Lentivirus-mediated RNAi silencing targeting ABCC2 increasing the sensitivity of a human nasopharyngeal carcinoma cell line against cisplatin

Abstract

Background: High resistance to drug is taken as a characteristic of human tumors, which is usually mediated by multidrug resistance-associated genes. ABCC2, an ATP-binding cassette multidrug resistance transporter, is found to be expressed in a variety of human cancers. In this study the effect of a RNAi construct targeting ABCC2 on the chemosensitivity of NPC cell line CNE2 against cisplatin was investigated.

Methods: Lentiviral vectors were constructed to allow an efficient expression of anti-ABCC2 siRNA. The effective target sequence comprised nucleotides 1707-1727 of the human ABCC2 mRNA. The cell clones expressing the construct were picked and expanded, followed by identification using qRT-PCR and western blot method. As control, lentiviral vector containing invalid RNAi sequence was transfected to CNE2 cells. In vitro, cellular accumulation of cisplatin was detected by HPLC. The capacity of cellular growth and sensitivity of cells against cisplatin were detected by MTT assay. In vivo, the sensitivity of the tumor tissues against cisplatin were evaluated by transplanted CNE2 nude mice model.

Results: Two CNE2 cell clones with reduced expression of targeted ABCC2 mRNA and protein for more than 70% by qRT-PCR and western blot were established, and no differences were shown in proliferation rates compared to control CNE2 cells by growth curves analysis. In vitro the accumulation of intracellular cisplatin in these CNE2 cell clones with reduced expression of ABCC2 increased markedly, accompanied by increased sensitivity against cisplatin. In vivo, the growth of CNE2 solid tumors with a stably transfected anti-ABCC2 siRNA construct was significantly inhibited by cisplatin in transplanted nude mice model.

Conclusion: Our investigation demonstrated that lentivirus-mediated RNAi silencing targeting ABCC2 might reverse the ABCC2-related drug resistance of NPC cell line CNE2 against cisplatin.

Figure 1

Special siRNA targeting ABCC2 silences the mRNA and protein expressions of ABCC2 in NPC cells. (A) Expression of ABCC2 mRNA in NP69 and human NPC cell lines by quantitative RT-PCR. N = 3, *P < 0.05 vs. NP69. (B and C)Analysis of ABCC2 expression levels in CNE2 cells treated with ABCC2-shRNA construct (ShABCC2-1 and shABCC2-2) and negative construct (ShABCC2-). (B) The expressions of ABCC2 mRNA detected by quantitative RT-PCR. (C) The expressions of ABCC2 protein detected by western blot. Data were expressed as mean ± SEM value. N = 3, *P < 0.05 vs. CNE2.

Figure 2

ABCC2 siRNA increased the intracellular accumulation of cisplatin. (A) A typical chromatogram for total analysis of cisplatin from CNE2 cells exposed to cisplatin for 2 h using HPLC determination. (B) Calibration curve for gradient concentration of cisplatin within the range of 5–80 μg/ml. A typical linear relationship (R² = 0.9965) was found between peak height and concentration of cisplatin. (C) The cellular accumulation of cisplatin in CNE2 cells treated with ABCC2-shRNA construct (ShABCC2-1 and shABCC2-2) and negative construct (ShABCC2-). The concentration of cisplatin were determined according to the calibration curve of cisplatin. Data were expressed as mean ± SEM value. N = 3, *P < 0.05 vs. CNE2.

Figure 3

Silencing of ABCC2 by siRNA increased the sensitivity of cisplatin in CNE2 without changing the cellular viability. (A) Cellular growth curve. The cell growth viability was assessed by MTT method for 7 days. (B) Modulation of sensitivity against cisplatin for CNE2 cells. MTT method was used to determined the IC50 value of cisplatin to CNE2 cells treated with ABCC2-shRNA construct (ShABCC2-1 and shABCC2-2) and ABCC2-shRNA negative construct (ShABCC2-). Data were expressed as mean ± SEM value. N = 3, * P < 0.05 vs. CNE2.

Figure 4

Silencing of ABCC2 by siRNA increased the inhibitory effects of cisplatin on growth of tumors in nude mice. (A) Expression of ABCC2 in tumor tissues (Immunohistochemistry method, DAB staining, 200×). CNE2, CNE2/shABCC2-1 cells were transplanted s.c. in nude mice, with ABCC2-shRNA negative construct (ShABCC2-) as control. (B) Efficacy of cisplatin on growth of tumors transplanted s.c. in nude mice. The efficacy was evaluated by relative tumor sizes (RTS). At the time of cisplatin administration, the weight of tumors were in the range of 50–200 mg. Data were expressed as mean ± SEM value. * P < 0.05. P value were shown in Tab 1. (C) The treated: control (T: C) ratio of tumor weight for s.c. tumors in nude mice. CNE2, CNE2 treated with shABCC2 construct (shABCC2-1) were used, with shABCC2 negative construct (shABCC2-) as control.