Abdullah A. Alkahtane

Chorein Sensitive Orai1 Expression and Store Operated Ca2+ Entry in Rhabdomyosarcoma Cells.

Background: Chorein, a protein encoded by VPS13A (vacuolar protein sorting-associated protein 13A), is defective in chorea acanthocytosis, a rare disease characterized by acanthocytosis of red blood cells and neuronal cell death with progressive hyperkinetic movement disorder, cognitive dysfunction, behavioral abnormalities and chronic hyperkalemia. Chorein is highly expressed in ZF rhabdomyosarcoma cells and counteracts apoptosis of those cells. Chorein is effective in part by interacting with and fostering stimulation of phosphoinositide-3-kinase (PI3K)-p85-subunit. PI3K dependent signaling includes the serum and glucocorticoid inducible kinase SGK1. The kinase activates NFκB with subsequent up-regulation of the Ca2+ channel subunit Orai1, which accomplishes store operated Ca2+ entry (SOCE). Orai1 and SOCE have been shown to confer survival of tumor cells. The present study thus explored whether chorein impacts on Orai1 expression and SOCE. Methods: In rhabdomyosarcoma cells chorein, Orai1, NFκB and SGK1 transcript levels were quantified by RT-PCR, Orai1 protein abundance by Western blotting, FACS analysis and confocal laser microscopy, [Ca2+]i utilizing Fura-2 fluorescence, and SOCE from the increase of [Ca2+]i following store depletion with extracellular Ca2+ removal and inhibition of the sarcoendoplasmatic reticular Ca2+ ATPase with thapsigargin. Results: The mRNA coding for chorein was most abundant in drug resistant, poorly differentiated human ZF rhabdomyosarcoma cells. Chorein silencing significantly decreased Orai1 transcript levels and Orai1 protein expression, as well as SGK1 and NFκB transcript levels. SOCE in ZF rhabdomyosarcoma cells was significantly blunted by chorein silencing, Orai1 inhibitor 2-APB (50 µM), SGK1 inhibitor EMD638683 (50 µM, 10 h) and NFκB inhibitor wogonin (50 µM, 24 h). Conclusion: Chorein is a stimulator of Orai1 expression and thus of store operated Ca2+ entry. The effect may involve SGK1 and NFκB.

Aminophthalocyanine-Mediated Photodynamic Inactivation of Leishmania tropica

ABSTRACT Photodynamic inactivation of Leishmania spp. requires the cellular uptake of photosensitizers, e.g., endocytosis of silicon(IV)-phthalocyanines (PC) axially substituted with bulky ligands. We report here that when substituted with amino-containing ligands, the PCs (PC1 and PC2) were endocytosed and displayed improved potency against Leishmania tropica promastigotes and axenic amastigotes in vitro. The uptake of these PCs by both Leishmania stages followed saturation kinetics, as expected. Sensitive assays were developed for assessing the photodynamic inactivation of Leishmania spp. by rendering them fluorescent in two ways: transfecting promastigotes to express green fluorescent protein (GFP) and loading them with carboxyfluorescein succinimidyl ester (CFSE). PC-sensitized Leishmania tropica strains were seen microscopically to lose their motility, structural integrity, and GFP/CFSE fluorescence after exposure to red light (wavelength, ∼650 nm) at a fluence of 1 to 2 J cm−2. Quantitative fluorescence assays based on the loss of GFP/CFSE from live Leishmania tropica showed that PC1 and PC2 dose dependently sensitized both stages for photoinactivation, consistent with the results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Leishmania tropica strains are >100 times more sensitive than their host cells or macrophages to PC1- and PC2-mediated photoinactivation, judging from the estimated 50% effective concentrations (EC50s) of these cells. Axial substitution of the PC with amino groups instead of other ligands appears to increase its leishmanial photolytic activity by up to 40-fold. PC1 and PC2 are thus potentially useful for photodynamic therapy of leishmaniasis and for oxidative photoinactivation of Leishmania spp. for use as vaccines or vaccine carriers.

A Steroidal Na + /K + ATPase Inhibitor Triggers Pro-apoptotic Signaling and Induces Apoptosis in Prostate and Lung Tumor Cells

Recently we have reported potent anti-cancer actions of various steroidal Na(+)/K(+) ATPase inhibitors in multiple cell lines. Furthermore, the most powerful compound identified in this study, the 3-[(R)-3-pyrrolidinyl]oxime derivative (3-R-POD), was highly effective in various tumor cell lines in vitro, and exhibited significant tumor growth inhibition in prostate and lung xenografts in vivo. In the present study we have addressed the molecular mechanisms implicated in the anti-cancer actions of 3-R-POD. We report here that 3-R-POD induces strong apoptotic responses in A549 lung- and in DU145 prostate- cancer cells. These effects are accompanied by significant upregulation of caspase-3 activity. Focussing on A549 cells, we further demonstrate late downregulation of BCL-2- and upregulation of c-Fos- gene transcription. In addition, the steroidal Na(+)/K(+) ATPase inhibitor induced late de-phosphorylation of Focal Adhesion Kinase (FAK) and activation of p38 MAPK. Our findings suggest that the steroidal Na(+)/K(+) ATPase inhibitor 3-R-POD induces apoptosis, paralleled by altered BCL-2 and c-Fos gene transcription, inhibition of the pro-survival FAK signalling, up-regulation of the pro-apoptotic p38 MAPK pathway and stimulation of caspase-3 activity.

Association of Toll-Like Receptor 3 Single-Nucleotide Polymorphisms and Hepatitis C Virus Infection

Toll-like receptor 3 (TLR3) plays a key role in innate immunity by recognizing pathogenic, double-stranded RNAs. Thus, activation of TLR3 is a major factor in antiviral defense and tumor eradication. Although downregulation of TLR3 gene expression has been mainly reported in patients infected with hepatitis C virus (HCV), the influence of TLR3 genotype on the risk of HCV infection, HCV-related cirrhosis, and/or hepatocellular carcinoma (HCC) remains to be determined. Single-nucleotide polymorphisms (SNPs) within the TLR3 gene and their associations with HCV-related disease risk were investigated in a Saudi Arabian population in this study. Eight TLR3 SNPs were analyzed in 563 patients with HCV, which consisted of 437 patients with chronic HCV infections, 88 with HCV-induced liver cirrhosis, and 38 with HCC. A total of 599 healthy control subjects were recruited to the study. Among the eight TLR3 SNPs studied, the rs78726532 SNP was strongly associated with HCV infection when compared to that in healthy control subjects. The rs5743314 was also strongly associated with HCV-related liver disease progression (cirrhosis and HCC). In summary, these results indicate that distinct genetic variants of TLR3 SNPs are associated with HCV infection and HCV-mediated liver disease progression in the Saudi Arabian population.

Up-regulation of Orai1 expression and store operated Ca 2+ entry following activation of membrane androgen receptors in MCF-7 breast tumor cells

BackgroundMembrane androgen receptors (mAR) are functionally expressed in a variety of tumor-cells including the breast tumor-cell line MCF-7. They are specifically activated by testosterone albumin conjugates (TAC). The mAR sensitive signaling includes activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and reorganization of the actin filament network. Signaling of tumor-cells may further involve up-regulation of pore forming Ca2+ channel protein Orai1, which accomplishes store operated Ca2+ entry (SOCE). This study explored the regulation of Orai1 abundance and SOCE by mAR.MethodsActin filaments were visualized utilizing confocal microscopy, Rac1 activity using GST-GBD assay, Orai1 transcript levels by RT-PCR and total protein abundance by western blotting, Orai1 abundance at the cell surface by confocal microscopy and FACS-analysis, cytosolic Ca2+ activity ([Ca2+]i) utilizing Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following readdition of Ca2+ after store depletion with thapsigargin (1 μM).ResultsTAC treatment of MCF-7 cells was followed by Rac1 activation, actin polymerization, transient increase of Orai1transcript levels and protein abundance, and transient increase of SOCE. The transient increase of Orai1 protein abundance was abrogated by Rac1 inhibitor NSC23766 (50 μM) and by prevention of actin reorganization with cytochalasin B (1 μM).ConclusionsmAR sensitive Rac1 activation and actin reorganization contribute to the regulation of Orai1 protein abundance and SOCE.

Isolation and evaluation of biological efficacy of quercetol in human hepatic carcinoma cells

Quercetol is a polyphenolic molecule present in vegetables and fruits, and is beneficial to human and animal health. The current work aimed to test cytotoxic and apoptotic effects of quercetol on HepG2 cells. Quercetol was isolated from Ocimum sanctum and characterized by gas chromatography–tandom mass spectrometry (GC-MS/MS), nuclear magnetic resonance spectroscopy, and Fourier transform infrared spectroscopy. Quercetol (50–600 μg/mL) was examined for cytotoxic activity by tetrazolium salt and neutral red uptake tests and comet assay for genotoxicity, using HepG2 cells, over 24 hours. Data from 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and neutral red uptake tests demonstrated quercetol-induced cytotoxicity in HepG2 cells in a concentration-dependent manner. With 4′,6-diamidino-2-phenylindole staining, a significant induction of chromosomal condensation was observed at 300 μg/mL of quercetol. DNA fragmentation analysis showed that quercetol produced cell death in HepG2 cells in a concentration-dependent manner. Thus, our study suggests that an environmentally relevant concentration of quercetol, which was a chemically standardized extract from O. sanctum, induced cell death and DNA damage in HepG2 cells.

Indium tin oxide nanoparticles-mediated DNA fragmentation and cell death by apoptosis in human lung epithelial cells

Indium tin oxide (ITO) nanoparticles (NP) have extensive applications in industrial fields, and concerns regarding their potential toxicity in humans and environmental impact have increased. Since exposure to ITO NP is mainly via skin and inhalation, this study was conducted utilizing human lung epithelial (A549) cell line. Cells were exposed to different concentrations of the ITO NP for 24 and 48 hr. A severe cytotoxic response of ITO NP was observed as evident by the (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and neutral red uptake assays after 48 hr exposure. ITO NP significantly reduced glutathione levels with a concomitant increase in lipid hydroperoxide levels, superoxide activity, and reactive oxygen species (ROS) generation after exposure. A significant induction in caspase activity and formation of condensed chromosomal bodies was also observed after ITO NP (10 or 25 µg/ml) exposure. Furthermore, a significant induction in DNA damage was observed by the Comet assay in cells exposed to ITO NP. Our data demonstrate that ITO NP display cytotoxic and genotoxic potential. However, increase in ROS levels and oxidative stress leading to oxidative DNA damage and condensed chromosomal bodies formation, suggests involvement of apotosis. Thus, ITO NP-mediated effects on cell viability indicate cytotoxicity, and therefore, exposures need to be carefully monitored in the industrial sector.

Detection of oxidative stress and DNA damage in freshwater snail Lymnea leuteola exposed to profenofos

Extensive production and use of organophosphate pesticide in agriculture, has risen concerned about its ecotoxicity and risk assessment of insecticides, which are more important. Therefore, the present investigation was aimed to study the induction of oxidative stress and DNA damage by organophosphate insecticide profenofos (PFF) in freshwater snail Lymnea luteola (L. luteola). The median lethal value (96 h LC50) of PFF was estimated as 1.26 mg/L for L. luteola in a semi-static system and on the basis of LC50 value three concentrations viz., 0.126 (1/10 of LC50, Sublethal I), 0.63 (1/2 of LC50, Sublethal II) and 0.84 mg/L (2/3 of LC50, Sublethal III) were determined. Snails were exposed to above-mentioned concentrations of PFF along with solvent control (acetone) and negative control for 96 h. The haemolymph was collected at 24 and 96 h of after treatment. In heamolymph of PFF exposed snail, lipid peroxide, glutathione reduced glutathione S transferase and superoxide dismutase activities at the tested concentrations significantly differ from those in the control. The genotoxicity induced in hemocytes of treated snails was measured by alkaline single cell gel electrophoresis assay. The data of this experiment demonstrated significantly enhancement of oxidative stress and DNA damage in the treated snails as compared to controls. Also, we observed statistically significant correlations of ROS with DNA damage (% tail DNA) (R2 = 0.9708) for 24 h and DNA damage (R2 = 0.9665) for 96 h.Results of the current experiment can be useful in risk evaluation of PFF among aquatic organisms. The study confirmed the use of comet assay for in vivo laboratory experiments using freshwater snail for selecting the toxic potential of industrial chemicals and environmental contaminants.

Nanosilica exerts cytotoxicity and apoptotic response via oxidative stress in mouse embryonic fibroblasts

Nanoscale silica is an important industrial material and extensively used in medicines. The objective of this study was to determine potential cytotoxicity and genotoxic effects attributed to nanosilica exposure in mouse embryonic fibroblasts (L929) cells. Nanosilica produced mild cytotoxicity in L929 cells. Results showed that nanosilica increased thiobarbituric acid reactive substance levels and enhanced superoxide dismutase activity but decreased levels of glutathione. This was accompanied by a concomitant generation of reactive oxygen species, loss of mitochondrial membrane potential, and activation of caspase-3 activity. In addition, in the single-cell gel test, nanosilica (50–300 μg/ml) at two treatment times 24 and 48 hr produced concentration- and time-dependent increase of DNA damage. Therefore, the obtained results indicate that nanosilica may induce genotoxic effects in cultured L929 cells associated with induction of oxidative stress.

ROS-dependent Bax/Bcl2 and caspase 3 pathway-mediated apoptosis induced by zineb in human keratinocyte cells

There are a large number of agricultural workers who are exposed to pesticides through skin and inhalation. The best approach to identify altered molecular pathways during dermal exposure to pesticides is relevant to risk-associated concern about skin safety. In this study, we investigated the cytotoxic effect of zineb, a fungicide, in human keratinocyte (HaCaT) cells. HaCaT cells were treated with zineb (1–40 µg/mL) for 24 hours. Cellular and molecular mechanisms of cell toxicity were investigated through MTT and neutral red-uptake assays. Zineb reduced viability of HaCaT cells and induced apoptosis in a concentration-dependent manner. Zineb increased levels of Bax and caspase 3 and inhibited the level of Bcl2, which subsequently induced apoptosis via the Bax/Bcl2 and caspase pathway. Therefore, zineb could have induced apoptosis through the mitochondrial pathway in HaCaT cells. Our study suggests that zineb is cytotoxic to HaCaT cells via the induction of apoptosis and oxidative stress in vitro.