Table 1.
The primer sequences used for quantitative RT-PCR.
Fig 1.
Lansoprazole activates the Nrf2/ARE pathway in RL34 cells.
A: Representative immunoblot for Nrf2 expression levels in the nuclear and cytoplasmic protein lysates obtained from cells treated with 100 μM of lansoprazole for 3 h; α-tubulin and histone H1 were loading controls for the cytoplasmic and nuclear lysates, respectively (left). Densitometric quantification of the representative immunoblots was performed using ImageJ, and values were normalized to each of the loading control protein level (right). B: Representative immunocytochemical images of the subcellular localization of Nrf2. RL34 cells were treated with 100 μM of lansoprazole for 3 h. Cells were fixed in 4% PFA for 15 min, followed by staining with an anti-Nrf2 antibody and an Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody. Counterstaining of the nuclei was performed with Hoechst 33342. The scale bar represents 50 μm. C and D: Relative mRNA expression levels of Nrf2-induced genes HO1, NQO1, and GSTA2 in cells treated with 100 μM lansoprazole for 3 h. RNA levels were normalized to those of β-actin (C) or GAPDH (D). E: Induction of the ARE-dependent luciferase reporter activity by lansoprazole. RL34 cells stably expressing an ARE-reporter gene were treated with 100 μM of lansoprazole for 3 h, and the luciferase activity in the cell lysates was measured. F: Degradation rate of the Nrf2 protein after CHX chase. Cells were treated with 100 μM of lansoprazole for 3 h and were then treated with 10 μM of CHX for the indicated periods. Cell lysates were used for immunoblotting analysis with an anti-Nrf2 and anti-β-actin antibody. Intensities of the Nrf2 bands were quantified and plotted on a semilog graph to obtain half-life values. Abbreviations used in the figure: LPZ, lansoprazole; CHX, cycloheximide. Data represent the mean ± SD of the three independent experiments performed in triplicate. Statistical analysis was performed by using Student’s t-test. *: p < 0.05 vs. DMSO-treated cells.
Fig 2.
Lansoprazole suppresses the cisplatin-induced cytotoxicity.
Cell viability of cisplatin-treated cells exposed to a lansoprazole pretreatment. RL34 cells were pretreated with 100 μM of lansoprazole for 3 h and then exposed to 20 μM of cisplatin for an additional 24 h. HO1 inhibitor SnMP was administered simultaneously with lansoprazole. Cell viability was quantified using an MTS assay. Data are expressed as a percentage of viability when compared with the viability of the DMSO-treated cells. Data represent the mean ± SD of three independent experiments performed in triplicate. Statistical analysis was performed by using ANOVA followed by Tukey’s test for multiple comparisons. *: p < 0.01. Abbreviations: LPZ, lansoprazole; SnMP, tin-mesoporphyrin IX; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt.
Fig 3.
Nrf2 is essential for the cytoprotective activity of lansoprazole.
A and B: Knockdown efficiency of siRNAs targeting Nrf2. RL34 cells were transfected with one of the two siRNAs targeting different sites in the rat Nrf2 mRNA (5 nM) or with control siRNA (5 nM) for 24 h. Nrf2 mRNA levels were measured using quantitative RT-PCR and were normalized to those of β-actin (A) or GAPDH (B). Results are expressed as a percentage of the mRNA levels of the samples treated with control siRNA. C and D: Effects of Nrf2 knockdown on the expression of the lansoprazole-induced gene HO1 mRNA. Cells were transfected with 5 nM of control or pooled Nrf2 siRNA and, 24 h later, were treated with 100 μM of lansoprazole for 3 h. HO1 mRNA levels were measured using quantitative RT-PCR; RNA levels were normalized to the β-actin (C) or GAPDH (D) mRNA level. Data represent the mean ± SD of three independent experiments performed in triplicate. Statistical analysis was performed using Student’s t-test. *: p < 0.05 vs. DMSO-treated control siRNA-transfected cells. E: Effects of Nrf2 knockdown on the expression of lansoprazole-induced Nrf2 and HO1 protein. Cells were transfected with 5 nM of control or pooled Nrf2 siRNA and then 24 h later were treated with 100 μM of lansoprazole for 3 h. Nrf2 and HO1 protein levels were measured using immunoblotting. β-actin served as a loading control. F: Effect of siRNA depletion of Nrf2 on the lansoprazole-mediated cytoprotection. siRNA-transfected cells were treated with 100 μM of lansoprazole for 3 h and then exposed to 20 μM of cisplatin for an additional 24 h. Cell viability was quantified using an MTS assay. Data are expressed as a percentage of viability when compared with DMSO-treated control siRNA-transfected cells. Data represent the mean ± SD of three independent experiments performed in triplicate. Statistical analysis was performed by using ANOVA and Dunnett’s test. *: p < 0.01 vs. cisplatin treatment group. Abbreviations: LPZ, lansoprazole; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt.
Fig 4.
Lansoprazole activates p38 MAPK, but not ERK1/2 or JNK in RL34 cells.
A: Detection of phosphorylated Nrf2 in the nucleus. RL34 cells were treated with lansoprazole for 3 h. Nuclear protein extracts from the cells were incubated with or without λPPase at 4°C or 30°C, for 30 min, and were analyzed using immunoblotting with an anti-Nrf2 antibody. B: Representative immunoblot for the phosphorylated and total p38 MAPK, ERK1/2, and JNK expression levels. RL34 cells were treated with 100 μM of lansoprazole for 3 h. Whole cell lysates from the cells were analyzed using immunoblotting with the indicated antibodies; β-actin served as a loading control (left). Densitometric quantification of representative immunoblots was performed using ImageJ. Values were normalized to β-actin protein levels. The ratio of the phosphorylated protein to total protein were also calculated (right). Data represent the mean ± SD of three independent experiments performed in triplicate. Statistical analysis was performed using Student’s t-test. *: p < 0.05 vs. DMSO-treated cells. Abbreviation: LPZ, lansoprazole.
Fig 5.
Activation of p38 MAPK is required for the lansoprazole-induced ARE/Nrf2 pathway in RL34 cells.
A: Effect of SB203580 on the induction of Nrf2 protein expression by lansoprazole. RL34 cells were treated with 10 μM of SB203580 (a specific p38 MAPK inhibitor) for 30 min, and then exposed to 100 μM of lansoprazole for an additional 3 h. Whole cell protein lysates were analyzed by immunoblotting with an anti-Nrf2 antibody; β-actin served as a loading control. B: Effect of SB203580 on the induction of the ARE-dependent reporter activity by lansoprazole. RL34 cells stably expressing the ARE-reporter gene were pretreated with 10 μM of SB203580 for 30 min, and then exposed to 100 μM of lansoprazole for an additional 3 h. Luciferase activity in the cell lysates was measured. C and D: Total RNA was isolated from the cells. Relative mRNA expression levels of HO1 were measured by quantitative RT-PCR. RNA levels were normalized to β-actin (C) and GAPDH (D) mRNA level. Data represent the mean ± SD of three independent experiments performed in triplicate. Student’s t-test was employed for statistical analysis. *: p < 0.05 vs. untreated cells. E: Representative immunocytochemical images of the subcellular localization of Nrf2. RL34 cells were treated with 10 μM of SB203580 for 30 min and then exposed to 100 μM of lansoprazole for an additional 3 h. Cells were fixed in 4% PFA for 15 min, followed by a staining with anti-Nrf2 antibody and an Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody; counterstaining of the nuclei was performed with Hoechst 33342. The scale bar represents 50 μm. Abbreviation: LPZ, lansoprazole.
Fig 6.
Activation of p38 MAPK is required for the cytoprotective activity of lansoprazole against cisplatin-induced toxicity.
Effect of SB203580 on the cytoprotective activity of lansoprazole. RL34 cells were treated with 100 μM of lansoprazole for 3 h in the presence or absence of 10 μM of an SB203580 (a specific p38 MAPK inhibitor) pretreatment for 30 min and were then exposed to 20 μM of cisplatin for an additional 24 h. Cell viability was quantified using an MTS assay. Data are expressed as a percentage of viability of nontreated cells. Data represent the mean ± SD of three independent experiments performed in triplicate. Statistical analysis was performed using ANOVA followed by Tukey’s test for multiple comparisons. *: p < 0.01. Abbreviations: LPZ, lansoprazole; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt.
Fig 7.
Lansoprazole activates the p38 MAPK/Nrf2/ARE pathway and suppresses cisplatin-induced cell death in RL34 cells.
Schematic model of the Nrf2-dependent cytoprotective signaling pathway by lansoprazole in RL34 cells. Lansoprazole suppresses cisplatin-induced cytotoxicity through the activation of the Nrf2 pathway, accompanied by induction of antioxidant genes such as that encoding HO1. These effects are dependent on the p38 MAPK pathway.