Borax affects cellular viability by inducing ER stress in hepatocellular carcinoma cells by targeting SLC12A5.

Hepatocellular carcinoma (HCC) presents a persistent challenge to conventional therapeutic approaches. SLC12A5 is implicated in an oncogenic capacity and facilitates the progression of cancer. The objective of this investigation is to scrutinize the inhibitory effects of borax on endoplasmic reticulum (ER)-stress and apoptosis mediated by SLC12A5 in HepG2 cells. Initially, we evaluated the cytotoxic impact of borax on both HL-7702 and HepG2 cell lines. Subsequently, the effects of borax on cellular morphology and the cell cycle of these lines were examined. Following this, we explored the impact of borax treatment on the mRNA and protein expression levels of SLC12A5, C/EBP homologous protein (CHOP), glucose-regulated protein-78 (GRP78), activating transcription factor-6 (ATF6), caspase-3 (CASP3), and cytochrome c (CYC) in these cellular populations. The determined IC50 value of borax for HL-7702 cells was 40.8 mM, whereas for HepG2 cells, this value was 22.6 mM. The concentrations of IC50 (22.6 mM) and IC75 (45.7 mM) of borax in HepG2 cells did not manifest morphological aberrations in HL-7702 cells. Conversely, these concentrations in HepG2 cells induced observable morphological and nuclear abnormalities, resulting in cell cycle arrest in the G1/G0 phase. Additionally, the levels of SLC12A5, ATF6, CHOP, GRP78, CASP3, and CYC were elevated in HepG2 cells in comparison to HL-7702 cells. Moreover, SLC12A5 levels decreased following borax treatment in HepG2 cells, whereas ATF6, CHOP, GRP78, CASP3, and CYC levels exhibited a significant increase. In conclusion, our data highlight the potential therapeutic effects of borax through the regulation of ER stress in HCC by targeting SLC12A5.

hUCMSC-derived exosomes protect against GVHD-induced endoplasmic reticulum stress in CD4+ T cells by targeting the miR-16-5p/ATF6/CHOP axis.

Exosomes generated from mesenchymal stem cells (MSCs) are thought to be a unique therapeutic strategy for several autoimmune deficiency illnesses. The purpose of this study was to elucidate the protective effects of human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-Exo) on CD4+ T cells dysfunction during graft-versus-host disease (GVHD) and to identify the underlying processes involved. Here, we showed that hUCMSC-Exo treatment can effectively attenuate GVHD injury by alleviating redox metabolism disorders and inflammatory cytokine bursts in CD4+ T cells. Furthermore, hUCMSC-Exo ameliorate ER stress and ATF6/CHOP signaling-mediated apoptosis in CD4+ T cells and promote the development of CD4+IL-10+ T cells during GVHD. Moreover, downregulating miR-16-5p in hUCMSC-Exo impaired their ability to prevent CD4+ T cells apoptosis and weakened their ability to promote the differentiation of CD4+IL-10+ T cells. Collectively, the obtained data suggested that hUCMSC-Exo suppress ATF6/CHOP signaling-mediated ER stress and apoptosis in CD4+ T cells, enhance the differentiation of CD4+IL-10+ T cells, and reverse the imbalance of immune homeostasis in the GVHD process by transferring miR-16-5p. Our study provided further evidence that GVHD patients can benefit from hUCMSC-Exo-mediated therapy.

CHOP-mediated IL-23 overexpression does not drive colitis in experimental spondyloarthritis.

HLA-B27 is a major risk factor for spondyloarthritis (SpA), yet the underlying mechanisms remain unclear. HLA-B27 misfolding-induced IL-23, which is mediated by endoplasmic reticulum (ER) stress has been hypothesized to drive SpA pathogenesis. Expression of HLA-B27 and human ß2m (hß2m) in rats (HLA-B27-Tg) recapitulates key SpA features including gut inflammation. Here we determined whether deleting the transcription factor CHOP (Ddit3-/-), which mediates ER-stress induced IL-23, affects gut inflammation in HLA-B27-Tg animals. ER stress-mediated Il23a overexpression was abolished in CHOP-deficient macrophages. Although CHOP-deficiency also reduced Il23a expression in immune cells isolated from the colon of B27+ rats, Il17a levels were not affected, and gut inflammation was not reduced. Rather, transcriptome analysis revealed increased expression of pro-inflammatory genes, including Il1a, Ifng and Tnf in HLA-B27-Tg colon tissue in the absence of CHOP, which was accompanied by higher histological Z-scores. RNAScope localized Il17a mRNA to the lamina propria of the HLA-B27-Tg rats and revealed similar co-localization with Cd3e (CD3) in the presence and absence of CHOP. This demonstrates that CHOP-deficiency does not improve, but rather exacerbates gut inflammation in HLA-B27-Tg rats, indicating that HLA-B27 is not promoting gut disease through ER stress-induced IL-23. Hence, CHOP may protect rats from more severe HLA-B27-induced gut inflammation.

[Ginsenoside Rg_1 ameliorates OGD/R-induced PC12 cell injury by inhibiting autography via IRE1-JNK-CHOP pathway].

This study investigated the protective effect of ginsenoside Rg_1(GRg_1) on oxygen and glucose deprivation/reoxygenation(OGD/R)-injured rat adrenal pheochromocytoma(PC12) cells and whether the underlying mechanism was related to the regulation of inositol-requiring enzyme 1(IRE1)-c-Jun N-terminal kinase(JNK)-C/EBP homologous protein(CHOP) signaling pathway. An OGD/R model was established in PC12 cells, and PC12 cells were randomly classified into control, model, OGD/R+GRg_1(0.1, 1, 10 µmol·L~(-1)), OGD/R+GRg_1+rapamycin(autophagy agonist), OGD/R+GRg_1+3-methyladenine(3-MA,autophagy inhibitor), OGD/R+GRg_1+tunicamycin(endoplasmic reticulum stress agonist), OGD/R+GRg_1+4-phenylbutyric acid(4-PBA, endoplasmic reticulum stress inhibitor), and OGD/R+GRg_1+3,5-dibromosalicylaldehyde(DBSA, IRE1 inhibitor) groups. Except the control group, the other groups were subjected to OGD/R treatment, i.e., oxygen and glucose deprivation for 6 h followed by reoxygenation for 6 h. Cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT) assay. Apoptosis was detected by Hoechst 33342 staining, and the fluorescence intensity of autophagosomes by the monodansylcadaverine(MDC) assay. Western blot was employed to determine the expression of autophagy-related proteins(Beclin1, LC3-Ⅱ, and p62) and the pathway-related proteins [IRE1, p-IRE1, JNK, p-JNK, glucose-regulated protein 78(GRP78), and CHOP]. The results showed that GRg_1 dose-dependently increased the viability of PC12 cells and down-regulated the expression of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, compared with the model group. Furthermore, GRg_1 decreased the apoptosis rate and MDC fluorescence intensity and up-regulated the expression of p62 protein. Compared with the OGD/R+GRg_1(10 µmol·L~(-1)) group, OGD/R+GRg_1+rapamycin and OGD/R+GRg_1+tunicamycin groups showed increased apoptosis rate and MDC fluorescence intensity, up-regulated protein levels of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, decreased relative cell survival rate, and down-regulated protein level of p62. The 3-MA, 4-PBA, and DBSA groups exerted the opposite effects. Taken together, GRg_1 may ameliorate OGD/R-induced PC12 cell injury by inhibiting autophagy via the IRE1-JNK-CHOP pathway.

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.

Esculin suppresses the PERK-eIF2α-CHOP pathway by enhancing SIRT1 expression in oxidative stress-induced rat chondrocytes, mitigating osteoarthritis progression in a rat model.

OBJECTIVE: Osteoarthritis (OA) is a degenerative disease characterized by the gradual degeneration of chondrocytes, involving endoplasmic reticulum (ER) stress. Esculin is a natural compound with antioxidant, anti-inflammatory and anti-tumor properties. However, its impact on ER stress in OA therapy has not been thoroughly investigated. We aim to determine the efficiency of Esculin in OA treatment and its underlying mechanism. METHODS: We utilized the tert-butyl hydroperoxide (TBHP) to establish OA model in chondrocytes. The expression of SIRT1, PERK/eIF2α pathway-related proteins, apoptosis-associated proteins and ER stress-related proteins were detected by Western blot and Real-time PCR. The apoptosis was evaluated by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. X-ray imaging, Hematoxylin & Eosin staining, Safranin O staining and immunohistochemistry were used to assess the pharmacological effects of Esculin in the anterior cruciate ligament transection (ACLT) rat OA model. RESULTS: Esculin downregulated the expression of PERK/eIF2α pathway-related proteins, apoptosis-associated proteins and ER stress-related proteins, while upregulated the expression of SIRT1 and Bcl2 in the TBHP-induced OA model in vitro. It was coincident with the results of TUNEL staining and flow cytometry. We further confirmed the protective effect of Esculin in the rat ACLT-related model. CONCLUSION: Our results suggest the potential therapeutic value of Esculin on osteoarthritis. It probably inhibits the PERK-eIF2α-ATF4-CHOP pathway by upregulating SIRT1, thereby mitigating endoplasmic reticulum stress and protecting chondrocytes from apoptosis.

Deciphering the role of FUS::DDIT3 expression and tumor microenvironment in myxoid liposarcoma development.

BACKGROUND: Myxoid liposarcoma (MLS) displays a distinctive tumor microenvironment and is characterized by the FUS::DDIT3 fusion oncogene, however, the precise functional contributions of these two elements remain enigmatic in tumor development. METHODS: To study the cell-free microenvironment in MLS, we developed an experimental model system based on decellularized patient-derived xenograft tumors. We characterized the cell-free scaffold using mass spectrometry. Subsequently, scaffolds were repopulated using sarcoma cells with or without FUS::DDIT3 expression that were analyzed with histology and RNA sequencing. RESULTS: Characterization of cell-free MLS scaffolds revealed intact structure and a large variation of protein types remaining after decellularization. We demonstrated an optimal culture time of 3 weeks and showed that FUS::DDIT3 expression decreased cell proliferation and scaffold invasiveness. The cell-free MLS microenvironment and FUS::DDIT3 expression both induced biological processes related to cell-to-cell and cell-to-extracellular matrix interactions, as well as chromatin remodeling, immune response, and metabolism. Data indicated that FUS::DDIT3 expression more than the microenvironment determined the pre-adipocytic phenotype that is typical for MLS. CONCLUSIONS: Our experimental approach opens new means to study the tumor microenvironment in detail and our findings suggest that FUS::DDIT3-expressing tumor cells can create their own extracellular niche.

ß-Cell-selective regulation of gene expression by nitric oxide.

Nitric oxide is produced at low micromolar levels following the induction of inducible nitric oxide synthase (iNOS) and is responsible for mediating the inhibitory actions of cytokines on glucose-stimulated insulin secretion by islets of Langerhans. It is through the inhibition of mitochondrial oxidative metabolism, specifically aconitase and complex 4 of the electron transport chain, that nitric oxide inhibits insulin secretion. Nitric oxide also attenuates protein synthesis, induces DNA damage, activates DNA repair pathways, and stimulates stress responses (unfolded protein and heat shock) in ß-cells. In this report, the time- and concentration-dependent effects of nitric oxide on the expression of six genes known to participate in the response of ß-cells to this free radical were examined. The genes included Gadd45α (DNA repair), Puma (apoptosis), Hmox1 (antioxidant defense), Hsp70 (heat shock), Chop (UPR), and Ppargc1α (mitochondrial biogenesis). We show that nitric oxide stimulates ß-cell gene expression in a narrow concentration range of ∼0.5-1 µM or levels corresponding to iNOS-derived nitric oxide. At concentrations greater than 1 µM, nitric oxide fails to stimulate gene expression in ß-cells, and this is associated with the inhibition of mitochondrial oxidative metabolism. This narrow concentration range of responses is ß-cell selective, as the actions of nitric oxide in non-ß-cells (α-cells, mouse embryonic fibroblasts, and macrophages) are concentration dependent. Our findings suggest that ß-cells respond to a narrow concentration range of nitric oxide that is consistent with the levels produced following iNOS induction, and that these concentration-dependent actions are selective for insulin-containing cells.

Endoplasmic reticulum stress enhances the expression of TLR3-induced TSLP by airway epithelium.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pleiotropic cytokine that regulates T-helper 2 (Th2) immune responses in the lung and plays a major role in severe uncontrolled asthma. Emerging evidence suggests a role for endoplasmic reticulum (ER) stress in the pathogenesis of asthma. In this study, we determined if ER stress and the unfolded protein response (UPR) signaling are involved in TSLP induction in the airway epithelium. For this, we treated human bronchial epithelial basal cells and differentiated primary bronchial epithelial cells with ER stress inducers and the TSLP mRNA and protein expression was determined. A series of siRNA gene knockdown experiments were conducted to determine the ER stress-induced TSLP signaling pathways. cDNA collected from asthmatic bronchial biopsies was used to determine the gene correlation between ER stress and TSLP. Our results show that ER stress signaling induces TSLP mRNA expression via the PERK-C/EBP homologous protein (CHOP) signaling pathway. AP-1 transcription factor is important in regulating this ER stress-induced TSLP mRNA induction, though ER stress alone cannot induce TSLP protein production. However, ER stress significantly enhances TLR3-induced TSLP protein secretion in the airway epithelium. TSLP and ER stress (PERK) mRNA expression positively correlates in bronchial biopsies from participants with asthma, particularly in neutrophilic asthma. In conclusion, these results suggest that ER stress primes TSLP that is then enhanced further upon TLR3 activation, which may induce severe asthma exacerbations. Targeting ER stress using pharmacological interventions may provide novel therapeutics for severe uncontrolled asthma.NEW & NOTEWORTHY TSLP is an epithelial-derived cytokine and a key regulator in the pathogenesis of severe uncontrolled asthma. We demonstrate a novel mechanism by which endoplasmic reticulum stress signaling upregulates airway epithelial TSLP mRNA expression via the PERK-CHOP signaling pathway and enhances TLR3-mediated TSLP protein secretion.

Targeting STING elicits GSDMD-dependent pyroptosis and boosts anti-tumor immunity in renal cell carcinoma.

While Stimulator-of-interferon genes (STING) is an innate immune adapter cruicial for sensing cytosolic DNA and modulating immune microenvironment, its tumor-promoting role in tumor survival and immune evasion remains largely unknown. Here we reported that renal cancer cells are exceptionally dependent on STING for survival and evading immunosurveillance via suppressing ER stress-mediated pyroptosis. We found that STING is significantly amplified and upregulated in clear cell renal cell carcinoma (ccRCC), and its elevated expression is associated with worse clinical outcomes. Mechanically, STING depletion in RCC cells specifically triggers activation of the PERK/eIF2α/ATF4/CHOP pathway and activates cleavage of Caspase-8, thereby inducing GSDMD-mediated pyroptosis, which is independent of the innate immune pathway of STING. Moreover, animal study revealed that STING depletion promoted infiltration of CD4+ and CD8+ T cells, consequently boosting robust antitumor immunity via pyroptosis-induced inflammation. From the perspective of targeted therapy, we found that Compound SP23, a PROTAC STING degrader, demonstrated comparable efficacy to STING depletion both in vitro and in vivo for treatment of ccRCC. These findings collectively unveiled an unforeseen function of STING in regulating GSDMD-dependent pyroptosis, thus regulating immune response in RCC. Consequently, pharmacological degradation of STING by SP23 may become an attractive strategy for treatment of advanced RCC.

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.

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.

Gene of the month: DDIT3.

DNA damage-inducible transcript 3 (DDIT3) gene, mapped to the human chromosome 12q13.3, encodes a protein that belongs to the CCAAT/enhancer-binding protein family of transcription factors. DDIT3 is involved in the proliferative control that responds to endoplasmic reticulum stress in normal conditions, dimerising other transcription factors with basic leucine zipper (bZIP) structural motifs. DDIT3 plays a significant role during cell differentiation, especially adipogenesis, arresting the maturation of adipoblasts. In disease, FUS/EWSR1::DDIT3 fusion is the pathogenic event that drives the development of myxoid liposarcoma. The amplification of DDIT3 in other adipocytic neoplasms mediates the presence of adipoblast-like elements. Another fusion, GLI1::DDIT3, has rarely been documented in other tumours. This paper reviews the structure and function of DDIT3, its role in disease-particularly cancer-and its use and pitfalls in diagnostic testing, including immunohistochemistry as a tissue-based marker.

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.

VPS34-IN1 induces apoptosis of ER+ breast cancer cells via activating PERK/ATF4/CHOP pathway.

VPS34-IN1 is a specific selective inhibitor of Class III Phosphatidylinositol 3-kinase (PI3K) and has been shown to exhibit a significant antitumor effect in leukemia and liver cancer. In current study, we focused on the anticancer effect and potential mechanism of VPS34-IN1 in estrogen receptor positive (ER+ ) breast cancer. Our results revealed that VPS34-IN1 inhibited the viability of ER+ breast cancer cells in vitro and in vivo. Flow cytometry and western blot analyses showed that VPS34-IN1 treatment induced breast cancer cell apopotosis. Interestingly, VPS34-IN1 treatment activated protein kinase R (PKR)-like ER kinase (PERK) branch of endoplasmic reticulum (ER) stress. Furthermore, knockdown of PERK by siRNA or inhibition of PERK activity by chemical inhibitor GSK2656157 could attenuate VPS34-IN1-mediated apoptosis in ER+ breast cancer cells. Collectively, VPS34-IN1 has an antitumor effect in breast cancer, and it may result from activating PERK/ATF4/CHOP pathway of ER stress to induce cell apoptosis. These findings broaden our understanding of the anti-breast cancer effects and mechanisms of VPS34-IN1 and provide new ideas and reference directions for the treatment of ER+ breast cancer.

[Clinical value of fluorescence in situ hybridization with MDM2 and DDIT3 probe in diagnosis of liposarcoma].

OBJECTIVE: To investigate the value of using MDM2 amplification probe and DDIT3 dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique in the diagnosis of liposarcoma. METHODS: In the study, 62 cases of liposarcoma diagnosed in Peking University First Hospital from January 2015 to December 2019 were analysed for clinicopathological information. Of these 62 cases of liposarcoma, all were analysed for MDM2 amplification and 48 cases were analysed for DDIT3 rearrangement using a FISH technique. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in liposarcoma and correlate it with diagnosis of different subtypes of liposarcoma. The subtypes of liposarcoma were classified according to the FISH results, combined with the relevant clinicopathological features. RESULTS: The patients aged 31-89 years (mean: 59 years) with a 1.75:1 male to female ratio. Histologically, there were 20 cases of atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLPS), 26 cases of dedifferentiated liposarcoma (DDLPS), 13 myxoid liposarcoma (MLPS) and 3 pleomorphic liposarcoma (PLPS). Tumors with DDLPS (23/26) and WDLPS (8/20) were localized retroperitoneally, while both tumours of MLPS and PLPS were localized extra-retroperitoneally, and the difference of sites among the four subtypes of liposarcoma was statistically significant (P < 0.05). Histologically, varied mucoid matrix could be observed in the four subtypes of liposarcoma, and the difference was statistically significant (P < 0.05). MDM2 gene amplification was demonstrated in all cases of ALT/WDLPS and DDLPS (100%, 20/20 and 26/26 respectively); DDIT3 gene rearrangement was noted only in MLPS (100%, 13/13); most cases of DDLPS (96.2%, 25/26) and ALT/WDLPS (83.3%, 5/6, 6 cases selected for detection) demonstrated the picture of amplification of the DDIT3 telomeric tag. According to the instructions of DDIT3 break-apart rearrangement probe, the 5' telomere probe and 3' centromere probe spanned but did not cover the DDIT3 gene itself, on the contrary, the 5' telomere probe covered the CDK4 gene, while the DDIT3 and CDK4 gene were located adjacent to each other on chromosome, therefore, when the amplification signal appeared on the telomeric tag of the DDIT3 rearrangement probe, it indeed indicated the CDK4 gene amplification rather than the DDIT3 gene rearrangement. Then the 10 cases with DDIT3 telomeric tag amplification were selected for CDK4 and DDIT3 gene amplification probe FISH tests, and all the cases showed CDK4 gene amplification (100%, 10/10) and two of the 10 cases demonstrated co-amplification of CDK4 and DDIT3 (20%, 2/10); DDIT3 polysomy detected by DDIT3 gene rearrangement probe was found in 1 case of DDLPS and 2 cases of PLPS (66.7%, 2/3) with morphology of high-grade malignant tumour and poor prognosis. CONCLUSION: Our results indicate that a diagnosis of different subtype liposarcoma could be confirmed based on the application of MDM2 and DDIT3 FISH, combined with clinicopathological findings. It is also noteworthy that atypical signals should be correctly interpreted to guide correct treatment of liposarcomas.

Interaction of Bmal1 and eIF2α/ATF4 pathway was involved in Shuxie compound alleviation of circadian rhythm disturbance-induced hepatic endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuxie Compound (SX) combines the composition and efficacy of Suanzaoren decoction and Huanglian Wendan decoction. It can soothe the liver, regulate the qi, nourish the blood and calm the mind. It is used in the clinical treatment of sleep disorder with liver stagnation. Modern studies have proved that circadian rhythm disorder (CRD) can cause sleep deprivation and liver damage, which can be effectively ameliorated by traditional Chinese medicine to soothe the liver stagnation. However, the mechanism of SX is unclear. AIM OF THE STUDY: This study was designed to demonstrate the impact of SX on CRD in vivo, and confirm the molecular mechanisms of SX in vitro. MATERIALS AND METHODS: The quality of SX and drug-containing serum was controlled by UPLC-Q-TOF/MS, which were used in vivo and in vitro experiments, respectively. In vivo, a light deprivation mouse model was used. In vitro, a stable knockdown Bmal1 cell line was used to explore SX mechanism. RESULTS: Low-dose SX (SXL) could restore (1) circadian activity pattern, (2) 24-h basal metabolic pattern, (3) liver injury, and (4) Endoplasmic reticulum (ER) stress in CRD mice. CRD decreased the liver Bmal1 protein at ZT15, which was reversed by SXL treatment. Besides, SXL decreased the mRNA expression of Grp78/ATF4/Chop and the protein expression of ATF4/Chop at ZT11. In vitro experiments, SX reduced the protein expression of thapsigargin (tg)-induced p-eIF2α/ATF4 pathway and increase the viability of AML12 cells by increasing the expression of Bmal1 protein. CONCLUSIONS: SXL relieved CRD-induced ER stress and improve cell viability by up-regulating the expression of Bmal1 protein in the liver and then inhibiting the protein expression of p-eIF2α/ATF4.

Selenium Protects Follicular Granulosa Cells from Apoptosis Induced by Mercury Through Inhibition of ATF6/CHOP Pathway in Laying Hens.

The purpose of this research was to explore the effect of selenium on mercury-mediated apoptosis of follicular granulosa cells in laying hens. Moreover, the ATF6/CHOP pathway was investigated to explore the mechanism in this progress. Hg, Se, and 4-phenyl butyric acid were used alone or in combination to treat the cells. Our results showed that the nuclear in cells became condensate after Hg exposure, while Se addition significantly alleviated this change. Hg exposure significantly induced the apoptosis and the reduction of mitochondrial membrane potential in cells (P < 0.05). Nevertheless, co-treatment of Se significantly inhibited these effects (P < 0.05). Additionally, Hg exposure dramatically elevated the gene expressions of Bax/Bcl-2 (P < 0.05), caspase-3 (P < 0.05), caspase-9 (P < 0.05), protein kinase RNA-like endoplasmic reticulum kinase (P < 0.05), activating transcription factor 6 (P < 0.05), C/EBP homologous protein (CHOP; P < 0.05), inositol-requiring enzyme 1α (P < 0.05), tumor necrosis factor-associated factor 2 (P < 0.05), activating transcription factor 6 (ATF6; P < 0.05), and apoptosis signal-regulating kinase 1 (P < 0.05) in cells, whereas Se addition avoided these changes. The exposure to Hg considerably boosted the expression of ATF6 and CHOP protein (P < 0.05), while Se addition significantly alleviated the above-mentioned enhancements (P < 0.05). In summary, Hg exposure induced apoptosis, which was considerably reduced alleviated by Se addition, which was linked to the ATF6/CHOP pathway in follicular granulosa cells in laying hens.

DHA Induces Cell Death through the Production of ROS and the Upregulation of CHOP in Fibroblast-like Synovial Cells from Human Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is an inflammatory disease marked by a massive proliferation of synovial cells in the joints. In this study, we investigated the pro-apoptotic effects of docosahexaenoic acid (DHA) in human fibroblast-like synovial cells from RA patients (RA-FLS). An in vitro study using MH7A cells showed that DHA treatment induced caspase-8-dependent apoptosis in a dose-dependent manner and reduced the TNF-α-mediated induction of MMP-9 and IL-1ß. DHA also induced the phosphorylation of eIF2α, the expression of the ER stress markers ATF4 and C/EBP homologous protein (CHOP), and death receptor 5 (DR5). The knockdown of CHOP or DR5 increased cell viability and reduced apoptosis in DHA-treated cells. Furthermore, the knockdown of CHOP reduced DHA-mediated DR5 expression, while the overexpression of CHOP increased DR5 expression. We also found that DHA treatment induced the accumulation of reactive oxygen species (ROS), and pretreatment with the anti-oxidant Tiron effectively abrogated not only the expression of CHOP and DR5, but also DHA-induced apoptosis. Under this condition, cell viability was increased, while PARP-1 cleavage and caspase-8 activation were reduced. All the findings were reproduced in human primary synovial cells obtained from RA patients. These results suggest that the DHA-mediated induction of ROS and CHOP induced apoptosis through the upregulation of DR5 in RA-FLSs, and that CHOP could be used as a therapy for RA.