Endoplasmic Reticulum Stress Stimulation Research Articles

Rab18 maintains homeostasis of subcutaneous adipose tissue to prevent obesity-induced metabolic disorders.

Metabolically healthy obesity refers to obese individuals who do not develop metabolic disorders. These people store fat in subcutaneous adipose tissue (SAT) rather than in visceral adipose tissue (VAT). However, the molecules participating in this specific scenario remain elusive. Rab18, a lipid droplet (LD)-associated protein, mediates the contact between the endoplasmic reticulum (ER) and LDs to facilitate LD growth and maturation. In the present study, we show that the protein level of Rab18 is specifically upregulated in the SAT of obese people and mice. Rab18 adipocyte-specific knockout (Rab18 AKO) mice had a decreased volume ratio of SAT to VAT compared with wildtype mice. When subjected to high-fat diet (HFD), Rab18 AKO mice had increased ER stress and inflammation, reduced adiponectin, and decreased triacylglycerol (TAG) accumulation in SAT. In contrast, TAG accumulation in VAT, brown adipose tissue (BAT) or liver of Rab18 AKO mice had a moderate increase without ER stress stimulation. Rab18 AKO mice developed insulin resistance and systematic inflammation. Rab18 AKO mice maintained body temperature in response to acute and chronic cold induction with a thermogenic SAT, similar to the counterpart mice. Furthermore, Rab18-deficient 3T3-L1 adipocytes were more prone to palmitate-induced ER stress, indicating the involvement of Rab18 in alleviating lipid toxicity. Rab18 AKO mice provide a good animal model to investigate metabolic disorders such as impaired SAT. In conclusion, our studies reveal that Rab18 is a key and specific regulator that maintains the proper functions of SAT by alleviating lipid-induced ER stress.

Sesamol as a potential small molecule in inhibiting the induction of neurotoxic amyloids fibrils involved in the pathophysiology of alzheimer's disease

The presence of possibly toxic forms of intracellular α-synuclein, binding of aggregated α-synuclein and monomeric Aβ, and the potential co-localization of α-synuclein with intracellular neurofibrillary tangles as well as Aβ plaques in the brain could be the main causes in serious clinical symptoms mostly developed in Alzheimer’s disease (AD) patients. Amyloid formation of α-synuclein can also induce neurotoxic effects through stimulation of endoplasmic reticulum stress (ERS). Therefore, the inhibition of protein aggregation can be of great importance for the control of pathophysiology of AD. Here, we assessed the use of sesamol as bioactive small molecule in inhibition of α-synuclein fibrillogenesis and neurotoxicity by different biophysical [ThT/Nile red/Congo red (CR)/circular dichroism (CD)] and cellular (MTT and qRT-PCR) analyses. From the spectroscopic analyses, we found that sesamol displays potential effect on the inhibiting the α-synuclein fibril formation. Also, sesamol showed no significant neurotoxicity and co-incubation of α-synuclein with sesamol apparently mitigated the ERS-mediated apoptosis induced by α-synuclein amyloids through regulation of IRE1, PERK, ATF6, and caspase-3 mRNA. Overall, sesamol-based compounds can be further developed and assessed for the regulation of pathophysiology of AD in which α-synuclein amyloids are predominantly involved.

Persistent Endoplasmic Reticulum Stress Stimulated by Peptide Assemblies for Sensitizing Cancer Chemotherapy.

Pharmacological targeting of endoplasmic reticulum (ER) stress represents one of important methods for disease therapy, which, however, is significantly suppressed by the ER homeostatic processe. Herein, a proof-of-concept strategy is reported for persistent stimulation of ER stress via preventing ER stress adaptation by utilizing multifunctional peptide assemblies. The strategy is established via creation of peptide assemblies with ER-targeting and chaperone glucose-regulated protein 78 (GRP78)-inhibiting functions. The peptides assemblies form well-defined nanofibers that are retrieved by ER organelles in human cervical cancer cell. The underlying mechanism studies unravel that the ER-accumulated peptide assemblies simultaneously stimulate ER stress and inhibit GRP78 refolding activity and thereby promoting endogenous protein aggregation. Combining the internalized peptide assemblies with the induced protein aggregates leads to the persistent stimulation of ER stress. The persistent ER stress induced by the peptide assemblies bestows their application in sensitizing cancer chemotherapy. Both in vitro and in vivo results confirm the enhanced cytotoxicity of drug toyocamycin against HeLa cells by peptide assemblies, thus efficiently inhibiting in vivo tumor growth. The strategy reported here discloses the fundamental keys for efficient promotion of ER stress, thus providing the guidance for development of ER-targeting-assisted cancer chemotherapy in the future.

RBM10 Regulates Embryonic Trophoblast Injury via Endoplasmic Reticulum Stress

Background: Previous studies shown that RNA binding motif proteins (RBM) participate in regulating various physiological processes such as cell autophagy, proliferation, and apoptosis, and are abnormally highly expressed in placental trophoblast cells intervened by hypoxia in vitro, but their molecular mechanisms regulating placental trophoblast damage remain unclear. This study aims to investigate the role and molecular mechanism of RBM10 in regulating hypoxia-induced placental trophoblast injury through endoplasmic reticulum stress. Methods: CCK-8 cell proliferation assay and Transwell cell invasion assay were applied to detect the proliferation and invasion ability of normal, hypoxic and RBM10 up-regulated plus hypoxic embryonic trophoblast cells, respectively. The expression of endoplasmic reticulum stress-related proteins (ERN-1) and C/EBP homologous protein (CHOP), apoptosis-related proteins B cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and Caspase-3, and autophagy-related proteins including microtubule-associated protein 1 light chain 3 (LC-3), Beclin-1 and P62 were also detected by western blot assays. The effects of hypoxia and overexpression of RBM10 on placental trophoblast apoptosis were examined using flow cytometry. Results: We found that the growth and invasion ability of placental trophoblast cells treated with hypoxia were significantly decreased (p < 0.05), and the upregulation of RBM10 further led to the decrease of the growth and invasion ability of hypoxic placental trophoblast cells. In addition, hypoxia promoted the expression of endoplasmic reticulum stress-related proteins (p < 0.05), which triggered apoptosis and autophagy of embryonic trophoblast cells. The data showed that embryonic trophoblast cells regulated cell injury by stimulating endoplasmic reticulum stress after hypoxia. after upregulation of RBM10 expression, the expression levels of endoplasmic reticulum stress-related proteins ERN-1 and CHOP were further increased (p < 0.05), and the apoptosis rate of embryonic trophoblast cells was further increased (p < 0.05). Conclusions: Overall, our findings suggest that post-hypoxia mediates autophagy in embryonic trophoblast cells through stimulation of endoplasmic reticulum stress, thereby promoting apoptosis. Overexpression of RBM10 levels regulates the proliferative, apoptotic capacity of trophoblast cells by affecting cellular endoplasmic reticulum stress. RBM10 plays an important role in regulating hypoxia-induced autophagy and apoptosis in trophoblast cells, and RBM10 upregulation can further stimulate endoplasmic reticulum stress-mediated autophagy and apoptosis in trophoblast cells.

The Acetyltransferase KAT5 Inhibitor NU 9056 Promotes Apoptosis and Inhibits JAK2/STAT3 Pathway in Extranodal NK/T Cell Lymphoma.

Extranodal natural killer/T cell lymphoma (ENKTL) is an aggressive malignant non- Hodgkin's lymphoma (NHL) with a poor prognosis. Therefore, novel therapeutic biomarkers and agents must be identified for the same. KAT5 inhibitor, NU 9056, is a small molecule that can inhibit cellular proliferation; however, its role in ENKTL has not been studied. The present study investigated the effect of NU 9056 in ENKTL cells and explored the possible molecular mechanism for its antitumour effect. The role of NU 9056 in ENKTL cells was investigated through the Cell Counting Kit-8 assay, flow cytometry, Western blot, and real-time quantitative polymerase chain reaction assay. NU 9056 inhibited ENKTL cell proliferation and induced G2/M phase arrest. NU 9056 also induced apoptosis by upregulating DR4, DR5, and caspase 8 expressions. Additionally, NU 9056 increased the expression of Bax, Bid, and cytochrome C and decreased the expression of Bcl-2, Mcl-1, and XIAP. Furthermore, NU 9056 activated endoplasmic reticulum (ER) stress and inhibited the JAK2/STAT3 signalling pathway. The p38 mitogen-activated protein kinase (MAPK) signalling pathway was also activated by NU 9056, and the ERK signalling pathway was suppressed in natural killer/T cell lymphoma cells. NU 9056 inhibited cell proliferation, arrested cell cycle in the G2/M phase, and induced apoptosis through the stimulation of ER stress, thus inhibiting the JAK2/STAT3 signalling pathway and regulating MAPK pathways in ENKTL cells.

DNA-Unresponsive Platinum(II) Complex Induces ERS-Mediated Mitophagy in Cancer Cells.

Mitophagy is a selective autophagic process that degrades dysfunctional mitochondria. Monofunctional platinum(II) complexes are candidates for anticancer drugs with the potential to circumvent the drug resistance and side effects of cisplatin and its analogues, but their mechanism of action is elusive. Complex Mono-Pt kills cancer cells through a mitophagic pathway. The mechanism involves the stimulation of endoplasmic reticulum stress (ERS) and activation of the unfolded protein response. Mono-Pt severely impairs the structure and function of mitochondria, including disruption of morphological integrity, dissipation of membrane potential, elevation of reactive oxygen species, inhibition of mtDNA transcription, and reduction of adenosine triphosphate (ATP), which ultimately leads to mitophagy. Mono-Pt does not react with nuclear DNA but exhibits potent antiproliferative activity against cancer cells, thus breaking the DNA-binding paradigm and classical structure-activity rules for platinum drugs. The ERS-mediated mitophagy provides an alternative mechanism for platinum complexes, which broadens the way for developing new platinum anticancer drugs.

Fine particulate matter induces endoplasmic reticulum stress-mediated apoptosis in human SH-SY5Y cells

Exposure to ambient fine particulate matter (PM2.5) may contribute to brain injury, however, the molecular mechanisms have not yet been fully described. In this study, the human SH-SY5Y cells were treated with PM2.5 with different concentrations (0, 25, 100, and 250 μg/mL) for 24 h to investigate the cell apoptosis mediated by endoplasmic reticulum (ER) stress. The ratio of apoptosis, Ca2+ level, biomarkers of ER stress and apoptosis were determined. The results revealed that PM2.5 triggered the increase of apoptosis ratio and cellular Ca2+ levels. Compared with control, the expression of GRP78 and phosphorylation of IER1α and p38 were enhanced significantly in the cells under the conditions of PM2.5 exposure for activating ER stress signals. Besides, the key genes (CHOP/DR5/Caspase8/Caspase12) in ER stress-induced apoptosis signals were up-regulated after the PM2.5 treatment compared to the control. The results suggested PM2.5 induced apoptosis in SH-SY5Y cells by the stimulation of ER stress, which may be the potential mechanism of neurological diseases incurred by PM2.5.

Promising therapeutic effect of thapsigargin nanoparticles on chronic kidney disease through the activation of Nrf2 and FoxO1.

Pathophysiological states cause misfolded protein accumulation in the endoplasmic reticulum (ER). Then, ER stress and the unfolded protein response (UPR) are activated. Targeting ER stress may enhance the adaptive UPR and then protect the cell against pathogenic environments. In the present study, we utilized nanotechnology to synthesize thapsigargin nanoparticles (TG NPs) which induced ER stress and the UPR pathway, to study the role of ER stress and autophagy in chronic kidney disease (CKD). We found that the mRNA levels of ER stress- and autophagy-related molecules were elevated in the renal tissue of CKD patients compared to those of healthy individuals. Furthermore, TG NPs induced the UPR pathway and autophagy in HK-2 human kidney tubular epithelial cells. TG NPs protected HK-2 cells against oxidative stress-induced cell death through the activation of Nrf2 and FoxO1. The siRNA-mediated inhibition of Nrf2 or FoxO1 resulted in enhanced oxidative stress-induced cytotoxicity in HK-2 cells. In a mouse model of adenine diet-induced CKD, TG NPs and KIM-1-TG NPs ameliorated renal injury through the stimulation of ER stress and its downstream pathways. Our findings suggest that the induction of ER stress using pharmacological agents may offer a promising therapeutic strategy for preventing or interfering with CKD progression.

Endoplasmic Reticulum Stress Impaired Uncoupling Protein 1 Expression via the Suppression of Peroxisome Proliferator-Activated Receptor γ Binding Activity in Mice Beige Adipocytes

Endoplasmic reticulum (ER) homeostasis is critical in maintaining metabolic regulation. Once it is disrupted due to accumulated unfolded proteins, ER homeostasis is restored via activation of the unfolded protein response (UPR); hence, the UPR affects diverse physiological processes. However, how ER stress influences adipocyte functions is not well known. In this study, we investigated the effect of ER stress in thermogenic capacity of mice beige adipocytes. Here, we show that the expression of uncoupling protein 1 (Ucp1) involved in thermoregulation is severely suppressed under ER stress conditions (afflicted by tunicamycin) in inguinal white adipose tissue (IWAT) both in vitro and in vivo. Further investigation showed that extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) were both activated after ER stress stimulation and regulated the mRNA levels of Ucp1 and peroxisome proliferator-activated receptor γ (Pparγ), which is known as a Ucp1 transcriptional activator, in vitro and ex vivo. We also found that Pparγ protein was significantly degraded, reducing its recruitment to the Ucp1 enhancer, thereby downregulating Ucp1 expression. Additionally, only JNK inhibition, but not ERK, rescued the Pparγ protein. These findings provide novel insights into the regulatory effect of ER stress on Ucp1 expression via Pparγ suppression in beige adipocytes.

Astragalus Polysaccharide Extends Lifespan via Mitigating Endoplasmic Reticulum Stress in the Silkworm, Bombyx mori.

The traditional Chinese medicine Astragalus polysaccharide (APS) has been widely used to improve glucose homeostasis and immunoregulator properties. In recent years, it has also been shown to extend the lifespan of Caenorhabditis elegans; however, the underlying molecular mechanisms are not fully understood. Here, our study shows that APS could significantly extend adult stage, mean, and maximum lifespan of the silkworm, Bombyx mori and increase body weight without affecting food intake and fecundity. Meanwhile, the activities of glutathione S-transferase and superoxide dismutase are significantly enhanced, and the reaction oxygen species content is reduced concomitantly. Moreover, the activity of lysozyme is increased dramatically. In addition, APS rescues the shortened lifespan by Bacillus thuringiensis infection in silkworm. Furthermore, the transcription of the crucial genes involved in endoplasmic reticulum stress is upregulated upon the endoplasmic reticulum stress stimulation. APS also significantly ameliorates endoplasmic reticulum stress in silkworm cell line and in vivo. Together, the results of this study indicate that APS can prolong the silkworm lifespan by mitigating endoplasmic reticulum stress. This study improves our understanding of the molecular mechanism of APS-induced lifespan extension and highlights the importance of the silkworm as an experimental animal for evaluating the effects and revealing the mechanisms in lifespan extension of traditional Chinese medicine.

MLN4924, a Protein Neddylation Inhibitor, Suppresses the Growth of Human Chondrosarcoma through Inhibiting Cell Proliferation and Inducing Endoplasmic Reticulum Stress-Related Apoptosis.

Chondrosarcoma, a heterogeneous malignant bone tumor, commonly produces cartilage matrix, which generally has no response to conventional therapies. Studies have reported that MLN4924, a NEDD8-activating enzyme inhibitor, achieves antitumor effects against numerous malignancies. In this study, the suppressive effects of MLN4924 on human chondrosarcoma cell lines were investigated using in vitro and in vivo assays, which involved measuring cell viability, cytotoxicity, apoptosis, proliferation, cell cycles, molecule-associated cell cycles, apoptosis, endoplasmic reticulum (ER) stress, and tumor growth in a xenograft mouse model. Our results demonstrated that MLN4924 significantly suppressed cell viability, exhibited cytotoxicity, and stimulated apoptosis through the activation of caspase-3 and caspase-7 in chondrosarcoma cell lines. Furthermore, MLN4924 significantly inhibited cell proliferation by diminishing the phosphorylation of histone H3 to cause G2/M cell cycle arrest. In addition, MLN4924 activated ER stress–related apoptosis by upregulating the phosphorylation of c-Jun N-terminal kinase (JNK), enhancing the expression of GRP78 and CCAAT-enhancer-binding protein homologous protein (CHOP, an inducer of endoplasmic ER stress–related apoptosis) and activating the cleavage of caspase-4. Moreover, MLN4924 considerably inhibited the growth of chondrosarcoma tumors in a xenograft mouse model. Finally, MLN4924-mediated antichondrosarcoma properties can be accompanied by the stimulation of ER stress–related apoptosis, implying that targeting neddylation by MLN4924 is a novel therapeutic strategy for treating chondrosarcoma.

Targeting PERK signaling with the small molecule GSK2606414 prevents neurodegeneration in a model of Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder, leading to the progressive decline of motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Accumulating evidence suggest that altered proteostasis is a salient feature of PD, highlighting perturbations to the endoplasmic reticulum (ER), the main compartment involved in protein folding and secretion. PERK is a central ER stress sensor that enforces adaptive programs to recover homeostasis through a block of protein translation and the induction of the transcription factor ATF4. In addition, chronic PERK signaling results in apoptosis induction and neuronal dysfunction due to the repression in the translation of synaptic proteins. Here we confirmed the activation of PERK signaling in postmortem brain tissue derived from PD patients and three different rodent models of the disease. Pharmacological targeting of PERK by the oral administration of GSK2606414 demonstrated efficient inhibition of the pathway in the SNpc after experimental ER stress stimulation. GSK2606414 protected nigral-dopaminergic neurons against a PD-inducing neurotoxin, improving motor performance. The neuroprotective effects of PERK inhibition were accompanied by an increase in dopamine levels and the expression of synaptic proteins. However, GSK2606414 treated animals developed secondary effects possibly related to pancreatic toxicity. This study suggests that strategies to attenuate ER stress levels may be effective to reduce neurodegeneration in PD.

Stellate ganglion block ameliorates vascular calcification by inhibiting endoplasmic reticulum stress

AimsVascular calcification (VC) underlies substantial cardiovascular morbidity and mortality. No clinically therapies have emerged presently. Stellate ganglion block (SGB) is one of the most often used sympathetic blockade procedure, and regulates vascular dilation. However, the effect of SGB on VC is still unknown. Therefore, we aimed to identify the ameliorative effect of SGB on VC. Key findingIn vivo VC was induced in rats by administering vitamin D3 plus nicotine (VDN), and in vitro calcification of rat aortic vascular smooth muscle cells (VSMC) was induced by β-glycerophosphate. In VDN rats, alkaline phosphatase (ALP) activity and Calcium contents were higher than that in control rats. The transformation of VSMC from contractile to osteoblast-like phenotype was observed in calcified aorta. SGB ameliorated the increase of ALP activity and Calcium content, and the transformation of VSMC in calcified aorta. The stimulation of endoplasmic reticulum stress (ERS) in calcified aorta was also attenuated by SGB treatment. The inducer of ERS, tunicamycin could block the beneficial effect of SGB on VC, and the ERS inhibitor, 4-PBA could mimic the amelioration of SGB. Furthermore, SGB attenuated the increased plasma levels of norepinephrine in VDN rats. In vitro experiments, norepinephrine exaggerated VSMC calcification, phenotype transformation and ERS. SignificanceThese results demonstrate that SGB could inhibit sympathetic nervous activity, and then prevent the activation of ERS followed by ameliorating VC. Sympathetic over-activation might play critical role in the pathogenesis of VC, which provides new strategy and target for therapy and prevention of VC.

The reproductive toxicology of male SD rats after PM2.5 exposure mediated by the stimulation of endoplasmic reticulum stress

Evidence has shown that exposure to fine particulate matter (PM) contributed to poor semen quality in males. However, the reproductive toxicity and relevant molecular mechanisms of Particular Matter 2.5 (PM2.5) from different seasons are not well understood. In the present work, we intend to investigate the toxic effects of PM2.5 during summer and winter on reproductive cells and tissues and focus on endoplasmic reticulum stress (ERS) to illustrate the possible molecular mechanisms. Sprague Dawley (SD) rats were exposed to PM2.5 from the summer season (0.2, 0.6, and 1.5 mg/kg b. w.) and winter (0.3, 1.5, and 2.7 mg/kg b. w.) through intratracheal instillation. The exposure was performed once every 3 days and continued for 2 months. Sperm and reproductive organs (testis and epididymis) were collected from the animals to conduct toxicity evaluation and mechanism analysis. The data showed that sperm relative motility rates were remarkably decreased, while sperm malformation rates were significantly increased with exposure to the summer and winter PM2.5. In particular, the reproductive toxicity of winter PM2.5 in the highest dose group was significantly greater than that in the other PM2.5 exposure groups. The pathological results also showed that the rats in the winter PM2.5 group of 2.7 mg/kg b. w. had severe testicular tissue injury, as determined by haematoxylin and eosin (HE) staining. The apoptotic results obtained by terminal dUTP nick-end labelling (TUNEL) further suggested that summer and winter PM2.5 exposure promoted the testicular germ cell apoptosis. The reproductive toxicity of winter PM2.5 in the testis was stronger than that of summer PM2.5. In addition, the expressions of GRP78 and XBP-1, biomarkers of ERS, was enhanced under the conditions of PM2.5 exposure, and ERS-mediated apoptosis through the upregulation of CHOP and Caspase-12 in the epididymis and testis was activated. In conclusion, PM2.5 exposure induced reproductive toxicity in male SD rats by the stimulation of ERS.

Cladosporium cladosporioides from the perspectives of medical and biotechnological approaches.

Fungi are important natural product sources that have enormous potential for the production of novel compounds for use in pharmacology, agricultural applications and industry. Compared with other natural sources such as plants, fungi are highly diverse but understudied. However, research on Cladosporium cladosporioides revealed the existence of bioactive products such as p-methylbenzoic acid, ergosterol peroxide (EP) and calphostin C as well as enzymes including pectin methylesterase (PME), polygalacturonase (PG) and chlorpyrifos hydrolase. p-Methylbenzoic acid has ability to synthesise 1,5-benzodiazepine and its derivatives, polyethylene terephthalate and eicosapentaenoic acid. EP has anticancer, antiangiogenic, antibacterial, anti-oxidative and immunosuppressive properties. Calphostin C inhibits protein kinase C (PKC) by inactivating both PKC-epsilon and PKC-alpha. In addition, calphostin C stimulates apoptosis in WEHI-231 cells and vascular smooth muscle cells. Based on the stimulation of endoplasmic reticulum stress in some types of cancer, calphostin C has also been evaluated as a potential photodynamic therapeutic agent. Methylesterase (PME) and PG have garnered attention because of their usage in the food processing industry and significant physiological function in plants. Chlorpyrifos, a human, animal and plant toxin, can be degraded and eliminated by chlorpyrifos hydrolase.

Periodontal disease level-butyric acid amounts locally administered in the rat gingival mucosa induce ER stress in the systemic blood

Periodontal diseases have long been postulated to contribute to systemic diseases and, likewise, it has been proposed that periodontal disease treatment may ameliorate certain systemic diseases. Short-chain fatty acids (SCFA) are major secondary metabolites produced by oral anaerobic bacteria and, among the SCFAs, butyric acid (BA) in high amounts contribute to periodontal disease development. Periodontal disease level-butyric acid (PDL-BA) is found among patients suffering from periodontal disease and has previously shown to induce oxidative stress, whereas, oxidative stress is correlated to endoplasmic reticulum (ER) stress. This would imply that PDL-BA may likewise stimulate ER stress, however, this was never elucidated. A better understanding of the correlation between PDL-BA and systemic ER stress stimulation could shed light on the possible systemic effects of PDL-BA-related periodontal diseases. Here, PDL-BA was injected into the gingival mucosa and the systemic blood obtained from the rat jugular was collected at 0, 15, 60, and 180 min post-injection. Collected blood samples were purified and only the blood cytosol was used throughout this study. Subsequently, we measured blood cytosolic GADD153, Ca2+, representative apoptotic and inflammatory caspases, and NF-κB amounts. We found that PDL-BA presence increased blood cytosolic GADD153 and Ca2+ amounts. Moreover, we observed that blood cytosolic caspases and NF-κB were activated only at 60 and 180 min post-injection in the rat gingival mucosa. This suggests that PDL-BA administered through the gingival mucosa may influence the systemic blood via ER stress stimulation and, moreover, prolonged PDL-BA retention in the gingival mucosa may play a significant role in ER stress-related caspase and NF-κB activation. In a periodontal disease scenario, we propose that PDL-BA-related ER stress stimulation leading to the simultaneous activation of apoptosis and inflammation may contribute to periodontal disease pathogenesis.

Arsenic trioxide induces programmed cell death through stimulation of ER stress and inhibition of the ubiquitin–proteasome system in human sarcoma cells

Sarcoma is a rare form of cancer that differs from the much more common carcinomas because it occurs in a distinct type of tissue. Many patients of sarcoma have poor response to chemotherapy and an increased risk for local recurrence. Arsenic trioxide (ATO) is used to treat certain types of leukemia. Recently, data have revealed that ATO induces sarcoma cell death in several types of solid tumor cell lines. In the present study, we investigated whether ATO induces cancer cell death and elucidated the underlying anti-cancer mechanisms. Our results showed that ATO caused concentration- and time-dependent cell death in human osteosarcoma and fibrosarcoma cells. The types of cell death that were induced by ATO were primarily autophagy and apoptosis. Furthermore, ATO activated p38, JNK and AMPK and inhibited the Akt/mTOR signaling pathways. Specifically, we found that ATO induced endoplasmic reticulum (ER) stress and suppressed proteasome activation in two types of sarcoma cell lines. However, the level of proteasome inhibition in osteosarcoma cells was lower than in fibrosarcoma cells. Thus, we used combined treatment with ATO and a proteasome inhibitor to examine the antitumor activity in fibrosarcoma cells. The data indicated showed that the combination treatment of ATO and MG132 (a proteasome inhibitor) resulted in synergistic cytotoxicity. In a fibrosarcoma xenograft mouse model, the combined treatment significantly reduced tumor progression. Immunohistochemical studies revealed that combined treatment induced autophagy and apoptosis. In summary, our results suggest a potential clinical application of ATO in sarcoma therapy and that combined treatment with a proteasome inhibitor can increase the therapeutic efficacy.

Stimulation of endoplasmic reticulum stress and inflammation by neuronal (pro)renin receptor is mediated by Toll‐like receptor 4 activation (686.32)

Our previous studies demonstrated that brain neuronal prorenin receptor (PRR) contributes to hypertension via NF‐KappB (KB)‐cytokine signaling. This finding coupled with the important role of toll like receptor 4 (TLR4) in inflammation and endoplasmic reticulum (ER) stress suggest that brain neuronal PRR activation, in part, stimulates TLR4 and subsequently induces ER stress and NF‐KB‐cytokine signaling. Therefore blocking TLR4 activation should attenuate the cytokines and ER stress response induced by PRR activation. To test this hypothesis, primary neuronal cultures from rat brain were incubated with 20 nM of renin for 6 hours. Real time PCR was performed to analyze the expression of inflammatory cytokines, NF‐KB and ER stress markers. Renin treatment resulted in significant increases in IL1β (1200‐fold), IL6 (33‐fold), TNFα (17‐fold), NF‐KB (6‐fold) and I‐kappa B kinase (IKK) (7‐fold). In addition, the mRNA levels of ER stress marker Ddit3 and Hspa3 increased by 47%and 41% in renin treated neurons. Quinazoline (10µM), a NF‐KB activation inhibitor, effectively blocked renin‐induced increases in IL1β, IL6, TNFα and NF‐KB, by more than 85%, but failed to inhibit IKK, Ddit3 and Hspa3. In contrast, renin stimulation on all these genes was almost entirely blocked (>90%) by a TLR4 inhibitor TAK‐242 (2 µM), indicating an important role of TRL4 in this newly identified PRR mediated activation of ER stress and inflammation signaling. Further study will probe whether NF‐KB‐inflammation is mediated by ER stress in the context of hypertension.Grant Funding Source: AHA 11SDG7420029

Synthetic induction of immunogenic cell death by genetic stimulation of endoplasmic reticulum stress

Cis-diamminedichloridoplatinum(II) (CDDP), commonly referred to as cisplatin, is a chemotherapeutic drug used for the treatment of a wide range of solid cancers. CDDP is a relatively poor inducer of immunogenic cell death (ICD), a cell death modality that converts dying cells into a tumor vaccine, stimulating an immune response against residual cancer cells that permits long-lasting immunity and a corresponding reduction in tumor growth. The incapacity of CDDP to trigger ICD is at least partially due to its failure to stimulate the premortem endoplasmic reticulum (ER)-stress response required for the externalization of the “eat-me” signal calreticulin (CRT) on the surface of dying cancer cells. Here, we developed a murine cancer cell line genetically modified to express the ER resident protein reticulon-1c (Rtn-1c) by virtue of tetracycline induction and showed that enforced Rtn-1c expression combined with CDDP treatment promoted CRT externalization to the surface of cancer cells. In contrast to single agent treatments, the tetracycline-mediated Rtn-1c induction combined with CDDP chemotherapy stimulated ICD as measured by the capacity of dying tumor cells, inoculated into syngenic immunocompetent mice, to mount an immune response to tumor re-challenge 1 week later. More importantly, established tumors, forced to constitutively express Rtn-1c in vivo by continuous treatment with tetracycline, became responsive to CDDP and exhibited a corresponding reduction in the rate of tumor growth. The combined therapeutic effects of Rtn-1c induction with CDDP treatment was only detected in the context of an intact immune system and not in nu/nu mice lacking thymus-dependent T lymphocytes. Altogether, these results indicate that the artificial or “synthetic” induction of immunogenic cell death by genetic manipulation of the ER-stress response can improve the efficacy of chemotherapy with CDDP by stimulating anticancer immunity.

Decreased vitamin B12 availability induces ER stress through impaired SIRT1-deacetylation of HSF1

Vitamin B12 (cobalamin) is a key determinant of S-adenosyl methionine (SAM)-dependent epigenomic cellular regulations related to methylation/acetylation and its deficiency produces neurodegenerative disorders by elusive mechanisms. Sirtuin 1 deacetylase (SIRT1) triggers cell response to nutritional stress through endoplasmic reticulum (ER) stress. Recently, we have established a N1E115 dopaminergic cell model by stable expression of a transcobalamin–oleosin chimera (TO), which impairs cellular availability of vitamin B12, decreases methionine synthase activity and SAM level, and reduces cell proliferation. In contrast, oleosin-transcobalamin chimera (OT) does not modify the phenotype of transfected cells. Presently, the impaired cellular availability of vitamin B12 in TO cells activated irreversible ER stress pathways, with increased P-eIF-2α, P-PERK, P-IRE1α, ATF6, ATF4, decreased chaperon proteins and increased pro-apoptotic markers, CHOP and cleaved caspase 3, through reduced SIRT1 expression and consequently greater acetylation of heat-shock factor protein 1 (HSF1). Adding either B12, SIRT1, or HSF1 activators as well as overexpressing SIRT1 or HSF1 dramatically reduced the activation of ER stress pathways in TO cells. Conversely, impairing SIRT1 and HSF1 by siRNA, expressing a dominant negative form of HSF1, or adding a SIRT1 inhibitor led to B12-dependent ER stress in OT cells. Addition of B12 abolished the activation of stress transducers and apoptosis, and increased the expression of protein chaperons in OT cells subjected to thapsigargin, a strong ER stress stimulator. AdoX, an inhibitor of methyltransferase activities, produced similar effects than decreased B12 availability on SIRT1 and ER stress by a mechanism related to increased expression of hypermethylated in cancer 1 (HIC1). Taken together, these data show that cellular vitamin B12 has a strong modulating influence on ER stress in N1E115 dopaminergic cells. The impaired cellular availability in vitamin B12 induces irreversible ER stress by greater acetylation of HSF1 through decreased SIRT1 expression, whereas adding vitamin B12 produces protective effects in cells subjected to ER stress stimulation.