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

Therapeutic applications of ceramide lipids for apoptosis induction

The emerging role of ceramide lipids in apoptosis and an increased understanding of their involvement in multidrug resistance (MDR) has revealed new opportunities for manipulating ceramide levels in order to achieve specific therapeutic objectives. The research presented in this thesis focused on the relationship between ceramide, MDR and apoptosis. Direct and indirect approaches for modulating intracellular ceramide levels were investigated in an attempt to chemosensitize MDR tumors and induce apoptosis. Inhibition of pro-apoptotic ceramide conversion to its non-cytotoxic glucosylceramide metabolite was shown to sensitize two human MDR breast cancer cell lines to the cytotoxic effects of tubulin-binding chemotherapy drugs. Enhanced sensitization was correlated with increased ceramide, suggesting that therapeutic manipulations aimed at increasing endogenous ceramide should promote apoptosis. On the basis of these results, the feasibility of delivering therapeutic amounts of exogenous ceramides to cells was then investigated. After evaluating different chain length ceramides it was determined that synthetic C₆-ceramide was internalized and cytotoxic to cells whereas naturally occurring C₁₆-ceramide was neither internalized nor cytotoxic. This difference established the importance of intracellular delivery as a prerequisite to apoptosis induction by exogenous ceramides. Liposome-based delivery systems were then introduced in an attempt to overcome the limitations associated with intracellular delivery of natural ceramide. Physically stable liposomes containing up to 50 mole percent C₁₆-ceramide in the lipid bilayer were successfully formulated. These liposomes were internalized by J774 macrophage cells in vitro and induced apoptosis with similar potency to free C6-ceramide. In order to translate this encouraging data to an in vivo model it was first necessary to evaluate the behavior of these liposomes in the circulation. Pharmacokinetic studies demonstrated in vivo stability over 24 hours following iv bolus administration. The antitumor activity of these liposomes was then evaluated in the J774 ascites tumor model. Optimal antitumor activity was observed following intraperitoneal administration of C₁₆-ceramide liposomes on days 1, 5, and 9. This corresponded to a statistically significant increase in animal survival of 43.5% over non-ceramide control liposomes. Taken together, this research provides evidence for the rational design of ceramide-based liposomes as a novel approach for cancer chemotherapy.Pharmaceutical Sciences, Faculty ofGraduat

Intracellular delivery of ceramide lipids via liposomes enhances apoptosis in vitro

AbstractCeramide lipids have emerged as important intracellular signalling molecules that mediate diverse cellular effects, of which programmed cell death, or apoptosis, has attracted significant interest. Although the exact mechanism(s) by which ceramides trigger apoptosis is not fully understood, there is considerable evidence that they are key mediators of this response. Exogenously applied, cell-permeable ceramides have been shown to induce apoptosis when incubated with cells in culture. We examined here the cytotoxicity of ceramides with varying acyl chain lengths in order to determine whether acyl chain length affects pro-apoptotic activity within the concentration range of 0–100 μM. We found that for C6-, C8-, C10-, C14- and C16-ceramide, the chain length was inversely proportional to cytotoxic activity, with C6-ceramide being most active (IC50 values in the 3–14 μM range) and C16-ceramide being least active (IC50 values in excess of 100 μM) in the MDA435/LCC6 human breast cancer and J774 mouse macrophage cell lines investigated. Using these two ceramide forms we were able to correlate the observed cytotoxicity with cellular uptake, and we observed that a lack of intracellular delivery may be responsible for the weak activity of C16-ceramide. We therefore investigated the possibility of incorporating ceramide lipids into liposome bilayers to enhance this delivery. We demonstrate that stable, ceramide-containing liposomes can be formulated, and that they are cytotoxic when taken up by cells in vitro. These results provide an increased understanding of the differences in cytotoxic activity of exogenous short- and long-chain ceramide lipids, and their incorporation into biologically active liposomal formulations opens new avenues for apoptosis induction