Copper-transporting P-type adenosine triphosphatase (ATP7A) is associated with platinum-resistance in non-small cell lung cancer (NSCLC)

Abstract

Background: Copper export protein ATP7A is important for maintaining copper homeostasis. Recent studies have shown that copper transporters are also involved in the transport of platinum. The goal of this study was to determine the role of ATP7A in the platinum-resistance of non-small cell lung cancer (NSCLC).

Methods: Sensitivities to platinums were detected by MTT assay and drug-resistance related genes were analyzed by real-time PCR and immunoblotting between DDP-sensitive A549 and the corresponding DDP-resistant cell subline (A549/DDP). ATP7A expression was evaluated by immunohistochemistry in tumor tissues of unresectable NSCLC patients who received cisplatin-basing chemotherapy.

Results: The expression of ATP7A was significantly higher in A549/DDP cell subline than in A549 cells at both mRNA and protein levels. The silencing of ATP7A expression in A549/DDP by siRNA partially reversed DDP-resistance (29.62%) and increased cell apoptosis. ATP7A expression was detected in 41.6%of NSCLC patients, but not in adjacent stroma nor normal lung tissues. ATP7A-positive patients had a significantly poorer histological grade (p = 0.039) and poorer response to platinum-basing chemotherapy (p = 0.001) compared with ATP7A-negative patients. Cox's proportional hazards analysis showed that ATP7A expression was an independent prognostic factor for overall survival (p = 0.045).

Conclusions: ATP7A overexpression played an important role in platinum-resistance of NSCLC, and was a negative prognostic factor of NSCLC patients treated with platinum-based chemotherapy.

Figure 1

Expression of ATP7A at RNA and protein levels in A549 and A549/DDP cells. a, total of RNA isolated from A549 and A549/DDP cells was subjected to real-time PCR for ATP7A b, ATP7A protein expression was determined by Western-blot analysis using anti-ATP7A antibody. Equal loading was confirmed with GAPDH as a control in real-time PCR and Western-Blot.

Figure 2

Effect of ATP7A-siRNA 80 h on ATP7A protein expression in A549/DDP ells. Equal loading was confirmed with GAPDH as a control. Western blot analysis of ATP7A in A549/DDP cells following treatment with control siRNA and different concentrations of targeted ATP7A siRNA (25 nM, 50 nM and 100 nM) for 80 h. Protein expression was quantified by Quantity One software. Results are the representative of three similar experiments.

Figure 3

Increasing DDP induced apoptosis after ATP7A's silence for 80 h by siRNA in A549/DDP cells. Apoptotic or necrotic cell death was tested by flow cytometric analysis. Control siRNA, added 100 nM nonsilencing siRNA sequences for 8 h and then added DDP for 72 h; ATP7A siRNA, added 100 nM SiRNA targeting ATP7A for 8 h and then added DDP for 72 h. Columns, mean of three independent experiments; bars, SD.

Figure 4

Immunohistochemical staining of normal and NSCLC human tissues for ATP7A (magnification, ×400). a, adult normal kidney tissue as positive control; b, adult normal lung tissue as negative Control; c, ATP7A high positive case, almost all of tumor cells showed strong positive reaction in the cytoplasm; d, ATP7A negative case, ATP7A expression was not detectable in the cytoplasm.

Figure 5

Overall survival curves of NSCLC treated with platinum-based chemotherapy. Comparison of survival curves for patients whose tumors stained positive for ATP7A with those patients whose tumors were classified as negative for ATP7A expression. Curves present the results for all patients. P value was conducted by Log-rank test.