H. Abdel-Aziz

Modulatory effects of melatonin and vitamin E on doxorubicin-induced cardiotoxicity in Ehrlich ascites carcinoma-bearing mice.

Doxorubicin (Dox), an anthracycline antibiotic, has a wide spectrum of antitumor activity with dose-limiting cardiotoxicity. The drugs toxicity is known to be closely related to the generation of active oxygen free radicals. In our study the normal cardiac tissue contents of total protein, glutathione (GSH) and malondialdehyde (MDA) were significantly decreased, by 25%, 33% and 92%, respectively, in the group of mice bearing Ehrlich ascites carcinoma (EAC) and treated with Dox (4 mg/kg/week x 2, ip). Administration of melatonin (5 mg/kg/day x 15, po) starting 24 hours prior to Dox treatment significantly increased the cardiac contents of total protein and GSH as well as the superoxide dismutase (SOD) activity, by 31%, 36% and 39%, respectively, compared to treatment with Dox only, while the content of MDA was decreased by 26%. Similarly, administration of vitamin E (250 mg/kg/day x 15, po) starting 24 hours prior to Dox treatment significantly increased the cardiac contents of total protein, GSH and SOD, by 23%, 26% and 42%, respectively, while the cardiac content of MDA was decreased by 35% compared with the Dox-only-treated group. As to the oncolytic activity of Dox, pretreatment of EAC-bearing mice with melatonin (5 mg/kg/day x 30, po) or vitamin E (250 mg/kg/day x 30, po) 24 hours prior to Dox administration (4 mg/kg/week x 4, ip) improved the antitumor activity of Dox as indicated by the increase in the average life span of the animals and the number of long-term survivors as well as the decrease in body weight loss induced by Dox treatment. It is clear from these results that administration of melatonin not only protects against the cardiotoxicity induced by Dox treatment but also enhances its antitumor activity to a more significant extent than does vitamin E.

CXCL16 Is Expressed in Podocytes and Acts as a Scavenger Receptor for Oxidized Low-Density Lipoprotein

Podocytes are a crucial cell type in the kidney and play an important role in the pathology of glomerular kidney diseases like membranous nephropathy (MN). The identification of new factors involved in the progression of glomerular kidney diseases is of great importance to the development of new strategies for the treatment of renal injury. Here we demonstrate that CXCL16 and ADAM10 are constitutively expressed in human podocytes in normal renal tissue. Proinflammatory cytokines like interferon-gamma and tumor necrosis factor-alpha induced the expression of cellular CXCL16 and the release of its soluble form from human podocytes. Using different metalloproteinase inhibitors, we provide evidence that ADAM10 is involved in the interferon-gamma- and tumor necrosis factor-alpha-induced shedding of CXCL16 from human podocytes. In addition, ADAM10 knockdown by siRNA significantly increased both CXCL16 levels and, surprisingly, its ADAM17-mediated release. Notably, targeting of CXCL16 in human podocytes both decreased the chemotaxis of CXCR6-expressing T cells and strongly reduced oxidized low-density lipoprotein uptake in human podocytes. Importantly, in kidney biopsies of patients with MN, increased glomerular CXCL16 expression was accompanied by high levels of oxidized low-density lipoprotein and decreased expression of ADAM10. In addition, we found increased glomerular ADAM17 expression in patients diagnosed with MN. In summary, we presume important roles for CXCL16, ADAM10, and ADAM17 in the development of MN, suggesting these proteins as new therapeutic targets in this glomerular kidney disease.

CXCL16 and oxLDL are induced in the onset of diabetic nephropathy.

Diabetic nephropathy (DN) is a major cause of end‐stage renal failure worldwide. Oxidative stress has been reported to be a major culprit of the disease and increased oxidized low density lipoprotein (oxLDL) immune complexes were found in patients with DN. In this study we present evidence, that CXCL16 is the main receptor in human podocytes mediating the uptake of oxLDL. In contrast, in primary tubular cells CD36 was mainly involved in the uptake of oxLDL. We further demonstrate that oxLDL down‐regulated α3‐integrin expression and increased the production of fibronectin in human podocytes. In addition, oxLDL uptake induced the production of reactive oxygen species (ROS) in human podocytes. Inhibition of oxLDL uptake by CXCL16 blocking antibodies abrogated the fibronectin and ROS production and restored α3 integrin expression in human podocytes. Furthermore we present evidence that hyperglycaemic conditions increased CXCL16 and reduced ADAM10 expression in podocytes. Importantly, in streptozotocin‐induced diabetic mice an early induction of CXCL16 was accompanied by higher levels of oxLDL. Finally immunofluorescence analysis in biopsies of patients with DN revealed increased glomerular CXCL16 expression, which was paralleled by high levels of oxLDL. In summary, regulation of CXCL16, ADAM10 and oxLDL expression may be an early event in the onset of DN and therefore all three proteins may represent potential new targets for diagnosis and therapeutic intervention in DN.

Effect of Glutathione Modulation On the Distribution and Transplacental Uptake of 2-[14C]-Chloroacetonitrile (Can) Quantitative Whole-Body Autoradiographic Study in Pregnant Mice

Chloroacetonitrile (CAN), a drinking water disinfectant by-product, has mutagenic and carcinogenic properties. CAN is known to deplete glutathione (GSH), and previous studies reported an enhanced molecular interaction of CAN after GSH depletion in the uterine and fetal tissues of mice. The present report may help to understand the potential mechanisms involved in such molecular interactions by examining the disposition, transplacental uptake and covalent interaction of the chemical in normal and GSH depleted pregnant mice (at 13th day of gestation). Both normal and GSH depleted (by administration of Diethylmaleate (DEM), 0.6 mL/kg, i.p.) pregnant mice were given an equitoxic i.v. dose of 2-[14C]-CAN (333 μCi/kg equivalent to 77 mg/kg). Animals were processed for whole-body autoradiography (WBA) at 1, 8 and 24 hr after treatment. Tissue distribution of radioactivity in the autoradiographs was quantitated using computer aided image analysis. With few exceptions, a rapid high uptake (at I hr) of radioactivity was observed in all major maternal (liver, lung, urinary bladder, gastrointestinal mucosa, cerebellum, uterine luminal fluid) and fetal (liver, brain) organs of both normal and GSH depleted mice. This pattern of distribution was observed, with lesser intensity, at 8 hr following treatment. At a later time period (24 hr), there was a significant higher retention and covalent interaction of radioactivity in GSH depleted mouse tissues especially in the liver as compared to normal mouse. This study suggests that 2-[14C]-CAN and/or its metabolites are capable of crossing the placental barrier. The observed higher uptake and retention of the radioactivity in the maternal liver, kidney, cerebellum, nasal turbinates and fetal liver may pose toxicity of the chemical to these organs. The increased covalent interaction of radioactivity in GSH depleted mice liver may indicate the potential utilization of GSH pathway by this organ in the detoxication of CAN derived metabolites and thus exerting hepatotoxicity.

Protective effect of N-acetylcysteine against carmustine-induced myelotoxicity in rats.

Carmustine (BCNU) is used to treat a variety of tumors, in particular gliomas. However, the success of such treatment is limited by severe myelosuppression. The role of N-acetylcysteine (NAC) in protection against BCNU-induced myelotoxicity is still needed to be explored. The aim of this work is to study the possible protective role of NAC against BCNU-induced myelotoxicity through evaluation of apoptosis, lipid peroxidation, antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase(CAT)) as well as glutathione (GSH) content in bone marrow cells of rats. Administration of BCNU in a single dose (30 mg/kg, i.p.) significantly decreased RBCs, WBCs and platelets counts as well as hemoglobin level. In addition, BCNU produced a significant apoptotic effect as well as a significant lipid peroxidation in bone marrow cells. Pretreatment of animals with NAC (150 mg/kg, i.p.) daily for 5 days significantly ameliorated the changes in oxidant and antioxidant parameters as well as apoptosis induced by BCNU. In addition the pattern of blood parameters was shifted markedly to normal values in animals pretreated with NAC when compared to BCNU-treated group. Conclusively, NAC could have a potential protective effect against BCNU-induced myelotoxicity; an effect that is mainly attributed to the antioxidant property.

All-trans retinoic acid potentiates cisplatin-induced kidney injury in rats: impact of retinoic acid signaling pathway

Cisplatin (cis-diammine dichloroplatinum (II), CDDP) is a widely used drug for treatment of various types of cancers. However, CDDP-induced nephrotoxicity remains the main dose-limiting side effect. Retinoids are a group of vitamin A-related compounds that exert their effects through retinoid receptors activation. In this study, we investigated the effect of CDDP treatment on retinoic acid receptor-α (RAR-α) and retinoid X receptor-α (RXR-α) expression. In addition, we investigated the possible modulatory effects of RAR agonist, all-trans retinoic acid (ATRA), on CDDP-induced nephrotoxicity. Rats were treated with saline, DMSO, CDDP, ATRA, or CDDP/ATRA. Twenty-four hours after the last ATRA injection, rats were killed; blood samples were collected; kidneys were dissected; and biochemical, immunohistochemical, and histological examinations were performed. Our results revealed that CDDP treatment significantly increased serum levels of creatinine and urea, with concomitant decrease in serum albumin. Moreover, reduced glutathione (GSH) content as well as superoxide dismutase (SOD) and catalase (CAT) activities were significantly reduced with concurrent increase in kidney malondialdehyde (MDA) content following CDDP treatment. Furthermore, CDDP markedly upregulated tubular RAR-α, RXR-α, fibrin, and inducible nitric oxide synthase (iNOS) protein expression. Although administration of ATRA to control rats did not produce marked alterations in kidney function parameters, administration of ATRA to CDDP-treated rats significantly exacerbated CDDP-induced nephrotoxicity. In addition, CDDP/ATRA co-treatment significantly increased RAR-α, RXR-α, fibrin, and iNOS protein expression compared to CDDP alone. In conclusion, we report, for the first time, the crucial role of retinoid receptors in CDDP-induced nephrotoxicity. Moreover, our findings indicate that co-administration of ATRA with CDDP, although beneficial on the therapeutic effects, their deleterious effects on the kidney may limit their clinical use.

Abstract 4054: Elongation factor-2 kinase (eEF-2K) promotes cell invasion and epithelial mesenchymal transition through regulation of TG2-mediated signaling in human pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) accounts for the majority of pancreatic cancer (PaCa) cases and is currently uncurable with poor prognosis/survival rates (∼4% 5-year survival). The extensive local tumor invasion, early metastasis/systemic dissemination and resistance to existing cancer therapies are the major characteristics of PaCa and the impediment to effective cure of this disease. Although PaCa has a well-defined spectrum of highly oncogenic lesions (e.g, mutations in K-RAS, p53, p16ink4a, and SMAD4/DPC4), effective therapies have not yet been developed. Therefore, novel molecular targets-based therapeutic strategies are urgently needed. Recently, we discovered that eukaryotic elongation factor-2 kinase (eEF-2K) is dramatically up-regulated in different PaCa cells, promoting their survival, and that targeted-silencing of eEF-2K led to significant cell death (Ashour et al, 2013). However, the role of eEF-2K in the pathways regulating invasion/metastasis and drug-resistance remains largely unknown. Here, we show that siRNA-mediated knockdown of eEF-2K markedly inhibits the invasion of PANC-1and MIAPaCa-2 cells. Furthermore, rottlerin, which we found to down-regulate eEF-2K expression, also significantly reduced the invasion of both cell lines. We later investigated the involvement of tissue transglutaminase (TG2), a multifunctional enzyme implicated in regulation of cell survival, migration and invasion. We found that eEF-2K regulates TG2 at the transcription level, as evidenced by eEF-2K knockdown-mediated down-regulation of TG2 protein and mRNA levels by Western blot and RT-PCR, respectively. Importantly, siRNA-mediated knock-down of eEF-2K or TG2 separately, suppresses the downstream key cellular pathways supporting PaCa invasion, and recapitulates rottlerin-induced invasion inhibition and correlated events. We also found that inhibition of eEF-2K/TG2 axis suppresses the epithelial-mesenchymal transition (EMT) through the modulation of ZEB1 zinc finger transcription factors, and tight junction proteins, claudins. Moreover, eEF-2K overexpression, by lentivirus-based expression system, led to increased TG2 expression, with concomitant induction in the invasion capability of PaCa cells. eEF-2K overexpression also rescued the cells from rottlerin-induced suppression of invasion and invasion-supporting machineries, suggesting that eEF-2K is a prominent target of rottlerin, and is a central regulator of PaCa cell invasion. We are currently treating PANC-1 tumor xenografts in nude mice with eEF-2K siRNA-nanoparticles to demonstrate the role of eEF-2K in invasion and tumor progression, in order to establish eEF-2K as a potential novel therapeutic target in PaCa. Collectively, our data suggest, for the first time, the pivotal role of eEF-2K in PaCa invasion/progression and possibly tumor growth. Citation Format: Ahmed A. Ashour, Sultan N. Alpay, Nilgun Gurbuz, Abdel-Aziz H. Abdel-Aziz, Ahmed M. Mansour, Bulent Ozpolat. Elongation factor-2 kinase (eEF-2K) promotes cell invasion and epithelial mesenchymal transition through regulation of TG2-mediated signaling in human pancreatic cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4054. doi:10.1158/1538-7445.AM2014-4054

Abstract 848: Inhibition of eukaryotic elongation factor-2 kinase mediates Rottlerin induced effects in apoptosis and cell proliferation inhibition in human pancreatic cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pancreatic cancer (PaCa) is one of the most aggressive cancers and currently incurable with less than 2% five-years survival rate. Among the ever-increasing list of naturally occurring anticancer agents, a Kmala Tree-derived anticancer compound, Rottlerin (ROT), a PKC-delta inhibitor, appears to have anti-proliferative activity due to its effects on several pathways and cell machineries involved in cell survival, apoptosis, autophagy and invasion. Recent studies suggest that ROT is not a specific PKC delta inhibitor and mediates its effects through other mechanisms. We have previously reported that inhibition of eukaryotic elongation factor-2 kinase (eEF-2K), one of the major kinases appears to be activated in rapidly proliferating malignant cells, leads to down-regulation of signaling pathways affecting growth, survival and chemotherapeutic resistance (Tekedereli et al, 2012). Thus, we hypothesized that ROT targets eEF-2K, and inhibition of eEF-2K signaling is responsible of mediating its effects in PaCa cells. We found that ROT treatment (4-10 μM) inhibits the expression of eEF-2K that was associated with apoptosis induction in PaCa cells in a dose and time-dependent manner as detected by Western blot analysis. We also found that ROT treatment inhibits PaCa cell proliferation and mitochondrial activity and modulates G1/S phase progression in these cells. Treating PaCa cells with ROT markedly induced expression of active caspase-9, caspase-3 and cleaved PARP. To demonstrate a direct link between eEF-2K inhibition and ROT-induced effects, we knocked-down eEF-2K by a specific siRNA. Such knockdown was associated with inhibition of cell growth, resulted in modulation of caspase-related events with induction of similar degree of apoptosis, increased the percentage of Annexin V positive apoptotic cells and resulted in induction of cellular shrinkage and blebbing. Moreover, we found that eEF-2K down-regulation is involved in ROT-induced stimulation of extrinsic apoptotic pathway in PaCa cells with concomitant induction of TNF related apoptosis inducing ligand (TRAIL) receptor DR4 and caspase-8 cleavage. In addition, our findings suggest that eEF-2K contributes to the regulation of G1/S phase progression, as in vitro down-modulation eEF-2K resulted in an increase in the expression of p27Kip1 cyclin-dependent kinase inhibitor (CDKI). Collectively, the results of our study demonstrate, for the first time, that down-regulation of eEF-2K contributes to rottlerin-induced effects including, growth inhibition and apoptosis in PaCa cells and clearly demonstrate novel mechanisms of ROT-induced potent antitumor effects. These findings show that the multi-targeted kinase inhibitor, ROT, represents a promising novel agent for PaCa treatment and eEF-2K could represent an attractive target for the future anticancer agents. Citation Format: Ahmed A. Ashour, Abdel-Aziz H. Abdel-Aziz, Ahmed M. Mansour, Sultan N. Alpay, Kevin Dalby, Bulent Ozpolat. Inhibition of eukaryotic elongation factor-2 kinase mediates Rottlerin induced effects in apoptosis and cell proliferation inhibition in human pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 848. doi:10.1158/1538-7445.AM2013-848