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Background The development of potent non-toxic chemotherapeutic drugs against castration resistant prostate cancer (CRPC) remains a major challenge. Corosolic acid (CA), a natural triterpenoid, has anti-cancer activity with limited side effects. However, CA anti-prostate cancer activities and mechanisms, particularly in CRPC, are not clearly understood. In this study, we investigated CA anti-tumor ability against human CRPC and its mechanism of action. Methods The cell apoptosis and proliferation effects were evaluated via MTT detection, colony formation assay and flow cytometry. Western blot, gene transfection and immunofluorescence assay were applied to investigate related protein expression of Endoplasmic reticulum stress. A xenograft tumor model was established to investigate the inhibitory effect of CA on castration resistant prostate cancer in vivo. Results The results showed that CA inhibited cell growth and induced apoptosis in human prostate cancer cell (PCa) line PC-3 and DU145, as well as retarded tumor growth in a xenograft model, exerting a limited toxicity to normal cells and tissues. Importantly, CA activated endoplasmic reticulum (ER) stress-associated two pro-apoptotic signaling pathways, as evidenced by increased protein levels of typical ER stress markers including IRE-1/ASK1/JNK and PERK/eIF2α/ATF4/CHOP. IRE-1, PERK or CHOP knockdown partially attenuated CA cytotoxicity against PCa cells. Meanwhile, CHOP induced expression increased Tribbles 3 (TRIB3) level, which lead to AKT inactivation and PCa cell death. CHOP silencing resulted in PCa cells sensitive to CA-induced apoptosis. Conclusion Our data demonstrated, for the first time, that CA might represent a novel drug candidate for the development of an anti-CRPC therapy.

Neuronal apoptosis is an important factor accounting for the poor outcomes of intracerebral hemorrhage (ICH). This study first showed that inhibition of activating transcription factor 6 (ATF6) could alleviate secondary brain injury through anti-apoptosis after ICH in rats. Melatonin, ATF6 and CCAAT/enhancer-binding protein homologous protein (CHOP) siRNAs were applied in this study. Brain edema, neurological functions, blood-brain barrier (BBB) integrity were evaluated at 24 h after ICH. Western blot analysis was used to evaluate the protein level of target proteins (ATF6, CHOP, Bip, Bcl-2, Bax, and cleaved caspase-3). Reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the mRNA level of ATF6, CHOP and cleaved caspase-3. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase-3 immunofluorescence staining were applied to evaluate the neuronal cell death. The results suggested that the levels of ATF6 and its downstream protein, CHOP, were upregulated and reached the peak at 24 h after ICH. ATF6 was highly expressed in neurons. The administration of melatonin significantly decreased the mRNA and protein levels of ATF6, and its downstream targets, CHOP and cleaved caspase-3, but increased the Bcl-2/Bax ratio, which ameliorated the neurological functions. The CHOP siRNA significantly reversed the pro-apoptotic effect induced by the increased ATF6 level after ICH. Melatonin could protect against neuronal apoptosis via suppression of ATF6/CHOP arm of ER-stress-response pathway.

Diabetes mellitus (DM) is associated with the increased risk of the central nervous system complications as cerebrovascular disease, impaired cognition, dementia and neurodegeneration. Curcumin is a polyphenol with anti-oxidant, anti-inflammatory, anti-hyperlipidemic, and anti-cancer effects. Therefore, the present study was aimed to focus on the mechanistic insights of diabetes-induced hippocampal neurodegeneration in addition to shedding the light on the modulatory effect of curcumin. Twenty-eight male Wistar rats were randomly divided into four groups. Type I DM was induced by a single intra-peritoneal injection of streptozotocin (STZ) (65 mg/kg b.w.). Curcumin (100 mg/kg b.w.) was given to the diabetic group after the induction and for eight weeks. Hippocampal glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF-4), Bcl2 and choline acetyl transferase (ChAT) genes expression were assessed. Heat shock protein 70 (HSP70), Bcl-2-Associated X protein (Bax), Interferon-γ (INF-γ) and CCAAT-enhancer-binding protein homologous protein (CHOP) levels in the hippocampus were immunoassayed, in addition to the assessment of glycemic and redox status. Curcumin significantly improved blood glucose level, redox status, cellular stress, and decreased INF-γ and Bax levels, down-regulated GRP78 and ATF-4 expression, meanwhile, up-regulated Bcl2 and ChAT expression in hippocampus. Histological findings proved the biochemical and molecular findings. Our results support curcumin as a potential neuro-protective agent against diabetes induced hippocampal neurodegeneration.

New and more effective therapies for canine cancer patients are urgently required and this necessitates advanced experimental research. Dogs are good models for studies in comparative oncology; however, canine cancer cell biology research is currently limited by low availability of validated antibody reagents and techniques. This study characterises the expression of key components of the unfolded protein response (UPR) in a panel of haematopoietic canine cancer cell lines using commercially available antibodies, and validates the methods used to study this pathway.The CLBL-1 canine lymphoma cell line and the GL-1 canine leukaemia cell line sourced externally and two counterparts established in house (CNK-89 and CLB70) were used as models of different lymphoma and leukaemia canine cell lines for the study. The human U2OS cell line served as the control. Antibodies were selected for identifying UPR proteins according to known canine cell reactivity and canine–murine and canine–human homology. Endoplasmic reticulum stress was induced with thapsigargin and MG132 in the cell lines. Etoposide was used to induce DNA damage in the cells. The techniques used for this validation analysis were RNA sequencing to observe the expression of UPR components in canine cell lines, Western blot to observe changes of protein expression levels after inducing ER stress in the cells, and flow cytometry in order to study cell death.Substantial variations in both the basic expression and agonist-induced activation of the UPR pathway were observed in canine cancer cell lines, although the biological significance of these differences requires further investigation.These findings will be a starting point for future studies on cancer biology in dogs. They will also contribute to developing novel anticancer therapies for canine patients and may provide new insights into human oncology.

Objective Free fatty acids (FFA) can increase the expression of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) in local tissues and organs. However, the mechanism underlying the effect of FFA on 11β-HSD1 expression remains unclear. Methods A total of 24 male Syrian golden hamsters (SPF grade) were selected and randomly divided into a control group (Con, n  = 8) fed a normal diet, and a high-fat diet group ( n  = 16) fed for 12 weeks. After successfully establishing the hyperlipidemia hamster model, the high-fat group was further divided into a high-fat group (HF) and a fenofibrate intervention group (Feno). Following an oral fat tolerance test (OFTT), blood lipids and FFA levels were measured. The expression levels of endoplasmic reticulum stress (ERS) marker GRP78, downstream key molecule CHOP, C/EBPα, and 11β-HSD1 were analyzed using Western blot and RT-PCR. Results After OFTT, FFA levels in all three groups initially decreased and then increased, with the highest levels observed in the HF group (Ps < 0.05). FFA levels in the Feno group were comparable to those in the Con group ( P  > 0.05). Hepatic FFA, 11β-HSD1, and corticosterone levels were highest in the HF group (Ps < 0.05), while the Feno group showed no significant difference compared to the Con group (Ps > 0.05). Hepatic 11β-HSD1 and corticosterone levels were positively correlated with FFA levels (Ps < 0.05). Western blot and RT-PCR results indicated higher GRP78, CHOP, C/EBPα, and 11β-HSD1 protein and mRNA expression in the HF group compared to the Con group (Ps < 0.05). Fenofibrate intervention reduced FFA levels and downregulated these indicators in the Feno group compared to the HF group (Ps < 0.05). Conclusion FFA may regulate the expression of hepatic 11β-HSD1 in high-fat-fed golden hamsters via the ERS-CHOP-C/EBPα signaling pathway, thereby affecting local corticosterone levels. Fenofibrate may downregulate the levels of 11β-HSD1 and corticosterone in local tissues by reducing FFA levels.

Apoptosis is a form of cell death by which the body maintains the homeostasis of the internal environment. Apoptosis is an initiative cell death process that is controlled by genes and is mainly divided into endogenous pathways (mitochondrial pathway), exogenous pathways (death receptor pathway), and apoptotic pathways induced by endoplasmic reticulum (ER) stress. The homeostasis imbalance in ER results in ER stress. Under specific conditions, ER stress can be beneficial to the body; however, if ER protein homeostasis is not restored, the prolonged activation of the unfolded protein response may initiate apoptotic cell death via the up-regulation of the C/EBP homologous protein (CHOP). CHOP plays an important role in ER stress-induced apoptosis and this review focuses on its multifunctional roles in that process, as well as its role in apoptosis during microbial infection. We summarize the upstream and downstream pathways of CHOP in ER stress induced apoptosis. We also focus on the newest discoveries in the functions of CHOP-induced apoptosis during microbial infection, including DNA and RNA viruses and some species of bacteria. Understanding how CHOP functions during microbial infection will assist with the development of antimicrobial therapies.

C/EBP homologous protein (CHOP), known also as DNA damage-inducible transcript 3 and as growth arrest and DNA damage-inducible protein 153 (GADD153), is induced in response to certain stressors. CHOP is universally acknowledged as a main conduit to endoplasmic reticulum stress-induced apoptosis. Ongoing research established the existence of CHOP-mediated apoptosis signaling networks, for which novel downstream targets are still being determined. However, there are studies that contradict this notion and assert that apoptosis is not the only mechanism by which CHOP plays in the development of pathologies. In this review, insights into the roles of CHOP in pathophysiology are summarized at the molecular and cellular levels. We further focus on the newest advances that implicate CHOP in human diseases including cancer, diabetes, neurodegenerative disorders, and notably, fibrosis.

Although tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) has shown efficacy in a phase 2 clinical trial, development of resistance to TRAIL by tumor cells is a major roadblock. We investigated whether quercetin, a flavonoid, can sensitize human ovarian cancer cells to TRAIL. Results indicate that quercetin sensitized cancer cells to TRAIL. The quercetin induced expression of death receptor DR5 but did not affect expression of DR4 in cancer cells. The induction of DR5 was mediated through activation of JNK and through upregulation of a transcription factor CCAAT enhancer‐binding protein homologous protein (CHOP); as silencing of these signaling molecules abrogated the effect of quercetin. Upregulation of DR5 was mediated through the generation of reactive oxygen species (ROS), as ROS scavengers reduced the effect of quercetin on JNK activation, CHOP upregulation, DR induction, TRAIL sensitization, downregulated the expression of cell survival proteins and upregulated the proapoptotic proteins. Furthermore, quercetin enhances TRAIL mediated inhibition of tumor growth of human SKOV‐3 xenograft was associated with induction of apoptosis, activation of caspase‐3, CHOP and DR5. Overall, our data suggest that quercetin enhances apoptotic death of ovarian cancer cells to TRAIL through upregulation of CHOP‐induced DR5 expression following ROS mediated endoplasmic reticulum‐stress. Mitochondrial proteins (ROS) are mediating the apoptotic process, downstream or in parallel, CHOP might be a crucial player for DR5 upregulation and cancer cell killing by quercetin. IRE1a‐JNK signaling could also be considered to be the major ER‐stress pathway for quercetin induced CHOP expression, thus contributing to the enhanced apoptotic death of ovarian cancer cells to TRAIL. Quercetin could be an attractive candidate for combined chemotherapy against cancer.

Background The endoplasmic reticulum stress (ER stress) has been involved in various musculoskeletal disorders including non-traumatic osteonecrosis of femoral head (NT-ONFH). Objective The current study aimed to investigate the association of glucose-regulated protein 78 (GRP78) as well as CCAAT/enhancer-binding protein homologous protein (CHOP) expressions in serum and femoral head (FH) tissues with NT-ONFH’s severity. Methods We enrolled NT-ONFH patients ( n  = 150) alongside healthy controls (HCs, n  = 150). Meanwhile, 49 patients with femoral neck fracture (FNF) were also enrolled. Serum CHOP and GRP78 levels were determined through enzyme linked immunosorbent assay (ELISA). Local CHOP and GRP78 expressions were detected by immunohistochemistry, western blot, alongside real-time polymerase chain reaction (RT-PCR). Radiographic severity was assessed by FICAT grading system. The visual analogue scale (VAS) together with Harris hip score (HHS) were utilized to determine symptomatic severity. Results Serum CHOP and GRP78 levels were markedly increased in NT-ONFH patients than HCs. NT-ONFH patients at FICAT stage 4 showed significant higher serum CHOP and GRP78 levels in contrast with those at stage 3. Furthermore, patients at stage 3 demonstrated higher serum CHOP and GRP78 levels than those at stage 2. There was a positive correlation observed between the serum CHOP and GRP78 levels and the severity of the FICAT stages. A total of 42 ONFH patients at FICAT stage 3, 40 patients at FICAT stage 4, and 49 FNF patients received total hip replacement (THR). The mRNA and protein levels of CHOP and GRP78 were elevated in necrotic area compared to the non-necrotic area of ONFH patients and the FH tissues of FNF patients with statistical significance. The expression levels of CHOP and GRP78 within the local tissues were significantly elevated in patients at FICAT stage 4 as opposed to those at stage 3. Besides, ROC curve analysis indicated that serum and local CHOP and GRP78 expressions may act as indicators of disease progression. The levels of CHOP and GRP78, both in serum and at the local site, were in a positive correlation with VAS scores but an inverse relationship with HHS. Conclusions Serum and local GRP78 as well as CHOP expressions were positively linked with disease progression in NT-ONFH patients. Potential therapeutics targeting ER stress related protein may serve as a method for alleviating NT-ONFH.

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