Access Type

Open Access Dissertation

Date of Award

1-1-2010

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Cancer Biology

First Advisor

Q. Ping Dou

Abstract

ACTIVATION OF TUMOR CELL DEATH PROGRAM BY TARGETING THE UBIQUITIN-PROTEASOME PATHWAY: SIGNIFICANCE IN CANCER TREATMENT AND PREVENTION

by

MICHAEL FREZZA

August 2010

Advisor: Dr. Q. Ping Dou

Major: Cancer Biology

Degree: Doctor of Philosophy

The ubiquitin-proteasome pathway serves as a quality control mechanism to regulate the degradation of intracellular proteins involved in a wide array of cellular processes including tumorigeneis. Thus targeting key features of protein turnover responsible for the growth and proliferation of cancer have emerged as a favorable approach in cancer therapy. Both in vitro and in vivo experimental and clinical results have demonstrated the potential use of proteasome inhibitors as novel anticancer drugs. The widespread clinical success of platinum-containing drugs served as a catalyst for investigating other metal complexes for the treatment of human cancer. In an effort to improve upon the limitations of platinum anticancer drugs, different metals and metal complexes that pose different mechanisms of action has been investigated as potential anticancer drugs. We have previously found that different metal containing complexes, including those of copper, zinc, gold, and tin could activate tumor cell death by inhibiting the proteasome. Therefore, it is proposed that gallium could similarly act as a proteasome inhibitor and apoptosis inducer in human tumor cells. The data presented in this dissertation strongly supports this hypothesis. We found that gallium-cotaining [NN'O] tridentate ligands appended with halogen substituents could inhibit the proteasomal activity in vitro and in human prostate cancer cells. Importantly, the most biologically active complex (5) could inhibit tumor growth in mice-bearing prostate cancer xenografts associated with inhibition of proteasomal activity and apoptosis. Based on the favorable cytotoxic activity of [NN'O] ligands complexed with gallium, subsequent studies relied on this model architecture complexed to different bivalent transistion metals to gain insight into the pharmacophore responsible for their proteasome-inhibitory effects. Since elevated levels of copper are a trademark of many tumors, targeting heightened levels of copper with copper-binding compounds as a means of tumor growth ablation was proposed. It was found that that these copper complexes (1-3), with distinct stoichiometries and protonation states, acted as proteasome inhibitiors and apoptosis inducers in cultured prostate cancer cells, and importantly the species [CuLI]+ as the minimal pharmacophore responsible for this effect. This hypothesis was further substantiated by the finding that Zinc-containing [NN'O] complex (2), but not Nickel (1) could inhibit the proteasomal activity of a purified 20S proteasome and culture prostate cancer cells associated with massive tumor cell death. These results strengthen our current working hypothesis that fast ligand dissociation (Zn-complex) is required to free up the [MLIA]+ capable of interaction with the proteasome. This is in agreement with previous studies showing that proteasome inhibition by zinc-containing dithiocarbamate derivatives is assoiciated with apoptosis induction. In contrast, chapters 5 and 6 were primarily focused on the mechanistic properties of proteasome inhibition and its downstream events. Since we have previously reported on a gold(III) complex showing potent in vitro and in vivo growth inhibitory activity associated with proteasome inhibition and apoptosis, two gold compounds that differ in the oxidation state of the metal were investigated to gain mechanistic insight into their biological activities. The data in this dissertation provides compelling evidence for the involvement of ROS-mediated proteasome inhibition by gold(III), but not gold(I), and highlights distinct mechanisms of action associated with their biological effects. Since it has been shown that proteasome inhibition is tightly linked to apoptosis in AR (+) prostate cancer cells, it is proposed that AR can influence the regulatory events associated with cell death in prostate cancer cells. Mechanistic studies from chapter 6 provide convincing evidence that proteasome inhibitor- or chemotherapy- induced cell death resulted in significantly higher levels of caspase-3 activity in AR independent prostate cancer cells compared to stably or transiently expressing AR cells. Interestingly, lower levels of caspase 3 activity were partially reversed with the addition of an AR antagonist in AR-dependent prostate cancer cells. These important findings could help facilitate the design of novel therapeutic strategies in the treatment of prostate cancer. Taken together, the studies presented in this dissertation could hold tremendous prognostic and therapeutic potential in the treatment of human cancer.

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