Investigation of the relationship between CTLA4 and the tumor suppressor RASSF1A and the possible mediating role of STAT4 in a cohort of Egyptian patients infected with hepatitis C virus with and without hepatocellular carcinoma.

The Ras association domain family 1 isoform A (RASSF1A), cytotoxic T lymphocyte antigen 4 (CTLA-4), and signal transducer and activator of transcription 4 (STAT4) genes play a role in regulating the cell cycle, apoptosis, and the autoimmune response against cancer. We investigated the genotype frequency and the possible association of the rs2073498 (RASSF1A), rs5742909 (CTLA-4) and rs7574865 (STAT4) genetic variants with hepatitis C virus (HCV)-G4-mediated hepatocellular carcinoma (HCC) progression in Egyptian patients. Fifty patients with HCV infection, 50 patients with HCV-mediated HCC, and 50 age- and sex-matched healthy controls were recruited. The investigated variants were genotyped based on polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). The Ser133 mutant G4 variant of the rs2073498 SNP in RASSF1A exhibited a positive correlation with HCC incidence risk (OR = 0.571, 95% CI = 0.175-1.865, P < 0.001). The rs7574865 variant in STAT4 (G/T) occurred frequently in both HCV groups, with a significant incidence risk (OR = 1.583, 95% CI = 1.123-2.232, P = 0.005). The rs5742909 change in CTLA4 (C/T) did not show a significant difference between HCV-mediated HCC cases and the control group (OR = 4.5, 95% CI = 1.326-15.277, P > 0.001). Activation of the immune checkpoint gene CTLA4 or polymorphism in the encoded CTLA4 protein causes phosphorylation of kinases needed for RAS gene activation. This in turn downregulates the tumor suppressor RASSF1, inhibiting apoptosis and leading to HCC development, indicating a negative impact of CTLA4 gene polymorphism on HCV-mediated HCC cases. A major determinant of disease progression could be immune system genetic variants, together with the presence of costimulatory factors. The rs2073498 and rs7574865 variations in the RASSF1A and STAT4 genes, respectively, could be genetic susceptibility factors for Egyptian patients with HCV-mediated HCC.

Influence of Incision Size on Dry Eye Symptoms in the Small Incision Lenticule Extraction Procedure.

PURPOSE: To evaluate the influence of incision size on dry eye symptoms in the small incision lenticule extraction (SMILE) procedure. METHODS: Ninety-four eyes of 47 patients with myopia and/or myopic astigmatism who had undergone the SMILE procedure were enrolled in this study. The patients were divided into 3 groups according to the incision size (2, 3, and 4 mm) applied during the SMILE procedure. RESULTS: There were no significant differences among the groups in respect to age and sex (P values 0.251 and 0.974, respectively) and in respect to preoperative, postoperative first day, first week, first month, third month, and sixth month values of the Ocular Surface Disease Index score, tear break-up time, ST1, and staining grades. CONCLUSIONS: There was no difference in dry eye symptoms with 2-, 3-, and 4-mm incisions. In the learning period of SMILE surgery, larger incision sites can be used to make the procedure easier.

Gene expression profile in squamous cell carcinoma of the urinary bladder using complementary deoxyribonucleic acid microarray.

To date, molecular evidence studies for bladder cancer, using the microarray technology, are focusing on the transitional cell carcinoma, however, similar fingerprinting studies have rarely been performed on the other molecular phenotypes of bladder cancer, squamous cell carcinoma (SCC). This study was conducted to monitor the gene expression profiles for bilharzial-related SCC of the bladder to be able to compare its data with transitional cell carcinoma microarray data. A total of 17 paired bilharzial urinary bladder SCC specimens and their corresponding normal urothelium were analyzed using the complementary deoxyribonucleic acid microarray hybridization approach to study the molecular basis of the development of SCC of the urinary bladder. Validation of the microarray results was performed using the Northern blotting technique. After supervised analysis of the microarray data, there was at least a 3-fold difference in the expression between SCC of the bladder and normal urothelium in 82 genes. A total of 38 genes were up-regulated in SCC of the bladder, including matrix degradation-related genes, growth factors, different oncogenes, and immunology related genes. Conversely, 44 genes were down-regulated in SCC of the bladder, including integrins, laminins, cadherins, nonmetastatic cell 1 (NM23) and apoptosis-related genes. Our findings can explain the aggressive behavior of SCC of the bladder. Such gene profiling studies will add to our understanding of the mechanisms of carcinogenesis, and may also improve our ability to diagnose and treat bladder cancer.

Transactivation of fetal liver kinase-1/kinase-insert domain-containing receptor by lysophosphatidylcholine induces vascular endothelial cell proliferation.

Lysophosphatidylcholine (LPC), a major lipid component of oxidized low-density lipoprotein, is a bioactive lipid molecule involved in numerous biological processes including the progression of atherosclerosis. Recently orphan G protein-coupled receptors were identified as high-affinity receptors for LPC. Although several G protein-coupled receptor ligands transactivate receptor tyrosine kinases, LPC-stimulated transactivation of receptor tyrosine kinase has not yet been reported. Here we observed for the first time that LPC treatment of human umbilical vein endothelial cells (HUVECs) induces tyrosyl phosphorylation of vascular endothelial growth factor receptor 2 [fetal liver kinase-1/kinase-insert domain-containing receptor, Flk-1/KDR)]. Flk-1/KDR transactivation by LPC was inhibited by vascular endothelial growth factor receptor tyrosine kinase inhibitors, SU1498 and 4-[(4'-chloro-2'-fluoro) phenylamino]6,7-dimethoxyquinazoline (VTKi) in immunoprecipitation. Furthermore, we examined the effects of the Src family kinases inhibitors, herbimycin A and 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2), on LPC-induced Flk-1/KDR transactivation. Results from Western blots, c-Src is involved in LPC-induced Flk-1/KDR transactivation because herbimycin A and PP2 inhibited this transactivation. Kinase-inactive (KI) Src transfection also inhibited LPC-induced Flk-1/KDR transactivation. In addition, results from Western blots, ERK1/2 and Akt, which are downstream effectors of Flk-1/KDR, were also activated by LPC, and this was inhibited by SU1498, VTKi, herbimycin A, PP2, and KI Src transfection in HUVECs. LPC-induced stimulation of HUVEC proliferation was shown to be secondary to transactivation because it was suppressed by SU1498, VTKi, herbimycin A, PP2, and KI Src transfection in dimethylthiazoldiphenyltetra-zoliumbromide assay. These findings suggest that LPC-induced Flk-1/KDR transactivation via c-Src may have important implications for the progression of atherosclerosis.

Differences in gene expression between noninvasive and invasive transitional cell carcinoma of the human bladder using complementary deoxyribonucleic acid microarray: preliminary results.

This study was performed to identify differences in gene expression between superficial noninvasive and invasive transitional cell carcinoma (TCC) of the bladder in human beings. We used complementary deoxyribonucleic acid microarrays containing 14,551 different genes to analyze gene expression among 6 cases of superficial and 6 cases of invasive TCC of the bladder to identify differences in gene expression, which might explain differences in the biology and clinical outcomes of these histologic subtypes of TCC. Quantitative real-time polymerase chain reaction was performed for selected genes to validate the microarray data. Significant up-regulation of 40 genes was associated with cases of superficial noninvasive, but not in invasive, TCC of the urinary bladder. This effect included genes involved in epithelial cell dedifferentiation and keratinization, as well as genes related to cell cycle, cell adhesion, transcription, and apoptosis. Conversely, significant up-regulation of 34 genes was associated with cases of invasive TCC, but not in superficial TCC, including genes related to extracellular matrix degradation, immune responses, cell cycling, and angiogenesis. This study shows the usefulness of complementary deoxyribonucleic acid microarray technology for identifying differences in gene expression among different histotypes of bladder cancer.

Pramipexole protects against H2O2-induced PC12 cell death.

Pramipexole, a novel non-ergot dopamine (DA) agonist, has been successfully applied to the treatment of Parkinson's disease (PD). Although the specific cause of PD remains unknown, recent studies have provided evidence that oxidative stress plays a role in the parthenogenesis of the disease. In the present study, we examined the effect of pramipexole on hydrogen peroxide (H2O2, 100 microM)-induced PC12 cell death, and the intracellular mechanism of this effect. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay revealed that pretreatment of PC12 cells with pramipexole (1-100 microM) resulted in significant protection against H2O2-induced cell death in a concentration-dependent manner. The protective effect of pramipexole was not affected by pretreatment with the DA receptor antagonists sulpiride, spiperone or domperidone, suggesting that the effect of pramipexole is not mediated by DA receptors. In PC12 cells, pramipexole inhibited H2O2-induced lactate dehydrogenase (LDH) leakage, as well as H2O2-induced cytochrome c release and caspase-3 activation with the resultant apoptosis. It was also observed in PC12 cells that H2O2 stimulated phosphorylation of mitogen-activated protein (MAP) kinases, i.e., extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase. Pramipexole inhibited H2O2-induced JNK and p38 MAP kinase, but not ERK1/2 phosphorylation. Furthermore, in these cells experiments with a fluorescent probe, 2-[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, revealed that pramipexole, the JNK inhibitor SP600125 and the p38 MAP kinase inhibitor SB203580 inhibited the generation of H2O2-induced reactive oxygen species. Caspase inhibitors Z-DEVD-FMK and Z-IETD-FMK, as well as SP600125 and SB203580, inhibited H2O2-induced PC12 cell death to a similar extent as pramipexole. These results suggest that pramipexole exerts a protective effect against oxidative stress-induced PC12 cell death in part through an inhibition of JNK and p38 MAP kinase.

Ebselen inhibits p38 mitogen-activated protein kinase-mediated endothelial cell death by hydrogen peroxide.

Ebselen (2-phenyl-1, 2-benzisoselenazol-3[2H]-one) is a seleno-organic compound exhibiting both glutathione peroxidase and antioxidant activity. Although it has been reported that ebselen is effective against hydrogen peroxide (H(2)O(2))-induced cell death in several cell types, its effect on endothelial cell damage has not yet been elucidated. In the present study, we examined the effect of ebselen on H(2)O(2)-induced human umbilical vein endothelial cells (HUVECs) death, and its intracellular mechanism. Our findings showed that pretreatment of HUVECs with ebselen resulted in a significant recovery from H(2)O(2)-induced cell death in a concentration-dependent manner. In addition to the inhibition of lactate dehydrogenase (LDH) leakage, ebselen inhibited H(2)O(2)-induced cytochrome c release and caspase-3 activation and the resultant apoptosis in HUVECs. Moreover, it was observed that H(2)O(2) significantly stimulated activation of mitogen-activated protein (MAP) kinases, i.e., p38 MAP kinase, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2). Ebselen inhibited H(2)O(2)-induced p38 MAP kinase, but not JNK or ERK1/2 activation. Furthermore, SB203580 (4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]-1H-imidazole), a specific p38 MAP kinase inhibitor, inhibited H(2)O(2)-induced p38 MAP kinase phosphorylation, cytochrome c release, caspase-3 activation, as well as cell death in HUVECs. These findings suggest that ebselen attenuates H(2)O(2)-induced endothelial cell death through the inhibition of signaling pathways mediated by p38 MAP kinase, caspase-3, and cytochrome c release. Thus, inhibition of p38 MAP kinase by ebselen may imply its usefulness for prevention and/or treatment of endothelial cell dysfunction, which was suggested to be the first step in the development of atherosclerosis.

Prostate stem cell antigen predicts tumour recurrence in superficial transitional cell carcinoma of the urinary bladder.

OBJECTIVES: To evaluate the relationship between prostate stem cell antigen (PSCA) expression level in transitional cell carcinoma (TCC) of the urinary bladder and various clinicopathological features, including stage and grade; and to determine whether PSCA mRNA expression predicts disease recurrence in superficial (not muscle-invasive) TCC of the bladder. PATIENTS AND METHODS: Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on 97 TCC tissue samples and in 36 samples of normal bladder urothelium; the findings were analysed in relation to clinicopathological factors. Immunohistochemical expression was examined using light and confocal immunofluorescence microscopy to validate the RT-PCR data. RESULTS: Twenty-seven patients developed disease recurrence, while the remaining 22 had no evidence of recurrence of superficial TCC of the bladder. There was significantly higher PSCA mRNA expression in TCC than in normal urothelium samples (P = 0.008). Superficial (TaT1) tumours had significantly higher PSCA expression than muscle-invasive (> or = pT2) tumours (P < 0.001). There was no significant difference between patients with G1-2 tumours and those with G3 tumours (P = 0.109). Immunohistochemical analysis showed markedly greater PSCA expression in superficial than invasive TCC. Notably, from a multivariate analysis, the expression level of PSCA was an independent predictor of disease recurrence in superficial TCC (P = 0.012). CONCLUSIONS: These findings suggest that the PSCA expression level measured by real-time RT-PCR could be a valuable prognostic marker for tumour recurrence in superficial TCC of the bladder.

The novel Src kinase inhibitor M475271 inhibits VEGF-induced vascular endothelial-cadherin and beta-catenin phosphorylation but increases their association.

M475271, 4-quinazolinamine, N-(2-chloro-5-methoxyphenyl)-6-methoxy-7-[(1-methyl-4-piperidinyl) methoxy]-(9Cl), is a new anilinoquinazoline derivative that displays selective inhibition of Src kinase activity and tumor growth in vivo. Vascular endothelial growth factor (VEGF)-induced angiogenesis plays a pivotal role in tumor growth and metastasis. Vascular endothelial (VE)-cadherin is an endothelial cell-specific adhesion molecule that can interact with the cytoskeleton via several anchoring molecules such as beta-catenin. Here, we examined the effect of M475271 on VE-cadherin and beta-catenin phosphorylation and association. We also examined its effect on VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation. The findings reveal pretreatment with M475271 significantly inhibits VEGF-induced VE-cadherin and beta-catenin phosphorylation. However, M475271 significantly increases VE-cadherin and beta-catenin association compared to the VEGF-treated group. Confocal laser microscopic examination confirmed the augmentation effect of M475271 on VE-cadherin and beta-catenin association. Finally, M475271 was shown to have inhibitory effects comparable to those of PP2 and Herbimycin A on VEGF-induced HUVEC proliferation, migration, and tube formation. These findings suggest that M475271 attenuates VEGF-induced angiogenesis by maintaining cell-cell junction stability. Although the involvement of other signaling molecules cannot be ruled out, M475271 has potential as a drug for the inhibition of the angiogenesis needed for tumor growth and metastasis.

A novel Src kinase inhibitor, M475271, inhibits VEGF-induced human umbilical vein endothelial cell proliferation and migration.

Vascular endothelial growth factor (VEGF) was reported to be a potent proangiogenic factor that plays a pivotal role in both physiological and pathological angiogenesis. M475271, 4-quinazolinamine, N-(2-chloro-5-methoxyphenyl)-6-methoxy-7-[(1-methyl-4-piperidinyl) methoxy]-(9Cl), is a new anilinoquinazoline derivative that showed selective inhibition of Src kinase activity and tumor growth in vivo. Here, we examined the effect of M475271 on VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration and their intracellular mechanisms. Our findings showed that M475271 pretreatment resulted in a significant inhibition of VEGF-induced HUVEC proliferation, [(3)H]thymidine incorporation, and migration. M475271 inhibited VEGF-induced Flk-1 and Src phosphorylation and their association. Confocal laser microscopic examination confirmed the inhibitory effect of M475271 on VEGF-induced Flk-1/Src association. M475271 inhibited VEGF-induced extracellular signal-regulated kinase1/2 (ERK1/2) and p38 but not Akt activation in a concentration-dependent manner. M475271, PI3-K inhibitor, and p38 inhibitor inhibited VEGF-induced HUVEC proliferation and migration. However, a MEK1/2 inhibitor inhibited VEGF-induced proliferation but not migration. These findings suggest that M475271 attenuates VEGF-induced HUVEC proliferation and migration through the inhibition of signaling pathways involving Src, ERK1/2, and/or p38. Taken together, these data indicate that M475271 may be a useful candidate for inhibition of endothelial cell proliferation and migration relevant to angiogenesis.

ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells.

Clinical evidence suggests a relationship between hypertension and insulin resistance, and cross-talk between angiotensin II (Ang II) and insulin signaling pathways may take place. We now report the effect of Ang II on insulin-induced glucose uptake and its intracellular mechanisms in vascular smooth muscle cells (VSMC). We examined the translocation of glucose transporter-4 (GLUT-4) and glucose uptake in rat aortic smooth muscle cells (RASMC). Mitogen-activated protein (MAP) kinases and Akt activities, and phosphorylation of insulin receptor substrate-1 (IRS-1) at the serine and tyrosine residues were measured by immunoprecipitation and immunoblotting. As a result, Ang II inhibited insulin-induced GLUT-4 translocation from cytoplasm to the plasma membrane in RASMC. Ang II induced extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation and IRS-1 phosphorylation at Ser307 and Ser616. Ang II-induced Ser307 and Ser616 phophorylation of IRS-1 was inhibited by a MEK inhibitor, PD98059, and a JNK inhibitor, SP600125. Ang II inhibition of insulin-stimulated IRS-1 tyrosyl phophorylation and Akt activation were reversed by PD98059 but not by SP600125. Ang II inhibited insulin-induced glucose uptake, which was also reversed by PD98059 but not by SP600125. It is shown that Ang II-induced ERK1/2 activation inhibits insulin-dependent glucose uptake through serine phophorylation of IRS-1 in RASMC.

Antioxidant effects of stereoisomers of N-acetylcysteine (NAC), L-NAC and D-NAC, on angiotensin II-stimulated MAP kinase activation and vascular smooth muscle cell proliferation.

We examined the effects of the stereoisomers of N-acetylcysteine (NAC), L-NAC and D-NAC, on cellular glutathione (GSH) concentration and whether NAC-regulated cellular GSH levels are directly associated with angiotensin II (Ang II)-induced intracellular signaling events in vascular smooth muscle cells (VSMC). Both L-NAC and D-NAC similarly increased intracellular GSH concentration. We found that L-NAC and D-NAC both inhibited Ang II-induced c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation and [(3)H]-thymidine incorporation in VSMC. Our present study indicates the comparable effects of NAC stereoisomers in regulating intracellular GSH and the redox-dependent intracellular signaling mechanisms in VSMC.

Src and Cas are essentially but differentially involved in angiotensin II-stimulated migration of vascular smooth muscle cells via extracellular signal-regulated kinase 1/2 and c-Jun NH2-terminal kinase activation.

Angiotensin II (Ang II) plays an important role in several cardiovascular diseases associated with vascular smooth muscle cell (VSMC) growth and migration. Src activity is known to be required for the migration of a number of cell types. p130Cas was reported to be essential for cell migration and actin filament reorganization. Mitogen-activated protein (MAP) kinases were also reported to be critical regulatory factors for growth and migration of VSMC. However, precise intracellular mechanisms involving c-Src, p130Cas, and MAP kinases in Ang II-stimulated migration of VSMC have not been well elucidated. Here we demonstrated that Ang II rapidly and significantly stimulated tyrosine phosphorylation of Src and Cas and their association in rat aortic smooth muscle cells (RASMC). Ang II-stimulated tyrosine phosphorylation of Src and Cas and activation of ERK1/2 and JNK, but not p38, were potently inhibited by Src family tyrosine kinase inhibitors, herbimycin A (HA) and PP2. Ang II-stimulated Src and Cas association, tyrosine phosphorylation of Cas, and activation of ERK1/2 and JNK were suppressed in kinase-inactive Src (KI Src)-overexpressed RASMC. Ang II-stimulated JNK activation but not ERK1/2 activation was blocked in substrate domain-deleted Cas (DeltaSD Cas)-overexpressed RASMC. In addition, HA, PP2, ERK1/2 inhibitor, 2'-amino-3'-methoxyflavone (PD98059) and JNK inhibitor, and anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) significantly inhibited Ang II-stimulated migration of RASMC. Ang II-induced colocalization of Src and Cas and migration were inhibited in both KI Src- and DeltaSD Cas-overexpressed RASMC. These findings suggest that Src and Cas are essentially but differentially involved in Ang II-stimulated migration of VSMC through the activation of ERK1/2 and JNK.

Ebselen inhibits tumor necrosis factor-alpha-induced c-Jun N-terminal kinase activation and adhesion molecule expression in endothelial cells.

Tumor necrosis factor-alpha (TNF-alpha) stimulates expression of endothelial cell (EC) genes that may promote atherosclerosis in part by an activation of mitogen-activated protein (MAP) kinases. Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one), a selenoorganic compound, is effective for acute ischemic stroke; however, its effect on EC has not yet been elucidated. We examined the effect of ebselen on TNF-alpha-induced MAP kinase activation and adhesion molecule expression in cultured human umbilical vein endothelial cells (HUVEC). Extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 were rapidly and significantly activated by TNF-alpha in HUVEC. TNF-alpha-induced JNK activation was inhibited by ebselen, whereas ERK1/2 and p38 were not affected. Apoptosis signal-regulated kinase 1 (ASK1) was suggested to be involved in TNF-alpha-induced JNK activation because transfection of kinase-inactive ASK1 inhibited TNF-alpha-induced JNK activation. Ebselen inhibited TNF-alpha-induced TNF receptor-associated factor 2 (TRAF2)-ASK1 complex formation and phosphorylation of stress-activated protein kinase ERK kinase 1 (SEK1), which is an upstream signaling molecule of JNK. Finally, TNF-alpha-induced activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) activation and resultant intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions were inhibited by ebselen. Specific inhibitors for JNK and NF-kappaB also inhibited TNF-alpha-induced ICAM-1 and VCAM-1 expressions in HUVEC. These findings suggest that ebselen prevents TNF-alpha-induced EC activation through the inhibition of TRAF2-ASK1-SEK1 signaling pathway, which leads to JNK activation. Inhibition of JNK by ebselen may imply its usefulness for the prevention of atherosclerosis relevant to EC activation.