R. Mohamed

The effect of Ginkgo biloba extract on 3-nitropropionic acid-induced neurotoxicity in rats.

3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase enzyme (SDH), induces neurodegeneration similar to that observed in Huntingtons disease (HD). Reduction of prepulse inhibition (PPI) of acoustic startle response, locomotor hypoactivity, bilateral striatal lesions as well as brain oxidative stress are major features of HD. The present study was designed to investigate neuroprotective effect of Ginkgo biloba extract (EGb 761) on 3-NP induced neurobehavioral changes and striatal lesions. Rats administered 3-NP (20mg/kg, s.c.) for five consecutive days exhibited PPI deficits and locomotor hypoactivity whereas, pretreatment of animals with EGb 761 (100mg/kg, i.p. for 15 days) ahead of and during the induction of HD by 3-NP (20mg/kg for 5 days starting at day 8) ameliorated 3-NP-induced neurobehavioral deficits. Administration of 3-NP increased the level of striatal malondialdehyde (MDA). This effect was prevented in animals pre-treated with EGb 761. Changes in the level of apoptotic regulatory gene expressions, following 3-NP treatment, were demonstrated as both an up-regulation and a down-regulation of the expression levels of striatal Bax and Bcl-xl genes, respectively. In addition, an up-regulation of the expression level of striatal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also observed. Pre-treatment with EGb 761 caused a down-regulation in striatal GAPDH and Bax together with an up-regulation of striatal Bcl-xl expression level as compared to the 3-NP treated group. Histochemical examination of striatal tissue showed that EGb 761 significantly prevented 3-NP induced inhibition of SDH activity. Histopathological examination further affirmed the neuroprotective effect of EGb 761 against 3-NP toxicity. Taken together, these results suggest that EGb 761 has a neuroprotective role in the current HD paradigm, which may be related to improvement of energy metabolism, antioxidant properties and antiapoptotic effects.

Assessment of the Prognostic Value of Methylation Status and Expression Levels of FHIT, GSTP1 and p16 in Non-Small Cell Lung Cancer in Egyptian Patients

BACKGROUND Methylation of tumor suppressor genes has been investigated in all kinds of cancer. Tumor specific epigenetic alterations can be used as a molecular markers of malignancy, which can lead to better diagnosis, prognosis and therapy. Therefore, the aim of this study was to evaluate the association between gene hypermethylation and expression of fragile histidine triad (FHIT), glutathione S-transferase P1 (GSTP1) and p16 genes and various clinicopathologic characteristics in primary non-small cell lung carcinomas (NSCLC). MATERIALS AND METHODS The study included 28 primary non-small cell lung carcinomas, where an additional 28 tissue samples taken from apparently normal safety margin surrounding the tumors served as controls. Methylation-specific polymerase chain reaction (MSP) was performed to analyze the methylation status of FHIT, GSTP1 and p16 while their mRNA expression levels were measured using a real-time PCR assay with SYBR Green I. RESULTS The methylation frequencies of the genes tested in NSCLC specimens were 53.6% for FHIT, 25% for GSTP1, and 0% for p16, and the risk of FHIT hypermethylation increased among patients with NSCLC by 2.88, while the risk of GSTP1 hypermethylation increased by 2.33. Hypermethylation of FHIT gene showed a highly significant correlation with pathologic stage (p<0.01) and a significant correlation with smoking habit and FHIT mRNA expression level (p<0.05). In contrast, no correlation was observed between the methylation of GSTP1 or p16 and smoking habit or any other parameter investigated (p>0.05). CONCLUSIONS RESULTS of the present study suggest that methylation of FHIT is a useful biomarker of biologically aggressive disease in patients with NSCLC. FHIT methylation may play a role in lung cancer later metastatic stages while GSTP1 methylation may rather play a role in the early pathogenesis.

The anti-apoptotic and anti-inflammatory properties of puerarin attenuate 3-nitropropionic-acid induced neurotoxicity in rats

Puerarin (Pur), an isoflavonoid extracted from the dried roots of Pueraria lobata, has been reported to be useful in the treatment of various diseases. This study was designed to evaluate the anti-apoptotic and anti-inflammatory activities of Pur against 3-nitropropionic acid (3-NP) induced neurotoxicity. For 5 consecutive days, male Wistar rats were given Pur (200 mg/kg body mass) 30 min before treatment with 20 mg/kg body mass of 3-NP. The striata, hippocampi, and cortices of the 3-NP treated group showed apoptotic damage, inflammation, and energy deficit as well as histopathological lesions. The 3-NP-induced alteration in apoptotic biomarkers (caspase-3 activity/level, cytosolic cytochrome c, Bax/Bcl-2 levels) were significantly ameliorated by Pur treatment. Moreover, Pur pretreatment blocked 3-NP-induced inflammatory biomarkers (NF-κB, TNF-α, and iNOS) and prevented the energy deficit (ATP reduction). Nissl staining further confirmed Purs neuroprotective effect. These results indicate that Pur may be a useful preventive approach to various neurodegenerative diseases with underlying apoptosis and neuroinflammation.

Umbelliferone and daphnetin ameliorate carbon tetrachloride-induced hepatotoxicity in rats via nuclear factor erythroid 2-related factor 2-mediated heme oxygenase-1 expression

Among various phytochemicals, coumarins comprise a very large class of plant phenolic compounds that have good nutritive value, in addition to their antioxidant effects. The purpose of the present study was to investigate the protective effects of two coumarin derivatives, umbelliferone and daphnetin, against carbon tetrachloride (CCl4)-induced hepatotoxicity in rats and elucidate the underlying mechanism. Treatment of rats with either umbelliferone or daphnetin significantly improved the CCl4-induced biochemical alterations. In addition, both compounds alleviated the induced-lipid peroxidation and boosted the antioxidant defense system. Moreover, the investigated compounds attenuated CCl4-induced histopathological alterations of the liver. Finally, umbelliferone and daphnetin induced the nuclear translocation of the nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), thereby inducing the expression and activity of the cytoprotective heme oxygenase-1 (HO-1). These results suggest that umbelliferone and daphnetin ameliorate oxidative stress-related hepatotoxicity via their ability to augment cellular antioxidant defenses by activating Nrf2-mediated HO-1 expression.

Abl Kinase Domain Mutations in Imatinib-treated Egyptian Patients with Chronic Myeloid Leukemia

Background: Point mutations within ABL kinase domain (AKD) of the BCR-ABL gene are the most common cause of resistance to Imatinib Mesylate (IM) treated Chronic Myeloid Leukemia (CML) patients. Objectives: To assess the frequency, type and impact of AKD mutations on prognosis in a cohort of Egyptian CML patients. Patients and methods: Serial measurements of BCR-ABL transcripts level in 175 IM treated CML patients were performed using real time quantitative polymerase chain reaction (RQ-PCR). Mutation screening was performed by allele specific oligonucleotide polymerase chain reaction (ASO-PCR) in 72 patients including all 42 non-optimal responders; 28 resistant patients, 18 suboptimal responders in addition to 30 patients randomly selected with stable/ decreasing transcript level representing an optimal responder category. Results: AKD mutations were detected in 16/28 resistant patients (57%) at time of >2-fold rise in BCR-ABL transcript and in none of the 44 optimal or suboptimal responders (0%) with decreasing or stable transcript levels. From 16 positive patients, P-loop mutations were detected in 9 patients; Q252H in 3 patients (19%), Y253H in 2 patients (12%), Y253F in 2 patients (12%) and E255K in 2 patients (12%). T315I was detected in 1/16 (6%) patient. Regarding non P-loop mutation; V299L was detected in one patient (6%), M351T in 4 patients (25%), F359V in 2 patients (12%). One patient had both Y253H and E255K mutations. Ten/16 (62%) patients carrying mutations experienced disease progression versus 1/56 (2%) in non mutation group (p=0.001). Median progression free survival (PFS) and overall survival (OS) of the mutation group was 13.5 months and 37.5 months, respectively versus 42.6 months in non mutation group (p=0.001). The estimated PFS and OS at 49 months in patients with mutations were 37.5% and 56.3% respectively versus 98.2% in non mutation carriers (p 2-folds in IM resistant patients may signal progression that implies testing for AKD mutations and early planning for second generation tyrosine kinase inhibitors (TKIs). P-loop mutations are significantly associated with advanced CML phases and poorer OS than non-P loop mutations. ASOPCR is a valuable tool for detection of mutations in countries where sequencing facilities are not available.

Transient and inducible expression of vaccinia/T7 recombinant viruses.

Recombinant DNA technology has made it possible to develop molecular cloning vectors that allow the expression of heterologous genes in a variety of animal viruses. This chapter discusses the use of vaccinia virus encoding bacteriophage T7 RNA polymerase as an expression vector system. A chosen gene is inserted into a plasmid vector designed to express genes under the control of the T7 promoter. Transient expression can then be achieved either by transfecting this plasmid into cells infected with the recombinant vaccinia virus expressing T7 RNA polymerase, vTF7-3 or by crossing this plasmid into the vaccinia virus genome and coinfecting cells with both viruses. Moreover, placement of lacO downstream of the vaccinia virus P11 late promoter regulating T7 RNA polymerase expression, and integration of lacI under vaccinia promoter control into the viral genome, vT7lacOI, yielded a recombinant virus capable of IPTG-inducible T7 promoter-controlled expression of foreign genes.

Proapoptotic and prepulse inhibition (PPI) disrupting effects of Hypericum perforatum in rats

ETHNOPHARMACOLOGICAL RELEVANCE St. Johns wort extract is commonly used as a wound healing, anti-inflammatory, anxiolytic, diuretic, antibiotic, antiviral and cancer chemoprotective agent. It also has nootropic and/or antiamnestic effects. AIM OF THE STUDY Prepulse inhibition (PPI) of startle response is a valuable paradigm for sensorimotor gating processes. A previous study indicated that single administration of St. Johns wort extract (500 mg/kg) caused PPI disruption in rats. The effect of antiamnestic doses of the extract on PPI has not been investigated despite the coexistence of impaired memory and PPI deficit in some neurological disorders. MATERIALS AND METHODS The effects of acute (500 mg/kg) and chronic (200mg/kg for 3 days) administration of St. Johns wort extract were investigated for its antiamnestic activity. The effects of administration of the antiamnestic dose of the extract and hyperforin, its main active component, were tested on PPI of an acoustic startle response in rats. This study also investigated the proapoptotic effect of hyperforin in animals, demonstrating PPI deficit, by electrophoresis of DNA isolated from selected brain areas. RESULTS Disruption of PPI resulted after treatment of rats with an antiamnestic dose of the extract (200mg/kg for 3 days) and with hyperforin. Gel electrophoresis showed DNA fragmentation of the cortices of hyperforin-treated animals exhibiting PPI deficit. CONCLUSIONS The exacerbating effect of St. Johns wort extract on PPI deficit may provide a limitation for using the extract to manage cognitive disturbance in psychotic and Huntingtons disease patients manifesting PPI deficit.

Copper (II)-Surfactant Complex and Its Nano Analog as Potential Antitumor Agents

The in vitro anticancer activity of copper cetyl trimethyl ammonium bromide (Cu-CTAB)-loaded cyclodextrin nanoparticles on Ehrlich ascites carcinoma (EAC), colon cancer cells (HCT 116), liver cancer cells (HepG-2), breast cancer cells (MCF-7), and cervix cancer cells (Hela) was investigated using MTT due assay. Cyclodextrin nanoparticles loaded with Cu-CTAB exerted in vitro anticancer activity against the previous human cancer cell lines comparable to the activity of free non-entrapped in nanoparticles macro-particle Cu-CTAB. The nano analog was synthesized by physical loading using grinding with ball mill. The ratio between Cu-CTAB and cyclodextrin oligosaccharide was 1 Cu-CTAB: 3 cyclodextrin. The particle size of the nano derivative was determined using the transmitted electron microscope (TEM).

Involvement of serotoninergic 5-HT1A/2A, alpha-adrenergic and dopaminergic d1 receptors in St. John's wort-induced prepulse inhibition deficit: a possible role of hyperforin.

Prepulse inhibition (PPI) of acoustic startle response is a valuable paradigm for sensorimotor gating processes. Previous research showed that acute administration of St. Johns wort extract (500 mg/kg, p.o.) to rats caused significant disruption of PPI while elevating monoamines levels in some brain areas. The cause-effect relationship between extract-induced PPI disruption and augmented monoaminergic transmission was studied using different serotoninergic, adrenergic and dopaminergic antagonists. The effects of hypericin and hyperforin, as the main active constituents of the extract, on PPI response were also tested. PPI disruption was prevented after blocking the serotoninergic 5-HT1A and 5-HT2A, alpha-adrenergic and dopaminergic D1 receptors. Results also demonstrated a significant PPI deficit after acute treatment of rats with hyperforin, and not hypericin. In some conditions manifesting disrupted PPI response, apoptosis coexists. Electrophoresis of DNA isolated from brains of hyperforin-treated animals revealed absence of any abnormal DNA fragmentation patterns. It is concluded that serotoninergic 5-HT1A and 5-HT2A, alpha-adrenergic and dopaminergic D1 receptors are involved in the disruptive effect of St. Johns wort extract on PPI response in rats. We can also conclude that hyperforin, and not hypericin, is one of the active ingredients responsible for St. Johns wort-induced PPI disruption with no relation to apoptotic processes.

Tissue CA125 and HE4 Gene Expression Levels Offer Superior Accuracy in Discriminating Benign from Malignant Pelvic Masses

BACKGROUND Ovarian cancer remains a major worldwide health care issue due to the lack of satisfactory diagnostic methods for early detection of the disease. Prior studies on the role of serum cancer antigen 125 (CA125) and human epididymis protein 4 (HE4) in detecting ovarian cancer presented conflicting results. New tools to improve the accuracy of identifying malignancy are urgently needed. We here aimed to evaluate the diagnostic utility of tissue CA125 and HE4 gene expression in comparison to serum CA125 and HE4 in discriminating benign from malignant pelvic masses. MATERIALS AND METHODS One-hundred Egyptian women were enrolled in this study, including 60 epithelial ovarian cancer (EOC) patients and 20 benign ovarian tumor patients, as well as 20 apparently healthy women. Preoperative serum levels of CA125 and HE4 were measured by immunoassays. Tissue expression levels of genes encoding CA125 and HE4 were determined by quantitative real time polymerase chain reaction (qRT-PCR). The diagnostic performance of CA125 and HE4, measured either as mRNA or protein levels, was evaluated by receiver operating characteristic (ROC) curves. RESULTS The serum CA125+HE4 combination and serum HE4, with area under the curve (AUC) values of 0.935 and 0.932, respectively, performed significantly better than serum CA125 (AUC=0.592; P<0.001). Tissue CA125 and HE4 (AUC=1) performed significantly better than serum CA125 (P<0.001), serum HE4 (P=0.016) and the serum CA125+HE4 combination (P=0.018). CONCLUSIONS Measurement of tissue CA125 and HE4 gene expression not only improves discriminatory performance, but also broadens the range of differential diagnostic possibilities in distinguishing EOC from benign ovarian tumors.