Endoplasmic reticulum stress mediates withaferin A-induced apoptosis in human renal carcinoma cells

The accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER) results in cellular stress that initiates a specialized response designated as the unfolded protein response. ER stress has been implicated in a variety of common diseases, such as diabetes, ischemia and neurodegenerative disorders. Withaferin A, a major chemical constituent of Withania somnifera, has been reported to inhibit tumor cell growth. We show that withaferin A induced a dose-dependent apoptotic cell death in several types of human cancer cells, as measured by FACS analysis and PARP cleavage. Treatment of Caki cells with withaferin A induced a number of signature ER stress markers, including phosphorylation of eukaryotic initiation factor-2α (eIF-2 α), ER stress-specific XBP1 splicing, and up-regulation of glucose-regulated protein (GRP)-78. In addition, withaferin A caused up-regulation of CAAT/enhancer-binding protein-homologous protein (CHOP), suggesting the induction of ER stress. Pretreatment with N-acetyl cysteine (NAC) significantly inhibited withaferin A-mediated ER stress proteins and cell death, suggesting that reactive oxygen species (ROS) mediate withaferin A-induced ER stress. Furthermore, CHOP siRNA or inhibition of caspase-4 activity attenuated withaferin A-induced apoptosis. Taken together, the present study provides strong evidence supporting an important role of the ER stress response in mediating withaferin A-induced apoptosis.     

The Endoplasmic Reticulum in Xenobiotic Toxicity

The endoplasmic reticulum (ER) is involved in an array of cellular functions that play important roles in xenobiotic toxicity. The ER contains the majority of cytochrome P450 enzymes involved in xenobiotic metabolism, as well as a number of conjugating enzymes. In addition to its role in drug bioactivation and detoxification, the ER can be a target for damage by reactive intermediates leading to cell death or immune-mediated toxicity. The ER contains a set of luminal proteins referred to as ER stress proteins (including GRP78, GRP94, protein disulfide isomerase, and calreticulin). These proteins help regulate protein processing and folding of membrane and secretory proteins in the ER, calcium homeostasis, and ER-associated apoptotic pathways. They are induced in response to ER stress. This review discusses the importance of the ER in molecular events leading to cell death following xenobiotic exposure. Data showing that the ER is important in both renal and hepatic toxicity will be discussed.     

The endoplasmic reticulum in xenobiotic toxicity

The endoplasmic reticulum (ER) is involved in an array of cellular functions that play important roles in xenobiotic toxicity. The ER contains the majority of cytochrome P450 enzymes involved in xenobiotic metabolism, as well as a number of conjugating enzymes. In addition to its role in drug bioactivation and detoxification, the ER can be a target for damage by reactive intermediates leading to cell death or immune-mediated toxicity. The ER contains a set of luminal proteins referred to as ER stress proteins (including GRP78, GRP94, protein disulfide isomerase, and calreticulin). These proteins help regulate protein processing and folding of membrane and secretory proteins in the ER, calcium homeostasis, and ER-associated apoptotic pathways. They are induced in response to ER stress. This review discusses the importance of the ER in molecular events leading to cell death following xenobiotic exposure. Data showing that the ER is important in both renal and hepatic toxicity will be discussed.     

Protective effects of HRD1 and 4-phenylbutyric acid against neuronal cell death

Endoplasmic reticulum-associated degradation (ERAD) is a system in which unfolded proteins drained from the ER lumen to the cytosol are ubiquitinated then degraded by 26S proteasome. We have identified and characterized human HRD1 as a ubiquitin ligase involved in ERAD that protects against ER stress-induced cell death. Accumulation of Pael receptor (Pael-R), a substrate of Parkin, has been proposed to lead to neuronal death in Autosomal Recessive Juvenile Parkinsonism (AR-JP). HRD1 co-localized with Pael-R in the ER and interacted with Pael-R through the proline-rich region of HRD1. HRD1 ubiquitinated and degraded Pael-R through its ubiqutin ligase activity. Furthermore, we found that ATF6 and XBP1 that induce HRD1 promoted the degradation of Pael-R. A class of compounds known as chemical chaperones, such as 4-phenylbutyric acid (4-PBA), has been demonstrated to repair unfolded proteins. We demonstrated that 4-PBA protected against ER stress-induced neuronal cell death. The tunicamycin-induced up-regulation of GRP78 and GRP94 and phosphorylation of PERK was suppressed by treatment with 4-PBA, indicating that 4-PBA suppresses ER stress responses by decreasing unfolded protein. Furthermore, 4-PBA suppressed ER stress induced by the overexpression of Pael-R. Thus, up-regulation of HRD1 and 4-PBA could decrease accumulation of Pael-R.     

Cyanobacteria-blooming water samples from Lake Taihu induce endoplasmic reticulum stress in liver and kidney of mice

To investigate whether endoplasmic reticulum (ER) stress was involved in apoptosis induced by cyanobacteria-blooming water, healthy male ICR mice were fed with water samples from cyanobacteria-blooming regions of Lake Taihu (China), including Meiliang Bay (M1 and M2), central lake region (H), macrophyte-dominated Xukou Bay (X), and tap water (control group) for three consecutive months. Hepatic and renal mRNA and protein expression of ER stress signaling molecules were measured with quantitative real-time PCR and western blotting. Compared to macrophyte-dominated and control water samples, cyanobacteria-blooming water changed hepatic ER stress signaling molecules. M1 water treatment increased the mRNA and protein levels of glucose regulation protein 78 (GRP78) and C/EBP homologous protein (CHOP), and decreased the mRNA levels of B-cell lymphoma 2 (Bcl-2). M2 water treatment up-regulated GRP78 mRNA and protein expression, whereas H water treatment up-regulated mRNA and protein expression of GRP78 and caspase-12. Cyanobacteria-blooming water exposure also changed mRNA and protein expression of ER stress signaling molecules in the kidneys. M1 water exposure up-regulated GRP78 mRNA and protein expression and CHOP mRNA expression, whereas M2 water treatment up-regulated caspase-12 and Bcl-2 mRNA expression. M1 and M2 cyanobacteria-blooming water exposure significantly increased relative liver weights, and induced hepatic cell apoptosis. However, cyanobacteria-blooming water treatment did not change kidney weights, and did not induce renal apoptosis compared to macrophyte-dominated and control water samples. Hence, cyanobacteria-blooming water induces hepatic apoptosis via ER stress, and ER stress may play an important role in the apparent anti-apoptotic effects on renal cells exposed to cyanobacteria-blooming water.     

The regulatory mechanism of 4-phenylbutyric acid against ER stress-induced autophagy in human gingival fibroblasts

Endoplasmic reticulum (ER) stress is closely connected to autophagy. When cells are exposed to ER stress, cells exhibit enhanced protein degradation and form autophagosomes. In this study, we demonstrate that the chemical chaperone, 4-phenylbutyric acid (4-PBA), regulates ER stressinduced cell death and autophagy in human gingival fibroblasts. We found that 4-PBA protected cells against thapsigargin-induced apoptotic cell death but did not affect the reduced cell proliferation. ER stress induced by thapsigargin was alleviated by 4-PBA through the regulation of several ER stress-inducible, unfolded protein response related proteins including GRP78, GRP94, C/EBP homologous protein, phospho-eIF-2α, eIF-2α, phospho-JNK1 (p46) and phospho-JNK2/3 (p54), JNK1, IRE-1α, PERK, and sXBP-1. Compared with cells treated with thapsigargin alone, cells treated with both 4-PBA and thapsigargin showed lower levels of Beclin-1, LC-3II and autophagic vacuoles, indicating that 4-PBA also inhibited autophagy induced by ER stress. This study suggests that 4-PBA may be a potential therapeutic agent against ER stress-associated pathologic situations.     

Induction of hepatic metallothionein synthesis by endoplasmic reticulum stress in mice

Metallothionein (MT) is a small sulfhydryl-rich protein whose levels are elevated by various inducers of organelle stresses, such as nuclear stress (cisplatin), mitochondrial stress (antimycin A, 2,4-dinitrophenol) and lysosomal stress (paraquat). Although abnormal folding of protein in the endoplasmic reticulum (ER) causes ER stress, induction of MT synthesis by ER stress has never been investigated. In this study, we examined the induction of MT by an inducer of ER stress, tunicamycin (Tun), which induces ER stress by inhibiting N-linked glycosylation of protein in the ER. Administration of Tun (0.5-1.5 mg/kg, sc) increased hepatic MT levels in C57BL/6J mice (3.1-fold). The maximal increase in hepatic MT was observed 48-96 h after the administration of Tun (1.0 mg/kg). Expressions of MT-I, II and glucose-regulated protein 78 (Bip/GRP78), which is a molecular chaperone induced by ER stress, mRNA were also detected by administration of Tun. Thapsigargin (Thap), a generator of ER stress by inhibiting ER Ca(2+)-ATPase, also increased both hepatic MT levels and expression of MT-I and -II mRNA. The level of expression of Bip/GRP78 mRNA induced by Tun administration in MT-null mice was greater than that in wild-type mice. Taken together, these findings suggest that inhibitors of ER are potent inducers of MT.     

The anti-cancer activity of dihydroartemisinin is associated with induction of iron-dependent endoplasmic reticulum stress in colorectal carcinoma HCT116 cells

Dihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, is among the artemisinin derivatives possessing potent anti-malarial and anti-cancer activities. In the present study, we found that DHA displayed significant anti-proliferative activity in human colorectal carcinoma HCT116 cells, which may be attributed to its induction of G1 phase arrest and apoptosis. To further elucidate the mechanism of action of DHA, a proteomic study employed two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed. Glucose-regulated protein 78 (GRP78), which is related with endoplasmic reticulum stress (ER stress), was identified to be significantly up-regulated after DHA treatment. Further study demonstrated that DHA enhanced expression of GRP78 as well as growth arrest and DNA-damage-inducible gene 153 (GADD153, another ER stress-associated molecule) at both mRNA and protein levels. DHA treatment also led to accumulation of GADD153 in cell nucleus. Moreover, pretreatment of HCT116 cells with the iron chelator deferoxamine mesylate salt (DFO) abrogated induction of GRP78 and GADD153 upon DHA treatment, indicating iron is required for DHA-induced ER stress. This result is consistent with the fact that the anti-proliferative activity of DHA is also mediated by iron. We thus suggest the unbalance of redox may result in DHA-induced ER stress, which may contribute, at least in part, to its anti-cancer activity.     

Increased expression of endoplasmic reticulum stress proteins following chronic valproate treatment of rat C6 glioma cells

The anticonvulsant sodium valproate has been shown to be an effective treatment for bipolar disorder, however, its precise mechanism of action has yet to be determined. It has been suggested that adaptational changes in gene expression are critical for valproate's prophylactic effects. Previous studies in our lab have shown that one gene that may be regulated by valproate is the 78-kilodalton glucose-regulated protein (GRP78). We report that treatment of rat C6 glioma cells with valproate can also increase the expression of additional endoplasmic reticulum stress proteins, GRP94 and calreticulin. All three proteins showed similar concentration-dependent increases in messenger RNA abundance. Chronic (seven days) treatment significantly increased GRP78 and GRP94 messenger RNA expression, whereas calreticulin expression increased after both acute and chronic treatment. Increases in mRNA expression corresponded to a similar increase in protein expression. The roles of GRP78, GRP94 and calreticulin as molecular chaperones and calcium binding proteins, suggest that these results might have functional relevance to the therapeutic action of valproate.     

Mycotoxin zearalenone induces apoptosis in mouse Leydig cells via an endoplasmic reticulum stress-dependent signalling pathway

Zearalenone (ZEN) is a Fusarium mycotoxin that causes several reproductive disorders and genotoxic effects. This study demonstrated the involvement of endoplasmic reticulum (ER) stress in ZEN-induced mouse Leydig cell death. Our study showed that ZEN reduced cell proliferation in a murine Leydig tumour cell line in a dose-dependent manner. The involvement of apoptosis as a major cause of ZEN-induced cell death was further confirmed by the results of a caspase-3 activity assay, which showed a ZEN dose-dependent increase in cell death. Treatment of MLTC-1 and primary mouse Leydig cells with ZEN upregulated the expression of the ER stress-typical markers GRP78, CHOP and caspase-12 protein. Further, pre-treating the cells with 4-phenylbutyrate or knocking down GRP78 using lentivirus-encoded shRNA significantly diminished ZEN-induced apoptosis and inhibited the expression of CHOP and caspase-12. In summary, these results suggest that the activation of an ER stress pathway plays a key role in ZEN-induced apoptosis in the mouse Leydig cells.     

毒胡萝卜素对人胚肺成纤维细胞凋亡影响及部分机制的初步探讨

目的研究内质网应激(endoplasmic reticulum stress,ERS)标识物葡萄糖调节蛋白(glucose regulated protein,GRP)和CCAAT增强子结合蛋白同源蛋白(CCAAT/enhancerbinding protein homologous protein,CHOP)在人胚肺成纤维细胞凋亡过程中的表达变化,初步探讨毒胡萝卜素对人胚肺成纤维细胞凋亡的作用及部分机制。方法体外用毒胡萝卜素(TG)诱导人胚肺成纤维细胞(MRC-5)后,采用流式细胞仪检测细胞的凋亡变化,RT-PCR检测细胞中GRP78、CHOPmRNA的表达变化,Western blot检测GRP、CHOP及Caspase-3蛋白的表达情况。结果与对照组比较,经TG作用24 h后,细胞凋亡率增加,并呈浓度依赖性,差异具有统计学意义(P<0.05)。TG组在24 h的GRP、CHOP表达均高于对照组,差异具有统计学意义(P<0.05,P<0.01);同时不同刺激组中Caspase-3的表达高于对照组,差异具有统计学意义(P<0.01)。结论毒胡萝卜素可诱导人胚肺成纤维细胞发生凋亡,该凋亡可能与ERS有关。     

Naltrexone changes the expression of lipid metabolism‐related proteins in the endoplasmic reticulum stress induced hepatic steatosis in mice

Endoplasmic reticulum (ER) stress is closely associated with several chronic diseases such as obesity, atherosclerosis, type 2 diabetes, and hepatic steatosis. Steatosis in hepatocytes may also lead to disorders such as nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), fibrosis, and possibly cirrhosis. Opioid peptides are involved in triglyceride and cholesterol dysregulation. Naltrexone also attenuates ER stress induced hepatic steatosis in mice. In this study, we evaluated the effects of naltrexone on the expression of lipid metabolism‐related nuclear factors and enzymes in the ER stress induced hepatic steatosis. C57/BL6 mice received saline, DMSO and naltrexone as control groups. In a fourth group, ER stress was induced by tunicamycin (TM) injection and in the last group, naltrexone was given before TM administration. Histopathological evaluations, real‐time RT‐PCR and western blot were performed. We found that GRP78, IRE1α, PERK and ATF6 gene expression and steatosis significantly reduced in naltrexone treated animals. Naltrexone alleviated the gene and protein expression of SREBP1c. Expression of ACAT1, apolipoprotein B (ApoB) and PPARα also increased after naltrexone treatment. In conclusion, this study, for the first time, shows that naltrexone has a considerable role in attenuation of ER stress‐induced liver injury.     

Modulation of AT-1R/CHOP-JNK-Caspase12 pathway by olmesartan treatment attenuates ER stress-induced renal apoptosis in streptozotocin-induced diabetic mice

There is evidence that the activation of renal angiotensin (Ang)-II plays a critical role in the pathogenesis of diabetic kidney diseases (DN) via the ER stress-induced renal apoptosis. Since, the potential negative role of Ang-II in the pathogenesis of ER stress-mediated apoptosis is poorly understood; we evaluated whether treatment of mice with AT-1R specific blocker, olmesartan is associated with the reduction of ER stress-induced renal apoptosis in streptozotocin (STZ)-induced diabetic animal model. We employed western blot analysis to measure the renal protein expressions level of NADPH oxidase subunits, ER chaperone GRP78 and the ER-associated apoptosis proteins. Furthermore, TUNEL staining was used to measure the renal apoptosis. Additionally, dihydroethidium staining and TBARS assay, and immunohistochemistry were performed to measure the renal superoxide radical production and lipid peroxidation, and activation of an Ang-II, respectively. The diabetic kidney mice were found to have increased protein expressions of NADPH oxidase subunits, GRP78 and ER-associated apoptosis proteins, such as TRAF2, IRE-1α, CHOP, p-JNK and procaspase-12, in comparison to normal mice, and which were significantly blunted by the olmesartan treatment in diabetic kidney mice. Furthermore, the diabetic kidney mice were found to have significant increment in renal apoptosis, superoxide radical production, MDA level and activation of an Ang-II and which were also attenuated by the olmesartan treatment. Considering all the findings, it is suggested that the AT-1R specific blocker-olmesartan treatment could be a potential therapy in treating ER stress-induced renal apoptosis via the modulation of AT-1R/CHOP-JNK-Caspase12 pathway in STZ-induced diabetic mice.     

Significance of GRP78 expression in acute myeloid leukemias

The GRP78 (glucose-regulated protein 78) is a major endoplasmic reticulum (ER) chaperone facilitating proper folding of the newly synthesized proteins. By the interaction with caspases, GRP78 has antiapoptotic properties allowing cells to survive under stress condition. GRP78 expression and its association with tumor proliferation, metastasis and resistance to chemotherapy were observed in solid tumors. There are limited data on the expression and impact of this protein on the clinical course and treatment response in acute myeloid leukemia (AML). The aim of this study was to evaluate the expression of GRP78 mRNA in patients with de novo AML. These results were compared to healthy controls, blast phenotype, molecular and cytogenetic status and clinical features of AML. 101 non-M3 AML patients and 26 healthy individuals were included in this study. The expression of GRP78 mRNA in bone marrow was analyzed by real-time quantitative polymerase chain reaction (RQ-PCR). We demonstrated increased GRP78 mRNA expression in AML patients compared to healthy controls, although this difference was statistically significant only in CD34⁺ leukemias. There was also no significant correlation between GRP78 mRNA expression and complete remission rate, relapse-free survival and overall survival. These results indicate that GRP78 expression is increased in CD34⁺ leukemias and has no prognostic impact on clinical outcome in AML.     

Calpain-Induced Endoplasmic Reticulum Stress and Cell Death following Cytotoxic Damage to Renal Cells

Calpains and endoplasmic reticulum (ER) stress have both beenimplicated in renal cell death following exposure to reactivechemical toxicants (RCTs). Therefore, we explored the link betweenER stress, calpain, and cell death in renal cell injury dueto model RCTs (iodoacetamide, menadione, tert-butyl hydroperoxide)and ER stress inducers (tunicamycin [TUN], thapsigargin [THAPS]).The calpain inhibitor, PD150606, significantly reduced the RCTand TUN-induced cell death in the renal cell line LLC-PK1, butnot death induced by THAPS. ER stress was confirmed by the significantinduction of GRP78 following exposure to RCTs and ER stressinducers. While GRP94 induction was observed following RCTsand TUN, it was not statistically significant because of variability.THAPS at 5µM significantly induced GRP94, while 20µMcaused a calpain-dependent cleavage of GRP94. Caspase-12 andm-calpain were variably induced and/or cleaved following exposureto all toxicants, supporting activation of these signaling pathways.Inhibition of calpain blocked the induction of GRP78 followingexposure to RCTs suggesting that calpain was contributing tothe observed ER stress following RCTs. In contrast, calpaininhibition did not block ER stress protein induction followingexposure to nontoxic concentrations of TUN or THAPS, indicatingthat calpain inhibition did not block the ER stress proteininduction pathways directly. These studies demonstrate a previouslyunappreciated link between calpain activation and ER stress–associatedcell death in renal cells. While further studies are requiredto clarify the molecular events involved, these results confirmthat calpain activation and the ER are important related playersin chemically induced renal cell damage.