Bupivacaine-induced apoptosis independently of WDR35 expression in mouse neuroblastoma Neuro2a cells

Abstract

Background: Bupivacaine-induced neurotoxicity has been shown to occur through apoptosis. Recently, bupivacaine was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in a human neuroblastoma cell line. We have reported that WDR35, a WD40-repeat protein, may mediate apoptosis through caspase-3 activation. The present study was undertaken to test whether bupivacaine induces apoptosis in mouse neuroblastoma Neuro2a cells and to determine whether ROS, p38 MAPK, and WDR35 are involved.

Results: Our results showed that bupivacaine induced ROS generation and p38 MAPK activation in Neuro2a cells, resulting in apoptosis. Bupivacaine also increased WDR35 expression in a dose- and time-dependent manner. Hydrogen peroxide (H(2)O(2)) also increased WDR35 expression in Neuro2a cells. Antioxidant (EUK-8) and p38 MAPK inhibitor (SB202190) treatment attenuated the increase in caspase-3 activity, cell death and WDR35 expression induced by bupivacaine or H(2)O(2). Although transfection of Neuro2a cells with WDR35 siRNA attenuated the bupivacaine- or H(2)O(2)-induced increase in expression of WDR35 mRNA and protein, in contrast to our previous studies, it did not inhibit the increase in caspase-3 activity in bupivacaine- or H(2)O(2)-treated cells.

Conclusions: In summary, our results indicated that bupivacaine induced apoptosis in Neuro2a cells. Bupivacaine induced ROS generation and p38 MAPK activation, resulting in an increase in WDR35 expression, in these cells. However, the increase in WDR35 expression may not be essential for the bupivacaine-induced apoptosis in Neuro2a cells. These results may suggest the existence of another mechanism of bupivacaine-induced apoptosis independent from WDR35 expression in Neuro2a cells.

Figure 1

Effect of bupivacaine on cell death, ROS generation and p38 MAPK activation. Neuro2a cells were treated with 2 mM bupivacaine for the time indicated. (A) Cell viability was measured with the MTT assay. (B) Caspase-3 activity was measured with a fluorometric substrate. (C) DNA extracted from the cells was analyzed by agarose gel electrophoresis. (D) Intracellular levels of ROS were measured with a fluorogenic probe. (E) Expression of p38 and phospho-p38 (P-p38) following cell exposure to bupivacaine (2 mM) or H₂O₂ (0.5 mM) was measured by Western blotting. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control (not treated with bupivacaine or H₂O₂, n = 6).

Figure 2

Effect of bupivacaine or H₂O₂ on WDR35 expression. (A) Neuro2a cells were treated with various concentrations of bupivacaine or H₂O₂ for 9 h. WDR35 mRNA expression was analyzed by qPCR and expressed relative to the expression of GAPDH mRNA. (B) WDR35 mRNA expression and (C) WDR35 protein expression were measured after treatment of Neuro2a cells with 2 mM bupivacaine or 0.5 mM H₂O₂ for the time indicated. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control (not treated with bupivacaine or H₂O₂, n = 6).

Figure 3

Effect of antioxidant and p38 MAPK inhibitor on bupivacaine- or H₂O₂-induced ROS level, caspase-3 activity and cell viability. Neuro2a cells were treated with EUK-8 (EUK, 100 μM) or SB202190 (SB, 10 μM) for 1 h, followed by bupivacaine (2 mM) or H₂O₂ (0.5 mM) for 9 h. (A) Intracellular levels of ROS, (B) caspase-3 activity, and (C) cell viability were measured (n = 6). **P < 0.01 and ***P < 0.001 vs. control (not treated with bupivacaine or H₂O₂), ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 vs. absence of EUK-8 or SB202190.

Figure 4

Effect of antioxidant and p38 MAPK inhibitor on bupivacaine- or H₂O₂-induced WDR35 expression. (A) Neuro2a cells were treated with various concentrations of EUK-8 for 1 h, followed by bupivacaine (2 mM) or H₂O₂ (0.5 mM) for 9 h (n = 6). (B) WDR35 protein expression with or without 100 μM EUK-8 was analyzed by Western blotting (n = 4). *P < 0.05 and ***P < 0.001 vs. control (not treated with bupivacaine or H₂O₂), ^#P < 0.05 and ^##P < 0.01 vs. absence of EUK-8. (C) Cells were treated with 10 μM SB202190 (SB) for 1 h, followed by bupivacaine (2 mM) or H₂O₂ (0.5 mM) for 9 h (n = 6). (D) WDR35 protein expression with or without SB202190 was analyzed by Western blotting (n = 4). *P < 0.05 and ***P < 0.001 vs. control (not treated with bupivacaine or H₂O₂), ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 vs. absence of SB202190.

Figure 5

Effect of WDR35 siRNA on bupivacaine- or H₂O₂-induced WDR35 expression, caspase-3 activity, and cell viability. Neuro2a cells were transfected with WDR35 siRNA (5 nM) for 24 h. Bupivacaine (2 mM) or H₂O₂ (0.5 mM) was then added. (A) WDR35 mRNA expression and (B) WDR35 protein expression were measured after treatment of Neuro2a cells with bupivacaine or H₂O₂ for 9h. (C) Caspase-3 activity and (D) cell viability were measured after treatment of Neuro2a cells with bupivacaine or H₂O₂ for the time indicated. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control (not treated with siRNA, bupivacaine, or H₂O₂), ^#P < 0.05 and ^###P < 0.001 vs. treated with bupivacaine or H₂O₂ alone (n = 4-6).