome c into the cytosol was promoted by V8. Furthermore, a clear alteration in endoplasmic reticulum (ER) stress was observed in cells treated with V8; upregulation of glucose-regulated protein (GRP) 78, GRP94, C/EBP homologous protein, cleavage of caspase-12, phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α) and activating transcription factor 4 (ATF4) was observed with qPCR and western blotting, suggesting that V8-induced apoptosis is involved in the ER stress response. Overall, the present results demonstrated that V8 induced apoptosis in human MM RPMI 8226 cells via the PERK-eIF2α-ATF4 ER stress response pathway, which may provide novel directions for exploiting this compound as a potential anti-neoplastic drug for MM therapy.

Polymorphisms of glucose-regulated protein 78 and clinical relevance of neuroblastoma: Risk and prognosis.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Glucose/regulated protein 78 (GRP78) is a stress-associated protein. It has been reported that overexpression of GRP78 occurs in various human cancers, and GRP78 polymorphisms have effect on the expression of GRP78 with possible function of predicting clinical outcome. Upregulation of GRP78 has been detected in NB. However, little is known about the relationship of GRP78 polymorphisms and the susceptibility of NB. To investigate whether GRP78 polymorphisms were associated with the risk and clinical characteristics of NB. Two GRP78 polymorphisms rs391957 (C>T) and rs430397 (G>A) were detected in 105 NB cases and 145 healthy controls using the polymerase chain reaction fragment length polymorphism technique. Compared with the CC genotype, carriers of CT and TT genotypes of rs391957 polymorphism had higher risks of NB. In NB cases, the variant T allele was significantly associated with tumor International Neuroblastoma Staging System stage (P = 0.027), but not with MYCN amplification (P = 0.056). Compared with the GG genotype, carriers of GA and AA genotypes of rs430397 polymorphism had higher risks of NB. The rs430397 polymorphism was not associated with the clinicopathological characteristics of NB. These data provide the first evidence that GRP78 rs391957 and rs430397 polymorphisms could serve as the markers to predict the risk and poor prognosis of NB.

GRP78 upregulation-induced increase in cisplatin sensitivity of SPCA1 lung cancer cells.

Glucose regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone, plays a critical role in chemotherapy resistance in a variety of cancers. In this study, we investigated the up-regulation of GRP78 induced by A23187 and its association with the chemotherapeutical sensibility to cisplatin in human lung cancer cell line SPCA1. SPCA1 cells were pretreated with A23187 at different concentrations. The expression of GRP78 at the mRNA level was analyzed by RT-PCR; the expression of GRP78 at the protein level was determined by Western blotting and immunofluorescence assay. Cell survival was determined by MTT assay. Cell apoptosis was analyzed by flow cytometry. The expression of GRP78 at both the mRNA and protein levels was obviously induced by A23187 in SPCA1 cells, with an elevation of GRP78 by 2.1-fold at the mRNA level and by 3.8-fold at the protein level compared to the control. There was a dose-dependent response. Survival curve analysis demonstrated that A23187 induction caused a significant reduction of survival for the cells subjected to cisplatin treatment (P < 0.05). After treatment by cisplatin, the percentage of apoptotic cells in the A23187 pretreated group increased about three fold compared with the control group ((27.53 ± 4.32)% vs. (9.25 ± 3.64)%, P < 0.05). A23187 treatment was fairly effective for the induction of GRP78 in SPCA1 cells at both the mRNA and protein levels. To a certain extent, GRP78 up-regulation by A23187 was associated with the enhancement of drug sensitivity to cisplatin in human lung cancer cell line SPCA1.

RU486 Induces Pro-Apoptotic Endoplasmic Reticulum Stress Through the Induction of CHOP Expression by Enhancing C/EBPδ Expression in Human Renal Carcinoma Caki Cells.

RU486 (Mifepristone) is known as an antagonist of the progesterone receptor and glucocorticoid receptor. Here, we investigated the mechanism underlying anti-tumor activity of RU486 in renal carcinoma Caki cells. Treatment of Caki cells with RU486 was found to induce several signature ER stress markers; including ER stress-specific XBP1 splicing, and the up-regulation of glucose-regulated protein (GRP)-78 and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. RU486-induced expression of CHOP involves the putative C/EBPδ site within the CHOP promoter region. Using a combination of C/EBPδ cDNA transfection, the luciferase assay with a mutated C/EBPδ binding site and siRNA-mediated C/EBPδ knockdown, we found that the C/EBPδ site is required for RU486-mediated activation of the CHOP promoter. In addition, RU486-induced CHOP expression is down-regulated by inhibition of the p38 MAPK and JNK signaling pathways at the post-translational levels. RU486 dose-dependently induced apoptotic cell death in renal carcinoma cells. Suppression of CHOP expression by CHOP siRNA attenuated RU486-induced apoptosis. Taken together, RU486 induces pro-apoptotic ER stress through the induction of CHOP expression.

Oxygen-Glucose Deprivation (OGD) Modulates the Unfolded Protein Response (UPR) and Inflicts Autophagy in a PC12 Hypoxia Cell Line Model.

Hypoxia is the lack of sufficient oxygenation of tissue, imposing severe stress upon cells. It is a major feature of many pathological conditions such as stroke, traumatic brain injury, cerebral hemorrhage, perinatal asphyxia and can lead to cell death due to energy depletion and increased free radical generation. The present study investigates the effect of hypoxia on the unfolded protein response of the cell (UPR), utilizing a 16-h oxygen-glucose deprivation protocol (OGD) in a PC12 cell line model. Expression of glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94), key players of the UPR, was studied along with the expression of glucose-regulated protein 75 (GRP75), heat shock cognate 70 (HSC70), and glyceraldehyde 3-phosphate dehydrogenase, all with respect to the cell death mechanism(s). Cells subjected to OGD displayed upregulation of GRP78 and GRP94 and concurrent downregulation of GRP75. These findings were accompanied with minimal apoptotic cell death and induction of autophagy. The above observation warrants further investigation to elucidate whether autophagy acts as a pro-survival mechanism that upon severe and prolonged hypoxia acts as a concerted cell response leading to cell death. In our OGD model, hypoxia modulates UPR and induces autophagy.