Phenylbutyric acid inhibits hypoxia-induced trophoblast apoptosis and autophagy in preeclampsia via the PERK/ATF-4/CHOP pathway.

Preeclampsia (PE) is a common pregnancy complication with a high mortality rate. Abnormally activated endoplasmic reticulum stress (ERS) is believed to be responsible for the destruction of key placental cells-trophoblasts. Phenylbutyric acid (4-PBA), an ERS inhibitor, is involved in regulating the development of ERS-related diseases. At present, how 4-PBA affects trophoblasts and its mechanisms is still unclear. In this study, PE cell models were established by stimulating HTR-8/SVneo cells with hypoxia. To verify the underlying mechanisms of 4-PBA on PE, CCT020312, an activator of PERK, was also used. The results showed that 4-PBA restored hypoxia-induced trophoblast viability, inhibited HIF-1α protein expression, inflammation, and PERK/ATF-4/CHOP pathway. Hoechst 33342 staining and flow cytometry results confirmed that 4-PBA decreased hypoxia-induced apoptosis in trophoblasts. The results of the JC-1 analysis and apoptosis initiation enzyme activity assay also demonstrated that 4-PBA inhibited apoptosis related to the mitochondrial pathway. Furthermore, by detecting autophagy in trophoblasts, an increased number of autophagic vesicles, damaged mitochondria, enhanced dansylcadaverine fluorescence, enhanced levels of autophagy proteins Beclin-1, LC3II, and decreased p62 were seen in hypoxia-stimulated cells. These changes were reversed by 4-PBA. Furthermore, it was observed that CCT020312 reversed the effects of 4-PBA on the viability, apoptosis, and autophagosome number of hypoxia-induced trophoblasts. In summary, 4-PBA reduces autophagy and apoptosis via the PERK/ATF-4/CHOP pathway and mitochondrial pathway, thereby restoring the viability of hypoxic trophoblasts. These findings provide a solid evidence base for the use of 4-PBA in PE treatment and guide a new direction for improving the outcomes of patients with PE.

CHOP regulated by METTL14-m6A affects cell cycle arrest and regorafenib sensitivity in HCC cells.

BACKGROUND: Regorafenib, a multi-targeted kinase inhibitor, has been used in the treatment of Hepatocellular carcinoma (HCC). The purpose of this study is to investigate the mechanism of Regorafenib in HCC. METHODS: Regorafenib's impact on the sensitivity of HCC cells was assessed using CCK8. Differential gene expression analysis was performed by conducting mRNA sequencing after treatment with Regorafenib. The m6A methylation status of CHOP and differential expression of m6A methylation-related proteins were assessed by RIP and Western Blot. To explore the molecular mechanisms involved in the therapeutic effects of Regorafenib in HCC and the impact of METTL14 and CHOP on Regorafenib treatment, we employed shRNA/overexpression approaches to transfect METTL14 and CHOP genes, as well as conducted in vivo experiments. RESULTS: Treatment with Regorafenib led to a notable decrease in viability and proliferation of SK-Hep-1 and HCC-LM3 cells. The expression level of CHOP was upregulated after Regorafenib intervention, and CHOP underwent m6A methylation. Among the m6A methylation-related proteins, METTL14 exhibited the most significant downregulation. Mechanistic studies revealed that Regorafenib regulated the cell cycle arrest in HCC through METTL14-mediated modulation of CHOP, and the METTL14/CHOP axis affected the sensitivity of HCC to Regorafenib. In vivo, CHOP enhanced the anticancer effect of Regorafenib. CONCLUSION: The inhibition of HCC development by Regorafenib is attributed to its modulation of m6A expression of CHOP, mediated by METTL14, and the METTL14/CHOP axis enhances the sensitivity of HCC to Regorafenib. These findings provide insights into the treatment of HCC and the issue of drug resistance to Regorafenib.

C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription.

BACKGROUND: Acute myeloid leukemia (AML) with biallelic (CEBPAbi) as well as single mutations located in the bZIP region is associated with a favorable prognosis, but the underlying mechanisms are still unclear. Here, we propose that two isoforms of C/EBPα regulate DNA damage-inducible transcript 3 (DDIT3) transcription in AML cells corporately, leading to altered susceptibility to endoplasmic reticulum (ER) stress and related drugs. METHODS: Human AML cell lines and murine myeloid precursor cell line 32Dcl3 cells were infected with recombinant lentiviruses to knock down CEBPA expression or over-express the two isoforms of C/EBPα. Quantitative real-time PCR and western immunoblotting were employed to determine gene expression levels. Cell apoptosis rates were assessed by flow cytometry. CFU assays were utilized to evaluate the differentiation potential of 32Dcl3 cells. Luciferase reporter analysis, ChIP-seq and ChIP-qPCR were used to validate the transcriptional regulatory ability and affinity of each C/EBPα isoform to specific sites at DDIT3 promoter. Finally, an AML xenograft model was generated to evaluate the in vivo therapeutic effect of agents. RESULTS: We found a negative correlation between CEBPA expression and DDIT3 levels in AML cells. After knockdown of CEBPA, DDIT3 expression was upregulated, resulting in increased apoptotic rate of AML cells induced by ER stress. Cebpa knockdown in mouse 32Dcl3 cells also led to impaired cell viability due to upregulation of Ddit3, thereby preventing leukemogenesis since their differentiation was blocked. Then we discovered that the two isoforms of C/EBPα regulate DDIT3 transcription in the opposite way. C/EBPα-p30 upregulated DDIT3 transcription when C/EBPα-p42 downregulated it instead. Both isoforms directly bound to the promoter region of DDIT3. However, C/EBPα-p30 has a unique binding site with stronger affinity than C/EBPα-p42. These findings indicated that balance of two isoforms of C/EBPα maintains protein homeostasis and surveil leukemia, and at least partially explained why AML cells with disrupted C/EBPα-p42 and/or overexpressed C/EBPα-p30 exhibit better response to chemotherapy stress. Additionally, we found that a low C/EBPα p42/p30 ratio induces resistance in AML cells to the BCL2 inhibitor venetoclax since BCL2 is a major target of DDIT3. This resistance can be overcome by combining ER stress inducers, such as tunicamycin and sorafenib in vitro and in vivo. CONCLUSION: Our results indicate that AML patients with a low C/EBPα p42/p30 ratio (e.g., CEBPAbi) may not benefit from monotherapy with BCL2 inhibitors. However, this issue can be resolved by combining ER stress inducers.

Hypoxia-inducible factor 1α inhibits heat stress-induced pig intestinal epithelial cell apoptosis through eif2α/ATF4/CHOP signaling.

Unstoppable global warming and increased frequency of extreme heat leads to human and animals easier to suffer from heat stress (HS), with gastrointestinal abnormalities as one of the initial clinical symptoms. HS induces intestinal mucosal damage owing to intestinal hypoxia and hyperthermia. Hypoxia-inducible factor 1α (HIF-1α) activates numerous genes to mediate cell hypoxic responses; however, its role in HS-treated intestinal mucosa is unknown. This work aimed to explore HIF-1α function and regulatory mechanisms in HS-treated pig intestines. We assigned 10 pigs to control and moderate HS groups. Physical signs, stress, and antioxidant levels were detected, and the intestines were harvested after 72 h of HS treatment to study histological changes and HIF-1α, heat shock protein 90 (HSP90), and prolyl-4-hydroxylase 2 (PHD-2) expression. In addition, porcine intestinal columnar epithelial cells (IPEC-J2) underwent HS treatment (42 °C, 5 % O2) to further explore the functions and regulatory mechanism of HIF-1α. The results of histological examination revealed HS caused intestinal villi damage and increased apoptotic epithelial cell; the expression of HIF-1α and HSP90 increased while PHD-2 showed and opposite trend. Transcriptome sequencing analysis revealed that HS activated HIF-1 signaling. To further explore the role of HIF-1α on HS induced IPEC-J2 apoptosis, the HIF-1α was interfered and overexpression respectively, and the result confirmed that HIF-1α could inhibited cell apoptosis under HS. Furthermore, HS-induced apoptosis depends on eukaryotic initiation factor 2 alpha (eif2α)/activating transcription factor 4 (ATF4)/CCAAT-enhancer-binding protein homologous protein (CHOP) pathway, and HIF-1α can inhibit this pathway to alleviate IPEC-J2 cell apoptosis. In conclusion, this study suggests that HS can promote intestinal epithelial cell apoptosis and cause pig intestinal mucosal barrier damage; the HIF-1α can alleviate cell apoptosis by inhibiting eif2α/ATF4/CHOP signaling. These results indicate that HIF-1α plays a protective role in HS, and offers a potential target for HS prevention and mitigation.

Integrated Bioinformatics Analysis Identified ASNS and DDIT3 as the Therapeutic Target in Castrate-Resistant Prostate Cancer.

Many studies have demonstrated the mechanisms of progression to castration-resistant prostate cancer (CRPC) and novel strategies for its treatment. Despite these advances, the molecular mechanisms underlying the progression to CRPC remain unclear, and currently, no effective treatments for CRPC are available. Here, we characterized the key genes involved in CRPC progression to gain insight into potential therapeutic targets. Bicalutamide-resistant prostate cancer cells derived from LNCaP were generated and named Bical R. RNA sequencing was used to identify differentially expressed genes (DEGs) between LNCaP and Bical R. In total, 631 DEGs (302 upregulated genes and 329 downregulated genes) were identified. The Cytohubba plug-in in Cytoscape was used to identify seven hub genes (ASNS, AGT, ATF3, ATF4, DDIT3, EFNA5, and VEGFA) associated with CRPC progression. Among these hub genes, ASNS and DDIT3 were markedly upregulated in CRPC cell lines and CRPC patient samples. The patients with high expression of ASNS and DDIT3 showed worse disease-free survival in patients with The Cancer Genome Atlas (TCGA)-prostate adenocarcinoma (PRAD) datasets. Our study revealed a potential association between ASNS and DDIT3 and the progression to CRPC. These results may contribute to the development of potential therapeutic targets and mechanisms underlying CRPC progression, aiming to improve clinical efficacy in CRPC treatment.

DDIT3 deficiency accelerates bone remodeling during bone healing by enhancing osteoblast and osteoclast differentiation through ULK1-mediated autophagy.

The coordination of osteoblasts and osteoclasts is essential for bone remodeling. DNA damage inducible script 3 (DDIT3) is an important regulator of bone and participates in cell differentiation, proliferation, autophagy, and apoptosis. However, its role in bone remodeling remains unexplored. Here, we found that Ddit3 knockout (Ddit3-KO) enhanced both bone formation and resorption. The increased new bone formation and woven bone resorption, i.e., enhanced bone remodeling capacity, was found to accelerate bone defect healing in Ddit3-KO mice. In vitro experiments showed that DDIT3 inhibited both osteoblast differentiation and Raw264.7 cell differentiation by regulating autophagy. Cell coculture assay showed that Ddit3-KO decreased the ratio of receptor activator of nuclear factor-κß ligand (RANKL) to osteoprotegerin (OPG) in osteoblasts, and Ddit3-KO osteoblasts inhibited osteoclast differentiation. Meanwhile, DDIT3 knockdown (DDIT3-sh) increased receptor activator of nuclear factor-κß (RANK) expression in Raw264.7 cells, and DDIT3-sh Raw264.7 cells promoted osteoblast differentiation, whereas, DDIT3 overexpression had the opposite effect. Mechanistically, DDIT3 promoted autophagy partly by increasing ULK1 phosphorylation at serine555 (pULK1-S555) and decreasing ULK1 phosphorylation at serine757 (pULK1-S757) in osteoblasts, thereby inhibiting osteoblast differentiation. DDIT3 inhibited autophagy partly by decreasing pULK1-S555 in Raw264.7 cells, thereby suppressing osteoclastic differentiation. Taken together, our data indicate that DDIT3 is one of the elements regulating bone remodeling and bone healing, which may become a potential target in bone defect treatment.

Licochalcone A induces endoplasmic reticulum stress-mediated apoptosis of endometrial cancer cells via upregulation of GRP78 expression.

Licochalcone A (LicA), a natural compound extracted from licorice root, has been shown to exert a variety of anticancer activities. Whether LicA has such effects on endometrial cancer (EMC) is unclear. This study aims to investigate the antitumor effects of LicA on EMC. Our results show that LicA significantly reduced the viability and induced apoptosis of EMC cells and EMC-7 cells from EMC patients. LicA was also found to induce endoplasmic reticulum (ER) stress, leading to increased expression of ER-related proteins (GRP78/PERK/IRE1α/CHOP) in EMC cell lines. Suppression of GRP78 expression in human EMC cells treated with LicA significantly attenuated the effects of LicA, resulting in reduced ER-stress mediated cell apoptosis and decreased expression of ER- and apoptosis-related proteins. Our findings demonstrate that LicA induces apoptosis in EMC cells through the GRP78-mediated ER-stress pathway, emphasizing the potential of LicA as an anticancer therapy for EMC.

DDIT3-amplified or low-polysomic pleomorphic sarcomas without MDM2 amplification: Clinicopathological review and immunohistochemical profile of nine cases.

Several high-grade pleomorphic sarcoma cases that cannot be classified into any existing established categories have been reported. These cases were provisionally classified into undifferentiated pleomorphic sarcoma (UPS). Some dedifferentiated liposarcoma (DDLS) cases may also have been classified into the UPS category due to the absence of MDM2 amplification or an atypical lipomatous tumor/well-differentiated liposarcoma component. We retrieved and reviewed 77 high-grade pleomorphic sarcoma cases, initially diagnosed as UPS in 66 cases and DDLS in 11 cases. Fluorescence in situ hybridization (FISH) analyses of DDIT3 and MDM2 were performed for available cases. Of the cases successfully subjected to DDIT3 FISH (n = 56), nine (7 UPS and 2 DDLS) showed DDIT3 amplification but no MDM2 amplification. Two UPS cases showed both telomeric (5') and centromeric (3') amplification of DDIT3 or low polysomy of chromosome 12, whereas 5 UPS and 2 DDLS cases showed 5'-predominant DDIT3 amplification. Histopathologically, all cases showed UPS-like proliferation of atypical pleomorphic tumor cells. Immunohistochemically, only one case showed focal nuclear positivity for DDIT3, supporting the previous finding that DDIT3 expression was not correlated with DDIT3 amplification. All three cases with focal MDM2 expression involved 5'-predominant amplification, two of which showed DDLS-like histological features. The majority of cases (7/9) showed decreased expression in p53 staining, suggesting that DDIT3 amplification regulates the expression of TP53 like MDM2. From a clinicopathological perspective, we hypothesize that DDIT3-amplified sarcoma, especially with 5'-predominant amplification, can be reclassified out of the UPS category.

Esketamine induces apoptosis of nasopharyngeal carcinoma cells through the PERK/CHOP pathway.

Nasopharyngeal carcinoma, a malignant tumor prevalent in southeast Asia and north Africa, still lacks effective treatment. Esketamine, an N-methyl-D-aspartatic acid (NMDA) receptor (NMDAR) antagonist, is widely used in clinical anesthesia. Emerging evidence suggests that esketamine plays an important role in inhibiting tumor cell activity. However, the underlying mechanisms of esketamine on nasopharyngeal carcinoma remain unknown. In this study, we found that esketamine inhibited the proliferation and migration of nasopharyngeal carcinoma cells. Mechanically, transcriptome sequencing and subsequent verification experiments revealed that esketamine promoted the apoptosis of nasopharyngeal carcinoma cells through endoplasmic reticulum stress PERK/ATF4/CHOP signaling pathway mediated by NMDAR. Additionally, when combined with esketamine, the inhibitory effect of cisplatin on the proliferation of nasopharyngeal carcinoma cells was significantly enhanced. These findings provide new insights into future anti-nasopharyngeal carcinoma clinical strategies via targeting the NMDAR/PERK/CHOP axis alone or in combination with cisplatin.

A beneficial adaptive role for CHOP in driving cell fate selection during ER stress.

Cellular stresses elicit signaling cascades that are capable of either mitigating the inciting dysfunction or initiating cell death. During endoplasmic reticulum (ER) stress, the transcription factor CHOP is widely recognized to promote cell death. However, it is not clear whether CHOP also has a beneficial role during adaptation. Here, we combine a new, versatile, genetically modified Chop allele with single cell analysis and with stresses of physiological intensity, to rigorously examine the contribution of CHOP to cell fate. Paradoxically, we find that CHOP promotes death in some cells, but proliferation-and hence recovery-in others. Strikingly, this function of CHOP confers to cells a stress-specific competitive growth advantage. The dynamics of CHOP expression and UPR activation at the single cell level suggest that CHOP maximizes UPR activation, which in turn favors stress resolution, subsequent UPR deactivation, and proliferation. Taken together, these findings suggest that CHOP's function can be better described as a "stress test" that drives cells into either of two mutually exclusive fates-adaptation or death-during stresses of physiological intensity.

Caspase-3/GSDME dependent pyroptosis contributes to offspring lung injury induced by gestational PFOS exposure via PERK/ATF4 signaling.

Perfluorooctane sulfonate (PFOS) is widely used in industry and consumer products. Previous studies have showed that PFOS gestational exposure is associated with offspring lung damage in rat. However, the underlying mechanisms remain poorly understood. In this study, we investigated the role of gasdermin E (GSDME) in lung injury of offspring and its underlying mechanisms using in vivo and in vitro approaches. Pregnant SD rats were exposed to PFOS (1 mg/kg BW/d) between gestational day 12-18, and the lung tissue of the offspring was evaluated on postnatal day 7. PFOS treated animals exhibited alveolar septal thickening and inflammation-related damages, with an increased expression of GSDME in alveolar type II epithelial cells (AECII). Furthermore, in vitro experiments demonstrated that PFOS exposure (with 225 µM and up) upregulated the caspase-3/GSDME signaling pathway in AECII. Also, ultrastructure analysis revealed significant changes in the endoplasmic reticulum (ER) structure in PFOS-induced pyroptotic cells, which is consistent with the ER stress detected in these cells. Additionally, PFOS exposure led to increased expression of ER stress-related proteins, including p-PERK, p-eIF2α, ATF4, and CHOP. Subsequently, using specific inhibitors, we found that the PERK/ATF4 pathway acted as an upstream signal regulating GSDME-dependent pyroptosis. Overall, our findings show that GSDME-dependent pyroptosis plays a crucial role in the lung injury induced by gestational PFOS exposure, and the PERK/ATF4 pathway may function as a possible mediator of this process.

Acetylshikonin induces apoptosis through the endoplasmic reticulum stress-activated PERK/eIF2α /CHOP axis in oesophageal squamous cell carcinoma.

Acetylshikonin (AS) is an active component of Lithospermum erythrorhizon Sieb. et Zucc that exhibits activity against various cancers; however, the underlying mechanisms of AS against oesophageal squamous carcinoma (ESCC) need to be elusive. The research explores the anti-cancer role and potential mechanism of AS on ESCC in vitro and in vivo, providing evidences for AS treatment against ESCC. In this study, we firstly demonstrated that AS treatment effectively inhibits cell viability and proliferation of ESCC cells. In addition, AS significantly induces G1/S phage arrest and promotes apoptosis in ESCC cell lines. Further studies reveal that AS induces ER stress, as observed by dose- and time-dependently increased expression of BIP, PDI, PERK, phosphorylation of eIF2α , CHOP and splicing of XBP1. CHOP knockdown or PERK inhibition markedly rescue cell apoptosis induced by AS. Moreover, AS treatment significantly inhibits ESCC xenograft growth in nude mice. Elevated expression of BIP and CHOP is also observed in xenograft tumours. Taken together, AS inhibits proliferation and induces apoptosis through ER stress-activated PERK/eIF2α /CHOP pathway in ESCC, which indicates AS represents a promising candidate for ESCC treatment.

Epigenetic Modulation of DDIT3 and MGMT Expression Acts Synergistically with Resistance to Imatinib towards CML Disease Progression: A Hospital based Study.

INTRODUCTION: Imatinib Mesylate is an authenticated drug that aids in the treatment of Chronic Myeloid Leukaemia and Philadelphia patients which is recognized as a BCR-ABL tyrosine kinase inhibitor. Indeed, DNA Methylation occupies a key role in the stability of chromosomes. OBJECTIVE: Changes in the methylation status of genes may impart to the advancement of Chronic Myeloid Leukaemia. The present investigation aims to assess the role of expression analysis and methylation status of DDIT3 and MGMT genes in imatinib-resistant and nonresistant cases. METHODS: The Imatinib resistance was screened through RFLP. In this case maximum number of patients were recorded in the chronic phase belonging to the age group 40-59 and the accelerated and blast phase is more common in elderly patients showing the progressive nature of the disease with age. Hemoglobin and platelet count are found to be higher in cases where WBC count was minimal. A history of long-term alcohol consumption is found to be associated with the progression of the disease. RESULTS: The maximum level of expression of the DDIT3 gene was recorded in the chronic phase regardless of upstream (67.8%) and downstream (57.9%) regulation. The highest MGMT expression regulation was also observed in the case of chronic phase in both upstream (78.9%) and downstream (44%) regulation. Further, the MGMT gene showed the highest methylation of 6.6% and DDIT3 showed 3.3% in CML cases. CONCLUSION: In the present study notable depletion of survivality was established in the Imatinib resistance patients manifesting genetic malfunction of BCR-ABL transcripts among the North East Indian inhabitants and advocating for the expansion of the disease.

Ascomylactam C Induces an Immunogenic Cell Death Signature via Mitochondria-Associated ER Stress in Lung Cancer and Melanoma.

Ascomylactam C (AsC) is a new 13-membered-ring macrocyclic alkaloid, which was first isolated and identified in 2019 from the secondary metabolites of the mangrove endophytic fungus Didymella sp. CYSK-4 in the South China Sea. AsC has been found to have a broad-spectrum cytotoxic activity. However, the antitumor effects in vivo and mechanisms of AsC remain unclear. The aim of this study was to describe the effects of AsC on lung cancer and melanoma cells and to explore the antitumor molecular mechanism of AsC. In vitro, we used plate colony formation experiments and demonstrated the ability of AsC to inhibit low-density tumor growth. An Annexin V/PI cell apoptosis detection experiment revealed that AsC induced tumor cell apoptosis. In vivo, AsC suppressed the tumor growth of LLC and B16F10 allograft significantly in mice, and promoted the infiltration of CD4+ T and CD8+ T cells in tumor tissues. Mechanistically, by analyses of Western blotting, immunofluorescence and ELISA analysis, we found that AsC increased ROS formation, induced endoplasmic reticulum (ER) stress, activated the protein kinase RNA-like ER kinase (PERK)/eukaryotic translation initiation factor (eIF2α)/activating transcription factor 4 (ATF4)/C/EBP homologous protein (CHOP) signaling pathway, and induced immunogenic cell death (ICD) of tumor cells. Our results suggest that AsC may be a potentially promising antitumor drug candidate.

CHOP upregulation and dysregulation of the mature form of the SNAT2 amino acid transporter in the placentas from small for gestational age newborns.

BACKGROUND: The placentas from newborns that are small for gestational age (SGA; birth weight < -2 SD for gestational age) may display multiple pathological characteristics. A key determinant of fetal growth and, therefore, birth weight is placental amino acid transport, which is under the control of the serine/threonine kinase mechanistic target of rapamycin (mTOR). The effects of endoplasmic reticulum (ER) stress on the mTOR pathway and the levels of amino acid transporters are not well established. METHODS: Placentas from SGA and appropriate for gestational age (AGA) newborns and the human placental BeWo cell line exposed to the ER stressor tunicamycin were used. RESULTS: We detected a significant increase in the levels of C/EBP homologous protein (CHOP) in the placentas from SGA newborns compared with those from AGA newborns, while the levels of other ER stress markers were barely affected. In addition, placental mTOR Complex 1 (mTORC1) activity and the levels of the mature form of the amino acid transporter sodium-coupled neutral amino acid transporter 2 (SNAT2) were also reduced in the SGA group. Interestingly, CHOP has been reported to upregulate growth arrest and DNA damage-inducible protein 34 (GADD34), which in turn suppresses mTORC1 activity. The GADD34 inhibitor guanabenz attenuated the increase in CHOP protein levels and the reduction in mTORC1 activity caused by the ER stressor tunicamycin in the human placental cell line BeWo, but it did not recover mature SNAT2 protein levels, which might be reduced as a result of defective glycosylation. CONCLUSIONS: Collectively, these data reveal that GADD34A activity and glycosylation are key factors controlling mTORC1 signaling and mature SNAT2 levels in trophoblasts, respectively, and might contribute to the SGA condition. Video Abstract.

Glycyrrhetinic acid attenuates endoplasmic reticulum stress-induced hepatocyte apoptosis via CHOP/DR5/Caspase 8 pathway in cholestasis.

Bile acid (BA)-induced apoptosis is a common pathologic feature of cholestatic liver injury. Glycyrrhetinic acid (GA) is the hepatoprotective constituent of licorice. In the present study, the anti-apoptotic potential of GA was investigated in wild type and macrophage-depleted C57BL/6 mice challenged with alpha-naphthyl isothiocyanate (ANIT), and hepatocytes stimulated with Taurocholic acid (TCA) or Tumor necrosis factor-alpha (TNF-α). Apoptosis was determined by TUNEL positive cells and expression of executioner caspases. Firstly, we found that GA markedly alleviated liver injury, accompanied with reduced positive TUNEL-staining cells, and expression of caspases 3, 8 and 9 in mice modeled with ANIT. Secondly, GA mitigated apoptosis in macrophage-depleted mice with exacerbated liver injury and augmented cell apoptosis. In vitro study, pre-treatment with GA reduced the expression of activated caspases 3 and 8 in hepatocytes stimulated with TCA, but not TNF-α. The ability of GA to ameliorate apoptosis was abolished in the presence of Tauroursodeoxycholic Acid (TUDCA), a chemical chaperon against Endoplasmic reticulum stress (ER stress). Furthermore, GA attenuated the over-expression of Glucose regulated protein 78 (GRP78), and blocked all three branches of Unfolded protein reaction (UPR) in cholestatic livers of mice induced by ANIT. GA also downregulated C/EBP homologous protein (CHOP) expression, accompanied with reduced expression of Death receptor 5 (DR5) and activation of caspase 8 in both ANIT-modeled mice and TCA-stimulated hepatocytes. The results indicate that GA inhibits ER stress-induced hepatocyte apoptosis in cholestasis, which correlates with blocking CHOP/DR5/Caspase 8 pathway.

The role of PERK-eIF2α-ATF4-CHOP pathway in sevoflurane induced neuroapoptosis and cognitive dysfunction in aged mice.

Postoperative cognitive dysfunction (POCD) is a common surgical complication that causes additional pain in patients and affects their quality of life. To address this problem, emerging studies have focused on the POCD. Recent studies have shown that aging and anesthetic exposure are the two major risk factors for developing POCD. However, few reports described the exact molecular mechanisms underlying POCD in elderly patients. In the previous studies, the endoplasmic reticulum (ER) stress and neuroapoptosis in the hippocampus were associated with inducing POCD; however, no further information on the related signaling pathways could be disclosed. The PERK-eIF2α-ATF4-CHOP pathway is identified as the main regulatory pathway involved in ER stress and cell apoptosis. Therefore, we assume that the occurrence of POCD induced by sevoflurane inhalation may potentially result from ER stress and neuroapoptosis in the hippocampus of aged mice mediated by the PERK-eIF2α-ATF4-CHOP pathway. In our study, we found a relationship between sevoflurane inhalation concentration and memory decline in aged mice, with a 'ceiling effect'. We have confirmed that POCD induced by sevoflurane results from ER stress and neuroapoptosis in the hippocampus of aged mice, which is regulated by the over-expression of PERK-eIF2α-ATF4-CHOP pathway. Furthermore, we also showed that the dephosphorylation inhibitor of eIF2α (salubrinal) could down-regulate PERK-eIF2α-ATF4-CHOP pathway expression to inhibit ER stress and enhance the cognitive function of aged mice. In general, our study has elucidated one of the molecular mechanisms of sevoflurane-related cognitive dysfunction in aged groups and provided new strategies for treating sevoflurane-induced POCD.

CYP2E1 triggered GRP78/ATF6/CHOP signaling axis inhibit apoptosis and promotes progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death worldwide. Cytochrome P450 2E1 (CYP2E1) is an enzyme, primarily involved in the metabolism of xenobiotics and procarcinogens. The present study was designed to investigate the potential role of CYP2E1 triggered endoplasmic reticulum stress in the progression of HCC through inhibition of apoptosis. In vitro CYP2E1 promotes HepG2 cell migration, reduced chromatin condensation, enhanced intracellular ROS accumulation and induce cell cycle progression. Conversely this effect was averted by CYP2E1 siRNA, selective inhibitor Diallyl sulphide (DAS) and antioxidants (vitamin C and E). In vivo Diethylnitrosamine (DEN) induced HCC rats showed decreased body weight and increased relative liver weight. Moreover, macro trabecular-massive HCC (MTM-HCC) histological subtyping showed pathological features like well-differentiated tumors, micro-trabecular and pseudo glandular patterns, megakaryocytes and cholestasis. Masson's trichrome staining revealed an intensive accumulation of collagen fibers in the extracellular matrix (ECM). Increased CYP2E1, VEGF and PCNA enhance the carcinogenicity as revealed in immunohistochemistry results. Immunoblot analysis showed reduced expression of copper-zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) in cytosolic as well as mitochondrial fraction of rat liver tissue respectively. Also, increased level of CYP2E1 stimulated the upregulation of unfolded proteins response (UPR) and ER stress-related proteins such as Glucose regulatory protein 78 (GRP78), activating transcription factor 6 (ATF6) and CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP). Meanwhile, CYP2E1 stimulated ER-stress reduces BCL2 and downregulates the cleaved caspase 3 thus suppresses apoptosis. in. Furthermore, immunofluorescence revealed increased expression level of α-SMA in the HCC rat liver tissue. The level of CYP2E1 mRNA was significantly increased. Altogether, these findings indicate that CYP2E1 has a dynamic role in the pathogenesis of HCC and might be a budding agent in liver carcinogenesis therapy.

ROS-mediated PERK-CHOP pathway plays an important role in cadmium-induced HepG2 cells apoptosis.

Cadmium (Cd) is a common heavy metal that is highly toxic to the liver, however, the exact mechanism underlying this damage accompanied by apoptosis has not been thoroughly demonstrated. In this study, we found that Cd exposure significantly reduced cell viability, including the increased populations of apoptotic cells and caspase-3/-7/-12 activation in HepG2 cells. Mechanistically, Cd initiated oxidative stress via elevating reactive oxygen species (ROS) levels, leading to oxidative damage in HepG2 cells. Simultaneously, Cd exposure induced endoplasmic reticulum (ER) stress via activating the protein kinase RNA-like ER kinase (PERK)-C/EBP homologous protein (CHOP) axis in HepG2 cells, and subsequently disturbed ER function as increased Ca2+ releasing from ER lumen. Intriguingly, further study revealed that oxidative stress is closely related with ER stress, as pretreatment with ROS scavenger, N-acetyl-l-cysteine (NAC) markedly reduced ER stress as well as protected ER function in Cd treated HepG2 cell. Collectively, these findings first revealed Cd exposure induced HepG2 cells death via a ROS-mediated PERK-CHOP-related apoptotic signaling pathway, which provides a novel insight into the mechanisms of Cd-induced hepatotoxicity. Furthermore, inhibitors for oxidative stress and ER stress might be considered as a new strategy to prevent or treat this disorder.

Depleted uranium induces thyroid damage through activation of ER stress via the thrombospondin 1-PERK pathway.

Depleted uranium (DU) can cause damage to the body, but its effects on the thyroid are unclear. The purpose of this study was to investigate the DU-induced thyroid damage and its potential mechanism in order to find new targets for detoxification after DU poisoning. A model of acute exposure to DU was constructed in rats. It was observed that DU accumulated in the thyroid, induced thyroid structure disorder and cell apoptosis, and decreased the serum T4 and FT4 levels. Gene screening showed that thrombospondin 1 (TSP-1) was a sensitive gene of DU, and the expression of TSP-1 decreased with the increase of DU exposure dose and time. TSP-1 knockout mice exposed to DU had more severe thyroid damage and lower serum FT4 and T4 levels than wild-type mice. Inhibiting the expression of TSP-1 in FRTL-5 cells aggravated DU-induced apoptosis, while exogenous TSP-1 protein alleviated the decreased viability in FRTL-5 cells caused by DU. It was suggested that DU may caused thyroid damage by down-regulating TSP-1. It was also found that DU increased the expressions of PERK, CHOP, and Caspase-3, and 4-Phenylbutyric (4-PBA) alleviated the DU-induced FRTL-5 cell viability decline and the decrease levels of rat serum FT4 and T4 caused by DU. After DU exposure, the PERK expression was further up-regulated in TSP-1 knockout mice, and the increased expression of PERK was alleviated in TSP-1 over-expressed cells, as well as the increased expression of CHOP and Caspase-3. Further verification showed that inhibition of PERK expression could reduce the DU-induced increased expression of CHOP and Caspase-3. These findings shed light on the mechanism that DU may activate ER stress via the TSP 1-PERK pathway, thereby leading to thyroid damage, and suggest that TSP-1 may be a potential therapeutic target for DU-induced thyroid damage.