Involvement of Intracellular Ca2+-and PI3K-Dependent ERK Activation in TCDD-Induced Inhibition of Cell Proliferation in SK-N-SH Human Neuronal Cells원문보기
2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) has previously shown to induce neurotoxicity through intracellular $Ca^{2+}$ increase in rat neurons. In this study we investigated the role and signaling pathway of intracellular $Ca^{2+}$ in TCDD-induced inhibition of neuronal cell p...
2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) has previously shown to induce neurotoxicity through intracellular $Ca^{2+}$ increase in rat neurons. In this study we investigated the role and signaling pathway of intracellular $Ca^{2+}$ in TCDD-induced inhibition of neuronal cell proliferation in SK-N-SH human neuronal cells. We found that TCDD(10nM) rapidly increased the level of intracellular $Ca^{2+}$, which was completely blocked by the extracellular $Ca^{2+}$ chelation with EGTA (1 mM) or by pretreatment of the cells with the non-selective cation channel blocker. flufenamic acid (200 ${\mu}M$). However, pretreatment of the cells with dantrolene (25 ${\mu}M$) and TMB-8(10 ${\mu}M$), intracellular $Ca^{2+}$-release blockers, or a voltage-sensitive $Ca^{2+}$ channel blocker, varapamil (100 ${\mu}M$), failed to block the TCDD-induced $Ca^{2+}$ increase in the cells. In addition, TCDD induced a rapid and transient activation of phatidvlinositol 3-kinase (PI3K) and extracellular signal-regulated kinase 1/2(ERK1/2), which was ingnificantly blocked by the pretreatment with BAPTA, an intracellular $Ca^{2+}$ chelator, and LY294002, a PI3K inhibitor. Furthermore, inhibitors of PI3K, ERK, or an intracellular $Ca^{2+}$ chelator further potentiated the anti-proliferative effect of TCDD in the cells. Collectively, the results suggest that intracellular $Ca^{2+}$ and PI3K-dependent activation of ERK 1/2 may be involved in the TCDD-induced inhibition of cell proliferation in SK-N-SH human neuronal cells.
2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) has previously shown to induce neurotoxicity through intracellular $Ca^{2+}$ increase in rat neurons. In this study we investigated the role and signaling pathway of intracellular $Ca^{2+}$ in TCDD-induced inhibition of neuronal cell proliferation in SK-N-SH human neuronal cells. We found that TCDD(10nM) rapidly increased the level of intracellular $Ca^{2+}$, which was completely blocked by the extracellular $Ca^{2+}$ chelation with EGTA (1 mM) or by pretreatment of the cells with the non-selective cation channel blocker. flufenamic acid (200 ${\mu}M$). However, pretreatment of the cells with dantrolene (25 ${\mu}M$) and TMB-8(10 ${\mu}M$), intracellular $Ca^{2+}$-release blockers, or a voltage-sensitive $Ca^{2+}$ channel blocker, varapamil (100 ${\mu}M$), failed to block the TCDD-induced $Ca^{2+}$ increase in the cells. In addition, TCDD induced a rapid and transient activation of phatidvlinositol 3-kinase (PI3K) and extracellular signal-regulated kinase 1/2(ERK1/2), which was ingnificantly blocked by the pretreatment with BAPTA, an intracellular $Ca^{2+}$ chelator, and LY294002, a PI3K inhibitor. Furthermore, inhibitors of PI3K, ERK, or an intracellular $Ca^{2+}$ chelator further potentiated the anti-proliferative effect of TCDD in the cells. Collectively, the results suggest that intracellular $Ca^{2+}$ and PI3K-dependent activation of ERK 1/2 may be involved in the TCDD-induced inhibition of cell proliferation in SK-N-SH human neuronal cells.
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대상 데이터
The SK-N-SH human neuroblastoma cell line was purchased from American Type Culture Collection (Rockville, MA). The powders for Eagle's minimum essential medium, trypsin solution, sodium pyruvate, MTT and all salt powders were obtained from Sigma Chemical CO.
데이터처리
All experiments were performed four times. Data were expressed as mean±standard error of the mean (SEM) and were analyzed using one way analysis of variance (ANOVA) and Student-Newman-Keurs test for individual comparisons. P values less than 0.
이론/모형
Cell viability was assessed by the MTT staining method (van de Loosdrecht et al., 1991). Cells from 4- to 5-day-old cultures were seeded in 24-well plates at the density of 5 x 104 cells/ well.
TCDD induces intracellular Ca2+ increase through the activation of nonselective cationic channels. Intracellular Ca2+ level was measured by Fura-2 fluorescence method. The arrow shows the time point for addition of TCDD (10 nM).
성능/효과
The present study also demonstrated that TCDD-mduced intracellular Ca2+ increase activated ERK and prevented further decrease in the cell growth induced by TCDD in the cells. Our results showing that TCDD increased PI3K activity and that pretreatement with a PI3K inhibitor not only suppressed ERK activation but enhanced the TCDD-induced decrease in cell proliferation indicate that intracellular Ca2+ and PI3K activation may, at least in part, suppress the anti-proliferative signaling induced by TCDD through ERK. This is contrary to the report that TCDD mimics the action of a growth factor through Ca2+-independent PI3K activity in promoting the proliferation of MCF-10A human mammary epithelial cells in the absence of insulin (Tannheimer et al.
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