The protective effect of imatinib against pancreatic β-cell apoptosis induced by dexamethasone via increased GSTP1 expression and reduced oxidative stress

Abstract

Glucocorticoids (GCs) are known to stimulate pancreatic beta (β)-cell apoptosis via several mechanisms, including oxidative stress. Our previous study suggested an increase in dexamethasone-induced pancreatic β-cell apoptosis via a reduction of glutathione S-transferase P1 (GSTP1), which is an antioxidant enzyme. Imatinib, which is a tyrosine kinase inhibitor, also exerts antioxidant effect. This study aims to test our hypothesis that imatinib would prevent pancreatic β-cell apoptosis induced by dexamethasone via increased GSTP1 expression and reduced oxidative stress. Our results revealed that dexamethasone significantly increased apoptosis in INS-1 cells when compared to the control, and that imatinib significantly decreased INS-1 cell apoptosis induced by dexamethasone. Moreover, dexamethasone significantly increased superoxide production in INS-1 cells when compared to the control; however, imatinib, when combined with dexamethasone, significantly reduced superoxide production in INS-1 cells. Dexamethasone significantly decreased GSTP1, p-ERK1/2, and BCL2 protein expression, but significantly increased p-JNK, p-p38, and BAX protein expression in INS-1 cells-all compared to control. Importantly, imatinib significantly ameliorated the effect of dexamethasone on the expression of GSTP1, p-ERK1/2, p-JNK, p-p38 MAPK, BAX, and BCL2. Furthermore-6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), which is a GSTP1 inhibitor, neutralized the protective effect of imatinib against pancreatic β-cell apoptosis induced by dexamethasone. In conclusion, imatinib decreases pancreatic β-cell apoptosis induced by dexamethasone via increased GSTP1 expression and reduced oxidative stress.

Keywords: Apoptosis; Dexamethasone; GSTP1; Glucocorticoid receptor; Pancreatic β-cells.

Figure 1

The effect of dexamethasone and RU486 on pancreatic β-cell apoptosis and GSTP1 protein expression. (A) Percent apoptosis in INS-1 cells at 72 h was evaluated by Annexin V-FITC/PI staining. (B) Representative Western blot analysis of GSTP1 and β-actin from INS-1 cells. The bar graph shows the GSTP1 protein level normalized to the β-actin protein. (C) Representative Western blot analysis of GSTP1 and β-actin from mouse islets. The bar graph shows the GSTP1 protein level normalized to the β-actin protein. (D) Representative Western blot analysis of GSTP1 and β-actin from INS-1 cells cultured with or without dexamethasone in the presence or absence of RU486. The bar graph shows the GSTP1 protein level normalized to the β-actin protein. Data are expressed as the mean ± SEM of at least 3 independent experiments, and a p-value less than 0.05 indicates statistical significance. (*p < 0.05 vs. dexamethasone; **p < 0.01 vs. dexamethasone; ***p < 0.001 vs. dexamethasone; ##p < 0.01 vs. control; and, ###p < 0.001 vs. control).

Figure 2

The effect of dexamethasone and NBDHEX on superoxide production and cell viability in INS-1 cells. (A) Cellular superoxide production was determined by NBT assay. (B) The effect of NBDHEX on cellular superoxide production was determined by NBT assay. (C) Cell viability was assessed by PrestoBlue assay. Data are expressed as the mean ± SEM of at least 3 independent experiments, and a p-value less than 0.05 indicates statistical significance. (**p < 0.01 vs. dexamethasone; ##p < 0.01 vs. control; *p < 0.05 vs. dexamethasone; and, #p < 0.05 vs. control).

Figure 3

The effect of dexamethasone and imatinib on p-JNK, p-p38, and p-ERK1/2 protein expression. (A) Representative Western blot analysis of p-JNK normalized to the JNK protein. (B) Representative Western blot analysis of p-p38 normalized to the p38 protein. (C) Representative Western blot analysis of p-ERK1/2 normalized to the ERK1/2 protein. Data are expressed as the mean ± SEM of at least 3 independent experiments, and a p-value less than 0.05 indicates statistical significance. (#p < 0.05 vs. control; *p < 0.05 vs. dexamethasone; and, **p < 0.01 vs. dexamethasone).

Figure 4

The effect of dexamethasone and imatinib on BAX and BCL2 protein expression. (A) Representative Western blot analysis of the proapoptotic protein BAX normalized to the β-actin protein in INS-1 cells at 48 h. (B) Representative Western blot analysis of BCL2 normalized to the β-actin protein in INS-1 cells at 48 h. Data are expressed as the mean ± SEM of at least 3 independent experiments, and a p-value less than 0.05 indicates statistical significance. (##p < 0.01 vs. control; ####p < 0.0001 vs. control; and, *p < 0.05 vs. dexamethasone) (C) The pictorially demonstrated proposed effect of imatinib is decreased dexamethasone-induced pancreatic β-cell apoptosis via the induction of GSTP1. Dexamethasone suppresses GSTP1 expression, which increases ROS and p-JNK. ROS also induces p-p38, but decreases p-ERK1/2, which causes apoptosis. Imatinib ameliorates the suppression of GSTP1, which results in a decrease in ROS, p-JNK, and p-p38 expression to control levels. These data strongly suggest that imatinib can protect against dexamethasone-induced pancreatic β-cell apoptosis in mouse INS-1 cells.