Endoplasmic Reticulum Stress-mediated Apoptosis Pathway Research Articles

Huangpu Tongqiao Capsule improves cognitive impairment in rats with Wilson disease by inhibiting endoplasmic reticulum stress-mediated apoptosis pathway

To investigate the neuroprotective effect of Huangpu Tongqiao Capsule (HPTQ) in a rat model of Wilson disease (WD) and explore the underlying mechanisms. SD rat models of WD were established by feeding of coppersupplemented chow diet and drinking water for 12 weeks, and starting from the 9th week, the rats were treated with low-, moderate- and high-dose HPTQ, penicillamine, or normal saline by gavage on a daily basis for 3 weeks. Copper levels in the liver and 24-h urine of the rats were detected, and their learning and memory abilities were evaluated using Morris water maze test. HE staining was used to observe morphological changes of CA1 region neurons in the hippocampus, and neuronal apoptosis was detected with TUNEL staining. Hippocampal expressions of endoplasmic reticulum stress (ERS)-mediated apoptosis pathway-related proteins GRP78, CHOP, caspase-12, cleaved caspase-9, and cleaved caspase-3 at both the mRNA and protein levels were detected using RT-qPCR, immunofluorescence assay or Western blotting. Compared with normal control rats, the rat models with copper overload-induced WD exhibited significantly increased copper levels in both the liver and 24-h urine, impaired learning and memory abilities, obvious hippocampal neuronal damage in the CA1 region and increased TUNEL-positive neurons (P<0.01), with also lowered mRNA and protein expressions of GRP78, CHOP, caspase-12, cleaved caspase-9, and cleaved caspase-3 in the hippocampus (all P<0.01). Treatments with HPTQ and penicillamine significantly lowered copper level in the liver but increased urinary copper level, improved learning and memory ability, alleviated neuronal damage and apoptosis in the hippocampus, and decreased hippocampal expressions of GRP78, CHOP, caspase-12, cleaved caspase-9, and cleaved caspase-3 in the rat models (P<0.01 or 0.05). HPTQ Capsule has neuroprotective effects in rat models of WD possibly by inhibiting ERS-mediated apoptosis pathway.

Evodiamine suppresses the progression of non-small cell lung carcinoma via endoplasmic reticulum stress-mediated apoptosis pathway in vivo and in vitro.

BackgroundEvodiamine (EVO) is one of the major components isolated from Evodia rutaecarpa (Juss.). Recent studies have shown that EVO has an anti-cancer effect. However, the pharmacological mechanism by which EVO impacts cancer is still poorly understood.ObjectivesThis study focused on asking the anti-cancer effect of EVO in human non-small cell lung carcinoma (NSCLC), and in particular to investigate whether EVO acts via modulating the endoplasmic reticulum stress (ERS)-mediated apoptosis pathway.Materials and MethodsA Lewis lung carcinoma (LLC) tumor-bearing mouse model was treated with low-dose EVO (5 mg/kg) and high-dose EVO (10 mg/kg) intraperitoneally for 14 d. The effects of EVO on tumor growth, apoptosis, and ERS were assessed. In addition, NSCLC A549 and LLC cells were treated with EVO in vitro. The effects of EVO on cell proliferation, apoptosis, and ERS were investigated. Finally, 4-phenylbutyric acid (4-PBA), an ERS inhibitor, was used to validate whether EVO induced apoptosis of NSCLC cells by modulating ERS.ResultsEVO treatment significantly inhibited tumor growth in LLC tumor-bearing mice. H&E staining indicated that EVO treatment reduced the number of tumor cells and the nucleo-plasmic ratio. Immunostaining showed that EVO treatment significantly decreased the expression of Ki-67. TUNEL staining revealed that EVO induced apoptosis in the tumor. Likewise, EVO treatment up-regulated the expression of apoptosis-related genes and proteins and increased activation of the ERS pathway in the tumor. Additionally, EVO inhibited cell proliferation and increased cell apoptotic rates in A549 and LLC cells. EVO also increased the expression levels of genes and proteins associated with ERS-mediated apoptosis pathway in vitro. The effects of EVO on apoptosis were abolished by 4-PBA treatment.ConclusionsOur study demonstrated that EVO suppresses the progression of NSCLC by modulating the ERS-mediated apoptosis pathway.

Dehydrocostus lactone inhibits cell proliferation and induces apoptosis by PI3K/Akt/Bad and ERS signalling pathway in human laryngeal carcinoma

The anti‐cancer effect of dehydrocostus lactone (DHL) derived from Saussurea costus (Falc.) Lipech against laryngeal carcinoma was assessed. The cytotoxic activity of DHL against laryngeal carcinoma is still obscure. Therefore, our study investigated the role of DHL in the growth inhibition of laryngeal carcinoma in vitro and in vivo, and the molecular mechanism of DHL‐induced apoptosis in cancer cells of the larynx. The results showed that DHL inhibits the viability, migration and proliferation of Hep‐2 and TU212 cells with little toxic effects on human normal larynx epithelial HBE cell line. Flow cytometry analysis (FAC) analysis and staining assay (Hoechst 33258) indicated that DHL stimulated Hep‐2 and TU212 cell apoptosis in a dose‐dependent manner. Mechanistically, DHL is capable of inhibiting Hep‐2 and TU212 cell viability via promoting p53 and P21 function, meanwhile DHL dose‐dependently induces Hep‐2 and TU212 cells apoptosis via activating mitochondrial apoptosis by inhibiting PI3K/Akt/Bad pathway and stimulating endoplasmic reticulum stress‐mediated apoptosis pathway. In vivo, DHL inhibited the growth of the Hep‐2 nude mouse xenograft model and observed no significant signs of toxicity in the organs of nude mice. In vivo experiments further confirmed the anti‐cancer effect of DHL on laryngeal carcinoma cells in vitro, and DHL‐treated nude mice can reduce the volume of tumours. Together, our study indicated that DHL has the potential to inhibit human laryngeal carcinoma via activating mitochondrial apoptosis pathway by inhibiting PI3K/Akt/Bad signalling pathway and stimulating endoplasmic reticulum stress‐mediated apoptosis pathway, providing a strategy for the treatment of human laryngeal carcinoma.

4-Phenylbutyrate Prevents Endoplasmic Reticulum Stress-Mediated Apoptosis Induced by Heatstroke in the Intestines of Mice.

ABSTRACTObjective:Heatstroke can induce serious physiological dysfunction in the intestine. However, the underlying mechanisms of this condition are unknown, and therapeutic strategies are not available. In this study, we explored the role of endoplasmic reticulum (ER) stress signaling in this process and assessed whether pretreating mice with an inhibitor of ER stress could alleviate intestinal damage.Methods:A heatstroke model was established in male mice. Mice were pretreated with 4-phenylbutyrate (4-PBA) before exposure to heat stress. Intestinal morphological changes were observed by hematoxylin and eosin (H&E) staining and transmission electron microscopy. The TUNEL assay was used to detect intestinal apoptosis. The expression of the ER stress-related proteins and apoptosis-related proteins was investigated by the Western blot assay.Results:Compared with control group, mice with heatstroke exhibited evidence of intestinal injury and epithelial apoptosis, accompanied by significantly increased expression of ER stress-related proteins in the intestines. The intestinal injury score and level of intestinal epithelial apoptosis were significantly reduced after administration of 4-PBA. Furthermore, the levels of the intestinal ER stress-related proteins GRP78, PERK, p-eIF2α, ATF4, and CHOP were decreased after 4-PBA treatment.Conclusions:Our results indicate that the ER stress-mediated apoptosis pathway is activated during heat stress-induced intestinal injury. 4-PBA can inhibit heatstroke-induced intestinal ER stress and attenuate intestinal injury. We provide evidence that the beneficial effect of 4-PBA is closely related to the inhibition of ER stress-mediated apoptosis. These findings suggest that ER stress may be a novel therapeutic target in patients with heatstroke.

Deficiency of lysyl hydroxylase 2 in mice causes systemic endoplasmic reticulum stress leading to early embryonic lethality

Lysyl hydroxylase 2 (LH2) is an endoplasmic reticulum (ER)-resident enzyme that catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens. This is a critical modification to determine the fate of collagen cross-linking pathway that contributes to the stability of collagen fibrils. Studies have demonstrated that the aberrant LH2 function causes various diseases including osteogenesis imperfecta, fibrosis, and cancer metastasis. However, surprisingly, a LH2-deficient animal model has not been reported. In the current study, to better understand the function of LH2, we generated LH2 gene knockout mice by CRISPR/Cas9 technology. LH2 deficiency was confirmed by genotyping polymerase chain reaction (PCR), reverse transcriptase-PCR, and immunohistochemical analyses. Homozygous LH2 knockout (LH2−/−) embryos failed to develop normally and died at early embryonic stage E10.5 with abnormal common ventricle in a heart, i.e., an insufficient wall, a thin ventricular wall, and loosely packed cells. In the LH2−/− mice, the ER stress-responsive genes, ATF4 and CHOP were significantly up-regulated leading to increased levels of Bax and cleaved caspase-3. These data indicate that LH2 plays an essential role in cardiac development through an ER stress-mediated apoptosis pathway.

Alginate oligosaccharide alleviates myocardial reperfusion injury by inhibiting nitrative and oxidative stress and endoplasmic reticulum stress-mediated apoptosis.

Alginate oligosaccharide (AOS) has recently demonstrated the ability to protect against acute doxorubicin cardiotoxicity and neurodegenerative disorders by inhibiting oxidative stress and endoplasmic reticulum (ER) stress-mediated apoptosis, which are both involved in myocardial ischemia/reperfusion (I/R) injury. In the present study, we investigated whether pretreatment with AOS protects against myocardial I/R injury in mice and explored potential cardioprotective mechanisms. AOS pretreatment significantly decreased the infarct size, reduced the cardiac troponin-I concentration, and ameliorated the cardiac dysfunction. Accompanied with the reduced cardiac injury, AOS pretreatment clearly decreased I/R-induced myocardial apoptosis. With regard to mechanism, AOS pretreatment markedly attenuated nitrative/oxidative stress, as evidenced by decreases in 3-nitrotyrosine content and superoxide generation, and downregulated inducible nitric oxide synthase, NADPH oxidase2, and 4-hydroxynonenal. Moreover, AOS pretreatment decreased myocardial apoptosis by inhibiting the ER stress-mediated apoptosis pathway, which is reflected by the downregulation of C/EBP homologous protein, glucose-regulated protein 78, caspase-12, and Bcl-2-associated X protein, and by the upregulation of the anti-apoptotic protein B-cell lymphoma-2. Collectively, these findings demonstrate that AOS renders the heart resistant to I/R injury, at least in part, by inhibiting nitrative/oxidative stress and ER stress-mediated apoptosis.

Palmitate induces myocardial lipotoxic injury via the endoplasmic reticulum stress‑mediated apoptosis pathway.

Increased free fatty acids in cardiomyocytes induce myocardial lipotoxic injury, but the underlying mechanisms are not well understood. Therefore, the aim of the present study was to explore the role of the endoplasmic reticulum (ER) stress‑mediated apoptosis pathway in palmitate (PA)‑induced cardiomyocyte lipotoxicity. H9c2 cells were treated with various doses (100, 200 and 400µM) of PA to mimic cardiomyocyte lipotoxicity invitro. Oil Red O staining was used to determine the accumulation of intracellular lipids. An MTT assay was used to determine the cell viability. Lactate dehydrogenase (LDH) activity was used to measure the injury of H9c2 cells. Flow cytometry analysis was used to detect apoptosis. Western blotting was used to evaluate the expression change of ER stress‑mediated apoptosis pathway proteins, including 78kDa glucose‑regulated protein (GRP78), eukaryotic initiation factor 2 α (eIF2α), protein kinase R‑like endoplasmic reticulum kinase (PERK), C/EBP homologous protein (CHOP) and cleaved caspase‑12. The results demonstrated that various doses of PA promoted excessive lipid deposition in cardiomyocytes and resulted in decreased cell viability, and increased the LDH activity and apoptosis rate in a dose‑dependent manner. Furthermore, the expression of GRP78, a marker of ER stress, and the phosphorylation of eIF2α and PERK were increased following treatment with PA. Notably, the levels of CHOP and cleaved caspase‑12, critical regulators of ER stress‑mediated apoptosis pathway, were also elevated, and this effect was reversed by a specific ER stress inhibitor (4‑phenyl butyric acid). In conclusion, the results of the current study demonstrated that PA induces myocardial lipotoxic injury by triggering ER stress and the ER stress‑mediated apoptosis pathway.

CSB ablation induced apoptosis is mediated by increased endoplasmic reticulum stress response.

The DNA repair protein Cockayne syndrome group B (CSB) has been recently identified as a promising anticancer target. Suppression, by antisense technology, of this protein causes devastating effects on tumor cells viability, through a massive induction of apoptosis, while being non-toxic to non-transformed cells. To gain insights into the mechanisms underlying the pro-apoptotic effects observed after CSB ablation, global gene expression patterns were determined, to identify genes that were significantly differentially regulated as a function of CSB expression. Our findings revealed that response to endoplasmic reticulum stress and response to unfolded proteins were ranked top amongst the cellular processes affected by CSB suppression. The major components of the endoplasmic reticulum stress-mediated apoptosis pathway, including pro-apoptotic factors downstream of the ATF3-CHOP cascade, were dramatically up-regulated. Altogether our findings add new pieces to the understanding of CSB mechanisms of action and to the molecular basis of CS syndrome.

Matrine Suppresses the ER-positive MCF Cells by Regulating Energy Metabolism and Endoplasmic Reticulum Stress Signaling Pathway.

Matrine (C15 H24 N2 O), an alkaloid that is one of the main active components from Sophora flavescens. Matrine has been demonstrated to have therapeutic effects on various solid tumors, including breast cancer, but the mechanism still needs further study. Endoplasmic reticulum (ER)-positive Michigan Cancer Foundation cells were cultured, and matrine was added in various amounts to measure the dose-dependent and time-dependent cytotoxicity. Hoechst 33258 staining was used to observed nuclear morphological changes. Apoptosis was measured by AnnexinV/PI double staining assay kit. Intracellular adenosine triphosphate and glycometabolism were detected by assay kit. The protein levels GRP78, p-eIF2α, CHOP, cytochrome c, and HexokinaseII were analyzed. Mechanistic investigations revealed that matrine treatment causes ER dilation and up-regulated the expression of ER stress markers GRP78, eIF2α, and CHOP, increases the levels of apoptotic in Michigan Cancer Foundation cells, subsequently, blocking the ER stress-mediated apoptosis pathway, significantly decreased matrine-induced apoptotic but still has significant difference between control group. In addition, matrine not only promoted the occurrence of ER stress but also inhibited the expression of hexokinase II, down-regulated energy metabolism. In summary, the present study suggests that the induction of ER stress-mediated apoptosis by matrine and down-regulated energy metabolism may account for its cytotoxic effects in human breast cancer cells. Copyright © 2017 John Wiley & Sons, Ltd.

Phenotypic and functional characterization of mesenchymal stem cells from decidua basils of human term placenta

The effect of hypercholesterolemia on myocardial ischemia reperfusion injury (MIRI) is in controversy and the underlying mechanism is still not well understood. In the present study, we firstly detected the effects of hypercholesterolemia on MIRI and the role of endoplasmic reticulum (ER) stress-mediated apoptosis pathway in this process. The infarct size was determined by TTC staining, and apoptosis was measured by the TUNEL method. The marker proteins of ER stress response and ER stress-mediated apoptosis pathway were detected by Western blot. The results showed that high cholesterol diet-induced hypercholesterolemia significantly increased the myocardial infarct size, the release of myocardium enzyme and the ratio of apoptosis, but did not affect the recovery of cardiac function. Moreover, hypercholesterolemia also remarkably up-regulated the expressions of ER stress markers (glucose-regulated protein 78 and calreticulin) and critical molecules in ER stress-mediated apoptosis pathway (CHOP, caspase 12, phospho-JNK). In conclusion, our study demonstrated that hypercholesterolemia enhanced myocardial vulnerability/sensitivity to ischemia reperfusion injury involved in aggravation the ER stress and activation of ER stress-mediated apoptosis pathway and it gave us a new insight into the underlying mechanisms associated with hypercholesterolemia-induced exaggerated MIRI and also provided a novel target for preventing MIRI in the presence of hypercholesterolemia.

Hypercholesterolemia aggravates myocardial ischemia reperfusion injury via activating endoplasmic reticulum stress-mediated apoptosis

The effect of hypercholesterolemia on myocardial ischemia reperfusion injury (MIRI) is in controversy and the underlying mechanism is still not well understood. In the present study, we firstly detected the effects of hypercholesterolemia on MIRI and the role of endoplasmic reticulum (ER) stress-mediated apoptosis pathway in this process. The infarct size was determined by TTC staining, and apoptosis was measured by the TUNEL method. The marker proteins of ER stress response and ER stress-mediated apoptosis pathway were detected by Western blot. The results showed that high cholesterol diet-induced hypercholesterolemia significantly increased the myocardial infarct size, the release of myocardium enzyme and the ratio of apoptosis, but did not affect the recovery of cardiac function. Moreover, hypercholesterolemia also remarkably up-regulated the expressions of ER stress markers (glucose-regulated protein 78 and calreticulin) and critical molecules in ER stress-mediated apoptosis pathway (CHOP, caspase 12, phospho-JNK). In conclusion, our study demonstrated that hypercholesterolemia enhanced myocardial vulnerability/sensitivity to ischemia reperfusion injury involved in aggravation the ER stress and activation of ER stress-mediated apoptosis pathway and it gave us a new insight into the underlying mechanisms associated with hypercholesterolemia-induced exaggerated MIRI and also provided a novel target for preventing MIRI in the presence of hypercholesterolemia.

Abstract 897: CHOP-deficiency promotes chronic inflammation-induced pancreatic fibrosis

Abstract Background and Aims: Endoplasmic reticulum (ER) stress is involved in the pathogenesis of various diseases, including neurodegenerative disease, diabetes mellitus, atherosclerosis, heart disease, and pancreatic disease. CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), also known as growth arrest- and DNA damage-inducible gene 153 (GADD153) is a transcriptional regulator induced by ER stress and a key factor in the ER stress-mediated apoptosis pathway. Chronic inflammation in the pancreas causes progressive pancreatic fibrosis that eventually leads to pancreatic ductal adenocarcinoma. The aim of our study is to prove the role of the ER stress-CHOP pathway in chronic pancreatitis. Methods: We generated gene engineered chronic pancreatitis model (Spink3 SPINK1/-) mice, and crossed them with CHOP-deficient (Chop-/-) mice. We assessed the pancreatic fibrosis by hematoxylin eosin and Sirius red staining, and performed Western blotting to measure alpha smooth muscle actin (α-SMA), which is a maker of the activated pancreatic stellate cells, transforming growth factor beta 1 (TGF-β, Desmin, amylase, and trypsinogen, which are makers of exocrine function, in wild-type (Chop+/+) and CHOP-deficient chronic pancreatitis model (Chop-/-; Spink3SPINK1/-) mice at 3-weeks-old. Results: There were no significant differences between the pancreases of Chop+/+ and Chop-/- mice. The body weight of Chop-/-; Spink3 SPINK1/- mice was not so different from that of Spink3 SPINK1/- mice at 2-weeks-old, but the body weight of Chop-/-; Spink3 SPINK1/- mice was lighter than that of Spink3 SPINK1/- mice at 4-weeks-old (p&amp;lt;0.001). However, there was no significant difference between the survival of Spink3 SPINK1/- mice than that of Chop-/-; Spink3 SPINK1/- mice. mRNA levels for inflammatory cytokines, such as TNFα, IL-6 and IL-1β increased in Chop-/-; Spink3 SPINK1/- mice than in Spink3 SPINK1/- mice at 2-weeks-old. We found the progression of pancreatic fibrosis and the activated pancreatic stellate cells in Chop-/-; Spink3 SPINK1/- mice compared with Spink3 SPINK1/- mice at 3-weeks-old. In addition, the expression levels of α-SMA, TGF-β and Desmin increased in Chop-/-; Spink3 SPINK1/- mice at 3-weeks-old. Apoptotic cells decreased in Chop-/-; Spink3 SPINK1/- mice compared with Chop-/-; Spink3 SPINK1/- mice at 3-weeks-old. Conclusions: These results indicated that the ER stress-CHOP pathway may have an essential role in the suppression of pancreatic fibrosis through the inactivation of pancreatic stellate cells in chronic pancreatitis. Therefore, anti-inflammatory treatment against chronic pancreatitis through the ER stress-CHOP pathway may reduce the risk of pancreatic cancer. Citation Format: Katsunobu Taki, Masaki Ohmuraya, Daisuke Hashimoto, Kota Arima, Hideaki Takeyama, Takayoshi Kaida, Takaaki Higashi, Hidetoshi Nitta, Hiromitsu Hayashi, Akira Chikamoto, Toru Beppu, Kimi Araki, Hideo Baba. CHOP-deficiency promotes chronic inflammation-induced pancreatic fibrosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 897. doi:10.1158/1538-7445.AM2015-897

Abstract 241: CHOP-deficiency promotes chronic inflammation-induced pancreatic fibrosis

Abstract Background and Aims: Endoplasmic reticulum (ER) stress is involved in the pathogenesis of various diseases, including neurodegenerative disease, diabetes mellitus, atherosclerosis, heart disease, and pancreatic disease. CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), also known as growth arrest- and DNA damage-inducible gene 153 (GADD153) is a transcriptional regulator induced by ER stress and a key factor in the ER stress-mediated apoptosis pathway. Chronic inflammation in the pancreas causes progressive pancreatic fibrosis that eventually leads to pancreatic ductal adenocarcinoma. The aim of our study is to prove the role of the ER stress-CHOP pathway in chronic pancreatitis. Methods: We generated gene engineered chronic pancreatitis model (Spink3 SPINK1/-) mice, and crossed them with CHOP-deficient (Chop-/-) mice. We assessed the pancreatic fibrosis by hematoxylin eosin and Sirius red staining, and performed Western blotting to measure α-SMA, which is a maker of the activated pancreatic stellate cells, transforming growth factor β, amylase, and trypsinogen, which are makers of exocrine function, in wild-type (Chop+/+) and CHOP-deficient chronic pancreatitis model (Chop-/-; Spink3 SPINK1/-) mice at 2-weeks- and 4-weeks-old. Results: There were no significant differences between the pancreases of Chop+/+ and Chop-/- mice. The body weight of Chop-/-; Spink3 SPINK1/- mice was not so different from that of Spink3 SPINK1/- mice at 2-weeks-old, but the body weight of Chop-/-; Spink3 SPINK1/- mice was lighter than that of Spink3 SPINK1/- mice at 4-weeks-old (p=0.001). However, there was no significant difference between the survival of Spink3 SPINK1/- mice than that of Chop-/-; Spink3 SPINK1/- mice. We found the progression of pancreatic fibrosis and the activated pancreatic stellate cells in Chop-/-; Spink3 SPINK1/- mice compared with Spink3 SPINK1/- mice at 2-weeks-old. In addition, the expression levels of α-SMA and Desmin increased in Chop-/-; Spink3 SPINK1/- mice at 2-weeks-old. Conclusions: These results indicated that the ER stress-CHOP pathway may have an essential role in the suppression of pancreatic fibrosis through the inactivation of pancreatic stellate cells in chronic pancreatitis. Therefore, anti-inflammatory treatment against chronic pancreatitis through the ER stress-CHOP pathway may reduce the risk of pancreatic cancer. Citation Format: Katsunobu Taki, Masaki Ohmuraya, Kazuya Sakata, Daisuke Hashimoto, Shinya Abe, Hidetoshi Nitta, Hiromitsu Hayashi, Akira Chikamoto, Toru Beppu, Hideo Baba. CHOP-deficiency promotes chronic inflammation-induced pancreatic fibrosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 241. doi:10.1158/1538-7445.AM2014-241

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

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

Abstract B21: Endoplasmic reticulum stress conditioned melanoma cell lines to chemotherapy-induced cell death.

Abstract Melanoma is among the most aggressive malignancies with increasing worldwide incidence and there is no effective treatment for the metastatic disease. The absence of an effective therapy may be due to adaptation and selection of melanoma cells to endoplasmic reticulum (ER) stress (Hersey P and Zhang XD). We showed that GADD153, one of the components of the ER stress-mediated apoptosis pathway, was mostly excluded from the nucleus of primary and metastatic melanoma cells compared with nevus cells. These data suggest that the unexpected GADD153 cellular localization could be involved in melanoma cell adaption to ER stress, since GADD153 accumulates in the nucleus during ER stress. Unfolded protein response (UPR) signaling induced in response to ER stress is a dual process that acts inducing a protective response to restore ER homeostasis or cell death in a severe or persistent ER stress induction. We investigated if induction of ER stress was a potential strategy to chemosensitize melanoma cells to genotoxic drugs by surpassing the adaptive levels to ER stress. We first treated human melanoma cells (SBCl2, Skmel28 and Mel85) with tunicamycin (TUN), an ER stress inducer before cis-diamminedichloroplatinum(II)(CDDP) treatment. CDDP is a low cost chemotherapeutic drug currently used in Brazil as a second line for melanoma treatment, especially in youngsters. We demonstrated an &amp;gt;50% increase in the percentage of cells in cell death process with TUN/CDDP treatment when compared to CDDP only. UPR markers, GRP78 and GADD153, were induced by TUN. One of the cellular mechanisms that are regulated by ER stress is autophagy. Accordingly, we observed an increase in the acidic vesicular organelles, assessed by acridine orange staining, in response to combined treatment. Autophagy inhibition with 3-Methyladenine increased TUN/CDDP induced-cell death, suggesting that autophagy plays a protective role in this response. Reactive oxygen species were involved in TUN and CDDP response, but more prominently in combined treatment response. However, the oxidative stress inhibition with N-acetylcysteine did not alter the sensitization effect of TUN pretreatment. We also tested temozolomide (TMZ), an equivalent drug to the active form of dacarbazine, the first line treatment of melanoma. The same sensitization capacity was observed in cells pretreated with TUN and further treated with TMZ. These results indicate that tumor cells could be preconditioned to cell death if exposed to a first ER stressor, such as TUN, which would compromise an effective adaptive response to a cell death inducer, as CDDP and TMZ. This combined approach may be a promising strategy for melanoma therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B21. Citation Format: Renata F. Saito, Andréia H. Otake, Margarita M. Cortes, Roger Chammas. Endoplasmic reticulum stress conditioned melanoma cell lines to chemotherapy-induced cell death. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B21.

Increased Endoplasmic Reticulum Stress Response Is Involved in Clopidogrel-Induced Apoptosis of Gastric Epithelial Cells

BackgroundThe widespread use of clopidogrel alone or in combination with aspirin may result in gastrointestinal mucosal injury, clinically represented as recurrent ulceration and bleeding complications. Our recent work suggested that clopidogrel significantly induced human gastric epithelial cell (GES-1) apoptosis and disrupted gastric mucosal barrier, and that a p38 MAPK inhibitor could attenuate such injury. However, their exact mechanisms are largely unknown.MethodsThe GES-1 cells were used as a model system, the effects of clopidogrel on the whole gene expression profile were evaluated by human gene expression microarray and gene ontology analysis, changes of the mRNA and protein expression were determined by real-time PCR and Western blot analysis, and cell viability and apoptosis were measured by MTT assay and flow cytometry analysis, respectively.ResultsGene microarray analysis identified 79 genes that were differentially expressed (P<0.05 and fold-change >3) when cells were treated with or without clopidogrel. Gene ontology analysis revealed that response to stress and cell apoptosis dysfunction were ranked in the top 10 cellular events being affected, and that the major components of endoplasmic reticulum stress-mediated apoptosis pathway – CHOP and TRIB3– were up-regulated in a concentration- and time-dependent manner when cells were treated with clopidogrel. Pathway analysis demonstrated that multiple MAPK kinases were phosphorylated in clopidogrel-treated GES-1 cells, but that only SB-203580 (a p38-specific MAPK inhibitor) attenuated cell apoptosis and CHOP over-expression, both of which were induced by clopidogrel.ConclusionsIncreased endoplasmic reticulum stress response is involved in clopidogrel-induced gastric mucosal injury, acting through p38 MAPK activation.

A Novel Monocarbonyl Analogue of Curcumin, (1E,4E)-1,5-Bis(2,3-dimethoxyphenyl)penta-1,4-dien-3-one, Induced Cancer Cell H460 Apoptosis via Activation of Endoplasmic Reticulum Stress Signaling Pathway

Endoplasmic reticulum (ER) stress-induced cancer cell apoptosis has become a novel signaling target for development of cancer therapeutic drugs. Curcumin exhibits growth-suppressive activity against a variety of cancer cells. We previously synthesized a series of monocarbonyl analogues of curcumin with strong cytotoxicity against tumor cells. In this study, we found that only compound 19 [(1E,4E)-1,5-bis(2,3-dimethoxyphenyl)penta-1,4-dien-3-one] can induce C/EBP-homologous protein (CHOP) expression in human lung cancer H460 cells. Treatment with 19 induced H460 cell apoptosis in a dose-responsive manner, and this effect was associated with corresponding increases in a series of key components in ER stress-mediated apoptosis pathway, followed by caspase cleavage and activation. However, curcumin at the same concentrations does not display such properties. CHOP knockdown by specific siRNA attenuated 19-induced cell apoptosis, further indicating that the apoptotic pathway is ER stress-dependent. In vivo, 19 showed a dramatic 53.5% reduction in H460 xenograft tumor size after 22 days of treatment. Taken together, these mechanistic insights on the novel compound 19, with nontoxicity, may provide us with a novel anticancer candidate.

Endoplasmic reticulum stress and related apoptosis in the lungs of a chronic obstructive pulmonary disease rat model

To study the endoplasmic reticulum stress (ERS) and the apoptosis of alveolar epithelial cells in a COPD rat model. Twenty-four Wistar rats were divided into a control group and a COPD group at random. The COPD rat model was established by intratracheal instillation of lipopolysaccharide (LPS) twice and exposure to cigarette smoke daily. The spirometry was conducted and the pathological changes were observed after the model was established. The levels of glucose regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). The protein expression of GRP78, CHOP and caspase-12 was detected by Western blot. TdT-mediated dUTP nick end labeling (TUNEL) was used to analyze alveolar epithelial cell apoptosis. Comparisons between the two groups were performed by t-test. Significant decrease of FEV(0.3)/FVC [(60 ± 6)%] and dynamic compliance of lung (CLdyn) [(0.17 ± 0.02) cm H2O×ml(-1)×s(-1)], and increase of airway resistance [(0.64 ± 0.07) ml/cm H2O] were found in the COPD group compared with the control group [(83 ± 7)%, (0.31 ± 0.03) cm H2O×ml(-1)×s(-1), (0.32 ± 0.03) ml/cm H2O] (t = -14.532 - 11.619, P < 0.05). GRP78 mRNA and CHOP mRNA densitometry [(0.65 ± 0.07), (0.79 ± 0.06)] were significantly increased in the COPD group compared with the control group [(0.21 ± 0.04), (0.07 ± 0.04), respectively] (t = -19.102 and -32.573, P < 0.05). GRP78, CHOP, and active caspase-12 protein densitometry (0.83 ± 0.06, 0.82 ± 0.06 and 0.81 ± 0.07) were significantly increased in the COPD group compared with the control group [(0.33 ± 0.05, 0.05 ± 0.03 and 0.24 ± 0.06), respectively] (t = -40.866 - -22.070, P < 0.05). More apoptotic alveolar epithelial cells were found in the COPD group [(32.4 ± 3.7)%] than in the control group [(6.2 ± 0.9)%] (t = -23.852, P < 0.05). ERS was triggered in the lung tissues of COPD rats, especially in the alveolar epithelial cells. Alveolar epithelial cell apoptosis was increased in the COPD group. The ERS mediated apoptosis pathway may participate in the alveolar epithelial cell apoptosis in COPD.

Polyunsaturated fatty acids synergize with lipid droplet binding thalidomide analogs to induce oxidative stress in cancer cells

BackgroundCytoplasmic lipid-droplets are common inclusions of eukaryotic cells. Lipid-droplet binding thalidomide analogs (2,6-dialkylphenyl-4/5-amino-substituted-5,6,7-trifluorophthalimides) with potent anticancer activities were synthesized.ResultsCytotoxicity was detected in different cell lines including melanoma, leukemia, hepatocellular carcinoma, glioblastoma at micromolar concentrations. The synthesized analogs are non-toxic to adult animals up to 1 g/kg but are teratogenic to zebrafish embryos at micromolar concentrations with defects in the developing muscle. Treatment of tumor cells resulted in calcium release from the endoplasmic reticulum (ER), induction of reactive oxygen species (ROS), ER stress and cell death. Antioxidants could partially, while an intracellular calcium chelator almost completely diminish ROS production. Exogenous docosahexaenoic acid or eicosapentaenoic acid induced calcium release and ROS generation, and synergized with the analogs in vitro, while oleic acid had no such an effect. Gene expression analysis confirmed the induction of ER stress-mediated apoptosis pathway components, such as GADD153, ATF3, Luman/CREB3 and the ER-associated degradation-related HERPUD1 genes. Tumor suppressors, P53, LATS2 and ING3 were also up-regulated in various cell lines after drug treatment. Amino-phthalimides down-regulated the expression of CCL2, which is implicated in tumor metastasis and angiogenesis.ConclusionsBecause of the anticancer, anti-angiogenic action and the wide range of applicability of the immunomodulatory drugs, including thalidomide analogs, lipid droplet-binding members of this family could represent a new class of agents by affecting ER-membrane integrity and perturbations of ER homeostasis.

The Endoplasmic Reticulum Stress-Mediated Apoptosis Signal Pathway Is Involved in Sepsis-Induced Abnormal Lymphocyte Apoptosis

Background/Aim: The mechanisms of abnormal lymphocyte apoptosis in sepsis are only partially defined. The present study was designed to investigate whether the endoplasmic reticulum (ER) is implicated in the extensive apoptosis of lymphocytes in sepsis. Methods: C57BL/6 mice were randomized into cecal ligation and puncture (CLP) and sham operation groups. Apoptosis was detected by the TUNEL method and flow cytometry. The expression of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) was detected by RT-PCR and Western blot. The splicing of X box-binding protein-1 (XBP1) mRNA was detected by RT-PCR. Results: A high degree of lymphocyte apoptosis was observed in the CLP group. Marked induction of GRP78 and accumulation of spliced XBP1 mRNA were observed in the splenocytes from septic mice, indicating activation of unfolded protein responses. Furthermore, both CHOP and its mRNA were markedly upregulated in the CLP group, suggesting that the ER stress response switched to a proapoptotic response. Conclusion: These data demonstrate activation of the unfolded protein response in lymphocytes and that ER stress may contribute to abnormal lymphocyte apoptosis during sepsis. Accordingly, the ER stress-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of sepsis-induced lymphocyte apoptosis.