Downregulation of Keap1 contributes to poor prognosis and Axitinib resistance of renal cell carcinoma via upregulation of Nrf2 expression

Abstract

Kelch‑like ECH‑associated protein 1 (Keap1)/nuclear factor erythroid 2‑related factor 2 (Nrf2) signaling has a protective effect on normal cells. A number of previous studies demonstrated that Keap1/Nrf2 signaling is associated with drug resistance in numerous tumors. The aim of the present study was to investigate the roles of Keap1 in renal cell carcinoma (RCC) and its effect on sensitivity to chemotherapy. Reverse transcription‑quantitative polymerase chain reaction was used to detect the mRNA expression of Keap1 in 45 cases of RCC tumors and adjacent normal tissues. A total of five randomly selected patients with RCC, five RCC cell lines and normal renal tubular cells were examined to detect the protein and mRNA expressions of Keap1. The 5‑year survival rate was analyzed by Kaplan‑Meier analysis. The cell viability was assessed by a Cell Counting kit‑8 assay. The cell apoptosis and reactive oxygen species (ROS) were determined by flow cytometry. The expressions of associated proteins were determined by western blot analysis. It was identified that in RCC tissues and RCC cell lines, the expression of Keap1 was downregulated, which was considered to be associated with poor prognosis. In total, 1 µM Axitinib significantly decreased cell viability, promoted ROS release and induced cell apoptosis in ACHN cells. Silencing Keap1 was able to reverse the inhibitory effect of Axitinib and enhance the protein expressions of Nrf2, NAD(P)H dehydrogenase [quinone] 1 and heme oxygenase 1. However, silencing Nrf2 increased the cell sensitivity to Axitinib. Under Axitinib condition, overexpressing Nrf2 was able to increase cell viability; however, overexpressing Keap1 resulted in an opposite effect. Keap1 serves as a tumor suppressor; its low expression was associated with poor prognosis and a decreased sensitivity of RCC cells to Axitinib. A possible mechanism underlying Axitinib resistance may involve Nrf2 overexpression.

Figure 1

Low expression of Keap1 in RCC is associated with a poor survival rate. (A) In total, 45 cases of patients with RCC were analyzed to determine the mRNA expression of Keap1 in normal and cancer tissues (P=0.001). (B) Kaplan-Meier survival curve was determined to assess the association between the expression of Keap1 and survival rate (P=0.140). A total of five patients with RCC were randomly selected to assess the expression of Keap1 at the (C) protein and (D) mRNA level. GAPDH served as an internal control. Data are presented as the mean ± standard deviation from three independent experiments. ^**P<0.01 vs. respective normal RCC tissue. (E) Western blotting was used to detect the protein expression in normal human renal tubular epithelial cells (HK-2 cells) and five RCC cell lines (ACHN, 769-P, A-498, 786-0 and Caki-1). (F) Reverse transcription-quantitative polymerase chain reaction was used to detect the mRNA expression in normal human renal tubular epithelial cells and five RCC cell lines. GAPDH served as an internal control. Data are presented as the mean ± standard deviation from three independent experiments. ^**P<0.01 vs. HK-2 cells. Keap1, Kelch-like ECH-associated protein 1; RCC, renal cell carcinoma.

Figure 2

Different concentrations of Axitinib are administered to renal cell carcinoma cell lines. Cell Counting kit-8 was used to detect the effect of different concentrations (0.1, 1, 10, 50 and 100 µM) of Axitinib on (A) ACHN and (B) 769-P cells for 24 and 48 h. Data are presented as the mean ± standard deviation from three independent experiments. ^*P<0.05, ^**P<0.01 vs. respective 0 µM Axitinib.

Figure 3

Transfection efficiencies of Keap1 and Nrf2 in ACHN cells. (A) Keap1 was silenced and overexpressed in cells and the mRNA expression of Keap1 was determined by RT-qPCR. (B) Nrf2 was silenced and overexpressed in cells and the mRNA expression of Nrf2 was determined by RT-qPCR. Data are presented as the mean ± standard deviation from three independent experiments. ^**P<0.01 vs. control. Keap1, Kelch-like ECH-associated protein 1; Nrf2, nuclear factor erythroid 2-related factor 2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; si, small interfering; NC, negative control.

Figure 4

Effect of silencing Keap1 on the cell viability, ROS level and protein expression of Nrf2 and ERK signaling. ACHN cells were transfected with siKeap1 and divided into five groups, which were the control, Ax (1 µM Ax), NC+Ax, siKeap1+Ax and siKeap1 groups. (A) Cell viability was determined by a Cell Counting kit-8 assay. (B) ROS levels are presented in a bar graph. (C) Flow cytometry was used to detect the ROS level. (D) Nrf2 protein expression was detected by western blotting. Lamin B1 served as an internal control. (E) Keap1 protein expression was detected by western blotting. GAPDH served as an internal control. (F) NQO1, HO1, p-ERK and ERK protein expressions were detected by western blotting. GAPDH served as an internal control. Data are presented as the mean ± standard deviation from three independent experiments. ^*P<0.05, ^**P<0.01 vs. control; ^#P<0.05, ^##P<0.01. Keap1, Kelch-like ECH-associated protein 1; ROS, reactive oxygen species; Nrf2, nuclear factor erythroid 2-related factor 2; ERK, extracellular signal-regulated kinase; Ax, Axitinib; NC, negative control; si, small interfering; NQO1, anti- NAD(P)H dehydrogenase [quinone] 1; HO1, heme oxygenase 1; p-, phosphorylated.

Figure 5

Effect of co-transfection of siKeap1 and siNrf2 on protein expression of Nrf2 and Keap1, cell viability and apoptosis. ACHN were transfected with siKeap1 and siNrf2 and were divided into six groups, which were the control, Ax (1 µM Ax), siKeap1+Ax, siKeap1+siNrf2+Ax, siNrf2+Ax and siNrf2 groups. (A) Nrf2 protein expression was detected by western blotting. Lamin B1 served as an internal control. (B) Keap1 protein expression was detected by western blotting. GAPDH served as an internal control. (C) Cell viability was assessed by a Cell Counting kit-8 assay. (D) Apoptosis rate is presented as a bar graph. (E) Flow cytometry was used to detect the apoptosis level. Data are presented as the mean ± standard deviation from three independent experiments. ^*P<0.05, ^**P<0.01 vs. control; ^#P<0.05, ^##P<0.01. Keap1, Kelch-like ECH-associated protein 1; Nrf2, nuclear factor erythroid 2-related factor 2; Ax, Axitinib; si, small interfering; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure 6

Effect of overexpressing Nrf2 on cell viability. Nrf2 was overexpressed in ACHN cells and were divided into three groups, which were the control, Mock+Ax and Nrf2+Ax groups. (A) Nrf2 protein expression was detected by western blotting. Lamin B1 served as an internal control. (B) Keap1 protein expression was detected by western blotting. GAPDH served as an internal control. (C) Cell viability was assessed by a Cell Counting kit-8 assay. Data are presented as the mean ± standard deviation from three independent experiments. ^*P<0.05, ^**P<0.01 vs. control; ^#P<0.05. Keap1, Kelch-like ECH-associated protein 1; Ax, Axitinib; Nrf2, nuclear factor erythroid 2-related factor 2.

Figure 7

Effect of overexpressing Keap1 on cell viability. Keap1 was overexpressed in ACHN cells and were divided into three groups, which were the control, Mock+Ax and Keap1+Ax groups. (A) Nrf2 protein expression was detected by western blotting. Lamin B1 served as an internal control. (B) Keap1 protein expression was detected by western blotting. GAPDH served as an internal control. (C) Cell viability was detected by a Cell Counting kit-8 assay. Data are presented as the mean ± standard deviation from three independent experiments. ^*P<0.05, ^**P<0.01 vs. control; ^##P<0.01. Keap1, Kelch-like ECH-associated protein 1; Ax, Axitinib; Nrf2, nuclear factor erythroid 2-related factor 2.