Tarek H. El-Metwally

The effectiveness of retinoic acid treatment in bladder cancer Impact on recurrence, survival and TGFα and VEGF as end-point biomarkers

Introduction and Aims: Being best-studied superficial bladder cancer (SBC) chemopreventives, retinoids’ negative studies and toxicity were stumbling. With proper understanding of retinoid metabolism, we aimed at investigating combined ketoconazole (a strong inhibitor of retinoic acid-catabolizing cytochrome P450s)-all-trans retinoic acid (Keto-atRA) SBC treatment. VEGF and TGF-α levels are end-point pathogenetic biomarkers involved in early SBC. Material and Methods: Seven days after TURT visible tumor(s), combined atRA 1 mg/kg for 5 days/week + Keto 200 mg twice daily for 5 days/week for 3 months were given to 16 patients with SBC stages Ta and T1 with various grades. 3 months follow up/20 months used white light cystoscopy and urinary cytology. Recurrence rate and survival time were compared to a retrospective group of 25 patients of comparable age, stage and grade with TURT as sole treatment for SBC. VEGF and TGF-α were measured in urine and serum of 12 normal subjects and treated patients. Samples were collected just before TURT, 1 week after TURT, at the end of 1 month and at the end of 3 months of treatment. Results: Keto-atRA treatment significantly improved survival time and decreased recurrence rate compared to control disease group, with tolerable and reversible side-effects. Treatment normalized induced levels of VEGF and TGF-α with a positive correlation between these cytokines. Conclusions: The combination and schedule used for Keto-atRA therapy effectively reduced recurrence rate and increased survival time of SBC patients probably through reduction of VEGF and TGF-α as major mitogenic/angiogenic factors; possibly by eliminating malignant cells that produce them.

Natural retinoids inhibit proliferation and induce apoptosis in pancreatic cancer cells previously reported to be retinoid resistant

Background: The anticancer ability of natural retinoids on pancreatic adenocarcinoma, an aggressive tumor, is still controversial. This investigation tested the hypothesis that all-trans retinoic acid can inhibit proliferation and induce apoptosis in pancreatic cancer cell lines. Materials and methods: Using our previously optimized conditions, the effect of all-trans retinoic acid (atRA, 0.001 - 10 µM) was tested in ten human pancreatic adenocarcinoma cell lines with various degrees of differentiation. Proliferation was monitored by cell number, [3H]-thymidine incorporation and cell cycle arrest. Apoptosis was investigated morphologically by light and electron microscopy and biochemically by tissue transglutaminase activity (TGase), mitochondrial membrane potential, cell cycle analysis of sub-G1 cells and detection of fragmented DNA (fragmentation of prelabeled DNA, agarose electrophoresis and TUNEL assays). Results: Retinoic acid caused potent concentration- and time-dependent inhibition of proliferation of all cell lines studied. Cell cycle was arrested at G1 or G2 with extensive reduction of number of cells at S-phase after 24 hours of treatment with apoptotic concentration of atRA. Complete inhibition of proliferation was followed by apoptosis as indicated by the progressive accumulation of sub-G1 apoptotic cells which was confirmed by the more specific DNA fragmentation assays. There were extensive apoptosis-indicative light and electron microscopic changes preceded by phenotypic redifferentiation. TGase was induced between 3 - 5-folds the control level and its inhibition partially reversed the antiproliferative effect of atRA. Cellular viability during the preapoptotic stage was confirmed by normal mitochondrial membrane potential in the first two days of treatment with the maximum atRA concentration used. However, the potential was progressively reduced with time as a preapoptotic change. Caspase 3-like activity was induced by the apoptotic concentrations of atRA at late time points. However, the redifferentiation indicative changes were not prevented by cotreatment with Ac-DEVE-CHO caspase 3 inhibitor. Conclusions: Together, our results demonstrated the efficient anticancer ability of natural retinoids on human pancreatic cancer cell lines tested, even those previously reported to be retinoid resistant.

Hypoxia Biomarkers, Oxidative Stress, and Circulating Microparticles in Pediatric Patients With Thalassemia in Upper Egypt:

This study aimed to investigate the oxidative stress, hypoxia biomarkers, and circulating microparticles (MPs) in β thalassemia major. The study included 56 children with thalassemia and 46 healthy controls. Hypoxia biomarkers, oxidative stress biomarkers, and total plasma fragmented DNA (fDNA) were detected by the standard methods. The MPs were assessed by flow cytometry. Hypoxia and oxidative stress biomarkers, fDNA, and MPs were higher and total antioxidant capacity (TAC) was lower in patients with thalassemia than the controls. In splenectomized patients and those who had complications, vascular endothelial growth factor (VEGF), malondialdehyde, fDNA, endothelial, platelet, and activated platelet MP levels were higher while, TAC was lower than the nonsplenectomized patients. In conclusion, the increased tissue hypoxia, oxidative stress in β thalassemia, and its relationship with DNA damage and MPs release could explain many complications of thalassemia and may have therapeutic implications. The VEGF could serve as an important indicator for adequacy of blood transfusion in thalassemia.

Retinoic acid can induce markers of endocrine transdifferentiation in pancreatic ductal adenocarcinoma: preliminary observations from an in vitro cell line model.

Background and hypothesis: The pancreatic ductal adenocarcinoma (HPAF) cells have a multipotent stem cell potential. It was hypothesised that all-trans-retinoic acid (atRA) can induce transdifferentiation of these cells into cells with an endocrine phenotype. Material and methods: To explore this hypothesis, an in vitro system of cells was established. Some cells were treated with atRA at concentrations of 100 nmol/l (non-apoptosis-inducing) and 5 μmol/l (apoptosis-inducing) and harvested. Cells were examined for cell cycle kinetics, apoptosis (terminal deoxynucleotidyl transferase assay and p53 protein expression) and immunomorphological features of redifferentiation (MUC1 and DUPAN-2) and endocrine transdifferentiation (insulin, somatostatin, glucagon, neurone-specific enolase) by using immunoperoxidase staining methods. Levels of insulin, transforming growth factor (TGF) β2, TGFα and epidermal growth factor receptor (EGFR) were measured by enzyme-linked immunosorbent assay (ELISA). The vehicle-treated cells served as a control group. Results: When compared with untreated cells, cells treated with 100 nmol/l and 5 μmol/l atRA were observed to show (1) decreased proliferative activity (cpm) as indicated by decreased incorporation of thymidine labelled with hydrogen-3; (2) cell cycle arrest; (3) increased apoptotic activity associated with p53 protein overexpression; (4) upregulated expression of the transdifferentiation and redifferentiation markers; (5) morphological changes indicative of transdifferentiation (increased cell size and appearance of dendrites); (6) decreased production of EGFR; (7) upregulation of TGFα and TGFβ2; and (8) increase in basal and glucose-induced insulin secretion. Conclusions: Functional endocrine transdifferentiation can be induced in HPAF lines by atRA. Further investigations are mandated to explore the underlying mechanisms of this transdifferentiation and to explore its in vivo extrapolation.

Dyslipidemia, oxidative stress and cardiac dysfunction in children with chronic renal failure: effects of L-carnitine supplementation.

BACKGROUND Secondary carnitine deficiency may develop in chronic renal failure (CRF) patients undergoing long-term hemodialysis (HD), with a resulting higher incidence of cardiovascular diseases, dyslipidemia and oxidative stress. We studied the efficacy of 12 months of L-carnitine supplementation on the amelioration of dyslipidemia, oxidative stress and cardiac dysfunction in 24 CRF children undergoing long-term HD compared with 24 age- and sex-matched controls. METHODS Plasma samples were analyzed spectrophotometerically before and after dialysis sessions and after 2-month supplementation with oral L-carnitine (50 mg/kg/day) for free carnitine (FC), the lipid profile, and oxidative stress markers. Echocardiography the day following dialysis measured cardiac diameters, wall thicknesses, left ventricular mass index (MI), end diastole and systole volume indices and functions. RESULTS The pre-dialysis FC concentration was substantially lower than controls and decreased significantly at the end of the dialysis session. Pre- and post-dialysis plasma levels of free fatty acids (FFAs), trigyleride (TG), total cholesterol (TC) and oxidative stress markers significantly increased while high-density lipoprotein cholesterol (HDL-C) and phospholipids significantly decreased compared to controls. Echocardiography detected a significant increase in cardiac diameters and thickness, and systolic and diastolic cardiac dysfunction. After L-carnitine supplementation, plasma levels of FC increased to normal levels. FFAs, TC and HDL-C returned to control levels while TG, phospholipids, and the oxidative stress markers decreased but remained significantly higher than controls. There was a significant decrease in cardiac diameters and an increase in left ventricular diastolic function (E/A ratio), but no correlation between FC levels and echocardiographic parameters. Pre-dialysis, post-dialysis and after treatment, plasma FC level showed a significant positive correlation with HDL-C and phospholipids and a significant negative correlation with each of oxidative stress markers, FFAs, TG and TC. On the other hand, FFAs showed a significant positive correlation with TG, TC, DC, NO and a significant negative correlation with HDL-C and phospholipids. CONCLUSION This study demonstrates that CRF children under regular HD suffer from a decrease in the level of plasma FC, dyslipidemia, oxidative stress, and an increase in cardiac diameters and thickness with impairment of cardiac functions. Oral L-carnitine supplementation at a dose of 50 mg/kg for 2 months can increase plasma FC level, improve dyslipidemia, decrease oxidative stress with reduction of cardiac diameters and increase in diastolic function.

Hypoxia and oxidative stress markers in pediatric patients undergoing hemodialysis: cross section study

BackgroundTissue injury due to hypoxia and/or free radicals is common in a variety of disease processes. This cross-sectional study aimed to investigate effect of chronic kidney diseases (CKD) and hemodialysis (HD) on hypoxia and oxidative stress biomarkers.MethodsForty pediatric patients with CKD on HD and 20 healthy children were recruited. Plasma hypoxia induced factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) were measured by specific ELISA kits while, total antioxidant capacity (TAC), total peroxide (TPX), pyruvate and lactate by enzymatic/chemical colorimetric methods. Oxidative stress index (OSI) and lactate/pyruvate (L/P) ratio were calculated.ResultsTAC was significantly lower while TPX, OSI and VEGF were higher in patients at before- and after-dialysis session than controls. Lactate and HIF-1α levels were significantly higher at before-dialysis session than controls. Before dialysis, TAC and L/P ratio were lower than after-dialysis. In before-dialysis session, VEGF correlated positively with pyruvate, HIF-1α and OSI correlated positively with TPX, but, negatively with TAC. In after-dialysis session, HIF-1α correlated negatively with TPX and OSI; while, OSI correlated positively with TPX.ConclusionsCKD patients succumb considerable tissue hypoxia with oxidative stress. Hemodialysis ameliorated hypoxia but lowered antioxidants as evidenced by decreased levels of HIF-1α and TAC at before- compared to after-dialysis levels.

Iron Chelation Therapy in Upper Egyptian Transfusion-dependent Pediatric Homozygous β-Thalassemia Major: Impact on Serum L-Carnitine/Free Fatty Acids, Osteoprotegerin/The Soluble Receptor Activator of Nuclear Factor-κβ Ligand Systems, and Bone Mineral Density

Bone disease in β-thalassemia major (βTM) remains poorly understood. Receptor activator of nuclear factor-κβ ligand (RANKL) regulates osteoclast formation and function. RANKL activity is balanced by interaction with its receptor (RANK) and binding to osteoprotegerin (OPG). L-Carnitine (LC) enhances osteoblastic activity by furnishing fuel. This study hypothesized that abnormal bone metabolism in βTM involves imbalanced RANKL/OPG and LC/free fatty acids (FFAs) metabolism. Sixty-nine transfusion-dependent βTM patients and 15 healthy controls were enrolled. One group of patients (n=34) received desferrioxamine (DFO) and the other (n=35) did not. Serum OPG, soluble RANKL (sRANKL), FFAs, LC [total LC (TC), free LC (FC), and esterified LC (EC)], calcium, and inorganic phosphate were measured by specific immuno and colorimetric assays; bone mineral density was examined by dual x-ray absorptiometry. Patients showed lower levels of OPG, TC, FC, EC and higher levels of sRANKL, sRANKL/OPG ratio, and FFAs than controls. Patients on DFO showed lower levels of OPG, TC, FC and higher levels of sRANKL, sRANKL/OPG ratio, and FFAs than those without chelation. In patients, sRANKL correlated negatively with TC and OPG and FC correlated positively with OPG and negatively with sRANKL, sRANKL/OPG ratio, and FFAs. In conclusion, altered bone metabolism owing to imbalanced osteoclastic bone resorption versus constructive osteoblastic activities in βTM pediatric patients could be due to abnormal sRANKL-OPG and LC-FFAs systems that were worsened by DFO.

Effect of All-Trans Retinoic Acid on the Pancreas of Streptozotocin-Induced Diabetic Rat.

All‐trans Retinoic acid (atRA) is instructive for the development of endocrine pancreas and is an integral component of β‐cell induction protocols. We showed that atRA induces glucose‐responsive endocrine transdifferentiation of pleomorphic pancreatic ductal adenocarcinoma cells in vitro. This study aimed to detect the role of atRA in improving the histological changes of the pancreas in diabetic rats. Forty young male Wistar rats were used and divided into three groups. Group I: normal vehicle control (N = 5). Group II: streptozotocin‐induced diabetic rats (N = 20) were followed up at 0.0, 1, 2, and 4 weeks. Group III: streptozotocin‐induced diabetic rats (N = 15) treated with atRA (2.5 mg/kg/day), were followed up at 1, 2, and 4 weeks. Specimens from the pancreas were processed for light, electron microscopy and pancreatic insulin mRNA expression. Blood samples were assayed for the levels of glucose, insulin, and total peroxides. In the atRA‐treated group, the number of the islets and the islet area significantly increased. Strong insulin‐immunoreactive endocrine‐like cells were observed nearby the pancreatic acini and the interlobular ducts. Interestingly, insulin‐positive cells seemed to arise from pancreatic acinar and ductal epithelium. Ultrastructurally, ß‐cells, acinar, and ductal cells restored their normal appearance. Pancreatic insulin mRNA and blood indices were almost normalized. AtRA improved the histological changes of the pancreas and the blood indices in diabetic rats. Anat Rec, 299:334–351, 2016.

Viability loss and ultrastructural changes on protoscolices of human hydatid cysts induced by retinoic acid

Surgical removal of intact hydatid cyst is the most effective treatment for hydatid disease. Recurrence of hydatid cyst is mainly due to dissemination of protoscolices (PSCs) rich fluid during the surgical operation. Therefore, preoperative instillation of a scolicidal agent into the cyst is a common practice with adverse side effects of the used drugs. All-trans retinoic acid (atRA) is the physiological mediator of most of the functions of vitamin A, particularly as a cellular differentiation and apoptosis regulating factor. We hypothesized that instillation of atRA could provide an alternative safe scolicidal approach. We tested the scolicidal effects and ultrastructural changes imposed by atRA on human hydatid cyst PSCs in vitro. Freshly isolated hydatid cyst PSCs were subjected to atRA (at 16.7, 1.67, 0.167 µM and 16.7 nM/L). Changes in protoscolices viability (0.1% eosin exclusion) and morphology (scanning and transmission electron microscopy; SEM and TEM) were investigated. Dose-dependent PSCs death within few minutes to 7 days of exposure to atRA was observed. SEM demonstrated ultrastructural damages including rosteller disorganization, loss of hooks and distortion of hooks morphology. TEM revealed loss of the integrity of the internal tissues of PSCs, an increased vacuolization, formation of large lipid droplets in the distal cytoplasm and aberrant, rounded abnormally large sized mitochondria. atRA is a promising alternative to the available synthetic and chemical scolicidal agents. However, in vivo scolicidal activities of atRA and the possible side effects necessitate further studies. Key words: Hydatid cyst, protoscolices, retinoic acid, ultrastructure, scolicidal activity.