Stromal Cell-Derived Factor (SDF) 2 and the Endoplasmic Reticulum Stress Response of Trophoblast Cells in Gestational Diabetes Mellitus and In vitro Hyperglycaemic Condition.

BACKGROUND AND AIM: The endoplasmic reticulum (ER) stress response and the unfolded protein response (UPR) are essential cellular mechanisms to ensure the proper functioning of ER in adverse conditions. However, activation of these pathways has also been associated with insulin resistance and cell death in pathological conditions such as diabetes mellitus. In the present study, we investigated whether stromal cell-derived factor 2 (SDF2)-an ER stress-responsive factor-is related to ER response in placental cells exposed to maternal gestational diabetes mellitus (GDM) or to a hyperglycaemic in vitro condition. OBJECTIVE: The study aimed to investigate the role of SDF2 in BeWo cells , a trophoblast cell line originating from choriocarcinoma , and in placental tissue under hyperglycaemic conditions. METHODS: Protein levels of SDF2 and UPR factors, glucose-related protein 78 (GRP78) and eukaryotic initiation factor 2 alpha (elF2 alpha) were evaluated in the placentae of pregnant women diagnosed with GDM and treated by diet-control (insulin was added when necessary). The mRNA expression of SDF2 and UPR factors CHOP and sXBP1 were assessed in cultured BeWo cells challenged with glucose and treated with or without insulin. RESULTS: SDF2 expression was increased in the placentae of GDM women treated with diet. However, its values were similar to those of normoglycemic controls when the GDM women were treated with insulin and diet. BeWo cells cultured with high glucose and insulin showed decreased SDF2 expression, while high glucose increased CHOP and sXBP1 expression, which was then significantly reverted with insulin treatment. CONCLUSION: Our findings extend the understanding of ER stress and SDF2 expression in placentae exposed to hyperglycaemia, highlighting the relevance of insulin in reducing the levels of ER stress factors in placental cells. Understanding the effect of ER stress partners such as SDF2 on signalling pathways involved in gestation, complicated by hyperglycaemia, is pivotal for basic biomedical research and may lead to new therapeutic possibilities.

RHBDD2 overexpression promotes a chemoresistant and invasive phenotype to rectal cancer tumors via modulating UPR and focal adhesion genes.

The current standard of care for locally advanced rectal cancer (RC) is neoadjuvant radio-chemotherapy (NRC) with 5-fluorouracil (5Fu) as the main drug, followed by surgery and adjuvant chemotherapy. While a group of patients will achieve a pathological complete response, a significant percentage will not respond to the treatment. The Unfolding Protein Response (UPR) pathway is generally activated in tumors and results in resistance to radio-chemotherapy. We previously showed that RHBDD2 gene is overexpressed in the advanced stages of colorectal cancer (CRC) and that it could modulate the UPR pathway. Moreover, RHBDD2 expression is induced by 5Fu. In this study, we demonstrate that the overexpression of RHBDD2 in CACO2 cell line confers resistance to 5Fu, favors cell migration, adhesion and proliferation and has a profound impact on the expression of both, the UPR genes BiP, PERK and CHOP, and on the cell adhesion genes FAK and PXN. We also determined that RHBDD2 binds to BiP protein, the master UPR regulator. Finally, we confirmed that a high expression of RHBDD2 in RC tumors after NRC treatment is associated with the development of local or distant metastases. The collected evidence positions RHBDD2 as a promising prognostic biomarker to predict the response to neoadjuvant therapy in patients with RC.

Mechanism of pingyangmycin-induced apoptosis of cultured human umbilical vein endothelial cells.

In this study, we investigated the effects of pingyangmycin (PYM) on the growth inhibition and apoptosis of human umbilical vein endothelial cells (HUVEC). In this study, we aimed to explore the optimal concentration of PYM to induce the apoptosis of HUVEC and to determine its mechanism of action. After treatment of HUVEC with different concentrations of PYM for 24 h, cell counting kit-8 (CCK-8) was used to detect growth inhibiting effects. Annexin V-FITC/propidium iodide stain was used to detect apoptosis, and western blot was used to detect the expression of glucose-related protein 78 (GPR78) and C/EBP homologous protein (CHOP) endoplasmic reticulum stress proteins. With increasing PYM concentration, the growth inhibition of HUVEC increased (P < 0.05), the apoptotic numbers of HUVEC increased (P < 0.05), with higher PYM concentrations inducing necrosis, and the protein expression of GRP78 and CHOP increased (P < 0.05). PYM could obviously inhibit the proliferation and promote the apoptosis of HUVEC. Necrotic cells were more prevalent than apoptotic cells at high PYM concentrations. This study helped to determine the proper concentration of PYM to induce more apoptosis than necrosis, which is critical to minimize inflammation, enhance the healing of the skin, and maintain safety for the patient. PYM might induce HUVEC apoptosis through the endoplasmic reticulum stress pathway.

15d-PGJ2 as an endoplasmic reticulum stress manipulator in multiple myeloma in vitro and in vivo.

Multiple myeloma (MM) is characterised by intense protein folding and, consequently endoplasmic reticulum (ER) stress. The prostaglandin 15d-PGJ2 is able to raise oxidative stress levels within the cell and potentially trigger cell death. The aim of this study was to evaluate the antineoplastic effect of 15d-PGJ2 on MM in vitro and in vivo via ER and oxidative stress pathways. MM.1R and MM.1S cell lines were treated with 15d-PGJ2 at 1-10µM and evaluated with regard to proliferation, mRNA expression of PRDX1, PRDX4, GRP78, GRP94, CHOP, BCL-2 and BAX. Stress data was validated via oxidized glutathione assays. MM.1R cells were inoculated into NOD/SCID mice, which were subsequently treated daily with 15d-PGJ2 at 4mg/kg or vehicle (control), with tumour volume being monitored for 14days. 15d-PGJ2 reduced cell proliferation, induced cell death and apoptosis at 5µM and 10µM and Stress-related genes were upregulated at the same doses. Oxidized glutathione levels were also increased. 15d-PGJ2 at 4mg/kg in vivo halted tumour growth. In conclusion, 15d-PGJ2 induced myeloma cell death via ER stress in vitro. 15d-PGJ2 in vivo also inhibited tumour growth.

Proapoptotic effects of heme oxygenase-1 inhibitor on Kasumi-1 cells via the ATF4/CHOP/Ire-1α pathway.

We evaluated the effects of down-regulated heme oxygenase (HO)-1 expression on the proliferation of the acute myelocytic leukemia Kasumi-1 cell line by using the HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) in combination with daunorubicin (DNR), and evaluated the mechanism. The proliferation rates of cells treated with 10 mg/mL DNR and 10 mM ZnPPIX individually or in combination for different time periods were detected using the MTT assay. The apoptotic outcomes of the blank control, ZnPPIX, DNR, and ZnPPIX groups in combination with the DNR group were detected by flow cytometry. The expression of HO-1, activating transcription factor 4, CCAAT-enhancer-binding protein homologous protein, and inositol-requiring enzyme-α mRNA and proteins were detected by fluorescent quantitative real-time polymerase chain reaction and western blotting, respectively. Combined administration inhibited the cells most potently and time-dependently, decreased the expression of HO-1, and significantly increased the expression of activating transcription factor 4, CCAAT-enhancer-binding protein homologous protein, and inositol-requiring enzyme-α expression levels. The cell apoptotic rates in the blank control, DNR, ZnPPIX, and combined administration groups were 8.32 ± 0.53, 39.16 ± 1.46, 10.46 ± 0.88, and 56.26 ± 2.24%, respectively. Inhibiting HO-1 expression can enhance the damaging effects of DNR on Kasumi-1 cells, providing experimental evidence for the improvement of therapeutic effects on acute myelocytic leukemia in clinical practice.

Implication of eIF2α kinase GCN2 in induction of apoptosis and endoplasmic reticulum stress-responsive genes by sodium salicylate.

OBJECTIVES: Sodium salicylate (NaSal) can disturb cell viability by affecting the activity of multiple cellular molecules. In this work, we investigated the involvement of stress-responsive kinase GCN2 in regulating cell death and expression of stress genes in mouse embryonic fibroblasts (MEFs) upon exposure to NaSal. METHODS: Cell viability was assayed using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) method, and apoptosis was evaluated by annexin V and propidium iodide staining. A polymerase chain reaction (PCR) array approach was used to analyse differential expression of a panel of 84 endoplasmic reticulum (ER) stress-associated genes. Gene reporter assays were carried out to determine activity of ER stress element (ERSE), and the protein levels of activating transcription factor 6 (ATF6) and C/EBP homologous protein (CHOP) were determined by western blot. KEY FINDINGS: NaSal treatment resulted in reduction of cellular viability and induction of apoptosis in wild-type but not Gcn2(-/-) cells. Many genes with important functions in protein synthesis/degradation, transcriptional regulation and apoptosis were induced by NaSal and most of these were dependent on GCN2. The activation of ERSE within Ddit3 and the production of CHOP and ATF6 induced by NaSal required GCN2. CONCLUSIONS: Our data provide evidence for the involvement of GCN2 in apoptosis and gene expression triggered by NaSal, and contributes to the understanding of molecular events occurring in NaSal-treated cells.

BRCA1 loss induces GADD153-mediated doxorubicin resistance in prostate cancer.

BRCA1 plays numerous roles in the regulation of genome integrity and chemoresistance. Although BRCA1 interaction with key proteins involved in DNA repair is well known, its role as a coregulator in the transcriptional response to DNA damage remains poorly understood. In this study, we show that BRCA1 plays a central role in the transcriptional response to genotoxic stress in prostate cancer. BRCA1 expression mediates apoptosis, cell-cycle arrest, and decreased viability in response to doxorubicin treatment. Xenograft studies using human prostate carcinoma PC3 cells show that BRCA1 depletion results in increased tumor growth. A focused survey of BRCA1-regulated genes in prostate carcinoma reveals that multiple regulators of genome stability and cell-cycle control, including BLM, FEN1, DDB2, H3F3B, BRCA2, CCNB2, MAD2L1, and GADD153, are direct transcriptional targets of BRCA1. Furthermore, we show that BRCA1 targets GADD153 promoter to increase its transcription in response to DNA damage. Finally, GADD153 depletion significantly abrogates BRCA1 influence on cell-cycle progression and cell death in response to doxorubicin treatment. These findings define a novel transcriptional pathway through which BRCA1 orchestrates cell fate decisions in response to genotoxic insults, and suggest that BRCA1 status should be considered for new chemotherapeutic treatment strategies in prostate cancer.

Nitric oxide reduces SLC29A1 promoter activity and adenosine transport involving transcription factor complex hCHOP-C/EBPalpha in human umbilical vein endothelial cells from gestational diabetes.

AIMS: Reduced expression of human equilibrative nucleoside transporter 1 (hENT1) results from nitric oxide (NO)-dependent reduced SLC29A1 transcriptional activity in human umbilical vein endothelial cells (HUVECs) from gestational diabetes. As expression of the transcription factor C/EBP homologous protein 10 (hCHOP, which forms heterodimers with C/EBPalpha transcription factor) is activated by NO and induced in diabetes mellitus, we hypothesize that hCHOP plays a role in the gestational diabetes-reduced hENT1 expression in HUVECs. METHODS AND RESULTS: HUVEC primary cultures from 42 normal and 42 gestational diabetic pregnancies were used for adenosine uptake assays. Real-time PCR (mRNA quantification), western blotting (protein abundance), and luciferase activity (SLC29A1 promoter activity) were used. hCHOP-C/EBPalpha activity was assayed by chromatin immunoprecipitation. Overlap extension mutagenesis was used to generate a mutated hCHOP-C/EBPalpha consensus site at the SLC29A1 promoter, and endothelial NO synthase (eNOS) siRNA recombinant adenovirus was used to knock down eNOS. hCHOP nuclear protein abundance and binding to DNA were higher in gestational diabetes, paralleled by reduced SLC29A1 promoter activity, hENT1 expression, and transport activity. These changes were blocked by hCHOP consensus sequence mutation (-1845G > T and -1844C > A), eNOS-siRNA-induced knockdown, and N(G)-nitro-L-arginine methyl ester (NOS inhibitor), and were mimicked by S-nitroso-N-acetyl-L, D-penicillamine (NO donor) in cells from normal pregnancies. hCHOP and C/EBPalpha overexpression mimicked gestational diabetes effects in cells from normal pregnancies, but did not alter SLC29A1 promoter activity or hENT1-adenosine transport in cells from gestational diabetes. CONCLUSION: The hCHOP-C/EBPalpha complex down-regulates SLC29A1 expression in an NO-dependent manner in HUVECs from gestational diabetes.

Detection of TLS/FUS-CHOP fusion transcripts in a case of oral liposarcoma.

OBJECTIVE: The aim of this study was to detect the chromosomal translocation t(12;16)(q13;p11) that leads to a gene fusion encoding a FUS-CHOP chimeric protein and has been shown to be highly characteristic of myxoid and round cell subtypes of liposarcoma, in a case of oral myxoid liposarcoma. METHOD AND MATERIALS: Nested reverse transcriptase-polymerase chain reaction to detect the TLS/FUS-CHOP fusion gene transcript was performed. A case of inflammatory fibrous hyperplasia and a case of oral lipoma were included as negative controls. RESULTS: Only the myxoid oral liposarcoma showed a 103-base pair product, specific of TLS/FUS-CHOP fusion type II transcript. CONCLUSION: The identification of FUS-CHOP transcript is potentially useful in the diagnosis and research of oral liposarcomas.