Piyajit Watcharasit

Pharmacological inhibition of GSK3 attenuates DNA damage-induced apoptosis via reduction of p53 mitochondrial translocation and Bax oligomerization in neuroblastoma SH-SY5Y cells.

Glycogen synthase kinase-3 (GSK3) and p53 play crucial roles in the mitochondrial apoptotic pathway and are known to interact in the nucleus. However, it is not known if GSK3 has a regulatory role in the mitochondrial translocation of p53 that participates in apoptotic signaling following DNA damage. In this study, we demonstrated that lithium and SB216763, which are pharmacological inhibitors of GSK3, attenuated p53 accumulation and caspase-3 activation, as shown by PARP cleavage induced by the DNA-damaging agents doxorubicin, etoposide and camptothecin. Furthermore, each of these agents induced translocation of p53 to the mitochondria and activated the mitochondrial pathway of apoptosis, as evidenced by the release of cytochrome C from the mitochondria. Both mitochondrial translocation of p53 and mitochondrial release of cytochrome C were attenuated by inhibition of GSK3, indicating that GSK3 promotes the DNA damage-induced mitochondrial translocation of p53 and the mitochondrial apoptosis pathway. Interestingly, the regulation of p53 mitochondrial translocation by GSK3 was only evident with wild-type p53, not with mutated p53. GSK3 inhibition also reduced the phosphorylation of wild-type p53 at serine 33, which is induced by doxorubicin, etoposide and camptothecin in the mitochondria. Moreover, inhibition of GSK3 reduced etoposide-induced association of p53 with Bcl2 and Bax oligomerization. These findings show that GSK3 promotes the mitochondrial translocation of p53, enabling its interaction with Bcl2 to allow Bax oligomerization and the subsequent release of cytochrome C. This leads to caspase activation in the mitochondrial pathway of intrinsic apoptotic signaling.

Estrogenic Activities of Sesame Lignans and Their Metabolites on Human Breast Cancer Cells

Sesame lignans (sesamin, sesamolin) and their metabolites (enterodiol, ED; enterolactone, EL; and sesamol) have been evaluated for their estrogenic activities. ED and EL have been indicated to have estrogenic/antiestrogenic properties on human breast cancer cells; however the estrogenic activities of sesamin, sesamolin and sesamol have not been reported. In the present study, estrogenic potencies of sesame lignans and their metabolites were determined by estrogen responsive element (ERE) luciferase reporter assay in T47D cells stably transfected with ERE-luc (T47D-KBluc cells) and quantifying pS2 and progesterone receptor gene expression in T47D cells. All tested compounds except ED possessed ability of ERE activation with a very low potency compared to estradiol (E2). These effects were abolished by coincubating tested compounds with 1 μM ICI 182 780, suggesting that estrogen receptors were directly involved in their ERE activations. Among tested compounds, sesamol showed the highest ability in ERE induction. The coincubation of increasing concentration of E2 (10(-12)-10(-6) M) with 10 μM of tested compounds resulted in a downward shift of E2-ERE dose-response curves. In contrast, at the low concentration of E2 (10(-12) M), sesamin and sesamol significantly exhibited additive effects on the E2 responses. The inhibitory effect in a dose-dependent manner was also observed when 1-100 μM sesamol was coincubated with 1 nM E2. Sesamin, sesamol and EL significantly induced pS2 gene expression whereas only sesamol could significantly induce progesterone receptor gene. The data obtained in this study suggested that sesame lignans and their metabolites possess weak estrogenic/antiestrogenic activity.

Arsenite promotes apoptosis and dysfunction in microvascular endothelial cells via an alteration of intracellular calcium homeostasis.

Vascular endothelium has been considered as a target for arsenic-induced cardiovascular toxicity. The present study demonstrated that arsenite caused slow and sustained elevation of intracellular free calcium levels ([Ca2+]i) in HMEC-1, a human microvessel-derived endothelial cell line, in a concentration-dependent manner. Pretreatment with U-73122 (a specific PLC inhibitor) or 2-APB (a specific IP3 receptor antagonist) attenuated this effect, suggesting that PLC/IP3 signaling cascade is involved in arsenite-induced elevation of [Ca2+]i. Cytotoxic concentrations of arsenite (5 and 10 μM) significantly enhanced endothelial nitric oxide synthase (eNOS) phosphorylation, nitric oxide (NO) production and apoptosis after 24-h exposure. Additionally, 2-APB attenuated eNOS phosphorylation and apoptosis induced by arsenite, indicating that Ca2+ -mediated eNOS activation participates in arsenite-induced endothelial cell apoptosis. Moreover, we also found that non-apoptotic concentrations of arsenite (0.5 and 1 μM) dramatically mitigated thrombin-induced rapid transient rise of [Ca2+]i, eNOS phosphorylation and NO production, suggesting functional disruption of endothelial by arsenite, and these effects occurred without an alteration of PLC-β1 and thrombin receptor levels. Altogether, the results reveal that arsenite induces apoptotic cell death and endothelial dysfunction as indicated by the reduction of thrombin responses, particularly related to an alteration of intracellular Ca2+ homeostasis.

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.

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. Copyright

Perfluorinated chemicals, PFOS and PFOA, enhance the estrogenic effects of 17β‐estradiol in T47D human breast cancer cells

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the two most popular surfactants among perfluorinated compounds (PFCs), with a wide range of uses. Growing evidence suggests that PFCs have the potential to interfere with estrogen homeostasis, posing a risk of endocrine‐disrupting effects. This in vitro study aimed to investigate the estrogenic effect of these compounds on T47D hormone‐dependent breast cancer cells. PFOS and PFOA (10−12 to 10−4 M) were not able to induce estrogen response element (ERE) activation in the ERE luciferase reporter assay. The ERE activation was induced when the cells were co‐incubated with PFOS (10−10 to 10−7 M) or PFOA (10−9 to 10−7 M) and 1 nM of 17β‐estradiol (E2). PFOS and PFOA did not modulate the expression of estrogen‐responsive genes, including progesterone (PR) and trefoil factor (pS2), but these compounds enhanced the effect of E2‐induced pS2 gene expression. Neither PFOS nor PFOA affected T47D cell viability at any of the tested concentrations. In contrast, co‐exposure with PFOS or PFOA and E2 resulted in an increase of E2‐induced cell viability, but no effect was found with 10 ng ml−1 EGF co‐exposure. Both compounds also intensified E2‐dependent growth in the proliferation assay. ERK1/2 phosphorylation was increased by co‐exposure with PFOS or PFOA and E2, but not with EGF. Collectively, this study shows that PFOS and PFOA did not possess estrogenic activity, but they enhanced the effects of E2 on estrogen‐responsive gene expression, ERK1/2 activation and the growth of the hormone‐deprived T47D cells. Copyright

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.

Sodium arsenite inhibited genomic estrogen signaling but induced pERα (Ser118) via MAPK pathway in breast cancer cells.

Arsenic (As) is considered a major environmental health threat worldwide due to its widespread contamination in drinking water. Recent studies reported that arsenic is a potential xenoestrogen as it interfered with the action of estrogen (E2) and estrogen receptor (ER) signaling. The present study investigated the effects of sodium arsenite (NaAsO2) on estrogen signaling in human breast cancer cells. The results demonstrated that NaAsO2 dose‐dependently increased viability of hormone‐dependent breast cancer MCF‐7 and T47D cells expressing both ERα and ERβ but not hormone‐independent MDA‐MB‐231 cells expressing ERβ. These suggested ERα contribution to NaAsO2‐stimulated breast cancer cells growth. NaAsO2 induced down‐regulation of ERα but up‐regulation of ERβ protein expressions in T47D cells. Moreover, NaAsO2 dose‐dependently inhibited E2‐induced ER transcriptional activity as it decreased E2‐mediated ERE‐luciferase transcription activation and PgR mRNA transcription but increased pS2 mRNA transcription. However, NaAsO2 induced both rapid and sustained activation of ERK1/2 and increased in phosphorylation of ERα at serine 118 residue, c‐fos and c‐myc protein expressions. These results indicated that NaAsO2 interferes the genomic estrogen‐signaling pathway but induces activation of a rapid nongenomic signal transduction through ERK1/2 pathway which may contribute to its proliferative effect on hormone‐dependent breast cancer cells.

Determination of levels of Mn, As, and other metals in water, sediment, and biota from Phayao Lake, Northern Thailand, and assessment of dietary exposure.

This study was undertaken to determine levels of contamination of toxic metals in water, sediment, and consumed fishery products from Phayao freshwater lake located in northern Thailand, which is a major water resource for drinking water, agriculture, and household use. Concentrations of Mn, As, and other metals were determined in water, sediment, fish tissues (Puntius gonionotus) and pond snails (Filopaludina martensi). Sampling was carried out in 3 periods (February, May, and August) in 2005. Metal analysis was performed by using inductively coupled plasma mass spectrometer (ICP-MS). Concentrations of Mn and As in lakewater ranged from 40–382 and 0.68–8.79 μ g L− 1 whereas the USEPA (Mn) and WHO (As) guidelines for drinking water are 50 and 10 μ g L− 1, respectively. Concentrations of some metals (Al, Cr, Mn, and Fe) in water were found to be higher in the area where water flowed into the lake from a small river than in other areas. The highest metal concentrations were found in the period of the dry season (May 2005). Among different sampling sites, the patterns of metal accumulations were different. Estimated fishery product consumption from the lake was calculated and the results indicated that the concentrations of metals in these products were lower than the recommended average daily dietary intake. Therefore, the consumption of fish and pond snail from this water resource may not pose a risk of metal toxicity. However, monitoring of the levels of Mn and As in lakewater should be carried out routinely so that appropriate prevention of contamination from these toxic metals can be implemented.

Src family kinase involvement in muscarinic receptor-induced tyrosine phosphorylation in differentiated SH-SY5Y cells.

Muscarinic receptor-mediated changes in protein tyrosine phosphorylation were examined in differentiated human neuroblastoma SH-SY5Y cells. Treatment of differentiated cells with 1 mM carbachol caused rapid increases in the tyrosine phosphorylation of focal adhesion kinase (FAK), Cas, and paxillin. The src family kinase-selective inhibitor PP1 reduced carbachol-stimulated tyrosine phosphorylation of FAK, Cas, and paxillin by 50 to 75%. In contrast, carbachol-stimulated activation of ERK1/2 was unaffected by PP1. Src family kinase activation by carbachol was further demonstrated by increased carbachol-induced tyrosine phosphorylation of the src-substrate, p120, and tyrosine phosphorylation of the src family kinase activation-associated autophosphorylation site. Site-specific FAK phosphotyrosine antibodies were used to determine that the carbachol-stimulated increase in the autophosphorylation of FAK was unaffected by pretreatment with PP1, whereas the carbachol-stimulated increase in the src family kinase-mediated phosphotyrosine of FAK was completely blocked by pretreatment with PP1. In SH-SY5Y cell lines stably overexpressing Fyn, the phosphotyrosine immunoreactivity of FAK was 625% that of control cells. Thus, muscarinic receptors activate protein tyrosine phosphorylation in differentiated cells, and the tyrosine phosphorylation of FAK, Cas, and paxillin, but not ERK1/2, is mediated by a src family tyrosine kinase activated in response to stimulation of muscarinic receptors.