Glutamine synthetase regulation by dexamethasone, RU486, and compound A in astrocytes derived from aged mouse cerebral hemispheres is mediated via glucocorticoid receptor.

Glucocorticoids (GCs) regulate astrocyte function, while glutamine synthetase (GS), an enzyme highly expressed in astrocytes, is one of the most remarkable GCs-induced genes. GCs mediate their effects through their cognate glucocorticoid receptor (GRα and GRß isoforms); however, the mechanism via which these isoforms regulate GS activity in astrocytes remains unknown. We used dexamethasone (DEX), a classical GRα/GRß agonist, RU486, which is a specific GRß ligand, and Compound A, a known "dissociated" ligand, to delineate the mechanism via which GR modulates GS activity. Aged Mouse Cerebral Hemisphere astrocytes were treated with DEX (1 µM), RU486 (1 nM-1 µM) or compound A (10 µM), alone or in combination with DEX. GS activity and expression, GR isoforms (mRNA and protein levels), and GRα subcellular trafficking were measured. DEX increased GS activity in parallel with GRα nuclear translocation. RU486 increased GS activity in absence of GRα nuclear translocation implicating thus a role of GRß-mediated mechanism compound A had no effect on GS activity implicating a GRα-GRE-mediated mechanism. None of the compounds affected whole-cell GRα protein content. DEX reduced GRα and GRß mRNA levels, while RU486 increased GRß gene expression. We provide evidence that GS activity, in astrocytes, is regulated via GRα- and GRß-mediated pathways with important implications in pathological conditions in which astrocytes are involved.

Cell-autonomous cytotoxicity of type I interferon response via induction of endoplasmic reticulum stress.

The interaction of IFN with specific membrane receptors that transduce death-inducing signals is considered to be the principle mechanism of IFN-induced cytotoxicity. In this study, the classic non-cell-autonomous cytotoxicity of IFN was augmented by cell-autonomous mechanisms that operated independently of the interaction of IFN with its receptors. Cells primed to produce IFN by 5-azacytidine (5-aza) underwent endoplasmic reticulum (ER) stress. The chemical chaperones tauroursodeoxycholate (TUDCA) and 4-phenylbutyrate (4-PBA), as well as the iron chelator ciclopirox (CPX), which reduces ER stress, alleviated the cytotoxicity of 5-aza. Ablation of CCAAT-enhancer-binding protein homologous protein (CHOP), the major ER stress-associated proapoptotic transcription factor, protected fibroblasts from 5-aza only when the cytotoxicity was examined cell autonomously. In a medium-transfer experiment in which the cell-autonomous effects of 5-aza was dissociated, CHOP ablation was incapable of modulating cytotoxicity; however, neutralization of IFN receptor was highly effective. Also the levels of caspase activation showed a distinct profile between the cell-autonomous and the medium-transfer experiments. We suggest that besides the classic paracrine mechanism, cell-autonomous mechanisms that involve induction of ER stress also participate. These results have implications in the development of anti-IFN-based therapies and expand the class of pathologic states that are viewed as protein-misfolding diseases.-Mihailidou, C., Papavassiliou, A. G., Kiaris, H. Cell-autonomous cytotoxicity of type I interferon response via induction of endoplasmic reticulum stress.

Ciclopirox enhances pancreatic islet health by modulating the unfolded protein response in diabetes.

Pancreatic dysfunction during diabetes is linked to the induction of endoplasmic reticulum (ER) stress on pancreatic beta (ß) cells. Our laboratory recently discovered that p21 protects from diabetes by modifying the outcome of ER stress response. In the present study, we explored the antidiabetic activity of ciclopirox (CPX), an iron chelator and recently described activator of p21 expression. The effects of CPX in beta cell survival and function were assessed in cultured islets in vitro as well as in diabetic mice in vivo. The consequences of CPX in high glucose-induced insulin release and reactive oxygen species (ROS) production were also evaluated. Islet survival assays confirmed the significance of p21 in the regulation of glucotoxicity and suggested that CPX counteracts glucotoxicity in a manner that depends on p21. In vivo, administration of CPX in wild-type (WT) diabetic mice restored glucose homeostasis. In WT-cultured islets, CPX suppressed the expression of ER stress markers BiP, GRP94, and CHOP and reduced the levels of ROS during culture at high glucose. This reduction of ER stress may be associated with the ability of CPX to inhibit insulin release. Iron citrate stimulated insulin release, which was inhibited by CPX that functions as an iron chelator. It is conceivable that inhibition of insulin production constrains ER stress in islets promoting their survival and thus protecting from diabetes in vivo. This unfolded protein response (UPR)-antagonizing activity of CPX suggests application for the management not only of diabetes but also of other conditions related to ER stress.

Systemic effects of AGEs in ER stress induction in vivo.

Emerging evidence indicates that accumulation of advanced glycation end products (AGEs) in human tissues may contribute to cell injury, inflammation and apoptosis through induction of endoplasmic reticulum (ER) stress. Human metabolism relies on ER homeostasis for the coordinated response of all metabolic organs by controlling the synthesis and catabolism of various nutrients. In vitro studies have demonstrated AGE-induced enhancement of unfolded protein response (UPR) in different cell types including endothelial, neuronal, pancreatic cells and podocytes, suggesting this crosstalk as an underlying pathological mechanism that contributes to metabolic diseases. In this minireview, we describe in vivo studies undertaken by our group and others that demonstrate the diverse systemic effects of AGEs in ER stress induction in major metabolic tissues such as brain, kidney, liver and pancreas of normal mice. Administration of high-AGEs content diet to normal mice for the period of 4 weeks upergulates the mRNA and protein levels of ER chaperone Bip (GRP78) indicative of UPR initiation in all major metabolic organs and induces activation of the pivotal transcription factor XBP1 that regulates glucose and lipid metabolism. Furthermore, animals with genetic ablation of UPR-activated transcription factor C/EBP homologous protein CHOP allocated in high-AGEs diet, exhibited relative resistance to UPR induction (BiP levels) and XBP1 activation in major metabolic organs. Since CHOP presents a critical mediator that links accumulation and aggregation of unfolded proteins with induction of oxidative stress and ER stress-related apoptosis, it is revealed as an important molecular target for the management of metabolic diseases.

CHOP-dependent regulation of p21/waf1 during ER stress.

The transcription factor CHOP/GADD153 is induced during the unfolded protein response (UPR) and is associated to the induction of ER stress-related apoptosis. However, how the transition between the pro-survival and the pro-apoptotic role of ER stress is being orchestrated remains poorly understood. Here we show that tunicamycin, an antibiotic promoting ER stress, suppresses the expression of p21, a tumor suppressor that induces cell cycle arrest and inhibits apoptosis. This suppression of p21 levels was independent of p53 that is the major transcriptional regulator of p21, but could be reproduced by forced expression of CHOP. Consistently with these findings, siRNA-mediated inhibition of p21 levels restored the sensitivity of CHOP-deficient cells to tunicamycin. Our findings are consistent with a CHOP-dependent role for p21 in the shift from the pro-survival to the pro-apoptotic function of UPR.

The Role of Adipokines in the Establishment and Progression of Head and Neck Neoplasms.

Adipokines constitute a family of protein factors secreted by white adipose tissue (WAT), that regulate the functions of WAT and other sites. Leptin, adiponectin and resistin, are the main adipokines present in serum and saliva, targeting several tissues and organs, including vessels, muscles, liver and pancreas. Besides body mass regulation, adipokines affect glucose homeostasis, inflammation, angiogenesis, cell proliferation and apoptosis, and other crucial cell procedures. Their involvement in tumor formation and growth is well established and deregulation of adipokine and adipokine receptors' expression is observed in several malignancies including those located in the head and neck region. Intracellular effects of adipokines are mediated by a plethora of receptors that activate several signaling cascades including Janus kinase/ Signal transducer and activator of transcription (JAK/ STAT pathway), Phospatidylinositol kinase (PI3/ Akt/ mTOR) and Peroxisome proliferator-activated receptor (PPAR). The present review summarizes the current knowledge on the role of adipokines family members in carcinogenesis of the head and neck region. The diagnostic and prognostic significance of adipokines and their potential role as serum and saliva biomarkers are also discussed.

Exosomes in lung cancer diagnosis and treatment. From the translating research into future clinical practice.

Lung cancer is one of the main causes of cancer-related death worldwide. Despite advances in lung cancer pathophysiology, diagnosis and prognosis, a better understanding of the disease is strongly needed in order to establish novel diagnostic and therapeutic approaches that should improve treatment outcomes. Exosomes are a type of cell-secreted extracellular vesicles, which transfer a wide variety of biomolecules, such as proteins, mRNAs, microRNAs, and lipids, are implicated in intercellular communication and modulate tumor-host interactions. The potential value of exosomes and their contents in lung cancer diagnosis, prognosis and prediction of treatment outcome is supported by ample literature. Growing attention has been drawn specifically to the critical role of exosomal miRNAs in lung cancer pathogenesis and their potential clinical utility, especially due to their ability to modulate gene expression post-transcriptionally. Owing to their universal presence in the blood and other bodily fluids, exosomes are considered candidate biomarkers. Furthermore, their ability to deliver biomolecules and drugs to recipient cells renders them possible drug delivery vehicles in lung cancer. Here we review the pathological functions of exosomes in cancer and discuss their possible clinical utility as biomarkers and therapeutic agents in the management of lung cancer.

Effect of steviol, steviol glycosides and stevia extract on glucocorticoid receptor signaling in normal and cancer blood cells.

The use of steviol glycosides as non-caloric sweeteners has proven to be beneficial for patients with type 2 diabetes mellitus (T2D), obesity, and metabolic syndrome. However, recent data demonstrate that steviol and stevioside might act as glucocorticoid receptor (GR) agonists and thus correlate with adverse effects on metabolism. Herein, we evaluated the impact of steviol, steviol glycosides, and a Greek-derived stevia extract on a number of key steps of GR signaling cascade in peripheral blood mononuclear cells (PBMCs) and in Jurkat leukemia cells. Our results revealed that none of the tested compounds altered the expression of primary GR-target genes (GILZ, FKPB5), GR protein levels or GR subcellular localization in PBMCs; those compounds increased GILZ and FKPB5 mRNA levels as well as GRE-mediated luciferase activity, inducing in parallel GR nuclear translocation in Jurkat cells. The GR-modulatory activity demonstrated by stevia-compounds in Jurkat cells but not in PBMCs may be due to a cell-type specific effect.

Superior efficacy of the antifungal agent ciclopirox olamine over gemcitabine in pancreatic cancer models.

Ciclopirox olamine (CPX) is an antifungal agent that has recently demonstrated promising anti-neoplastic activity against hematologic and solid tumors. Here, we evaluated CPX compared with gemcitabine alone as well as their combination in human pancreatic cancer cell lines; BxPC-3, Panc-1, and MIA PaCa-2 and in humanized xenograft mouse models. We also examined the preclinical pharmacodynamic activity of CPX. CPX caused a pronounced decrease in cell proliferation and clonogenic growth potential. These inhibitory effects were accompanied by induction of reactive oxygen species (ROS), which were strongly associated with reduced Bcl-xL and survivin levels and activation of a panel of caspases, especially caspase-3, and finally resulted in apoptotic death. CPX-induced apoptosis was associated with reduced pEGFR (Y1068) and pAkt (Ser473) protein levels. Additionally, decreased proliferation was observed in CPX-treated xenografts tumors, demonstrating unique tumor regression and a profound survival benefit. Finally, we showed that CPX significantly abrogated gemcitabine-induced ROS levels in pancreatic tissues. These pre-clinical results have verified the superior antitumor efficacy of CPX over gemcitabine alone, while their combination is even more effective, providing the rationale for further clinical testing of CPX plus gemcitabine in pancreatic cancer patients.

Modulation of Pancreatic Islets' Function and Survival During Aging Involves the Differential Regulation of Endoplasmic Reticulum Stress by p21 and CHOP.

AIMS: Although endoplasmic reticulum (ER) stress is recognized as a major mechanism causing pancreatic dysfunction in diabetes, little is known on how aging modulates the process. Here, we compared the response with ER stress, viability, and insulin release from pancreatic islets of young (6 weeks) or aged (14 months) mice. RESULTS: Islets from aged mice were more sensitive to ER stress than their younger counterparts; they exhibited more pronounced unfolded protein response (UPR) and caspase activation and displayed compromised insulin release after high-glucose stimulation. Genetic ablation of p21 sensitized the islets to ER stress, especially in the aged group, whereas CHOP ablation was protective for islets from both aged and younger animals. Ciclopirox (CPX), an iron chelator that stimulates p21 expression, protected islets from glucotoxicity and mice from diet-induced diabetes, especially in the aged group in a manner that was both p21 and CHOP dependent. INNOVATION: For the first time, the study shows that age-dependent susceptibility to diet-induced diabetes is associated with the activity of p21 and CHOP in pancreatic islets and that CPX protects islets from glucotoxicity and mice from diabetes in an age-dependent manner. CONCLUSIONS: Our results identify ER stress as an age-dependent modifier of islet survival and function by mechanisms implicating enhancement of CHOP activity and inhibition of the protective activity of p21. These findings suggest that interventions restoring the homeostatic activity of ER stress, by agents such as CPX, may be particularly beneficial for the management of diabetes in the elderly. Antioxid. Redox Signal. 27, 185-200.

Co-targeting of EGFR and autophagy signaling is an emerging treatment strategy in metastatic colorectal cancer.

The epidermal growth factor receptor (EGFR) and its associated pathway is a critical key regulator of CRC development and progression. The monoclonal antibodies (MoAbs) cetuximab and panitumumab, directed against EGFR, represent a major step forward in the treatment of metastatic colorectal cancer (mCRC), in terms of progression-free survival and overall survival in several clinical trials. However, the activity of anti-EGFR MoAbs appears to be limited to a subset of patients with mCRC. Studies have highlighted that acquired-resistance to anti-EGFR MoAbs biochemically converge into Ras/Raf/Mek/Erk and PI3K/Akt/mTOR pathways. Recent data also suggest that acquired-resistance to anti-EGFR MoAbs is accompanied by inhibition of EGFR internalization, ubiqutinization, degradation and prolonged downregulation. It is well established that autophagy, a self-cannibalization process, is considered to be associated with resistance to the anti-EGFR MoAbs therapy. Additionally, autophagy induced by anti-EGFR MoAbs acts as a protective response in cancer cells. Thus, inhibition of autophagy after treatment with EGFR MoAbs can result in autophagic cell death. A combination therapy comprising of anti-EGFR MoAbs and autophagy inhibitors would represent a multi-pronged approach that could be evolved into an active therapeutic strategy in mCRC patients.

Co-targeting c-Met and DNA double-strand breaks (DSBs): Therapeutic strategies in BRCA-mutated gastric carcinomas.

Gastric cancer (GC) is a threatening malignancy characterized by heterogeneity. Current therapies use DNA damaging agents, for example, chemotherapeutic agents and ionizing radiation (IR). However, a significant portion of GC patients develops therapeutic resistance to DNA damage response (DDR) - inducing agents. An important mechanism is the stimulation of the c-MET RTK, which is a tyrosine kinase receptor and its ligand hepatocyte growth factor (HGF), which facilitates cell survival by boosting DNA damage repair pathways and via escaping cell cycle arrest. A small subgroup of GC diagnosed patients has defects in BRCA1 and BRCA2 as mediators of DNA repair proteins. BRCA1/2 related-tumors acquire resistance to chemotherapy through the DSBs (DNA double strand breaks) repair pathways. However, BRCA2-deficient cells, are vulnerable to PARP [poly (ADP-ribose) polymerase] inhibitors as the replication forks collapse and the DNA-induced damage is not reversed. Herein, we pose that taking into consideration the defective DDR machinery can trigger GC cell sensitization to therapies via inhibition of DNA repair response. Inhibition of DNA damage response axis may designate cancer cells with BRCAness (BRCA-mutant cells) more vulnerable to DNA-damaging mediators, such as c-Met inhibitors.

Crosstalk between C/EBP homologous protein (CHOP) and glucocorticoid receptor in lung cancer.

Loss of homeostasis triggers the endoplasmic reticulum (ER) stress response and activates the unfolded protein response (UPR) resulting in the induction of the CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP). Glucocorticoids (GCs), via the glucocorticoid receptor (GR), regulate numerous physiological processes in an effort to maintain homeostasis. Previous studies demonstrated that glucocorticoids suppress ER stress by enhancing correct folding of secreted proteins and degradation of misfolded proteins. Here, we describe a novel crosstalk between ER-stress and the glucocorticoid receptor signaling. We showed that treatment of wild type mice with Tunicamycin (inducer of ER-stress) increased GR protein levels in the lungs. Treatment of A549 cells (human lung cancer cells) with ER stress inducers modulated the Dexamethasone-induced subcellular localization of GR and the phosphorylated forms of GR (pGRSer211 and pGRSer203) with concomitant changes in the expression of primary GR-target genes. We demonstrated a significant protein-protein interaction between GR and CHOP, (GR-CHOP heterocomplex formation) under ER stress conditions. The functional consequences of ER stress- GR signaling crosstalk were assessed and demonstrated that long time exposure (24-48 h) of A549 cells to dexamethasone (10(-6) M) reversed the Tunicamycin-induced cell death, a phenomenon associated with parallel increases in GR protein content, increases in cell survival parameters and decreases in cell apoptosis-related parameters. Our study provides evidence that there is a cross talk between ER-stress and GR signaling, this being associated with mutual functional antagonism between CHOP and GR-mediated pathways in lung cells with important implications in lung cell function.

Regulation of P21 during diabetes-associated stress of the endoplasmic reticulum.

Endoplasmic reticulum (ER) stress plays a major role in the pathogenesis of diabetes by inducing ß-cell apoptosis in the islets of Langerhans. In this study, we show that the transcription factor CHOP, which is instrumental for the induction of ER-stress-associated apoptosis and the pancreatic dysfunction in diabetes, regulates the expression of P21 (WAF1), a cell cycle regulator with anti-apoptotic activity that promotes cell survival. Deficiency of P21 sensitizes pancreatic ß-cells to glucotoxicity, while in mice genetic ablation of P21 accelerates experimental diet-induced diabetes, results indicative of a protective role for P21 in the development of the disease. Conversely, pharmacological stimulation of P21 expression by nutlin-3a, an inhibitor of P53-MDM2 interaction, restores pancreatic function and facilitates glucose homeostasis. These findings indicate that P21 acts as an inhibitor of ER-stress-associated tissue damage and that stimulation of P21 activity can be beneficial for the management of diabetes and probably of other conditions in which ER-stress-associated death is undesirable.

Improvement of chemotherapeutic drug efficacy by endoplasmic reticulum stress.

Tunicamycin (TUN), an inhibitor of protein glycosylation and therefore a potent stimulator of endoplasmic reticulum (ER) stress, has been used to improve anticancer drug efficacy, but the underlying mechanism remains obscure. In this study, we show that acute administration of TUN in mice induces the unfolded protein response and suppresses the levels of P21, a cell cycle regulator with anti-apoptotic activity. The inhibition of P21 after ER stress appears to be C/EBP homologous protein (CHOP)-dependent because in CHOP-deficient mice, TUN not only failed to suppress, but rather induced the expression of P21. Results of promoter-activity reporter assays using human cancer cells and mouse fibroblasts indicated that the regulation of P21 by CHOP operates at the level of transcription and involves direct binding of CHOP transcription factor to the P21 promoter. The results of cell viability and clonogenic assays indicate that ER-stress-related suppression of P21 expression potentiates caspase activation and sensitizes cells to doxorubicin treatment, while administration of TUN to mice increases the therapeutic efficacy of anticancer therapy for HepG2 liver and A549 lung cancers.

A crosstalk between p21 and UPR-induced transcription factor C/EBP homologous protein (CHOP) linked to type 2 diabetes.

Type 2 diabetes (T2D) is a disease that is characterized by raised levels of glucose in the blood combined with insulin resistance and relative insulin deficiency. The pathogenesis of type 2 diabetes is associated with the induction of the unfolded protein response (UPR). While UPR aims to restore tissue homeostasis following stress of the endoplasmic reticulum (ER), prolonged ER stress triggers apoptosis at least in part through the unfolded protein response (UPR)-activated transcription factor C/EBP (CCAAT/enhancer binding protein) homologous protein (CHOP). CHOP has elevated as a critical mediator connecting accumulation and aggregation of unfolded proteins in the ER and oxidative stress and also contributes to the induction of apoptosis in ß-cell (beta-cell) - cells under conditions of increased insulin demand. p21 is a cell cycle regulator that is implicated in the regulation of the UPR by various mechanisms involving inhibition of apoptosis and facilitation of the regeneration capacity of the ß cells. In this review we summarize the role of ER stress in the pathogenesis of type 2 diabetes which is associated with the induction of the unfolded protein response (UPR). We also review recent evidence associating p21 activity with ß cell health and regenerative capacity by mechanisms that may interfere with the effects of p21 in the UPR or operate independently of ER stress. Most likely understanding the molecular details of the pathogenesis of type 2 diabetes will be beneficial for the management of the disease.