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Copyright of Journal of Prevention & Treatment For Stomatological Diseases is the property of Journal of Prevention & Treatment For Stomatological Diseases Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective To investigate the effect of calcium ions on the expression of kallikrein-4 (KLK4) and cell growth of ameloblast, and to provide an experimental basis for calcium ion promoting normal mineralization of enamel. Methods ALC cells were treated with 0, 2.0, 2.5, 3.0, and 3.5 mmol/L CaCl2 for 24 and 48 h. KLK4 expression was analyzed by qRT-PCR and Western blot analysis. The viability of ALC cells was determined by using CCK-8. AnnexinV-FITC/PI dual staining combined with flow cytometry and Hoechst 33342 staining were used to detect the ALC cell cycle and cell apoptosis. The protein expression level of glucose-regulated protein 78 (GRP78) was measured by Western blot analysis. Results After 24 h of treatment with 2.5, 3.0, and 3.5 mmol/L CaCl2, the expression of KLK4 mRNA was increased (P2, the expression of KLK4 protein was increased (P2, the expression of KLK4 mRNA and protein was increased (P2 (P2. Hoechst 33342 staining results showed that 3.0 mmol/L and 3.5 mmol/L CaCl2 may promote apoptosis in ALC cells. Flow cytometry showed that the proportion of G2/M phase cells and the apoptosis rate increased after 3.5 mmol /L CaCl2 treatment for 24 h (P2 groups. After 24 h of treatment with 3.0 mmol/L and 3.5 mmol/L CaCl2, the expression of GRP78 protein was reduced (P2, the expression of GRP78 protein was reduced (P

• High-intensity interval training (HIIT) avoided liver steatosis in high-fat-fed mice. • HIIT (thrice weekly) prevented hyperglycemia and endoplasmic reticulum (ER) stress. • HIIT preserved mitochondrial ultrastructure and enhanced beta-oxidation. • HIIT targeted ER and mitochondrial homeostasis to prevent liver steatosis. Metabolic dysfunction-associated steatotic liver disease (MASLD) has attracted increasing attention from the scientific community because of its severe but silent progression and the lack of specific treatment. Glucolipotoxicity triggers endoplasmic reticulum (ER) stress with decreased beta-oxidation and enhanced lipogenesis, promoting the onset of MASLD, whereas regular physical exercise can prevent MASLD by preserving ER and mitochondrial function. Thus, the hypothesis of this study was that high-intensity interval training (HIIT) could prevent the development of MASLD in high-fat (HF)-fed C57BL/6J mice by maintaining insulin sensitivity, preventing ER stress, and promoting beta-oxidation. Forty male C57BL/6J mice (3 months old) comprised 4 experimental groups: the control (C) diet group, the C diet + HIIT (C-HIIT) group, the HF diet group, and the HF diet + HIIT (HF-HIIT) group. HIIT sessions lasted 12 minutes and were performed 3 times weekly by trained mice. The diet and exercise protocols lasted for 10 weeks. The HIIT protocol prevented weight gain and maintained insulin sensitivity in the HF-HIIT group. A chronic HF diet increased ER stress-related gene and protein expression, but HIIT helped to maintain ER homeostasis, preserve mitochondrial ultrastructure, and maximize beta-oxidation. The increased sirtuin-1/peroxisome proliferator-activated receptor-gamma coactivator 1-alpha expression implies that HIIT enhanced mitochondrial biogenesis and yielded adequate mitochondrial dynamics. High hepatic fibronectin type III domain containing 5 /irisin agreed with the antilipogenic and anti-inflammatory effects observed in the HF-HIIT group, reinforcing the antisteatotic effects of HIIT. Thus, we confirmed that practicing HIIT 3 times per week maintained insulin sensitivity, prevented ER stress, and enhanced hepatic beta-oxidation, impeding MASLD development in this mouse model even when consuming high energy intake from saturated fatty acids. HIIT prevented overweight and liver steatosis by mitochondrial and ER enhancement. Abbreviations: ACOX1, acyl-coenzyme A oxidase 1; Atf4 , activating transcription factor 4; ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78; Chop , C/EBP homologous protein; Fgf21 , fibroblast growth factor 21; Gadd45 , growth arrest and DNA damage-inducible 45; Ppar , peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (Pgc1-a); Xbp-a , X-box binding protein 1. Created with www.biorender.com. [Display omitted] [ABSTRACT FROM AUTHOR]

Objective To study the effect of low concentrations of sodium fluoride on the osteogenic/odontogenic differentiation of human dental pulp cells (hDPCs) in vitro. Methods This study was reviewed and approved by the Ethics Committee. hDPCs were cultured using a modified tissue explant technique in vitro. The effects of different concentrations of sodium fluoride on the proliferation of hDPCs were measured by methylthiazol tetrazolium (MTT) assay. Appropriate concentrations were added to the osteogenic/odontogenic differentiation induction medium, and the cells were induced in vitro. Alizarin red S staining was used to detect the osteoblastic/odontogenic differentiation ability of the cells, and the mRNA expression of the key differentiation factors was detected by RT-qPCR. Moreover, the expression of key molecules of endoplasmic reticulum stress (ERS) was detected by RT-qPCR and Western blot. The data were analyzed with the SPSS 18.0 software package. Results Low concentration of NaF (0.1 mmol/L) could stimulate cell proliferation in vitro, while a high concentration (5-10 mmol/L) could inhibit cell proliferation (P

Objectives To construct human breast cancer cell lines HS578T and MDA-MB-231 that overexpress the endoplasmic reticulum stress marker glucose-regulated protein 78（GRP78), and to provide a basis for exploring the mechanism of GRP78 in the development and progression of breast cancer. Methods The lentiviral vector pCDH-CMVGRP78-HA-EF1-Puro plasmid expressing GRP78 was constructed by molecular cloning technology, and the plasmid was amplified by PCR method. Then the plasmid was cut by EcoRⅠ and NotⅠ restriction enzymes, and the digestion products were identified by sequencing. The pCDH-CMV-GRP78-HA-EF1-Puro plasmid was constructed successfully. 293FT cell line （clonal isolate） in the logarithmic growth phase was added into a culture dish with pCDH-CMV-GRP78-HA-EF1-Puro mixture, and was cultured for 48 h； the supernate containing GRP78 lentiviral particles was collected and stored for later use. In addition, human breast cancer cell lines HS578T and MDA-MB-231 in the logarithmic growth phase were cultured in the medium containing supernate of GRP78 lentiviral particles. After 48 h culture, 1：500 diluted purinycin was added to the medium, and the cell lines of HS578T and MDA-MB-231 with overexpression of GRP78 were screened. The GRP78 of HS578T and MDA-MB-231 cells was detected by Western blotting after being cultured with purinamycin for 48 h. The GRP78 protein in HS578T and MDA-MB-231 cells was localized by confocal laser microscopy. Results The cell lines of HS578T and MDA-MB-231 with overexpression of GRP78 showed obvious GPR78 protein electrophoretic bands around 78 kd. There was GRP78 protein expression in HS578T cells, which was mainly distributed in the cytoplasm. Conclusions We successfully constructed MDA-MB-231 and HS578T cell lines with GRP78 overexpression, and GRP78 was mainly ex‐ pressed in the cytoplasm. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Prevention & Treatment For Stomatological Diseases is the property of Journal of Prevention & Treatment For Stomatological Diseases Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Endoplasmic reticulum (ER) stress has been shown to play an important role in osteoarthritis (OA). Objective: This study was aimed at assessing the relationship of endoplasmic reticulum (ER) stress-related glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) concentrations in the serum/synovial fluid (SF) with disease severity of primary knee osteoarthritis (pkOA). Methods: Patients with pkOA together with healthy individuals were consecutively recruited from our hospital. The levels of GRP78 and CHOP in serum / SF were detected using enzyme-linked immunosorbent assay. The levels of IL-6 and MMP-3 were also examined. Radiographic progression of pkOA was evaluated based on Kellgren-Lawrence (K-L) grades. Receiver Operating Characteristic (ROC) curves were used to assess the diagnostic value of GRP78/CHOP levels with regard to K-L grades. The assessment of clinical severity was conducted using the visual analogue scale (VAS), Oxford knee score (OKS), and Lequesne algofunctional index (LAI). Results: A total of 140 pkOA patients and 140 healthy individuals were included. Serum GRP78 and CHOP levels in pkOA patients were not significantly different from those in healthy individuals. The SF GRP78 and CHOP levels in healthy controls were not detected due to ethical reasons. Compared to those with K-L grade 2 and 3, the pkOA patients with K-L grade 4 had higher GRP78 and CHOP levels in the SF with statistical significance. In addition, the pkOA patients with K-L grade 3 exhibited drastically upregulated GRP78 and CHOP concentrations in the SF compared to those with K-L grade 2. Positive correlations of GRP78 and CHOP levels with K-L grades, IL-6, and MMP-3 levels in the SF were observed. ROC curve analysis indicated that both GRP78 and CHOP levels may act as decent indicators with regard to OA. GRP78 and CHOP concentrations in the SF were positively correlated with VAS/LAI score and negatively associated with OKS score. Conclusion: The study indicated that GRP78 and CHOP levels in the SF but not the serum were positively correlated with disease severity of pkOA. [ABSTRACT FROM AUTHOR]

The construction of multifunctional oncotherapy nanoplatforms that combine diagnosis and treatment remains challenging. Nanodroplets (NDs), which simultaneously enhance ultrasound imaging and therapeutic effects, are a potential strategy for non-invasive drug delivery. To achieve the goals of precise medicine, novel SP94 peptide-modified and doxorubicin-loaded ultrasonic NDs (SP94-DOX-NDs) for castration-resistant prostate cancer (CRPC) targeting and treatment were constructed in this study. The characteristics, contrast-enhanced ultrasound imaging (CEUI), targeting ability to glucose-regulated protein 78 (GRP78)-overexpressing CRPC and anticancer effect of the SP94-DOX-NDs were assessed. The desired SP94-NDs were successfully prepared using the nanoemulsification method using a certain proportion of SP94-PEG-chitosan, perfluoropentane (PFP), Tween 20, and lecithin. SP94-NDs with a size of ∼300 nm showed great biocompatibility and CEUI ability. Compared with blank NDs, SP94-NDs exhibited higher tumor-specific targeting ability due to conjugation between the SP94 peptide and GRP78-overexpressing 22RV1 cells. Most importantly, in vitro and in vivo investigations showed that SP94-DOX-NDs combined with ultrasound could specifically deliver DOX into 22RV1 cells and thereby demonstrated a stronger anticancer effect than DOX-NDs and DOX. Thus, SP94-DOX-NDs may provide an efficient approach for the real-time imaging of tumors and triggered, accurate drug delivery to tumors.

Hypochlorous acid (HOCl) is an essential signal molecule in cancer cells. Activated GRP78 ATPase by a HOCl probe named ZBM-H inhibits lung cancer cell growth. However, the role and underlying mechanism of GRP78 ATPase in lung cancer cell migration have not been established. Here, we reported that activation of GRP78 ATPase by ZBM-H suppressed A549 cell migration and inhibited EMT process. Notably, ZBM-H time-dependently decreased the protein level of integrin β4 (ITGB4) in A549 cells. Combinatorial treatment of 3BDO (an autophagy inhibitor) and ZBM-H partially rescued the protein level of ITGB4. Consistently, 3BDO partially reversed ZBM-H-inhibited cell migration. Furthermore, ZBM-H promoted the interaction between ANXA7 and Hsc70, which participated in the regulation of selective autophagy and degradation of ITGB4.

Simple Summary: Ovarian cancer (OVCA) is a fatal gynecological disease for which there is no early detection test. Glucose-regulated protein 78 (GRP78), a protein marker of stress, increases during chronic stress. Chronic stress has been suggested as a hallmark of cancer development. This study examined whether expression of GRP78 is associated with development of OVCA and whether GRP78 can predict OVCA at early stage. This study found GRP78 expression and its secretion in blood increased during OVCA development and progression. This study also developed a GRP78-targeted ultrasound scanning agent that detected ovarian tumors at early stages. Thus, a woman with high levels of GRP78 in her blood may be referred to have targeted-ultrasound scanning for confirming if she has ovarian tumors. These results will be a foundation for a clinical study to examine the feasibility of GRP78 as a potential marker of blood and ultrasound scanning for early detection of OVCA. Background: Understanding malignant transformation associated with ovarian cancer (OVCA) is important to establish early detection tests. This study examined whether expression of glucose-regulated protein 78 (GRP78, marker of cellular stress) increases during OVCA development, and whether GRP78 can be detected by targeted-transvaginal ultrasound (TVUS) imaging. Methods: Normal ovaries (n = 10), benign (n = 10) and malignant ovarian tumors at early (n = 8) and late stages (n = 16), hens with and without ovarian tumors at early and late stages (n = 10, each) were examined for GRP78 expression during OVCA development by immunohistochemistry, immunoblotting, gene expression and immunoassay. Feasibility of GRP78-targeted TVUS imaging in detecting early OVCA was examined. Results: Compared with normal ovaries and benign tumors, intensity of GRP78 expression was higher (p < 0.0001) in OVCA patients. Compared with normal (9007.76 ± 816.54 pg/mL), serum GRP78 levels were significantly higher (p < 0.05) in patients with early (12,730.59 ± 817.35 pg/mL) and late-stage OVCA (13,930.12 ± 202.35) (p < 0.01). Compared with normal (222.62 ± 181.69 pg/mL), serum GRP78 levels increased (p < 0.05) in hens with early (590.19 ± 198.18 pg/mL) and late-stage OVCA (1261.38 ± 372.85) (p < 0.01). Compared with non-targeted, GRP78-targeted imaging enhanced signal intensity of TVUS (p < 0.0001). Conclusions: Tissue and serum levels of GRP78 increase in association with OVCA. GRP78 offers a potential serum and imaging marker for early OVCA detection. [ABSTRACT FROM AUTHOR]

Background/Purpose: To overcome liver failure, we focused on liver regeneration mechanisms by the activation of hepatic stellate cells (HSCs) and Kupffer cells (KCs). It is known that the HSC‐secreted Mac‐2‐binding protein glycan isomer (M2BPGi) activates KC in the fibrotic liver. However, its importance for liver regeneration of the HSCs/M2BPGi/KCs axis after hepatectomy is still unknown. The aim of this study was to clarify whether the HSC‐derived M2BPGi can activate KCs after hepatectomy, and elucidate the new molecular mechanism of liver regeneration. Methods: We examined the effect of M2BPGi on human hepatocytes and KCs, and explored secretory factors from M2BPGi‐activated KCs using proteomics. Furthermore, the effect on liver regeneration of glucose‐regulated protein 78 (GRP78) as one of the M2BPGi‐related secreted proteins was examined in vitro and in murine hepatectomy models. Results: Although M2BPGi had no hepatocyte‐promoting effect, M2BPGi promoted the production of GRP78 in KCs. The KC‐driven GRP78 promoted hepatocyte proliferation. GRP78 administration facilitated liver regeneration after 70% hepatectomy and increased the survival rate after 90% hepatectomy in mice. Conclusions: The M2BPGi‐activated KCs secrete GRP78, which facilitates liver regeneration and improves the survival in a lethal mice model. Our data suggest that the new hepatotrophic factor GRP78 may be a promising therapeutic tool for lethal liver failure. [ABSTRACT FROM AUTHOR]

Background and aims: The destruction of endoplasmic reticulum (ER) homeostasis leads to heart failure (HF), which further aggravates ER stress. Limited data are available on the levels of ER stress markers in HF patients in clinical practice. This study aimed to determine the clinical significance of the ER stress markers, glucose-regulated protein 78 (GRP78), Caspase-3, and C/EBP homologous protein (CHOP), in predicting HF and its severity. Materials and methods: A total of 62 patients with HF and 44 healthy controls were enrolled in the study, and all participants were followed-up for 2 years. Results: Serum GRP78, Caspase-3, and CHOP levels were significantly higher in patients with HF than those in healthy controls. The level of GRP78 increased with the severity of HF. GRP78 levels were negatively correlated with left ventricular ejection fraction, and positively correlated with N-terminal B-type natriuretic peptide, D-dimer, and lactic acid. Serum GRP78 and Caspase-3 levels showed moderate predictive values for HF patients. Conclusion: ER stress markers, GRP78 and Caspase-3, had a certain predictive value in HF and can be used as serum biomarkers for the diagnosis of HF. Additionally, GRP78 showed a certain predictive value in HF severity.

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Cellular senescence is linked with chemotherapy resistance. Based on previous studies, GRP78 is a signal transducer in senescent cells. However, the association between GRP78 and stem cell phenotype remains unknown. Cisplatin treatment was clarified to induce cellular senescence leading to stemness induction via GRP78/Akt signal transduction. H460 cells were treated with 5 μM of cisplatin for 6 days to develop senescence. The colony formation assay and cell cycle analysis were performed. SA-β-galactosidase staining indicated senescence. Western blot analysis and RT-PCR were operated. Immunoprecipitation (IP) and immunocytochemistry assays (ICC) were also performed. Colony-forming activity was completely inhibited, and 87.07% of the cell population was arrested in the G2 phase of the cell cycle. mRNA of p21 and p53 increased approximately by 15.91- and 19.32-fold, respectively. The protein level of p21 and p53 was elevated by 9.57- and 5.9-fold, respectively. In addition, the c-Myc protein level was decreased by 0.2-fold when compared with the non-treatment control. Even though, the total of GRP78 protein was downregulated after cisplatin treatment, but the MTJ1 and downstream regulator, p-Akt/Akt ratio were upregulated by approximately 3.38 and 1.44-fold, respectively. GRP78 and MTJ1 were found at the cell surface membrane. Results showed that the GRP78/MTJ1 complex and stemness markers, including CD44, CD133, Nanog, Oct4, and Sox2, were concomitantly increased in senescent cells. MTJ1 anchored GRP78, facilitating the signal transduction of stem-like phenotypes. The strategy that could interrupt the binding between these crucial proteins or inhibit the translocation of GRP78 might beuseful for cancer therapy. [ABSTRACT FROM AUTHOR]

Purpose: To determine the effect of miR-30a-5p on hepatoma cell radiosensitivity and elucidate the underlying mechanism. Methods: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure miR-30a-5p expression in HepG2 and THLE-3 cells. After 4-Gy X-ray irradiation or miR-30a-5p mimic transfection, the miR-30a-5p level in HepG2 cells was determined using qRT-PCR. Luciferase reporter assay was used to confirm the correlation between miR-30a-5p and glucose-regulated protein 78 (GRP78) levels, while the effects of miR-30a-5p on the viability of HepG2 cells were determined using clone formation and 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assays. Apoptotic cells were evaluated by flow cytometry whereas the protein levels of GRP78, B-cell lymphoma-2 (Bcl-2), BCL2-Associated X Protein (Bax), and cleaved-caspase-9 were quantified by immunoblotting. Results: MicroRNA-30a-5p expression was decreased in HepG2 cells but reduced after 4-Gy x-ray treatment, while miR-30a-5p mimic transfection upregulated miR-30a-5p expression (p < 0.05). Cell viability was inhibited after x-ray irradiation or miR-30a-5p mimic transfection and further inhibited by irradiation + miR-30a-5p (p < 0.05). Irradiation or miR-30a-5p transfection triggered cell apoptosis; however, irradiation + miR-30a-5p induced more apoptosis, upregulated Bax and cleaved-caspase-9 expression, and reduced Bcl-2 expression (p < 0.05). MicroRNA-30a-5p also suppressed GRP78 expression. Conclusion: MicroRNA-30a-5p may enhance HCC x-ray radiosensitivity by inhibiting GRP78., and may be useful in developing treatment strategies for HCC patients. [ABSTRACT FROM AUTHOR]

To investigate the effects of quercetin on oxidative stress and endoplasmic retic‑ ulum stress of human umbilical vein endothelial cells（HUVECs）induced by high glucose and their pos‑ sible mechanism. Methods HUVECs were cultured in vitro，and the cells were divided into the control group（Control group），high glucose（HG）group，and quercetin groups with different concentrations （20 µmol/L，50 µmol/L，and 100 µmol/L，named as Q20，Q50，Q100 groups），respectively. HU‑ VECs in the Q20 group were pretreated with 20 µmol/L quercetin for 1 h，and then were added with 10 mg/L glucose for culture. HUVECs in the Q50 group were pretreated with 50 µmol/L quercetin for 1 h，and then were added with glucose for culture. HUVECs in the Q100 group were pretreated with 100 µmol/L quercetin for 1 h，and then were added with 10 mg/L glucose for culture. In the HG group， 10 mg/L HG was added to the culture medium，and the cells in the Control group were cultured in the low-glucose DMEM medium. After 24 hours of intervention，the cell viability was detected by cell count‑ ing Kit-8（CCK-8）method. The apoptosis rate and reactive oxygen species（ROS）level were detected by flow cytometry. The degree of cell membrane damage was evaluated by detecting lactate dehydrogenase （LDH）activity in the media. Western blotting was used to detect the expression levels of glucose-regulat‑ ed protein 78 （GRP78），CCAAT/enhancer-binding protein homologous protein （CHOP） and Cas‑ pase12. Results Compared with the Control group，the activity of endothelial cells in the HG group de‑ creased after 24 h（P<0. 05）. Compared with the HG group，the activity of endothelial cells in the Q20， Q50 and Q100 groups increased，and the cell viability increased with the increased concentrations of quercetin（all P<0. 05）. Compared with the Control group，the apoptosis rate of the HG group increased （P<0. 05），while the apoptosis rates of the Q20，Q50 and Q100 groups decreased，and the apoptosis rate decreased with the increased concentrations of quercetin（all P<0. 05）. Compared with the Control group，the ROS in the HG group increased（P<0. 05）；compared with the HG group，the ROS in the Q20，Q50 and Q100 groups decreased，and the ROS decreased with the increased concentrations of quer‑ cetin（all P<0. 05）. Compared with the Control group，the LDH activity in the cell culture medium of the HG group increased（P<0. 05）. Compared with the HG group，the LDH activity in the cell culture medi‑ um of the Q50 group and Q100 group decreased （both P<0. 05）. Compared with the Q50 group， the LDH activity in the cell culture medium of the Q100 group decreased（P<0. 05）. Compared with the Control group，the expression levels of GRP78，CHOP，and Caspase 12 in the endothelial cells of the HG group increased（all P<0. 01）. Compared with the HG group，the expression levels of GRP78， CHOP，and Caspase 12 decreased in the endothelial cells of the Q20，Q50 and Q100 groups（all P< 0. 01）. Conclusions Quercetin can increase endothelial cell activity，reduce LDH activity，inhibit the apoptosis，reduce ROS content，and inhibit the expression levels of GRP78，CHOP and Caspase 12 of endothelial cells in the high glucose solution，and its inhibitory effect is enhanced with the increased con‑ centrations of quercetin. Quercetin may play a protective effect on endothelial cells by inhibiting endothe‑ lial cell oxidative stress，endoplasmic reticulum stress and reducing apoptosis under the condition of high glucose. [ABSTRACT FROM AUTHOR]

The 78‐kDa glucose‐regulated protein (GRP78) has extracellular, anti‐inflammatory properties that can aid resolving inflammation. It has been established previously that GRP78 induced myeloid CD11c+ cell differentiation into distinct tolerogenic cells. This tolerance induction makes GRP78 a potential therapeutic agent for transplanted allogeneic grafts and autoimmune diseases, such as type 1 diabetes. In this research, it is revealed that rmGRP78‐treated NOD mice bone marrow‐derived CD11c+ cells (GRP78‐DCs) highly expressed B7‐H4 but down‐regulated CD86 and CD40, and retained a tolerogenic signature even after stimulation by LPS. In the assessment of in vivo therapeutic efficacy after the adoptive transfer of GRP78‐DCs into NOD mice, fluorescent imaging analyses revealed that the transfer specifically homed in inflamed pancreases, promoting β‐cell survival and alleviating insulitis in NOD mice. The adoptive transfer of GRP78‐DCs also helped reduce Th1, Th17, and CTL, suppressing inflammatory cytokine production in vivo. The findings suggest that adoptive GRP78‐DC transfer is critical to resolving inflammation in NOD mice and may have relevance in a clinical setting. [ABSTRACT FROM AUTHOR]

Cellular senescence is linked with chemotherapy resistance. Based on previous studies, GRP78 is a signal transducer in senescent cells. However, the association between GRP78 and stem cell phenotype remains unknown. Cisplatin treatment was clarified to induce cellular senescence leading to stemness induction via GRP78/Akt signal transduction. H460 cells were treated with 5 μM of cisplatin for 6 days to develop senescence. The colony formation assay and cell cycle analysis were performed. SA-β-galactosidase staining indicated senescence. Western blot analysis and RT-PCR were operated. Immunoprecipitation (IP) and immunocytochemistry assays (ICC) were also performed. Colony-forming activity was completely inhibited, and 87.07% of the cell population was arrested in the G2 phase of the cell cycle. mRNA of p21 and p53 increased approximately by 15.91- and 19.32-fold, respectively. The protein level of p21 and p53 was elevated by 9.57- and 5.9-fold, respectively. In addition, the c-Myc protein level was decreased by 0.2-fold when compared with the non-treatment control. Even though, the total of GRP78 protein was downregulated after cisplatin treatment, but the MTJ1 and downstream regulator, p-Akt/Akt ratio were upregulated by approximately 3.38 and 1.44-fold, respectively. GRP78 and MTJ1 were found at the cell surface membrane. Results showed that the GRP78/MTJ1 complex and stemness markers, including CD44, CD133, Nanog, Oct4, and Sox2, were concomitantly increased in senescent cells. MTJ1 anchored GRP78, facilitating the signal transduction of stem-like phenotypes. The strategy that could interrupt the binding between these crucial proteins or inhibit the translocation of GRP78 might beuseful for cancer therapy.

Previous studies have shown that resveratrol, a bioactive substance found in many plants, can reduce early brain injury after subarachnoid hemorrhage, but how it acts is still unclear. This study explored the mechanism using the experimental subarachnoid hemorrhage rat model established by injecting autologous blood into the cerebellomedullary cistern. Rat models were treated with an intraperitoneal injection of 60 mg/kg resveratrol 2, 6, 24 and 46 hours after injury. At 48 hours after injury, their neurological function was assessed using a modified Garcia score. Brain edema was measured by the wet-dry method. Neuronal apoptosis in the prefrontal cortex was detected by terminal deoxyribonucleotidyl transferase-mediated biotin-16-dUTP nick-end labeling assay. Levels of reactive oxygen species and malondialdehyde in the prefrontal cortex were determined by colorimetry. CHOP, glucose-regulated protein 78, nuclear factor-erythroid 2-related factor 2 and heme oxygenase-1 mRNA expression levels in the prefrontal cortex were measured by reverse transcription polymerase chain reaction. Tumor necrosis factor-alpha content in the prefrontal cortex was detected by enzyme linked immunosorbent assay. Immunohistochemical staining was used to detect the number of positive cells of nuclear factor-erythroid 2-related factor 2, heme oxygenase 1, glucose-regulated protein 78, CHOP and glial fibrillary acidic protein. Western blot assay was utilized to analyze the expression levels of nuclear factor-erythroid 2-related factor 2, heme oxygenase 1, glucose-regulated protein 78 and CHOP protein expression levels in the prefrontal cortex. The results showed that resveratrol treatment markedly alleviated neurological deficits and brain edema in experimental subarachnoid hemorrhage rats, and reduced neuronal apoptosis in the prefrontal cortex. Resveratrol reduced the levels of reactive oxygen species and malondialdehyde, and increased the expression of nuclear factor-erythroid 2-related factor 2, heme oxygenase-1 mRNA and protein in the prefrontal cortex. Resveratrol decreased glucose-regulated protein 78, CHOP mRNA and protein expression and tumor necrosis factor-alpha level. It also activated astrocytes. The results suggest that resveratrol exerted neuroprotective effect on subarachnoid hemorrhage by reducing oxidative damage, endoplasmic reticulum stress and neuroinflammation. The study was approved by the Animals Ethics Committee of Shandong University, China on February 22, 2016 (approval No. LL-201602022).

Studies have confirmed a strong association between activation of the endoplasmic reticulum stress pathway and cerebral ischemia/reperfusion (I/R) injury. In this study, three key proteins in the endoplasmic reticulum stress pathway (glucose-regulated protein 78, caspase-12, and C/EBP homologous protein) were selected to examine the potential mechanism of endoplasmic reticulum stress in the neuroprotective effect of G protein-coupled estrogen receptor. Female Sprague-Dawley rats received ovariectomy (OVX), and then cerebral I/R rat models (OVX + I/R) were established by middle cerebral artery occlusion. Immediately after I/R, rat models were injected with 100 μg/kg E2 (OVX + I/R + E2), or 100 μg/kg G protein-coupled estrogen receptor agonist G1 (OVX + I/R + G1) in the lateral ventricle. Longa scoring was used to detect neurobehavioral changes in each group. Infarct volumes were measured by 2,3,5-triphenyltetrazolium chloride staining. Morphological changes in neurons were observed by Nissl staining. Terminal dexynucleotidyl transferase-mediated nick end-labeling staining revealed that compared with the OVX + I/R group, neurological function was remarkably improved, infarct volume was reduced, number of normal Nissl bodies was dramatically increased, and number of apoptotic neurons in the hippocampus was decreased after E2 and G1 intervention. To detect the expression and distribution of endoplasmic reticulum stress-related proteins in the endoplasmic reticulum, caspase-12 distribution and expression were detected by immunofluorescence, and mRNA and protein levels of glucose-regulated protein 78, caspase-12, and C/EBP homologous protein were determined by polymerase chain reaction and western blot assay. The results showed that compared with the OVX + I/R group, E2 and G1 treatment obviously decreased mRNA and protein expression levels of glucose-regulated protein 78, C/EBP homologous protein, and caspase-12. However, the G protein-coupled estrogen receptor antagonist G15 (OVX + I/R + E2 + G15) could eliminate the effect of E2 on cerebral I/R injury. These results confirm that E2 and G protein-coupled estrogen receptor can inhibit the expression of endoplasmic reticulum stress-related proteins and neuronal apoptosis in the hippocampus, thereby improving dysfunction caused by cerebral I/R injury. Every experimental protocol was approved by the Institutional Ethics Review Board at the First Affiliated Hospital of Shihezi University School of Medicine, China (approval No. SHZ A2017-171) on February 27, 2017.

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) chaperone that has been shown that is overexpressed in cancer cells. Overexpression of GRP78 on cancer cells makes this molecule a suitable candidate for cancer detection and targeted therapy. VHH is the binding fragment of camelid heavy-chain antibodies also known as "nanobody." The aim of this study is to isolate and produce a new recombinant nanobody using phage display technique to detect cancer cells. Using the c-terminal domain of GRP78 (CGRP) as an antigen, four rounds of biopanning were performed, and high-affinity binders were selected by ELISA. Their affinity and functionality were characterized by surface plasmon resonance (SPR) cell ELISA and immunocytochemistry. A unique nanobody named V80 was purified. ELISA and SPR showed that this antibody had high specificity and affinity to the GRP78. Immunofluorescence analysis showed that V80 could specifically bind to the HepG2 and A549 cancer cell lines. This novel recombinant nanobody could bind to the cell surface of different cancer cells. After further evaluation, this nanobody can be used as a new tool for cancer detection and tumor therapy. [ABSTRACT FROM AUTHOR]

Objective: To analyze the clinical application value of serum heme oxygenase (HO)-1expression level in non-alcoholic fatty liver disease (NAFLD) and, based on that, establish a diagnostic model combined with glucose regulatory protein 78 (GRP78) so as to clarify its diagnostic effectiveness and application value. Methods: A total of 210 NAFLD patients diagnosed by abdominal B-ultrasound and liver elastography were included, and at the same time, 170 healthy controls were enrolled. The general clinical data, peripheral blood cell counts, and biochemical indicators of the research subjects were collected. The expression levels of HO-1 and GRP78 were detected using an enzyme-linked immunosorbent assay. Multivariate analysis was used to screen independent risk factors for NAFLD. Visual output was performed through nomogram diagrams, and the diagnostic model was constructed. Receiver operating characteristic curve (ROC), calibration curve, and decision curve analysis (DCA) were used to evaluate the diagnostic effectiveness of NAFLD. Measurement data were analyzed using a t -test or Mann-Whitney U rank sum test to detect data differences between groups. Enumeration data were analyzed using the Fisher's exact probability test or the Pearson χ (2) test. Results: Compared with the healthy control group, the white blood cell count, aspartate aminotransferase (AST), alanine aminotransferase, gamma-glutamyl transferase (GTT), fasting blood glucose (Glu), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), serum HO-1, and GRP78 levels were significantly increased in the NAFLD group patients ( P  < 0.05). Binary logistic analysis results showed that AST, TG, LDL-C, serum HO-1, and GRP78 were independent risk factors for NAFLD ( P  < 0.05). A nomogram clinical predictive model HGATL was established using HO-1 (H), GRP78 (G) combined with AST (A), TG (T), and LDL-C (L), with the formula P =-21.469+3.621×HO-1+0.116 ×GRP78+0.674×AST+6.250×TG+4.122 ×LDL-C. The results confirmed that the area under the ROC curve of the HGATL model was 0.965 8, with an optimal cutoff value of 81.69, a sensitivity of 87.06%, a specificity of 92.82%, a P  < 0.05, and the diagnostic effectiveness significantly higher than that of a single indicator. The calibration curve and DCA both showed that the model had good diagnostic performance. Conclusion: The HGATL model can be used as a novel, non-invasive diagnosis model for NAFLD and has a positive application value in NAFLD diagnosis and therapeutic effect evaluation. Therefore, it should be explored and promoted in clinical applications.

To achieve a cure for metastatic breast cancer, further understanding of molecular drivers of the metastatic cascade is essential. Currently, chemotherapy regimens include doxorubicin and paclitaxel which act in part by inducing the unfolded protein response (UPR). The master regulator of the UPR, glucose regulated protein 78 (GRP78), localizes on the surface of tumor cells and is associated with metastatic disease. Cyclic AMP responsive element binding protein 3-like 1 (CREB3L1), a member of the UPR, is a breast cancer metastasis suppressor that acts on cyclic AMP to promote the expression of target genes including GRP78. The aim of the present study was to evaluate the effects of chemotherapy on CREB3L1 and cell-surface GRP78 expression and its association with the development of breast cancer metastasis. For this purpose, we use breast cancer cells migration in vitro assays and an in vivo metastatic mouse model. The results showed that chemotherapy activated CREB3L1 and enhanced cell-surface GRP78 expression specifically in triple-negative breast cancer cells (TNBC), reducing their migration and metastatic potential. CREB3L1 knockout (KO) in the triple negative MDAMB231 cell line using CRISPR/Cas9 technology led to inhibition of GRP78 expression and abrogation of the CREB3L1 metastatic suppression function. Inoculation of CREB3L1-KO MDAMB231 cells into a mouse metastatic model induced a massive metastatic profile which chemotherapy failed to prevent. These findings elucidate a potential pathway to the development of a novel treatment strategy for metastatic TNBC based on modulating CREB3L1 and cell-surface GRP78 expression by chemotherapy and GRP78-targeted drugs.

Yong Wang,1 Jie-Sen Zhou,1 Xu-Chen Xu,1 Zhou-Yang Li,1 Hai-Pin Chen,1 Song-Min Ying,1 Wen Li,1 Hua-Hao Shen,1,2 Zhi-Hua Chen1 1Department of Respiratory and Critical Care Medicine, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 2State Key Laboratory of Respiratory Disease, Guangzhou, People’s Republic of China Introduction: Bronchial epithelial cell death and airway inflammation induced by cigarette smoke (CS) have been involved in the pathogenesis of COPD. GRP78, belonging to heat shock protein 70 family, has been implicated in cell death and inflammation, while little is known about its roles in COPD. Here, we demonstrate that GRP78 regulates CS-induced necroptosis and injury in bronchial epithelial cells.Materials and methods: GRP78 and necroptosis markers were examined in human bronchial epithelial (HBE) cell line, primary mouse tracheal epithelial cells, and mouse lungs. siRNA targeting GRP78 gene and necroptosis inhibitor were used. Expression of inflammatory cytokines, mucin MUC5AC, and related signaling pathways were detected.Results: Exposure to CS significantly increased the expression of GRP78 and necroptosis markers in HBE cell line, primary mouse tracheal epithelial cells, and mouse lungs. Inhibition of GRP78 significantly suppressed CS extract (CSE)-induced necroptosis. Furthermore, GRP78–necroptosis cooperatively regulated CSE-induced inflammatory cytokines such as interleukin 6 (IL6), IL8, and mucin MUC5AC in HBE cells, likely through the activation of nuclear factor (NF-κB) and activator protein 1 (AP-1) pathways, respectively.Conclusion: Taken together, our results demonstrate that GRP78 promotes CSE-induced inflammatory response and mucus hyperproduction in airway epithelial cells, likely through upregulation of necroptosis and subsequent activation of NF-κB and AP-1 pathways. Thus, inhibition of GRP78 and/or inhibition of necroptosis could be the effective therapeutic approaches for the treatment of COPD. Keywords: cigarette smoke, airway epithelium, glucose-regulated protein 78, necroptosis, airway injury

Cervical cancer is one of the most common gynecological malignancies, and it has become a crucial public health problem. In the present study, the expression profiles of cervical cancer and normal cervical tissues were downloaded from the Gene Expression Omnibus and The Cancer Genome Atlas databases. Subsequently, the dysregulated long non-coding RNAs (lncRNAs) in cervical cancer were identified using R software Differentially expressed lncRNAs in cervical cancer that were associated with glucose-regulated protein 78 (GRP78) were screened out and the results demonstrated that eight lncRNAs were strongly positively correlated with GRP78. In order to confirm the relationship between GRP78 and candidate lncRNAs, GRP78 small interfering RNA (siRNA) was transfected into HeLa cells. The target lncRNAs that were regulated by GRP78 were then identified by reverse transcription-quantitative PCR and it was revealed that LINC00294 was significantly downregulated following GRP78-knockdown. Subsequently, Gene Set Enrichment Analysis demonstrated that LINC00294 was mainly enriched in regulating the cell cycle and the Hedgehog pathway. Following transfection of HeLa and SiHa cells with LINC00294 siRNA, the cell cycle was arrested at the G0/G1 phase. Western blotting suggested that LINC00294-knockdown downregulated the expression of cell cycle-associated factors (cyclin D, cyclin E and cyclin Dependent kinase 4) and upregulated cell cycle inhibitory factors (p16 and p21). The Hedgehog pathway was inhibited following knockdown of LINC00294 in HeLa and SiHa cells. In summary, LINC00294 induced by GRP78 promoted the progression of cervical cancer by regulating the cell cycle via Hedgehog pathway. [ABSTRACT FROM AUTHOR]

To achieve a cure for metastatic breast cancer, further understanding of molecular drivers of the metastatic cascade is essential. Currently, chemotherapy regimens include doxorubicin and paclitaxel which act in part by inducing the unfolded protein response (UPR). The master regulator of the UPR, glucose regulated protein 78 (GRP78), localizes on the surface of tumor cells and is associated with metastatic disease. Cyclic AMP responsive element binding protein 3-like 1 (CREB3L1), a member of the UPR, is a breast cancer metastasis suppressor that acts on cyclic AMP to promote the expression of target genes including GRP78. The aim of the present study was to evaluate the effects of chemotherapy on CREB3L1 and cell-surface GRP78 expression and its association with the development of breast cancer metastasis. For this purpose, we use breast cancer cells migration in vitro assays and an in vivo metastatic mouse model. The results showed that chemotherapy activated CREB3L1 and enhanced cell-surface GRP78 expression specifically in triple-negative breast cancer cells (TNBC), reducing their migration and metastatic potential. CREB3L1 knockout (KO) in the triple negative MDAMB231 cell line using CRISPR/Cas9 technology led to inhibition of GRP78 expression and abrogation of the CREB3L1 metastatic suppression function. Inoculation of CREB3L1-KO MDAMB231 cells into a mouse metastatic model induced a massive metastatic profile which chemotherapy failed to prevent. These findings elucidate a potential pathway to the development of a novel treatment strategy for metastatic TNBC based on modulating CREB3L1 and cell-surface GRP78 expression by chemotherapy and GRP78-targeted drugs. [ABSTRACT FROM AUTHOR]

Aims: Glucose-regulated protein 78 (GRP-78) is one of the basic markers of endoplasmic reticulum (ER) stress in tissues. It is known that ER stress develops in the livers of patients infected with hepatitis C. In this study, the aim was to assess serum GRP78 levels which have not previously been investigated as a stress marker in chronic hepatitis C patients (CHC). Methods: This case control study includes patients with chronic hepatitis C (CHC) infection in our Infectious Diseases clinic (n=60) and a healthy control group without any additional chronic disease (n=60). Serum GRP78 levels were measured with enzyme-linked immunosorbent assay (ELISA), then correlation analysis was performed for serum GRP78 levels with alanine aminotransferase (ALT), aspartate aminotransferase (AST) and HCV-RNA levels. Results: A significant positive correlation was observed between HCV-RNA, ALT and AST levels in CHC patients (P<0.001 and P=0.008, respectively). Serum GRP78 was identified at similar levels in both the control and HCV subgroups. While a significant positive correlation was identified between serum GRP78 and AST levels (P=0.046), no significant correlation was detected for serum ALT levels. Conclusion: Though liver injury induced by HCV is shown to cause ER stress, our results showed there was no significant increase in serum GRP78 levels during chronic HCV infection. Amaç: Glikozla düzenlenen protein 78 (GRP-78) dokudaki endoplazmik retikulum (ER) stresinin temel göstergelerinden birisidir. Hepatit C ile enfekte hastalarda karaciğerde ER stresinin geliştiği bilinmektedir. Bu çalışmada kronik hepatit C (KHC) hastalarında bir stres belirteci olarak daha önce incelenmemiş olan serum GRP78 düzeylerinin değerlendirilmesi amaçlanmıştır. Yöntemler: Çalışmamız, Enfeksiyon Hastalıkları polikliniğimize başvuran Kronik Hepatit C (KHC) enfeksiyonu tanısı almış hasta grubu (n=60) ve ek kronik hastalığı olmayan sağlıklı kontrol (n=60) grubundan oluşan bir vaka kontrol çalışmasıdır. Serum GRP78 seviyesi Enzyme-Linked Immuno Sorbent Assay (ELISA) ile ölçülmüş, ardından GRP78 ile alanin aminotransferaz (ALT), aspartat aminotransferaz (AST), HCV-RNA düzeyleri arasında korelasyon analizi yapılmıştır. Bulgular: KHC hastalarında HCV-RNA düzeyleri ile ALT ve AST düzeyleri arasında anlamlı bir ilişki izlenmiştir (sırasıyla P<0,001 ve P=0,008). Serum GRP78 hem kontrol hem de HCV alt gruplarında benzer seviyelerde saptanmıştır. Serum GRP78 seviyesi ile AST düzeyleri arasında anlamlı bir pozitif korelasyon saptanırken (P=0,046), serum ALT düzeyleri ile anlamlı bir ilişki saptanamamıştır. Sonuç: HCV ile indüklenen karaciğer hasarında ER stresinin geliştiği gösterilmiş olmasına rağmen, sonuçlarımız kronik HCV enfeksiyonu sırasında serum GRP 78 düzeyinde anlamlı bir artış olmadığını göstermektedir. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress plays a pivotal role in the progression of steatohepatitis. 5-aminolevulinic acid (5-ALA), a precursor in the heme biosynthetic pathway, has recently been reported to induce heme oxygenase (HO)-1. HO-1 exerts important cytoprotective actions. In this study, we aimed to explore the therapeutic potential of 5-ALA on palmitate-induced ER stress and lipoapoptosis. Huh-7 cells were treated with palmitic acid (PA) (800 μM) to induce steatosis for eight hours. Steatosis was evaluated by Lipi-green staining. 5-ALA (200 μM) was added with PA. The gene expression levels of the nuclear factor erythroid 2–related factor 2 (NRF2), HO-1, Glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), C/EBP homologous protein (CHOP), and B-cell lymphoma 2 (BCL-2) were evaluated by RT-PCR. Caspase-3/7 activity was evaluated by fluorescein active Caspase-3/7 staining. Cell death was evaluated by Annexin V/SYTOX green staining. PA significantly induced steatosis and increased GRP78 expression in Huh-7 cells. 5-ALA significantly induced HO-1 and decreased GRP78 expression. ATF6 was subsequently decreased. However, NRF2 and CHOP expression were not altered. Anti-apoptotic BCL-2 expression significantly increased, and Caspase 3/7 activity and cell death also decreased. 5-ALA has a therapeutic potential on hepatic steatosis by suppressing ER stress and lipoapoptosis by attenuating GRP78 via HO-1 induction.

Abstract Background Hepatitis 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. Methods GRP78 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. Results GRP78 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. Conclusions GRP78 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.

Numerous studies have shown that topical application of mitomycin C after surgical decompression effectively reduces scar adhesion. However, the underlying mechanisms remain unclear. In this study, we investigated the effect of mitomycin C on the proliferation and apoptosis of human epidural scar fibroblasts. Human epidural scar fibroblasts were treated with various concentrations of mitomycin C (1, 5, 10, 20, 40 μg/mL) for 12, 24 and 48 hours. Mitomycin C suppressed the growth of these cells in a dose- and time-dependent manner. Mitomycin C upregulated the expression levels of Fas, DR4, DR5, cleaved caspase-8/9, Bax, Bim and cleaved caspase-3 proteins, and it downregulated Bcl-2 and Bcl-xL expression. In addition, inhibitors of caspase-8 and caspase-9 (Z-IETD-FMK and Z-LEHD-FMK, respectively) did not fully inhibit mitomycin C-induced apoptosis. Furthermore, mitomycin C induced endoplasmic reticulum stress by increasing the expression of glucose-regulated protein 78, CAAT/enhancer-binding protein homologous protein (CHOP) and caspase-4 in a dose-dependent manner. Salubrinal significantly inhibited the mitomycin C-induced cell viability loss and apoptosis, and these effects were accompanied by a reduction in CHOP expression. Our results support the hypothesis that mitomycin C induces human epidural scar fibroblast apoptosis, at least in part, via the endoplasmic reticulum stress pathway.

Accumulation of misfolded proteins results in cellular stress, and is detected by specific sensors in the endoplasmic reticulum, collectively known as the unfolded protein response (UPR). It has been prominently proposed that the UPR is involved in the pathophysiology of Parkinson’s disease (PD). In the present study, the levels of the UPR proteins and mRNA transcripts were quantified in post mortem brain tissue from PD patients and matched controls. The level of a key mediator of the UPR pathway, glucose-regulated protein 78 (GRP78), was significantly decreased in temporal cortex and cingulate gyrus, whereas there were no significant changes in the caudate nucleus, prefrontal, or parietal cortex regions. On the other hand, GRP78 mRNA level was significantly increased in caudate nucleus, cingulate gyrus, prefrontal, and parietal cortex regions. GRP78 protein level was also measured in plasma and cerebrospinal fluid, but there were no differences in these levels between PD patients and control subjects. Furthermore, immunofluorescence labeling of the CD4+ T cells from PD patients showed that GRP78 protein is found in the cytoplasm. However, GRP78 level in PD patients was not significantly different from control subjects. Unlike the previous Lewy body dementia study, the present investigation reports reduced cortical protein, but increased transcript levels of GPR78 in PD. In summary, these data provide further evidence that GRP78 regulation is dysfunctional in the brains of PD patients.

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.

Accumulation of misfolded proteins results in cellular stress, and is detected by specific sensors in the endoplasmic reticulum, collectively known as the unfolded protein response (UPR). It has been prominently proposed that the UPR is involved in the pathophysiology of Parkinson's disease (PD). In the present study, the levels of the UPR proteins and mRNA transcripts were quantified in post mortem brain tissue from PD patients and matched controls. The level of a key mediator of the UPR pathway, glucose-regulated protein 78 (GRP78), was significantly decreased in temporal cortex and cingulate gyrus, whereas there were no significant changes in the caudate nucleus, prefrontal, or parietal cortex regions. On the other hand, GRP78 mRNA level was significantly increased in caudate nucleus, cingulate gyrus, prefrontal, and parietal cortex regions. GRP78 protein level was also measured in plasma and cerebrospinal fluid, but there were no differences in these levels between PD patients and control subjects. Furthermore, immunofluorescence labeling of the CD4+ T cells from PD patients showed that GRP78 protein is found in the cytoplasm. However, GRP78 level in PD patients was not significantly different from control subjects. Unlike the previous Lewy body dementia study, the present investigation reports reduced cortical protein, but increased transcript levels of GPR78 in PD. In summary, these data provide further evidence that GRP78 regulation is dysfunctional in the brains of PD patients. [ABSTRACT FROM AUTHOR]

Diabetic cardiomyopathy (DCM), one of the common diabetic complications, causes a high rate of mortality in patients with diabetes. Tanshinone IIA (TSIIA), one of the components of Salvia miltiorrhiza (Danshen), has anti-oxidative stress activity and is widely used to treat diabetes-associated diseases. However, its efficacy on DCM remains unclear. The present study aimed to investigate the potential therapeutic function of TSIIA on DCM in an experimental diabetic rat model. Streptozotocin (STZ)-induced diabetic rats were intraperitoneally injected with TSIIA for 6 weeks. The present results indicated that blood glucose concentration was slightly reduced in the low-dose TSIIA treatment group. TSIIA injection was also noted to improve cardiac function, and restore loss of mitochondrial cristae, swollen mitochondrial matrix and disorganized myofibrils in myocardial cells, which are thought to be characteristics of apoptosis. Furthermore, TSIIA injection could increase the activity of superoxide dismutase in STZ-induced diabetic rats, and suppress the endoplasmic reticulum (ER) stress signaling pathway via reducing the expression of glucose-regulated protein 78 and C/EBP homologous protein. These results provide evidence that TSIIA may ameliorate DCM in diabetic rats, possibly via suppressing oxidative stress and ER stress activation. [ABSTRACT FROM AUTHOR]

As a central regulator for endoplasmic reticulum (ER) stress, glucose-regulated protein 78 (GRP78), controls the activation of ER-transmembrane signaling mechanisms by inducing unfolded protein response (UPR) in response to ER stress. Although limited glucose availability often occurs in poorly vascularized solid cancers, cancer cells often initiate the UPR to support cellular homeostasis and survival under stress conditions. Therefore, targeting GRP78 expression and UPR pathway activation may provide a new strategy for anticancer therapy. Based on this premise, we investigated the molecular mechanisms of a synthetic quinolone derivative, 2-hexyl-3-methyl-4(1H)-quinolinone (HHQ-4), in regulating the GRP78 expression and UPR transcriptional program under glucose deprivation or 2-deoxy- d -glucose (2-DG)-stressed conditions. We found that HHQ-4 suppressed the transcriptional and translational expression of GRP78 gene in glucose-deprived breast cancer cells. HHQ-4 also showed selective antiproliferative activity against glucose-deprived breast cancer cells. Constitutive expression of GRP78 completely prevented breast cancer cells from HHQ-4-mediated proliferation inhibition during glucose starvation, stressing the important role of suppression of the GRP78 in HHQ-4-mediated cell proliferation inhibition. HHQ-4 was also found to exert inhibitory activity against breast cancer cell proliferation by suppressing three survival arms of the UPR, including PERK/eIF2α/ATF4, IRE1/XBP1, and ATF6, which orchestrate an intricate signaling network to modulate GRP78 gene transcription under glucose-deprived stress. Furthermore, HHQ-4 combined with 2-DG synergistically inhibited breast cancer cell proliferation. Our findings show HHQ-4 could be a promising candidate, alone or in combination with 2-DG, for selectively inhibiting breast cancer cell proliferation by down-regulating the transcription and expression of GRP78 under stressful microenvironments. Unlabelled Image • HHQ-4 acts as a GRP78 down-regulator during glucose deprivation. • HHQ-4 shows antiproliferative activity against glucose-deprived breast cancer cells. • Overexpressing GRP78 prevents HHQ-4-induced cell proliferation inhibition. • Disruption of UPR signaling is responsible for down-regulation of GRP78 by HHQ-4. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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The occurrence of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVD-19), represents a catastrophic threat to global health. Protruding from the viral surface is a densely glycosylated spike (S) protein, which engages angiotensin-converting enzyme 2 (ACE2) to mediate host cell entry. However, studies have reported viral susceptibility in intra- and extrapulmonary immune and non-immune cells lacking ACE2, suggesting that the S protein may exploit additional receptors for infection. Studies have demonstrated interactions between S protein and innate immune system, including C-lectin type receptors (CLR), toll-like receptors (TLR) and neuropilin-1 (NRP1), and the non-immune receptor glucose regulated protein 78 (GRP78). Recognition of carbohydrate moieties clustered on the surface of the S protein may drive receptor-dependent internalization, accentuate severe immunopathological inflammation, and allow for systemic spread of infection, independent of ACE2. Furthermore, targeting TLRs, CLRs, and other receptors (Ezrin and dipeptidyl peptidase-4) that do not directly engage SARS-CoV-2 S protein, but may contribute to augmented anti-viral immunity and viral clearance, may represent therapeutic targets against COVID-19.

Keywords: lung cancer; lung adenocarcinoma; epithelial mesenchymal transition; hypoxia; glucose-regulated protein 78; GRP78 EN lung cancer lung adenocarcinoma epithelial mesenchymal transition hypoxia glucose-regulated protein 78 GRP78 N.PAG N.PAG 3 12/18/20 20201215 NES 201215 In the original article, there was a mistake in Figure 1 and 2 as published. * P < 0.05, compared with A549 cells in the normal oxygen environments, the Smad2/3, Src, and MAPK proteins of A549 cells are highly regulated and activated in hypoxia environments. * P < 0.05, compared with A549 cells in the normal oxygen environments, the EMT process of A549 cells under hypoxia is activated; # P < 0.05, compared with A549 cells in the hypoxia environments, the EMT process of A549 cells under hypoxia is inhibited separately by Smad2/3, Src, p38, ERK, and JNK inhibitors. [Extracted from the article]

Since 2010, outbreak and spread of tembusu virus (TMUV) caused huge losses to the breeding industry of waterfowl in several provinces of China. In this study, we identify the glucose-regulated protein 78 (GRP78) as a receptor in BHK-21 cells for duck TMUV infection. Using cell membrane from BHK-21 cells, a TMUV-binding protein of approximately 70 kDa was observed by viral overlay protein binding assay (VOPBA). LC-MS/MS analysis and co-immunoprecipitation identified GRP78 as a protein interacting with TMUV. Antibody against GRP78 inhibited the binding of TMUV to the cell surface of BHK-21 cells. Indirect immunofluorescence studies showed the colocalization of GRP78 with TMUV in virus-infected BHK-21 cells. We found that GRP78 over-expression increased TMUV infection, whereas GRP78 knockdown by using a specific small interfering RNA inhibited TMUV infection in BHK-21 cells. Taken together, our results indicate that GRP78 is a novel host factor involved in TMUV entry.

Background: Breast infiltrating ductal carcinoma (BIDC) represents the largest heterotypic tumor group, and an in-depth understanding of the pathogenesis of BIDC is key to improving its prognosis., Aim: To analyze the expression profiles and clinical implications of forkhead box M1 (FOXM1), cyclooxygenase-2 (COX-2), and glucose-regulated protein 78 (GRP78) in BIDC., Methods: A total of 65 BIDC patients and 70 healthy controls who presented to our hospital between August 2019 and May 2021 were selected for analysis. The peripheral blood FOXM1, COX-2, and GRP78 levels in both groups were measured and the association between their expression profiles in BIDC was examined. Additionally, we investigated the diagnostic value of FOXM1, COX-2, and GRP78 in patients with BIDC and their correlations with clinicopathological features. Furthermore, BIDC patients were followed for 1 year to identify factors influencing patient prognosis., Results: The levels of FOXM1, COX-2, and GRP78 were significantly higher in BIDC patients compared to healthy controls ( P < 0.05), and a positive correlation was observed among them ( P < 0.05). Receiver operating characteristic analysis demonstrated that FOXM1, COX-2, and GRP78 had excellent diagnostic value in predicting the occurrence of BIDC ( P < 0.05). Subsequently, we found significant differences in FOXM1, COX-2, and GRP78 levels among patients with different histological grades and metastasis statuses (with vs without) ( P < 0.05). Cox analysis revealed that FOXM1, COX-2, GRP78, increased histological grade, and the presence of tumor metastasis were independent risk factors for prognostic death in BIDC ( P < 0.001)., Conclusion: FOXM1, COX-2, and GRP78 exhibit abnormally high expression in BIDC, promoting malignant tumor development and closely correlating with prognosis. These findings hold significant research implications for the future diagnosis and treatment of BIDC., Competing Interests: Conflict-of-interest statement: We have no financial relationships to disclose., (©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.)

Objective To explore the effect of drug-containing serum of Zhishi (immature hitter orange) on endoplasmic reticulum stress (ERS) injury in rat gastric interstitial cells of Cajal (ICCs). Methods ICCs were isolated by collagenase digestion and identified by immunofluorescence staining. Tunicamycin (Tm) was used to induce ICCs to establish ERS models, the 4-Phenylbutyric acid (4-PBA) was used to block autophagy. Rat drug-containing serum of Zhishi was prepared by using the serum pharmacology routine method. ICCs were randomly divided into four groups after subculture, including the blank group (normal culture), ERS group (the ICCs were stimulated with 1 μg /mL Tm), 4-PBA group (the ICCs were stimulated with 1 μg /mL Tm and 10 mmol/L 4-PBA), and Zhsihi group (the ICCs were stimulated with 1 μg /mL Tm and 20% drug-containing serum), and we continued to cultivate ICCs for 24 h. The ultrastructure of endoplasmic reticulum in ICCs was observed by transmission electron microscopy. The mRNA expression levels of GRP78 and ATF6 were detected by real-time fluorescent quantitative PCR. Results We isolated and identified ICCs successfully. Under transmission electron microscope, we observed that the endoplasmic reticulum of ICCs was normal and well-arranged in the blank group; the number of organelles reduced, and endoplasmic reticulum swelling, dilation, vesiculation changes and disordered arrangement were found in the ERS group; ICCs had endoplasmic reticulum swelling and limitation expansion in the Zhsihi group. Compared with the ICCs group, the mRNA expression of GRP78 and ATF6 increased in the ERS group (both P <0.05). Compared with the ERS group, the mRNA expression of GRP78 and ATF6 decreased in the Zhishi group (both P < 0.05). Conclusion The drug-containing serum of Zhishi can reduce the ERS injury of ICCs possibly by regula-ting the ATF6 signaling pathway of ERS. [ABSTRACT FROM AUTHOR]

Abstract Endoplasmic reticulum (ER) stress, together with unfolded protein response (UPR), can remove unfolded proteins and promote survival. However, severe and prolonged ER stress leads to cell death, tissue injury, and many serious diseases. Therefore, it is essential to identify drugs that can attenuate ER stress for ER-related disease treatment. A great deal of research shows that selenoprotein S (SelS) is a sensitive and ideal marker of ER stress. Here, we used a firefly luciferase reporter driven by SelS gene promoter to screen natural compounds that can attenuate ER stress. Then we identified compound 20(S)-25-methoxyl-dammarane-3β,12β,20-triol (25-OCH 3 -PPD) could inhibit the promoter activity of SelS, further results showed that 25-OCH 3 -PPD effectively inhibited tunicamycin (TM) induced up-regulation of SelS expression in both mRNA and protein levels. Moreover, 25-OCH 3 -PPD significantly inhibited glucose-regulated protein 78 (GRP78; the major ER stress marker) expression in TM-induced ER stress in HepG2 and HEK293T cells, suggesting that 25-OCH 3 -PPD could attenuate ER stress in these cells. Mechanism studies showed that 25-OCH 3 -PPD significantly activated ERK/MAPK signaling pathway, and the inhibition of ERK/MAPK by U0126 dramatically abolished the inhibitory effect of 25-OCH 3 -PPD on ER stress, suggesting that 25-OCH 3 -PPD attenuated ER stress at least partially through activation of ERK/MAPK signaling pathway. Taken together, our studies indicate that 25-OCH 3 -PPD is a novel small molecular compound reducing ER stress, and a potential drug for treating diseases associated with ER stress. [ABSTRACT FROM AUTHOR]

Objective To explore the effect of curcumin on endoplasmic reticulum stress of alveolar epithelial cells in a rat model of chronic obstructive pulmonary disease (COPD). Methods Male SD rats were randomly divided into normal control, COPD model, curcumin-treated and solvent control groups. The rat model of COPD was induced by cigarette smoke exposure combined with intratracheal administration of lipopolysaccharide, and rats were further treated according to different groups. The numbers of total cells, neutrophils and macrophages in bronchoalveolar lavage fluid (BALF) were counted. Pathological changes of lung tissues were studied by HE staining. The endoplasmic reticulum morphology of alveolar epithelial cells was observed using transmission electron microscopy. The protein expression of glucose-regulated protein 78 (GRP78) in alveolar epithelial cells was detected by immunohistochemistry. Results Compared with those in normal control group, the numbers of total cells, neutrophils and macrophages in BALF from rats in COPD model group were all significantly increased (P<0.05). The numbers of total cells, neutrophils and macrophages in BALF from rats in curcumin-treated group were significantly lower than those in COPD model group (P<0.05). Light microscopy and transmission electron microscopy showed enlarged alveolar space and swollen endoplasmic reticulum of alveolar epithelial cells in rats from COPD model group. The damage of alveolar epithelial cells and endoplasmic reticulum in COPD rats was attenuated by curcumin treatment. Compared with that in normal control group, the expression of GRP78 in alveolar epithelial cells in rats from COPD model group was significantly increased (P<0.05). And the protein expression level of GRP78 in alveolar epithelial cells in COPD rats was significantly down-regulated by curcumin treatment (P<0.05). Conclusion Curcumin plays a protective effect against COPD possibly by inhibiting endoplasmic reticulum stress of alveolar epithelial cells. [ABSTRACT FROM AUTHOR]