Abstract A112: Enhanced accumulation of anticancer drug in tumor cells through albumin interactions: A novel drug delivery mechanism.

Abstract

Abstract While chelation therapy was traditionally employed for the treatment of iron overload disease, several studies including clinical trials have demonstrated that certain classes of chelators possess anti-cancer activity. In an effort to develop increasingly active anti-cancer chelators, structure-activity relationship studies led to the synthesis of di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), which possess potent in vitro and in vivo anti-tumor activity1,2. Knowledge of membrane transport is crucial in the development of successful chemotherapeutics. This is because the membrane acts as barrier preventing drug accumulation within tumor cells, inhibiting the interaction between drugs and their intracellular targets. Furthermore, cancer cells can develop drug resistance through the impairment of drug uptake. Additionally, an understanding of drug membrane transport has implications in the development of targeted drug delivery. Consequently, Bp4eT and Dp44mT were labeled with 14C to assess membrane transport through uptake experiments using human SK-N-MC neuroepithelioma cells. These studies demonstrated that these two chelators have contrasting drug delivery mechanisms despite possessing similar structures. 14C-Bp4eT enters cancer cells through passive diffusion, as demonstrated through it possessing an energy- and temperature-independent and non-saturable linear uptake. Whereas, the saturable, energy- and temperature-dependent uptake process of Dp44mT implies a carrier-mediated transport system. Albumin is known to bind drugs and hence affect drug delivery. Additionally, higher levels of albumin reside within the tumor interstitium as compared to normal tissue, a phenomena known as the enhanced permeability and retention effect3. Considering this, the concentration of albumin in the medium was altered to model the tumor microenvironment. Human and bovine serum albumin significantly (p<0.01) decreased the cellular uptake of 14C-Bp4eT. Similarly, the uptake of 14C-Dp44mT was also considerably (p<0.01) reduced in the presence of bovine serum albumin. However, human serum albumin significantly (p<0.01) increased 14C-Dp44mT uptake suggesting a specific interaction with this protein. In order to determine the mechanisms by which albumin effects membrane transport, albumin-drug binding was assessed through equilibrium dialysis, fluorescence and UV-visible spectroscopy and molecular modeling. Interestingly, these studies confirmed that both Bp4eT and Dp44mT bind directly to both bovine and human serum albumin. In conclusion, Bp4eT binds to human and bovine serum albumin, thereby decreasing the levels of free drug available to passively diffuse into cells. Conversely, the interactions of Dp44mT with human serum albumin, but not bovine, enhances chelator uptake into cancer cells. These results suggest the possibility of a novel human albumin receptor that facilitates Dp44mT uptake. Furthermore, the enhancement of Dp44mT tumor accumulation encourages the development of albumin-Dp44mT carriers in order to improve tumor drug delivery and increase its therapeutic window.