Nuclear expression of DDIT3 distinguishes high-grade myxoid liposarcoma from other round cell sarcomas.

Myxoid liposarcoma (MLPS) is a malignant adipocytic neoplasm with predilection for the extremities. MLPS is genetically defined by a t(12;16) translocation leading to FUS-DDIT3 (95%) or more rarely t(12;22) leading to EWSR1-DDIT3. Low-grade MLPS is characterized by bland spindle cells within a myxoid matrix containing delicate "chicken-wire" vasculature, whereas high-grade ("round cell") MLPS may be indistinguishable from other round cell sarcomas. In many cases, cytogenetic or molecular genetic techniques are applied to confirm the diagnosis. A recent study documented the utility of DDIT3 immunohistochemistry (IHC) in the differential diagnosis of adipocytic and myxoid soft tissue tumors. The purpose of this study was to evaluate DDIT3 IHC as a surrogate for molecular testing in high-grade MLPS. IHC was performed using a mouse monoclonal antibody directed against the N-terminus of DDIT3 on whole tissue sections from 50 high-grade MLPS cases and 319 histologic mimics used as controls (170 on whole tissue sections and 149 on a tissue microarray). Histologic mimics included Ewing sarcoma, CIC-rearranged sarcoma, sarcomas with BCOR genetic alterations, poorly differentiated synovial sarcoma, alveolar and embryonal rhabdomyosarcomas, mesenchymal chondrosarcoma, desmoplastic small round cell tumor, and neuroblastoma. Nuclear staining in >5% of cells was considered positive. By IHC, 48 (96%) high-grade MLPS showed strong diffuse nuclear staining for DDIT3. Of the controls, 2% of cases were positive, with no more than 25% nuclear staining. An additional 19% of control cases displayed less than 5% nuclear staining. Overall, DDIT3 IHC showed 96% sensitivity and 98% specificity for high-grade MLPS; strong, diffuse staining is also 96% sensitive but is 100% specific. IHC using an antibody directed against the N-terminus of DDIT3 is highly sensitive and specific for high-grade MLPS among histologic mimics and could replace molecular genetic testing in many cases, although limited labeling may be seen in a range of other tumor types.

DNA damage-inducible transcript 3 immunohistochemistry is highly sensitive for the diagnosis of myxoid liposarcoma but care is required in interpreting the significance of focal expression.

AIMS: Myxoid liposarcoma (MLPS) is characterised by DNA damage-inducible transcript 3 (DDIT3) gene rearrangements, confirmation of which is commonly used diagnostically. Recently, DDIT3 immunohistochemistry (IHC) has been reported to be highly sensitive and, when strict criteria are employed, specific for the diagnosis of MLPS. The aim of this study was to independently investigate DDIT3 IHC as a diagnostic marker for MLPS. METHODS AND RESULTS: DDIT3 IHC was performed on 52 MLPS and on 152 mimics on whole sections, and on 515 non-MLPS sarcomas in tissue microarray format. Only one MLPS (which had undergone acid-based decalcification) was completely negative. With inclusion of this case if any nuclear expression is considered to indicate positivity, the overall sensitivity of DDIT3 is 98% (51 of 52 cases) and the specificity is 94% (633 of 667 non-MLPS cases are negative). If a cut-off of >10% of neoplastic cells is required for positivity, then the sensitivity remains 98% (51/52) and the specificity is 98.5% (657 of 667 non-MLPS cases are negative). If a cut-off of >50% of cells is required for positivity, then the sensitivity is 96% (50 of 52 cases) but the specificity improves to 100%. CONCLUSIONS: Diffuse nuclear DDIT3 expression occurs in the overwhelming majority of MLPSs, and can be used to confirm the diagnosis in most cases without the need for molecular testing. A complete absence of expression argues strongly against MLPS, and almost completely excludes this diagnosis, particularly if there is consideration of technical factors such as decalcification. The significance of focal DDIT3 expression should be interpreted in the morphological and clinical context, although most tumours showing only focal expression are not MLPS.

DDIT3 Immunohistochemistry Is a Useful Tool for the Diagnosis of Myxoid Liposarcoma.

Myxoid liposarcoma is a malignant adipogenic neoplasm characterized by prominent arborizing capillaries, occasional lipoblasts, and primitive-appearing spindle cells in a myxoid background. A recurrent translocation in myxoid liposarcoma results in an oncoprotein consisting of full-length DDIT3 (CHOP) fused to an N-terminal segment of either FUS (TLS) or, less often, EWSR1. Here, we explore the diagnostic significance of DDIT3 expression in myxoid liposarcoma using a mouse monoclonal antibody recognizing an epitope in the N-terminal region. Studying a total of 300 tumors, we find diffuse, moderate-to-strong nuclear-localized anti-DDIT3 immunoreactivity in all 46 cases of myxoid liposarcoma representing 36 unique tumors, including 6 cases with high-grade (round cell) morphology. DDIT3 immunohistochemistry also highlighted a distinctive vasculocentric growth pattern in 7 myxoid liposarcomas treated with neoadjuvant radiation. In contrast, the vast majority of other examined lipomatous and myxoid neoplasms exhibited no DDIT3 expression; limited, weak immunoreactivity in <10% of cells was infrequently observed in dedifferentiated liposarcoma (6/39, 15%), solitary fibrous tumor (3/12, 25%), pleomorphic liposarcoma (1/15, 7%), and high-grade myxofibrosarcoma (2/17, 12%). Although this minimal DDIT3 expression did not correlate with DDIT3 amplification or myxoid liposarcoma-like morphology in dedifferentiated liposarcoma, there was evidence among sarcomas (excluding myxoid liposarcoma) of a relationship between expression and exposure to neoadjuvant radiation or cytotoxic chemotherapy. The constellation of findings indicates that DDIT3 immunohistochemistry may have utility in the evaluation of myxoid and lipomatous neoplasms to support the diagnosis of myxoid liposarcoma.

Effect of uremic serum on Th17/Treg cell balance and endoplasmic reticulum stress in rats.

BACKGROUND/AIMS: The imbalance of T helper 17 (Th17) and regulatory T (Treg) cells exists in the occurrence and development of various diseases. Endoplasmic reticulum stress (ERS) is an important self-protective cellular response to harmful stimuli, such as uremic environment. The objective of this study was to investigate the Th17/Treg cell balance and ERS in a uremic environment and analyze the relationship between them. METHODS: (1) The rat spleen lymphocytes were extracted and treated with thapsigargin (inducer of ERS) and sodium citrate. The proportion of Th17 and Treg cells were then detected. (2) The uremic serum-cultured lymphocytes were used and divided into three groups: non-uremic serum group, uremic serum group, and uremic serum + sodium citrate group. Afterward, the proportion of Th17/Treg cells and the expression of ERS-related proteins (GRP78 and CHOP) were detected. RESULTS: Thapsigargin had no significant effect on the proportion of Th17 cells within a limited concentration range, but it could reduce the proportion of Treg cells, sodium citrate had a negative influence on the deviation of Th17/Treg cells treated with thapsigargin. Uremic serum treatment reduced the proportion of Treg cells, resulting in an increase of the Th17/Treg ratio. However, sodium citrate had no influence on the deviation of Th17/Treg cells treated by uremic serum. Sodium citrate reduced the elevation of ERS-related proteins induced by uremic serum. CONCLUSIONS: Uremic serum can lead to the imbalance of Th17/Treg cells as well as ERS, suggesting that ERS is one of the mechanisms of the imbalance of Th17/Treg cells induced by uremic serum. Sodium citrate can inhibit ERS induced by uremic serum.

The critical role of PPARα in the binary switch between life and death induced by endoplasmic reticulum stress.

Endoplasmic reticulum stress (ER stress) just like a double-edged sword depending on different conditions in the development of multiple hepatic diseases. But the molecular mechanisms of functional conversion during ER stress have not been fully elucidated. In this study, we aim to illustrate the role of PPARα and the subtle mechanism in the functional conversion of ER stress. Tunicamycin (TM) and thapsigargin (TG), as ER stress inducers, were used to induce ER stress in AML12 cells. During the ER stress, qRT-PCR and immunoblotting was used to measure the expression levels of GRP78 and CHOP which show a gradually increasing trend, while PPARα and autophagy was significantly activated in the early stage but was inhibited in the late stage. Moreover, PPARα inhibition by siRNA promoted cell injury in the mild-ER stress and PPARα activation by WY-14643 reduced cell apoptosis in the serious ER stress. In the mild-ER stress with PPARα knocked down, activation of autophagy by rapamycin significantly improved cell survival, in the serious ER stress with PPARα activation, inhibition of autophagy by 3-MA aggravate cell injury. In addition, in the mild-ER stress with PPARα knocked down, CHOP knocked down by siRNA reduced cell apoptosis, in the serious ER stress activated PPARα, CHOP over-expression mediated by lentiviral vector contributed to serious cell injury. Furthermore, C57BL/6 mice was used to induce ER stress with TM intraperitoneal injection, PPARα and autophagy was upregulated in the mild-ER stress while downregulated in the serious ER stress, measured by qRT-PCR and immunoblotting, further confirmed the finding in vitro. Our results firstly demonstrated that PPARα is a key molecule in the functional conversion of ER stress: protective effects in the mild ER stress was mediated by PPARα-autophagy pathway and destructive effects in the serious ER stress was mediated by PPARα-CHOP pathway.

Improvement of immunodetection of the transcription factor C/EBP homologous protein by western blot.

Accumulation of misfolded proteins within the endoplasmic reticulum (ER) induces an unfolded protein response (UPR) that either restores homeostasis or triggers apoptosis in case of adaptation failure. The three activated branches of UPR lead to IRE1-, PERK- and ATF6- dependent transcriptional induction of the gene encoding the transcription factor C/EBP homologous protein (CHOP) which plays an important role in apoptosis induction. In conventional immunoblotting conditions, detection of CHOP is a difficult task. Using a fixation step, we have optimized the detection of CHOP and this method provides a valuable tool to decipher CHOP involvement in UPR.

Gambogic Acid Shows Anti-Proliferative Effects on Non-Small Cell Lung Cancer (NSCLC) Cells by Activating Reactive Oxygen Species (ROS)-Induced Endoplasmic Reticulum (ER) Stress-Mediated Apoptosis.

BACKGROUND Gambogic acid (AG) is believed to be a potent anti-cancer agent. ER (endoplasmic reticulum) stress-induced cell apoptosis was identified as one of the anti-proliferative mechanisms of several anti-cancer agents. In this study, we investigated the involvement of ER stress-induced apoptosis in the anti-proliferative effect of GA on NSCLC (non-small cell lung cancer) cells. MATERIAL AND METHODS GA at 0, 0.5, and 1.0 µmol/l was used to treat A549 cells. We also used the ER stress-specific inhibitor 4-PBA (4-phenylbutyric acid) (1 µmol/l) to co-treat the cells incubated with GA. Cell viability was assessed by MTT (methyl thiazolyl tetrazolium) assay. Cell apoptosis was evaluated by MTT (methyl thiazolyl tetrazolium) assay. Intracellular ROS (reactive oxygen species) production was detected by DCFH-DA (2,7- dichloro-dihydrofluorescein diacetate) florescent staining. Western blotting was used to assess the expression and phosphorylation levels of protein. RESULTS GA treatment significantly reduced cell viabilities of NSCLC cells in a concentration-dependent manner. GA treatment increased intracellular ROS level, expression levels of GRP (glucose-regulated protein) 78, CHOP (C/EBP-homologous protein), ATF (activating transcription factor) 6 and caspase 12, as well as the phosphorylation levels of PERK (protein kinase R-like ER kinase) and IRE (inositol-requiring enzyme) 1alpha. Co-treatment of 4-PBA dramatically impaired the inhibitory effect of GA on cell viability. 4PBA co-treatment also decreased expression levels of GRP78, CHOP, ATF6, and caspase12, as well as the phosphorylation levels of PERK and IRE1alpha, in GA-treated NSCLC cells, without affecting ROS levels. CONCLUSIONS GA inhibited NSCLC cell proliferation by inducing ROS-induced ER stress-medicated apoptosis of NSCLC cells.

[Pathological significance of NY-ESO-1 expression in the diagnosis of myxoid liposarcoma].

Objective: To detect the expression of New York esophageal squamous cell carcinoma antigen 1 (NY-ESO-1) in common types of mesenchymal myxoid tumors, and to investigate its significance in the diagnosis and differential diagnosis of myxoid liposarcoma. Methods: A total of 43 formalin-fixed paraffin-embedded samples of mesenchymal myxoid tumors from the Affiliated Hospital of Qingdao University and Qingdao Municipal Hospital ranging between 2010 and 2017 were selected. NY-ESO-1 expression was detected by immunohistochemical staining. DDIT3 gene status was detected by fluorescence in situ hybridization (FISH). NY-ESO-1 mRNA was detected by reverse transcription-PCR (RT-PCR). Results: Histopathology and FISH results confirmed that there were 11 cases of myxoid liposarcoma and 32 other types (including 7 cases of well-differentiated liposarcoma, 1 dedifferentiated liposarcoma, 3 lipomas, 2 lipoblastomas and 19 non-adipocytic tumors). Immunohistochemical staining showed that the positive expression propotion of NY-ESO-1 in myxoid liposarcoma was 11/11, and the positive location was the cytoplasm and nucleus of lipoblast cells. The expression intensity is higher in regions with round cell differentiation. Among the 32 cases of other mesenchymal myxoid tumors, only one well-differentiated liposarcoma showed positive immunoreactivity for NY-ESO-1. RT-PCR confirmed that 7 cases of myxoid liposarcoma (7/11) and one well-differentiated liposarcoma (1/7) had NY-ESO-1 mRNA expression. Conclusions: NY-ESO-1 is positively expressed in myxoid liposarcoma. It can be served as a useful marker for the diagnosis and differential diagnosis of myxoid liposarcoma.

Endoplasmic reticulum stress induced autophagy in 6-OHDA-induced Parkinsonian rats.

Both endoplasmic reticulum (ER) stress and autophagy involve in the pathological process of Parkinson's disease (PD). But the relationship between them is not clear in PD. A 6-OHDA-induced parkinsonian rat is recognized as a standard model for many years, and it can be used in experimental study. The glucose regulated protein 78 (GRP78) is a master regulator of ER stress, and the C/EBP homologous binding protein (CHOP) is an indicator of the UPR signaling. Besides, the Beclin-1 is also well known as a regulator of autophagy, and P62 is a specific marker to monitor autophagy. Therefore, we investigated the expressions of GRP78, CHOP, Beclin-1 and P62 in 6-OHDA-induced parkinsonian rat. Unilateral 6-OHDA injection into medial forebrain bundle was used except sham-operated rats. The rats were randomly divided into 6 groups: a sham-operated group; a model group; a 3-methyladenine (3-MA) group, administered 3-MA---autophagy inhibitor; a rapamycin group, administered rapamycin---autophagy inducer; a 4-phenylbutyric acids (4-PBA) group, administered 4-PBA---ER stress inhibitor; a tunicamycin (TM) group, administered TM---ER stress inducer. The results showed that the expressions of GRP78, CHOP and Beclin-1 increased, P62 decreased in model group; the expressions of GRP78 and CHOP were unchanged in 3-MA group and rapamycin group; but the expression of Beclin-1 decreased and P62 increased in 4-PBA group, while the expression of Beclin-1 increased and P62 decreased in TM group. These data suggest that ER stress and autophagy occurred in 6-OHDA-induced parkinsonian rat, and ER stress might induce autophagy. The result is important for the pathological mechanism of PD.

Induction of ER Stress-Mediated Apoptosis by the Major Component 5,7,4'-Trimethoxyflavone Isolated from Kaempferia parviflora Tea Infusion.

Kaempferia parviflora (KP) is a famous medicinal plant from Thailand, and is a rich source of various kinds of methoxyflavones (MFs). Many kinds of food products such as tea, capsule, and liquor are manufactured from the rhizomes of KP. In this study, KP infusions were prepared with different brewing conditions, and the amounts of three major methoxylflavones, 5,7-dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF), and 3,5,7,3',4'-pentamethoxyflavone (PMF), were analyzed. The antiproliferative activities of DMF, TMF, and PMF isolated from the brewed tea samples were evaluated. TMF was discovered to be significantly effective at inhibiting proliferation of SNU-16 human gastric cancer cells in a concentration dependent manner. TMF induced apoptosis, as evidenced by increments of sub-G1 phase, DNA fragmentation, annexin-V/PI staining, the Bax/Bcl-xL ratio, proteolytic activation of caspase-3,-7,-8, and degradation of poly (ADP-ribose) polymerase (PARP) protein. Furthermore, it was found that TMF induced apoptosis via ER stress, verified by an increase in the level of C/EBP homologous protein (CHOP), glucose regulated protein 78 (GRP78), inositol-requiring enzyme 1 α (IRE1α), activating transcription factor-4 (ATF-4), and the splice isoform of X-box-binding protein-1 (XBP-1) mRNA.

Postconditioning with sevoflurane ameliorates spatial learning and memory deficit via attenuating endoplasmic reticulum stress induced neuron apoptosis in a rat model of hemorrhage shock and resuscitation.

Hemorrhage shock could initiate endoplasmic reticulum stress (ERS) and then induce neuronal apoptosis. The aim of this study was to investigate whether sevoflurane postconditioning could attenuate brain injury via suppressing apoptosis induced by ERS. Seventy male rats were randomized into five groups: sham, shock, low concentration (sevo1, 1.2%), middle concentration (sevo2, 2.4%) and high concentration (sevo3, 3.6%) of sevoflurane postconditioning. Hemorrhage shock was induced by removing 40% of the total blood volume during an interval of 30 min. 1 h after the completion of bleeding, the animals were reinfused with shed blood during the ensuing 30 min. The spatial learning and memory ability of rats were measured by Morris water maze (MWM) test three days after the operation. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells in the hippocampus CA1 region were assessed after the MWM test. The expression of C/EBP-homologousprotein (CHOP) and glucose-regulated protein 78 (GRP78) in the hippocampus were measured at 24 h after reperfusion. We found that sevoflurane postconditioning with the concentrations of 2.4% and 3.6% significantly ameliorated the spatial learning and memory ability, decreased the TUNEL-positive cells, and reduced the GRP78 and CHOP expression compared with the shock group. These results suggested that sevoflurane postconditioning with the concentrations of 2.4% and 3.6% could ameliorate spatial learning and memory deficit after hemorrhage shock and resuscitation injury via suppressing apoptosis induced by ERS.

[Protective effect of prostaglandin E1 against brain injury induced by hyperoxia in neonatal rats].

OBJECTIVE: To investigate the protective effect of prostaglandin E1 (PGE-1) against brain injury induced by hyperoxia in neonatal rats and observe the changes in the expression of glucose-regulated protein 78 (GRP78) and cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), and to provide a theoretical basis for the clinical application of PGE-1 in the treatment of neonatal brain injury induced by hyperoxia. METHODS: Sixty neonatal Wistar rats were randomly divided into air control group, hyperoxic brain injury model group, and hyperoxic brain injury+PGE-1 group. All rats except those in the air control group were treated to establish a hyperoxic brain injury model. From the first day of modeling, the rats in the hyperoxia brain injury+PGE-1 group were intraperitoneally injected with PGE-1 2 µg/kg daily for 7 consecutive days, while the other two groups were treated with normal saline instead. The water content of brain tissue was measured; the pathological changes of brain tissue were evaluated by hematoxylin-eosin staining; the apoptosis of brain cells was assessed by nuclear staining combined with TUNEL staining; the protein expression of GRP78 and CHOP in brain tissue was measured by Western blot. RESULTS: The water content of brain tissue in the hyperoxic brain injury model group was significantly higher than that in the hyperoxic brain injury+PGE-1 group and air control group (P<0.05); the water content of brain tissue in the hyperoxic brain injury+PGE-1 group was significantly higher than that in the air control group (P<0.05). The pathological section of brain tissue showed inflammatory cell infiltration and mild cerebrovascular edema in the brain parenchyma in the hyperoxic brain injury model group; the periparenchymal inflammation and edema in the hyperoxic brain injury+PGE-1 group were milder than those in the hyperoxic brain injury model group. The apoptosis index of brain tissue in the hyperoxic brain injury model group was significantly higher than that in the hyperoxic brain injury+PGE-1 group and air control group (P<0.05); the apoptosis index of brain tissue in the hyperoxic brain injury+PGE-1 group was significantly higher than that in the air control group (P<0.05). The protein expression of GRP78 and CHOP in brain tissue was significantly higher in the hyperoxic brain injury model group than in the hyperoxic brain injury+PGE-1 group and air control group (P<0.05); the protein expression of GRP78 and CHOP was significantly higher in the hyperoxic brain injury+PGE-1 group than in the air control group (P<0.05). CONCLUSIONS: PGE-1 has a protective effect against hyperoxia-induced brain injury in neonatal rats, which may be related to the inhibition of cell apoptosis by down-regulating the expression of GRP78 and CHOP.

In vitro efficacy of a first-generation valosin-containing protein inhibitor (CB-5083) against canine lymphoma.

Valosin-containing protein (VCP), through its critical role in the maintenance of protein homeostasis, is a promising target for the treatment of several malignancies, including canine lymphoma. CB-5083, a first-in-class VCP inhibitor, exerts cytotoxicity through the induction of irreversible proteotoxic stress and possesses a broad spectrum of anticancer activity. Here, we determined the cytotoxicity CB-5083 in canine lymphoma cells and its mechanism of action in vitro. Canine lymphoma cell lines were treated with varying concentrations of CB-5083 and assessed for viability by trypan blue exclusion and apoptosis by caspase activity assays. The mechanism of CB-5083 action was determined by immunoblotting and RT-qPCR analyses of Lys48 ubiquitination and markers of ER stress (DDIT3), autophagy (SQSTM1, MAP1LC3A) and DNA damage (γH2AX). Unfolded protein response markers were also evaluated by immunoblotting (eIF2α, P-eIF2α) and RT-qPCR (ATF4). CB-5083 treatment resulted in preferential cytotoxicity in canine lymphoma cell lines over control peripheral blood mononuclear cells. CB-5083 rapidly disrupted the ubiquitin-dependent protein degradation system, inducing sustained ER stress as indicated by a dramatic increase in DDIT3. Activation of the unfolded protein response occurred through the increase eIF2α phosphorylation and increased transcription of ATF4, but did not re-establish protein homeostasis. Cells rapidly underwent apoptosis through activation of the caspase cascade. These results further validate VCP as an attractive target for the treatment of canine lymphoma and identify CB-5083 as a novel therapy with clinical potential for this malignancy.

High NRF2 expression controls endoplasmic reticulum stress induced apoptosis in multiple myeloma.

Multiple myeloma (MM) is an incurable disease characterized by clonal plasma cell proliferation. The stress response transcription factor Nuclear factor erythroid 2 [NF-E2]-related factor 2 (NRF2) is known to be activated in MM in response to proteasome inhibitors (PI). Here, we hypothesize that the transcription factor NRF2 whose physiological role is to protect cells from reactive oxygen species via the regulation of drug metabolism and antioxidant gene plays an important role in MM cells survival and proliferation. We report for the first time that NRF2 is constitutively activated in circa 50% of MM primary samples and all MM cell lines. Moreover, genetic inhibition of constitutively expressed NRF2 reduced MM cell viability. We confirm that PI induced further expression of NRF2 in MM cell lines and primary MM. Furthermore, genetic inhibition of NRF2 of PI treated MM cells increased ER-stress through the regulation of CCAAT-enhancer-binding protein homologous protein (CHOP). Finally, inhibition of NRF2 in combination with PI treatment significantly increased apoptosis in MM cells. Here we identify NRF2 as a key regulator of MM survival in treatment naive and PI treated cells.

Chronic Intermittent Hypobaric Hypoxia Improves Cardiac Function through Inhibition of Endoplasmic Reticulum Stress.

We investigated the role of endoplasmic reticulum stress (ERS) in chronic intermittent hypobaric hypoxia (CIHH)-induced cardiac protection. Adult male Sprague-Dawley rats were exposed to CIHH treatment simulating 5000 m altitude for 28 days, 6 hours per day. The heart was isolated and perfused with Langendorff apparatus and subjected to 30-min ischemia followed by 60-min reperfusion. Cardiac function, infarct size, and lactate dehydrogenase (LDH) activity were assessed. Expression of ERS molecular chaperones (GRP78, CHOP and caspase-12) was assayed by western blot analysis. CIHH treatment improved the recovery of left ventricular function and decreased cardiac infarct size and activity of LDH after I/R compared to control rats. Furthermore, CIHH treatment inhibited over-expression of ERS-related factors including GRP78, CHOP and caspase-12. CIHH-induced cardioprotection and inhibition of ERS were eliminated by application of dithiothreitol, an ERS inducer, and chelerythrine, a protein kinase C (PKC) inhibitor. In conclusion CIHH treatment exerts cardiac protection against I/R injury through inhibition of ERS via PKC signaling pathway.

The effect of bovine rotavirus and its nonstructural protein 4 on ER stress-mediated apoptosis in HeLa and HT-29 cells.

Endoplasmic reticulum (ER) plays important roles in multiple cellular processes as well as cell survival and apoptosis. Perturbation of ER functions leads to ER stress and unfolded protein response (UPR). The primary goal of this response is cell survival, but severe ER stress can trigger apoptosis signaling. In tumor cells, chronically activated UPR response provides tumor growth. So, apoptosis induced by the ER stress has been the target for anti-cancer therapy. In this in vitro study, we examined the apoptotic effect associated with ER stress of bovine rotavirus and its nonstructural protein 4 (NSP4) alone in two cancer cell lines. The plasmid pcDNA3.1 encoding NSP4 protein of bovine rotavirus transfected with lipofectamine 2000 into the HeLa and HT-29 cells for protein production. MTT, flow cytometry, and Western blot were used to evaluate the cell viability, apoptosis, and expression level of C/EBP-homologous protein (CHOP) and activated caspase-4. In parallel, the apoptotic effect of the bovine rotavirus associated with ER stress in the infected cells was examined too. The cytotoxic and apoptotic effect of NSP4 protein on the cells were statistically significant compared to the control groups. However, Western blot showed that the expression of the NSP4 protein by recombinant plasmid did not lead to high expression of CHOP and activation of caspase-4. Interestingly, rotavirus not only induced significant apoptosis but also caused an increase in CHOP expression and caspase-4 activation in the infected cells compared to control. As a result, NSP4 protein and bovine rotavirus can be considered a potential novel bio-therapeutic strategy for cancer treatment.

Endoplasmic reticulum stress and oxidative stress are involved in ZnO nanoparticle-induced hepatotoxicity.

Zinc oxide nanoparticles (Nano-ZnO) are widely used in sunscreens, clothes, medicine and electronic devices. However, the potential risks of human exposure and the potential for adverse health impacts are not well understood. Previous studies have demonstrated that exposure to Nano-ZnO caused liver damage and hepatocyte apoptosis through oxidative stress, but the molecular mechanisms that are involved in Nano-ZnO-induced hepatotoxicity are still unclear. Endoplasmic reticulum (ER) is sensitive to oxidative stress, and also plays a crucial role in oxidative stress-induced damage. Previous studies showed that ER stress was involved in many chemical-induced liver injuries. We hypothesized that exposure to Nano-ZnO caused oxidative stress and ER stress that were involved in Nano-ZnO-induced liver injury. To test our hypothesis, mice were gavaged with 200 mg/kg or 400 mg/kg of Nano-ZnO once a day for a period of 90 days, and blood and liver tissues were obtained for study. Our results showed that exposure to Nano-ZnO caused liver injury that was reflected by focal hepatocellular necrosis, congestive dilation of central veins, and significantly increased alanine transaminase (ALT) and aspartate transaminase (AST) levels. Exposure to Nano-ZnO also caused depletion of glutathione (GSH) in the liver tissues. In addition, our electron microscope results showed that ER swelling and ribosomal degranulation were observed in the liver tissues from mice treated with Nano-ZnO. The mRNA expression levels of ER stress-associated genes (grp78, grp94, pdi-3, xbp-1) were also up-regulated in Nano-ZnO-treated mice. Nano-ZnO caused increased phosphorylation of RNA-dependent protein kinase-like ER kinase (PERK) and eukaryotic initiation factor 2α (eIF2α). Finally, we found that exposure to Nano-ZnO caused increased ER stress-associated apoptotic protein levels, such as caspase-3, caspase-9, caspase-12, phosphorylation of JNK, and CHOP/GADD153, and up-regulation of pro-apoptotic genes (chop and bax). These results suggest that oxidative stress and ER stress-induced apoptosis are involved in Nano-ZnO-induced hepatotoxicity in mice.

Modulation of the unfolded protein response impedes tumor cell adaptation to proteotoxic stress: a PERK for hepatocellular carcinoma therapy.

BACKGROUND: Functional disturbances of the endoplasmic reticulum (ER) lead to activation of the unfolded protein response (UPR), which is involved in the consecutive steps of carcinogenesis. In human hepatocellular carcinoma (HCC), the UPR is shown to be activated; however, little is known about the UPR kinetics and effects of UPR modulation in HCC. METHODS: We sequentially monitored the UPR over time in an orthotopic mouse model for HCC and explored the effects of UPR modulation on cell viability and proliferation in vitro and in the mouse model. RESULTS: The expression of ER-resident chaperones peaked during tumor initiation and increased further during tumor progression, predominantly within the nodules. A peak in Ire1 signaling was observed during tumor initiation. The Perk pathway was activated during tumor progression, and the proapoptotic target Chop was upregulated from week 5 and continued to rise, especially in the tumors. The Atf6 pathway was modestly activated only after tumor initiation. Consistent with the UPR activation, electron microscopy demonstrated ER expansion and reorganization in HCC cells in vivo. Strikingly, under ER stress or hypoxia, the Perk inhibitor and not the Ire1 inhibitor reduced cell viability and proliferation via escalating proteotoxic stress in vitro. Notably, the Perk inhibitor significantly decreased tumor burden in the mouse model. CONCLUSION: We provide the first evaluation of the UPR dynamics in a long-term cancer model and identified a small molecule inhibitor of Perk as a promising strategy for HCC therapy.

The effect of glucose concentration and sodium phenylbutyrate treatment on mitochondrial bioenergetics and ER stress in 3T3-L1 adipocytes.

While the 3T3-L1 adipocyte model is routinely used for the study of obesity and diabetes, the mitochondrial respiratory profile in normal versus high glucose has not been examined in detail. We matured adipocytes in normal (5mM) or high (30 mM) glucose and insulin and examined the mitochondrial bioenergetics. We also assessed the requirement for the Unfolded Protein Response (UPR) and ER stress under these conditions. Basal respiration was ~1.7-fold greater in adipocytes that had matured in 30 mM glucose; however, their ability to increase oxygen consumption in response to stress was impaired. Adipogenesis proceeded in both normal and high glucose with concomitant activation of the UPR, but only high glucose was associated with increased levels of ER stress and mitochondrial stress as observed by parallel increases in CHOP and protein succination. Treatment of adipocytes with sodium phenylbutyrate relieved mitochondrial stress through a reduction in mitochondrial respiration. Our data suggests that mitochondrial stress, protein succination and ER stress are uniquely linked in adipocytes matured in high glucose.

Transcriptional factor ATF6 is involved in odontoblastic differentiation.

ATF6 is an endoplasmic reticulum (ER) membrane-bound transcription factor that regulates various cellular functions. The purpose of this study was to investigate the role of ATF6 in odontoblast differentiation. Rat tooth germs were isolated, changes in gene expression were evaluated over time, and localization of ATF6 was determined by immunohistochemistry. Human dental pulp cells (HDPCs) were cultured with 50 µg/mL ascorbic acid and 5 mmol/L ß-glycerophosphate or 100 ng/mL bone morphogenetic protein 2 to induce differentiation. Translocation of ATF6 was observed by immunofluorescence and confocal microscopy. Overexpression of ATF6 was performed with an adenoviral vector. Matrix mineralization was evaluated by alizarin red staining. Immunoreactivity to anti-ATF6 was observed in the odontoblastic layer of the molar tooth germ, and expressions of ATF6, dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) increased gradually during tooth germ development. When HDPCs were cultured in differentiation media, ATF6, DSPP, and DMP1 expression increased with the expression of unfolded protein response (UPR) markers, BiP and CHOP. Immunofluorescence results showed that ATF6 protein moved from cytoplasm to nucleus when cells were exposed to differentiation media. Notably, overexpression of ATF6 increased DSPP and DMP1 expression, alkaline phosphatase (ALP) activity, and matrix mineralization in HDPC cultures. Inhibition of ATF6 decreased ALP activity and mineralization. These results suggest that ER membrane-bound transcriptional factor ATF6 may be involved in odontoblastic differentiation.