Increased ABCC2 expression predicts cisplatin resistance in non-small cell lung cancer

Abstract

Long-term use of platinum-based drugs can cause non-small cell lung cancer (NSCLC) to develop extremely strong drug resistance. Increasing the drug dosage does not have better treatment effects and could lead to serious complications. High levels of drug resistance are considered to be characteristic of human tumours and are usually mediated by genes related to multidrug resistance. Multidrug resistance-associated protein 2 (ABCC2), an ATP-binding cassette multidrug resistance transporter, was found to be overexpressed in various human cancers. In this study, we found that ABCC2 was also upregulated in cisplatin (DDP)-resistant A549 cells (A549/DDP). Functional studies demonstrated that ABCC2 knockdown reversed DDP resistance and promoted G1 phase arrest in A549/DDP cells, and PARP and caspase-3 were activated in A549/DDP cells following ABCC2 knockdown. In vivo, ABCC2 knockdown enhanced the cytotoxicity of DDP to subcutaneous A549 tumours. Together, these results suggest that ABCC2 may be a potential therapeutic strategy for overcoming DDP resistance in NSCLC patients. SIGNIFICANCE OF THE STUDY: In this study, we investigated the role of ABCC2 in cisplatin resistance of NSCLC cells. Our data show that ABCC2 expression was associated with resistance to cisplatin and that knockdown ABCC2 could reverse cisplatin resistance in NSCLC cells. Taken together, our study suggests that reducing the expression of ABCC2 could become an important strategy for enhancing the sensitivity of NSCLC cells to cisplatin.

Keywords: ABCC2; NSCLC; apoptosis; cell cycle; cisplatin-resistance.

FIGURE 1

Upregulation of ABCC2 is related to lung cancer prognosis. Association between ABCC2 expression and OS, A, and PFS, B. OS, overall survival; PFS, progression‐free survival

FIGURE 2

High expression of ABCC2 gene increases resistance of A549/DDP cells to DDP. A,B, A549 and A549/DDP cells treated with DDP were measured using CCK8 after 48 hours. The IC₅₀ in A549 cells and A549/DDP cells was 2.93 and 38.57 μM, respectively. The concentrations of DDP in A549 cells were 1.25, 2.5, 5, 10, 20 and 40 μM. The concentrations of DDP in A549/DDP were 5, 10, 20, 40, 60 and 120 μM. C,D, RNA sequencing showed that the expression of cell membrane transporter family related genes increased. Among them, expression of ABCC2 increased most significantly, reaching 12.351 times that of A549 cells. E, PCR results confirmed that a significant increase in ABCC2 expression was consistent with the RNA sequencing results. F, Western blot results showed that the expression of ABCC2 in DDP‐resistant cell lines also increased significantly at the protein level. G, Quantitative analysis of western blot in Fig. 2F. ***p < 0.001

FIGURE 3

Knockdown of ABCC2 partially reversed the resistance of A549/DDP to DDP. A‐C, ABCC2 expression was analysed using qRT‐PCR and western blot assays after A549/DDP cells were transfected with sh1‐ABCC2, sh2‐ABCC2 and sh‐control. D,E, The IC₅₀ in A549/DDP transfected with sh‐control or sh1‐ABCC2 were 36.25 and 18.74 μM, respectively. The concentrations of DDP in A549/DDP were 5, 10, 20, 40, 60, and 120 μM. F,G, A549 cells were treated with DDP (2 μM) for 48 hours. A549/DDP cells were treated with DDP (10 μM) for 48 hours. Cell cycle distribution assays were performed in A549 and A549/DDP cells using flow cytometry (n = 3, each group). *p < 0.05, **p < 0.01,***p < 0.001

FIGURE 4

Knockdown of ABCC2 promotes A549/DDP cell apoptosis by activating the caspase‐3 signalling pathway. A,B, Apoptosis of A549/DDP cells was examined using TUNEL assay. Nuclei labelled with red fluorescence represent early apoptotic cells. The TUNEL results reveal that the apoptosis rate was increased in the sh1‐ABCC2 + DDP (10 μM) group (scale bar = 20 μm). C,D, A549/DDP sh‐control and sh1‐ABCC2 cells were treated with DDP (10 μM) for 48 hours. Western blot revealed that the expression of apoptosis‐related genes, cleaved caspase‐3 and cleaved PARP, was increased. *p < 0.05, **p < 0.01

FIGURE 5

ABCC2 inhibits tumour growth in vivo. A, A549/DDP cell‐derived subcutaneous neoplasms on day 20 (four groups, n = 4). B, The volumes of the tumours were measured from day 0 to day 20 in each group (formula: volume = width² × length/2; units: mm³). C, Tumour weights of A549/DDP xenografts were measured at day 20 after DDP treatment. D, The expression of Ki‐67 was detected using immunohistochemistry. scale bar = 50 μm. E, Apoptosis in subcutaneous tumours was detected using TUNEL staining. Scale bar = 50 μm. *p < 0.05