Year of Publication

2008

Document Type

Dissertation

College

Pharmacy

Department

Pharmaceutical Sciences

First Advisor

Russell Mumper

Second Advisor

Michael Jay

Abstract

The significantly increased copper and oxidative stress levels are characteristic hallmarks of cancer cells. These differences provide a unique opportunity for selective targeting of cancer cells. D-penicillamine (D-pen) has been proposed to generate reactive oxygen species (ROS) in presence of copper. Therefore, these studies were aimed at investigating the potential application of a currently marketed copper chelator, D-pen, as a novel cytotoxic anti-cancer agent. D-pen was shown to produce ROS, specifically hydrogen peroxide (H2O2), in the presence of cupric sulfate through a copper catalyzed oxidation reaction. During this process D-pen was converted to D-pen disulfide. The experimental proof of the H2O2 generation was conclusively shown with the aid of a novel High Performance Liquid Chromatography (HPLC) assay. The in-vitro cytotoxicity of D-pen co-incubated with cupric sulfate was examined in human beast cancer (MCF-7 and BT474) and leukemia cells (HL-60, HL-60/VCR, and HL-60/ADR). D-pen was shown to cause concentration dependent cytotoxicity in both leukemia and breast cancer cells. A direct correlation between the detection of intracellular ROS and cytotoxicity was established. The treatment of D-pen plus cupric sulfate resulted in a significant reduction in the intracellular thiol content. D-pen is highly hydrophilic and is rapidly eliminated from the body; therefore to improve the intracellular uptake and to protect the thiol group of D-pen, we carried out the synthesis and the in-vitro characterization of a novel gelatin-D-pen conjugate. It was shown that D-pen alone does not enter cells. Confocal microscopy was employed to exhibit the uptake of the novel gelatin-D-pen conjugate by cancer cells. As the cancer cells in-vitro do not accumulate the same levels of copper as reported for cancer cells in-vivo, cancer cells were pre-treated with cupric sulfate to simulate the elevated copper levels. The cupric sulfate pretreatment resulted in reduced thiol level and significantly increased cellular copper content compared to untreated cells. Whereas both free D-pen and gelatin-D-pen conjugate lacked cytotoxicity in un-treated cells, both agents caused concentration dependent cytotoxicity in cupric sulfate pre-treated leukemia cells. Therefore, it was shown that the administration of D-pen as polymer conjugate would potentially provide cytotoxicity and specificity in the treatment of cancer.

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