ß-catenin involvement in arsenite-induced VEGF expression in neuroblastoma SH-SY5Y cells.

Arsenic is a widespread contaminant in the environment especially in drinking water. Although it is a known carcinogen in human, the mechanism by which arsenic induces carcinogenesis is not well understood. Among several effects of arsenic, it has been suggested that arsenic-induced vascular endothelial growth factor (VEGF) expression plays a critical role in arsenic carcinogenesis. In the present study, we demonstrated that arsenite induced VEGF expression in neuroblastoma SH-SY5Y cells without induction of HIF-1α, a well-known transcriptional activator for VEGF suggesting that arsenite-induced VEGF expression in SH-SY5Y cells may not require HIF-1α activation. It has been reported that VEGF expression is regulated by multiple transcription factors including ß-catenin. We therefore investigated whether ß-catenin was involved in arsenite-induced VEGF expression in SH-SY5Y cells. Treatment of arsenite caused ß-catenin accumulation in the nucleus. Additionally, arsenite treatment decreased the activity of GSK3, an enzyme that phosphorylates and targets ß-catenin for degradation by proteasome, without activation of its upstream kinase, Akt. Inhibition of PI3K/Akt which negatively regulates GSK3 activity by LY294002 resulted in a decrease in arsenite-mediated ß-catenin nuclear accumulation, and VEGF expression. These results suggested that ß-catenin plays a role in arsenite-induced VEGF in SH-SY5Y cells, and the induction of ß-catenin by arsenite is mediated by inhibition of GSK3 without activating its upstream kinase Akt.

Potentiation of TRAIL-Induced Apoptosis in TRAIL-Resistant Cholangiocarcinoma Cells by Curcumin through the Induction of DR5 Membrane Localization and Disruption of the Anti-Apoptotic Complex DR5/DDX3/GSK3ß.

OBJECTIVE: Cholangiocarcinoma (CCA) is a cancer of the bile duct with a poor prognosis. The present study examined the ability of curcumin to sensitize apoptosis in the TNF-related apoptosis-inducing ligand (TRAIL)-resistant CCA cell lines of HuCCA-1 and KKU-213A. METHODS: Apoptosis was measured using a TUNEL assay. Protein expression was determined by immunoblotting. Membrane death receptor 5 (DR5) was detected by flow cytometry. Protein complex was examined by co-immunoprecipitation. RESULT: Curcumin potentiated TRAIL-induced apoptosis in both cell lines, indicating the sensitization to TRAIL-induced apoptosis by curcumin. Additionally, curcumin increased DR5 expression and membrane localization; however, the curcumin/TRAIL combination did not result in further increases in DR5 expression and membrane localization in either cell line. Moreover, the curcumin/TRAIL combination reduced DR5/decoy receptor 2 (DcR2) complexes in both cell lines, suggesting that curcumin may enhance TRAIL-induced apoptosis by disrupting DR5/DcR2 interaction. In addition, levels of the anti-apoptotic complex DR5/ DDX3/GSK3ß were reduced by the curcumin/TRAIL combination in HuCCA-1 but not in KKU-213A cells. This study also demonstrated that the DR5/DcR2 and DR5/DDX3/GSK3ß complexes could be observed under basal conditions, suggesting that these anti-apoptotic complexes may contribute to TRAIL-resistant phenotypes in both cell lines. Pretreatment with the antioxidant N-acetylcysteine attenuated curcumin-enhanced apoptosis by TRAIL, indicating that curcumin sensitized TRAIL-induced apoptosis through an oxidative stress-dependent mechanism. CONCLUSION: The present study demonstrates the potential of using curcumin in combination with TRAIL to yield better TRAIL therapy outcomes in TRAIL-resistant CCA.

Acrylonitrile induced apoptosis via oxidative stress in neuroblastoma SH-SY5Y cell.

Acrylonitrile (ACN) is a chemical that is widely used in the production of plastics, acrylic fibers, synthetic rubbers and resins. It has been reported that ACN can cause oxidative stress, a condition which is well recognized as an apoptotic initiator; however, information regarding ACN-induced apoptosis is limited. This present study investigated whether ACN induces apoptosis in human neuroblastoma SH-SY5Y cells, and whether its apoptotic induction involves oxidative stress. The results showed that ACN caused activation of caspase-3, a key enzyme involved in apoptosis, in a dose- and time-dependent manner. Detection of sub-G1 apoptotic cell death and apoptotic nuclear condensation revealed that ACN caused an increase in the number of apoptotic cells indicating ACN induces apoptosis in SH-SY5Y cells. ACN dose- and time-dependently increased the level of proapoptotic protein, Bax. Pretreatment with N-acetylcysteine (NAC), an antioxidant, attenuated caspase-3 activation by ACN, as evidenced by a reduction in proteolysis of PARP, a known caspase-3 substrate, as well as in the number of sub-G1 apoptotic cells. Moreover, induction of Bax by ACN was abolished by NAC. Taken together, the results indicate that ACN induces apoptosis in SH-SY5Y cells via a mechanism involving generation of oxidative stress-mediated Bax induction.

Sodium arsenite exposure impairs B cell proliferation and enhances vascular inflammation in Plasmodium berghei mouse model.

Arsenic exposure has been linked to an impaired immune response and inflammation. Our study investigated the effects of sodium arsenite on host immune response and vascular inflammation during malarial infection. Mice were divided into three groups: control (C), Plasmodium berghei infection (I) and sodium arsenite exposure with Plasmodium berghei infection (As-I). The results showed that splenocyte proliferation stimulated by lipopolysaccharide (LPS) and pokeweed mitogen (PWM) was suppressed in the I group, and the suppression was more pronounced in the As-I group, suggesting that acquired immunity in infected mice was worsening following arsenic exposure. ICAM-1, an adhesion protein involved in parasite-infected red blood cell (iRBC) binding to endothelium, and HIF-1α, a hypoxia marker protein in the descending aorta, were increased in the As-I group compared to the I group. Collectively, our results suggest that arsenic may increase host susceptibility to malaria through suppression of B cell proliferation and enhancement of adhesion between iRBC and endothelium by increasing ICAM-1.

Arsenic impairs insulin signaling in differentiated neuroblastoma SH-SY5Y cells.

A strong correlation between chronic arsenic exposure and neuropsychological disorders leads to a growing concern about a potential risk of arsenic related neurodegeneration. Evidently, brain insulin signaling contributes to physiological effects, including energy homeostasis, and learning and memory. Arsenic has been shown to impair insulin signaling in adipocytes and myocytes, however, this impairment has not yet been explored in neurons. Here we showed that NaAsO2 caused significant reduction in basal levels of glucose, plasma membrane glucose transporter, GLUT 3 and Akt phosphorylation in differentiated human neuroblastoma SH-SY5Y cells. NaAsO2 significantly decreased insulin-mediated glucose uptake, as well as GLUT1 and 3 membrane translocation. Furthermore, the ability of insulin to increase Akt phosphorylation, a well-recognized insulin signaling response, was significantly lessened by NaAsO2 treatment. In addition, the classical tyrosine phosphorylation response of insulin was reduced by NaAsO2, as evidenced by reduction of insulin-induced tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1(IRS-1). Moreover, NaAsO2 lowered the ratio of p110, a catalytic subunit to p85, a regulatory subunit of PI3K causing an imbalance between p110 and p85, the conditions reported to contribute to insulin sensitivity. Additionally, increment of IRS-1 interaction with GSK3ß, and p85-PI3K were observed in NaAsO2 treated cells. These molecular modulations may be mechanistically attributed to neuronal insulin signaling impairment by arsenic.

Low arsenite concentrations induce cell proliferation via activation of VEGF signaling in human neuroblastoma SH-SY5Y cells.

Arsenic widely contaminates the environment, especially in drinking water. Although it is a known carcinogen in humans, its carcinogenic mechanism has not yet been clarified. Here, we demonstrated that a low concentration of arsenite treatment induced proliferation of human neuroblastoma SH-SY5Y cells as indicated by increases in cell viability and BrdU incorporation. Additionally, arsenite increased VEGF expression and secretion. Inhibition of VEGF-induced signaling by SU4312, the inhibitor of VEGF receptor 2 kinase, and by treatment with anti-VEGF antibody blocked arsenite-induced increases in cell proliferation. Moreover, arsenite caused activation of ERK, a key signaling molecule involved in cell proliferation, and this activation was attenuated by SU4312, suggesting that ERK activation contributes to VEGF-mediated cell proliferation induced by arsenite. Collectively, the present study reveals that a mechanism underlying arsenic-induced cell proliferation may be through induction and activation of VEGF signaling, and this may subsequently contribute to tumor formation.

Safety evaluation of longan seed extract: acute and repeated oral administration.

Longan seed extract (LSE) contains high levels of beneficial polyphenolic compounds. The present study evaluated acute and repeated dose (4 and 13 weeks) toxicological effects of aqueous extract of longan seed to ensure the safety of utilization of this extract. Our study revealed that all treated animals survived through the whole experimental periods without adverse effects observed in either sex of animals after acute and repeated dose (4 and 13 weeks) oral administration of LSE. Likewise, growth pattern (body weights, food consumption, and relative organ weights), hematology analysis, and clinical biochemistry analysis in all LSE-treated animals were in normal physiological ranges. Moreover, histopathological finding of LSE-treated animals in repeated dose studies demonstrated no obvious alterations. Although the significant increment in food consumption of female rats (100 mg/kg, Week 4) and % eosinophil of male rats (400 mg/kg), and decrement in food consumption of male rats (250 mg/kg, Week 3 and 9) were observed, these alterations were not dose- and time-response relationships. Therefore, we concluded that acute and repeated dose (4 and 13 weeks) oral administration of LSE has no significant toxicological effects;hence it may be safe to use with caution pending its chronic toxicity study and/or clinical trial.

Immunomodulatory effects of cadmium and Gynostemma pentaphyllum herbal tea on rat splenocyte proliferation.

Gynostemma pentaphyllum Makino (GP) is a herbal tea widely grown in Southeast Asia. However, this herbal tea can be contaminated with some heavy metals, especially cadmium (Cd), from agricultural areas, which may affect human health. The objective of this study is to evaluate the immunomodulatory effects of Cd contaminated in GP herbal tea and inorganic Cd on rat splenocytes. Rats were divided into groups and treated with drinking water (control), high CdCl 2 in drinking water (HCd; 0.05 mg/L), GP herbal tea containing 0.05 mg/L Cd (GP-HCd) for 4 months, low CdCl 2 in drinking water (LCd; 0.006 mg/L), and GP herbal tea containing 0.006 mg/L Cd (GP-LCd) for 6 months. After the treatments, Cd accumulation in organs and blood was detected by using a graphite furnace atomic absorption spectrophotometer. In spleen, HCd-treated rats had 4-fold higher Cd accumulations than GP-HCd-treated rats. Cd accumulation in liver and kidney in the HCd group also increased significantly. There were no significant changes in total leucocyte and lymphocyte counts; however, these parameters tended to decrease slightly in LCd, GP-LCd, and GP-HCd groups. The HCd group (ex vivo) significantly produced suppressive effects on T cell mitogen-induced splenocyte proliferation, with 1 mug/mL Con A and PHA-P. In addition, 0.5 mug/mL PWM-induced B cell proliferation, through T cell functions, was also significantly inhibited by HCd as compared to the control group, while GP-HCd had no effects. However, both GP-LCd- and LCd-treated rats had a slight increase in Con A-stimulated splenocyte proliferation. This study indicated that high Cd contamination in drinking water alone had suppressive effects on T cell functions, but these effects could not be found with the same Cd level contamination in GP herbal tea.