GRP78 via releasing ß-catenin from adherens junctions facilitates its interaction with STAT3 in mediating retinal neovascularization.

Retinopathy due neovascularization is one of the major causes of vision loss. To understand the mechanisms underlying retinal neovascularization, using oxygen-induced retinopathy (OIR) model, we performed 2D gel-MALDI-TOF/TOF analysis of normoxic and 24-h post OIR mice pups' retinas. 2D gel analysis revealed that GRP78 is one of the several molecules induced by OIR in the retinal ECs. VEGFA also induced GRP78 expression independent of ER stress response in HRMVECs and depletion of its levels reduced VEGFA-induced EC angiogenic responses. Consistent with these observations, EC-specific deletion of GRP78 inhibited OIR-induced retinal neovascularization. In exploring the mechanisms, we found that GRP78 binds with VE-cadherin and releases adherens junction's but not Wnt-mediated ß-catenin and that ß-catenin, in turn, via interacting with STAT3 triggers cyclin D1 expression. Furthermore, depletion of ß-catenin or cyclin D1 levels negated VEGFA-induced EC angiogenic responses and OIR-induced retinal neovascularization. EC-specific deletion of GRP78 also suppressed OIR-induced vascular leakage. In elucidating the upstream signaling, we found that ATF6 mediates GRP78 induction in the modulation of VEGFA-induced EC angiogenic responses and OIR-induced retinal neovascularization. Together, these observations reveal that GRP78 independent of its response to ER stress is involved in mediating EC angiogenic responses by VEGFA and retinal neovascularization by OIR. In view of these findings, it appears that GRP78 could be a desirable target for drug development against diabetic retinopathy.

Effect of kangaroo mother care and white noise on physiological-stress parameters in heel lancing: randomized controlled study.

BACKGROUND: Newborns are exposed to varying degrees of stressful interventions due to procedures such as heel lancing used in routine metabolic screenings. It is an examination of the effects of white noise and kangaroo care on some physiological parameters and stress markers (cortisol and glucose-regulated protein 78-GRP78) in heel lancing in newborns. METHODS: Randomized controlled study was conducted at a gynecology service of a hospital between January and September 2023. 90 babies were divided into three groups: 30 babies in the Kangaroo Care Group (KCG), 30 babies in the White Music Group (WMG), and 30 babies in the Control Group (CG). All babies were randomly divided into groups. Stress parameters were measured by saliva collection method and physiological parameters by saturation device. RESULTS: A statistically significant difference was determined between the total crying time, pulse and saturation values ​​according to the groups (p < 0.001; p = 0.001). A statistically significant difference was determined between the mean values ​​of cortisol and GRP78 measurements according to group and time interaction (p < 0.001). KCG was more effective in reducing total crying time and stabilizing pulse, saturation, salivary cortisol, GRP-78 values compared to WNG and CG. CONCLUSION: It was concluded that white noise and kangaroo care help reduce newborns' stress in the case of heel lancing. PRACTICAL IMPLICATIONS: The practice of kangaroo care and the use of white noise methods may assist healthcare professionals as supportive methods in stress management during invasive procedures. TRIAL REGISTRATION: NCT06278441, registered on 19/02/2024.

Targeting stress induction of GRP78 by cardiac glycoside oleandrin dually suppresses cancer and COVID-19.

BACKGROUND: Despite recent therapeutic advances, combating cancer resistance remains a formidable challenge. The 78-kilodalton glucose-regulated protein (GRP78), a key stress-inducible endoplasmic reticulum (ER) chaperone, plays a crucial role in both cancer cell survival and stress adaptation. GRP78 is also upregulated during SARS-CoV-2 infection and acts as a critical host factor. Recently, we discovered cardiac glycosides (CGs) as novel suppressors of GRP78 stress induction through a high-throughput screen of clinically relevant compound libraries. This study aims to test the possibility that agents capable of blocking stress induction of GRP78 could dually suppress cancer and COVID-19. RESULTS: Here we report that oleandrin (OLN), is the most potent among the CGs in inhibiting acute stress induction of total GRP78, which also results in reduced cell surface and nuclear forms of GRP78 in stressed cells. The inhibition of stress induction of GRP78 is at the post-transcriptional level, independent of protein degradation and autophagy and may involve translational control as OLN blocks stress-induced loading of ribosomes onto GRP78 mRNAs. Moreover, the human Na+/K+-ATPase α3 isoform is critical for OLN suppression of GRP78 stress induction. OLN, in nanomolar range, enhances apoptosis, sensitizes colorectal cancer cells to chemotherapeutic agents, and reduces the viability of patient-derived colon cancer organoids. Likewise, OLN, suppresses GRP78 expression and impedes tumor growth in an orthotopic breast cancer xenograft model. Furthermore, OLN blocks infection by SARS-CoV-2 and its variants and enhances existing anti-viral therapies. Notably, GRP78 overexpression mitigates OLN-mediated cancer cell apoptotic onset and suppression of virus release. CONCLUSION: Our findings validate GRP78 as a target of OLN anti-cancer and anti-viral activities. These proof-of-principle studies support further investigation of OLN as a readily accessible compound to dually combat cancer and COVID-19.

Interaction Between the PERK/ATF4 Branch of the Endoplasmic Reticulum Stress and Mitochondrial One-Carbon Metabolism Regulates Neuronal Survival After Intracerebral Hemorrhage.

Recent investigations have revealed that oxidative stress can lead to neuronal damage and disrupt mitochondrial and endoplasmic reticulum functions after intracerebral hemorrhage (ICH). However, there is limited evidence elucidating their role in maintaining neuronal homeostasis. Metabolomics analysis, RNA sequencing, and CUT&Tag-seq were performed to investigate the mechanism underlying the interaction between the PERK/ATF4 branch of the endoplasmic reticulum stress (ERS) and mitochondrial one-carbon (1C) metabolism during neuronal resistance to oxidative stress. The association between mitochondrial 1C metabolism and the PERK/ATF4 branch of the ERS after ICH was investigated using transcription factor motif analysis and co-immunoprecipitation. The findings revealed interactions between the GRP78/PERK/ATF4 and mitochondrial 1C metabolism, which are important in preserving neuronal homeostasis after ICH. ATF4 is an upstream transcription factor that directly regulates the expression of 1C metabolism genes. Additionally, the GRP78/PERK/ATF4 forms a negative regulatory loop with MTHFD2 because of the interaction between GRP78 and MTHFD2. This study presents evidence of disrupted 1C metabolism and the occurrence of ERS in neurons post-ICH. Supplementing exogenous NADPH or interfering with the PERK/ATF4 could reduce symptoms related to neuronal injuries, suggesting new therapeutic prospects for ICH.

Regulation of lipid metabolism in grass carp primary hepatocytes by exosomes derived from fatty hepatocytes though GRP78.

Exosomes regulate lipid metabolism by carrying miRNAs, nucleic acids, and proteins, thereby influencing the function of receptor cells. Glucose-regulated protein 78 (GRP78) is also involved in the regulation of lipid metabolism. However, it remains unclear whether exosomes derived from fatty hepatocytes (OA-Exo) regulate lipid metabolism through the enrichment of GRP78. In this study, we observed the expression of GRP78 was significantly increased in fatty hepatocytes (incubating hepatocytes with oleic acid (OA) for 24 h) and OA-Exo (P < 0.05). In addition, OA-Exo (50 µg/mL) and GRP78 protein (1 µg/mL) significant increased the content of triacylglycerol (TG) and total cholesterol (TC), as well as up-regulated the expression of GRP78 and inositol-requiring enzyme-1alpha (IRE1α) protein (P < 0.05). We further used YUM70 (an inhibitor of GRP78) to inhibit endogenous GRP78, and compared with the YUM70 group, OA-Exo reversed the effect of YUM70 and increased the content of TG, TC, and the expression of GRP78 protein in hepatocytes (P < 0.05). Furthermore, the inhibition of the IRE1α pathway with 4µ8C resulted in a significant decrease in TG content compared to the control group (P < 0.05). However, when compared with the 4µ8C group, OA-Exo and GRP78 reversed the effect of 4µ8C and significantly increased TG content (P < 0.05). Taken together, these results indicated that OA-Exo activated IRE1α to promote lipid accumulation in hepatocytes through the enrichment of GRP78. This study provided a new perspective for further exploration of exosomal lipid metabolism in fish.

Small Molecules Targeting GRP78 Mitigate Anti- GRP78 Autoantibody-Mediated Tissue Factor Procoagulant Activity in Cultured Endothelial Cells.

BACKGROUND: The 78-kDa glucose-regulated protein (GRP78) expressed on the cell surface (csGRP78) has been reported to regulate tissue factor procoagulant activity (TF PCA) in lesion-resident endothelial cells (ECs), which is further enhanced by circulating anti-GRP78 autoantibodies that bind to the Leu98-Leu115 epitope in GRP78. OBJECTIVES: Determine the effects of the engagement of the anti-GRP78 autoantibody to cell surface GRP78 on endothelial cells and the underlying mechanisms that impact TF PCA. METHODS: Immunofluorescent staining was used to determine the presence of csGRP78 in TNFα-treated endothelial cells. An established TF PCA assay was used to evaluate human ECs following treatment with anti-GRP78 autoantibodies. The Fura 2-AM assay was used to quantify changes in intracellular Ca2+ levels. Small molecules predicted to bind csGRP78 were identified using artificial intelligence. ELISAs were used to assess the ability of these GRP78 binders to mitigate TF activity and interfere with the autoantibody/csGRP78 complex. RESULTS: In TNFα-treated ECs, anti-GRP78 autoantibodies increased TF PCA. This observation was further enhanced by ER-stress-induced elevation of csGRP78 levels. Anti-GRP78 autoantibody treatment increased intracellular Ca2+ levels. Sequestering the anti-GRP78 autoantibody with a conformational peptide or blocking with heparin molecules attenuated anti-GRP78 autoantibody-induced TF PCA. We identified B07*, a GRP78 binder that diminished anti-GRP78 autoantibody-induced TF PCA on ECs. CONCLUSIONS: These findings show how anti-GRP78 autoantibodies enhance TF PCA that contributes to thrombosis and identify novel GRP78 binders that represent a potential novel therapeutic strategy for treating and managing atherothrombotic disease.

Eurycomanone inhibits osteosarcoma growth and metastasis by suppressing GRP78 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Osteosarcoma (OS) is characterized by rapid growth and frequent pulmonary metastasis. Eurycoma longifolia Jack, a flowering plant primarily found in Southeast Asian countries, is commonly used in traditional herbal medicine. Its root extract is mainly used for against cancer, malaria, parasites and other conditions. The active compound in its root extract, eurycomanone (EUR), has been proven to inhibit lung and liver cancer proliferation. AIM OF THE STUDY: Our research aimed to investigate the inhibitory effect and underlying molecular mechanism of EUR on OS growth and metastasis. MATERIALS AND METHODS: In vitro experiments: western blotting (WB) screened 41 compounds that inhibited GRP78 expression and evaluated the protein levels of GRP78, PARP, cleaved-PARP, MMP2, and MMP9. Cell proliferation was evaluated using CCK-8, EdU, colony formation assay, and cell apoptosis was assessed by flow cytometry. Transwell, wound healing, and tube formation assays were performed to determine the effect of EUR on tumor invasion, migration, and angiogenesis, respectively. Quantitative real-time polymerase chain (qRT-PCR) and dual-luciferase activity assays detected GRP78 mRNA stability and transcription levels post-EUR and thapsigargin treatment. RNA-Seq identified signaling pathways inhibited by EUR. In vivo experiments: effects of EUR in mice were evaluated by H&E staining to detect lung metastasis and potential toxic effects in tissues. Immunohistochemical (IHC) staining detected the expression of Ki-67, CD31, and cleaved caspase-3 in tumors. RESULTS: GRP78 is highly expressed in OS and correlated with poor prognosis. In vitro, eurycomanone (EUR) significantly downregulated GRP78 expression, inhibited cell proliferation, migration, invasion, tube formation, and induced apoptosis. Moreover, it enhanced trichostatin A (TSA) sensitivity and exhibited inhibitory effects on other cancer types. Mechanistically, EUR decreased GRP78 mRNA stability and transcription. In vivo, EUR inhibited proliferation and invasion in tibial and PDX models. CONCLUSIONS: Our study demonstrated that EUR inhibits the growth and metastasis of OS by reducing GRP78 mRNA stability and inhibiting its transcription, which offers a novel approach for clinical treatment of OS.

BRD4 absence inactivates endoplasmic reticulum stress to retard dehydroepiandrosterone-triggered ovarian granular cell apoptosis in polycystic ovary syndrome via GRP78.

Polycystic ovary syndrome (PCOS) is a hormonal disorder and significantly affects reproductive and metabolic function. Bromodomain-containing protein 4 (BRD4) is reported to promote ovarian fibrosis in PCOS. The present work was conducted to investigate the detailed role of BRD4 and the corresponding functional mechanism in PCOS. Functional experiments including CCK-8 method, EDU staining and TUNEL staining were used to detect the key cellular processes. Western blot examined the expression of BRD4, apoptosis- and endoplasmic reticulum stress (ERS)-associated proteins. HDOCK server predicted the binding of BRD4 with Glucose-Regulated Protein 78 (GRP78), which was validated by Co-IP assay. BRD4 expression was increased and ERS was activated in dehydroepiandrosterone (DHEA)-induced KGN cells. Inhibition of BRD4 improved the viability whereas it inhibited the apoptosis and ERS of KGN cells induced by DHEA. In addition, BRD4 bound to GRP78. GRP78 elevation or ERS activator tunicamycin (TM) partly abolished the impacts of BRD4 silencing on the ERS, proliferation and apoptosis in DHEA-treated KGN cells. Anyway, knockdown of BRD4 may reduce DHEA-induced ovarian granular cell damage in PCOS via inactivating GRP78-mediated ERS.

Grp78 regulates NLRP3 inflammasome and participates in Sjogren's syndrome.

OBJECTIVE: The purpose of the present study was to potential effects of forsythiaside A (FA) on Sjogren's syndrome (SS). METHODS: Enzyme linked immunosorbent assay for detecting cytokines and Western blotting was used for detecting related protein expression. RESULTS: FA effectively reduced the secretion of inflammatory cytokines, the expression of Caspase-1 and NLRP3 proteins and the expression of p65 in SS. FA also effectively inhibited the high expression of Grp78 in SS. When Grp78 expression was silenced, it effectively reduced the secretion of inflammatory cytokines, the expression of Caspase-1 and NLRP3 proteins and the expression of p65 in the nucleus in SS. FA effectively inhibit the secretion of inflammatory cytokines induced by overexpression of Grp78, the expression of Caspase-1 and NLRP3 proteins and the expression of p65 in the nucleus in SS. CONCLUSION: FA induces the degradation of Grp78 protein, regulates the NF-κB signaling pathway in SS and inhibited NLRP3 inflammasome activation and reduced the release of inflammatory cytokines to alleviate SS.

Allicin affects immunoreactivity of osteosarcoma cells through lncRNA CBR3-AS1.

Objective: To analyze the effect of allicin on the immunoreactivity of osteosarcoma (OS) cells and further explore whether its mechanism is related to the long non-coding Ribonucleic Acid (lncRNA) CBR3-AS1/miR-145-5p/GRP78 axis, so as to provide clinical evidence. Methods: The human OS cell line Saos-2 was treated with allicin at 25, 50, and 100 µmol/L, respectively, to observe changes in cell biological behaviors. Subsequently, CBR3-AS1 abnormal expression vectors were constructed and transfected into Saos-2 to discuss their influence on OS. Furthermore, the regulatory relationship between allicin and the CBR3-AS1/miR-145-5p/GRP78 axis was validated by rescue experiments. Finally, a nude mice tumorigenesis experiment was carried out to analyze the effects of allicin and CBR3-AS1/miR-145-5p/GRP78 axis on the growth of living tumors. Alterations in T-lymphocyte subsets were also detected to assess the effect of allicin on OS immunoreactivity. Results: With the increase of allicin concentration, Saos-2 activity decreased and apoptosis increased (P < 0.05). In addition, the expression of CBR3-AS1 and GRP78 decreased after allicin intervention, while miR-145-5p increased (P < 0.05). Silencing CBR3-AS1 led to reduced Saos-2 activity, enhanced apoptosis, and activated mitophagy and endoplasmic reticulum stress (P < 0.05). In the rescue experiment, the effect of CBR3-AS1 on OS cells was reversed by silencing miR-145-5p, while the impact of miR-145-5p was reversed by GRP78. Finally, the tumorigenesis experiment in nude mice confirmed the regulatory effects of allicin and CBR3-AS1/miR-145-5p/GRP78 on tumor growth in vivo. Meanwhile, it was seen that allicin activated CD4+CD8+ in OS mice, confirming that allicin has the effect of activating OS immunoreactivity. Conclusions: Allicin activates OS immunoreactivity and induces apoptosis through the CBR3-AS1/miR-145-5p/GRP78 molecular axis.

A New Histology-Based Prognostic Index for Aggressive T-Cell lymphoma: Preliminary Results of the "TCL Urayasu Classification".

Background: Aggressive mature T-cell lymphoma (TCL) is a disease that carries a poor prognosis. Methods: We analyzed the expression of 22 tumor cell functional proteins in 16 randomly selected patients with TCL. Immunohistochemistry was performed in paraffin-embedded tumor tissue sections to determine the protein expression statuses in tumor cells. Results: Glucose-regulated protein 94 (GRP94), a protein that serves as a pro-survival component under endoplasmic reticulum (ER) stress in the tumor microenvironment, was significantly associated with a shortened survival. Furthermore, significant differences were observed when GRP94 was combined with six other factors. The six factors were (1) programmed cell death-ligand 1 (PD-L1); (2) programmed cell death 1 (PD-1); (3) aldo-keto reductase family 1 member C3 (AKR1C3); (4) P53, a tumor suppressor; (5) glucose-regulated protein 78 (GRP78), an ER stress protein; and (6) thymidine phosphorylase (TP). Based on the combination of GRP94 and the six other factors expressed in the tumors, we propose a new prognostic classification system for TCL (TCL Urayasu classification). Group 1 (relatively good prognosis): GRP94-negative (n = 6; median OS, 88 months; p < 0.01); Group 2 (poor prognosis): GRP94-positive, plus expression of two of the six factors mentioned above (n = 5; median OS, 25 months; p > 0.05); and Group 3 (very poor prognosis): GRP94-positive, plus expression of at least three of the six factors mentioned above (n = 5; median OS, 10 months; p < 0.01). Conclusions: Thus, the TCL Urayasu prognostic classification may be a simple, useful, and innovative classification that also explains the mechanism of resistance to treatment for each functional protein. If validated in a larger number of patients, the TCL Urayasu classification will enable a targeted treatment using selected inhibitors acting on the abnormal protein found in each patient.

Immunoexpression Pattern of Autophagy-Related Proteins in Human Congenital Anomalies of the Kidney and Urinary Tract.

The purpose of this study was to evaluate the spatiotemporal immunoexpression pattern of microtubule-associated protein 1 light chain 3 beta (LC3B), glucose-regulated protein 78 (GRP78), heat shock protein 70 (HSP70), and lysosomal-associated membrane protein 2A (LAMP2A) in normal human fetal kidney development (CTRL) and kidneys affected with congenital anomalies of the kidney and urinary tract (CAKUT). Human fetal kidneys (control, horseshoe, dysplastic, duplex, and hypoplastic) from the 18th to the 38th developmental week underwent epifluorescence microscopy analysis after being stained with antibodies. Immunoreactivity was quantified in various kidney structures, and expression dynamics were examined using linear and nonlinear regression modeling. The punctate expression of LC3B was observed mainly in tubules and glomerular cells, with dysplastic kidneys displaying distinct staining patterns. In the control group's glomeruli, LAMP2A showed a sporadic, punctate signal; in contrast to other phenotypes, duplex kidneys showed significantly stronger expression in convoluted tubules. GRP78 had a weaker expression in CAKUT kidneys, especially hypoplastic ones, while normal kidneys exhibited punctate staining of convoluted tubules and glomeruli. HSP70 staining varied among phenotypes, with dysplastic and hypoplastic kidneys exhibiting stronger staining compared to controls. Expression dynamics varied among observed autophagy markers and phenotypes, indicating their potential roles in normal and dysfunctional kidney development.

The regulation of the thermal stability and affinity of the HSPA5 (Grp78/BiP) by clients and nucleotides is modulated by domains coupling.

The HSPA5 protein (BiP/Grp78) serves as a pivotal chaperone in maintaining cellular protein quality control. As a member of the human HSP70 family, HSPA5 comprises two distinct domains: a nucleotide-binding domain (NBD) and a peptide-binding domain (PBD). In this study, we investigated the interdomain interactions of HSPA5, aiming to elucidate how these domains regulate its function as a chaperone. Our findings revealed that HSPA5-FL, HSPA5-T, and HSPA5-N exhibit varying affinities for ATP and ADP, with a noticeable dependency on Mg2+ for optimal interactions. Interestingly, in ADP assays, the presence of the metal ion seems to enhance NBD binding only for HSPA5-FL and HSPA5-T. Moreover, while the truncation of the C-terminus does not significantly impact the thermal stability of HSPA5, experiments involving MgATP underscore its essential role in mediating interactions and nucleotide hydrolysis. Thermal stability assays further suggested that the NBD-PBD interface enhances the stability of the NBD, more pronounced for HSPA5 than for the orthologous HSPA1A, and prevents self-aggregation through interdomain coupling. Enzymatic analyses indicated that the presence of PBD enhances NBD ATPase activity and augments its nucleotide affinity. Notably, the intrinsic chaperone activity of the PBD is dependent on the presence of the NBD, potentially due to the propensity of the PBD for self-oligomerization. Collectively, our data highlight the pivotal role of allosteric mechanisms in modulating thermal stability, nucleotide interaction, and ATPase activity of HSPA5, underscoring its significance in protein quality control within cellular environments.

GRP78 inhibitor YUM70 upregulates 4E-BP1 and suppresses c-MYC expression and viability of oncogenic c-MYC tumors.

The 78-kDa glucose regulated protein (GRP78) commonly upregulated in a wide variety of tumors is an important prognostic marker and a promising target for suppressing tumorigenesis and treatment resistance. While GRP78 is well established as a major endoplasmic reticulum (ER) chaperone with anti-apoptotic properties and a master regulator of the unfolded protein response, its new role as a regulator of oncoprotein expression is just emerging. MYC is dysregulated in about 70 % of human cancers and is the most commonly activated oncoprotein. However, despite recent advances, therapeutic targeting of MYC remains challenging. Here we identify GRP78 as a new target for suppression of MYC expression. Using multiple MYC-dependent cancer models including head and neck squamous cell carcinoma and their cisplatin-resistant clones, breast and pancreatic adenocarcinoma, our studies revealed that GRP78 knockdown by siRNA or inhibition of its activity by small molecule inhibitors (YUM70 or HA15) reduced c-MYC expression, leading to onset of apoptosis and loss of cell viability. This was observed in 2D cell culture, 3D spheroid and in xenograft models. Mechanistically, we determined that the suppression of c-MYC is at the post-transcriptional level and that YUM70 and HA15 treatment potently upregulated the eukaryotic translation inhibitor 4E-BP1, which targets eIF4E critical for c-MYC translation initiation. Furthermore, knock-down of 4E-BP1 via siRNA rescued YUM70-mediated c-MYC suppression. As YUM70 is also capable of suppressing N-MYC expression, this study offers a new approach to suppress MYC protein expression through knockdown or inhibition of GRP78.

Short and long-term 2100 MHz radiofrequency radiation causes endoplasmic reticulum stress in rat testis.

Long-term radiofrequency radiation (RFR) exposure, which adversely affects organisms, deteriorates testicular functions. Misfolding or unfolding protein accumulation in the endoplasmic reticulum (ER) initiates an intracellular reaction known as ER stress (ERS), which activates the unfolded protein response (UPR) for proteostasis. Since both RFR exposure and ERS can cause male infertility, we hypothesized that RFR exposure causes ERS to adversely affect testicular functions in rats. To investigate role of ERS in mediating RFR effects on rat testis, we established five experimental groups in male rats: control, short-term 2100-megahertz (MHz) RFR (1-week), short-term sham (sham/1-week), long-term 2100-MHz RFR (10-week), and long-term sham (sham/10-week). ERS markers Grp78 and phosphorylated PERK (p-Perk) levels and ERS-related apoptosis markers Chop and caspase 12 were investigated by immunohistochemistry, immunoblotting, and quantitative real-time polymerase chain reaction (qPCR). Long-term RFR exposure increased Grp78, p-Perk, and Chop levels, while short-term RFR exposure elevated Chop and caspase 12 levels. Chop expression was not observed in spermatogonia and primary spermatocytes, which may protect spermatogonia and primary spermatocytes against RFR-induced ERS-mediated apoptosis, thereby allowing transmission of genetic material to next generations. While short and long-term RFR exposures trigger ERS and ERS-related apoptotic pathways, further functional analyses are needed to elucidate whether this RFR-induced apoptosis has long-term male infertility effects.

Flow shear force destabilizes carotid plaques by affecting CHOP and GRP78 proteins.

BACKGROUND: Various factors, including blood, inflammatory, infectious, and immune factors, can cause ischemic stroke. However, the primary cause is often the instability of cervical arteriosclerosis plaque. It is estimated that 18-25% of ischemic strokes are caused by the rupture of carotid plaque.1 Plaque stability is crucial in determining patient prognosis. Developing a highly accurate, non-invasive, or minimally invasive technique to assess carotid plaque stability is crucial for diagnosing and treating stroke.Previous research by our group has demonstrated that the expression levels of CHOP (C/EBP homologous protein) and GRP78 (glucose-regulated protein 78) are correlated with the stability of atherosclerotic plaques.2 OBJECT: This research assesses changes in GRP78 and CHOP expressions in human umbilical vein endothelial cells(HUVEC) following experiments within the hemodynamic influencing factors test system. Additionally, it includes conducting an empirical study on the impact of blood flow shear force on the stability of human carotid atherosclerotic plaques. The objective is to explore the implications of blood flow shear force on the stability of carotid atherosclerotic plaques. METHOD: The hemodynamic influencing factors test bench system was configured with low (Group A, 4 dyns/cm²), medium (Group B, 8 dyns/cm²), and high shear force groups (Group C, 12 dyns/cm²). Relative expression levels of GRP78 and CHOP proteins in human umbilical vein endothelial cells were measured using Western blot analysis, and quantitative analysis of GRP78 and CHOP mRNA was conducted using RT-qPCR. Meanwhile, plaques from 60 carotid artery patients, retrieved via Carotid Endarterectomy (CEA), were classified into stable (S) and unstable (U) groups based on pathological criteria. Shear force at the carotid bifurcation was measured preoperatively using ultrasound. Western blot and RT-qPCR were used to analyze the relative expression levels of GRP78 and CHOP proteins and mRNA, respectively, in the plaque specimens from both groups. RESULT: Expression levels of GRP78, CHOP proteins, and their mRNAs were assessed in groups A, B, and C via Western blot and RT-qPCR. Results showed that in the low-shear group, all markers were elevated in group A compared to groups B and C. Statistical analysis revealed significantly lower shear forces at the carotid bifurcation in group U compared to group S. In group U plaques, GRP78 and CHOP expressions were significantly higher in group U than in group S. CONCLUSION: Blood flow shear forces variably affect the expression of GRP78 and CHOP proteins, as well as their mRNA levels, in vascular endothelial cells. The lower the shear force and fluid flow rate, the higher the expression of GRP78 and CHOP, potentially leading to endoplasmic reticulum stress(ERS), which may destabilize the plaque.

Metformin augments major cytoplasmic organization except for spindle organization in oocytes cultured under hyperglycemic and hyperlipidemic conditions: An in vitro study.

The present study aimed to investigate the role of antidiabetic drug metformin on the cytoplasmic organization of oocytes. Germinal vesicle (GV) stage oocytes were collected from adult female Swiss albino mice and subjected to in vitro maturation (IVM) in various experimental groups- control, vehicle control (0.3% ethanol), metformin (50 µg/mL), high glucose and high lipid (HGHL, 10 mM glucose; 150 µM palmitic acid; 75 µM stearic acid and 200 µM oleic acid in ethanol), and HGHL supplemented with metformin. The metaphase II (MII) oocytes were analyzed for lipid accumulation, mitochondrial and endoplasmic reticulum (ER) distribution pattern, oxidative and ER stress, actin filament organization, cortical granule distribution pattern, spindle organization and chromosome alignment. An early polar body extrusion was observed in the HGHL group. However, the maturation rate at 24 h did not differ significantly among the experimental groups compared to the control. The HGHL conditions exhibited significantly higher levels of oxidative stress, ER stress, poor actin filament organization, increased lipid accumulation, altered mitochondrial distribution, spindle abnormalities, and chromosome misalignment compared to the control. Except for spindle organization, supplementation of metformin to the HGHL conditions improved all the parameters (non-significant for ER and actin distribution pattern). These results show that metformin exposure in the culture media helped to improve the hyperglycemia and hyperlipidemia-induced cytoplasmic anomalies except for spindle organization. Given the crucial role of spindle organization in proper chromosome segregation during oocyte maturation and meiotic resumption, the implications of metformin's limitations in this aspect warrant careful evaluation and further investigation.

p20BAP31 promotes cell apoptosis via interaction with GRP78 and activating the PERK pathway in colorectal cancer.

Colorectal cancer (CRC) is the second most deadly cancer worldwide. Although various treatments for CRC have made progress, they have limitations. Therefore, the search for new effective molecular targets is important for the treatment of CRC. p20BAP31 induces apoptosis through diverse pathways and exhibits greater sensitivity in CRC. Therefore, a comprehensive exploration of the molecular functions of p20BAP31 is important for its application in anti-tumor therapy. In this study, we showed that exogenous p20BAP31 was still located in the ER and significantly activated the unfolded protein response (UPR) through the PERK pathway. The activation of the PERK pathway is prominent in p20BAP31-induced reactive oxygen species (ROS) accumulation and apoptosis. We found, for the first time, that p20BAP31 leads to ER stress and markedly attenuates tumor cell growth in vivo. Importantly, mechanistic investigations indicated that p20BAP31 competitively binds to GRP78 from PERK and causes hyperactivation of the UPR. Furthermore, p20BAP31 upregulates the expression of GRP78 by promoting HSF1 nuclear translocation and enhancing its binding to the GRP78 promoter. These findings reveal p20BAP31 as a regulator of ER stress and a potential target for tumor therapy, and elucidate the underlying mechanism by which p20BAP31 mediates signal transduction between ER and mitochondria.

Inhibition of endoplasmic reticulum stress and excessive autophagy by Jiedu Tongluo Tiaogan Formula via a CaMKKß/AMPK pathway contributes to protect pancreatic ß-cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiedu Tongluo Tiaogan Formula (JTTF), a traditional Chinese herbal decoction, exhibits the potential to treat type 2 diabetes mellitus (T2DM) by inhibiting endoplasmic reticulum stress (ERS) and excessive autophagy, which are the risk factors for the abnormal development and progression of ß cells. AIM OF THE STUDY: We aimed to assess the effect of JTTF on pancreatic glucotoxicity by inhibiting ERS and excessive autophagy, for which db/db mice and INS-1 insulinoma cells were used. MATERIALS AND METHODS: The chemical composition of the JTTF was analyzed by UPLC-Q/TOF-MS. Diabetic (db/db) mice were treated with distilled water or JTTF (2.4 and 7.2 g/kg/day) for 8 weeks. Furthermore, INS-1 cells induced by high glucose (HG) levels were treated with or without JTTF (50, 100, and 200 µg/mL) for 48 h to elucidate the protective mechanism of JTTF on glucose toxicity. The experimental methods included an oral glucose tolerance test, hematoxylin-eosin staining, immunohistochemistry, western blotting, RT-qPCR, and acridine orange staining. RESULT: 28 chemical components of JTTF were identified. Additionally, treatment with JTTF significantly decreased the severity of glycemic symptoms in the db/db mice. Moreover, the treatment partially restored glucose homeostasis in the db/db mice and protected the pancreatic ß-cell function. JTTF protected INS-1 cells from HG injury by upregulating GSIS and PDX1, MafA mRNA expression. Further, treatment with JTTF downregulated GRP78 and ATF6 expression, whereas it inhibited Beclin-1 and LC3 activation. The treatment protected the cells from HG-induced ERS and excessive autophagy by downregulating the CaMKKß/AMPK pathway. CONCLUSIONS: The present study findings show that JTTF may protects ß-cells by inhibiting the CaMKKß/AMPK pathway, which deepens our understanding of the effectiveness of JTTF as a treatment strategy against T2DM.

PBI-05204, a supercritical CO2 extract of Nerium oleander, suppresses glioblastoma stem cells by inhibiting GRP78 and inducing programmed necroptotic cell death.

Successful treatment of glioblastoma multiforme (GBM), an aggressive form of primary brain neoplasm, mandates the need to develop new therapeutic strategies. In this study, we investigated the potential of PBI-05204 in targeting GBM stem cells (GSCs) and the underlying mechanisms. Treatment with PBI-05204 significantly reduced both the number and size of tumor spheres derived from patient-derived GSCs (GBM9, GSC28 and TS543), and suppressed the tumorigenesis of GBM9 xenografts. Moreover, PBI-05204 treatment led to a significant decrease in the expression of CD44 and NANOG, crucial markers of progenitor stem cells, in GBM9 and GSC28 GSCs. This treatment also down-regulated GRP78 expression in both GSC types. Knocking down GRP78 expression through GRP78 siRNA transfection in GBM9 and GSC28 GSCs also resulted in reduced spheroid size and CD44 expression. Combining PBI-05204 with GRP78 siRNA further decreased spheroid numbers compared to GRP78 siRNA treatment alone. PBI-05204 treatment led to increased expression of pRIP1K and pRIP3K, along with enhanced binding of RIPK1/RIPK3 in GBM9 and GSC28 cells, resembling the effects observed in GRP78-silenced GSCs, suggesting that PBI-05204 induced necroptosis in these cells. Furthermore, oleandrin, a principle active cardiac glycoside component of PBI-05204, showed the ability to inhibit the self-renewal capacity in GSCs. These findings highlight the potential of PBI-05204 as a promising candidate for the development of novel therapies that target GBM stem cells.