CHOP siRNA Research Articles

Regulatory mechanisms underlying endoplasmic reticulum stress involvement in the development of gestational diabetes mellitus entail the CHOP-PPARα–NF–κB pathway

ObjectiveWe investigated the proinflammatory functions of endoplasmic reticulum stress and peroxisome proliferator-activated receptor α (PPARα) in the development of gestational diabetes mellitus (GDM) and their relationship in regulating inflammation in GDM. MethodsThis study was performed on placentas of normal pregnant women, women with GDM, and HTR8 cells. Transmission electron microscopy, immunohistochemistry, Western blot analysis, and RT-PCR were performed to analyze ERS and PPARα expression on both normal and GDM pregnancy placentas. ELISA was performed to analyze inflammatory biomarkers. To generate models of the GDM-like state, placentas of normal pregnancy were treated with LPS and polyinosinic-polycytidylic acid (poly [I:C]). TG, CHOP plasmid, and CHOP siRNA were assessed as to their regulation of HTR8 cells to discern the relationship between ERS and PPARα in regulating the inflammation associated with GDM. ResultsERS was elevated in GDM placentas, induced the secretion of IL-6 and TNF-α, and attenuated the expression of GLUT-4. PPARα was diminished in GDM placentas and inhibited the inflammatory responses via the NF-κB nuclear-transport process. 4-PBA reduced CHOP and augmented PPARα, and it decreased IL-6 and TNF-α in our GDM-like explant. However, with both 4-PBA and MK886 treatment, we noted no significant difference in CHOP expression. The level of PPARα was reduced, and that of NF-κB p65 in the nucleus was elevated with TG treatment in the HTR8/Svneo. Knockdown of CHOP increased PPARα and reduced NF-κB p65, while expression of PPARα declined, and that of NF-κB p65 rose with the application of CHOP when HTR8 cells were treated with TG. ConclusionsERS contributes to the pathophysiology of GDM in pregnancy via the CHOP-PPARα–NF–κB-signalling pathway by inducing aberrant activation of inflammation and insulin resistance.

Exploring the molecular mechanisms underlie the endoplasmic reticulum stress-mediated methylmercury-induced neuronal developmental damage

Methylmercury (MeHg) is a ubiquitous environmental pollutant, which can cross the placenta and blood brain barrier, thus affecting fetal growth and development. Although previous studies have demonstrated that MeHg induces endoplasmic reticulum (ER) stress in rat cerebral cortex and primary neurons, the role of ER stress in MeHg-induced neurodevelopmental toxicity remains unclear. Here, we used ICR pregnant mice and hippocampal neurons cells (HT22 cells) to investigate the molecular mechanism by which MeHg exposure during pregnancy affects neurodevelopment. We found that prenatal MeHg exposure caused developmental delay in offspring, accompanied with ER stress, cell apoptosis, cell cycle arrest and abnormal DNA methylation. Then, we used ER stress specific inhibitor 4-PBA and CHOP siRNA to investigate the role of ER stress on HT22 cells damage caused by MeHg. The results showed that 4-PBA pretreatment restored MeHg-induced axonal shortening and alleviated apoptosis, cell cycle arrest and DNA methylation. At the same time, the activation of CHOP/c-Jun/GADD45A signaling pathway was inhibited, and the interaction between CHOP and c-Jun was weakened. In addition, CHOP siRNA reduced the expression of c-Jun and GADD45A, and relieved DNA methylation levels to some extent. In summary, our study suggested that ER stress induced by MeHg mediated cell apoptosis and cell cycle arrest, and may affect DNA methylation through activation of CHOP/c-Jun/GADD45A signaling pathway, thus leading to neuronal damage.

Salvianolic Acid B Suppresses ER Stress-Induced NLRP3 Inflammasome and Pyroptosis via the AMPK/FoxO4 and Syndecan-4/Rac1 Signaling Pathways in Human Endothelial Progenitor Cells.

Mounting evidence demonstrates uncontrolled endoplasmic reticulum (ER) stress responses can activate the inflammasome, which generally results in endothelial dysfunction, a major pathogenetic factor of chronic inflammatory diseases such as atherosclerosis. Salvianolic acid B (SalB), produced by Radix Salviae, exerts antioxidative and anti-inflammatory activities in multiple cell types. However, SalB's effects on ER stress-related inflammasome and endothelial dysfunction remain unknown. Here, we showed SalB substantially abrogated ER stress-induced cell death and reduction in capillary tube formation, with declined intracellular reactive oxygen species (ROS) amounts and restored mitochondrial membrane potential (MMP), as well as increased expression of HO-1 and SOD2 in bone marrow-derived endothelial progenitor cells (BM-EPCs). ER stress suppression by CHOP or caspase-4 siRNA transfection attenuated the protective effect of SalB. Additionally, SalB alleviated ER stress-mediated pyroptotic cell death via the suppression of TXNIP/NLRP3 inflammasome, as evidenced by reduced cleavage of caspase-1 and interleukin- (IL-) 1β and IL-18 secretion levels. Furthermore, this study provided a mechanistic basis that AMPK/FoxO4/KLF2 and Syndecan-4/Rac1/ATF2 signaling pathway modulation by SalB substantially prevented BM-EPCs damage associated with ER stress by decreasing intracellular ROS amounts and inducing NLRP3-dependent pyroptosis. In summary, our findings identify that ER stress triggered mitochondrial ROS release and NLRP3 generation in BM-EPCs, while SalB inhibits NLRP3 inflammasome-mediated pyroptotic cell death by regulating the AMPK/FoxO4/KLF2 and Syndecan-4/Rac1/ATF2 pathways. The current findings reveal SalB as a potential new candidate for the treatment of atherosclerotic heart disease.

Salvianolic Acid B Suppresses ER Stress-Induced NLRP3 Inflammasome and Pyroptosis via AMPK/FoxO4 and Syndecan-4/Rac1 Signal Pathways in Human Endothelial Progenitor Cells

Increasing evidence demonstrates that uncontrolled endoplasmic reticulum (ER) stress responses can activate the inflammasome, which often leads to endothelial dysfunction that underlies the pathogenesis of chronic inflammatory diseases such as atherosclerosis. Salvianolic acid B (SalB), a bioactive compound isolated from Radix Salviae, has been reported to exert anti-oxidative and anti-inflammatory activities in various types of cells. However, the effect of SalB on ER stress-related inflammasome and endothelial dysfunction has not been studied. In the present study, we demonstrated that SalB substantially abrogated ER stress-induced cell death and reduction in capillary tube formation that was linked to declined intracellular reactive oxygen species (ROS) levels, restored mitochondrial membrane potential (MMP), and increased expression of HO-1 and SOD2 in bone marrow-derived endothelial progenitor cells (BM-EPCs). Inhibition of ER stress by CHOP or caspase-4 siRNA transfection attenuated the protective effect of SalB. Furthermore, SalB alleviates ER stress-mediated pyroptotic cell death via suppression of TXNIP/NLRP3 inflammasome activation, as evidenced by decrease in cleavage of caspase-1 and downstream mature interleukin (IL)-1β and IL-18 secretion. Furthermore, our results provide mechanistic evidence that modulation of AMPK/FoxO4/KLF2 and Syndecan-4/Rac1/ATF2 signaling pathways by SalB significantly protected BM-EPCs against cell injury caused by ER stress via reduction of intracellular ROS levels and NLRP3-dependent pyroptosis. In summary, our findings identify that ER stress-induced mitochondrial ROS release and NLRP3 assembly in BM-EPCs, while SalB inhibit NLRP3 inflammasome-mediated pyroptotic cell death via regulating novel AMPK/FoxO4/KLF2 and Syndecan-4/Rac1/ATF2 signaling pathways. These findings suggest SalB to be a potential new agent with applications in the fields of atherosclerotic heart disease. Funding Information: The project was supported by grants from the National Natural Science Foundation of China (No. 81503281), Natural Science Foundation of Guangdong Province (No. 2021A1515011257), Science and Technology Planning Project of Guangzhou (No. 201904010109), Joint Funds of Natural Science Foundation of Guangdong Province (No. 2020B1515120094), and the Talent Grant of Sun Yat-sen University (No. 18ykpy11). Declaration of Interests: The authors declare that there is no conflict of interest to reveal. Ethics Approval Statement: Informed consent for bone marrow collection was obtained from healthy volunteers. All procedures were performed in accordance with the guidance and approval of the local institutional review board (approval no. 2016[130]).

Nao-Fu-Cong ameliorates diabetic cognitive dysfunction by inhibition of JNK/CHOP/Bcl2-mediated apoptosis in vivo and in vitro

Chinese herbal compound Nao-Fu-Cong (NFC) has been mainly used to treat cognitive disorders in Traditional Chinese Medicine (TCM). The present study aimed to investigate whether its neuroprotective effects might be related to the inhibition of JNK/CHOP/Bcl2-mediated apoptosis pathway or not. We randomly assigned STZ (60 mg·kg-1)-induced diabetic rats into control group, diabetic model group and NFC groups (low-dose, medium-dose and high-dose). The primary culture of hippocampal neurons were transferred into different culture media on the third day. The cells were then divided into control group, high-glucose group, NFC (low-dose, medium-dose and high-dose) groups, CHOP si-RNA intervention group, JNK pathway inhibitor SP600125 group and oxidative stress inhibitor N-acetylcysteine (NAC) group. NFC significantly improved the cognitive function of diabetic rats, and had neuroprotective effect on hippocampal neurons cultured in high glucose. Further research results showed that NFC could reduce the apoptosis of hippocampal neurons in rats with diabetic cognitive dysfunction. NFC had inhibitory effects on CHOP/JNK apoptosis pathway induced by high glucose, and also decreased the levels of ROS and increased the mitochondrial membrane potential. These suggested that the neuroprotective effect of NFC might be related to the inhibition of CHOP and JNK apoptotic signaling pathways, and the cross pathway between oxidative stress and mitochondrial damage pathway.

Pristimerin Exacerbates Cellular Injury in Conditionally Reprogrammed Patient-Derived Lung Adenocarcinoma Cells by Aggravating Mitochondrial Impairment and Endoplasmic Reticulum Stress through EphB4/CDC42/N-WASP Signaling.

Lung cancer is the most common and lethal malignant disease for which the development of efficacious chemotherapeutic agents remains an urgent need. Pristimerin (PRIS), a natural bioactive component isolated from various plant species in the Celastraceae and Hippocrateaceae families, has been reported to exhibit outstanding antitumor effects in several types of cells. However, the underlying mechanisms involved remain poorly understood. Here, we reported the novel finding that PRIS significantly suppressed lung cancer growth in conditionally reprogrammed patient-derived lung adenocarcinoma cells (CRLCs). We demonstrated that PRIS inhibited the cell viabilities, migrative and invaded abilities, and capillary structure formation of CRLCs. Furthermore, our results clarified that PRIS induced mitochondrial dysfunction through reactive oxygen species (ROS) generation, activation of caspase-9, caspase-3, and caspase-4, and expression of endoplasmic reticulum (ER) stress-associated proteins. Inhibition of ER stress by 4-PBA (4-phenylbutyric acid, a specific ER stress inhibitor) or CHOP siRNA transfection ameliorated PRIS-induced loss of mitochondrial membrane potential and intrinsic apoptosis. The present study also provides mechanistic evidence that PRIS suppressed the EphB4/CDC42/N-WASP signaling pathway, which is required for mitochondrial-mediated intrinsic apoptosis, activation of ER stress, and stimulation of caspase-4 induced by PRIS, and consequently resulting in suppressed cell viability, migration, and angiogenesis in CRLCs. Taken together, by providing a mechanistic insight into the modulation of ER stress-induced cell death in CRLCs by PRIS, we suggest that PRIS has a strong potential of being a new antitumor therapeutic agent with applications in the fields of human lung adenocarcinoma.

Small Molecule Inhibitor Adjuvant Surfactant Therapy Attenuates Ventilator- and Hyperoxia-Induced Lung Injury in Preterm Rabbits.

BackgroundInvasive mechanical ventilation (IMV) has become one of the mainstays of therapy in NICUs worldwide, as a result of which premature babies with extremely low birth weight have been able to survive. Although lifesaving, IMV can result in lung inflammation and injury. Surfactant therapy is considered a standard of care in preterm infants with immature lungs. Recently, small molecule inhibitors like siRNAs and miRNAs have been used for therapeutic purposes. Ddit3 (CHOP), Ang2 and miR34a are known to be upregulated in experimental lung injury. We wanted to test whether inhibitors for these molecules (CHOP siRNA, Ang2 siRNA, and miR34a antagomir) if used alone or with a combination with surfactant (Curosurf®) would help in reducing ventilation and hyperoxia-induced injury in an experimental lung injury model.MethodsPreterm rabbits born by cesarean section were intratracheally instilled with the three small molecule inhibitors with or without Curosurf® prior to IMV and hyperoxia exposure. Prior to testing the inhibitors in rabbits, these small molecule inhibitors were transfected in mouse lung epithelial cells (MLE12 and AECII) and delivered to neonatal mouse pups intranasally as a proof of concept that surfactant (Curosurf®) could be used as an effective vehicle for administration of such drugs. Survival, pulmonary function tests, histopathology, immunostaining, quantitative PCR and western blotting were done to see the adjuvant effect of surfactant with these three small molecule inhibitors.ResultsOur data shows that Curosurf® can facilitate transfection of small molecules in MLE12 cells with the same and/or increased efficiency as Lipofectamine. Surfactant given alone or as an adjuvant with small molecule inhibitors increases survival, decreases IMV and hyperoxia-induced inflammation, improves pulmonary function and lung injury scores in preterm rabbit kits.ConclusionOur study shows that Curosurf® can be used successfully as an adjuvant therapy with small molecule inhibitors for CHOP/Ang2/miR34a. In this study, of the three inhibitors used, miR34a inhibitor seemed to be the most promising compound to combat IMV and hyperoxia-induced lung injury in preterm rabbits.

Lactobacillus paracasei-derived extracellular vesicles attenuate the intestinal inflammatory response by augmenting the endoplasmic reticulum stress pathway

Lactobacillus paracasei is a major probiotic and is well known for its anti-inflammatory properties. Thus, we investigated the effects of L. paracasei-derived extracellular vesicles (LpEVs) on LPS-induced inflammation in HT29 human colorectal cancer cells and dextran sulfate sodium (DSS)-induced colitis in C57BL/6 mice. ER stress inhibitors (salubrinal or 4-PBA) or CHOP siRNA were utilized to investigate the relationship between LpEV-induced endoplasmic reticulum (ER) stress and the inhibitory effect of LpEVs against LPS-induced inflammation. DSS (2%) was administered to male C57BL/6 mice to induce inflammatory bowel disease, and disease activity was measured by determining colon length, disease activity index, and survival ratio. In in vitro experiments, LpEVs reduced the expression of the LPS-induced pro-inflammatory cytokines IL-1α, IL-1β, IL-2, and TNFα and increased the expression of the anti-inflammatory cytokines IL-10 and TGFβ. LpEVs reduced LPS-induced inflammation in HT29 cells and decreased the activation of inflammation-associated proteins, such as COX-2, iNOS and NFκB, as well as nitric oxide. In in vivo mouse experiments, the oral administration of LpEVs also protected against DSS-induced colitis by reducing weight loss, maintaining colon length, and decreasing the disease activity index (DAI). In addition, LpEVs induced the expression of endoplasmic reticulum (ER) stress-associated proteins, while the inhibition of these proteins blocked the anti-inflammatory effects of LpEVs in LPS-treated HT29 cells, restoring the pro-inflammatory effects of LPS. This study found that LpEVs attenuate LPS-induced inflammation in the intestine through ER stress activation. Our results suggest that LpEVs have a significant effect in maintaining colorectal homeostasis in inflammation-mediated pathogenesis.

Honokiol induces endoplasmic reticulum stress-mediated apoptosis in human lung cancer cells

AimsHonokiol is a hydroxylated biphenyl natural product and displays potent antitumor activity against several cancers including prostate cancer, melanoma, leukemia, and colorectal cancer. The present study was to investigate the in vitro activity of honokiol against A549 and 95-D human lung cancer cells. Main methodsA549 and 95-D cells were used with honokiol treatment. Cell viability was determined by CCK-8 assay. The cell migration and apoptosis were evaluated by wound healing assay and TUNEL staining method respectively. The expressions of ER-related proteins were analyzed by western blot and the CHOP siRNA was used to downregulate the CHOP expression. Key findingsThe results demonstrated that treatment of A549 and 95-D cells with honokiol significantly reduced cell viability in a dose- and time-dependent manner. Furthermore, honokiol treatment decreased cell migration and enhanced cell apoptosis, which is accompanied by the upregulation of the expressions of ER stress-induced apoptotic signaling molecules such as GRP78, phosphorylated PERK, phosphorylated eIF2α, CHOP, Bcl-2, Bax, and cleaved Caspase 9. Honokiol treatment-induced increase of ER stress-related signaling molecules and apoptotic proteins in A549 and 95-D cells were reversed by CHOP siRNA. SignificanceCollectively, we conclude that ER stress may participate in the action of the anticancer activity of honokiol in A549 and 95-D cells and induction of ER stress-related apoptosis may represent a novel therapeutic intervention for human lung cancer.

PERK/eIF-2α/CHOP Pathway Dependent ROS Generation Mediates Butein-induced Non-small-cell Lung Cancer Apoptosis and G2/M Phase Arrest.

Butein, a member of the chalcone family, is a potent anticarcinogen against multiple cancers, but its specific anti-NSCLC mechanism remains unknown. The present study examined the effects of butein treatment on NSCLC cell lines and NSCLC xenografts. Butein markedly decreased NSCLC cell viability; inhibited cell adhesion, migration, invasion, and colony forming ability; and induced cell apoptosis and G2/M phase arrest in NSCLC cells. Moreover, butein significantly inhibited PC-9 xenograft growth. Both in vivo and in vitro studies verified that butein exerted anti-NSCLC effect through activating endoplasmic reticulum (ER) stress-dependent reactive oxygen species (ROS) generation. These pro-apoptotic effects were reversed by the use of 4- phenylbutyric acid (4-PBA), CHOP siRNA, N-acetyl-L-cysteine (NAC) and Z-VAD-FMK (z-VAD) in vitro. Moreover, inhibition of ER stress markedly reduced ROS generation. In addition, in vivo studies further confirmed that inhibition of ER stress or oxidative stress partially abolished the butein-induced inhibition of tumor growth. Therefore, butein is a potential therapeutic agent for NSCLC, and its anticarcinogenic action might be mediated by ER stress-dependent ROS generation and the apoptosis pathway.

High Glucose-Induced Podocyte Injury Involves Activation of Mammalian Target of Rapamycin (mTOR)-Induced Endoplasmic Reticulum (ER) Stress

Background/Aims: The mechanisms by which high glucose (HG) results in podocyte damage remains unclear. We investigated the potential role of endoplasmic reticulum (ER) stress and mTOR signaling in HG injured podocyte. Methods: In cultured mouse podocytes, cellular apoptosis was assessed using FITC-Annexin V and propidium iodide staining followed by flow cytometry analysis. Apoptosis-related proteins as well as the ER stress and the mTOR signals were evaluated using immunoblot assay. Results: Compared to normal glucose (NG) and osmotic mannitol (MN) control, the percentage of apoptotic cells was increased significantly in HG-treated podocytes. The levels of CHOP, Grp78, phospho-PERKThr982, and caspase-12 were increased significantly following HG treatment. The downstream effects of ER stress were obtained in HG-treated podocytes, showing upregulation of Bax, Bak and cytochrome c, and downregulation of Bcl-2. HG-induced increase of cytochrome c, Bax and active caspase-3 was prevented by both ER inhibitor sodium 4-phenylbultyrate (PBA) and CHOP siRNA (siCHOP). PBA and CHOP knockdown remarkably decreased HG-induced apoptosis. In addition, the levels of phospho-mTORSer2448 and phospho- p70S6kThr389 as well as phospho-AMPKα (a sensor of energy consumption) were increased significantly in HG-treated cells. Moreover, the Erk inhibitor U0126 prevented HG-induced mTOR activation. Increased phospho-AMPKα, CHOP and Grp78 as well as cellular apoptosis were prevented by mTOR inhibitor rapamycin in HG-treated podocytes. Conclusion: Our data demonstrate that the activated mTOR by Erk1/2 results in energy consumption, which in turn leads to ER stress signaling and thus induces apoptosis in HG-treated podocytes.

2,4,6-Trinitrotoluene Induces Apoptosis via ROS-Regulated Mitochondrial Dysfunction and Endoplasmic Reticulum Stress in HepG2 and Hep3B Cells

2,4,6-trinitrotoluene (TNT) has been reported to cause numerous adverse effects. However, the detailed molecular mechanisms underlying TNT-induced liver toxicity need to be elucidated. In this study, we used HepG2 (p53wt) and Hep3B (p53null) cell lines to investigate the cytotoxic effects of TNT. At first, we found that TNT significantly decreased cell viability and induced DNA damage. Thereafter, through transcriptomic analysis, we observed that the diverse biological functions affected included mitochondrial dysfunction and endoplasmic reticulum (ER) stress. Mitochondrial dysfunction was evidenced by the loss of mitochondrial membrane potential, increased expression of cleaved-caspase-9&-3 and increased caspase-3/7 activity, indicating that apoptosis had occurred. In addition, the expressions of some ER stress-related proteins had increased. Next, we investigated the role of reactive oxygen species (ROS) in TNT-induced cellular toxicity. The levels of DNA damage, mitochondrial dysfunction, ER stress and apoptosis were alleviated when the cells were pretreated with N-acetyl-cysteine (NAC). These results indicated that TNT caused the ROS dependent apoptosis via ER stress and mitochondrial dysfunction. Finally, the cells transfected with CHOP siRNA significantly reversed the TNT-induced apoptosis, which indicated that ER stress led to apoptosis. Overall, we examined TNT-induced apoptosis via ROS dependent mitochondrial dysfunction and ER stress in HepG2 and Hep3B cells.

Pterostilbene exerts anticancer activity on non-small-cell lung cancer via activating endoplasmic reticulum stress

Pterostilbene (PT), the natural dimethylated analog of resveratrol (RSV), is a potent anticarcinogen for non-small-cell lung cancer (NSCLC), but its anti-NSCLC mechanisms remain unclear. In this study, we show that PT treatment time- and dose-dependently enhanced the endoplasmic reticulum stress (ERS) signaling (i.e., p-PERK, IRE1, ATF4, CHOP), thus decreasing the cell viability and inducing apoptosis in human PC9 and A549 NSCLC cell lines. Moreover, the decreased migratory and adhesive abilities, downregulation of intracellular glutathione (GSH) level, enhanced reactive oxygen species (ROS) generation, Caspase 3 activity and mitochondrial membrane depolarization were observed in NSCLC cells treated with PT. These effects were reversed by CHOP siRNA which inhibited the ERS signaling pathway, but were promoted by thapsigargin (a classical ERS inducer) in vitro. Besides, in vivo studies also verify that PT exerted anticancer activity by mobilizing ERS signaling and apoptosis-related proteins, and these effects were enhanced by thapsigargin. Therefore, ERS activation may represent a new mechanism of anti-NSCLC action by PT, and a novel therapeutic intervention for lung cancer.

Taraxacum mongolicum extract induced endoplasmic reticulum stress associated-apoptosis in triple-negative breast cancer cells

BackgroundTriple-negative breast cancer (TNBC) is an aggressive and deadly breast cancer subtype with limited treatment options. It is necessary to seek complementary strategies for TNBC management. Taraxacum mongolicum, commonly named as dandelion, is a herb medicine with anti-cancer activity and has been utilized to treat mammary abscess, hyperplasia of mammary glands from ancient time in China, but the scientific evidence and action mechanisms still need to be studied. Aim of the studyThis study was intended to investigate the therapeutic effect and molecular mechanisms of dandelion extract in TNBC cell line. MethodologyDandelion extract was prepared and purified, and then its chemical composition was determined. Cell viability was evaluated by MTT assay. Analysis of cell apoptosis and cell cycle was assessed by flow cytometry. The expression levels of mRNA and proteins were determined by real-time PCR and Western blotting, respectively. Caspase inhibitor Z-VAD-FMK and CHOP siRNA were used to confirm the cell apoptosis induced by dandelion extract. ResultsDandelion extract significantly decreased MDA-MB-231cell viability, triggered G2/M phase arrest and cell apoptosis. Concurrently, it caused a markedly increase of cleaved caspase-3 and PARP proteins. Caspase inhibitor Z-VAD-FMK abolished the apoptosis triggered by dandelion extract. The three ER stress-related signals were strongly induced after dandelion treatment, including increased mRNA expressions of ATF4, ATF6, XBP1s, GRP78 and CHOP genes, elevated protein levels of phosphorylated PERK, eIF-2α, IRE1, as well as the downstream molecules of CHOP and GRP78. MDA-MB-231 cells transfected with CHOP siRNA significantly reduced apoptosis induced by dandelion extract. The underlying mechanisms at least partially ascribe to the strong activation of PERK/p-eIF2α/ATF4/CHOP axis. ConclusionER stress related cell apoptosis accounted for the anti-cancer effect of dandelion extract, and these findings support dandelion extract might be a potential therapeutic approach to treat TNBC.

The unfolded protein response in relation to mitochondrial biogenesis in skeletal muscle cells

Mitochondria comprise both nuclear and mitochondrially encoded proteins requiring precise stoichiometry for their integration into functional complexes. The augmented protein synthesis associated with mitochondrial biogenesis results in the accumulation of unfolded proteins, thus triggering cellular stress. As such, the unfolded protein responses emanating from the endoplasmic reticulum (UPRER) or the mitochondrion (UPRMT) are triggered to ensure correct protein handling. Whether this response is necessary for mitochondrial adaptations is unknown. Two models of mitochondrial biogenesis were used: muscle differentiation and chronic contractile activity (CCA) in murine muscle cells. After 4 days of differentiation, our findings depict selective activation of the UPRMT in which chaperones decreased; however, Sirt3 and UPRER markers were elevated. To delineate the role of ER stress in mitochondrial adaptations, the ER stress inhibitor TUDCA was administered. Surprisingly, mitochondrial markers COX-I, COX-IV, and PGC-1α protein levels were augmented up to 1.5-fold above that of vehicle-treated cells. Similar results were obtained in myotubes undergoing CCA, in which biogenesis was enhanced by ~2-3-fold, along with elevated UPRMT markers Sirt3 and CPN10. To verify whether the findings were attributable to the terminal UPRER branch directed by the transcription factor CHOP, cells were transfected with CHOP siRNA. Basally, COX-I levels increased (~20%) and COX-IV decreased (~30%), suggesting that CHOP influences mitochondrial composition. This effect was fully restored by CCA. Therefore, our results suggest that mitochondrial biogenesis is independent of the terminal UPRER Under basal conditions, CHOP is required for the maintenance of mitochondrial composition, but not for differentiation- or CCA-induced mitochondrial biogenesis.

Thapsigargin sensitizes human esophageal cancer to TRAIL-induced apoptosis via AMPK activation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent for esophageal squamous cell carcinoma (ESCC). Forced expression of CHOP, one of the key downstream transcription factors during endoplasmic reticulum (ER) stress, upregulates the death receptor 5 (DR5) levels and promotes oxidative stress and cell death. In this study, we show that ER stress mediated by thapsigargin promoted CHOP and DR5 synthesis thus sensitizing TRAIL treatment, which induced ESCC cells apoptosis. These effects were reversed by DR5 siRNA in vitro and CHOP siRNA both in vitro and in vivo. Besides, chemically inhibition of AMPK by Compound C and AMPK siRNA weakened the anti-cancer effect of thapsigargin and TRAIL co-treatment. Therefore, our findings suggest ER stress effectively sensitizes human ESCC to TRAIL-mediated apoptosis via the TRAIL-DR5-AMPK signaling pathway, and that activation of ER stress may be beneficial for improving the efficacy of TRAIL-based anti-cancer therapy.

Pterostilbene Inhibits the Growth of Human Esophageal Cancer Cells by Regulating Endoplasmic Reticulum Stress

Background/Aims: Pterostilbene (PTE), a natural dimethylated resveratrol analog from blueberries, is known to have diverse pharmacological activities, including anticancer properties. In this study, we investigated the anticancer activity of PTE against human esophageal cancer cells both in vitro and in vivo and explored the role of endoplasmic reticulum (ER) stress (ERS) signaling in this process. Methods: Cell viability, the apoptotic index, Caspase 3 activity, adhesion, migration, reactive oxygen species (ROS) levels, and glutathione (GSH) levels were detected to explore the effect of PTE on human EC109 esophageal cancer cells. Furthermore, siRNA transfection and a chemical inhibitor were employed to confirm the role of ERS. Results: PTE treatment dose- and time-dependently decreased the viability of human esophageal cancer EC109 cells. PTE also decreased tumor cell adhesion, migration and intracellular GSH levels while increasing the apoptotic index, Caspase 3 activity and ROS levels, which suggest the strong anticancer activity of PTE. Furthermore, PTE treatment increased the expression of ERS-related molecules (GRP78, ATF6, p-PERK, p-eIF2α and CHOP), upregulated the pro-apoptosis-related protein PUMA and downregulated the anti-apoptosis-related protein Bcl-2 while promoting the translocation of cytochrome c from mitochondria to cytosol and the activation of Caspase 9 and Caspase 12. The downregulation of ERS signaling by CHOP siRNA desensitized esophageal cancer cells to PTE treatment, whereas upregulation of ERS signaling by thapsigargin (THA) had the opposite effect. N-Acetylcysteine (NAC), a ROS scavenger, also desensitized esophageal cancer cells to PTE treatment. Conclusions: Overall, the results indicate that PTE is a potent anti-cancer pharmaceutical against human esophageal cancer, and the possible mechanism involves the activation of ERS signaling pathways.

Curcumin analog WZ35 induced cell death via ROS-dependent ER stress and G2/M cell cycle arrest in human prostate cancer cells.

BackgroundProstate cancer is the most commonly diagnosed malignancy among men. The Discovery of new agents for the treatment of prostate cancer is urgently needed. Compound WZ35, a novel analog of the natural product curcumin, exhibited good anti-prostate cancer activity, with an IC50 of 2.2 μM in PC-3 cells. However, the underlying mechanism of WZ35 against prostate cancer cells is still unclear.MethodsHuman prostate cancer PC-3 cells and DU145 cells were treated with WZ35 for further proliferation, apoptosis, cell cycle, and mechanism analyses. NAC and CHOP siRNA were used to validate the role of ROS and ER stress, respectively, in the anti-cancer actions of WZ35.ResultsOur results show that WZ35 exhibited much higher cell growth inhibition than curcumin by inducing ER stress-dependent cell apoptosis in human prostate cells. The reduction of CHOP expression by siRNA partially abrogated WZ35-induced cell apoptosis. WZ35 also dose-dependently induced cell cycle arrest in the G2/M phase. Furthermore, we found that WZ35 treatment for 30 min significantly induced reactive oxygen species (ROS) production in PC-3 cells. Co-treatment with the ROS scavenger NAC completely abrogated the induction of WZ35 on cell apoptosis, ER stress activation, and cell cycle arrest, indicating an upstream role of ROS generation in mediating the anti-cancer effect of WZ35.ConclusionsTaken together, this work presents the novel anticancer candidate WZ35 for the treatment of prostate cancer, and importantly, reveals that increased ROS generation might be an effective strategy in human prostate cancer treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1851-3) contains supplementary material, which is available to authorized users.

Remote ischemic postconditioning alleviates cerebral ischemic injury by attenuating endoplasmic reticulum stress-mediated apoptosis.

Remote ischemic postconditioning (RIPostC) has been proved to protect the brain from stroke, but the precise mechanism remains not fully understood. In the present study, we aimed to investigate whether RIPostC attenuates cerebral ischemia-reperfusion injury by abating endoplasmic reticulum (ER) stress response. CHOP, a multifunctional transcription factor in ER stress, regulates the expression of genes related to apoptosis, such as Bim and Bcl-2. Male SD rats were subjected to right middle cerebral artery occlusion (MCAO) for 2h followed by reperfusion, and RIPostC was induced by three cycles of 10min ischemia and 10min reperfusion on bilateral femoral arteries immediately after ischemia. CHOP siRNA (CHOPi) and control siRNA (Coni) were injected into the right lateral ventricle 30min before the beginning of ischemia. RIPostC, CHOPi, or RIPostC + CHOPi application reduced infarct volume, improved the neurological function, and decreased cell apoptosis. RIPostC increased the protein level of glucose-regulated protein 78 (GRP78) and decreased the protein level of phosphorylated-EIF2α, caspase-12, and CHOP. Furthermore, the expression of CHOP, Bim and cleaved-caspase-3 was decreased, while Bcl-2 expression was increased in response to application of RIPostC, CHOPi, or RIPostC + CHOPi. In sum, RIPostC protects against ischemia-reperfusion brain injury in rats by attenuating ER stress response-induced apoptosis.

Ampelopsin Induces Cell Growth Inhibition and Apoptosis in Breast Cancer Cells through ROS Generation and Endoplasmic Reticulum Stress Pathway

Ampelopsin (AMP), a major bioactive constituent of Ampelopsis grossedentata, exerts a number of biological effects. In this study, we investigated its anti-cancer activity in human breast cancer cell lines, and explored the underlying mechanism of this action. Our results showed that treatment with AMP dose-dependently inhibited cell viability and induced apoptosis in MCF-7 and MDA-MB-231 breast cancer cells without cytotoxicity in human normal breast epithelial cells MCF-10A. Meanwhile, AMP dose- dependently triggered reactive oxygen species (ROS) generation in both breast cancer cells. The ROS scavenger N-acetyl-L-cysteine (NAC) strongly attenuated AMP-induced ROS production, along with cell growth inhibition and apoptosis. Furthermore, AMP was observed to activate endoplasmic reticulum (ER) stress, as evidenced by the up-regulation of ER stress-related proteins, including GRP78, p-PERK, p-elF2α, cleaved ATF6α and CHOP, while knockdown of ATF6α or PERK markedly down-regulated AMP-induced CHOP expression. Blocking ER stress using 4-phenylbutyric acid not only down-regulated AMP-induced GRP78 and CHOP expression, but also significantly decreased AMP-induced cell growth inhibition and apoptosis, whereas ER stress inducer thapsigargin played opposing effects. Additionally, NAC inhibited AMP-induced ER stress by down-regulating GRP78 and CHOP expression. Conversely, blocking ER stress using CHOP siRNA decreased AMP-induced ROS production and cell apoptosis. Taken together, these results demonstrate that AMP has anti-tumor effects against breast cancer cells through ROS generation and ER stress pathway, which therefore provide experimental evidences for developing AMP as a new therapeutic drug for breast cancer.