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Original Article

Curcumin inhibits oxidative stress-induced TRPM2 channel activation, calcium ion entry and apoptosis values in SH-SY5Y neuroblastoma cells: Involvement of transfection procedure

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Pages 76-88 | Received 01 Dec 2016, Accepted 20 Feb 2017, Published online: 01 Jun 2017

Figures & data

Figure 1. Effects of curcumin on TRPM2 channel activity in SH-SY5Y neuroblastoma cells. (A) Control record: original recordings from SH-SY5Y cells. (B) Non-transfected control group, was tested whether H2O2 cause on the cellular cationic currents or not. (C) Non-transfected but curcumin incubated records of cells were to test any relationship between curcumin incubation and H2O2 induction on the currents. (D) TRPM2 currents in the transfected SH-SY5Y cells were stimulated by H2O2 (10 mM) in the chamber (bath) solution and they were inhibited by ACA (25 μM) in the bath. (E) I–V curve of panel D: current-voltage relationships of whole-cell currents in the presence of various extracellular cations, activators and inhibitors as indicated (same experiments as in panel D). (F) TRPM2 currents inhibited by curcumin incubation in the transfected + curcumin group. The cells were incubated with curcumin (5 μM) after transfection for 24 h and then they were not gated by H2O2 administration (in the patch chamber). The holding potential was -60 mV. NMDG+ time is shown where the normal bath solution (140 nM Na+) was exchanged to a solution with NMDG+ as main cation (150 mM, Ca2+ free solution). W.C.: Whole cell.

Figure 1. Effects of curcumin on TRPM2 channel activity in SH-SY5Y neuroblastoma cells. (A) Control record: original recordings from SH-SY5Y cells. (B) Non-transfected control group, was tested whether H2O2 cause on the cellular cationic currents or not. (C) Non-transfected but curcumin incubated records of cells were to test any relationship between curcumin incubation and H2O2 induction on the currents. (D) TRPM2 currents in the transfected SH-SY5Y cells were stimulated by H2O2 (10 mM) in the chamber (bath) solution and they were inhibited by ACA (25 μM) in the bath. (E) I–V curve of panel D: current-voltage relationships of whole-cell currents in the presence of various extracellular cations, activators and inhibitors as indicated (same experiments as in panel D). (F) TRPM2 currents inhibited by curcumin incubation in the transfected + curcumin group. The cells were incubated with curcumin (5 μM) after transfection for 24 h and then they were not gated by H2O2 administration (in the patch chamber). The holding potential was -60 mV. NMDG+ time is shown where the normal bath solution (140 nM Na+) was exchanged to a solution with NMDG+ as main cation (150 mM, Ca2+ free solution). W.C.: Whole cell.

Figure 2. Effects of curcumin on TRPM2 cation channel current densities in SH-SY5Y neuroblastoma cells. The cells were in vitro treated with H2O2 (10 mM) and ACA (25 μM). For each of the experiments, the initial current density (divided to the cell capacitance, which is a measure of cell size) was calculated after administration of H2O2 (mean ± SD). The numbers in parentheses indicate n number of groups. Significant stimulation and inhibition of currents were indicated with letters a; p < 0.001 vs control group, b; p < 0.001 vs curcumin group, c; p < 0.001 vs transfected group. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 2. Effects of curcumin on TRPM2 cation channel current densities in SH-SY5Y neuroblastoma cells. The cells were in vitro treated with H2O2 (10 mM) and ACA (25 μM). For each of the experiments, the initial current density (divided to the cell capacitance, which is a measure of cell size) was calculated after administration of H2O2 (mean ± SD). The numbers in parentheses indicate n number of groups. Significant stimulation and inhibition of currents were indicated with letters a; p < 0.001 vs control group, b; p < 0.001 vs curcumin group, c; p < 0.001 vs transfected group. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 3. A. Calcium release from four groups of SH-SY5Y cells following stimulation with 25 μM cumene hydroperoxide (CHPx). Original time course chart recordings showing [Ca2+]i transients in SH-SY5Y cells. B. Bar charts showing mean ± SD data for [Ca2+]i from H2O2 stimulated groups of SH-SY5Y cells (n = 6 for each). Note that the significant elevation in [Ca2+]i for SH-SY5Y cells compared to control group. a; p < 0.05 vs control group, b; p < 0.05 vs transfected group. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 3. A. Calcium release from four groups of SH-SY5Y cells following stimulation with 25 μM cumene hydroperoxide (CHPx). Original time course chart recordings showing [Ca2+]i transients in SH-SY5Y cells. B. Bar charts showing mean ± SD data for [Ca2+]i from H2O2 stimulated groups of SH-SY5Y cells (n = 6 for each). Note that the significant elevation in [Ca2+]i for SH-SY5Y cells compared to control group. a; p < 0.05 vs control group, b; p < 0.05 vs transfected group. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 4. Effects of TRPM2 transfection and/or curcumin incubation on the TRPM2 channel protein (A), PARP1 enzyme (B), procaspase 9 and procaspase 3 enzymes expression levels (C) relative densities represented of these protein levels respectively in SH-SY5Y cells. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 4. Effects of TRPM2 transfection and/or curcumin incubation on the TRPM2 channel protein (A), PARP1 enzyme (B), procaspase 9 and procaspase 3 enzymes expression levels (C) relative densities represented of these protein levels respectively in SH-SY5Y cells. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 5. Effects of curcumin administration on ROS production levels in SH-SY5Y cells (mean ± SD; n = 8). The values were expressed as fold increase over the control level (experimental/control). a; p < 0.001 vs control group, b; p < 0.001 vs curcumin group, c; p < 0.001 vs transfected group. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 5. Effects of curcumin administration on ROS production levels in SH-SY5Y cells (mean ± SD; n = 8). The values were expressed as fold increase over the control level (experimental/control). a; p < 0.001 vs control group, b; p < 0.001 vs curcumin group, c; p < 0.001 vs transfected group. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 6. Effects of curcumin on mitochondrial membrane depolarization levels in transfected and non-transfected SH-SY5Y cells (mean ± SD; n = 8). The values were expressed as fold increase over the control level (experimental/control). a; p < 0.05 vs control group, b; p < 0.05 vs curcumin group, c; p < 0.05 vs transfected group. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 6. Effects of curcumin on mitochondrial membrane depolarization levels in transfected and non-transfected SH-SY5Y cells (mean ± SD; n = 8). The values were expressed as fold increase over the control level (experimental/control). a; p < 0.05 vs control group, b; p < 0.05 vs curcumin group, c; p < 0.05 vs transfected group. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 7. Effects of curcumin on caspase 3 (A) and caspase 9 enzyme activity values (B) in SH-SY5Y neuroblastoma cells (mean ± SD; n = 8). The values were expressed as fold increase over the control level (experimental/control). a; p < 0.05 vs control group, b; p < 0.05 vs transfected group. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 7. Effects of curcumin on caspase 3 (A) and caspase 9 enzyme activity values (B) in SH-SY5Y neuroblastoma cells (mean ± SD; n = 8). The values were expressed as fold increase over the control level (experimental/control). a; p < 0.05 vs control group, b; p < 0.05 vs transfected group. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 8. Effects of curcumin on cell viability levels in SH-SY5Y cells (mean ± SD; n = 8). The values were expressed as fold increase over the control level (experimental/control). a; p < 0.05 vs control group, b; p < 0.05 vs curcumin group, c; p < 0.05 vs transfected group. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 8. Effects of curcumin on cell viability levels in SH-SY5Y cells (mean ± SD; n = 8). The values were expressed as fold increase over the control level (experimental/control). a; p < 0.05 vs control group, b; p < 0.05 vs curcumin group, c; p < 0.05 vs transfected group. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 9. Effects of curcumin on apoptosis levels in SH-SY5Y cells (mean ± SD; n = 8). The values were expressed as fold increase over the control level (experimental/control). a; p < 0.05 vs control group, b; p < 0.05 vs curcumin group, c; p < 0.001 vs transfected group. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

Figure 9. Effects of curcumin on apoptosis levels in SH-SY5Y cells (mean ± SD; n = 8). The values were expressed as fold increase over the control level (experimental/control). a; p < 0.05 vs control group, b; p < 0.05 vs curcumin group, c; p < 0.001 vs transfected group. Cur: curcumin; Trans: transfected; Trans + Cur: express transfected + curcumin group.

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