CHOP mRNA Levels Research Articles

N-Acetylcysteine increases corneal endothelial cell survival in a mouse model of Fuchs endothelial corneal dystrophy

The present study evaluated survival effects of N-acetylcysteine (NAC) on cultured corneal endothelial cells exposed to oxidative and endoplasmic reticulum (ER) stress and in a mouse model of early-onset Fuchs endothelial corneal dystrophy (FECD). Cultured bovine corneal endothelial cell viability against oxidative and ER stress was determined by CellTiter-Glo® luminescent reagent. Two-month-old homozygous knock-in Col8a2L450W/L450W mutant (L450W) and C57/Bl6 wild-type (WT) animals were divided into two groups of 15 mice. Group I received 7 mg/mL NAC in drinking water and Group II received control water for 7 months. Endothelial cell density and morphology were evaluated with confocal microscopy. Antioxidant gene (iNos) and ER stress/unfolded protein response gene (Grp78 and Chop) mRNA levels and protein expression were measured in corneal endothelium by real time PCR and Western blotting. Cell viability of H2O2 and thapsigargin exposed cells pre-treated with NAC was significantly increased compared to untreated controls (p < 0.01). Corneal endothelial cell density (CD) was higher (p = 0.001) and percent polymegathism was lower (p = 0.04) in NAC treated L450W mice than in untreated L450W mice. NAC treated L450W endothelium showed significant upregulation of iNos, whereas Grp78 and Chop were downregulated compared to untreated L450W endothelium by real time PCR and Western blotting. NAC increases survival in cultured corneal endothelial cells exposed against ER and oxidative stress. Systemic NAC ingestion increases corneal endothelial cell survival which is associated with increased antioxidant and decreased ER stress markers in a mouse model of early-onset FECD. Our study presents in vivo evidence of a novel potential medical treatment for FECD.

HKH40A downregulates GRP78/BiP expression in cancer cells.

HKH40A, the 8-methoxy analog of WMC79, is a synthetic agent with promising in vitro and in vivo antitumor activity, especially against solid tumors. However, molecular mechanisms underlying its antitumor effects are poorly understood. Here, we report that HKH40A markedly reduces the level of GRP78/BiP protein in cancer cell lines of various origin. In this study, we show that HKH40A not only downregulates transcription of GRP78 but also directly binds to the isolated protein and induces its proteosomal degradation. Knockdown of BiP increased the efficacy of the drug and overexpression of BiP diminished its activity. BiP is generally highly elevated in solid tumors having a pivotal role in cancer cell survival and chemoresistance, and has been suggested as a novel target for therapeutic intervention. We show that reduction of BiP level by HKH40A impairs its function and induces unfolded protein response as evidenced by the activation of IRE1α, ATF6 and PERK. This leads to a series of downstream events, including sustained eIF2α phosphorylation, increased abundance of spliced XBP1 mRNA and protein levels of ATF4 and CHOP. We also demonstrate that HKH40A inhibited tumor formation in an in vivo xenograft tumor model. Collectively, our data show that HKH40A reduces BiP levels and this could have an important role in the activity of HKH40A against cancer cells.

Calnexin Silencing in Mouse Neonatal Cardiomyocytes Induces Ca2+ Cycling Defects, ER Stress, and Apoptosis

Calnexin (CNX) is an endoplasmic reticulum (ER) quality control chaperone that has been implicated in ER stress. ER stress is a prominent pathological feature of various pathologic conditions, including cardiovascular diseases. However, the role of CNX and ER stress has not been studied in the heart. In the present study, we aimed to characterize the role of CNX in cardiomyocyte physiology with respect to ER stress, apoptosis, and cardiomyocyte Ca(2+) cycling. We demonstrated significantly decreased CNX mRNA and protein levels by LentiVector mediated transduction of targeting shRNAs. CNX silenced cardiomyocytes exhibited ER stress as evidenced by increased GRP78 and ATF6 protein levels, increased levels of spliced XBP1 mRNA, ASK-1, ERO1a, and CHOP mRNA levels. CNX silencing also led to significant activation of caspases-3 and -9. This activation of caspases was associated with hallmark morphological features of apoptosis including loss of sarcomeric organization and nuclear integrity. Ca(2+) imaging in live cells showed that CNX silencing resulted in Ca(2+) transients with significantly larger amplitudes but decreased frequency and Ca(2+) uptake rates in the basal state. Interestingly, 5 mM caffeine stimulated Ca(2+) transients were similar between control and CNX silenced cardiomyocytes. Finally, we demonstrated that CNX silencing induced the expression of the L-type voltage dependent calcium channel (CAV1.2) but reduced the expression of the sarcoplasmic reticulum ATPase (SERCA2a). In conclusion, this is the first study to demonstrate CNX has a specific role in cardiomyocyte viability and Ca(2+) cycling through its effects on ER stress, apoptosis and Ca(2+) channel expression.

Toll-Like Receptor Activation Promotes Multiple Myeloma Cell Growth and Survival By Suppression Of Endoplasmic Reticulum Stress Factor CHOP

Multiple Myeloma (MM) patients are vulnerable to infections, which remain a major cause of death, including early death. During infection, human immune cells sense the presence of invading pathogens through the toll-like receptor family (TLR) of receptors. TLRs detect microbes to activate transcriptional programs that orchestrate adaptive responses to specific insults. This means that they induce the endoplasmic reticulum (ER) unfolded protein response (UPR) to accommodate essential protein translation. If the UPR fails to resolve the protein-folding defect, due to severe and prolonged ER stress, apoptosis is activated. However, the timing sequence of the prolonged ER stress has probably implications for ER stress-induced apoptosis that might help cells to adapt under these conditions rather than driving them to apoptosis. Studies have shown that prolonged ER stress occurs in response to microbes and specifically when cells are exposed to lipopolysaccharide (LPS), a TLR4 activator. The prolonged stress, possibly arising from a massive increase in protein synthesis, has shown to suppress CHOP, an apoptosis biomarker, in ER-stressed macrophages, while low levels of CHOP expression promotes B cell survival. Expression and function of TLRs in MM has recently become the focus of several studies and although the regulatory role of TLRs in MM plasma cells has been reported, the underlying molecular mechanisms remain unclear. It has been shown that human myeloma cell lines (HMCL) and primary myeloma cells express high levels of TLRs and specifically of TLR-4 and TLR-9. The aim of our study was to investigate TLR4 signaling in myeloma cells and to explore possible implications with endoplasmic reticulum unfolded protein response as a potential mechanism of drug resistance.We initially investigated whether TLR-4 is expressed in human myeloma cell lines and primary myeloma cells and we found that TLR-4 mRNA is expressed at increased levels (2-10 fold) both in HMCLs and primary cells. To test the hypothesis that TLR-4 signaling may suppress CHOP expression during sustained UPR response, two myeloma cell lines, H929 and U266, were pre-treated with low dose LPS (1 ng/mL) and then subjected to ER stress conditions by treatment with tunicamycin (TM). LPS pre-treatment significantly decreased CHOP mRNA expression after 24 hours. Despite the marked suppression of CHOP, LPS pre-treatment of these myeloma cell lines did not suppress ATF4 mRNA levels which also were not altered by TM treatment. LPS pre-treatment did not also suppress XBP-1 splicing compared to the control ER-stressed cells. To test the specificity of these effects, the same set of experiments where performed on other cancer tissues such as ovarian cancer cell lines. Interestingly, although LPS pre-treatment increased TLR-4 mRNA expression in SKOV3 ovarian cancer cell line, CHOP mRNA levels remained intact prior and after treatment while TM treatment did not make any difference in CHOP mRNA expression. These results suggest the relevance of exploring this pathway in tissues such as plasma cells which are highly dependent on the UPR as a repair mechanism. Pre-treated LPS and TM samples of HMCLs were also subjected to Annexin-PI staining to determine the amount of apoptosis. As expected, pre-treated LPS myeloma cells which were exposed to TM had 30% lower Annexin-FITC stained cells compared to the TM-stressed cells only. These data suggest that blockage of CHOP by TLR4 ligands may promote the growth and survival of MM cells. We then examined the impact of therapy with bortezomib on TLR4 and CHOP mRNA expression in primary tumors cells which were collected before and at day 7 after bortezomib-based therapy from 6 myeloma patients. In 5 out of 6 cases TLR-4 expression was significantly up-regulated and was accompanied with a coupled down-regulation of CHOP mRNA expression.In conclusion, our data suggest that the TLR-4 signaling pathway might provide a translational control pathway which enables cells to carry out essential protein synthesis and avoid CHOP-induced apoptosis. Further exploration of this pathway is needed to establish its role as a potential mechanism of drug resistance. Disclosures:No relevant conflicts of interest to declare.

Abstract LB-250: 2-Deoxy-D-glucose cytotoxicity relies on both glycolysis inhibition and ER stress induction, and is efficacious in a variety of human lymphoma models

Abstract Introduction: 2-Deoxy-D-glucose (2DG) is a glucose analogue reported to be efficacious in a variety of cancers. Its mechanism of cytotoxicity has been proposed to lie in its inhibition of glycolysis as well as interfering with N-linked glycosylation, which results in protein misfolding and activation of the ER stress response. Previous work indicates that ER stress induction is important in 2DG toxicity. However, these studies do not clarify the relative role of glycolysis inhibition in its toxicity. In addition, little is known about the efficacy of 2DG in human lymphoma. Materials and Methods: Eight human lymphoma lines, murine B cell lymphoma lines and peripheral blood samples of patients with chronic lymphocytic leukemia (CLL) were utilized in these studies. Q-PCR was used to measure CHOP induction by 2DG as a standard marker of ER stress. Four halogenated derivatives of 2DG as well as oxamate, a known glycolysis inhibitor, and tunicamycin, a known inhibitor of N-linked glycosylation, were used to isolate and study the individual mechanisms (glycolysis vs. N-linked glycosylation inhibition) of 2DG activity. Mannose was also used in co-treatment with these drugs as it overcomes the inhibition of N-linked glycosylation caused by these agents. Results and Conclusions: We found that a variety of human lymphoma cell lines and CLL samples exhibited varying degrees of sensitivity to 2DG. The human cell lines showed a 4-12 fold elevation of CHOP RNA following 24 hrs of 2DG treatment, with the sensitive lines showing an even larger increase in CHOP mRNA levels when compared to the resistant lines. Consistent with an important role of ER stress in 2DG toxicity, mannose protected against 2DG treatment. CLL patient samples showed significant cell death caused by 2DG but only at extended time points (6 days), which is consistent with the fact that CLL cells are slowly dividing with low metabolism. Two of the halogenated 2DG derivatives, 2-chloro or 2-bromo-2-deoxy-D-mannose were not toxic, did not inhibit ATP levels or activate ER stress in any of the cells. In contrast, 2-chloro or 2-bromo-2-deoxy-D-glucose showed intermediate toxicity, reduction in ATP levels, but did not significantly upregulate CHOP. This indicates the halogenated glucose derivatives inhibit glycolysis similar to 2DG but do not activate ER stress. Consistent with this conclusion, mannose did not protect cells from treatment with these derivatives. Combining these glucose derivatives with tunicamycin, an inducer of ER stress, showed cooperative activity in cell killing. Similarly, oxamate alone showed minimal toxicity but cooperated with tunicamycin in causing cell death. In summary, our results suggest that 2DG is uniquely effective because it inhibits two critical biochemical pathways - both of which appear to contribute to its cytotoxic activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-250. doi:1538-7445.AM2012-LB-250

The role of the CNOT1 subunit of the CCR4-NOT complex in mRNA deadenylation and cell viability

The human CCR4-NOT deadenylase complex consists of at least nine enzymatic and non-enzymatic subunits. Accumulating evidence suggests that the non-enzymatic subunits are involved in the regulation of mRNA deadenylation, although their precise roles remain to be established. In this study, we addressed the function of the CNOT1 subunit by depleting its expression in HeLa cells. Flow cytometric analysis revealed that the sub G(1) fraction was increased in CNOT1-depleted cells. Virtually, the same level of the sub G1 fraction was seen when cells were treated with a mixture of siRNAs targeted against all enzymatic subunits, suggesting that CNOT1 depletion induces apoptosis by destroying the CCR4-NOT-associated deadenylase activity. Further analysis revealed that CNOT1 depletion leads to a reduction in the amount of other CCR4-NOT subunits. Importantly, the specific activity of the CNOT6L immunoprecipitates-associated deadenylase from CNOT1-depleted cells was less than that from control cells. The formation of P-bodies, where mRNA decay is reported to take place, was largely suppressed in CNOT1-depleted cells. Therefore, CNOT1 has an important role in exhibiting enzymatic activity of the CCR4-NOT complex, and thus is critical in control of mRNA deadenylation and mRNA decay. We further showed that CNOT1 depletion enhanced CHOP mRNA levels and activated caspase-4, which is associated with endoplasmic reticulum ER stress-induced apoptosis. Taken together, CNOT1 depletion structurally and functionally deteriorates the CCR4-NOTcomplex and induces stabilization of mRNAs, which results in the increment of translation causing ER stress-mediated apoptosis. We conclude that CNOT1 contributes to cell viability by securing the activity of the CCR4-NOT deadenylase.

CNOT2 depletion disrupts and inhibits the CCR4-NOT deadenylase complex and induces apoptotic cell death

Eukaryotic mRNA decay is initiated by shortening of the poly (A) tail; however, neither the molecular mechanisms underlying deadenylation nor its regulation is well understood. The human CCR4-NOT complex is a major cytoplasmic deadenylase consisting of a combination of at least nine subunits, four of which have deadenylase activity. The roles of the other subunits remain obscure. Here, we show that CNOT2 depletion by siRNA induces apoptosis. We also show that CNOT2 depletion destabilizes the complex, resulting in the formation of a complex smaller than that formed in control siRNA-treated cells. The deadenylase activity of the CNOT6L subunit-containing complex prepared from CNOT2-depleted cells was less than that from control cells. Intriguingly, the formation of P-bodies, where mRNA degradation supposedly takes place, was largely suppressed in CNOT2-depleted cells. Furthermore, CNOT2 depletion enhanced CHOP mRNA levels, suggesting that endoplasmic reticulum (ER) stress was occurring, which causes apoptosis in a caspase-dependent manner. These results suggest that CNOT2 is important for controlling cell viability through the maintenance of the structural integrity and enzymatic activity of the CCR4-NOT complex.

Autophagy Is a Key Myeloma Survival Pathway That Can Be Manipulated Therapeutically to Enhance Apoptosis

AbstractAbstract 4083In normal cells, autophagy is up-regulated under conditions of stress ensuring cell survival, suggesting that if it is inhibited cell death will ensue. In some cancer settings however it has been shown to enhance cell death. Therefore if autophagy is to be a target for anti-cancer therapy, it is important to determine whether to promote or inhibit the process. Cellular entry to autophagy can be manipulated via the PI3K/AKT/mTOR pathway, a pathway known to be important for myeloma cell growth and survival. In this study we investigated the effects of PI-103, a dual Class I PI3K and mTOR inhibitor, which activates autophagy, and used it as a tool to investigate the interaction of autophagy with other myeloma therapies. As the PI3K/AKT/mTOR pathway has been suggested to be a central pathway controlling entry into autophagy, we first determined the basal expression of key members of the pathway in a panel of myeloma cell lines. PI3K alpha, beta delta and gamma isoforms were expressed to varying degrees and constitutive activation of the pathway, (pAKT or pMTOR), was seen in the majority of cell lines. As predicted, treatment with PI-103 induced autophagy in myeloma cells as demonstrated by an increase in cellular inclusions staining positively with acridine orange, cleavage of the autophagosome marker, LC3, and a decrease in p62. PI-103 was shown to inhibit proliferation of all the myeloma cell lines and patient cells tested to varying degrees, although 100% growth inhibition was not seen. Bone marrow stromal cells were unaffected. The main mode of action of PI-103 was autophagy activation, and in keeping with this the extent of cell death measured by Annexin V/PI binding and trypan blue exclusion, was minimal. Cell cycle analysis demonstrated an increase in G0-G1 phase. The unfolded protein response (UPR) is important in myeloma cells and UPR activation and autophagy have been reported to be interlinked. Following exposure to PI-103, splicing of XBP1 mRNA to its active form, XBP1s, was seen, CHOP and ATF4 mRNA levels were also increased, consistent with activation of at least two branches of the UPR in response to PI3K/mTOR inhibition and autophagy induction. As myeloma cells activate autophagy as a pro-survival pathway following PI3-kinase inhibition, we were interested to understand the effect of blocking autophagy in this context. When PI-103 was combined with the autophagy inhibitor, Bafilomycin greatly enhanced apoptosis was seen. This increased apoptosis was seen in cells constitutively expressing p-AKT, with a complete loss of both phospho- and total levels of AKT and mTOR, an increase in the cleaved forms of caspase 3 and Bcl2, and massive activation of the IRE1 and PERK branches of the UPR. This effect was not seen in cells lacking p-AKT, a phenomenon described as ‘context-dependent oncogene addiction’ suggesting that measurement of p-AKT may be a useful predictive marker for response to joint PI3K/autophagy inhibition. Importantly the pro-apoptotic effects of the combination are not overcome by the presence of bone marrow cytokines, a more representative model of the physiological situation in vivo. In conclusion our data highlights the interplay between known myeloma growth and survival pathways and autophagy and suggests that combining PI3K inhibitors, with agents that target autophagy, may be beneficial for the treatment of myeloma, particularly in >50% of patients that express p-AKT. Disclosures:No relevant conflicts of interest to declare.

ER Stress Induced By Hydrogen Peroxide In C2C12 Cells

Oxidative stress and endoplasmic reticulum stress (ER stress) are both involved in a number of diseases amongst which insulin resistance, Parkinson syndrome and atherosclerosis. Although, those stresses are often regarded in a separately way, it is known that they can be interrelated. Oxidative stress could lead to ER stress and vice versa as demonstrated by studies focused on the potential cross talk between the signaling pathways triggered by each of them in various cell types including mouse fibroblast cells, Chinese hamster ovary cells and mesencephalic cells. Skeletal muscle is a great provider of reactive oxygen species (ROS) and ER stress has been found to be present in skeletal muscle in certain conditions such as high-fat feeding and extreme endurance exercise. In agreement with those observations we have investigated the consequences of oxidative stress on ER stress in C2C12 myotubes. PURPOSE: To establish if oxidative stress, induced by hydrogen peroxide (H2O2), can lead to ER stress in C2C12 myotubes. METHODS: Myogenic C2C12 cells were grown until confluence and further differentiated for 4 days. Preliminary experiments were carried out to determine the optimal H2O2 dose and incubation period for inducing ER stress. A dose of 200μM and incubation periods of 4h and 17h were the most potent conditions for triggering ER stress and were used subsequently. Several downstream targets of the IRE1α and PERK pathways were analyzed at the protein level by western blot and at the mRNA level by qPCR. Three independent experiments were carried out. Unpaired Student t-test was used for the statistical analysis. RESULTS: After 4h H2O2 incubation, P-eIF2 α increased 7.7 +/- 1.3 fold (p 0.05). At the mRNA level, bip and atf4 increased 1.9 +/- 0.2 and 2.4 +/- 0.4 fold, respectively (p<0.05), whereas spliced xbp1 (0.90 +/- 0.2 fold) and unspliced xbp1 (0.8 +/- 0.2 fold) were not altered (ns). Finally chop and trb3 mRNA levels augmented drastically with H2O2 (17.9 +/- 4.8 and 34.4 +/- 6.3 fold, respectively, p<0.05). CONCLUSION: Hydrogen peroxide induces ER stress in C2C12 myotubes through at least the PERK pathway.

Effect of mTOR inhibitors on sorafenib-induced endoplasmic reticulum stress and apoptosis in melanoma cells.

e19027 Background: In melanoma, the RAF-MEK-ERK (MAPK) and the PI3K-AKT-mTOR (AKT) signaling pathways are constitutively activated and have key functions in tumor progression. In our previous studies, combination of the nonselective RAF inhibitor sorafenib with the mTOR inhibitor sirolimus significantly potentiated growth inhibition and led to an approximately 2-fold increase of apoptosis of melanoma cells in monolayer culture compared with sorafenib monotreatment (p<0.05). Moreover, sorafenib in combination with sirolimus completely suppressed invasive melanoma growth in organotypic culture. Methods: To investigate the molecular mechanisms involved in the antitumoral action of this drug combination we analyzed the gene expression profile of melanoma cells in response to sorafenib or/and sirolimus. Results: Data analysis showed a series of genes (p8, ATF4/3, CHOP, TRB3) that were upregulated after treatment with sorafenib and sirolimus and have been proposed to be responsible for the execution of endoplasmic reticulum (ER) stress-induced apoptosis. Using real-time quantitative PCR, we confirmed that sorafenib and sirolimus upregulate p8, ATF4/3, CHOP and TRB3 mRNA levels in melanoma cells. Likewise, western blot analysis showed that sorafenib and sirolimus increase p8 and CHOP protein levels. Furthermore, the farnesyl transferase inhibitor lonafarnib inhibiting mTOR signaling also augmented sorafenib-induced apoptosis and upregulation of the ER stress-related transcription factor p8. Of note, growth inhibitory effects and upregulation of ER stress-related genes by sorafenib and sirolimus or lonafarnib did not depend on BRAF or NRAS mutation status. By contrast, the BRAFV600E kinase inhibitor PLX4032 potently inhibited growth and upregulated the ER stress-related transcription factor p8 preferentially in BRAFV600E melanoma cells. Conclusions: These data suggest that mTOR inhibitors enhance sorafenib-induced endoplasmic reticulum stress and apoptosis in melanoma cells. No significant financial relationships to disclose.

Glyceollins, one of the phytoalexins derived from soybeans under fungal stress, enhance insulin sensitivity and exert insulinotropic actions in vitro and in vivo

Glyceollins are known to anti‐estrogenic phytoalexins but they have not been studied for anti‐diabetic properties. We hypothesized that glyceollins play an important role in glucose homeostasis through regulating glucose utilization in adipocytes and by improving ¥â‐cell function and survival in vitro and in vivo. Glyceollins improved insulin‐stimulated glucose uptake in 3T3‐L1 adipocytes without activating the peroxisome proliferator‐activated receptor‐gamma‐¥ã agonist. In addition, glyceollins slightly improved glucose‐stimulated insulin secretion without palmitate treatment in Min6 cells, and they potentiated insulinotropic actions when 500¥ìM palmitate treatment was used to induce ¥â‐cell dysfunction. This was associated with decreased ¥â‐cell apoptosis due to attenuation of ER stress, determined by mRNA levels of XBP‐1, ATF4, ATF6 and CHOP. The 8‐week oral consumption of the fraction containing glyceollins improved glucose tolerance during oral glucose tolerance test in diabetic mice induced by low‐dose multiple injections of streptozotocin. It also potentiated glucose‐stimulated insulin secretion and prevented ¥â‐cell apoptosis in the mice. In conclusion, glyceollins help normalize glucose homeostasis by potentiating ¥â‐cell function and survival and by improving glucose utilization in adipocytes.

Glyceollins, One of the Phytoalexins Derived from Soybeans under Fungal Stress, Enhance Insulin Sensitivity and Exert Insulinotropic Actions

Glyceollins are a category of phytoalexins that are produced by soybeans under fungal stress, but their effects on glucose homeostasis remain unknown. We hypothesized that glyceollins play an important role in glucose homeostasis by regulating glucose utilization in adipocytes and improving beta-cell function and survival. Glyceollins improved insulin-stimulated glucose uptake in 3T3-L1 adipocytes without activating the peroxisome proliferator-activated receptor-gamma agonist. They decreased triacylglycerol accumulation in adipocytes. In addition, glyceollins slightly improved glucose-stimulated insulin secretion without palmitate treatment in Min6 cells, and they potentiated insulinotropic actions when 500 microM palmitate was used to induce beta-cell dysfunction. This was associated with decreased beta-cell apoptosis because of the attenuation of endoplasmic reticulum stress, as determined by mRNA levels of XBP-1, ATF-4, ATF-6, and CHOP. Glyceollins also potentiated GLP-1 secretion to enhance insulinotropic actions in enteroendocrine cells. In conclusion, glyceollins help normalize glucose homeostasis by potentiating beta-cell function and survival and improving glucose utilization in adipocytes.

Amyloid formation in human IAPP transgenic mouse islets and pancreas, and human pancreas, is not associated with endoplasmic reticulum stress.

Supraphysiological levels of the amyloidogenic peptide human islet amyloid polypeptide have been associated with beta cell endoplasmic reticulum (ER) stress. However, in human type 2 diabetes, levels of human IAPP are equivalent or decreased relative to matched controls. Thus, we sought to investigate whether ER stress is induced during amyloidogenesis at physiological levels of human IAPP. Islets from human IAPP transgenic mice that develop amyloid, and non-transgenic mice that do not, were cultured for up to 7 days in 11.1, 16.7 and 33.3 mmol/l glucose. Pancreases from human IAPP transgenic and non-transgenic mice and humans with or without type 2 diabetes were also evaluated. Amyloid formation was determined histologically. ER stress was determined in islets by quantifying mRNA levels of Bip, Atf4 and Chop (also known as Ddit3) and alternate splicing of Xbp1 mRNA, or in pancreases by immunostaining for immunoglobulin heavy chain-binding protein (BIP), C/EBP homologous protein (CHOP) and X-box binding protein 1 (XBP1). Amyloid formation in human IAPP transgenic islets was associated with reduced beta cell area in a glucose- and time-dependent manner. However, amyloid formation was not associated with significant increases in expression of ER stress markers under any culture condition. Thapsigargin treatment, a positive control, did result in significant ER stress. Amyloid formation in vivo in pancreas samples from human IAPP transgenic mice or humans was not associated with upregulation of ER stress markers. Our data suggest that ER stress is not an obligatory pathway mediating the toxic effects of amyloid formation at physiological levels of human IAPP.

Insulin‐like growth factor stimulation increases radiosensitivity of a pancreatic cancer cell line through endoplasmic reticulum stress under hypoxic conditions

Tumor hypoxia is an obstacle to radiotherapy. Radiosensitivity under hypoxic conditions is determined by molecular oxygen levels, as well as by various biological cellular responses. The insulin-like growth factor (IGF) signaling pathway is a widely recognized survival signal that confers radioresistance. However, under hypoxic conditions the role of IGF signaling in radiosensitivity is still poorly understood. Here, we demonstrate that IGF-II stimulation decreases clonogenic survival under hypoxic conditions in the pancreatic cancer cell lines AsPC-1 and Panc-1, and in the human breast cancer cell line MCF-7. IGF treatment under hypoxic conditions suppressed increased radiation sensitivity in these cell lines by pharmacologically inhibiting the phosphoinositide 3-kinase-mammalian target of rapamycin pathway, a major IGF signal-transduction pathway. Meanwhile, IGF-II induced the endoplasmic reticulum stress response under hypoxia, including increased protein levels of CHOP and ATF4, mRNA levels of CHOP, GADD34, and BiP, as well as splicing levels of XBP-1. The response was suppressed by inhibiting phosphoinositide 3-kinase and mammalian target of rapamycin activity. Overexpression of CHOP in AsPC-1 cells increased radiation sensitivity by IGF-II simulation under hypoxic conditions, whereas suppression of CHOP expression levels with small hairpin RNA or a dominant negative form of a proline-rich extensin-like receptor protein kinase in hypoxia decreased IGF-induced radiosensitivity. IGF-induced endoplasmic reticulum stress contributed to radiosensitization independent of cell cycle status. Taken together, IGF stimulation increased radiosensitivity through the endoplasmic reticulum stress response under hypoxic conditions.

Insulin induces chaperone and CHOP gene expressions in adipocytes

Adipocyte secretes bioactive proteins called adipocytokines, and biosynthesis of secretory proteins requires molecular chaperones and folding enzymes in endoplasmic reticulum (ER). ER chaperones are known to be induced by unfolded protein response (UPR) and growth factors, however, it has not been determined how ER chaperones expression is regulated in adipocytes. Here we show that insulin treatment induced GRP78 and ERO1L mRNA levels in 3T3-L1 adipocytes. Insulin also upregulated CHOP mRNA levels, but did not induce phosphorylation of eIF2α. Pretreatment with insulin protected 3T3-L1 adipocytes against thapsigargin-mediated phosphorylation of eIF2α but did not against DTT-mediated one. In vivo mice study showed that GRP78 and CHOP expressions were regulated by feeding conditions. These results suggest that insulin signaling is important to induce mRNA expressions of GRP78 and CHOP, and may have a protective role against UPR.

Increased Endoplasmic Reticulum Stress in Atherosclerotic Plaques Associated With Acute Coronary Syndrome

Acute coronary syndrome (ACS) represents a series of indications of sudden cardiac ischemia that involve a variety of causes, including acute myocardial infarction and unstable angina.1 ACS usually is associated with thrombus formation in the coronary artery or coronary artery vasospasm, resulting in myocardial ischemia. 2 Thrombus formation may involve platelet adhesion and degranulation on damaged or dysfunctional endothelium overlying the intact or ruptured atherosclerotic plaque, as well as microthrombi.3 Atherosclerotic plaque rupture represents a series of deleterious events linked to the breakdown of the fibrous cap. This process may occur as a result of the increased secretion of matrix metalloproteinases by lesion resident macrophages or enhanced apoptotic cell death within the fibrous cap. However, viable smooth muscle cells (SMCs) within the fibrous cap synthesize interstitial collagen fibers that enhance the structural integrity of the plaque and prevent its rupture.4

Hypoxic Regulation of Id-1 and Activation of the Unfolded Protein Response Are Aberrant in Neuroblastoma

The Id proteins play an important role in proliferation, differentiation and tumorigenesis. Many tumors are hypoxic, but it is unknown if expression of Id proteins is regulated in hypoxic cells. Here we show that Id-1 is down-regulated in multiple primary, immortalized, and neoplastic hypoxic cell lines, and the transcriptional repressor ATF-3 is both necessary and sufficient for this hypoxia-induced repression of Id-1. Hypoxic up-regulation of ATF-3 is due in part to activation of the unfolded protein response, a cellular stress response. Remarkably, we observe that the unfolded protein response is de-regulated in all neuroblastoma cell lines tested. Indeed, in the absence of ATF-3 the hypoxia-induced transcription factor HIF-1 up-regulates Id-1 in hypoxic neuroblastoma cells. Hypoxic neuroblastoma cells diminish expression of some neuronal differentiation markers, and forced expression of ATF-3 in hypoxic neuroblastoma cells represses Id-1 and prevents the loss of these markers. The divergent regulation of Id proteins in distinct hypoxic cells may explain some of the varied effects hypoxia has on cellular differentiation and proliferation.