Pro-apoptotic Endoplasmic Reticulum Stress Research Articles

Antenatal Ureaplasma Infection Causes Colonic Mucus Barrier Defects: Implications for Intestinal Pathologies.

Chorioamnionitis is a risk factor for necrotizing enterocolitis (NEC). Ureaplasma parvum (UP) is clinically the most isolated microorganism in chorioamnionitis, but its pathogenicity remains debated. Chorioamnionitis is associated with ileal barrier changes, but colonic barrier alterations, including those of the mucus barrier, remain under-investigated, despite their importance in NEC pathophysiology. Therefore, in this study, the hypothesis that antenatal UP exposure disturbs colonic mucus barrier integrity, thereby potentially contributing to NEC pathogenesis, was investigated. In an established ovine chorioamnionitis model, lambs were intra-amniotically exposed to UP or saline for 7 d from 122 to 129 d gestational age. Thereafter, colonic mucus layer thickness and functional integrity, underlying mechanisms, including endoplasmic reticulum (ER) stress and redox status, and cellular morphology by transmission electron microscopy were studied. The clinical significance of the experimental findings was verified by examining colon samples from NEC patients and controls. UP-exposed lambs have a thicker but dysfunctional colonic mucus layer in which bacteria-sized beads reach the intestinal epithelium, indicating undesired bacterial contact with the epithelium. This is paralleled by disturbed goblet cell MUC2 folding, pro-apoptotic ER stress and signs of mitochondrial dysfunction in the colonic epithelium. Importantly, the colonic epithelium from human NEC patients showed comparable mitochondrial aberrations, indicating that NEC-associated intestinal barrier injury already occurs during chorioamnionitis. This study underlines the pathogenic potential of UP during pregnancy; it demonstrates that antenatal UP infection leads to severe colonic mucus barrier deficits, providing a mechanistic link between antenatal infections and postnatal NEC development.

Mitigation of Acute Hepatotoxicity Induced by Cadmium Through Morin: Modulation of Oxidative and Pro-apoptotic Endoplasmic Reticulum Stress and Inflammatory Responses in Rats.

Cadmium (Cd) is a toxic heavy metal with significant environmental health hazards. It enters the body through various routes with tissue accumulation. The relatively longer half-life with slow body clearance significantly results in hepatotoxicity during its liver detoxification. Therefore, researchers are exploring the potential use of herbal-derived phytocomponents to mitigate their toxicity. Here, we investigated, for the first time, the possible ameliorative effect of the phytochemical Morin (3,5,7,29,49-pentahydroxyflavone) against acute Cd-induced hepatotoxicity while resolving its underlying cellular mechanisms in a rat animal model. The study involved 50 adult male Sprague-Dawley rats weighing 200-250g. The animals were divided into five equal groups: control, Cd, Morin100 + Cd, Morin200 + Cd, and Morin200. The 2nd, 3rd, and 4th groups were intraperitoneally treated with Cd (6.5mg/kg), while the 3rd, 4th, and 5th groups were orally treated with Morin (100 and 200mg/kg) for 5 consecutive days. On the 6th day, hepatic function (serum ALT, AST, ALP, LDH enzyme activities, and total bilirubin level) testing, transcriptome analysis, and immunohistochemistry were performed to elucidate the ameliorative effect of Morin on hepatotoxicity. In addition to restoring liver function and tissue injury, Morin alleviated Cd-induced hepatic oxidative/endoplasmic reticulum stress in a dose-dependent manner, as revealed by upregulating the expression of antioxidants (SOD, GSH, Gpx, CAT, and Nrf2) and decreasing the expression of ER stress markers. The expression of the proinflammatory mediators (TNF-α, IL-1-β, and IL-6) was also downregulated while improving the anti-inflammatory (IL-10 and IL-4) expression levels. Morin further slowed the apoptotic cascades by deregulating the expression of pro-apoptotic Bax and Caspase 12 markers concomitant with an increase in anti-apoptotic Blc2 mRNA expression. Furthermore, Morin restored Cd-induced tissue damage and markedly suppressed the cytoplasmic expression of JNK and p-PERK immunostained proteins. This study demonstrated the dose-dependent antioxidant hepatoprotective effect of Morin against acute hepatic Cd intoxication. This effect is likely linked with the modulation of upstream p-GRP78/PERK/ATF6 pro-apoptotic oxidative/ER stress and the downstream JNK/BAX/caspase 12 apoptotic signaling pathways.

Avocado Seeds-Mediated Alleviation of Cyclosporine A-Induced Hepatotoxicity Involves the Inhibition of Oxidative Stress and Proapoptotic Endoplasmic Reticulum Stress.

Previous studies reported disrupted hepatic function and structure following the administration of cyclosporine A (CsA) in humans and animals. Recently, we found that avocado seeds (AvS) ameliorated CsA-induced nephrotoxicity in rats. As a continuation, herein we checked whether AvS could also attenuate CsA-induced hepatotoxicity in rats. Subcutaneous injection of CsA (5 mg/kg) for 7 days triggered hepatotoxicity in rats, as indicated by liver dysfunction, redox imbalance, and histopathological changes. Oral administration of 5% AvS powder for 4 weeks ameliorated CsA-induced hepatotoxicity, as evidenced by (1) decreased levels of liver damage parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and bilirubin), (2) resumed redox balance in the liver (reduced malondialdehyde (MDA) and increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)), (3) downregulated hepatic expression of endoplasmic reticulum (ER) stress-related genes (X-box binding protein 1 (XBP1), binding immunoglobulin protein (BIP), C/EBP homologous protein (CHOP)), and apoptosis-related genes (Bax and Casp3), (4) upregulated expression of the anti-apoptotic gene Bcl2, (5) reduced DNA damage, and (6) improved liver histology. These results highlight the ability of AvS to ameliorate CsA-induced hepatotoxicity via the inhibition of oxidative stress and proapoptotic ER stress.

Selenium Yeast and Fish Oil Combination Diminishes Cancer Stem Cell Traits and Reverses Cisplatin Resistance in A549 Sphere Cells.

Cisplatin is a prevalent chemotherapeutic agent used for non-small cell lung cancer (NSCLC) that is difficult to treat by targeted therapy, but the emergence of resistance severely limits its efficacy. Thus, an effective strategy to combat cisplatin resistance is required. This study demonstrated that, at clinically achievable concentrations, the combination of selenium yeast (Se-Y) and fish oil (FO) could synergistically induce the apoptosis of cancer stem cell (CSC)-like A549 NSCLC sphere cells, accompanied by a reversal of their resistance to cisplatin. Compared to parental A549 cells, sphere cells have higher cisplatin resistance and possess elevated CSC markers (CD133 and ABCG2), epithelial–mesenchymal transition markers (anexelekto (AXL), vimentin, and N-cadherin), and cytoprotective endoplasmic reticulum (ER) stress marker (glucose-regulated protein 78) and increased oncogenic drivers, such as yes-associated protein, transcriptional coactivator with PDZ-binding motif, β-catenin, and cyclooxygenase-2. In contrast, the proapoptotic ER stress marker CCAAT/enhancer-binding protein homologous protein and AMP-activated protein kinase (AMPK) activity were reduced in sphere cells. The Se-Y and FO combination synergistically counteracted the above molecular features of A549 sphere cells and diminished their elevated CSC-like side population. AMPK inhibition by compound C restored the side population proportion diminished by this nutrient combination. The results suggest that the Se-Y and FO combination can potentially improve the outcome of cisplatin-treated NSCLC with phenotypes such as A549 cells.

Ketamine-induced neurotoxicity is mediated through endoplasmic reticulum stress in vitro in STHdhQ7/Q7 cells

Ketamine has traditionally been used as a dissociative anesthetic agent and more recently as a treatment for treatment-resistant depression. However, there is growing concern over the increased use of ketamine in recreational and therapeutic settings due to the potential neurotoxic effects. Recent studies have demonstrated that ketamine is cytotoxic in several cell types, such as fibroblasts, hepatocytes, uroepithelial cells, and adult induced pluripotent stem cells (iPSCs). Ketamine has been shown to dysregulate calcium signalling, increase reactive oxygen species (ROS) production, and impair mitochondrial function, ultimately leading to apoptosis. However, it is unclear whether endoplasmic reticulum (ER) stress plays a role in ketamine associated neurotoxicity in striatal neurons. Disruption to ER homeostasis can initiate ER-mediated cell death, which has been implicated in several neurodegenerative diseases. Thus, the purpose of this study was to determine whether ketamine's neurotoxic effects involve an ER stress-dependent pathway and to elucidate the underlying mechanisms involved in its neurotoxic effects. Mouse striatal cells were treated with various concentrations of ketamine (10 μM, 100 μM, 1 mM) or DMEM for 9–72 hrs. Cell viability was assessed using the MTT assay, and changes in gene expression of ER stress markers were evaluated using RT-qPCR. MTT results revealed that 1 mM ketamine decreased cell viability in striatal cells after 24 h of treatment. Gene expression studies complemented these findings such that ketamine upregulated pro-apoptotic ER stress markers, including X-box binding protein 1 (XBP1), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) and downregulated pro-survival ER stress proteins such as GRP78, MANF and CDNF. Ketamine activated all three stress sensing pathways including PERK, IRE1, and ATF6. Taken together, our results show that ketamine-induced neurotoxicity is mediated through an ER stress-dependent apoptotic pathway.

Immunosuppressive calcineurin inhibitor cyclosporine A induces proapoptotic endoplasmic reticulum stress in renal tubular cells

Current immunosuppressive strategies in organ transplantation rely on calcineurin inhibitors cyclosporine A (CsA) or tacrolimus (Tac). Both drugs are nephrotoxic, but CsA has been associated with greater renal damage than Tac. CsA inhibits calcineurin by forming complexes with cyclophilins, whose chaperone function is essential for proteostasis. We hypothesized that stronger toxicity of CsA may be related to suppression of cyclophilins with ensuing endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in kidney epithelia. Effects of CsA and Tac (10 µM for 6 h each) were compared in cultured human embryonic kidney 293 (HEK 293) cells, primary human renal proximal tubule (PT) cells, freshly isolated rat PTs, and knockout HEK 293 cell lines lacking the critical ER stress sensors, protein kinase RNA–like ER kinase or activating transcription factor 6 (ATF6). UPR was evaluated by detection of its key components. Compared with Tac treatment, CsA induced significantly stronger UPR in native cultured cells and isolated PTs. Evaluation of proapoptotic and antiapoptotic markers suggested an enhanced apoptotic rate in CsA-treated cells compared with Tac-treated cells as well. Similar to CsA treatment, knockdown of cyclophilin A or B by siRNA caused proapoptotic UPR, whereas application of the chemical chaperones tauroursodeoxycholic acid or 4-phenylbutyric acid alleviated CsA-induced UPR. Deletion of protein kinase RNA–like ER kinase or ATF6 blunted CsA-induced UPR as well. In summary, inhibition of cyclophilin chaperone function with ensuing ER stress and proapoptotic UPR aggravates CsA toxicity, whereas pharmacological modulation of UPR bears potential to alleviate renal side effects of CsA.

Cyclosporine A but Not Tacrolimus Promotes Pro-Apoptotic Endoplasmic Reticulum Stress in Cultured Kidney Cells

Cyclosporine A but Not Tacrolimus Promotes Pro-Apoptotic Endoplasmic Reticulum Stress in Cultured Kidney Cells

Cardiomyocyte Stim1 Deficiency Exacerbates Doxorubicin Cardiotoxicity by Magnification of Endoplasmic Reticulum Stress.

IntroductionDoxorubicin (Dox) is an effective anticancer agent; however, its cardiotoxicity remains a challenge. Dysfunction of intracellular calcium ion (Ca2+) is implicated in the process of Dox-induced cardiomyocyte apoptosis. Although store-operated Ca2+ entry (SOCE) is suggested to be responsible for Ca2+ entry in cardiomyocytes, the direct role of store-operated Ca2+ channels in Dox-related cardiomyocyte apoptosis is unknown.Materials and MethodsCardiomyocyte Stim1-specific knockout or overexpression mice were treated with Dox. Cardiomyocytes were pretreated with Stim1 adenovirus or siRNA followed by Dox incubation in vitro. Cardiac function and underlying mechanisms echocardiography were assessed via immunofluorescence, flow cytometry, real-time PCR, Western blotting and immunoprecipitation.ResultsWe observed the inhibition of Stim1 expression, association of Stim1 to Orai1 or Trpc1, and SOCE in Dox-treated mouse myocardium and cardiomyocytes. Orai1 and Trpc1 expression remained unchanged. Cardiomyocyte-specific deficiency of Stim1 exacerbated Dox-induced cardiac dysfunction and myocardial apoptosis. However, specific overexpression of Stim1 in the myocardium was associated with amelioration of cardiac dysfunction and myocardial apoptosis. In vitro, STIM1 knockdown potentiated Dox-induced AC16 human cardiomyocyte apoptosis. This apoptosis was attenuated by STIM1 upregulation. Moreover, STIM1 downregulation enhanced Dox-induced endoplasmic reticulum (ER) stress in cardiomyocytes. In contrast, STIM1 overexpression inhibited the activation of the above molecular markers of ER stress. Immunoprecipitation assay showed that STIM1 interacted with GRP78 in cardiomyocytes. This interaction was attenuated in response to Dox treatment.ConclusionOur data demonstrate that cardiomyocyte STIM1 binding to GRP78 ameliorates Dox cardiotoxicity by inhibiting pro-apoptotic ER stress.

Dextromethorphan Dampens Neonatal Astrocyte Activation and Endoplasmic Reticulum Stress Induced by Prenatal Exposure to Buprenorphine.

Prenatal exposure to buprenorphine renders offspring vulnerable to cerebral impairments. In this study, our data demonstrate, for the first time, that prenatal exposure to buprenorphine escalates astrocyte activation concurrent with indications of endoplasmic reticulum (ER) stress in the hippocampi of neonates, and this can be prevented by the coadministration of dextromethorphan with buprenorphine. Furthermore, dextromethorphan can inhibit the accumulation of GPR37 in the hippocampus of newborns caused by buprenorphine and is accompanied by the proapoptotic ER stress response that involves the procaspase-3/CHOP pathway. Primary astrocyte cultures derived from the neonates of the buprenorphine group also displayed aberrant ER calcium mobilization and elevated basal levels of cyclooxygenase-2 (COX-2) at 14 days in vitro while showing sensitivity to lipopolysaccharide-activated expression of COX-2. Similarly, these long-lasting defects in the hippocampus and astrocytes were abolished by dextromethorphan. Our findings suggest that prenatal exposure to buprenorphine might instigate long-lasting effects on hippocampal and astrocytic functions. The beneficial effects of prenatal coadministration of dextromethorphan might be, at least in part, attributed to its properties in attenuating astrocyte activation and hippocampal ER stress in neonates.

Protective effects of tetramethylpyrazine on dysfunction of the locus coeruleus in rats exposed to single prolonged stress by anti-ER stress mechanism.

Post-traumatic stress disorder (PTSD) is a serious stress-related neuropsychiatric disorder caused by major traumatic events. Abnormal activity of the locus coeruleus (LC)-noradrenergic system is related to the development of PTSD-like symptoms. Our previous studies have indicated that endoplasmic reticulum (ER) stress induced neuronal apoptosis of LC in rats with PTSD. The purpose of this study was to further investigate the role of ER stress pathways in LC neuronal dysfunction and elucidate the effect of the bioactive component tetramethylpyrazine (TMP) against ER stress response. We used an acute exposure to single prolonged stress (SPS) to model PTSD in rats. There were higher norepinephrine (NE) levels in the brain, increased tyrosine hydroxylase expression in LC, and enhanced anxiety-like behaviors in rats exposed to SPS, which were observed by enzyme-linked immunosorbent assay, western blot analysis and elevated plus maze test, respectively. In addition, the three major pathways of ER stress were activated by SPS exposure, which may be involved in the dysregulation of the LC-noradrenergic system of rats with PTSD. Furthermore, we found that TMP administration significantly suppressed the increased responsiveness of LC-noradrenergic system, effectively reduced the anxiety response of SPS rats, and selectively attenuated the activation of pro-apoptotic ER stress pathways. The results suggest that TMP was efficient in improving the LC-NE dysfunction induced by excessive ER stress. TMP exhibited a significant neuroprotective effect and potential therapeutics on PTSD-like symptoms.

Cytisine is neuroprotective in female but not male 6-hydroxydopamine lesioned parkinsonian mice and acts in combination with 17-β-estradiol to inhibit apoptotic endoplasmic reticulum stress in dopaminergic neurons.

Apoptotic endoplasmic reticulum (ER) stress is a major mechanism for dopaminergic (DA) loss in Parkinson's disease (PD). We assessed if low doses of the partial α4β2 nicotinic acetylcholine receptor agonist, cytisine attenuates apoptotic ER stress and exerts neuroprotection in substantia nigra pars compacta (SNc) DA neurons. Alternate day intraperitoneal injections of 0.2mg/kg cytisine were administered to female and male mice with 6-hydroxydopamine (6-OHDA) lesions in the dorsolateral striatum, which caused unilateral degeneration of SNc DA neurons. Cytisine attenuated 6-OHDA-induced PD-related behaviors in female, but not in male mice. We also found significant reductions in tyrosine hydroxylase (TH) loss within the lesioned SNc of female, but not male mice. In contrast to female mice, DA neurons within the lesioned SNc of male mice showed a cytisine-induced pathological increase in the nuclear translocation of the pro-apoptotic ER stress protein, C/EBP homologous protein (CHOP). To assess the role of estrogen in cytisine neuroprotection in female mice, we exposed primary mouse DA cultures to either 10nM 17-β-estradiol and 200nM cytisine or 10nM 17-β-estradiol alone. 17-β-estradiol reduced expression of CHOP, whereas cytisine exposure reduced 6-OHDA-mediated nuclear translocation of two other ER stress proteins, activating transcription factor 6 and x-box-binding protein 1, but not CHOP. Taken together, these data show that cytisine and 17-β-estradiol work in combination to inhibit all three arms (activating transcription factor 6, x-box-binding protein 1, and CHOP) of apoptotic ER stress signaling in DA neurons, which can explain the neuroprotective effect of low-dose cytisine in female mice.

Abstract 257: Calreticulin Regulates Acetylation in Cardiac Cells During Endoplasmic Reticulum Stress

Background: Myocardial infarction (MI) is exacerbated by endoplasmic reticulum (ER) stress, which contributes to apoptosis signaling and tissue death in cardiomyocytes. Although the calcium-handing protein calreticulin is upregulated during MI, the role of calreticulin in the progression of ER stress remains controversial. We hypothesized that calreticulin expression accelerates ER stress via regulation of the acetylation status of ER proteins, and knockdown reduces pro-apoptotic ER stress. Methods: We examined the expression of calreticulin in human cardiac cells in response to ischemia or thapsigargin-mediated ER stress. To test the effect of calreticulin modulation on cell mortality, we generated a stable partial calreticulin knockdown line in proliferating human cardiomyocyte-derived cells. We further examined the induction and acetylation of ER stress signaling proteins, including proteins in each of the three major pathways that account for ER-stress mediated cell death. Results and Conclusions: Calreticulin is upregulated in human ischemic heart failure cardiac tissue, as well as simulated hypoxia and reoxygenation and thapsigargin-mediated ER stress. It was found that partial knockdown protects against the expression of caspase 12, CHOP, and reduces thapsigargin-driven TUNEL staining. In addition, significant changes in the acetylation of ER stress proteins and the expression of the deacetylase SIRT1 were observed. These data shed light on the role that calreticulin plays in apoptosis signaling during ER stress in cardiac cells.

Dexmedetomidine inhibits neuronal apoptosis by inducing Sigma-1 receptor signaling in cerebral ischemia-reperfusion injury.

Dexmedetomidine is known to alleviate cerebral ischemia-reperfusion injury (CIRI). We established a rat model of CIRI, which exhibited higher neurological deficit scores and a greater number of apoptotic cells in the cerebral ischemic penumbra than controls. Dexmedetomidine reversed the neuronal apoptosis and improved neurological function in this model. We then examined Sigma-1 receptor (Sig-1R) expression on the endoplasmic reticulum (ER) in brain tissues at different reperfusion time points. Sig-1R expression increased with CIRI and decreased with increasing reperfusion times. After 24 hours of reperfusion, dexmedetomidine upregulated Sig-1R expression, and ER stress proteins (GRP78, CHOP, JNK and Caspase-3) were detected in brain tissues with Western blotting. Moreover, GRP78 expression followed a pattern similar to Sig-1R. Dexmedetomidine induced GRP78 expression but inhibited CHOP, Caspase-3 and phosphorylated-JNK expression in brain tissues. A Sig-1R-specific inhibitor reduced GRP78 expression and partially inhibited the upregulation of GRP78 by dexmedetomidine. The inhibitor also increased CHOP and Caspase-3 expression and partially reversed the inhibitory effects of dexmedetomidine on these pro-apoptotic ER stress proteins. These results suggest that dexmedetomidine at least partially inhibits ER stress-induced apoptosis by activating Sig-1R, thereby attenuating brain damage after 24 hours of ischemia-reperfusion.

HIF-1\u03b1 triggers ER stress and CHOP-mediated apoptosis in alveolar epithelial cells, a key event in pulmonary fibrosis

Endoplasmic Reticulum (ER) stress of alveolar epithelial cells (AECs) is recognized as a key event of cell dysfunction in pulmonary fibrosis (PF). However, the mechanisms leading to AECs ER stress and ensuing unfolded protein response (UPR) pathways in idiopathic PF (IPF) remain unclear. We hypothesized that alveolar hypoxic microenvironment would generate ER stress and AECs apoptosis through the hypoxia-inducible factor-1α (HIF-1α). Combining ex vivo, in vivo and in vitro experiments, we investigated the effects of hypoxia on the UPR pathways and ER stress-mediated apoptosis, and consecutively the mechanisms linking hypoxia, HIF-1α, UPR and apoptosis. HIF-1α and the pro-apoptotic ER stress marker C/EBP homologous protein (CHOP) were co-expressed in hyperplastic AECs from bleomycin-treated mice and IPF lungs, not in controls. Hypoxic exposure of rat lungs or primary rat AECs induced HIF-1α, CHOP and apoptosis markers expression. In primary AECs, hypoxia activated UPR pathways. Pharmacological ER stress inhibitors and pharmacological inhibition or silencing of HIF-1α both prevented hypoxia-induced upregulation of CHOP and apoptosis. Interestingly, overexpression of HIF-1α in normoxic AECs increased UPR pathways transcription factors activities, and CHOP expression. These results indicate that hypoxia and HIF-1α can trigger ER stress and CHOP-mediated apoptosis in AECs, suggesting their potential contribution to the development of IPF.

(-)- Epigallocatechin-3-gallate induces GRP78 accumulation in the ER and shifts mesothelioma constitutive UPR into proapoptotic ER stress.

GRP78 is a molecular chaperone of the endoplasmic reticulum (ER) that aids proper folding of nascent polypeptides. When unfolded proteins accumulate, GRP78 triggers unfolded protein response (UPR), involving activation of transcription factors like XBP1 and CHOP that may restore cell homeostasis. Increased expression of GRP78 and mild UPR can be constitutive in cancer cells, hindering apoptosis, and promoting cell survival, for example, by GRP78 relocation to the plasma membrane that activates MAPK and PI3 K/AKT pathways. These processes are thought to favor the insurgence of chemoresistance and worsen patient outcome. We have previously shown that (-)-epigallocatechin-3-gallate (EGCG) enhances ROS production and alters Ca2+ homeostasis in cell lines deriving from therapy-recalcitrant malignant mesothelioma (MMe). We consider here the EGCG impact on GRP78 and downstream factors by using qRT-PCR, Western blot, immunofluorescence, caspase assays, GRP78 siRNA silencing, and cell surface ELISA. MMe cells were found to display mild constitutive UPR, as shown by increased levels of GRP78, and presence of the protein at the cell surface, linked to AKT activation. Exposure to EGCG further increased GRP78 in the ER, and induced ATF4, spliced XBP1, CHOP, and EDEM expressions, combined with a reduction of cell surface GRP78 and a rise in caspase 3 and 8 activities. We propose that GRP78 accumulation in the ER, caused by EGCG, converts constitutive UPR of MMe cells into proapoptotic ER stress. This argues for a possible therapeutic use of EGCG in the treatment of MMe as a co-drug able to abolish chemoresistance to conventional drugs at tolerable doses.

Induction of Pro-Apoptotic Endoplasmic Reticulum Stress in Multiple Myeloma Cells by NEO214, Perillyl Alcohol Conjugated to Rolipram.

Despite the introduction of new therapies for multiple myeloma (MM), many patients are still dying from this disease and novel treatments are urgently needed. We have designed a novel hybrid molecule, called NEO214, that was generated by covalent conjugation of the natural monoterpene perillyl alcohol (POH), an inducer of endoplasmic reticulum (ER) stress, to rolipram (Rp), an inhibitor of phosphodiesterase-4 (PDE4). Its potential anticancer effects were investigated in a panel of MM cell lines. We found that NEO214 effectively killed MM cells in vitro with a potency that was over an order of magnitude stronger than that of its individual components, either alone or in combination. The cytotoxic mechanism of NEO214 involved severe ER stress and prolonged induction of CCAAT/enhancer-binding protein homologous protein (CHOP), a key pro-apoptotic component of the ER stress response. These effects were prevented by salubrinal, a pharmacologic inhibitor of ER stress, and by CHOP gene knockout. Conversely, combination of NEO214 with bortezomib, a drug in clinical use for patients with MM, resulted in synergistic enhancement of MM cell death. Combination with the adenylate cyclase stimulant forskolin did not enhance NEO214 impact, indicating that cyclic adenosine 3′,5′-monophosphate (AMP) pathways might play a lesser role. Our study introduces the novel agent NEO214 as a potent inducer of ER stress with significant anti-MM activity in vitro. It should be further investigated as a potential MM therapy aimed at exploiting this tumor’s distinct sensitivity to ER stress.

Metformin attenuates albumin-induced alterations in renal tubular cells in vitro.

Proteinuria (albuminuria) plays a crucial role in the etiology of chronic kidney disease (CKD) via alteration of multiple signaling pathways and cellular process in renal cells. The objectives of this study are to investigate the effects of activation of the energy-sensing molecule AMP-activated kinase (AMPK) in renal cells using metformin on endoplasmic reticulum (ER) stress, AKT, mTOR, epithelial-to-mesenchymal transition (EMT), autophagy, and apoptosis that are thought to mediate renal cell injury during proteinuria, and to dissect the AMPK- and non-AMPK mediated effects of metformin using an in vitro model of albumin-induced renal cell injury. Rat renal proximal tubular (NRK-52E) cells were exposed to 10 and 15 mg/ml of albumin for 72 h in the presence of 1 mM Metformin and/or 0.5 µM compound C, and assessed for alterations in the aforementioned pathways. Metformin treatment restored AMPK phosphorylation and augmented autophagy in renal cells exposed to albumin. In addition, metformin treatment attenuated the albumin-induced phosphorylation of AKT and the downstream targets of mTOR, and prevented albumin-mediated inductions of EMT marker (α-SMA), pro-apoptotic ER stress marker CHOP, and apoptotic caspases -12 and -3 in renal cells. Blockade of metformin-induced AMPK activation with compound C blunted the ER defense response and autophagy but had no effect on the markers of EMT and apoptosis in our model. Our studies suggest that metformin protects renal cells against proteinuric cytotoxicity via suppression of AKT and mTOR activation, inhibition of EMT and apoptosis, and augmentation of autophagy and ER defense response through AMPK-independent and AMPK-dependent mechanisms, respectively.

Structure-activity relationship of piperine and its synthetic amide analogs for therapeutic potential to prevent experimentally induced ER stress in vitro.

Endoplasmic reticulum (ER) is the key organelle involved in protein folding and maturation. Emerging studies implicate the role of ER stress in the development of chronic kidney disease. Thus, there is an urgent need for compounds that could ameliorate ER stress and prevent CKD. Piperine and its analogs have been reported to exhibit multiple pharmacological activities; however, their efficacy against ER stress in kidney cells has not been studied yet. Hence, the goal of this study was to synthesize amide-substituted piperine analogs and screen them for pharmacological activity to relieve ER stress using an in vitro model of tunicamycin-induced ER stress using normal rat kidney (NRK-52E) cells. Five amide-substituted piperine analogs were synthesized and their chemical structures were elucidated by pertinent spectroscopic techniques. An in vitro model of ER stress was developed using tunicamycin, and the compounds of interest were screened for their effect on cell viability, and the expression of ER chaperone GRP78, the pro-apoptotic ER stress marker CHOP, and apoptotic caspases 3 and 12 (via western blotting). Our findings indicate that exposure to tunicamycin (0.5μg/mL) for 2h induces the expression of GRP78 and CHOP, and apoptotic markers (caspase-3 and caspase-12) and causes a significant reduction in renal cell viability. Pre-treatment of cells with piperine and its cyclohexylamino analog decreased the tunicamycin-induced upregulation of GRP78 and CHOP and cell death. Taken together, our findings demonstrate that piperine and its analogs differentially regulate ER stress, and thus represent potential therapeutic agents to treat ER stress-related renal disorders. Graphical Abstract Piperine (PIP) reduces the expression of ER stress markers (GRP78 and CHOP) induced by pathologic stimuli and consequently decreases the activation of apoptotic caspase-12 and caspase-3; all of which contributes to its chemical chaperone and cytoprotective properties to protect renal cells against ER stress and ER stress-induced cell death, and would ultimately prevent the development of chronic kidney disease.

P31 Endoplasmic reticulum stress drives high selenium induced endothelial dysfunction

Selenium is associated with insulin resistance and may therefore affect endothelial function, increasing type II diabetes and its associated cardiovascular-disease risk. However the molecular mechanisms underpinning this association are not clear. High selenium doses cause apoptosis in cancer cells through the induction of the endoplasmic reticulum (ER) stress response, a mechanism implicated in the pathogenesis of insulin resistance and endothelial dysfunction (ED). Thus we hypothesised that high selenium intake could cause ED via ER stress induction. Endothelial cells were treated with selenite (0.5–10 µM) in the presence or absence of the chemical chaperone, 4-phenylbutyric acid (PBA). Supra-nutritional concentrations (5–10 µM of selenite) but not physiological concentration (0.5 µM) of selenite induced expression of several pro-apoptotic ER stress markers; such as activating transcription factor-4 (ATF4) and X-binding box-1 (XBP–1). Griess assay showed that high selenite treatment (5–10 µM) reduced nitric oxide production. Moreover, high selenium induced the production of reactive oxygen species as shown by flow cytometry and impaired endothelial cell function as assessed by in-vitro angiogenesis assay. Interestingly, pre-incubation with PBA completely reversed these effects of high selenium suggesting ER stress drives the response to selenite. Overall, we hereby show that high selenium causes endothelial dysfunction and cell death through the activation of ER stress. These results highlight the importance of a balanced selenium intake for maximal health benefits and prevention of cardiovascular disease. These findings also indicate that cancer patients supplemented with high amounts of selenium as part of their chemotherapeutic regimen should be monitored for cardiovascular disease risk.

The Herbal Medicine Cordyceps sinensis Protects Pancreatic Beta Cells from Streptozotocin-Induced Endoplasmic Reticulum Stress.

Our previous work found that Cordyceps sinensis (CS) improves the activity and secretory function of pancreatic islet beta cells. The objective was to observe a further possible role of CS in the protection of insulin-secreting cells. A rat model of type 2 diabetes mellitus was developed with streptozotocin (STZ) and a high-energy fat diet (HFD). CS was administered in the successful model of rats with type 2 diabetes. After 4 weeks, the biochemistry index of blood samples was measured, and pathologic observation was performed by immunohistochemistry. In the rats with type 2 diabetes induced by a HFD and STZ, the levels of fasting blood glucose and fasting insulin were elevated, and the insulin sensitivity index was decreased. Pathologic examination found an increased number of apoptotic cells, an elevated protein expression of pro-apoptotic C/EBP homologous protein (CHOP) and an increased c-Jun level by means of JNK phosphorylation, responsive to the endoplasmic reticulum stress of islet beta cells. With treatment by CS for 4 weeks, the elevated levels of both fasting blood glucose and fasting insulin in the rats with type 2 diabetes were significantly lower, and the decreased insulin sensitivity index was reversed. Compared to the control rats with type 2 diabetes, CS application significantly reduced the number of apoptotic cells and decreased protein expression of both CHOP and c-Jun. The herbal compound CS could protect pancreatic beta cells from the pro-apoptotic endoplasmic reticulum stress induced by HFD-STZ. This suggests an alternative approach to treating type 2 diabetes.