Complete Reversal of ABCG2-Depending Atypical Multidrug Resistance by RNA Interference in Human Carcinoma Cells

Abstract

In the chemotherapeutic treatment of patients with disseminated neoplasms, multidrug resistance (MDR) is a major obstacle. ABCG2 (BCRP/MXR), a member of the superfamily of adenosine triphosphate-binding cassette (ABC) transporters, was demonstrated to be associated with "atypical" forms of multidrug-resistant phenotypes of cancer cells. To overcome the ABCG2-depending MDR, two specific anti-ABCG2 small interfering RNAs (siRNAs) were designed for transient triggering of the gene-silencing RNA interference (RNAi) pathway in the human gastric carcinoma cell line EPG85-257RNOV, exhibiting an atypical MDR phenotype. Because both siRNAs showed biological activity, for stable inhibition of ABCG2 corresponding short hairpin RNA (shRNA) expression vectors were constructed. By treatment of EPG85-257RNOV cells with these constructs, expression of the targeted ABCG2-encoding mRNA and transport protein was inhibited completely. Furthermore, anti-ABCG2 shRNA-treated cells increased cellular drug accumulation to the same level measured in drug-sensitive parental cells. These effects were accompanied by complete reversal of the drug-resistant phenotype. Thus, the data indicate that siRNA- and shRNA-mediated RNAi-based gene therapy may be applicable in preventing and reversing ABCG2-depending atypical MDR.

Breast cancer resistance protein (ABCG2) and drug disposition: intestinal expression, polymorphisms and sulfasalazine as an in vivo probe

“…Cells expressing or overexpressing BCRP are known to be resistant to chemotherapeutic agents [30][31][32][33] and therefore, decreased BCRP mediated transport in tumors of patients, who harbor the 421C > A polymorphism, would be expected to result in greater tumor sensitivity to chemotherapy. Our results would suggest that patients with the 421C > A polymorphism would likely be more prone to side effects during chemotherapy with BCRP substrate chemotherapeutic agents owing to higher drug exposure and C max .…”

Identification of the Efflux Transporter of the Fluoroquinolone Antibiotic Ciprofloxacin in Murine Macrophages: Studies with Ciprofloxacin-Resistant Cells

“…This may reflect the fact that the expression of Mrp2 is too low, even in ciprofloxacin-resistant cells, to affect ciprofloxacin accumulation or that ciprofloxacin is not a substrate for Mrp2. siRNAs have already been used to reverse multidrug resistance in human cancer cell lines due to overexpression of P-glycoprotein (19), MRP1 (12), MRP2 (21), MRP4 (31), or BCRP (30). The incomplete decreased expression observed here (about 70%) is in line with what is commonly observed with this type of approach for other transporters (12,21,41) and could be accounted for by the very large amount of protein present (41) and/or its long half-life (as is the case for other transporters, such as P-glycoprotein [26], MRP1 [1], or MRP2 [16]), as well as the short half-life of the siRNAs.…”

Reversal of ABC Drug Transporter-Mediated Multidrug Resistance in Cancer Cells: Evaluation of Current Strategies

“…The half-life of the ABCB1 mRNA (4 hours), and protein (16 hours) [107], allows for an increase in transcript and protein level to original levels 7 days after siRNA administration. To extend the silencing effects of siRNA, others have engineered an H1-RNA gene promoter-driven expression vector encoding anti-ABCB1 [108], anti-ABCC2 [109] and anti-ABCG2 [110] short hairpin RNA (shRNA), which shows the highest efficacy to date in gene-silencing. The efficiency of gene knock-down depends on the delivery into the cell, and studies report that an adenoviral-based delivery of shRNA is superior to adenoviral delivery of ribozymes [111].…”