Figure 3: Inhibition of p38-MAPK impairs MDR1 expression and function of P-gp in SGC7901/VCR cells. SGC7901/VCR cells were treated with DMSO or SB202190 (10 μM). (A) Protein levels of P-gp were detected by Western-blot analysis. A representative example of an experiment that was repeated three times is shown. (B) SGC7901/VCR cells were treated with DMSO and SB202190. Expression of MDR1 mRNA was assessed by RT-PCR. β-actin mRNA levels were measured as positive internal controls. (C) The MDR1 mRNA expression levels were normalized to those of β-actin and are the means ± SD of at least three independent experiments. Significant differences are indicated by asterisks. *, P < 0.05. (D)Effects of SB202190 on Rh123 accumulation (left) and retention (right) in SGC7901 and SGC7901/VCR cells. (left) Cells treated with SB202190 (10 μM) or vehicle control (0.1% DMSO). Rh123 (1 μg/mL) was added, and the cells were incubated for 120 min. (right) Cells were incubated with Rh123 for 120 min, washed, and resuspended in medium with SB202190 (10 μM)or vehicle control (0.1% DMSO) for 120 min. Rh123 fluorescence was measured using FAC scan. Means ± SD from three independent experiments. *P < 0.05 vs SGC7901 cells. **P < 0.05 vs vehicle control.

Mentions: Our previous data clearly showed that the p38-MAPK/AP-1 pathway was activated in SGC7901/VCR cells. Moreover, AP-1 was reported to be involved in MDR1 upregulation [11]. We then investigated the effects of p38-MAPK inhibition on MDR1 expression and function of P-gp. Western-blot analysis showed that inhibition of p38-MAPK by SB202190 markedly downregulated P-gp expression in SGC7901/VCR cells. This effect was confirmed by RT-PCR analysis (Fig. 3A–C). To examine the effect of SB202190 on function of P-gp, Rh123 accumulation and efflux studies were chosen. As shown in Figure 3D, Rh123 accumulation and retention in SGC7901/VCR cells were obviously less than that in SGC7901 cells. After treatment with SB202190 (10 μM), Rh123 accumulation and retention in SGC7901/VCR cells were increased; however, there was no change in SGC7901 cells(Fig. 3D). Thus, our results clearly showed that inhibition of p38-MAPK reduced MDR1 gene expression and function of P-gp in SGC7901/VCR cells.

Guo X, Ma N, Wang J, Song J, Bu X, Cheng Y, Sun K, Xiong H, Jiang G, Zhang B, Wu M, Wei L - BMC Cancer (2008)

Bottom Line: Ap-1 activity and cell apoptosis were detected by Dual-Luciferase Reporter Assay and annexin V-PI dual staining.The vincristine-resistant SGC7901/VCR cells with increased expression of the multidrug-resistance 1 (MDR1) gene were resistant to P-gp-related drug and P-gp-unrelated drugs.Constitutive increases of phosphorylated p38-MAPK and AP-1 activities were also found in the drug-resistant cells.Inhibition of p38-MAPK by SB202190 reduced activator protein-1 (AP-1) activity and MDR1 expression levels and increased the sensitivity of SGC7901/VCR cells to chemotherapy.Activation of the p38-MAPK pathway might be responsible for the modulation of P-glycoprotein-mediated and P-glycoprotein-unmediated multidrug resistance in the SGC7901/VCR cell line.

Affiliation: Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, the Second Military Medical University, Shanghai, PR China. guoguo1188@gmail.com

Abstract: Chemoresistance is one of the main obstacles to successful cancer therapy and is frequently associated with Multidrug resistance (MDR). Many different mechanisms have been suggested to explain the development of an MDR phenotype in cancer cells. One of the most studied mechanisms is the overexpression of P-glycoprotein (P-gp), which is a product of the MDR1 gene. Tumor cells often acquire the drug-resistance phenotype due to upregulation of the MDR1 gene. Overexpression of MDR1 gene has often been reported in primary gastric adenocarcinoma.This study investigated the role of p38-MAPK signal pathway in vincristine-resistant SGC7901/VCR cells. P-gp and MDR1 RNA were detected by Western blot analysis and RT-PCR amplification. Mitgen-activated protein kinases and function of P-gp were demonstrated by Western blot and FACS Aria cytometer analysis. Ap-1 activity and cell apoptosis were detected by Dual-Luciferase Reporter Assay and annexin V-PI dual staining.The vincristine-resistant SGC7901/VCR cells with increased expression of the multidrug-resistance 1 (MDR1) gene were resistant to P-gp-related drug and P-gp-unrelated drugs. Constitutive increases of phosphorylated p38-MAPK and AP-1 activities were also found in the drug-resistant cells. Inhibition of p38-MAPK by SB202190 reduced activator protein-1 (AP-1) activity and MDR1 expression levels and increased the sensitivity of SGC7901/VCR cells to chemotherapy.Activation of the p38-MAPK pathway might be responsible for the modulation of P-glycoprotein-mediated and P-glycoprotein-unmediated multidrug resistance in the SGC7901/VCR cell line.