Endoplasmic Reticulum Stress Unfolded Protein Response Research Articles

MiRNAs associated with endoplasmic reticulum stress and unfolded protein response during decidualization

Do microRNAs (miRNAs) play a role in regulating endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) in decidualized cells and endometrium associated with reproductive failures? Endometrial stromal cell line St-T1b was decidualized in vitro with 8-Br-cAMP over 5 days, or treated with the ERS inducer thapsigargin. Expression of ERS sensors, UPR markers and potential miRNA regulators was analysed by quantitative PCR. Endometrial biopsies from patients with recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF) were investigated for the location of miRNA expression. Decidualization of St-T1b cells resulted in increased expression of ERS sensors including ATF6α, PERK and IRE1α, and the UPR marker, CHOP. TXNIP, which serves as a link between the ERS pathway and inflammation, as well as inflammasome NLRP3 and interleukin 1β expression increased in decidualized cells. An in-silico analysis identified miR-17-5p, miR-21-5p and miR-193b-3p as miRNAs potentially involved in regulation of the ERS/UPR pathways and inflammation associated with embryo implantation. Their expression decreased significantly (P ≤ 0.0391) in non-decidualized cells in the presence of thapsigargin. Finally, expression of the selected miRNAs was localized by in-situ hybridization in stromal and glandular epithelial cells in endometrial samples from patients with RPL and RIF. Expression in stroma cells from patients with RPL was lower in comparison with stroma cells from patients with RIF. Decidualization in St-T1b cells is accompanied by ERS/UPR processes, associated with an inflammatory response that is potentially influenced by miR-17-5p, miR-21-5p and miR-193b-3p. These miRNAs are expressed differentially in stromal cells from patients with RPL and RIF, indicating an alteration in regulation of the ERS/UPR pathways.

Ac-SDKP inhibits Tunicamycin-induced ER stress UPR's sensors and subsequently inflammation and fibrosis in human cardiac fibroblasts

Background: Endoplasmic Reticulum (ER) stress, chronic inflammation and cardiac fibrosis are very common pathophysiologic processes found in hypertension and other cardiovascular diseases. Prolonged ER stress disturbs ER functional capacity, resulting from accumulated unfolded or misfolded proteins in ER lumen. Thereby, cells activate an adaptive mechanism ‘unfolded protein response (UPR)’ to restore ER normal function. Consequently, a series of downstream signaling pathways are activated. N-acetyl-seryl-aspartyl-proline (Ac-SDKP) is an endogenous tetrapeptide released from its precursor thymosin-β4 and found in circulation and organs including heart and kidneys. Previously, our in-vivo studies showed that Ac-SDKP inhibits inflammation and fibrosis through the mechanism that remains elusive. Methods and Results: Quiescent human Cardiac Fibroblast (hCFB) cells were treated with tunicamycin (TM; 0.25 μg/mL) in absence or presence of Ac-SDKP (10 nM) and Tauroursodeoxycholic acid (TUDCA; 500 μg/mL). Ac-SDKP inhibited TM-induced expression of ER stress and downstream mediated UPRs sensors IRE1ɑ, XBP1, ATF4 and CHOP encoding molecular chaperons and protein processing enzymes, at the transcriptional and translational levels [ IRE1α; Ctl (1.0 ± 0.39)***, TM (2.99 ± 0.34), Ac-SDKP +TM (1.9 ± 0.10)*** Tudca +TM (2.5 ± 0.20)***. XBP1; Ctl (1.0 ± 0.36)***, TM (8.56 ± 0.77), Ac-SDKP +TM (4.57 ± 0.4)***, Tudca +TM (5.1 ± 0.6)***. ATF4; Ctl (1.0 ± 0.17)***, TM (5.35 ± 0.63), Ac-SDKP +TM (3.0 ± 0.31)**, Tudca +TM (2.9 ± 0.36)**. CHOP; Ctl (1.0 ± 0.17)***, TM (3.1 ± 0.21), Ac-SDKP +TM (1.75 ± 0.24)***, Tudca +TM (2.0 ± 0.18)**]. The inhibition of ER and UPRs sensors subsequently prevented proinflammatory mediator NF-kB activation, cytokine IL-6 and fibrotic Collagen-I (Coll I) expression [ NF-κB; Ctl (1.0 ± 0.10)*, TM (1.6 ± 0.13), Ac-SDKP +TM (1.08 ± 0.11)†, Tudca +TM (1.09 ± 0.14)†. IL-6; Ctl (1.0 ± 0.18)***, TM (6.8 ± 1.4), Ac-SDKP +TM (1.24 ± 0.23***), Tudca +TM (1.3 ± 0.35)***. Coll I; Ctl (1.0 ± 0.02)*** TM (2.5 ± 0.43), Ac-SDKP +TM (1.13 ± 0.13)***, Tudca +TM (1.22 ± 0.18)***]. TM also induced collagen production (Hydroxyproline assay), which was significantly inhibited by Ac-SDKP. The common ER stress inhibitor, TUDCA, exhibited similar inhibitory profile as Ac-SDKP. Conclusion: Ac-SDKP prevents inflammation and fibrosis in hCFB, likely by blocking the overexpressed detrimental ER stress UPRs sensors and its downstream molecular chaperon. Notes: Data are shown a mean ± SEM, p values were calculated using one-way ANOVA compared to CTL. All the experiments are representative of n = 5 – 8.***:p ≤ 0.0001 vs TM; **:p ≤ 0.001 vs TM; *: p ≤ 0.05 vs TM; †:p ≤ 0.05 vs TM. NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aggregative Perivascular Tumor Cell Growth Pattern of Primary Central Nervous System Lymphomas Is Associated with Hypoxia-Related Endoplasmic Reticulum Stress.

Primary central nervous system lymphomas (PCNSLs) often present a unique histopathological feature of aggregative perivascular tumor cells (APVT). Our previous studies showed that patients of PCNSL with APVTs exhibited poor long-term outcomes and increased expression of the endoplasmic reticulum stress (ERS) factor X-box-binding protein (XBP1). However, very little is known about molecular mechanism of the APVT formation in PCNSLs. The aim of this study is to determine if hypoxia-induced ERS is related to the APVT formation in PCNSLs. In this study, cell culture was used to observe the interplay between diffuse large B cell lymphoma (DLBCL) tumor cells and human brain microvascular endothelial cells (HBMECs) in different oxygen conditions. The expression of XBP1, CXCR and CD44 was manipulated by molecular cloning and siRNA technology. Mouse in vivo experiments and clinical studies were conducted to confirm our hypothesis. Our results showed that activated B-cell type-DLBCL cells easily migrated and invaded, and expressed high levels of XBP1 and stromal molecules CXCR4 and CD44 during hypoxia-induced ERS and dithiothreitol unfolded protein response (UPR). The gene upregulation (using overexpression vector) and downregulation (siRNA gene knock-out) in cultured cells and in mouse models further confirmed a close relation of the expression of XBP1, CXCR4, and CD44 with APVT formation, which is coincided with our clinical observation that increased expression of XBP1, CXCR4, and CD44 in the APVT cells in PCNSLs were associated with poor clinical outcomes. The results suggest that hypoxia-induced ERS and UPR might be associated with APVTs formation in PCNSL and its poor clinical outcomes. The results will help us better understand the progression of PCNSL with APVTs feature in daily pathological work and could be valuable for future target treatment of PCNSLs.

Oleic acid ameliorates palmitic acid-induced ER stress and inflammation markers in naive and cerulein-treated exocrine pancreas cells.

Dietary fat overload (typical to obesity) increases the risk of pancreatic pathologies through mechanisms yet to be defined. We previously showed that saturated dietary fat induces pancreatic acinar lipotoxicity and cellular stress. The endoplasmic reticulum (ER) of exocrine pancreas cells is highly developed and thus predisposed to stress. We studied the combination of saturated and unsaturated FAs in metabolic and pancreatitis like cerulein (CER)-induced stress states on cellular ER stress.Exocrine pancreas AR42J and rat primary exocrine acinar cells underwent acute (24 h) challenge with different FAs (saturated, monounsaturated) at different concentrations (250 and 500 µM) and in combination with acute CER-induced stress, and were analyzed for fat accumulation, ER stress unfolded protein response (UPR) and immune and enzyme markers. Acute exposure of AR42J and pancreatic acinar cells to different FAs and their combinations increased triglyceride accumulation. Palmitic acid significantly dose-dependently enhanced the UPR, immune factors and pancreatic lipase (PL) levels, as demonstrated by XBP1 splicing and elevation in UPR transcripts and protein levels (Xbp1,Atf6, Atf4, Chop, Tnfα, Tgfβ and Il-6). Exposure to high palmitic levels in a CER-induced stress state synergistically increased ER stress and inflammation marker levels. Exposure to oleic acid did not induce ER stress and PL levels and significantly decreased immune factors in an acute CER-induced stress state. Combination of oleic and palmitic acids significantly reduced the palmitic-induced ER stress, but did not affect the immune factor response. We show that combination of monounsaturated and saturated FAs protects from exocrine pancreatic cellular ER stress in both metabolic and CER-induced stress.

Defective interplay between mTORC1 activity and endoplasmic reticulum stress-unfolded protein response in uremic vascular calcification.

Vascular calcification increases the risk of cardiovascular disease and death in patients with chronic kidney disease (CKD). Increased activity of mammalian target of rapamycin complex 1 (mTORC1) and endoplasmic reticulum (ER) stress-unfolded protein response (UPR) are independently reported to partake in the pathogenesis of vascular calcification in CKD. However, the association between mTORC1 activity and ER stress-UPR remains unknown. We report here that components of the uremic state [activation of the receptor for advanced glycation end products (RAGE) and hyperphosphatemia] potentiate vascular smooth muscle cell (VSMC) calcification by inducing persistent and exaggerated activity of mTORC1. This gives rise to prolonged and excessive ER stress-UPR as well as attenuated levels of sestrin 1 ( Sesn1) and Sesn3 feeding back to inhibit mTORC1 activity. Activating transcription factor 4 arising from the UPR mediates cell death via expression of CCAAT/enhancer-binding protein (c/EBP) homologous protein (CHOP), impairs the generation of pyrophosphate, a potent inhibitor of mineralization, and potentiates VSMC transdifferentiation to the osteochondrocytic phenotype. Short-term treatment of CKD mice with rapamycin, an inhibitor of mTORC1, or tauroursodeoxycholic acid, a bile acid that restores ER homeostasis, normalized mTORC1 activity, molecular markers of UPR, and calcium content of aortas. Collectively, these data highlight that increased and/or protracted mTORC1 activity arising from the uremic state leads to dysregulated ER stress-UPR and VSMC calcification. Manipulation of the mTORC1-ER stress-UPR pathway opens up new therapeutic strategies for the prevention and treatment of vascular calcification in CKD.

Pancreatic stellate cell activation is regulated by fatty acids and ER stress

IntroductionPancreatic pathologies are characterized by a progressive fibrosis process. Pancreatic stellate cells (PSC) play a crucial role in pancreatic fibrogenesis. Endoplasmic reticulum (ER) stress emerges as an important determinant of fibrotic remodeling. Overload of fatty acids (FA), typical to obesity, may lead to lipotoxic state and cellular stress. AimTo study the effect of different lipolytic challenges on pancreatic ER stress and PSC activation. MethodsPrimary PSCs were exposed to different FAs, palmitate (pal) and oleate (ole), at pathophysiological concentrations typical to obese state, and in acute caerulein-induced stress (cer). PSC activation and differentiation were analyzed by measuring fat accumulation (oil-red staining and quantitation), proliferation (cells count) and migration (wound- healing assay). PSC differentiation markers (α-sma, fibronectin, tgf-β and collagen secretion), ER stress unfolded protein response and immune indicators (Xbp1, CHOP, TNF-α, IL-6) were analyzed at the transcript and protein expression levels (quantitative RT-PCR and western blotting). ResultsPSC exposure to pal and ole FAs (500µM) increased significantly fat accumulation. Proliferation and migration analysis demonstrated that ole FA retained PSC activation, while exposure to pal FA significantly halted proliferation rate and delayed migration. Cer significantly augmented PSC differentiation markers α- sma, fibronectin and collagen, and ER stress and inflammation markers including Xbp1, CHOP, TNF-α and IL-6. The ole FA treatment significantly elevated PSC differentiation markers α-sma, fibronectin and collagen secretion. PSC ER stress was demonstrated following pal treatment with significant elevation of Xbp1 splicing and CHOP levels. ConclusionExposure to pal FA halted PSC activation and differentiation and elevated ER stress markers, while cer and ole exposure significantly induced activation, differentiation and fibrosis. Thus, dietary FA composition should be considered and optimized to regulate PSC activation and differentiation in pancreatic pathologies.

The role of the Amyloid Precursor Protein mutations and PERKdependent signaling pathways in the pathogenesis of Alzheimer’s disease

Alzheimer’s disease (AD) is a highly complex, progressive, age-related neurodegenerative human disease entity. The genetic basis of AD is strictly connected with occurrence of mutations in Amyloid Precursor (APP) gene on chromosome 21. Molecular mechanism that leads to AD development still remains unclear. Recent data reported that it is closely correlated with Endoplasmic Reticulum (ER) stress conditions, which subsequently activate Unfolded Protein Response (UPR) signaling pathways, via the induction of protein kinase RNA-like endoplasmic reticulum kinase (PERK), as a self-protective, adaptive response to adverse stress conditions. That results in the attenuation of global protein synthesis and, on the contrary, selective translation of Activating Transcriptor Factor 4 (ATF4) and secretase β. Interestingly, under prolonged, severe ER stress UPR may switch its signal into apoptotic cell death. That ensues by ATF4-CHOP-mediated activation of a range of pro-apoptotic genes and, on the other hand, downregulation of the expression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) genes. Current investigations suggest that inhibitions of PERK activity may contribute to the attenuation of the deposition of toxic senile plaques in the brain tissue and, as a result, prevent degeneration of neurons and decline in cognitive abilities.

MP84-10 ERG INDUCES ENDOPLASMIC RETICULUM STRESS (ER) AND UNFOLDED PROTEIN RESPONSE (UPR) TO INITIATE PROSTATE CANCER CARCINOGENESIS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2016MP84-10 ERG INDUCES ENDOPLASMIC RETICULUM STRESS (ER) AND UNFOLDED PROTEIN RESPONSE (UPR) TO INITIATE PROSTATE CANCER CARCINOGENESIS Taduru L. Sreenath, Shiela S. Macalindong, Natallia Mikhalkevich, Shashwat Sharad, Parameet Kumar, Denise Young, Rishita Gupta, Shilpa Katta, Ahmed Mohamed, Shyh-Han Tan, Albert Dobi, Gyorgy Petrovics, Isabell A. Sesterhenn, Charles J. Bieberich, Peter Nelson, David G. McLeod, Valeri Vasioukhin, and Shiv Srivastava Taduru L. SreenathTaduru L. Sreenath More articles by this author , Shiela S. MacalindongShiela S. Macalindong More articles by this author , Natallia MikhalkevichNatallia Mikhalkevich More articles by this author , Shashwat SharadShashwat Sharad More articles by this author , Parameet KumarParameet Kumar More articles by this author , Denise YoungDenise Young More articles by this author , Rishita GuptaRishita Gupta More articles by this author , Shilpa KattaShilpa Katta More articles by this author , Ahmed MohamedAhmed Mohamed More articles by this author , Shyh-Han TanShyh-Han Tan More articles by this author , Albert DobiAlbert Dobi More articles by this author , Gyorgy PetrovicsGyorgy Petrovics More articles by this author , Isabell A. SesterhennIsabell A. Sesterhenn More articles by this author , Charles J. BieberichCharles J. Bieberich More articles by this author , Peter NelsonPeter Nelson More articles by this author , David G. McLeodDavid G. McLeod More articles by this author , Valeri VasioukhinValeri Vasioukhin More articles by this author , and Shiv SrivastavaShiv Srivastava More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2234AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES High levels of the ERG expression due to gene fusions (predominantly TMPRSS2-ERG) is frequently detected in prostate cancer (CaP) in Western countries. Better understanding of the roles of the ERG oncogenic functions in CaP initiation and/or progression will enhance its utility as the therapeutic target. Transgenic mice generated to understand ERG’s role in the development of prostate adenocarcinoma have shown focal proliferative and dysplastic PIN lesions and later developed adenocarcinoma in older mice. Mice with ERG and PTEN alterations exhibit more accelerated malignant progression in prostate. The present study was aimed towards understanding the biological functions of ERG in early stages oncogenic transformation of prostate epithelium. METHODS Morphological analyses of mouse prostate glands were analyzed by H&E staining and by electron microscopy. Luminal cell surface markers were analyzed by FACS analysis. Luminal cells were analyzed for their potential to proliferate and form spheres by prostate sphere formation assay. ER stress and UPR marker proteins were analyzed by Western blot analysis. Analysis of representative ER stress pathway in ERG-positive human prostate cancer was analyzed in CPDR-80 gene-chip array data set representing well- and poor differentiated tumors. RESULTS Histological examination of ERG-Tg mouse prostates revealed increased luminal cell death due to apoptosis. Subsequently, TEM analysis revealed significant morphological differences such as increased numbers of lysosomes, autophagosomes, concentric membrane bodies with ribosomes, and lipid droplets in the cytoplasm of transgenic secretory luminal epithelial cells. Epithelial cells of ERG-Tg mouse prostates showed ~70% increase in CD49f (low) and Sca-1 (med) population with increased sphere formation capability and resistance to induced cell death. Western blots of ERG-Tg mouse and LNCaP-ERG transfectants showed increased expression of ER stress sensors and UPR proteins. A strong positive correlation was also observed between ERG and P4HB/PDI, one of the ER stress response proteins in human prostate tumors. CONCLUSIONS Cumulative data from experimental models of ERG and human CaP suggests that ERG mediates ER stress resulting into the activation of UPR, cell death with eventual biological selection for cell survival. Taken together these data indicate a critical biological function for ERG in prostate cancer initiation. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1093 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Taduru L. Sreenath More articles by this author Shiela S. Macalindong More articles by this author Natallia Mikhalkevich More articles by this author Shashwat Sharad More articles by this author Parameet Kumar More articles by this author Denise Young More articles by this author Rishita Gupta More articles by this author Shilpa Katta More articles by this author Ahmed Mohamed More articles by this author Shyh-Han Tan More articles by this author Albert Dobi More articles by this author Gyorgy Petrovics More articles by this author Isabell A. Sesterhenn More articles by this author Charles J. Bieberich More articles by this author Peter Nelson More articles by this author David G. McLeod More articles by this author Valeri Vasioukhin More articles by this author Shiv Srivastava More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Uterine endoplasmic reticulum stress-unfolded protein response regulation of gestational length is caspase-3 and -7–dependent

We previously identified myometrial caspase-3 (CASP3) as a potential regulator of uterine quiescence. We also determined that during pregnancy, the functional activation of uterine CASP3 is likely governed by an integrated endoplasmic reticulum stress response (ERSR) and is consequently limited by an increased unfolded protein response (UPR). The present study examined the functional relevance of uterine UPR-ERSR in maintaining myometrial quiescence and regulating the timing of parturition. In vitro analysis of the human uterine myocyte hTERT-HM cell line revealed that tunicamycin (TM)-induced ERSR modified uterine myocyte contractile responsiveness. Accordingly, alteration of in vivo uterine UPR-ERSR using a pregnant mouse model significantly modified gestational length. We determined that "normal" gestational activation of the ERSR-induced CASP3 and caspase 7 (CASP7) maintains uterine quiescence through previously unidentified proteolytic targeting of the gap junction protein, alpha 1(GJA1); however, surprisingly, TM-induced uterine ERSR triggered an exaggerated UPR that eliminated uterine CASP3 and 7 tocolytic action precociously. These events allowed for a premature increase in myometrial GJA1 levels, elevated contractile responsiveness, and the onset of preterm labor. Importantly, a successful reversal of the magnified ERSR-induced preterm birth phenotype could be achieved by pretreatment with 4-phenylbutrate, a chaperone protein mimic.

Rg1 exhibits neuroprotective effects by inhibiting the endoplasmic reticulum stress-mediated c-Jun N-terminal protein kinase apoptotic pathway in a rat model of Alzheimer's disease.

The neuroprotective agents currently used to treat Alzheimer's disease (AD) often only target one aspect of the disease process. Therefore, identifying effective drug targets associated with the pathogenesis of AD is critical for the production of novel AD therapeutic strategies. The present study aimed to investigate the underlying mechanisms of the neuroprotective effects of Rg1 on a rat model of AD. A double transgenic β‑amyloid (Aβ) precursor protein/PS1 rat model was established, which co‑expressed mutations associated with AD. Aβ plaques and neurofibrillary tangles (NFTs) were detected by immunohistochemistry. The detection of the protein expression levels of caspase‑3 and terminal deoxynucleotidyl‑transferase‑mediated dUTP nick end labeling (TUNEL) staining were used to determine the level of apoptosis in the brain tissue. The expression levels of the endoplasmic reticulum (ER) stress biomarker, glucose‑regulated protein 78 (Grp78), and the mitochondrial apoptosis biomarkers, B‑cell lymphoma 2 (Bcl‑2) and Bcl‑2‑associated X protein (Bax), were analyzed by western blotting. Furthermore, the expression of the proteins associated with the ER stress unfolded protein response (UPR) was determined, in order to examine the levels of ER stress. The mRNA expression of downstream genes of UPR were also detected by reverse transcription‑polymerase chain reaction. The protein expression levels of the apoptosis‑associated phosphorylated‑c‑Jun N‑terminal protein kinase (p‑JNK), caspase‑12 and cAMP response element‑binding transcription factor homologous protein were determined by western blotting. The results of the present study indicated that the accumulation of NFTs and Aβ plaques was significantly decreased in the Rg1‑treated AD rats, compared with untreated AD rats. The expression of caspase‑3 and the number of TUNEL‑positive cells were also significantly decreased in the Rg1‑treated rats, as compared with the AD rats. Furthermore, treatment with Rg1 significantly reduced the expression of Grp78, and triggered inositol‑requiring enzyme‑1 (IRE‑1) and phosphorylated protein kinase RNA‑like ER kinase‑associated ER stress. The IRE‑1 UPR pathway downstream gene, tumor necrosis factor receptor‑associated factor 2, was significantly decreased in rats treated with Rg1, compared with untreated AD rats. Furthermore, the activation of p‑JNK was also inhibited when AD rats were treated with Rg1. In conclusion, Rg1 was shown to function as an important factor that inhibits the accumulation of NFTs and Aβ via inhibition of the ER stress‑mediated pathway. Blocking of this pathway was triggered by the IRE‑1 and TRAF2 pathway, as a result of inhibition of the expression of p‑JNK.

Nelfinavir and other protease inhibitors in cancer: mechanisms involved in anticancer activity.

To review the mechanisms of anti-cancer activity of nelfinavir and other protease inhibitors (PIs) based on evidences reported in the published literature. We extensively reviewed the literature concerning nelfinavir (NFV) as an off target anti-cancer drug and other PIs. A classification of PIs based on anti-cancer mode of action was proposed. Controversies regarding nelfinavir mode of action were also addressed. The two main mechanisms involved in anti-cancer activity are endoplasmic reticulum stress-unfolded protein response pathway and Akt inhibition. However there are many other effects, partially dependent and independent of those mentioned, that may be useful in cancer treatment, including MMP-9 and MMP-2 inhibition, down-regulation of CDK-2, VEGF, bFGF, NF-kB, STAT-3, HIF-1 alfa, IGF, EGFR, survivin, BCRP, androgen receptor, proteasome, fatty acid synthase (FAS), decrease in cellular ATP concentration and upregulation of TRAIL receptor DR5, Bax, increased radiosensitivity, and autophagy. The end result of all these effects is slower growth, decreased angiogenesis, decreased invasion and increased apoptosis, which means reduced proliferation and increased cancer cells death. PIs may be classified according to their anticancer activity at clinically achievable doses, in AKT inhibitors, ER stressors and Akt inhibitors/ER stressors. Beyond the phase I trials that have been recently completed, adequately powered and well-designed clinical trials are needed in the various cancer type settings, and specific trials where NFV is tested in association with other known anti-cancer pharmaceuticals should be sought, in order to find an appropriate place for NFV in cancer treatment. The analysis of controversies on the molecular mechanisms of NFV hints to the possibility that NFV works in a different way in tumor cells and in hepatocytes and adipocytes.

Nelfinavir and other protease inhibitors in cancer: mechanisms involved in anticancer activity

To review the mechanisms of anti-cancer activity of nelfinavir and other protein inhibitors (PIs) based on evidences reported in the published literature. We extensively reviewed the literature concerning nelfinavir (NFV) as an off target anti-cancer drug and other PIs. A classification of PIs based on anti-cancer mode of action was proposed. Controversies regarding nelfinavir mode of action were also addressed. The two main mechanisms involved in anti-cancer activity are endoplasmic reticulum stress-unfolded protein response pathway and Akt inhibition. However there are many other effects, partially dependent and independent of those mentioned, that may be useful in cancer treatment, including MMP-9 and MMP-2 inhibition, down-regulation of CDK-2, VEGF, bFGF, NF-kB, STAT-3, HIF-1 alfa, IGF, EGFR, survivin, BCRP, androgen receptor, proteasome, fatty acid synthase (FAS), decrease in cellular ATP concentration and upregulation of TRAIL receptor DR5, Bax, increased radiosensitivity, and autophagy. The end result of all these effects is slower growth, decreased angiogenesis, decreased invasion and increased apoptosis, which means reduced proliferation and increased cancer cells death. PIs may be classified according to their anticancer activity at clinically achievable doses, in AKT inhibitors, ER stressors and Akt inhibitors/ER stressors. Beyond the phase I trials that have been recently completed, adequately powered and well-designed clinical trials are needed in the various cancer type settings, and specific trials where NFV is tested in association with other known anti-cancer pharmaceuticals should be sought, in order to find an appropriate place for NFV in cancer treatment. The analysis of controversies on the molecular mechanisms of NFV hints to the possibility that NFV works in a different way in tumor cells and in hepatocytes and adipocytes.

Calcium-independent Phospholipase A2γ Enhances Activation of the ATF6 Transcription Factor during Endoplasmic Reticulum Stress

Injury of visceral glomerular epithelial cells (GECs) causes proteinuria in many glomerular diseases. We reported previously that calcium-independent phospholipase A2γ (iPLA2γ) is cytoprotective against complement-mediated GEC injury. Because iPLA2γ is localized at the endoplasmic reticulum (ER), this study addressed whether the cytoprotective effect of iPLA2γ involves the ER stress unfolded protein response (UPR). In cultured rat GECs, overexpression of the full-length iPLA2γ, but not a mutant iPLA2γ that fails to associate with the ER, augmented tunicamycin-induced activation of activating transcription factor-6 (ATF6) and induction of the ER chaperones, glucose-regulated protein 94 (GRP94) and glucose-regulated protein 78 (GRP78). Augmented responses were inhibited by the iPLA2γ inhibitor, (R)-bromoenol lactone, but not by the cyclooxygenase inhibitor, indomethacin. Tunicamycin-induced cytotoxicity was reduced in GECs expressing iPLA2γ, and the cytoprotection was reversed by dominant-negative ATF6. GECs from iPLA2γ knock-out mice showed blunted ATF6 activation and chaperone up-regulation in response to tunicamycin. Unlike ATF6, the two other UPR pathways, i.e. inositol-requiring enzyme 1α and protein kinase RNA-like ER kinase pathways, were not affected by iPLA2γ. Thus, in GECs, iPLA2γ amplified activation of the ATF6 pathway of the UPR, resulting in up-regulation of ER chaperones and cytoprotection. These effects were dependent on iPLA2γ catalytic activity and association with the ER but not on prostanoids. Modulating iPLA2γ activity may provide opportunities for pharmacological intervention in glomerular diseases associated with ER stress.

ER stress and unfolded protein response in amyotrophic lateral sclerosis-a controversial role of protein disulphide isomerase.

Accumulation of proteins in aberrant conformation occurs in many neurodegenerative diseases. Furthermore, dysfunctions in protein handling in endoplasmic reticulum (ER) and the following ER stress have been implicated in a vast number of diseases, such as amyotrophic lateral sclerosis (ALS). During excessive ER stress unfolded protein response (UPR) is activated to return ER to its normal physiological balance. The exact mechanisms of protein misfolding, accumulation and the following ER stress, which could lead to neurodegeneration, and the question whether UPR is a beneficial compensatory mechanism slowing down the neurodegenerative processes, are of interest. Protein disulphide isomerase (PDI) is a disulphide bond-modulating ER chaperone, which can also facilitate the ER-associated degradation (ERAD) of misfolded proteins. In this review we discuss the recent findings of ER stress, UPR and especially the role of PDI in ALS.

Retraction note to: "Human leptin triggers proliferation of A549 cells via blocking endoplasmic reticulum stress-related apoptosis" [Biochemistry (Moscow), 78, No. 12, 1333-1341 (2013)

Lung cancer is a disease characterized by uncontrolled cell growth in tissues of the lung. Leptin is a pleiotropic hormone with antiapoptotic and proliferative roles involved in several systems. However, there is no known antiapoptotic mechanism of leptin in non-small cell lung cancer (NSCLC). So, we investigated the antiapoptotic mechanism of leptin in NSCLC. Proliferation, apoptosis, and the specific mechanism of leptin-transfected cells were analyzed in this study. Leptin, p-Perk, IRE1, cleaved ATF6, spliced XBP1, eIF2-α, TRAF2, CHOP, and caspase 12 proteins were detected by Western blot, and endoplasmic reticulum (ER) stress-related mRNA was detected by semi-quantitative reverse transcription PCR (RT-PCR). Leptin in A549 and transfected cells inhibited cisplatin-activated ER stress-associated mRNA transcription and activation of proteins. ER stress unfolded protein response (UPR) proteins, PERK and ATF6, were involved in leptin-triggered apoptosis. XBP1 and TRAF2 were increased significantly when treated with cisplatin in A549-siLPT and non-transfected cells. CHOP expression was blocked in A549 and transfected cells (LPT-PeP and LPT-EX cells). In conclusion, leptin can promote the proliferation of A549 cells through blocking ER stress-mediated apoptosis. This blocking is mediated by the p-Perk and ATF6 pathway through blocking activation of CHOP.

Human leptin protein activates the growth of HepG2 cells by inhibiting PERK‑mediated ER stress and apoptosis.

Current treatment modalities for various types of hepatic cancer, which has an increasing incidence rate, are inadequate and novel therapies are required. Therefore, identifying targets for liver cancer is becoming increasingly valuable to develop novel methods for therapy. The aim of the present study was to examine the growth activation mechanism of the leptin protein in the liver cancer cell line HepG2. The effects of the leptin protein on cell death were investigated by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide analysis. DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end labeling analysis were also performed to detect cell apoptosis. The expression of leptin and three endoplasmic reticulum (ER) stress unfolded protein response (UPR) proteins, including activating transcription factor 6, phosphorylated‑PKR‑like ER kinase (p‑PERK) and inositol requiring protein 1, were investigated for the examination of ER stress. The mRNA UPR proteins were also detected by reverse transcription polymerase chain reaction. The apoptosis‑associated caspase 12 and C/EBP homologous protein (CHOP) was detected by western blot analysis. The expression of or incubation with the leptin protein was able to activate cell growth and inhibit cell death and apoptosis. In cells that expressed leptin or were incubated with leptin protein (pep-LPT), cisplatin‑induced ER stress‑associated mRNA transcription and protein activation were inhibited. Levels of the ER stress UPR pathway protein, PERK, increased significantly in leptin‑silenced cells when treated with cisplatin as compared with those in the leptin‑expressing or pep-LPT cells. Furthermore, caspase 12 activation was inhibited in ex‑LPT, pep‑LPT and HepG2 cells. In conclusion, human leptin protein is involved in promoting the proliferation of HepG2 cells through inhibiting the ER stress‑associated apoptotic pathway. The PERK UPR pathway and the apoptotic factor caspase 12 were found to be involved in the inhibition of apoptosis and enhancement of proliferation.

Liraglutide alleviates diabetic cardiomyopathy by blocking CHOP-triggered apoptosis via the inhibition of the IRE-α pathway

Clinically, diabetes mellitus is closely associated with and induces certain cardiovascular diseases. The aim of this study was to investigate endoplasmic reticulum (ER) stress-associated apoptosis of diabetic cardiomyopathy (DCM), and explore the protective mechanism of liraglutide. The DCM model was established with a high-fat diet and streptozotocin (STZ). Cardiac function was detected by echocardiogram examination and hematoxylin-eosin staining. ER stress unfolded protein response (UPR) hallmarks [inositol-requiring enzyme-α (IRE-α), p-Perk and ATF6] and transcription factors were detected with western blotting. Apoptosis inducers CHOP, c-Jun amino terminal kinase (JNK) and casapse-12 were also examined with western blotting. The results indicated that liraglutide is capable of improving cardiac function in DCM rats (P<0.05). IRE-α expression was significantly increased in the DCM group compared with the control group (P<0.05), and liraglutide is capable of decreasing IRE-α expression. X-box transcription factor-1 (XBP-1) was significantly spliced in the model group, and downregulated in the liraglutide-treated group. CHOP protein was upregulated in the DCM group, but inactivated by liraglutide treatment. In conclusion, liraglutide is capable of protecting DCM and blocking CHOP-mediated ER stress by inhibiting the IRE-α UPR pathway.

Melittin protein inhibits the proliferation of MG63 cells by activating inositol-requiring protein-1α and X-box binding protein 1-mediated apoptosis

The aim of the present study was to explore the pro-apoptotic effect and specific mechanism of action of melittin (MEL) in humans. The effects of MEL on apoptosis in osteosarcoma and fetal osteoblast cells were investigated, and the mechanism that induced MG63 cell growth was also explored. The effects of MEL on cell proliferation were detected by a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide analysis. Apoptosis was detected by flow cytometric analysis. MEL protein, inositol-requiring protein-1 (IRE-α), phosphorylated-protein kinase R-like endoplasmic reticulum (ER) kinase, spliced X-box-1 (XBP1), eukaryotic translation initiation factor-2α, cleaved activating transcription factor-6, caspase-12 and C/EBP homology protein (CHOP) were detected in three groups and two cell lines by western blot analysis. The results indicated that the expression or incubation of MEL in the MG63 cells triggered apoptosis and the inhibition of proliferation. One protein from the ER stress unfolded protein response pathway, IRE-α, was involved in the MEL-induced apoptosis in MG63 cells. Furthermore, spliced XBP1 protein was significantly increased in the MEL peptide incubated and MEL expressing groups of MG63 cells. Furthermore, CHOP protein expression was activated in MG63 cells following being incubated with or expressing MEL. In conclusion, MEL serves as an effective factor that inhibits the proliferation of MG63 cells via activating the ER stress-mediated apoptosis pathway. This activation is triggered by the IRE-α pathway mediated by inducing CHOP protein expression.

RETRACTED ARTICLE: Human leptin triggers proliferation of A549 cells via blocking endoplasmic reticulum stress-related apoptosis

Lung cancer is a disease characterized by uncontrolled cell growth in tissues of the lung. Leptin is a pleiotropic hormone with antiapoptotic and proliferative roles involved in several systems. However, there is no known antiapoptotic mechanism of leptin in non-small cell lung cancer (NSCLC). So, we investigated the antiapoptotic mechanism of leptin in NSCLC. Proliferation, apoptosis, and the specific mechanism of leptin-transfected cells were analyzed in this study. Leptin, p-Perk, IRE1, cleaved ATF6, spliced XBP1, eIF2-α, TRAF2, CHOP, and caspase 12 proteins were detected by Western blot, and endoplasmic reticulum (ER) stress-related mRNA was detected by semi-quantitative reverse transcription PCR (RT-PCR). Leptin in A549 and transfected cells inhibited cisplatin-activated ER stress-associated mRNA transcription and activation of proteins. ER stress unfolded protein response (UPR) proteins, PERK and ATF6, were involved in leptin-triggered apoptosis. XBP1 and TRAF2 were increased significantly when treated with cisplatin in A549-siLPT and non-transfected cells. CHOP expression was blocked in A549 and transfected cells (LPT-PeP and LPT-EX cells). In conclusion, leptin can promote the proliferation of A549 cells through blocking ER stress-mediated apoptosis. This blocking is mediated by the p-Perk and ATF6 pathway through blocking activation of CHOP.

Human Leptin Protein Induces Proliferation of A549 Cells via Inhibition of PKR-Like ER Kinase and Activating Transcription Factor-6 Mediated Apoptosis

PurposeTo investigate the anti-apoptotic mechanism of leptin in non-small cell lung cancer.Materials and MethodsThe influences of leptin on apoptosis were investigated, analyzing the mechanism that triggers growth of A549 cells. The effects of leptin on cell proliferation were examined by XTT analysis. Leptin, C/EBP homologous protein (CHOP), phosphorylated-PKR-like ER kinase (p-Perk), inositol requiring proteins-1, spliced X-box transcription factor-1 (XBP1), cleaved activating transcription factor-6 (ATF6), eukaryotic translation initiation factor-2α, caspase-12 and CHOP protein were detected in four groups by western blot, and endoplasmic reticulum (ER) stress related mRNA were detected by reverse transcription PCR.ResultsThe expression of leptin in A549 and leptin transfected cells inhibited cisplatin activated ER stress-associated mRNA transcription and protein activation. Two ER stress unfolded protein response pathways, PERK and ATF6, were involved, and XBP1 and tumor necrosis factor receptor-associated factor 2 (TRAF2) were increased significantly when treated with cisplatin in A549-siRNA against leptin cells. Furthermore, CHOP expression was inhibited upon leptin expression in A549, LPT-PeP and LPT-EX cells.ConclusionLeptin serves as an important factor that promotes the growth of A549 cells through blocking ER stress-mediated pathways. This blocking is triggered by p-Perk and ATF6 via inhibition of CHOP expression.