In vivo RNA interference-mediated ablation of MDR1 P-glycoprotein

Abstract

Multidrug resistance (MDR) remains a major obstacle to successful chemotherapeutic treatment of cancer and can be caused by overexpression of P-glycoprotein, the MDR1 gene product. To further validate a knockdown approach for circumventing MDR, we developed a P-glycoprotein inhibition strategy using short hairpin RNA interference (shRNAi) and now show efficacy and target specificity in vivo. Two of eight tested shRNAi constructs targeted against human MDR1 mRNA inhibited expression of P-glycoprotein by >90%, whereas control shRNAi had no effect. Ablation of P-glycoprotein in cells stably transduced with retroviral-mediated shRNAi was documented by Western blot and functionally confirmed by increased sensitivity of MDR1-transfected cells toward the cytotoxic drugs vincristine, paclitaxel, and doxorubicin as well as by transport of (99m)Tc-Sestamibi. shRNAi-mediated down-regulation of P-glycoprotein transport activity both in cultured cells and in tumor implants in living animals could be followed by direct noninvasive bioluminescence imaging using the Renilla luciferase fluorophore, coelenterazine, a known P-glycoprotein transport substrate. Furthermore, after somatic gene transfer by hydrodynamic infusion of a MDR1-Firefly luciferase (MDR1-FLuc) fusion construct into mouse liver, the effect of shRNAi delivered in vivo on P-glycoprotein-FLuc protein levels was documented with bioluminescence imaging using d-luciferin. ShRNAi against MDR1 reduced bioluminescence output of the P-glycoprotein-FLuc reporter 4-fold in vivo compared with mice treated with control or scrambled shRNAi. Targeted down-regulation of a somatically transferred P-glycoprotein-eGFP fusion reporter also was observed using fluorescence microscopy. Our results show that shRNAi effectively inhibited MDR1 expression and function in cultured cells, tumor implants and mammalian liver, documenting the feasibility of a knockdown approach to reversing MDR in vivo.