Endoplasmic Reticulum Stress-related Cell Death Research Articles

Evaluating the effect of basic fibroblast growth factor on the progression of NASH disease by inhibiting ceramide synthesis and ER stress-related pathways

Non-alcoholic steatohepatitis (NASH) is associated with intrahepatic lipid accumulation, inflammation, and hepatocyte death. Several studies have indicated that high-fat diets increase ceramide synthases-6 (CerS-6) expression and a concomitant elevation of C16-ceramides, which can modulate endoplasmic reticulum (ER) stress and further contribute to the progression of NASH. Ceramide levels have reportedly been impacted by basic fibroblast growth factor (bFGF) in various diseases. This study looked into the role of bFGF on CerS6/C16-ceramide and ER stress-related pathways in a mouse model of NASH. Male C57BL/6J mice were fed a western diet (WD) combined with carbon tetrachloride (CCl4) for eight weeks. Next, bFGF was injected into the NASH mice for seven days of continuous treatment. The effects of bFGF on NASH endpoints (including steatosis, inflammation, ballooning, and fibrosis), ceramide levels and ER-stress-induced inflammation, reactive oxygen species (ROS) production, and apoptosis were evaluated. Treatment with bFGF significantly reduced CerS-6/C16-ceramide. Further, the inflammatory condition was alleviated with reduction of nuclear factor-kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6) gene expression. ROS level was also reduced. ER stress-related cell death diminished by reducing C/EBP homologous protein (CHOP) mRNA expression and caspase 3 activity. Furthermore, activation of the hepatic stellate cells was inhibited in the bFGF-treated mice by lowering the amount of alpha-smooth muscle actin (α-SMA) at the mRNA and protein level. According to our findings, CerS-6/C16-ceramide alteration impacts ER stress-mediated inflammation, oxidative stress, and apoptosis. The bFGF treatment effectively attenuated the development of NASH by downregulating CerS-6/C16-ceramide and subsequent ER stress-related pathways.

51-OR: Loss of Critical ER Chaperone GRP78 Leads to Beta-Cell Death by a JNK Dependent Mechanism

Endoplasmic reticulum (ER) health is an important determinant of pancreatic beta cell function or failure. Adaptive ER stress leads to expansion of beta cell number through proliferation, but decompensated ER stress results in beta cell loss and contributes to both T1D and T2D. Therefore, understanding the mechanisms of ER-stress-induced beta cell death could help identify novel strategies for the prevention or treatment of diabetes. Glucose responsive protein GRP78/HSPA5 is a key ER chaperone. Loss of GRP78 causes cell death in many cell types; including, in our own prior work, the beta cell. To study the mechanism by which GRP78 loss leads to beta cell death we devised an ex vivo model of GRP78 knockdown by transducing Grp78-flox mouse islets with adenovirus expressing Cre recombinase. To study human islets, we used adenovirus expressing shRNA against GRP78. Using these tools, we successfully reduced GRP78 RNA and protein in primary mouse and human islet cells. Loss of GRP78 strongly activated the ATF6, IRE1 and PERK pathways of the unfolded protein response (UPR). GRP78 knockdown decreased beta cell number due to increased cell death as measured by Annexin V and TUNEL staining. Probing the mechanisms of cell loss, we found that loss of GRP78 activated multiple death-related pathways, notably autophagy and apoptosis; cell death effectors Chop, caspase 9 and cleaved caspase 3 were increased. Additionally, GRP78 deletion increased stress kinase JNK (c-Jun-N-terminal kinase) which has been implicated in cellular proliferation as well as cell death. Inhibition of JNK kinase using JNK-IN-8 inhibitor during GRP78 loss conditions decreased phosphorylation of JNK and rescued beta cells from cell death as measured with Annexin V and TUNEL. Therefore, we conclude that ER stress related cell death induced by loss of GRP78 (ER stress) involves JNK activation, and that modulation of JNK under certain circumstances may help maintain beta cell mass to protect against diabetes. Disclosure R.B. Sharma: None. C.O. Darko: None. B. Gablaski: None. A.S. Lee: None. L.C. Alonso: Consultant; Self; Fairbanks Pharmaceuticals Inc. Research Support; Self; American Diabetes Association. Funding National Institute of Diabetes and Digestive and Kidney Diseases (5R01DK113300-02)

Guanabenz Acetate Induces Endoplasmic Reticulum Stress–Related Cell Death in Hepatocellular Carcinoma Cells

BackgroundDevelopment of chemotherapeutics for the treatment of advanced hepatocellular carcinoma (HCC) has been lagging. Screening of candidate therapeutic agents by using patient-derived preclinical models may facilitate drug discovery for HCC patients.MethodsFour primary cultured HCC cells from surgically resected tumor tissues and six HCC cell lines were used for high-throughput screening of 252 drugs from the Prestwick Chemical Library. The efficacy and mechanisms of action of the candidate anti-cancer drug were analyzed via cell viability, cell cycle assays, and western blotting.ResultsGuanabenz acetate, which has been used as an antihypertensive drug, was screened as a candidate anti-cancer agent for HCC through a drug sensitivity assay by using the primary cultured HCC cells and HCC cell lines. Guanabenz acetate reduced HCC cell viability through apoptosis and autophagy. This occurred via inhibition of growth arrest and DNA damage-inducible protein 34, increased phosphorylation of eukaryotic initiation factor 2α, increased activating transcription factor 4, and cell cycle arrest.ConclusionsGuanabenz acetate induces endoplasmic reticulum stress–related cell death in HCC and may be repositioned as an anti-cancer therapeutic agent for HCC patients.

Effects of Heat Stress Treatment on Age-dependent Unfolded Protein Response in Different Types of Skeletal Muscle.

Mitochondrial and endoplasmic reticulum (ER) stress, and subsequently activated responses (mitochondrial/ER unfolded protein responses; UPRmt/UPRER), are involved in the pathogenesis of sarcopenia. To extend both basic and translational knowledge, we examined (i) whether age-induced mitochondrial and ER stress depend on skeletal muscle type in mice and (ii) whether heat stress treatment, a suggested strategy for sarcopenia, improves age-induced mitochondrial and ER stress. Aged (21-month-old) mice showed more severe mitochondrial stress and UPRmt than young (12-week-old) mice, based on increased oxidative stress, mitochondrial proteases, and mitochondrial E3 ubiquitin ligase. The aged mice also showed ER stress and UPRER, based on decreased ER enzymes and increased ER stress-related cell death. These changes were much more evident in soleus muscle than in gastrocnemius and plantaris muscles. After daily heat stress treatment (40 °C chamber for 30 minutes per day) for 4 weeks, mice showed remarkable improvements in age-related changes in soleus muscle. Heat stress had only minor effects in gastrocnemius and plantaris muscles. Based on these findings, age-associated mitochondrial stress, ER stress, and UPRmt/ER vary qualitatively with skeletal muscle type. Our results suggest a molecular basis for the beneficial effects of heat stress on muscle atrophy with age in soleus muscle.

Blocking autophagy enhances meloxicam lethality to hepatocellular carcinoma by promotion of endoplasmic reticulum stress.

Meloxicam, a selective cyclooxygenase-2 (COX-2) inhibitor, has been demonstrated to exert anti-tumour effects against various malignancies. However, up to now, mechanisms involved in meloxicam anti-hepatocellular carcinoma effects have remained unclear. Cell viability and apoptosis were assessed by CCK-8 and flow cytometry. Endoplasmic reticulum (ER) stress and autophagy-associated molecules were analysed by western blotting and immunofluorescence assay. GRP78 and Atg5 knock-down by siRNA or chemical inhibition was used to investigate cytotoxic effects of meloxicam treatment on HCC cells. We found that meloxicam led to apoptosis and autophagy in HepG2 and Bel-7402 cells via a mechanism that involved ER stress. Up-regulation of GRP78 signalling pathway from meloxicam-induced ER stress was critical for activation of autophagy. Furthermore, autophagy activation attenuated ER stress-related cell death. Blocking autophagy by 3-methyladenine (3-MA) or Atg5 siRNA knock-down enhanced meloxicam lethality for HCC by activation of ER stress-related apoptosis. In addition, GRP78 seemed to lead to autophagic activation via the AMPK-mTOR signalling pathway. Blocking AMPK with a chemical inhibitor inhibited autophagy suggesting that meloxicam-regulated autophagy requires activation of AMPK. Our results revealed that both ER stress and autophagy were involved in cell death evoked by meloxicam in HCC cells. This inhibition of autophagy to enhance meloxicam lethality, suggests a novel therapeutic strategy against HCC.

Abstract P6-08-27: Glucose-regulated protein 78 and C/-EBP homologous protein predict disease-free survival and responsiveness to chemotherapy in breast cancer

Abstract Background Cancer cells are generally under endoplasmic reticulum(ER) stress. Notably, the ability of cells to respond to ER stress is critical for cell survival, and chronic or unresolved ER stress can lead to apoptosis. Glucose-regulated protein (GRP) 78 and C/-EBP homologous protein (CHOP) are commonly used as markers of endoplasmic reticulum (ER) stress. As an ER chaperone, GRP78 functions as a potent anti-apoptotic factor and confers drug resistance, whereas CHOP is a key initiating factor of ER stress-related cell death. The clinical implications of GRP78 and CHOP, however, have not been fully studied in breast cancer. In this study, we aimed to investigate the predictive value of GRP78 and CHOP in breast cancer patients who underwent adjuvant chemotherapy. Methods: An immunohistochemistry screen for GRP78 and CHOP was performed using a tissue microarray (TMA) containing 250 tumors from female patients diagnosed with invasive ductal breast carcinoma at the Fudan University Shanghai Cancer Center. The staining results were scored semi-quantitatively, and a prediction model was constructed to verify the hypothesis. Results: In this retrospective study cohort, positive GRP78 staining was detected in 52.6% (n = 112; 52.6% positive, 47.4% negative) of tumors, and CHOP staining was present in 56.3% (n = 120; 56.3% positive, 43.7% negative) of cases. In Kaplan-Meier analysis, CHOP correlated with prolonged disease-free survival (DFS; P = 0.001), whereas GRP78 showed an opposite association (P < 0.001). Moreover, in a GRP78-positive subset, CHOP overexpression correlated with a lower risk of recurrence. A further multivariate COX analysis revealed that positive GRP78 staining cases exhibited a higher likelihood for disease events (HR = 4.573; 95% CI: 2.291-9.128; P < 0.001), while CHOP positivity was indicative of lower risk for recurrence (HR = 0.385, 95% CI: 0.215-0.688; P = 0.001). In the receiver operating characteristic (ROC) analysis, the prediction capability of the predictive model combining the above two markers surpassed that of a traditional model (P = 0.0085 for the area under the curve comparison). Within the anthracycline-treatment subgroup, the combined GRP78 and CHOP exhibited similar predictive significance. Conclusions: Collectively, our findings suggest a tight association between ER stress markers and clinical outcomes for female patients diagnosed with invasive ductal breast carcinoma. Adding these two ER stress markers to traditional prognostic factors provides a more sensitive and accurate predictive model for female patients with invasive ductal breast cancer. Moreover, the combination of GRP78 and CHOP has predictive value for responsiveness to anthracycline-based adjuvant breast cancer chemotherapy. Thus, our findings provide insights into further applications of GRP78 and CHOP, potentially providing additional predictive information for oncologists with regard to choosing treatment regimens. Citation Format: Yi-Zi Zheng, Zhi-Gang Cao, Xin Hu, Zhi-Ming Shao. Glucose-regulated protein 78 and C/-EBP homologous protein predict disease-free survival and responsiveness to chemotherapy in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-27.

The endoplasmic reticulum stress markers GRP78 and CHOP predict disease-free survival and responsiveness to chemotherapy in breast cancer.

Glucose-regulated protein (GRP) 78 and C/-EBP homologous protein (CHOP) are commonly used as markers of endoplasmic reticulum (ER) stress. As an ER chaperone, GRP78 functions as a potent anti-apoptotic factor and confers drug resistance, whereas CHOP is a key initiating factor of ER stress-related cell death. We aimed at investigating the predictive values of GRP78 and CHOP in breast cancer patients who underwent adjuvant chemotherapy. An immunohistochemistry screen for GRP78 and CHOP was performed using a tissue microarray containing 250 tumors from female patients diagnosed with invasive ductal breast carcinoma at the Fudan University Shanghai Cancer Center. The staining results were scored semi-quantitatively, and a prediction model was constructed to verify the hypothesis. In this retrospective cohort study, CHOP correlated with prolonged disease-free survival (HR=0.385, 95% CI 0.215-0.688; P=0.001), whereas GRP78 showed an opposite association (HR=4.573; 95% CI 2.291-9.128; P<0.001). Moreover, in a GRP78-positive subset, CHOP overexpression correlated with a lower risk of recurrence. In the receiver operating characteristic analysis, the prediction capability of the predictive model combining the above two markers surpassed that of the traditional model (P=0.0085 for the area under the curve comparison). Within the anthracycline-treatment subgroup, the combined GRP78 and CHOP exhibited similar predictive significance. Cumulatively, our findings suggest a tight association between ER stress markers and clinical outcomes for patients with breast cancer.

Enhanced autophagic flux by endoplasmic reticulum stress in human hepatocellular carcinoma cells contributes to the maintenance of cell viability

Endoplasmic reticulum (ER) stress and autophagy are important adaptive responses in eukaryotes. The aim of this study was to investigate the autophagic responses in hepatocellular carcinoma (HCC) cells under ER stress and the effect of autophagy on cell survival and death. The human HCC cell line HepG2 was stimulated with tunicamycin to induce ER stress. Cell viability was detected using the Cell Counting Kit-8. The accumulation of autophagic compartments was observed using transmission electron microscopy. The expression of ER and autophagy-related proteins was assessed by western blotting. Autophagic flux was assessed by microtubule-associated protein 1-light chain 3 (MAP1-LC3) turnover assay in the presence of chloroquine to inhibit lysosomes. HepG2 cells subjected to the ER stress presented a significant accumulation of autophagosomes and increased conversion of LC3-I to LC3-II as well as enhanced autophagic flux as detected by the LC3 turnover assay. Inhibition of autophagy with 3-methyladenine facilitated ER stress-related cell death. We conclude that ER stress enhances the autophagic flux in HepG2 cells, which may contribute to the maintenance of cell viability.

Diabetes-Induced Hepatic Pathogenic Damage, Inflammation, Oxidative Stress, and Insulin Resistance Was Exacerbated in Zinc Deficient Mouse Model

ObjectivesZinc (Zn) deficiency often occurs in the patients with diabetes. Effects of Zn deficiency on diabetes-induced hepatic injury were investigated.MethodsType 1 diabetes was induced in FVB mice with multiple low-dose streptozotocin. Hyperglycemic and age-matched control mice were treated with and without Zn chelator, N,N,N′,N′-tetrakis (2-pyridylemethyl) ethylenediamine (TPEN), at 5 mg/kg body-weight daily for 4 months. Hepatic injury was examined by serum alanine aminotransferase (ALT) level and liver histopathological and biochemical changes.ResultsHepatic Zn deficiency (lower than control level, p<0.05) was seen in the mice with either diabetes or TPEN treatment and more evident in the mice with both diabetes and TPEN. Zn deficiency exacerbated hepatic injuries, shown by further increased serum ALT, hepatic lipid accumulation, inflammation, oxidative damage, and endoplasmic reticulum stress-related cell death in Diabetes/TPEN group compared to Diabetes alone. Diabetes/TPEN group also showed a significant decrease in nuclear factor-erythroid 2-related factor 2 (Nrf2) expression and transcription action along with significant increases in Akt negative regulators, decrease in Akt and GSK-3β phosphorylation, and increase in nuclear accumulation of Fyn (a Nrf2 negative regulator). In vitro study with HepG2 cells showed that apoptotic effect of TPEN at 0.5–1.0 µM could be completely prevented by simultaneous Zn supplementation at the dose range of 30–50 µM.ConclusionsZn is required for maintaining Akt activation by inhibiting the expression of Akt negative regulators; Akt activation can inhibit Fyn nuclear translocation to export nuclear Nrf2 to cytoplasm for degradation. Zn deficiency significantly enhanced diabetes-induced hepatic injury likely through down-regulation of Nrf2 function.

GLP-1 Analogs Reduce Hepatocyte Steatosis and Improve Survival by Enhancing the Unfolded Protein Response and Promoting Macroautophagy

BackgroundNonalcoholic fatty liver disease (NAFLD) is a known outcome of hepatosteatosis. Free fatty acids (FFA) induce the unfolded protein response (UPR) or endoplasmic reticulum (ER) stress that may induce apoptosis. Recent data indicate ER stress to be a major player in the progression of fatty liver to more aggressive lesions. Autophagy on the other hand has been demonstrated to be protective against ER stress- induced cell death. We hypothesized that exendin-4 (GLP-1 analog) treatment of fat loaded hepatocytes can reduce steatosis by autophagy which leads to reduced ER stress-related hepatocyte apoptosis.Methodology/Principal FindingsPrimary human hepatocytes were loaded with saturated, cis- and trans-unsaturated fatty acids (palmitic, oleic and elaidic acid respectively). Steatosis, induced with all three fatty acids, was significantly resolved after exendin-4 treatment. Exendin-4 sustained levels of GRP78 expression in fat-loaded cells when compared to untreated fat-loaded cells alone. In contrast, CHOP (C/EBP homologous protein); the penultimate protein that leads to ER stress-related cell death was significantly decreased by exendin-4 in hepatocytes loaded with fatty acids. Finally, exendin-4 in fat loaded hepatocytes clearly promoted gene products associated with macroautophagy as measured by enhanced production of both Beclin-1 and LC3B-II, markers for autophagy; and visualized by transmission electron microscopy (TEM). Similar observations were made in mouse liver lysates after mice were fed with high fat high fructose diet and treated with a long acting GLP-1 receptor agonist, liraglutide. Conclusions/SignificanceGLP-1 proteins appear to protect hepatocytes from fatty acid-related death by prohibition of a dysfunctional ER stress response; and reduce fatty acid accumulation, by activation of both macro-and chaperone-mediated autophagy. These findings provide a novel role for GLP-1 proteins in halting the progression of more aggressive lesions from underlying steatosis in humans afflicted with NAFLD.

The Unfolded Protein Response Is Triggered by a Plant Viral Movement Protein

Infection with Potato virus X (PVX) in Nicotiana benthamiana plants leads to increased transcript levels of several stress-related host genes, including basic-region leucine zipper 60 (bZIP60), SKP1, ER luminal binding protein (BiP), protein disulfide isomerase (PDI), calreticulin (CRT), and calmodulin (CAM). bZIP60 is a key transcription factor that responds to endoplasmic reticulum (ER) stress and induces the expression of ER-resident chaperones (BiP, PDI, CRT, and CAM). SKP1 is a component of SCF (for SKP1-Cullin-F box protein) ubiquitin ligase complexes that target proteins for proteasomal degradation. Expression of PVX TGBp3 from a heterologous vector induces the same set of genes in N. benthamiana and Arabidopsis (Arabidopsis thaliana) leaves. Virus-induced gene silencing was employed to knock down the expression of bZIP60 and SKP1, and the number of infection foci on inoculated leaves was reduced and systemic PVX accumulation was altered. Silencing bZIP60 led to the suppression of BiP and SKP1 transcript levels, suggesting that bZIP60 might be an upstream signal transducer. Overexpression of TGBp3 led to localized necrosis, but coexpression of TGBp3 with BiP abrogated necrosis, demonstrating that the unfolded protein response alleviates ER stress-related cell death. Steady-state levels of PVX replicase and TGBp2 (which reside in the ER) proteins were unaltered by the presence of TGBp3, suggesting that TGBp3 does not contribute to their turnover. Taken together, PVX TGBp3-induced ER stress leads to up-regulation of bZIP60 and unfolded protein response-related gene expression, which may be important to regulate cellular cytotoxicity that could otherwise lead to cell death if viral proteins reach high levels in the ER.

Deletion of the BH3-only protein puma protects motoneurons from ER stress-induced apoptosis and delays motoneuron loss in ALS mice

BH3-only proteins couple diverse stress signals to the evolutionarily conserved mitochondrial apoptosis pathway. Previously, we reported that the activation of the BH3-only protein p53-up-regulated mediator of apoptosis (Puma) was necessary and sufficient for endoplasmic reticulum (ER) stress- and proteasome inhibition-induced apoptosis in neuroblastoma and other cancer cells. Defects in protein quality control have also been suggested to be a key event in ALS, a fatal neurodegenerative condition characterized by motoneuron degeneration. Using the SOD1(G93A) mouse model as well as human post mortem samples from ALS patients, we show evidence for increased ER stress and defects in protein degradation in motoneurons during disease progression. Before symptom onset, we detected a significant up-regulation of Puma in motoneurons of SOD1(G93A) mice. Genetic deletion of puma significantly improved motoneuron survival and delayed disease onset and motor dysfunction in SOD1(G93A) mice. However, it had no significant effect on lifespan, suggesting that other ER stress-related cell-death proteins or other factors, such as excitotoxicity, necrosis, or inflammatory injury, may contribute at later disease stages. Indeed, further experiments using cultured motoneurons revealed that genetic deletion of puma protected motoneurons against ER stress-induced apoptosis but showed no effect against excitotoxic injury. These findings demonstrate that a single BH3-only protein, the ER stress-associated protein Puma, plays an important role during the early stages of chronic neurodegeneration in vivo.