Tunicamycin Research Articles

High-Density Lipoprotein Prevents Endoplasmic Reticulum Stress-Induced Downregulation of Liver LOX-1 Expression

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a specific cell-surface receptor for oxidized-low-density lipoprotein (ox-LDL). The impact of high-density lipoprotein (HDL) on endoplasmic reticulum (ER) stress-mediated alteration of the LOX-1 level in hepatocytes remains unclear. We aimed to investigate the impact on LOX-1 expression by tunicamycin (TM)-induced ER stress and to determine the effect of HDL on TM-affected LOX-1 expression in hepatic L02 cells. Overexpression or silencing of related cellular genes was conducted in TM-treated cells. mRNA expression was evaluated using real-time polymerase chain reaction (PCR). Protein expression was analyzed by western blot and immunocytochemistry. Lipid uptake was examined by DiI-ox-LDL, followed by flow cytometric analysis. The results showed that TM induced the upregulation of ER chaperone GRP78, downregulation of LOX-1 expression, and lipid uptake. Knock down of IRE1 or XBP-1 effectively restored LOX-1 expression and improved lipid uptake in TM-treated cells. HDL treatment prevented the negative impact on LOX-1 expression and lipid uptake induced by TM. Additionally, 1–10 μg/mL HDL significantly reduced the GRP78, IRE1, and XBP-1 expression levels in TM-treated cells. Our findings reveal that HDL could prevent the TM-induced reduction of LOX-1 expression via inhibiting the IRE1/XBP-1 pathway, suggesting a new mechanism for beneficial roles of HDL in improving lipid metabolism.

Evidence that Vascular Endothelial Derived Endothelin‐1 Promotes Development of Tunicamycin‐Induced Endoplasmic Reticulum Stress in Renal Vessels

Levels of the vasoconstrictor peptide endothelin-1 (ET-1) are elevated in cardiovascular disease. Endoplasmic reticulum (ER) stress is one of the known mechanisms for cellular apoptosis. Our group has recently implicated the ET-1 system in the activation of renal ER stress pathways, although it is unclear which cells are involved. To clarify the role of ET-1 in the development of renal vascular ER stress, we hypothesized that lack of ET-1 in endothelial cells attenuates tunicamycin (TM)-induced ER stress in renal vessels. Female mice lacking ET-1 in vascular endothelial cells (VEET KO) and the corresponding flox controls (n=5-9/group) received a single i.p. injection of either TM (1.5 mg/kg) or saline. 24 hours post-injection, renal vessels were isolated, ER stress genes determined by qRT-PCR, and renal apoptosis detected by TUNEL staining. TM significantly increased the expression of ER stress markers in renal vessels from flox mice (saline vs. TM, fold change/flox+saline, p<0.05; GRP94: 1.0±0.3 vs. 16.5±6.6, ATF-6: 1.0±0.5 vs. 6.8±3.5, and CHOP: 1.4±0.4 vs. 12.5±1.9). TM treatment led to increased outer medullary, non-vascular TUNEL staining in flox mice as well. TM failed to increase renal vascular ER stress markers or renal apoptosis in VEET KO mice. These results indicate that ET-1 is critical for the development of renal vascular ER stress and apoptosis in response to TM, and highlight the possibility of targeting the ET-1 system as a therapeutic approach against ER stress-induced renal vascular damage. Funded by NIH T32 DK007545 to CDM and P01 HL95499 to DMP and JSP.

0379: Cytoarchitectural and metabolic alterations induced by ER stress in heart

The rough endoplasmic reticulum (rER) is the site for synthesis, folding and quality control of secreted and membrane proteins. Impairment of ER function in response to stresses such as oxidative stress, disruption of calcium homeostasis or ischemia causes the accumulation of misfolded proteins in the rER lumen, resulting in ER stress. Over the past decade, ER stress has emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases including heart failure. However, the molecular mechanisms underlying the contribution of ER stress to cardiac dysfunction remain poorly understood. In the present study, we evaluated the effect of the ER stressor tunicamycin (TN) on cardiac function in mice. TN injection (2mg/kg, 72h) induced a significant impairment of systolic function as indicated by the decrease in ejection fraction and fractional shortening. However, the heart rate, left ventricular internal diameters in diastole and systole and wall thickness were not affected. Transmission electron microscopy analysis revealed that TN induced an important ultrastructural remodeling of the cardiomyocytes with an increase in the occurrence of rER. Whereas rER was essentially located near the nucleus in cardiomyocytes of control mice, we observed an expansion of the rER network near sarcomeres and around T-tubules and mitochondrial clusters after TN treatment. In addition, mitochondrial structure and network were also disorganized. When measured in skinned fibers, the rate of mitochondrial oxidation was slower and an impairment of the function of the creatine kinase energy shuttle was observed in response to TN. In addition, ER stress triggered a metabolic remodeling characterized by a shift from fatty acid to glycogenic substrates consumption. Taken together our results show for the first time that the cytoarchitectural and metabolic alterations of cardiomyocytes contribute to the cardiac injury induced by ER stress.

High MUC2 Production in Goblet Cells Induces ER Stress and Exhibit Increase Susceptibility to Apoptosis

MUC2 is a large glycoprotein produced by goblet cells (GC) that form the protective mucus blanket overlying the intestinal epithelium. During pathological conditions such as IBD or colonic cancers, MUC2 biosynthesis and secretion are accelerated. Little information is known on how MUC2 production is regulated and whether GC undergoes endoplasmic reticulum (ER) stress following high MUC2 biosynthesis. We studied the role of MUC2 in GC stress using a high MUC2 producing GC line, HT29‐H, and a clone of HT29‐H (HT29‐L) in which MUC2 was stably silenced using lentivirus shRNA. Cells were treated with the ER stressor, tunicamycin (TUN) and markers for ER stress (GRP78, ATF4, CHOP, sXBP1) and apoptosis (caspase 3 and PARP cleavage) quantified by western blotting and RT‐qPCR. Compared to HT29‐L, HT29‐H cells showed significant increase in ER stress and apoptosis in response to TUN. Over expressing MUC2 in non‐MUC2‐producing SKCO15 cells also increased ER stress and apoptosis confirming specificity of MUC2. Pre‐treatment of cells with reactive oxygen species (ROS) inhibitor, diphenyleneiodonium, abrogated basal stress levels, TUN‐induced ER stress and apoptosis. HT29‐H constitutively produced significantly more ROS than HT29‐L cells indicating that high MUC2 production specifically increases ROS production that drives ER stress and apoptosis. These findings were corroborated in isolated colonic epithelial cells and colonic tissues from Wt and Muc2‐/‐ mice that showed significant increase in ER stress in Wt as compared to Muc2‐/‐ mice. We conclude that high MUC2 production by GC induces ER stress and apoptosis that could subsequently lead to diminished mucus barrier function in disease pathogenesis.Grant: CIHR

The Role of Cross‐links Between Endoplasmic Reticulum Stress, Oxidative stress and Mitochondrial dysfunction in Cardiomyocytes and H9c2 Cells under Hyperglycemia

Under physiological condition, endoplasmic reticulum (ER) is responsible basically from protein synthesis and secretion as well as intracellular Ca2+ signalling while sarcoplasmic reticulum (SR) plays basical role in excitation‐contraction coupling of cardiomyocytes.A change or differientiation in ER related functions can induced by ER stress via affecting signaling pathways. Various studies showed that the cross‐relations between ER stress, increased ROS/RNS, and mitochondrial (MitC) dysfunction induced cardiomyocyte dysfunction via affecting intracellular free Ca2+and Zn2+ homeostasis. Therefore, we aimed to investigate the cross‐links in cardiomyocytes and H9c2 cells under hyperglycemia, ER‐stress induced by tunicamycin (TM) and increased cytosolic Zn2+due to increased ROS/RNS. Freshly isolated ventricular cardiomyocytes from male rats under hypergycemia and increased cytosolic Zn2+ conditions exibited significant increases in the levels of ER stress chaperon proteins such as GRP78 and calregulin while only hyperglycemia and ER‐stress induced these changes in the levels of calnexin and Gadd153. We also show the levels of those proteins in H9c2 cells after incubation of TM for 18 h. And also, our data show ER and MitC interaction, the levels of PML and some proteins associated with apoptosis as Bcl‐2 and Bax are examined. Furthermore, these conditions induced marked changes in the membrane potential of MitC. These data with functional data demonstrate that increased ROS/RNS observed under these conditions in cardiomyocytes and H9c2 cells underlie both ER stress and MitC dysfunction thereby inducing cardiac dysfunction in diabetic subjects (Supported by TUBITAK‐SBAG 113S466).

A Cell Type-Specific Role of Protein Tyrosine Phosphatase Non-Receptor Type 2 in Regulating ER Stress Signalling

Background/Aims: Genetic polymorphisms within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) have been associated with inflammatory bowel disease (IBD). A recent study demonstrated that PTPN2 regulates ER stress signalling in pancreatic β-cells. Therefore, we investigated whether PTPN2 regulates ER stress pathways, apoptosis and cytokine secretion in human intestinal epithelial cells (IECs) and monocytes. Methods: THP-1 and HT-29 IECs were stimulated with 2 µg/ml tunicamycin (TNM) for the indicated periods of time. For knockdown experiments, cells were transfected using a mixture of three different PTPN2-specific siRNA oligonucleotides. Cell lysates were analysed by Western blot and real-time PCR. Cytokine secretion was studied by ELISA measurements of cell culture supernatant. Results: TNM treatment reduced PTPN2 protein levels in HT-29 IECs and THP-1 monocytes. Knockdown of PTPN2 in THP-1 monocytes led to an exaggerated induction of phospho-eIF2α, enhanced PARP cleavage, indicative of apoptosis, and attenuated IL-8 and TNF secretion upon TNM stimulation. In HT-29 cells PTPN2 deficiency caused reduced phosphorylation of eIF2α and PARP cleavage under ER stress conditions. Conclusions: Whereas the knockdown of PTPN2 made THP-1 cells more susceptible to ER stress, PTPN2 deficiency reduced ER stress responses in HT-29 IECs. This suggests that PTPN2 regulates adaptation to ER stress in a cell type-specific manner.

Stress of endoplasmic reticulum modulates differentiation and lipogenesis of human adipocytes

BackgroundAdipocytes are cells specialized for storage of neutral lipids. This storage capacity is dependent on lipogenesis and is diminished in obesity. The reason for the decline in lipogenic activity of adipocytes in obesity remains unknown. Recent data show that lipogenesis in liver is regulated by pathways initiated by endoplasmic reticulum stress (ERS). Thus, we aimed at investigating the effect of ERS on lipogenesis in adipose cells. MethodsPreadipocytes were isolated from subcutaneous abdominal adipose tissue from obese volunteers and in vitro differentiated into adipocytes. ERS was induced pharmacologically by thapsigargin (TG) or tunicamycin (TM). Activation of Unfolded Protein Response pathway (UPR) was monitored on the level of eIF2α phosphorylation and mRNA expression of downstream targets of UPR sensors. Adipogenic and lipogenic capacity was evaluated by Oil Red O staining, measurement of incorporation of radio-labelled glucose or acetic acid into lipids and mRNA analysis of adipogenic/lipogenic markers. ResultsExposition of adipocytes to high doses of TG (100 nM) and TM (1 μg/ml) for 1–24 h enhanced expression of several UPR markers (HSPA5, EDEM1, ATF4, XBP1s) and phosphorylation of eIF2α. This acute ERS substantially inhibited expression of lipogenic genes (DGAT2, FASN, SCD1) and glucose incorporation into lipids. Moreover, chronic exposure of preadipocytes to low dose of TG (2.5 nM) during the early phases of adipogenic conversion of preadipocytes impaired both, lipogenesis and adipogenesis. On the other hand, chronic low ERS had no apparent effect on lipogenesis in mature adipocytes. ConclusionsAcute ERS weakened a capacity of mature adipocytes to store lipids and chronic ERS diminished adipogenic potential of preadipocytes.

Supplementation of tauroursodeoxycholic acid during IVC did not enhance in vitro development and quality of buffalo IVF embryos but combated endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress, a dysfunction in protein-folding capacity of ER, is involved in many pathologic and physiological responses including embryonic development. This study investigated the effect of supplementation of IVC medium with an ER stress inducer, tunicamycin (TM), and an inhibitor, tauroursodeoxycholic acid (TUDCA), on the developmental competence, apoptosis, and gene expression in buffalo embryos produced by IVF. Treatment of presumed zygotes with TM resulted in a significant (P < 0.01) decrease in the blastocyst rate, whereas TUDCA supplementation did not improve the blastocyst development rate. Further, presence of TUDCA could not ameliorate the adverse effects of TM in terms of the blastocyst rate in combined (TM + TUDCA) treatment. Tunicamycin treatment increased (P < 0.01) the apoptotic index and reduced the total cell number, whereas TUDCA did not affect them significantly. However, TUDCA reduced the extent of TM-mediated apoptosis during combined (TM + TUDCA) treatment. Tunicamycin treatment increased (P < 0.01) and TUDCA treatment decreased (P < 0.01) the expression level of ER chaperones, GRP78 and GRP94. In the combined TM + TUDCA treatment, TUDCA decreased their expression level compared to that in the controls. A similar pattern was observed in the case of proapoptotic gene BAX. We did not find any significant difference in the expression level of BCl-XL, BID, P53, and CASPASE 3 after TM and TUDCA supplementation. In conclusion, our study reported that TM induces ER stress in buffalo embryos produced in vitro resulting in a decrease in the blastocyst rate and an increase in the level of apoptosis and that these actions are mediated by modulating the expression of apoptosis-related genes and ER chaperones. Tauroursodeoxycholic acid did not improve the developmental potential of buffalo embryos; however, it attenuated the TM-induced apoptosis by downregulating BAX and ER chaperones.

Improvement of chemotherapeutic drug efficacy by endoplasmic reticulum stress.

Tunicamycin (TUN), an inhibitor of protein glycosylation and therefore a potent stimulator of endoplasmic reticulum (ER) stress, has been used to improve anticancer drug efficacy, but the underlying mechanism remains obscure. In this study, we show that acute administration of TUN in mice induces the unfolded protein response and suppresses the levels of P21, a cell cycle regulator with anti-apoptotic activity. The inhibition of P21 after ER stress appears to be C/EBP homologous protein (CHOP)-dependent because in CHOP-deficient mice, TUN not only failed to suppress, but rather induced the expression of P21. Results of promoter-activity reporter assays using human cancer cells and mouse fibroblasts indicated that the regulation of P21 by CHOP operates at the level of transcription and involves direct binding of CHOP transcription factor to the P21 promoter. The results of cell viability and clonogenic assays indicate that ER-stress-related suppression of P21 expression potentiates caspase activation and sensitizes cells to doxorubicin treatment, while administration of TUN to mice increases the therapeutic efficacy of anticancer therapy for HepG2 liver and A549 lung cancers.

Hepatocyte autophagy is linked to C/EBP-homologous protein, Bcl2-interacting mediator of cell death, and BH3-interacting domain death agonist gene expression

BackgroundEndoplasmic reticulum (ER) stress and autophagy each play important roles in hepatocyte cell injury. We hypothesized that gene expression of C/EBP-homologous protein (CHOP) and the BH3 proteins Bcl2-interacting mediator of cell death (BIM) and BH3-interacting domain death agonist (BID) are involved in a complex interplay that regulates ER stress-induced autophagy and cell death. Materials and methodsHepatocytes were cultured from lean Zucker rats. Confluent hepatocytes were incubated with single or combined small interfering RNA for CHOP, BIM, and/or BID for 24 h providing gene inhibition. Incubation with tunicamycin (TM) for another 24 h stimulated ER stress. Quantitative real-time polymerase chain reaction determined the expression levels of CHOP, BIM, and BID. Immunostaining with microtubule-associated protein 1 light chain 3 measured autophagy activity. Trypan blue exclusion determined the cell viability. ResultsTM treatment increased the messenger RNA levels of CHOP and BIM but decreased the messenger RNA levels of BID. TM increased autophagy and decreased cell viability. Individual inhibition of CHOP, BIM, or BID protected against autophagy and cell death. However, simultaneous treatment with any combination of CHOP, BIM, and BID small interfering RNAs reduced autophagy activity but increased cell death independent of ER stress induction. ConclusionsAutophagy in hepatocytes results from acute ER stress and involves interplay, at the gene expression level, of CHOP, BIM, and BID. Inhibition of any one of these individual genes during acute ER stress is protective against cell death. Conversely, inhibition of any two of the three genes results in increased nonautophagic cell death independent of ER stress induction. This study suggests interplay between CHOP, BIM, and BID expression that can be leveraged for protection against ER stress-related cell death. However, disruption of the CHOP/BH3 gene expression homeostasis is detrimental to cell survival independent of other cellular stress.

Sialylation by β-galactoside α-2,6-sialyltransferase and N-glycans regulate cell adhesion and invasion in human anaplastic large cell lymphoma.

The interaction between cell surface glycans and extracellular matrix (ECM) including galectins is known to be closely associated with tumor cell adhesion, invasion and metastasis. We analyzed the roles of cell surface sialylation or glycosylation in galectin or ECM-mediated cell adhesion and invasion of human malignant lymphoma cells. Neuraminidase from Arthrobacter ureafaciens (AU) treatment resulted in reduction of cell adhesion to galectin-8 in human anaplastic large cell lymphoma (H-ALCL) which was established in our laboratory. The knockdown of β-galactoside α-2,6-sialyltransferase (ST6Gal1) by siRNA showed inhibition of ST6Gal1 expression in the cytoplasm of H-ALCL cells on immunohistochemical findings, and showed dramatic enhancement of cell adhesion to galectin-8. On the other hand, α-2,3-specific neuraminidase treatment resulted in moderate enhancement of cell adhesion to galectin-8. We performed chemically artificial modification of cell surface O-glycans by treatment of benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside (Bz-α-GalNAc) in H-ALCL. Cell adhesion to galectin-8 was enhanced by treatment of Bz-α-GalNAc suggesting that inhibition of elongation of O-glycans may enhance cell adhesion to galectin-8 in H-ALCL cells. On the other hand inhibition of elongation of N-glycosylation by tunicamycin (TM) resulted in inhibition of Phaseolus vulgaris-L (L-PHA) lectin-binding activity and inhibited cell adhesion to galectin-8,laminin and fibronectin. Neuraminidase treatment enhanced cell adhesion to laminin, and knockdown of ST6Gal1 resulted in enhancement of cell adhesion to laminin, but not to fibronectin, collagen type 1 and 4. Galectin-8 pre-treatment dramatically enhanced cell adhesion to laminin and neuraminidase treatment also enhanced cell adhesion to laminin in combination with galectin-8. Rho inhibitor, C3-transferase pre-treatment resulted in inhibition of cell invasion to galectin-8. Phosphatidylinositol 3-phosphate kinase (PI3K) inhibitor, wortmannin inhibits the cell invasive capacity to galectin-8. Neuraminidase treatment induces growth inhibition of lymphoma cells by galectin-8.

A possible involvement of endoplasmic reticulum stress in biliary epithelial autophagy and senescence in primary biliary cirrhosis.

Deregulated autophagy followed by cellular senescence in biliary epithelial cells (BECs) may be closely related to the abnormal expression of mitochondrial antigens and following autoimmune pathogenesis in primary biliary cirrhosis (PBC). We examined an involvement of endoplasmic reticulum (ER) stress in the deregulated autophagy and cellular senescence in PBC. We examined the degree of ER stress using markers; glucose-regulated protein 78 (GRP78) and protein disulfide isomerases (PDI), autophagy and cellular senescence in cultured BECs treated with an ER stress inducer, tunicamycin (TM), glycochenodeoxycholic acid (GCDC), and palmitic acid (PA), and the effect of pretreatment with tauroursodeoxycholic acid (TUDCA). We examined the expression of PDI and GRP78 in livers taken from the patients with PBC (n = 43) and 75 control livers. The expression of ER stress markers was significantly increased in cultured BECs treated with TM, GCDC or PA in BECs (p < 0.05), and pretreatment with TUDCA significantly suppressed the induced ER stress (p < 0.05). Autophagy, deregulated autophagy, and cellular senescence were induced in BECs treated with TM, GCDC, or PA. Pretreatment with TUDCA further increased autophagy in BECs treated with PA and suppressed cellular senescence caused by treatments with TM, GCDC, or PA (p < 0.05). A granular expression of PDI and GRP78 was significantly more extensive in small bile ducts in PBC, compared with control livers (p < 0.05). The expression of GRP78 was seen in senescent BECs in PBC. ER stress may play a role in the pathogenesis of deregulated autophagy and cellular senescence in biliary epithelial lesions in PBC.

Activation of AMP-activated protein kinase inhibits ER stress and renal fibrosis.

It has been suggested that endoplasmic reticulum (ER) stress facilitates fibrotic remodeling. Therefore, modulation of ER stress may serve as one of the possible therapeutic approaches to renal fibrosis. We examined whether and how activation of AMP-activated protein kinase (AMPK) suppressed ER stress induced by chemical ER stress inducers [tunicamycin (TM) and thapsigargin (TG)] and also nonchemical inducers in tubular HK-2 cells. We further investigated the in vivo effects of AMPK on ER stress and renal fibrosis. Western blot analysis, immunofluorescence, small interfering (si)RNA experiments, and immunohistochemical staining were performed. Metformin (the best known clinical activator of AMPK) suppressed TM- or TG-induced ER stress, as shown by the inhibition of TM- or TG-induced upregulation of glucose-related protein (GRP)78 and phosphorylated eukaryotic initiation factor-2α through induction of heme oxygenase-1. Metformin inhibited TM- or TG-induced epithelial-mesenchymal transitions as well. Compound C (AMPK inhibitor) blocked the effect of metformin, and 5-aminoimidazole-4-carboxamide-1β riboside (another AMPK activator) exerted the same effects as metformin. Transfection with siRNA targeting AMPK blocked the effect of metformin. Consistent with the results of cell culture experiments, metformin reduced renal cortical GRP78 expression and increased heme oxygenase-1 expression in a mouse model of ER stress-induced acute kidney injury by TM. Activation of AMPK also suppressed ER stress by transforming growth factor-β, ANG II, aldosterone, and high glucose. Furthermore, metformin reduced GRP78 expression and renal fibrosis in a mouse model of unilateral ureteral obstruction. In conclusion, AMPK may serve as a promising therapeutic target through reducing ER stress and renal fibrosis.

Wogonin protects rat dorsal root ganglion neurons against tunicamycin-induced ER stress through the PERK-eIF2α-ATF4 signaling pathway.

Endoplasmic reticulum (ER) stress has been demonstrated to contribute to neurodegeneration in multiple nervous system diseases. Wogonin is a flavonoid isolated from Scutellaria baicalensis root and has multiple pharmacological effects, including anti-inflammatory, antioxidant, and anticancer effects. It has a protective role in nervous system diseases; however, the pharmacological function of wogonin in the spinal cord is still with limited acquaintance. In the present study, rat dorsal root ganglion (DRG) neurons were pretreated with different concentrations of wogonin (0-100μM) before inducing ER stress using tunicamycin (TUN) (0.75μg/ml). Wogonin pretreatment at 75 and 100μM had a cytoprotective effect on cells against TUN-induced toxicity. Wogonin also decreased the number of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive DRG neurons and increased expression of superoxide dismutase (SOD), which was accompanied by decreased malondialdehyde (MDA) level. The induction of apoptosis was prevented with reduction in expression level of Bax and concomitant increase in B cell lymphoma 2 (Bcl-2) level. Furthermore, wogonin downregulated expression level of ER stress genes coding for glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), active caspase 12, transcription factor 4 (ATF4), and phosphorylation of pancreatic ER stress kinase (PERK) and eukaryotic initiation factor 2 alpha (eIF2α). The current study indicated that wogonin modulated stress-responsive genes, helping DRG neurons prevent TUN-induced ER stress through the PERK-eIF2α-ATF4 signaling pathway.

Tunicamycin-induced neutrophil extracellular trap (NET)-like structures in cultured human myeloid cell lines.

The mechanism of neutrophil extracellular trap cell death (NETosis), a regulated cell death pathway relevant to infection, autoimmunity and sepsis, is not completely known. The reason for this, at least in part, is the lack of an in vitro system that recapitulates the NETosis pathway using established human cell lines. We show that exposure of a human promyelocytic leukemia cell line HL-60 to the glycosyltransferase inhibitor tunicamycin (TM) resulted in extrusion of decompacted genomic DNAs to extracellular space, morphologically similar to NETs. Immunostaining using antibodies against NET marker proteins and bacterial trapping assay showed biochemical similarities between the TM-induced extracellular DNA structures and NETs. The NET-like structures were also generated on exposure of TM to other myeloid cell lines, such as U937 and THP-1. Thus, our findings provide an experimental setting to induce NET-like structures using cultured human myeloid cell lines, which may help our understanding of the regulation and function of NETosis.

Endoplasmic reticulum stress protects human thyroid carcinoma cell lines against ionizing radiation-induced apoptosis.

Radiotherapy is one of the most effective forms of cancer treatment, used in the treatment of a number of malignant tumors. However, the resistance of tumor cells to ionizing radiation remains a major therapeutic problem and the critical mechanisms determining radiation resistance are poorly defined. In the present study, a cellular endoplasmic reticulum (ER) stress microenvironment was established through the pretreatment of cultured thyroid cancer cells with tunicamycin (TM) and thapsigargin (TG), in order to mimic the ER stress response in a tumor microenvironment. This microenviroment was confirmed through the X‑box binding protein 1 splice process, glucose‑regulated protein 78 kD and ER degradation‑enhancing α‑mannosidase‑like mRNA expression. A clonogenic assay was used to measure cancer cell resistance to 60Co‑γ following TM pretreatment; in addition, human C/EBP homologous protein (CHOP) mRNA expression was determined and apoptosis assays were performed. The results showed that TM or TG pretreatment inhibited CHOP expression and reduced the apoptotic rate of cells. Furthermore, the results demonstrated that the induced ER stress response rendered cancer cells more resistant to ionizing radiation‑induced apoptosis. Therefore, the ER stress pathway may be a potential therapeutic target in order to improve the clinical efficiency of radiotherapy.

Wogonin prevents rat dorsal root ganglion neurons death via inhibiting tunicamycin-induced ER stress in vitro.

Wogonin is a natural flavonoid isolated from the root of Scutellaria baicalensis Georgi, which has been widely used in various research areas for its anti-oxidant, anti-inflammatory, and anti-cancer activities. It also presents a neuroprotective effect in the brain while encounters stress conditions, but the mechanisms controlling the neuroprotective effect of wogonin are not clear. In this study, we investigated the biomechanism underlying the neuroprotective effect of wogonin on rat dorsal root ganglion (DRG) neurons. Wogonin pre-treatment at 75μM significantly increased the cell viability of DRG neurons and decreased the number of the propidium iodide-positive DRG neurons before the endoplasmic reticulum (ER) stress is being induced by tunicamycin (TUN) (0.75μg/mL). In addition, Wogonin also inhibited the release of LDH and up-regulated the level of GSH. Furthermore, wogonin decreased the activation of ER stress-related molecules, including glucose-regulated protein 78 (GRP78), GRP94, C/EBP-homologous protein, active caspase12 and active caspase3, phosphorylation of pancreatic ER stress kinase, and eukaryotic initiation factor 2 alpha (eIF2α). In summary, our results indicated that wogonin could protect DRG neurons against TUN-induced ER stress.

L-Carnitine attenuates H2O2-induced neuron apoptosis via inhibition of endoplasmic reticulum stress

Both oxidative stress and endoplasmic reticulum stress (ER stress) have been linked to pathogenesis of neurodegenerative diseases. Our previous study has shown that l-carnitine may function as an antioxidant to inhibit H2O2-induced oxidative stress in neuroblastoma SH-SY5Y cells. To further explore the neuroprotection of l-carnitine, here we study the effects of l-carnitine on the ER stress response in H2O2-induced SH-SY5Y cell injury. Our results showed that l-carnitine pretreatment could increase cell viability; inhibit apoptosis and ROS accumulation caused by H2O2 or tunicamycin (TM). l-carnitine suppress the endoplasmic reticulum dilation and activation of ER stress-associated proteins including glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein-homologous protein (CHOP), JNK, Bax and Bim induced by H2O2 or TM. In addition, H2O2-induced cell apoptosis and activation of ER stress can also be attenuated by antioxidant N-acetylcysteine (NAC), CHOP siRNA and the inhibitor of ER stress 4-phenylbutyric acid (4-PBA). Taken together, our results demonstrated that H2O2 could trigger both oxidative stress and ER stress in SH-SY5Y cells, and ER stress participated in SH-SY5Y apoptosis mediated by H2O2-induced oxidative stress. CHOP/Bim or JNK/Bim-dependent ER stress signaling pathways maybe related to the neuroprotective effects of l-carnitine against H2O2-induced apoptosis and oxidative injury.

Endoplasmic reticulum stress-induced human chemoresistance to adrimycin in hepatocellular cancer cells

Objective To study the mechanism of chemoresistance to adrimycin in hepatocellular cancer cells.Methods Endoplasmic reticulum (ER) stress was established in SMMC7721 and HepG2 hepatocellular cancer cells using ER stress inducer tunicamycin (5 mg/L).The expression levels of ER stress indicators immunoglobulin-binding protein (Bip) and X-box binding protein 1 spliced (XBP1s) were examined by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR).SMMC7721 and HepG2 hepatocellular cancer cells were divided into 4 groups as follows:untreated control group,tunicamycin-treated (5 mg/L) group,doxorubicin-treated (10 mg/L) group,and tunicamycin (5 mg/L) plus doxorubicin-treated (10 mg/L) group.The inhibition rate of cell growth and apoptosis rate were detected by methyl thiazol tetrazolium (MTT) assay and flow cytometry,respectively.Results Data from Western blotting and RT-PCR showed that the expression levels of Bip and XBP1s were dramatically increased after tunicamycin incubation.MTT data showed that the relative growth inhibition rate in tunicamycin-treated group,doxorubicin-treated group and tunicamycin plus doxorubicin-treated group in SMMC7721 and HepG2 cells were (15.1±0.7)％,(57.3±1.5)％ and (36.7±2.1)％; (16.2±0.4)％,(54.9± 2.1) ％ and (30.8 ± 1.4) ％,respectively.As compared with doxorubicin-treated group,the relative cell growth inhibition rate in tunicamycin plus doxorubicin-treated group was decreased significantly in both cell lines.Flow cytometry data showed that the apoptosis rate in untreated control group,tunicamycin-treated group and doxorubicin-treated group and tunicamycin plus doxorubicin-treated group in SMMC7721 and HepG2 cells were (2.1 ±0.5)％,(13.2±1.6)％,(47.6 ±2.4)％ and (9.8 ±2.0)％; (1.9±0.8)％,(11.7±1.1)％,(42.1 ±2.9)％ and (26.9±1.8)％,respectively.As compared with the untreated control group,the apoptosis rate in treated groups was significantly increased.As compared with doxorubicin-treated group,the apoptosis rate in tunicamycin plus doxorubicin-treated group was decreased significantly in both cell lines.Conclusion ER stress could induce chemoresistance to adriamycin in hepatocellular cancer cells. Key words: Endoplasmic reticulum stress ; Carcinoma, hepatocellular ; Adrimycin ; Chemoresistance

Inhibition of autophagy promotes caspase-mediated apoptosis by tunicamycin in HepG2 cells

Tunicamycin (TM) causes accumulation of unfolded protein in endoplasmic reticulum (ER) lumen and introduces from elsewhere ER stress. This study was to assess the apoptosis and autophagy effect induced by TM on HepG2 cells and the role of autophagy in the system. The viability of HepG2 cells was significantly inhibited by TM in a dose-dependent manner detected by MTT assay. Then, the apoptotic morphology change, increasing apoptotic cell rate suggested that apoptosis was induced by TM in a time- and dose-dependent manner. To further determine the involvement of caspase-dependent pathway in TM-induced apoptosis, we discover that the activity of caspase-3/7, 8, 9 and cleavage of PARP markedly increased after TM treatment and the apoptosis was effectively attenuated by using caspase-9 and pan caspase inhibitor. Moreover, provided the rising stained acidic vacuoles and an increased level of LC3II and activation of Beclin1, we concluded that autophagy could be triggered by TM in a time- and dose-dependent manner. In addition, the inhibition of autophagy efficiently promoted TM-induced cell death identified by MTT assay. Meanwhile, the apoptotic cell rate and caspase-3 activation increased significantly after autophagy blockage. In conclusion, we found that TM initiated apoptosis and autophagy both in a time- and dose-dependent manner in HepG2 cells; and inhibition of autophagy may promote TM-induced cell death through enhancing apoptosis.