Nodal is involved in chemoresistance of renal cell carcinoma cells via regulation of ABCB1

Abstract

Renal cell carcinoma (RCC) is the third most frequent malignancy within urological oncology. Understanding mechanisms of chemoresistance in RCC cell is important for therapy and drug development. We established cisplatin (CDDP) resistant RCC cells by treating cells with increasing concentrations of CDDP. Nodal, an important embryonic morphogen, was increased in RCC/CDDP cells. Targeted inhibition of Nodal via its siRNA or neutralization antibody restored sensitivity of RCC resistant cells to CDDP treatment. It was due to that si-Nodal can decrease expression of P-glycoprotein (P-gp, encoded by ABCB1), one important ATP-binding cassette (ABC) membrane transporter for drug efflux. si-Nodal can decrease the transcription and promoter activity of ABCB1. Mechanistically, si-Nodal can decrease the phosphorylation of p65, which can bind to the promoter of ABCB1 and then trigger its transcription. Further, CDDP treatment decreased the expression of Nodal in culture medium of RCC cells. Collectively, we found that Nodal can regulate chemoresistance of RCC cells via regulating transcription of ABCB1.

Keywords: Nodal; P-gp; RCC; p65; transcription.

Figure 1

The establish of RCC/CDDP cells. GRC-1/CDDP (A), ACHN/CDDP (B) and their parental cells were treated with increasing concentrations of CDDP for 48 h, the cell viability was tested.

Figure 2

Nodal was increased in RCC/CDDP cells. The mRNA levels of measured cytokines in GRC-1/CDDP (A) and ACHN/CDDP (B) cells as compared to their corresponding control cells; The relative levels of Nodal in medium (C) or intracellular (D) of RCC/CDDP and RCC cells were measured by ELISA and western blot analysis, respectively.

Figure 3

Nodal regulated CDDP resistance of RCC cells. (A) Cells were transfected with si-NC or si-Nodal for 24 h, the expression of Nodal was tested by western blot analysis; GRC-1/CDDP (B) or ACHN/CDDP (C) cells were pre-transfected with si-NC or si-Nodal for 24 h and then further treated with increasing concentrations of CDDP for 48 h; GRC-1/CDDP (D) or ACHN/CDDP (E) cells were pre-treated with or without anti-Nodal (100 ng/ml) for 12 h and then further treated with increasing concentrations of CDDP for 48 h.

Figure 4

Nodal regulated expression of P-gp in RCC/CDDP cells. GRC-1/CDDP (A) or ACHN/CDDP (B) cells were transfected with si-NC or si-Nodal for 24 h, the mRNA expression of ABC transporters was checked; (C) The expression of P-gp in cells transfected with si-NC or si-Nodal for 24 h were tested; GRC-1 or ACHN cells were treated with or without rNodal (100 ng/ml) for 24 h, the mRNA (D) and protein (E) expression of P-gp was checked.

Figure 5

Nodal regulated promoter activity of ABCB1 in RCC/CDDP cells. (A) GRC-1/CDDP cells were pre-transfected with si-NC or si-Nodal for 24 h and then further treated with Act-D for the indicated times periods, the mRNA stability of ABCB1 was checked; (B) Cells were transfected with si-NC or si-Nodal for 24 h and precursor mRNA of ABCB1 was checked; (C) Both parental and resistant RCC cells were transfected pGL-ABCB1 and pRL-TK for 24 h, the luciferase activities of promoter were measured by dual-luciferase assay; (D) Cell pre-transfected with si-NC or si-Nodal were further transfected pGL-ABCB1 and pRL-TK for 24 h, the luciferase activities of promoter were measured.

Figure 6

NF-κB/p65 was involved in Nodal-regulated P-gp expression. (A) Cells pre- transfected pGL-ABCB1 and pRL-TK for 12 h were further treated with or without BAY (10 µM) for 24 h, the promoter activity of ABCB1 was checked by luciferase assay; (B) Cells were treated with or without BAY (10 µM) for 24 h, the mRNA expression of ABCB1 was checked; (C) Cells were treated with or without anti-Nodal for 30 min, the phosphorylation and total expression of p65 were checked; (D) GRC-1 cells were pre-treated with control or ACHP (10 µM) for 90 min and then treated with rNodal (100 ng/ml) for 30 min. p-p65 and p65 were measured by western blot analysis; GRC-1/CDDP cells were pre-transfected with vector control (pcDNA3.1) or pcDNA/p65 for 12 h and then further treated with or without anti-Nodal for 24 h, the mRNA (E) or protein (F) levels of P-gp were measured.

Figure 7

CDDP increased Nodal expression in RCC cells. (A) Cells were treated with or without 0.2 µM CDDP for 24 h, the expression of Nodal in medium was checked; (B) GRC-1 cells were treated with increasing concentrations of CDDP for 24 h, the expression of Nodal in medium was checked; Cells were treated with or without 0.2 µM CDDP for 24 h, the mRNA expression (C) or promoter activity (D) of Nodal were checked; GRC-1 (E) or ACHN (D) cells were treated with or without 0.2 µM CDDP for 24 h, the binding of Nodal promoter and transcription factors were checked by ChIP-PCR.