Blockade of TRPM7 Channel Activity and Cell Death by Inhibitors of 5-Lipoxygenase (original) (raw)
Figure 6
The effects of 5-LOX inhibitors on cell viability.
(A) Overexpression of TRPM7 in HEK-293 cells cultured in low extracellular divalent cations (0.5 mM Ca2+ and 0 mM Mg2+) increased cell death compared to expressing cells grown in HBSS containing 1.8 mM Ca2+ and 1 mM Mg2+. Application of AA861 (10 µM) and NDGA (10 µM) but not zileuton (10 µM) increased the cell viability of TRPM7-expressing cells in response to reduced extracellular divalent cations. Cell viability was measured after 48 hours treatment by manual cell counting and trypan blue staining. (B) Knockdown of TRPM7 in HEK-293 cells rendered the cells more resistant to cell stress. Cell viability of TRPM7-knockdown 293-M7shRNA2 and the control 293-shRNA-C cell line in response to 48 hours treatment with the apoptotic stimulating agents doxorubicin (DOX) at 0.25 µM, cycloheximide (CHX) at 1 µg/ml, and staurosporine (STS) at 0.25 µM. Cell viability was measured using the MTT assay. (C) Overexpression of TRPM7 in 293-TRPM7 cells increased their sensitivity to cell death by DOX, STS, and CHX. 293-TRPM7 cells were treated with 0.0625 µM DOX, 1 µg/ml CHX, and 0.25 µM STS for 48 hours with or without pre-treatment with TET for 18 hours to induce protein expression. Cell viability was determined using the MTT assay. (D) Application of the 5-LOX inhibitor AA861 (10 µM), but not zileuton (10 µM), to 293-shRNA-C cells increased their resistance to cell death by DOX (0.25 µM), CHX (1 µg/ml) and STS (0.25 µM) to a level similar to 293-M7shRNA2 cells not treated with AA861. Cell viability was determined by manual cell counting and trypan blue staining. All data are presented as the mean ± standard deviation of three independent experiments. The asterisk indicates a significant difference in cell viability between two different treatments using Student's t test (p<0.05).