The phosphodiesterase-5 inhibitor vardenafil is a potent inhibitor of ABCB1/P-glycoprotein transporter

Abstract

One of the major causes of chemotherapy failure in cancer treatment is multidrug resistance (MDR) which is mediated by the ABCB1/P-glycoprotein. Previously, through the use of an extensive screening process, we found that vardenafil, a phosphodiesterase 5 (PDE-5) inhibitor significantly reverses MDR in ABCB1 overexpressing cancer cells, and its efficacy was greater than that of tadalafil, another PDE-5 inhibitor. The present study was designed to determine the reversal mechanisms of vardenafil and tadalafil on ABC transporters-mediated MDR. Vardenafil or tadalafil alone, at concentrations up to 20 µM, had no significant toxic effects on any of the cell lines used in this study, regardless of their membrane transporter status. However, vardenafil when used in combination with anticancer substrates of ABCB1, significantly potentiated their cytotoxicity in ABCB1 overexpressing cells in a concentration-dependent manner, and this effect was greater than that of tadalafil. The sensitivity of the parenteral cell lines to cytotoxic anticancer drugs was not significantly altered by vardenafil. The differential effects of vardenafil and tadalafil appear to be specific for the ABCB1 transporter as both vardenafil and tadalafil had no significant effect on the reversal of drug resistance conferred by ABCC1 (MRP1) and ABCG2 (BCRP) transporters. Vardenafil significantly increased the intracellular accumulation of [(3)H]-paclitaxel in the ABCB1 overexpressing KB-C2 cells. In addition, vardenafil significantly stimulated the ATPase activity of ABCB1 and inhibited the photolabeling of ABCB1 with [(125)I]-IAAP. Furthermore, Western blot analysis indicated the incubation of cells with either vardenafil or tadalafil for 72 h did not alter ABCB1 protein expression. Overall, our results suggest that vardenafil reverses ABCB1-mediated MDR by directly blocking the drug efflux function of ABCB1.

Figure 1. The effect of vardenafil and tadalafil on the accumulation (A), and efflux (B) of [³H]-paclitaxel, and ABCB1 expression (C) in ABCB1 overexpressing cells.

(A) The accumulation of [³H]-paclitaxel was measured after cells were pre-incubated with or without vardenifil, tadalafil, or verapamil for 1 h at 37°C and then incubated with 0.1 µM [³H]-paclitaxel for another 2 h at 37°C. (B) The percentage of the paclitaxel released was plotted as a function of time. After 1 h of incubation of the vardenifil, [³H]-paclitaxel was co-incubated in KB-3-1 and KB-C2 cells with or without vardenafil or verapamil. Data points represent the means ± SD of triplicate determinations. * and ** represent p<0.05 and p<0.01, respectively, for values versus those in the control group. (C) Effect of vardenafil (left panel) or tadalafil (right panel) on the expression of ABCB1 for 36 and 72 h, respectively. Independent experiments were performed at least three times, and a representative experiment is shown.

Figure 2. The effect of vardenafil or tadalafil on the ATPase activity of ABCB1 (A) and the photoaffinity labeling of ABCB1 with [¹²⁵I]-IAAP. (B).

(A) The Vi-sensitive ATPase activity of ABCB1 in membrane vesicles was determined with different concentrations of vardenafil (closed circles) or tadalafil (closed squares) as described previously . The concentration required for 50% stimulation with vardenafil was 2.69±0.72 µM; whereas the stimulation with tadalafil was not saturable. (B) The photoaffinity labeling of ABCB1 with [¹²⁵I]-IAAP was performed in the presence or absence of different concentrations of vardenafil (A) or tadalafil (B). The radioactivity incorporated into ABCB1 was determined by exposing the gel to an X-ray film at −70°C. Mean values are given, and the error bars represent standard error from at least three independent experiments.

Figure 3

(A) The ribbon diagram of open to the cytoplasm-3D structural conformation of a homology model of human ABCB1 based on the crystal structure coordinates of mouse ABCB1. The docked poses of vardenafil (Green), tadalafil (Brown) and IAAP (Purple) as a ball and stick model are shown within the large hydrophobic cavity of ABCB1 at different inhibitor biding sites. (B) The XP-Glide predicted binding mode of vardenafil (left panel) and tadalafil (right panel). Important amino acids are depicted as sticks with the atoms colored as carbon – green, hydrogen – white, nitrogen – blue, oxygen – red and sulfur – yellow) whereas the inhibitor is shown as a ball and stick model with the same color scheme as above except carbon atoms are represented in orange. The dotted black line indicates hydrogen bonding interaction whereas a dotted red line indicates interacting distance.