Stable suppression of MDR-1 gene using siRNA expression vector to reverse drug resistance in a human uterine sarcoma cell line

Abstract

Objective.

Chemotherapy is highly effective in treating a number of gynecologic malignancies; however, its effectiveness diminishes with repeated exposures due to the emergence of multi-drug resistance (MDR). The aim of this study was to establish a permanent MDR gene knockdown model via infection with the siRNA-hairpin expression vector. The impact of transfecting the RNAi upon MDR-1 mRNA and P-glycoprotein expression as well as resultant chemotherapy resistance was assessed.

Methods.

Multi-drug resistant cell line MES-SA/DX5 was transfected with the siRNA-hairpin expression vector (pSMDR-HYG) designed to target MDR-1 mRNA. A negative control was established utilizing a vector lacking the anti-sense component (pSCON-HYG). The LD₅₀ of doxorubicin for the stable transfectants was determined utilizing a cytotoxic MTT assay. The mRNA expression of MDR-1 gene among those cell lines was evaluated by semi-quantitative RT-PCR. The product of P-glycoprotein (P-gp) was examined by Western blotting hybridization and immunostaining.

Results.

Two stable transfected cell lines: MES-SA/DX5-M (with pSMDR-HYG) and MES-SA/DX5-C (with pSCON-HYG) were established. The cell line MES-SA/DX5-M was nearly 7 times more sensitive to doxorubicin than MES-SA/DX5-C and its parent cell line MES-SA/DX5 (P < 0.01). The mRNA expression of the MDR-1 gene in MES-SA/DX5-M was also statistically significantly lower than in the other 2 cell lines (P < 0.01) as assessed by semi-quantitative RT-PCR. A barely detectable signal for P-gp (170 kDa) was observed in MES-SA/DX5-M. The vast majority of MES-SA/DX5-M cells were immunohistochemically negative for P-gp.

Conclusions.

Stable, sequence-specific MDR-1 gene silencing can be demonstrated by inducing the endogenous expression of hairpin siRNA. Hairpin-siRNA-based MDR-1 gene silencing correlated with decreased levels of MDR-1 mRNA and P-gp, thereby restoring permanent native chemosensitivity. This methodologic strategy may have significant clinical impact in reversing chemo-resistance, especially the multi-drug-resistant phenotype, in the treatment of gynecologic malignancies.

Introduction

Chemotherapy is highly effective in treating a number of gynecologic malignancies; however, its effectiveness often diminishes with repeated exposure due to the emergence of multi-drug resistance (MDR) [1]. A multi-drug-resistant phenotype (MDR) has been observed both in vitro and in vivo [2], [3], [4]. MDR is frequently associated with the overexpression of a 170-kDa membrane protein, known as P-glycoprotein (P-gp) [2]. This protein belongs to the super-family of ATP binding cassette (ABC) transporters and functions as an ATP-dependent drug-efflux pump. A common feature of multi-drug resistance is the reduced accumulation of several classes of chemotherapy drugs including vinca alkaloids, anthracyclines, epipodophyllotoxin, and taxanes via P-gp. Multiple attempts have been undertaken to circumvent drug resistance by restoring native chemosensitivity of resistant clones [8], [9]. Treatment modalities include using high-dose chemotherapy with or without the transplantation of progenitor cells as well as using inhibitors of proteins encoded by drug resistance genes [5], [6], [7]. Both anti-sense oligodeoxynucleotide and hammerhead ribozyme technologies have been employed to modulate P-gp-dependent mRNA turnover [8], [9]. However, these methods have had limited applicability and success. Only a transient effect is achieved by the anti-sense method. Reduction in mRNA with single-unit ribozyme is variable and dependent on ribozyme activity and intracellular level.

A new process termed RNA interference (RNAi) has been described in which double-stranded RNA targets homologous mRNA resulting in endo-nucleolytic cleavage and degradation [10], [11], [12]. This process, first discovered in non-mammalian systems, effectively blocks gene expression at the post-transcriptional level and is several orders of magnitude more efficient than anti-sense or ribozyme treatment methods [11]. RNA interference (RNAi) is the process of gene silencing whereby double-stranded RNA induces the homology-dependent degradation of its cognate mRNA. This novel technology is superior to knockout genetics in that it can be easily adapted to study homologous gene function in a wide variety of organisms. Other groups have employed RNA interference (RNAi) by using synthesized small RNA to suppress MDR mRNA, but the effects were transient [13], [14]. siRNA ready vectors can be engineered to express small hairpin RNA in mammalian cells and then processed into siRNA-like molecules capable of carrying out gene-specific silencing which importantly is stable [15], [16], [17], [18]. In this study, we utilized this novel siRNA expression vector silencing technology to target MDR-1 mRNA in the doxorubicin-resistant human uterine sarcoma cell line MES-SA/DX5. This cell line is known to overexpress MDR-1 mRNA and P-glycoprotein [19]. The impact of transfecting the RNAi upon MDR-1 mRNA levels, P-glycoprotein expression, and chemotherapy resistance was assessed.