Phosphodiesterase Type5Inhibitors Reverse MRP7-mediated Multidrug Resistance Mechanism

Background and Objective: Chemotherapy is the most important treatment toconquer cancer. However, tumor cells will get resistance to chemotherapeutic drugs,which is the main reason to affect the efficacy of chemotherapy and cause the failureof chemotherapy. The drug resistance of tumor cells according to the spectrum of thedrug can be divided into primary drug resistance (PDR) and multidrug resistance(MDR). MDR is a phenomenon of resistance not only to certain chemotherapeuticdrug, but also to mechanically and structurally unrelated drugs after exposure to onechemotherapeutic drug for a long time. MDR is a major obstacle in the success ofcancer chemotherapy. One of the major mechanisms behind the simultaneousresistance is the efflux of different drugs mediated by ATP-binding cassette (ABC)transporters that depend on the energy released by ATP hydrolysis. ABC transportersuperfamily is a group of transmembrane proteins which are grouped into sevensubfamilies (A~G) based on genome sequence similarities. The P-glycoprotein(P-gp/ABCB1), multidrug resistance-associated proteins (MRPs/ABCCs) and breastcancer resistance protein (BCRP/ABCG2) have been reported as the major players tobe involved in mediating MDR. P-gp is the first discovered human ABC drugtransporter, which transports a wide variety of hydrophobic compounds, includingsome of the most common anticancer drugs such as taxanes, anthracyclines and vinca alkaloids. The MRP7(ABCC10), a member of MRP subfamily, is able to conferresistance to several natural product chemotherapeutic drugs including taxanes andvinca alkaloids which are also substrates of P-gp. MRP7and P-gp may togetherparticipate in the formation of resistance to similar anti-cancer drug in tumor cells.It is recognized that developing inhibitors to ABC transporters may blocktransporter-mediated drug efflux function and re-sensitize MDR cancer cells toanticancer drugs. In the past three decades, numerous broad-spectrum or specificinhibitors of ABC transporters have been discovered and tested in in vitro and in vivostudies. However, most of the ABC transporter inhibitors that are used aschemosensitizers have not been successfully used in clinical cancer chemotherapybecause of either adverse effects or toxic pharmacokinetic issues. Another strategy forthe reversal agent development is discovering new functions of the drugs that areclinically approved, which can avoid their side effects in clinical use. Recently, ourgroup reported that phosphodiesterase-5(PDE5) inhibitors could reverseP-gp-mediated MDR. In the present study, we performed experiments to determinewhether PDE5inhibitors such as sildenafil, vardenafil and tadalafil could modulateMRP7-mediated MDR.Methods:1. The MTT assay was used to evaluate drug cytotoxicity.2. Drug accumulation and efflux experiments were measured by liquid scintillationcounter to detect the intracellular radioactive label [3H]-paclitaxel.3. Western blotting was used to determine the MRP7protein expression level.4. Immunofluorescence analysis was used to detect the MRP7protein localization inthe membrane.5. All experiments were repeated at least three times and data were described as mean±SD. The differences between groups were determined by using the Student’s t-testwith SPSS19.0according to data feature. The statistical significance was determinedat P <0.05. Results:1. Sildenafil, vardenafil and tadalafil at5mol/L could significantly reversethe resistance of HEK293-MRP7cells to paclitaxel from9.21fold-resistancesdecreased to1.15-,1.06-and5.87-fold, to docetaxel from11.31fold-resistancesdecreased to1.28-,1.19-and8.35-fold and to vinblastine from5.03fold-resistancesdecreased to1.28-,1.19-and3.63-fold, respectively.2. In drug accumulation experiments, sildenafil, vardenafil and tadalafil at5mol/Lcould increase the intracellular accumulation of [3H]-paclitaxel in HEK293-MRP7cells and the accumulation level of [3H]-paclitaxel was significantly increased by3.34-,3.72-and2.08-fold, respectively.3. The efflux percentages of [3H]-paclitaxel in HEK293-MRP7cells weresignificantly higher than that of HEK293-pcDNA3.1. The efflux percentages of[3H]-paclitaxel in HEK293-MRP7cells were increased with increasing time at30,60and120min. Sildenafil, vardenafil and tadalafil at5mol/L could significantlydecreased the efflux percentages of [3H]-paclitaxel in HEK293-MRP7cells.4. The reversal strength on MRP7-mediated MDR and the efficacy on inhibitionMRP7function of tadalafil are weaker than that of sildenafil and vardenafil.5. Sildenafil, vardenafil and tadalafil at5mol/L do not alter the expression andlocalization of MRP7in HEK293-MRP7cells.Conclusions:1. Sildenafil and vardenafil could significantly reverse the resistance ofHEK293/MRP7cells to paclitaxel, docetaxel and vinblastine.2. Sildenafil and vardenafil could increase the intracellular accumulation of[3H]-paclitaxel in the HEK/MRP7cells.3. Sildenafil and vardenafil could inhibit the efflux of [3H]-paclitaxel mediated byMRP7in HEK/MRP7cells.4. Our findings demonstrate for the first time that sildenafil and vardenafil are able toreverse MRP7-mediated MDR by directly blocking drug efflux function of MRP7without altering MRP7protein expression and localization from the plasmamembranes. These findings can provide a theoretical basis for overcoming the MRP7-mediated MDR.5. Sildenafil and vardenafil are already in clinical use, which makes them idealcandidates to be considered as an adjuvant to anticancer chemotherapy, especially inMRP7and/or P-gp-mediated MDR.