Upregulation Of GRP78 Research Articles

Aβ1-42 induces cell damage via RAGE-dependent endoplasmic reticulum stress in bEnd.3 cells

Blood-brain barrier (BBB) breakdown has been determined to play a critical role in the pathogenesis of Alzheimer's disease (AD). However, the underlying mechanisms of BBB disruption in AD remain unclear. Our previous study suggested that the receptor for advanced glycation end-products (RAGE) functioned as a signal transduction receptor in Aβ1–42-induced damage in endothelial cells. In our present study, we revealed that RAGE-mediated endoplasmic reticulum stress (ERS) is essential for Aβ-induced endothelial cell damage. Here, we found that Aβ1–42 activated ERS by upregulation of Grp78, xbp-1 and CHOP in endothelial cells and that Aβ1–42-resulted lesions, including the upregulations of caspase-12 and caspase-3, the augment of bax/bcl-2 ratio, and the downregulations of ZO-1 and Occludin in bEnd.3 cells, were ameliorated by the pretreatment of salubrinal, an ERS inhibitor. Furthermore, the expressions of Grp78, xbp-1 and CHOP induced by Aβ1–42 were blocked by transfection of RAGE small interfering RNA (siRNA), which indicated that Aβ1–42 activated ERS in a RAGE-dependent manner. Additionally, bEnd.3 cells transfected with RAGE siRNA showed lower expressions of caspase-12 and caspase-3, decreased bax/bcl-2 ratio, and higher expressions of ZO-1 and Occludin following Aβ1-42 treatment, comparing to control cells. In conclusion, our data demonstrated that Aβ1–42 induced endothelial cells damage via activation of ERS in a RAGE-dependent manner.

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.

MiR-181a Inhibits Cervical Cancer Development via Downregulating GRP78.

Cervical cancer is among the most common cancers inflicting women worldwide. Understanding the pathological mechanisms of cervical cancer development is critical for identifying novel targets for cervical cancer treatment. MicroRNAs (miRs) have various roles in regulating cancer development. In this study, we investigated the potential role of miR-181a and its target in regulating cervical cancer development and chemotherapy resistance. The expression of miR-181a was evaluated and modulated in several human cervical cancer cell lines. The role of miR-181a in regulating cervical cancer growth and chemotherapy sensitivity was investigated in cell culture models and mouse tumor xenograft models. The target of miR-181a and its function were identified in cervical cancer models. We found a distinct expression profile for miR-181a in cervical cancer cell lines. Low expression of miR-181a was closely related to cervical cancer growth and oxaliplatin resistance. HSPA5/GRP78 was identified as a target of miR-181a in cervical cancer cells. Upregulation of GRP78 led to a high cell proliferation rate and oxaliplatin resistance in cervical cancer models. In a retrospective cervical cancer cohort, high GRP78 expression was correlated with poor survival. miR-181a suppressed cervical cancer development via downregulating GRP78. High expression of GRP78 is a tumor-promoting factor in cervical cancer and is thus a potential target for novel treatment.

GRP78 Participates in PCA3-regulated Prostate Cancer Progression.

The human prostate cancer antigen 3 (PCA3) is a long non-coding RNA (lncRNA) commonly used as a diagnostic marker for prostate cancer (PCa). Herein we investigated the cellular function of PCA3 in PCa and its potential mechanism. PCA3 was overexpressed in a PC3 cell line (PC3PCA+) and cell proliferation, migration, invasion and apoptosis were compared to those of control cells (PC3NC). Differentially expressed proteins were identified by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. Overexpression of PCA3 significantly increased cell proliferation rate, migration and invasion, while inhibited apoptosis in PC3 cells. Three proteins were found down-regulated and 7 proteins up-regulated in PC3PCA+ cells compared to PC3NC cells, including GRP78. Higher GRP78 was also found in PCa clinical specimens. This study confirmed that in PCa, PCA3 plays a pro-cancer role through promoting cell proliferation, migration and invasion while inhibiting cell apoptosis. This process might involve the up-regulation of GRP78.

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.

Ibutilide treatment protects against ER stress induced apoptosis by regulating calumenin expression in tunicamycin treated cardiomyocytes.

BackgroundIbutilide, a class III antiarrhythmic agent has been shown to be cardioprotective in treating atrial fibrillation, promoting cardioconversion and recently this agent has been shown to protect against ER stress induced apoptosis in cardiomyocytes. In this study we begin to identify the mechanism by which ibutilide exerts its cardioprotection in tunicamycin treated cardiomyocytes. We examined ER stress markers including calumenin; a calcium binding ER chaperone protein that has recently been linked to ER stress in cardiomyocytes, in our treated cells.MethodsTo assess the effect of ibutilide we used the well characterized in vitro model of ER stress induced apoptosis in rat neonatal cardiomyocytes (RNC). RNC were treated with tunicamycin and the degree of ER stress was assessed by quantifying mRNA and protein levels of GRP78, GRP94 and calumenin, and examined the extent of apoptosis by assessing the protein levels of caspase-3/9/12, CHOP, ATF6, p-PERK, spliced XBP-1, the ratio of Bax/Bcl-2 and the percentage of deoxynucleotidyl-transferase- mediated dUTP nick end labeling (TUNEL) positive cells.ResultsWe demonstrate ibutilide attenuated the up-regulation of ER stress markers GRP78 and GRP94 and rescued the decline in calumenin mRNA and protein levels in tunicamycin treated cardiomyocytes. The up-regulation of apoptotic markers caspase-3, CHOP, ATF6, p-PERK, spliced XBP-1, the ratio of Bax/Bcl-2 and the percentage of TUNEL positive cells were also attenuated after ibutilide treatment while the protein levels of Caspase-9 and Caspase-12 were unaffected.ConclusionsThis study suggests another cardioprotective effect of the antiarrhythmic agent ibutilide whereby pretreatment leads to the attenuation of ER stress induced apoptosis by regulating calumenin expression. This study provides further evidence for the role of calumenin in the cardiomyocyte ER stress response.

Structure-activity relationship of piperine and its synthetic amide analogs for therapeutic potential to prevent experimentally induced ER stress in vitro.

Endoplasmic reticulum (ER) is the key organelle involved in protein folding and maturation. Emerging studies implicate the role of ER stress in the development of chronic kidney disease. Thus, there is an urgent need for compounds that could ameliorate ER stress and prevent CKD. Piperine and its analogs have been reported to exhibit multiple pharmacological activities; however, their efficacy against ER stress in kidney cells has not been studied yet. Hence, the goal of this study was to synthesize amide-substituted piperine analogs and screen them for pharmacological activity to relieve ER stress using an in vitro model of tunicamycin-induced ER stress using normal rat kidney (NRK-52E) cells. Five amide-substituted piperine analogs were synthesized and their chemical structures were elucidated by pertinent spectroscopic techniques. An in vitro model of ER stress was developed using tunicamycin, and the compounds of interest were screened for their effect on cell viability, and the expression of ER chaperone GRP78, the pro-apoptotic ER stress marker CHOP, and apoptotic caspases 3 and 12 (via western blotting). Our findings indicate that exposure to tunicamycin (0.5μg/mL) for 2h induces the expression of GRP78 and CHOP, and apoptotic markers (caspase-3 and caspase-12) and causes a significant reduction in renal cell viability. Pre-treatment of cells with piperine and its cyclohexylamino analog decreased the tunicamycin-induced upregulation of GRP78 and CHOP and cell death. Taken together, our findings demonstrate that piperine and its analogs differentially regulate ER stress, and thus represent potential therapeutic agents to treat ER stress-related renal disorders. Graphical Abstract Piperine (PIP) reduces the expression of ER stress markers (GRP78 and CHOP) induced by pathologic stimuli and consequently decreases the activation of apoptotic caspase-12 and caspase-3; all of which contributes to its chemical chaperone and cytoprotective properties to protect renal cells against ER stress and ER stress-induced cell death, and would ultimately prevent the development of chronic kidney disease.

MiR-199a-5p and miR-495 target GRP78 within UPR pathway of lung cancer

IntroductionNon-small cell lung cancer (NSCLC) is the most common subtype of lung cancer. One of the signal transduction pathways related to NSCLC is Unfolded Protein Response (UPR), which is mainly regulated by GRP78 (HSPA5, Gene ID: 3309). The aim of this study was to employ bioinformatics tools to predict microRNAs (miRNAs) affecting GRP78 expression, experimentally validate interaction of these miRNAs with GRP78 and also evaluating the expression correlation of GRP78 and its predicted miRNAs in clinical samples. Materials and methodsVarious software were used to predict miRNAs that simultaneously target all upstream and downstream components of GRP78 in the UPR, as well as the main components of PI3K/AKT, MAPK, ErbB and calcium pathways. For experimental analysis, 36 pairs of Formalin-Fixed Paraffin-Embedded (FFPE) lung tumor and non-tumor tissue samples were obtained. Additionally, A549 and QU-DB lung cancer cell lines were used for expression determination of GRP78 and its predicted targeting miRNAs. We also employed a luciferase assay to evaluate interactions between candidate miRNAs with the 3′-UTR of GRP78. Resultshsa-miR-495 and hsa-miR-199-5p were chosen based on several criteria including thermodynamic binding features of miRNAs to the target transcripts, number of recognition sites, and conservation of binding sites within the 3′-UTR of GRP78. RT-qPCR data revealed a significant up-regulation of GRP78 (3.87 times, P=0.002) and down-regulation of miR-199a-5p (0.13 times, P=0.0001) and miR-495 (0.085 times, P=0.0001) in tumor samples. Luciferase assay confirmed an interaction of hsa-miR-199a-5p and hsa-miR-495 with the 3′-UTR of GRP78 transcript. In addition, over-expression and competitive inhibition of the aforementioned miRNAs, significantly altered the expression of GRP78 and spliced XBP1 level. ConclusionOur data revealed a significant up-regulation of GRP78 and a concomitant down-regulation of miR-495 and miR-199a-5p in NSCLC. Accordingly, our data suggest a causative role for miR-199-5p and miR-495 in tumorgenesis of lung and probably other cancer types.

Calcium release induced by 2-pyridinecarboxaldehyde thiosemicarbazone and its copper complex contributes to tumor cell death.

Thiosemicarbazones display significant antitumor activity and their copper complexes also exhibit enhanced biological activities in most situations, but the underlying mechanism is poorly understood. Therefore, investigation of the mechanism involved in the change upon chelation is required to extend our understanding of the effects of thiosemicarbazones. In the present study, the inhibitory effect of 2-pyridinecarboxaldehyde thiosemicarbazone (PCT) and its copper complex (PCT-Cu) on cell proliferation was investigated. The copper chelate exhibited a 3- to 10-fold increase in antitumor activity (with an IC50 <5µM). The results showed that both PCT and PCT-Cu induced reactive oxygen species (ROS) generation invitro and invivo, caused cellular DNA fragmentation, depolarization of the mitochondrial membrane and cell cycle arrest. Western blotting showed that both PCT and PCT-Cu induced apoptosis. Upregulation of GRP78 in HepG2 cells following treatment with the agents indicated that endoplasmic reticulum (ER) stress occurred. Furthermore calcium release was revealed in this study, suggesting that PCT and PCT-Cu disturbed calcium homeostasis. It was noted that PCT-Cu sensitized thapsigargin‑stimulated calcium release from the ER, which was correlated with the ROS level they induced, implying that the antitumor activity of PCT and PCT-Cu partly stemmed from calcium mobilization, a situation that was reported in few studies. Our findings may significantly contribute to the understanding of the anti‑proliferative effect of the derivatives of thiosemicarbazones along with their antitumor mechanism.

Proteomic Profiling Reveals the Induction of UPR in Addition to DNA Damage Response in HeLa Cells Treated With the Thiazolo[5,4-b]Quinoline Derivative D3ClP.

9-[(3-chloro)phenylamine]-2-[3-(diethylamine)propylamine]thiazolo[5,4-b]quinolone (D3ClP) is a bioisostere of N-(4-(acridin-9-ylamino)-3-methoxyphenyl)methanesulfonamide (m-AMSA) a DNA topoisomerase II inhibitor with proven cytotoxic activity and known to induce DNA damage and apoptotic cell death in K562 cells. However, recent evidence is not consistent with DNA topoisomerase II (DNA TOP2) as the primary target of D3ClP, in contrast to m-AMSA. We provide evidence of histone γH2AX phosphorylation at Ser135 in HeLa cells treated with D3ClP, a marker of DNA double strand repair through Mre11-Rad50-Nbs1 (MRN) pathway. Using two-dimensional gel electrophoresis and mass spectrometry, the upregulation of the protein GRP78, the cleavage of Cytokeratin 18, and the downregulation of prothymosine, calumenin, and the α chain of the nascent polypeptide associated complex were observed in HeLa cells treated with D3ClP. An increase in GRP78 has been related with the onset and progression of the unfolded protein response (UPR), a process aimed to reduce endoplasmic reticulum (ER) stress and protein misfolding. The IRE1-α dependent splicing of mRNA encoding X-box binding protein 1 was detected. Microtubule-associated Proteins 1A/1B, Light Chain 3-II (LC3b-II) accumulation was observed, and suggest some involvement of autophagy. The production of the pro-apoptotic protein DNA-damage-inducible protein 153 (GADD-153) was also detected. These results, are consistent with the induction of the UPR and the DNA-Damage Response in D3ClP-treated HeLa cells, and are also consistent with a concurrent apoptotic cell death. J. Cell. Biochem. 118: 1164-1173, 2017. © 2016 Wiley Periodicals, Inc.

P4HB promotes HCC tumorigenesis through downregulation of GRP78 and subsequent upregulation of epithelial-to-mesenchymal transition.

P4HB and GRP78 are molecular chaperones involved in cellular response to ER stress. They have been linked to cancer progression; however, their roles in hepatocellular carcinoma (HCC) are largely unclear. In this study, we found that P4HB is overexpressed in human HCC tissues and cell lines. Higher tumoral P4HB levels are correlated with more advanced disease and poorer survival. GRP78 expression is inversely correlated with P4HB in human HCC tissues, and downregulated by P4HB in HCC cell lines. P4HB overexpression promotes HCC cell growth, migration, invasion and epithelial-to-mesenchymal transition (EMT) in vitro. GRP78 overexpression not only inhibits HCC cell growth, migration, invasion and EMT, but also antagonizes the oncogenic effects of P4HB overexpression. Furthermore, P4HB silencing inhibits HCC tumorigenesis in vivo. Taken together, our results provided evidence that P4HB promotes HCC progression through downregulation of GRP78 and subsequent upregulation of EMT.

Equol Attenuates Atherosclerosis in Apolipoprotein E-Deficient Mice by Inhibiting Endoplasmic Reticulum Stress via Activation of Nrf2 in Endothelial Cells.

The development of atherosclerosis is closely related to excessive endoplasmic reticulum stress (ERs). Equol reportedly protects against cardiovascular disease; however, the underlying mechanism for this protection remains unknown. Herein, the mechanisms contributing to the atheroprotective effect of equol were addressed using apolipoprotein E knockout (apoE-/-) mice fed a high-fat diet (HFD) with or without equol. Equol intervention reduced atherosclerotic lesions in the aorta in HFD-fed apoE-/- mice. Plasma lipid analysis showed that equol intervention reduced triglycerides, total cholesterol and LDL-cholesterol and increased HDL-cholesterol. Additionally, equol administration decreased lipid accumulation in the liver. Simultaneously, equol treatment inhibited cell apoptosis induced by t-BHP and thapsigargin in human umbilical vein endothelial cells (HUVECs). Furthermore, equol treatment attenuated palmitate, t-BHP or thapsigargin-induced upregulation of ER stress markers, including p-PERK, p-eIF2α, GRP78, ATF6 and CHOP proteins expression. The same tendency was also observed in aortic lysates in apoE-/- mice fed with equol plus HFD compared with HFD alone. Moreover, equol treatment dose dependently activated the Nrf2 signaling pathway under oxidative stress. Additionally, elevation of Nrf2 induction was found in aortic lysates in apoE-/- mice fed with a HFD diet containing equol compared with a HFD diet without equol. Importantly, Nrf2 siRNA interference induced CHOP and attenuated the effect of equol to inhibit t-BHP mediated CHOP induction, furthermore, abrogated cell apoptosis induced by t-BHP, suggesting a role for Nrf2 in the protective effect of equol in HUVECs. Collectively, these findings implicate that the improvement of atherosclerosis by equol through attenuation of ER stress is mediated, at least in part, by activating the Nrf2 signaling pathway.

Tauroursodeoxycholic Acid Attenuates Angiotensin II Induced Abdominal Aortic Aneurysm Formation in Apolipoprotein E-deficient Mice by Inhibiting Endoplasmic Reticulum Stress

Abdominal aortic aneurysm (AAA) is characterised by the infiltration of smooth muscle cell (SMC) apoptosis, inflammatory cells, neovascularisation, and degradation of the extracellular matrix. Previous work has shown that endoplasmic reticulum (ER) stress and SMC apoptosis were increased both in a mouse model and human thoracic aortic aneurysm. However, whether the ER stress is activated in AAA formation and whether suppressing ER stress attenuates AAA is unknown. Human AAA and control aorta samples were collected. Expression of ER stress chaperones glucose-regulated protein (GRP)-78 and GRP-94 was detected by immunohistochemical staining. The effect of ER stress inhibitor tauroursodeoxycholic acid (TUDCA) on AAA formation in angiotensin (Ang) II induced apolipoprotein E-/- mice was explored. Elastin staining was used to observe the rupture of elastic fragmentation. Immunohistochemistry and Western blot analysis were performed, to detect the protein expression of ER stress chaperones and apoptosis molecules. There was significant upregulation of GRP-78 and GRP-94 in aneurysmal areas of human AAA and Ang II induced ApoE-/- mice (p<.05). TUDCA significantly attenuated the maximum diameters of abdominal aortas in Ang II induced ApoE-/- mice (p<.05). TUDCA significantly reduced expression of ER stress chaperones and the apoptotic cell numbers (p<.05). Furthermore, TUDCA significantly reduced expression of apoptosis molecules, such as caspase-3, caspase-12, C/EBP homologous protein, c-Jun N-terminal kinase activating transcription factor 4, X-box binding protein, and eukaryotic initiation factor 2α in Ang II induced ApoE-/- mice (p<.05). The results suggest that ER stress is involved in human and Ang II induced AAA formation in ApoE-/- mice. TUDCA attenuates Ang II induced AAA formation in ApoE-/- mice by inhibiting ER stress mediated apoptosis.

Bifidobacteria Prevent Tunicamycin-Induced Endoplasmic Reticulum Stress and Subsequent Barrier Disruption in Human Intestinal Epithelial Caco-2 Monolayers

Endoplasmic reticulum (ER) stress is caused by accumulation of unfolded and misfolded proteins in the ER, thereby compromising its vital cellular functions in protein production and secretion. Genome wide association studies in humans as well as experimental animal models linked ER stress in intestinal epithelial cells (IECs) with intestinal disorders including inflammatory bowel diseases. However, the mechanisms linking the outcomes of ER stress in IECs to intestinal disease have not been clarified. In this study, we investigated the impact of ER stress on intestinal epithelial barrier function using human colon carcinoma-derived Caco-2 monolayers. Tunicamycin-induced ER stress decreased the trans-epithelial electrical resistance of Caco-2 monolayers, concomitant with loss of cellular plasma membrane integrity. Epithelial barrier disruption in Caco-2 cells after ER stress was not caused by caspase- or RIPK1-dependent cell death but was accompanied by lysosomal rupture and up-regulation of the ER stress markers Grp78, sXBP1 and Chop. Interestingly, several bifidobacteria species inhibited tunicamycin-induced ER stress and thereby diminished barrier disruption in Caco-2 monolayers. Together, these results showed that ER stress compromises the epithelial barrier function of Caco-2 monolayers and demonstrate beneficial impacts of bifidobacteria on ER stress in IECs. Our results identify epithelial barrier loss as a potential link between ER stress and intestinal disease development, and suggest that bifidobacteria could exert beneficial effects on this phenomenon.

Targeting HSP70 and GRP78 in canine osteosarcoma cells in combination with doxorubicin chemotherapy.

Heat shock proteins (HSPs) are molecular chaperones subdivided into several families based on their molecular weight. Due to their cytoprotective roles, these proteins may help protect cancer cells against chemotherapy-induced cell death. Investigation into the biologic activity of HSPs in a variety of cancers including primary bone tumors, such as osteosarcoma (OSA), is of great interest. Both human and canine OSA tumor samples have aberrant production of HSP70. This study assessed the response of canine OSA cells to inhibition of HSP70 and GRP78 by the ATP-mimetic VER-155008 and whether this treatment strategy could sensitize cells to doxorubicin chemotherapy. Single-agent VER-155008 treatment decreased cellular viability and clonogenic survival and increased apoptosis in canine OSA cell lines. However, combination schedules with doxorubicin after pretreatment with VER-155008 did not improve inhibition of cellular viability, apoptosis, or clonogenic survival. Treatment with VER-155008 prior to chemotherapy resulted in an upregulation of target proteins HSP70 and GRP78 in addition to the co-chaperone proteins Herp, C/EBP homologous transcription protein (CHOP), and BAG-1. The increased GRP78 was more cytoplasmic in location compared to untreated cells. Single-agent treatment also revealed a dose-dependent reduction in activated and total Akt. Based on these results, targeting GRP78 and HSP70 may have biologic activity in canine osteosarcoma. Further studies are required to determine if and how this strategy may impact the response of osteosarcoma cells to chemotherapy.

Targeting bortezomib-induced aggresome formation using vinorelbine enhances the cytotoxic effect along with ER stress loading in breast cancer cell lines.

The ubiquitin-proteasome and autophagy-lysosome pathways are two major self-digestive systems for cellular proteins. Ubiquitinated misfolded proteins are degraded mostly by proteasome. However, when ubiquitinated proteins accumulate beyond the capacity of proteasome clearance, they are transported to the microtubule-organizing center (MTOC) along the microtubules to form aggresomes, and subsequently some of them are degraded by the autophagy-lysosome system. We previously reported that macrolide antibiotics such as azithromycin and clarithromycin block autophagy flux, and that concomitant treatment with the proteasome inhibitor bortezomib (BZ) and macrolide enhances endoplasmic reticulum (ER) stress-mediated apoptosis in breast cancer cells. As ubiquitinated proteins are concentrated at the aggresome upon proteasome failure, we focused on the microtubule as the scaffold of this transport pathway for aggresome formation. Treatment of metastatic breast cancer cell lines (e.g., MDA-MB-231 cells) with BZ resulted in induction of aggresomes, which immunocytochemistry detected as a distinctive eyeball-shaped vimentin-positive inclusion body that formed in a perinuclear lesion, and that electron microscopy detected as a sphere of fibrous structure with some dense amorphous deposit. Vinorelbine (VNR), which inhibits microtubule polymerization, more effectively suppressed BZ-induced aggresome formation than paclitaxel (PTX), which stabilizes microtubules. Combined treatment using BZ and VNR, but not PTX, enhanced the cytotoxic effect and apoptosis induction along with pronounced ER stress loading such as upregulation of GRP78 and CHOP/GADD153. The addition of azithromycin to block autophagy flux in the BZ plus VNR-containing cell culture further enhanced the cytotoxicity. These data suggest that suppression of BZ-induced aggresome formation using an inhibitory drug such as VNR for microtubule polymerization is a novel strategy for meta-static breast cancer therapy.

Tumor Necrosis Factor-α Attenuates the Osteogenic Differentiation Capacity of Periodontal Ligament Stem Cells by Activating PERK Signaling.

Human periodontal ligament stem cells (PDLSCs) display efficient osteogenic differentiation capacity but fail to rescue bone breakdown associated with periodontitis. Endoplasmic reticulum (ER) stress and the unfolded protein response have recently been linked to inflammation and osteogenic differentiation. Therefore, the role of the double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) pathway in the impaired osteogenic differentiation ability of PDLSCs treated with tumor necrosis factor (TNF)-α was investigated. PDLSCs were isolated and stimulated with osteogenic media containing 1, 10, or 20 ng/mL TNF-α. Assessment included: 1) expression of runt-related transcription factor 2 and osteocalcin; 2) mRNA expression and activity of alkaline phosphatase; and 3) formation of mineralization nodules. Furthermore, expression of PERK pathway-related factors: 1) glucose-regulated protein (GRP) 78; 2) PERK; 3) activating transcription factor (ATF) 4; and 4) CCAAT-enhancer-binding proteins (C/EBP) homologous protein were also measured. Osteogenic differentiation and inhibition of the PERK pathway were also examined in cells pretreated with an inhibitor of ER stress, 4-phenylbutyric acid (PBA), followed by TNF-α stimulation. Finally, PERK small interfering RNA was used to examine osteogenic differentiation attenuated by TNF-α. Higher concentrations of TNF-α (10 and 20 ng/mL) impaired osteogenic differentiation of PDLSCs but activated the PERK pathway. Pretreatment of PDLSCs with lower concentrations of 4-PBA prevented the TNF-α-induced upregulation of GRP78, PERK, and ATF4 and recovered differentiation ability. Finally, PERK knockdown also restored osteogenic differentiation. TNF-α attenuates osteogenic differentiation ability of PDLSCs through activation of the PERK pathway.

Renoprotective Effects of 4‐Phenylbutyrate Against Palmitate‐Induced Renal Cell Injury

BackgroundIncreased dietary intake of fats rich in saturated fatty acids and dyslipidemia have been shown to significantly aggravate the risk of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). Although multiple mechanisms including endoplasmic reticulum (ER) stress and oxidative stress are implicated in the pathophysiology of CKD, the effects of elevated free fatty acid (FFA) levels per se on renal cells is still unclear. Hence, we investigated the effects of palmitic acid (a saturated fatty acid) on rat renal proximal tubular cells in vitro, and tested the renoprotective effects of 4‐phenylbutyrate (4‐PBA), a chemical chaperone shown to inhibit ER stress in vitro and in vivo, in our model.MethodsSubconfluent rat renal proximal tubular (NRK‐52E) cells were exposed to 100 mM palmitic acid in presence and absence of 5 mM 4‐PBA for 24 hours. Cells were then assessed for alterations in cell viability (using MTT assay), oxidative stress (via Dichlorofluorescein (DCF) staining) and the expression of ER stress markers (i.e., glucose‐regulated protein, GRP78 and C/EBP homologous protein, CHOP) and apoptosis (caspase‐3 and ‐12) using western blotting. Flow cytometry for cell cycle analysis and immunoblotting for p27 (an inhibitor of cell cycle) were performed to determine the effects of palmitate and 4‐PBA on the cell cycle in renal cells.ResultsRenal cells exposed to palmitate for 24 hours showed decreased viability (76.9% to that of control), increased DCF staining (1.3‐fold increase over control), and marked upregulation of GRP78 and CHOP. Similarly, the expression of active forms of caspase‐3 (1.8‐fold increase over control) and caspase‐12 (3‐fold increase over control) were also increased in renal cells treated with palmitate. These changes were significantly attenuated in cells co‐treated with 4‐PBA. Intriguingly, exposure to palmitate did not alter the cell cycle in renal cells, whereas treatment with 4‐PBA caused downregulation of p27 (54% to that of control) and increased the transition of cells from G1 to S‐phase and G2/M phase.ConclusionsOur findings indicate that exposure to palmitate induces ER stress as well as oxidative stress in renal cells, and results in renal cell apoptosis. Co‐treatment with 4‐PBA protects renal cells against the perturbations induced by palmitate and improves renal cell viability, which underscores the renoprotective effects of 4‐PBA against fatty acid‐induced renal cell injury. Further studies are required to understand the mechanisms by which 4‐PBA prevents oxidative stress and alters cell cycle in renal cells.Support or Funding InformationSupported by student research grants # QUST‐CPH‐FALL‐12/13‐6 and QUST‐CPH‐SPR‐12/13‐3 from the Office of Academic Research, Qatar University.

Hyperglycemia attenuates remifentanil postconditioning-induced cardioprotection against hypoxia/reoxygenation injury in H9c2 cardiomyoblasts

BackgroundHyperglycemia is proposed to be an independent risk factor for cardiovascular morbidity and mortality. Preclinical studies suggest that diabetes mellitus exacerbates myocardial ischemia/reperfusion injury and attenuates the effects of cardioprotective strategies. The cardioprotective effects of postconditioning with the opioid analgesic remifentanil against ischemia/reperfusion injury under the hyperglycemic condition remain contradictory. Therefore, the aim of this study was to investigate the mechanisms by which hyperglycemia affects cardioprotection induced by remifentanil postconditioning. Materials and methodsH9c2 cardiomyoblasts were cultured under the normoglycemic or hyperglycemic condition. Cells were exposed to hypoxia/reoxygenation (H/R) injury followed by hypoxia postconditioning (HPC group) or remifentanil postconditioning (RPC group). Cell viability, injury, and apoptosis were measured after each postconditioning treatment. Activation of endoplasmic reticulum stress (ERS) was analyzed by examining the protein levels of GRP78, CHOP, cleaved caspase-12 and cleaved caspase-3. ResultsRPC significantly increased cell viability and reduced apoptosis in normoglycemic cardiomyoblasts, but not in hyperglycemic cardiomyoblasts. HPC and RPC markedly decreased the upregulation of GRP78, CHOP, cleaved caspase 12, and cleaved caspase 3 in response to H/R injury under the normoglycemic condition. Hyperglycemia significantly increased these ERS-associated biomarkers and apoptosis, which could not be reduced by HPC or RPC. ConclusionsRemifentanil postconditioning protected cardiomyoblasts from H/R injury under normoglycemia, at least in part, through inhibiting ERS-induced apoptosis. Hyperglycemia attenuated the cardioprotection conferred by remifentanil postconditioning, likely as a result of the exacerbated ERS. Inhibiting the ERS response may be an attractive strategy to enhance the cardioprotective effects of postconditioning in diabetic patients.

Costunolide induces lung adenocarcinoma cell line A549 cells apoptosis through ROS (reactive oxygen species)-mediated endoplasmic reticulum stress.

Costunolide is an active sesquiterpene lactone derived from many herbal medicines. It has a broad spectrum of bioactivities, including anti-inflammatory and potential anti-tumor effects. The aims of the present study were to evaluate the inhibitory effects of costunolide on A549 cell growth and to explore the underlying molecular mechanisms. Annexin V-FITC/PI flow cytometry analysis revealed that costunolide induced apoptosis. To study the mechanism, we found that costunolide exposure activated the unfolded protein response (UPR) signaling pathways, as shown by the up-regulation of GRP78 and IRE1α and the activation of ASK1 and JNK. Meanwhile, siRNA knockdown of IRE1α significantly attenuated costunolide-induced apoptosis and partly restored the mitochondrial membrane potential. ER stress-activated JNK phosphorylated Bcl-2 at Ser70, which changes the anti-apoptotic function of Bcl-2, resulting in mitochondrial dysfunction and leading to mitochondrial activation of apoptosis. Furthermore, costunolide induced ROS generation, while the antioxidant N-acetyl cysteine (NAC) effectively blocked ER stress and apoptosis activation, suggesting that ROS acts as an upstream signaling molecule in triggering ER stress and mitochondrial apoptotic pathways. Taken together, our research demonstrates that costunolide exhibits its anti-tumor activity though inducing apoptosis, which is mediated by ER stress. We further confirm that Bcl-2 is a key molecule connecting the ER stress and mitochondrial pathways.