The Phosphodiesterase-5 Inhibitor Vardenafil Is a Potent Inhibitor of ABCB1/P-Glycoprotein Transporter

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 [3H]-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 [125I]-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

Data from: The extracellular and cytoplasmic domains of Syndecan cooperate postsynaptically to promote synapse growth at the Drosophila neuromuscular junction

The heparan sulfate proteoglycan (HSPG) Syndecan (Sdc) is a crucial regulator of synapse development and growth in both vertebrates and invertebrates. In Drosophila, Sdc binds via its extracellular heparan sulfate (HS) sidechains to the receptor protein tyrosine phosphatase LAR to promote the morphological growth of the neuromuscular junction (NMJ). To date, however, little else is known about the molecular mechanisms by which Sdc functions to promote synapse growth. Here we show that all detectable Sdc found at the NMJ is provided by the muscle, strongly suggesting a post-synaptic role for Sdc. We also show that both the cytoplasmic and extracellular domains of Sdc are required to promote synapse growth or to rescue Sdc loss of function. We report the results of a yeast two-hybrid screen using the cytoplasmic domains of Sdc as bait, and identify several novel candidate binding partners for the cytoplasmic domains of Sdc. Together, these studies provide new insight into the mechanism of Sdc function at the NMJ, and provide enticing future directions for further exploring how Sdc promotes synapse growth

Sdc’s cytoplasmic domain influences Sdc’s subcellular localization.

<p>(A) In wildtype larvae, Sdc colocalizes with HRP at the 3^rd instar larval NMJ on muscles 6/7. (B) Muscle expression of a Sdc ΔCyto construct has diminished synaptic localization whereas expression of a construct lacking either C1 (C) or C2 (D) show intracellular accumulation of protein (arrows) in addition to the synaptic localization. Both the TM Swap construct (E) and the ΔEcto construct (F) exhibit similar subcellular localization to Sdc-FL.</p

Detergent-free staining shows all Sdc constructs localize to the synapse.

<p>Using a detergent-free staining protocol to visualize only the cell-surface distribution of the expressed Sdc transgenes, all Sdc constructs appear to localize to the synapse (A-F) although there may be a slight decrease in staining intensity with the ΔC1 construct (C).</p

Candidate binding partners from yeast two-hybrid screen.

<p>Candidate binding partners from yeast two-hybrid screen.</p

The cytoplasmic and extracellular domains are required for Sdc function.

<p>(A) Schematic diagram of the Sdc constructs used in this study. The Tm domain of the TM swap construct was made from human platelet derived growth factor receptor (hPDGFR). *—site of 5xMyc tag used for construct detection. (B) Muscle overexpression of the UAS-Sdc constructs shown in (A) using the 24B-Gal4 driver. Overexpression of either Sdc-FL or TM Swap generated a significant increase in the number of boutons per NMJ when compared to Canton S controls (p < .01 n = 21; p < .05 n = 21). Overexpression of ΔCyto caused a significant reduction in the number of boutons per NMJ (p < .05; n = 23). (C) Sdc mutants had a significant decrease in boutons per NMJ as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151621#pone.0151621.ref005" target="_blank">5</a>]. The UAS-Sdc constructs shown in (A) were expressed in muscles in a Sdc mutant background (P2377/Df48; ubi-Sara) using the 24B-Gal4 driver. Both Sdc-FL and TM Swap constructs generated a significant rescue of the number of boutons per NMJ when compared to Sdc mutants (p < .01 n = 40; p < .01 n = 23).</p

Sdc at the larval NMJ is provided postsynaptically.

<p>(A) In wildtype larvae, Sdc protein colocalizes with Fasciclin II at the 3^rd instar larval NMJ on muscles 6/7. (B) Neural expression of a Sdc RNAi construct using elav-Gal4 has no effect on synaptic Sdc, but muscle Sdc RNAi expression using 24B-Gal4 (C) completely abolishes the synaptic localization of Sdc. (D) Wildtype larvae have no detectable anti-Myc immunoreactivity. (E) When a myc-tagged UAS-SdcFL transgene is overexpressed in neurons using elav-Gal4, the protein does not localize to the larval NMJ. (F) When expressed in muscles using 24B-Gal4, this construct recapitulates the wildtype distribution of Sdc. (G) Postsynaptic, but not presynaptic, expression of a Sdc RNAi construct decreases synapse size when compared to Canton S controls (p < .01 n = 33; p > .05 n = 28). Bouton numbers for all graphs are normalized to Canton S (100).</p