Abstract
Rapamycin (Rapa) is a highly potent drug; however, its clinical potential is limited by poor solubility, bioavailability, and cytotoxicity. To improve Rapa delivery, our team has fused the cognate protein receptor for Rapa, FKBP12, to high molecular weight elastin-like polypeptides (ELPs). One construct, FAF, includes an FKBP domain at each termini of an ELP. In a recent report, FAF/Rapa outperformed a family of related carriers with higher tumor accumulation and efficacy. Despite apparent efficacy, an explanation for how FAF carries Rapa into cells has not been elucidated. This manuscript explores the intracellular fate of FAF in MDA-MB-468, a triple negative (ER-/PR-/HER2-) breast cancer line. Based on a lack of displacement by excess unlabeled FAF, no evidence was found for the involvement of a receptor in cell-surface binding. Cellular association showed no dose-dependent saturation at concentrations up to 100 μM, which is consistent with uptake through fluid phase endocytosis. FAF does colocalize with dextran, a marker of fluid phase endocytosis. Upon internalization, both FAF and dextran target low pH intracellular compartments similarly. Despite likely exposure to lysosomal pH and proteolytic activity, intracellular FAF is eliminated from cells with a relatively long half-life of 17.7 and 19.0 h by confocal microscopy and SDS-PAGE respectively. A split luciferase reporter assay demonstrated that FAF delays the cytosolic access of Rapa in comparison to free drug by 30 min. A specific macropinocytosis inhibitor, amiloride, completely inhibits the cytosolic delivery of Rapa from FAF. Each of these results are consistent with macropinocytosis as the mechanism of cellular uptake necessary for the hand-off of Rapa from FKBP-based drug carriers like FAF to endogenous FKBP12 in the cytosol.
Highlights
Rapamycin (Rapa), known as Sirolimus is an immunosuppressive agent approved for the prophylaxis of organ rejection during transplantations, especially renal transplants (Dumont and Su, 1995; MacDonald, 2001)
Rapa is not approved for the treatment of cancer, the closely related rapalogue known as Everolimus has been FDA approved for multiple indications in cancer
Bacteria were pelleted by centrifuging at 5000 g for 15 min, and the pellet was re-suspended in phosphate buffered saline (PBS) (Caisson labs, Smithfield, UT, United States)
Summary
Rapamycin (Rapa), known as Sirolimus is an immunosuppressive agent approved for the prophylaxis of organ rejection during transplantations, especially renal transplants (Dumont and Su, 1995; MacDonald, 2001). Rapalogues continue to be evaluated in multiple clinical trials, in combination with other drugs. Treatment is associated with adverse effects including painful oral ulcers (incidence > 30%) (de Oliveira et al, 2011), severe anemia (incidence > 20%), hyperlipidemia, hypercholesteremia, pulmonary and renal toxicities (Marti and Frey, 2005). This limits patient compliance and makes it difficult to maintain patients on Rapa therapy (Gomez-Fernandez et al, 2012), thereby restricting usage to low dosage regimens (Pham et al, 2004)
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