Glutathione Peroxidase 1 Promotes NSCLC Resistance to Cisplatin via ROS-Induced Activation of PI3K/AKT Pathway

Abstract

Purpose: Reactive oxygen species (ROS)-induced cytotoxicity is an important mechanism by which cisplatin kills tumor cells. Glutathione peroxidase family (GPXs) is an important member of antioxidant system which metabolizes intracellular ROS and maintains homeostasis of cells. Altered expressions of GPXs enzymes, especially GPX1, have been described in a variety of human cancers. However, their functional roles in cisplatin-based chemoresistance in human malignancies including non-small cell lung cancer have never been explored.

Methods: A panel of NSCLC cell lines were selected for this study. GPX1 expression was detected using quantitative RT-PCR and Western blot. Cisplatin-induced cell killing was analyzed by CCK8 assay. Intracellular ROS levels were detected by fluorescence-based flow cytometry analysis. In vitro overexpression and knockdown of GPX1 expression were performed using GPX1 expression vector and siRNA approaches. Protein levels of PTEN, NF-κB, BCL2, Bax, and phosphorylated AKT were detected with western blot analysis using specific antibodies.

Results: GPX1 expression was upregulated in a subset of NSCLC cell lines resistant to cisplatin treatment. Expression vector-mediated forced overexpression of GPX1 significantly increased cisplatin resistance in NSCLC cell lines, whereas RNA inference-mediated downregulation of GPX1 could restore sensitivity to cisplatin. Overexpression of GPX1 significantly suppressed elevation of intracellular ROS and activation of AKT pathway when NSCLC cell lines were exposed to different concentrations of cisplatin. Activation of the AKT pathway inhibited proapoptotic cascade and subsequently led to cisplatin resistance in NSCLC cells. Inhibition of NF-κB by its chemical inhibitor BAY can significantly downregulate GPX1 expression and restore the cisplatin sensitivity of the cell lines resistant to cisplatin.

Conclusions: Our findings suggested that overexpression of GPX1 is a novel molecular mechanism for cisplatin-based chemoresistance in NSCLC. GPX1 overexpression blocks cisplatin-induced ROS intracellular accumulation, activates PI3K-AKT pathway by increased AKT phosphorylation, and further leads to cisplatin resistance in NSCLC cells. Inhibition of NF-κB signaling may be an alternative approach for restoring cisplatin sensitivity for NSCLC cells resistant to cisplatin-based chemotherapy.

Figure 1

The relationship of the expression of GPX1 and IC50 in NSCLC cell lines. (a) The relative expression of GPX1 in 9 NSCLC cell lines was detected by quantitative RT-PCR. (b) The IC50 of 9 NSCLC cell lines to cisplatin. (c) The relationship of the expression of GPX1 and IC50 in NSCLC cell lines.

Figure 2

Increased expression of GPX1 promotes cisplatin resistance in NSCLC cells. ((a), (b)) To confirm the transfection efficiency, we used quantitative RT-PCR (a) and western blotting (b). Decreasing ((c) and (d)) or increasing ((e) and (f)) GPX1 expression influenced the cisplatin resistance in NSCLC cells. Reduced GPX1 expression downregulates cisplatin resistance, and increased GPX1 expression enhances cisplatin resistance (^∗P<0.05). Highly significant effects were observed at cisplatin concentrations of 2.5 and 5 μmol/L.

Figure 3

Increased GPX1 expression reduced intracellular ROS accumulation in NSCLC cells. The H460 (a) and H1650 (b) were transfected with GPX1 expression vector, and GEO mean value was detected by a flow cytometer after cells were treated with different concentrations of cisplatin for 72 h in culture. Along with the increased concentrations of cisplatin, intracellular ROS (GEO mean) in GPX1 vector transfected cells decreased as compared to empty vector transfected control cells ((c) and (d)). The difference of intracellular ROS levels between GPX1 vector and empty vector transfected cells is statistically significant (P<0.05).

Figure 4

GPX1 could affect the PI3K-AKT pathway in NSCLC. (a) In cisplatin sensitive cell lines H460 and H1650, forced expression of GPX1 increased the level of activated AKT (detected using antibody against phosphorylated AKT) whereas downregulation of GPX1 by siRNA reduced the level of activated AKT. (b) Overexpression of GPX1 increased BCL2 level and reduced Bax expression, whereas downregulation of GPX1 inhibited BCL2 expression and increased Bax expression.

Figure 5

GPX1 could mediate cisplatin resistance in NSCLC via the PI3K-AKT pathway. (a) Treatment of GPX1 downregulated A549 cells with EGF, a PI(3)K activator, increased their sensitivity to cisplatin compared to A549 cells treated with siGPX1 alone. Western blot analysis demonstrating an inhibition of AKT phosphorylation in response to LY294002 in GPX1-downregulated A549 cells and regaining the expression of that by treatment of EGF. (b) Treatment of GPX1 upregulated H1650 cells with LY294002, a PI(3)K inhibitor, decreased their sensitivity to cisplatin compared to H1650 cells treated with GPX1 vector alone. (∗: LY294002a, LY294002 with siGPX1; EGFa, EGF with siGPX1; LY294002b, LY294002 with GPX1 vector; EGFb, EGF with GPX1 vector.)

Figure 6

BAY 11-7082, a NF-κB inhibitor, downregulated intracellular ROS and reduced cisplatin resistance. (a) A549 and H1975 NSCLC cells treated with BAY 11-7082 showed downregulation of GPX1 expression, and correspondingly AKT phosphorylation was inhibited. The effects of BAY 11-7082 were similar to the effects observed in the same cell lines treated with GPX1 siRNA. ((b), (c), and (d)) In H1975 cells, suppression of GPX1 expression by both siGPX1 and BAY 11-7082 enhanced intracellular ROS accumulation and subsequently restored the sensitivity of the treated cells to cisplatin. ((e), (f), and (g)) A549 cells showed the same findings as H1975 cells when the cells were treated with siGPX1 and BAY 11-7082.