C/EBP Homologous Protein Induction Research Articles

DHA Induces Cell Death through the Production of ROS and the Upregulation of CHOP in Fibroblast-like Synovial Cells from Human Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is an inflammatory disease marked by a massive proliferation of synovial cells in the joints. In this study, we investigated the pro-apoptotic effects of docosahexaenoic acid (DHA) in human fibroblast-like synovial cells from RA patients (RA-FLS). An in vitro study using MH7A cells showed that DHA treatment induced caspase-8-dependent apoptosis in a dose-dependent manner and reduced the TNF-α-mediated induction of MMP-9 and IL-1β. DHA also induced the phosphorylation of eIF2α, the expression of the ER stress markers ATF4 and C/EBP homologous protein (CHOP), and death receptor 5 (DR5). The knockdown of CHOP or DR5 increased cell viability and reduced apoptosis in DHA-treated cells. Furthermore, the knockdown of CHOP reduced DHA-mediated DR5 expression, while the overexpression of CHOP increased DR5 expression. We also found that DHA treatment induced the accumulation of reactive oxygen species (ROS), and pretreatment with the anti-oxidant Tiron effectively abrogated not only the expression of CHOP and DR5, but also DHA-induced apoptosis. Under this condition, cell viability was increased, while PARP-1 cleavage and caspase-8 activation were reduced. All the findings were reproduced in human primary synovial cells obtained from RA patients. These results suggest that the DHA-mediated induction of ROS and CHOP induced apoptosis through the upregulation of DR5 in RA-FLSs, and that CHOP could be used as a therapy for RA.

Betulinic acid and its ionic derivatives impaired growth of prostate cancer cells without induction of GRP78 and CHOP

Abstract Prostate cancer (PCa) is the most common invasive malignancy for men in the USA. The incidence and mortality rates of PCa are significantly higher among African American men, as compared to those in Caucasian men. Betulinic acid (BA) is a penta-cyclic triterpenoid that is often found in the bark of several species of plants. It possesses a variety of biological activities, including anti-cancer activities. We examined the cytotoxic effects and endoplasmic reticulum (ER) stress induced by BA and its ionic derivatives with PCa cells derived from African Americans and Caucasian men. The viability of all PCa cells was reduced by the BA compounds, and the cytotoxicity of these BA compounds was independent of ethnicity and androgen dependency. The BA compounds induced modest effects on ER stress proteins when compared with ER stress inducers, tunicamycin and thapsigargin. The induction of glucose regulated protein 78 (GRP78) was largely correlated with the expression of C/EBP homologous protein (CHOP) and cleaved poly [ADP-ribose] polymerase (PARP)/caspase-3 in the PCa cells. In summary, our data demonstrated that BA compounds impaired the growth of PCa cells regardless of ethnicity – through GRP78- and CHOP-independent pathways.

Activation of mTORC1 by Free Fatty Acids Suppresses LAMP2 and Autophagy Function via ER Stress in Alcohol-Related Liver Disease.

Alcohol-related liver disease (ALD) is characterized by accumulation of hepatic free fatty acids (FFAs) and liver injury. The present study aimed to investigate if mechanistic target of rapamycin complex 1 (mTORC1) plays a role in FFA-induced organelle dysfunction, thereby contributing to the development of ALD. Cell studies were conducted to define the causal role and underlying mechanism of FFA-activated mTORC1 signaling in hepatocellular cell injury. C57BL/6J wild-type mice were subjected to chronic alcohol feeding with or without rapamycin to inhibit mTORC1 activation. We revealed that palmitic acid (PA)-induced ER stress and suppression of LAMP2 and autophagy flux were mTORC1-dependent as rapamycin reversed such deleterious effects. C/EBP homologous protein (CHOP) was downstream of ATF4 which partially modulated LAMP2. Supplementation with rapamycin to alcohol-fed mice attenuated mTORC1 activation and ER stress, restored LAMP2 protein, and improved autophagy, leading to amelioration of alcohol-induced liver injury. Induction of mTORC1 signaling and CHOP were also detected in the liver of patients with severe alcoholic hepatitis. This study demonstrates that hepatic FFAs play a crucial role in the pathogenesis of ALD by activating mTORC1 signaling, thereby inducing ER stress and suppressing LAMP2-autophagy flux pathway, which represents an important mechanism of FFA-induced hepatocellular injury.

A glaucoma‐ and ALS‐associated mutant of OPTN induces neuronal cell death dependent on Tbk1 activity, autophagy and ER stress

Mutations in OPTN are associated with glaucoma, an eye disease, and also with amyotrophic lateral sclerosis (ALS), a motor neuron disease. A 2-bp insertion in OPTN (691_692insAG or 2bpIns-OPTN) is associated with both glaucoma and ALS. This mutation results in frame shift after 127 amino acids, giving rise to a protein with C-terminal aberrant sequence. We have explored the mechanism of induction of cell death by this mutant in a motor neuron cell line, NSC-34, and also in a retinal cell line, 661W. Compared to wild-type OPTN, this mutant induced more cell death in NSC-34 and 661W cells. This mutant localizes predominantly in the nucleus whereas normal OPTN localizes in the cytoplasm. Deletion analysis of 2bpIns-OPTN showed that the aberrant sequence was not essential for cell death induction. This mutant interacts with TANK-binding kinase 1 (Tbk1) but not with OPTN and activates Tbk1. This mutant induced ER stress in NSC-34 cells as seen by induction of C/EBP homologous protein (CHOP) and some other genes. Induction of CHOP, autophagosomal protein LC3-II and cell death by this mutant were abrogated by Tbk1 knockdown and also by 4-phenylbutyric acid, that inhibits ER stress. Induction of CHOP and cell death by 2bpIns-OPTN was autophagy dependent as shown by the effect of Atg5 knockdown. This mutant caused increased formation of LC3-positive aggregates. Treatment of cells with autophagy inducer rapamycin reduced LC3-positive aggregates, CHOP and cell death induced by 2bpIns-OPTN. These results suggest that constitutive activation of Tbk1 by 2bpIns-OPTN leads to impaired autophagy that results in ER stress and cell death.

Soat1 mediates the mouse strain effects on cholesterol loading-induced endoplasmic reticulum stress and CHOP expression in macrophages

We previously demonstrated that AKR vs. DBA/2 mouse bone marrow derived macrophages have higher levels of free cholesterol and lower levels of esterified cholesterol after cholesterol loading, and that AKR, but not DBA/2, macrophages induced C/EBP homologous protein (CHOP) expression after cholesterol loading. We earlier determined that the free and esterified cholesterol level effect is due to a truncation in the sterol O-acyltransferase 1 (Soat1) gene, encoding acetyl-coenzyme A acetyltransferase 1 (ACAT1). Here we examined the mechanism for the differential induction of CHOP by cholesterol loading. CHOP was induced in both strains after incubation with tunicamycin, indicating both strains have competent endoplasmic reticulum stress pathways. CHOP was induced when DBA/2 macrophages were cholesterol loaded in the presence of an ACAT inhibitor, indicating that the difference in free cholesterol levels were responsible for this strain effect. This finding was confirmed in macrophages derived from DBA/2 embryonic stem cells. Cholesterol loading of Soat1 gene edited cells, mimicking the AKR allele, led to increased free cholesterol levels and restored CHOP induction. The upstream pathway of free cholesterol induced endoplasmic reticulum stress was investigated; and, RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1 α protein kinase (IRE1α) pathways were required for maximal CHOP expression.

Induction of ER Stress in Acute Lymphoblastic Leukemia Cells by the Deubiquitinase Inhibitor VLX1570.

The proteasome is a validated target of cancer therapeutics. Inhibition of proteasome activity results in the activation of the unfolded protein response (UPR) characterized by phosphorylation of eukaryotic initiation factor 2α (eIF2α), global translational arrest, and increased expression of the proapoptotic CHOP (C/EBP homologous protein) protein. Defects in the UPR response has been reported to result in altered sensitivity of tumor cells to proteasome inhibitors. Here, we characterized the effects of the deubiquitinase (DUB) inhibitor VLX1570 on protein homeostasis, both at the level of the UPR and on protein translation, in acute lymphoblastic leukemia (ALL). Similar to the 20S inhibitor bortezomib, VLX1570 induced accumulation of polyubiquitinated proteins and increased expression of the chaperone Grp78/Bip in ALL cells. Both compounds induced cleavage of PARP (Poly (ADP-ribose) polymerase) in ALL cells, consistent with induction of apoptosis. However, and in contrast to bortezomib, VLX1570 treatment resulted in limited induction of the proapoptotic CHOP protein. Translational inhibition was observed by both bortezomib and VLX1570. We report that in distinction to bortezomib, suppression of translation by VXL1570 occurred at the level of elongation. Increased levels of Hsc70/Hsp70 proteins were observed on polysomes following exposure to VLX1570, possibly suggesting defects in nascent protein folding. Our findings demonstrate apoptosis induction in ALL cells that appears to be uncoupled from CHOP induction, and show that VLX1570 suppresses protein translation by a mechanism distinct from that of bortezomib.

Xanthatin induces glioma cell apoptosis and inhibits tumor growth via activating endoplasmic reticulum stress-dependent CHOP pathway.

Xanthatin is a natural sesquiterpene lactone purified from Xanthium strumarium L., which has shown prominent antitumor activity against a variety of cancer cells. In the current study, we investigated the effect of xanthatin on the growth of glioma cells in vitro and in vivo, and elucidated the underlying mechanisms. In both rat glioma C6 and human glioma U251 cell lines, xanthatin (1–15 μM) dose-dependently inhibited cell viability without apparent effect on the cell cycle. Furthermore, xanthatin treatment dose-dependently induced glioma cell apoptosis. In nude mice bearing C6 glioma tumor xenografts, administration of xanthatin (10, 20, 40 mg·kg−1·d−1, ip, for 2 weeks) dose-dependently inhibited the tumor growth, but did not affect the body weight. More importantly, xanthatin treatment markedly increased the expression levels of the endoplasmic reticulum (ER) stress-related markers in both the glioma cell lines as well as in C6 xenografts, including glucose-regulated protein 78, C/EBP-homologous protein (CHOP), activating factor 4, activating transcription factor 6, spliced X-box binding protein-1, phosphorylated protein kinase R-like endoplasmic reticulum kinase, and phosphorylated eukaryotic initiation factor 2a. Pretreatment of C6 glioma cells with the ER stress inhibitor 4-phenylbutyric acid (4-PBA, 7 mM) or knockdown of CHOP using small interfering RNA significantly attenuated xanthatin-induced cell apoptosis and increase of proapoptotic caspase-3. These results demonstrate that xanthatin induces glioma cell apoptosis and inhibits tumor growth via activating the ER stress-related unfolded protein response pathway involving CHOP induction. Xanthatin may serve as a promising agent in the treatment of human glioma.

Capsaicin Induces ATF4 Translation with Upregulation of CHOP, GADD34 and PUMA.

Primary Effusion Lymphoma (PEL) is a rare and aggressive B-lymphoma caused by Kaposi's sarcoma-associated herpes virus (KSHV) infection that occurs in immunocompromised patients. PEL patients have a poor prognosis. KSHV modulates various cellular signaling pathways to maintain latent infection, and causes malignant conversion of host cells. We previously reported that capsaicin suppressed extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling and induced apoptosis in PEL. Generally, cellular stress such as nutrient starvation, oxidation and virus infection induce CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) expression by activating transcription factor 4 (ATF4), however endoplasmic reticulum (ER) stress induces CHOP expression by both ATF4 and ATF6. CHOP is associated with apoptosis induction and upregulates growth arrest and DNA damage-inducible protein 34 (GADD34) and p53 up-regulated modulator of apoptosis (PUMA) mRNA expression. In this study, we found a new mechanism in which capsaicin induces apoptosis via ATF4-CHOP-PUMA. Capsaicin promoted transcriptional activation of CHOP, which increased mRNA expression of GADD34 and PUMA, resulting in PEL apoptosis. Furthermore, capsaicin increased ATF4 protein levels by promoting ATF4 translation, not transcription, and had no effect on ATF6-dependent transcriptional activation. In sum, capsaicin promotes ATF4 translation and transcriptional induction of CHOP, which results in PUMA expression and apoptosis in PEL cells.

Inhibition of TRIB3 Protects Against Neurotoxic Injury Induced by Kainic Acid in Rats.

Epilepsy refers to a group of neurological disorders of varying etiologies characterized by recurrent seizures, resulting in brain dysfunction. Endoplasmic reticulum (ER) stress is highly activated in the process of epilepsy-related brain injury. However, the mechanisms by which ER stress triggers neuronal apoptosis remain to be fully elucidated. Tribbles pseudokinase 3 (TRIB3) is a pseudokinase that affects a number of cellular functions, and its expression is increased during ER stress. Here, we sought to clarify the role of TRIB3 in neuronal apoptosis mediated by ER stress. In the kainic acid (KA) (10 mg/kg)-induced rat seizure model, we characterized neuronal injury and apoptosis after KA injection. KA induced an ER stress response, as indicated by elevated expression of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP). TRIB3 protein was upregulated concomitantly with the downregulation of phosphorylated-protein kinase B (p-AKT) in rats following KA administration. In rat cortical neurons treated with KA, TRIB3 knockdown by siRNA reduced the number of dying neurons, decreased the induction of GRP78 and CHOP and the activation of caspase-3, and blocked the dephosphorylation of AKT after KA treatment. Our findings indicate that TRIB3 is involved in neuronal apoptosis occurring after KA-induced seizure. The knockdown of TRIB3 effectively protects against neuronal apoptosis in vitro, suggesting that TRIB3 may be a potential therapeutic target for the treatment of epilepsy.

Berberine mitigates IL-21/IL-21R mediated autophagic influx in fibroblast-like synoviocytes and regulates Th17/Treg imbalance in rheumatoid arthritis.

In our previous study, we explored the therapeutic effect of berberine (BBR) against IL-21/IL-21R mediated inflammatory proliferation of adjuvant-induced arthritic fibroblast-like synoviocytes (AA-FLS) through the PI3K/Akt pathway. The current study was designed to explore the therapeutic potential of BBR (15-45µM) against IL-21/IL-21R mediated autophagy in AA-FLS mediated through PI3K/Akt signaling and Th17/Treg imbalance. Upon IL-21 stimulation, AA-FLS expressed elevated levels of autophagy-related 5 (Atg5), Beclin-1 and LC3-phosphatidylethanolamine conjugate 3-II (LC3-II) through the utilization of p62 and inhibition of C/EBP homologous protein (CHOP). BBR (15-45µM) inhibited autophagy in AA-FLS cells mediated through PI3K/Akt signaling via suppressing autophagic elements, p62 sequestration and induction of CHOP in a dose-dependent manner. Moreover, IL-21 promoted the uncontrolled proliferation of AA-FLS through induction of B cell lymphoma-2 (Bcl-2) and diminished expression of Bcl-2 associated X protein (BAX) via PI3K/Akt signaling. BBR inhibited the proliferation of AA-FLS via promoting apoptosis through increased expression of BAX and diminished Bcl-2 transcription factor levels. Furthermore, T cells stimulated with IL-21 induced CD4+ CD196+ Th17 cells proliferation through RORγt activation mediated in a PI3K/Akt dependent manner. BBR inhibited the proliferation of Th17 cells through downregulation of RORγt in a concentration-dependent manner. BBR also promoted the differentiation of CD4+ CD25+ Treg cells through induction of forkhead box P3 (Foxp3) activation via aryl hydrocarbon receptor (AhR) and upregulation of cytochrome P450 family 1, subfamily A, polypeptide 1 (CYP1A1). Collectively, we conclude that BBR might attenuate AA-FLS proliferation through inhibition of IL-21/IL-21R dependent autophagy and regulates the Th17/Treg imbalancein RA.

Regulation and role of the ER stress transcription factor CHOP in alveolar epithelial type-II cells

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by type-II alveolar epithelial cell (AECII) injury and fibroblast hyperproliferation. Severe AECII endoplasmic reticulum (ER) stress is thought to underlie IPF, but is yet incompletely understood. We studied the regulation of C/EBP homologous protein (CHOP), a proapoptotic ER-stress-related transcription factor (TF) in AECII-like cells. Interestingly, single or combined overexpression of the active ER stress transducers activating transcription factor-4 (Atf4) and activating transcription factor-6 (p50Atf6) or spliced x-box-binding protein-1 (sXbp1) in MLE12 cells did not result in a substantial Chop induction, as compared to the ER stress inducer thapsigargin. Employing reporter gene assays of distinct CHOP promoter fragments, we could identify that, next to the conventional amino acid (AARE) and ER stress response elements (ERSE) within the CHOP promoter, activator protein-1 (AP-1) and c-Ets-1 TF binding sites are necessary for CHOP induction. Serial deletion and mutation analyses revealed that both AP-1 and c-Ets-1 motifs act in concert to induce CHOP expression. In agreement, CHOP promoter activity was greatly enhanced upon combined versus single overexpression of AP-1 and c-Ets-1. Moreover, combined overexpression of AP-1 and c-Ets-1 in MLE12 cells alone in the absence of any other ER stress inducer was sufficient to induce Chop protein expression. Further, AP-1 and c-Ets-1 were upregulated in AECII under ER stress conditions and in human IPF. Finally, Chop overexpression in vitro resulted in AECII apoptosis, lung fibroblast proliferation, and collagen-I production. We propose that CHOP activation by AP-1 and c-Ets-1 plays a key role in AECII maladaptive ER stress responses and consecutive fibrosis, offering new therapeutic prospects in IPF.Key messagesOverexpression of active ER stress sensors Atf4, Atf6, and Xbp1 does not induce Chop.AP-1 and c-Ets-1 TFs are necessary for induction of the ER stress factor Chop.AP-1 and c-Ets-1 alone induce Chop expression in the absence of any ER stress inducers.AP-1 and c-Ets-1 are induced in AECII under ER stress conditions and in human IPF.Chop expression alone triggers AECII apoptosis and consecutive profibrotic responses.

MON-201 ER Stress Activated by Androgen Enhances Apoptosis of Granulosa Cells via Induction of Death Receptor 5 in PCOS

PCOS is associated with hyperandrogenism and growth arrest of antral follicles. Excess androgens work as a stage-specific inhibitor of a follicular growth suppressing the growth of late-stage follicles. Previously, we found that endoplasmic reticulum (ER) stress is activated in granulosa cells of antral follicles in PCOS (1). This is evidenced by the activation of various unfolded protein response (UPR) genes, including a pro-apoptotic UPR transcription factor, C/EBP homologous protein (CHOP). Death receptor (DR) 5, which harbors a CHOP-binding site in its promoter region, plays a crucial role in ER stress-induced apoptosis by increasing autocrine death-ligand signal, independent of its extracellular ligand TNF-related apoptosis-inducing ligand (TRAIL) (2). Based on these observations, we hypothesized that ER stress is activated by androgen in granulosa cells of antral follicles in PCOS and activated ER stress promotes apoptosis via induction of CHOP and subsequent activation of DR5. Using RT-PCR (qPCR), we showed that testosterone induced the mRNA expression of various UPR factors including XBP1(S), HSPA5, ATF4 and CHOP along with DR5 in cultured human granulosa-lutein cells (GLCs). Treatment with an ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), inhibited testosterone-induced mRNA and protein expression of DR5 and CHOP, with a concomitant reduction in apoptosis examined by qPCR, western blotting and flow cytometry respectively. Furthermore, knockdown of CHOP or DR5 by RNA interference inhibited testosterone-induced apoptosis and knockdown of CHOP reduced testosterone-induced DR5 mRNA expression. Treatment with an androgen receptor (AR) antagonist, flutamide, as well as the knockdown of AR, decreased testosterone-induced DR5 and CHOP mRNA expression and apoptosis. In addition, expression of DR5 and CHOP was upregulated in GLCs of PCOS patients and granulosa cells of antral follicles in ovarian sections obtained from both PCOS patients and dehydroepiandrosterone (DHEA)-induced PCOS mice, proven by qPCR and immunohistochemistry (IHC). Treatment of PCOS mice with TUDCA decreased apoptosis and DR5 expression in granulosa cells of antral follicles, with a concomitant reduction in CHOP expression, shown by TUNEL and IHC staining. Our findings indicate that ER stress activated by hyperandrogenism in PCOS promotes apoptosis of granulosa cells of antral follicles via induction of DR5. Reference: (1) Takahashi N, Harada M, et al. Sci Rep 2017, 7:10824 (2) Lu M et al. Science 2014;345:98-101.

Modulation of the unfolded protein response pathway as an antiviral approach in airway epithelial cells

IntroductionRhinovirus (RV) infection is a major cause of cystic fibrosis (CF) lung morbidity with limited therapeutic options. Various diseases involving chronic inflammatory response and infection are associated with endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response (UPR), an adaptive response to maintain cellular homeostasis. Recent evidence suggests impaired ER stress response in CF airway epithelial cells, this might be a reason for recurrent viral infection in CF. Therefore, assuming that ER stress inducing drugs have antiviral properties, we evaluated the activation of the UPR by selected ER stress inducers as an approach to control virus replication in the CF bronchial epithelium. MethodsWe assessed the levels of UPR markers, namely the glucose-regulated protein 78 (Grp78) and the C/EBP homologous protein (CHOP), in primary CF and control bronchial epithelial cells and in a CF and control bronchial epithelial cell line before and after infection with RV. The cells were also pretreated with ER stress-inducing drugs and RV replication and shedding was measured by quantitative RT-PCR and by a TCID50 assay, respectively. Cell death was assessed by a lactate dehydrogenate (LDH) activity test in supernatants. ResultsWe observed a significantly impaired induction of Grp78 and CHOP in CF compare to control cells following RV infection. The ER stress response could be significantly induced in CF cells by pharmacological ER stress inducers Brefeldin A, Tunicamycin, and Thapsigargin. The chemical induction of the UPR pathway prior to RV infection of CF and control cells reduced viral replication and shedding by up to two orders of magnitude and protected cells from RV-induced cell death. ConclusionRV infection causes an impaired activation of the UPR in CF cells. Rescue of the ER stress response by chemical ER stress inducers reduced significantly RV replication in CF cells. Thus, pharmacological modulation of the UPR might represent a strategy to control respiratory virus replication in the CF bronchial epithelium.

Vernodalol enhances TRAIL-induced apoptosis in diffuse large B-cell lymphoma cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent anti-tumor agent that triggers apoptosis in cells from multiple types of carcinoma but not in normal cells. However, diverse mechanisms are associated with insensitivity to TRAIL in various cancers. TRAIL efficacy may be enhanced by combining TRAIL with a sensitizer. In this study, vernodalol, a sesquiterpene lactone, sensitized diffuse large B-cell lymphoma (DLBCL) cells to TRAIL-induced apoptosis. Vernodalol increased the expression of death receptor (DR) 5, and silencing of DR5 with a small interfering RNA (siRNA) reduced the effect of vernodalol on TRAIL-mediated apoptosis. Additionally, vernodalol up-regulated the expression of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), a transcription factor. Inhibition of CHOP with a siRNA diminished DR5 expression and vernodalol-induced sensitization to the TRAIL treatment. In addition, a c-Jun N-terminal kinase (JNK) inhibitor blocked the vernodalol-induced up-regulation of DR5, indicating that the effect depended on JNK activation. Furthermore, the down-regulation of induced myeloid leukaemia cell differentiation protein (Mcl-1) played an important role in vernodalol/TRAIL-induced apoptosis, as Mcl-1 overexpression prevented this apoptotic effect. Moreover, the vernodalol/TRAIL combination inhibited tumor growth in a xenograft model. Based on our results, vernodalol enhanced TRAIL-induced apoptosis by down-regulating Mcl-1 and up-regulating DR5, and the effects of DR5 depended on JNK activation and CHOP induction. Therefore, combining TRAIL with vernodalol, a naturally occurring agent, may represent a promising therapeutic approach for DLBCL.

α-Asarone blocks 7β-hydroxycholesterol-exposed macrophage injury through blocking elF2α phosphorylation and prompting beclin-1-dependent autophagy.

Macrophage apoptosis is salient in advanced atherosclerotic lesions and is induced by several stimuli including endoplasmic reticulum (ER) stress. This study examined that a-asarone present in purple perilla abrogated macrophage injury caused by oxysterols via ER stress- and autophagy-mediated mechanisms. Nontoxic a-asarone at 1-20 M attenuated 7β-hydroxycholesterol-induced activation of eukaryotic initiation factor 2a in macrophages leading to C/EBP homologous protein (CHOP) expression and apoptosis due to sustained ER stress. The a-asarone treatment increased the formation of autophagolysosomes localizing in perinuclear regions of 7β-hydroxycholesterol-exposed macrophages. Consistently, this compound promoted the induction of the key autophagic proteins of beclin-1, vacuolar protein sorting 34 and p150 responsible for vesicle nucleation, and prompted the conversion of microtubule-associated protein 1A/1B-light chain 3 and the induction of p62, neighbor of BRCA1 and autophagy-related (Atg) 12-Atg5-Atg16L conjugate involved in phagophore expansion and autophagosome formation. Additionally, a-asarone increased ER phosphorylation of bcl-2 facilitating beclin-1 entry to autophagic process. Furthermore, the deletion of Atg5 or beclin-1 gene enhanced apoptotic CHOP induction. Collectively, a-asarone-stimulated autophagy may be potential multi-targeted therapeutic avenues in treating ER stress-associated macrophage apoptosis.

α‐Asarone augments autophagophore elongation in oxysterol‐challenged macrophages

Macrophage apoptosis plays a major role in the advanced atherosclerotic lesions, which is induced by several stimuli including endoplasmic reticulum (ER) stress and oxidative stress. In the recent studies, autophagic responses make cells escape from cellular apoptosis through promoting phagocytosis of misfolded proteins. α‐Asarone is isolated from Perilla frutescens, a traditional herb in the East‐Asia, and this compound possesses several effects on allergic inflammation. This study investigated whether α‐asarone alleviated macrophage apoptosis by promoting autophagic responses. To induce lipotoxicity in macrophages, this study treated 7β‐hydroxycholesterol in richly present in oxidized LDL. 7β‐Hydroxycholesterol induced cellular apoptosis by decreasing cell viability by ≈70% within 18 h, which were recovered by treating 10 μM α‐asarone. To induce ER stress by this oxysterol, the activation and induction of pancreatic ER kinase (PERK), translation initiation factor (eIF) 2α, activating transcription factor (ATF) 4 were determined. α‐Asarone down‐regulated the activation and induction of these ER stress‐related proteins. In addition, the oxysterol induced C/EBP homologous‐protein (CHOP) and DNA damage‐inducible protein (GADD34), which was dose‐dependently ameliorated by supplementing α‐asarone. Furthermore, to reveal a relationship between cellular apoptosis and autophagy, autophagy protein (ATG) 5 siRNA was employed in oxysterol‐challenged macrophages. The CHOP induction was up‐regulated in ATG5‐deleted macrophages free of autophagy, indicating that cellular apoptosis and autophagy was reciprocal in oxysterol‐challenged macrophages. The oxysterol enhanced the formation of ATG5/12/16L complex by instigating autophagy and increased LC3 induction for the autophagosome formation. α‐Asarone attenuated the formation autophagosomes through disturbing the formation of ATG5/12/16L complex and the LC3 conversion to LC3II for mature. Therefore, α‐asarone is a protective agent inhibiting atherosclerosis by ameliorating oxysterol‐mediated macrophage apoptosis and by augmenting autophagosome formation.Support or Funding InformationThis study was supported by Food, Agriculture, Forestry and Fisheries (112085‐03‐1‐SB010) and Brain Korea 21 plus program by Ministry of Education of Korea (22A20130012421).

Differential expression of endoplasmic reticulum stress-response proteins in different renal tubule subtypes of OVE26 diabetic mice.

Regulation of the endoplasmic reticulum (ER) stress-response pathway during the course of diabetes specifically in renal tubules is unclear. Since tubule cell dysfunction is critical to progression of diabetic nephropathy, this study analyzed markers of ER stress response and ER chaperones at different stages of diabetes and in different renal tubule subtypes of OVE26 type-1 diabetic mice. ER stress-responseinduced chaperones GRP78, GRP94, and protein disulfide isomerase (PDI) were increased in isolated cortical tubules of older diabetic mice, while PDI was decreased in tubules of young diabetic mice. Immunofluorescence staining of kidneys from older mice showed GRP78 and PDI upregulation in all cortical tubule segments, with substantial induction of PDI in distal tubules. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation was increased in cortical tubules of young diabetic mice, with no differences between older diabetic and control mice. Expression of ER stress-induced PERK inhibitor p58IPK was decreased and then increased in all tubule subtypes of young and older mice, respectively. Knockdown of PERK by small interfering RNA (siRNA) increased fibronectin secretion in cultured proximal tubule cells. Tubules of older diabetic mice had significantly more apoptotic cells, and ER stress-induced proapoptotic transcription factor C/EBP homologous protein (CHOP) was increased in proximal and distal tubules of diabetic mice and diabetic humans. CHOP induction in OVE26 mice was not altered by severity of proteinuria. Overexpression of CHOP in cultured proximal tubule cells increased expression of fibronectin. These findings demonstrate differential ER stress-response signaling in tubule subtypes of diabetic mice and implicate a role for PERK and CHOP in tubule cell matrix protein production.

Fluvoxamine alleviates paclitaxel-induced neurotoxicity

Paclitaxel (Px) is an effective chemotherapeutic agent for the treatment of various cancers. However, it is often associated with neurological side effects, including chemotherapy-associated cognitive impairment (CACI), such as “chemobrain”. Previously, we reported that endoplasmic reticulum (ER) stress is involved in Px-induced neurotoxicity, and immunoglobulin heavy chain binding protein (BiP) inducer X (BIX) alleviates Px-induced neurotoxicity. However, BIX has not been used in clinical practice yet. We recently reported that fluvoxamine (Flv) alleviates ER stress via induction of sigma-1 receptor (Sig-1R). The purpose of this study was to investigate whether Flv could alleviate Px-induced neurotoxicity in vitro. SK-N-SH cells were pre-treated for 12h with or without 10μg/ml Flv followed by treatment with 1μM Px with or without co-existence of 10μg/ml Flv for 24h. To investigate the involvement of Sig-1R in alleviation effect on Px-induced neurotoxicity,1μM NE100, an antagonist of Sig-1R, was added for 24h. Neurotoxicity was assessed using the MTS viability assay and ER stress-mediated neurotoxicity was assessed by evaluating the expression of C/EBP homologous protein (CHOP), cleaved caspase 4, and cleaved caspase 3.Pre-treatment with Flv significantly alleviated the induction of CHOP, cleaved caspase 4, and cleaved caspase 3 in SK-N-SH cells. At the same time, pre-treatment with Flv significantly induced Sig-1R in SK-N-SH cells. In addition, viability was significantly higher in Flv-treated cells than in untreated cells, which was reversed by treatment with NE100.Our results suggest that Flv alleviates Px-induced neurotoxicity in part through the induction of Sig-1R. Our findings should contribute to one of the novel approaches for the alleviation of Px-induced neurotoxicity, including chemobrain.

Identification of 5-nitrofuran-2-amide derivatives that induce apoptosis in triple negative breast cancer cells by activating C/EBP-homologous protein expression

The transcription factor C/EBP-homologous protein (CHOP) is a key component of the terminal unfolded protein response (UPR) that mediates unresolvable endoplasmic reticulum stress-induced apoptosis. CHOP induction is known to cause cancer cell death. Chemicals that induce CHOP expression would thus be valuable as potential cancer therapeutics and as research tools. Here, we identified 5-nitrofuran-2-amide derivatives as small molecule activators of CHOP expression that induced apoptosis in triple negative breast cancer (TNBC) cells. Our preliminary structure–activity relationship studies indicated that compounds with an N-phenyl-5-nitrofuran-2-carboxamide skeleton were particularly potent inducers of TNBC cell apoptosis. The compounds activate CHOP expression via the PERK–eIF2α–ATF4 branch of the UPR. These results indicate that small molecule activators of CHOP expression may have therapeutic potential for TNBC.

L-type amino-acid transporter 1 (LAT1): a therapeutic target supporting growth and survival of T-cell lymphoblastic lymphoma/T-cell acute lymphoblastic leukemia.

The altered metabolism of cancer cells is a treasure trove to discover new antitumoral strategies. The gene (SLC7A5) encoding system L amino-acid transporter 1 (LAT1) is overexpressed in murine lymphoma cells generated via T-cell deletion of the pten tumor suppressor, and also in human T-cell acute lymphoblastic leukemia (T-ALL)/lymphoma (T-LL) cells. We show here that a potent and LAT1 selective inhibitor (JPH203) decreased leukemic cell viability and proliferation, and induced transient autophagy followed by apoptosis. JPH203 could also alter the in vivo growth of luciferase-expressing-tPTEN-/- cells xenografted into nude mice. In contrast, JPH203 was nontoxic to normal murine thymocytes and human peripheral blood lymphocytes. JPH203 interfered with constitutive activation of mTORC1 and Akt, decreased expression of c-myc and triggered an unfolded protein response mediated by the C/EBP homologous protein (CHOP) transcription factor associated with cell death. A JPH203-resistant tPTEN-/-clone appeared CHOP induction deficient. We also demonstrate that targeting LAT1 may be an efficient broad spectrum adjuvant approach to treat deadly T-cell malignancies as the molecule synergized with rapamycin, dexamethasone, doxorubicin, velcade and l-asparaginase to alter leukemic cell viability.