Browsing by Subject "Ctr1"

Copper is an essential element due to its unique ability to cycle between redox states under biological conditions. This property makes copper an ideal catalytic co-factor for enzymes that function in energy generation, iron acquisition, oxygen transport, cellular metabolism, peptide hormone maturation, blood clotting, signal transduction and a variety of other cellular processes. However, excess copper can lead to free radical mediated membrane damage, protein oxidation, and DNA cleavage along with improper metallation of Fe-S clusters. The inability to properly acquire and handle copper is associated with severe genetic diseases of both deficiency and overload as exemplified in Menkes and Wilsons diseases, respectively. Thus, characterizing the acquisition, regulation, and homeostasis of this essential metal is imperative for our understanding of human biology.

The studies presented here utilize a diverse array of techniques including yeast and mouse genetics, recombinant protein expression, purification and biochemical characterization of copper homeostasis proteins, evolutionary genetic analysis, in vitro copper transport assays, and cell culture studies to decipher novel aspects of mammalian copper biology. Critical findings include the discovery of a previously unknown regulator of Copper transporter 1 (Ctr1), and analysis of the evolutionary history of mammalian Ctr proteins, the development of biochemical techniques to probe copper transport mechanisms in a purified system, and the identification of a novel polymorphism in the human Ctr1 gene that has functional consequences. These unique insights lay the foundation for a greater understanding of the basic mechanisms for copper homeostasis in both healthy and diseased states.

Mechanisms of copper homeostasis are of great interest partly due to their connection to debilitating genetic and neurological disorders. The family of high-affinity copper transporters (Ctr) is responsible for extracellular copper acquisition and internalization in yeast, plants, and mammals, including human. The extracellular domain of the human high-affinity copper transporter (hCtr1) contains essential Cu-binding methionine-rich MXXM and MXM (Mets) motifs that are important for copper acquisition and transport. The hCtr1 extracellular domain also contains potential copper binding histidine (His) clusters, including a high-affinity Cu(II) ATCUN site. As of yet, extracellular His clusters have no established significance for hCtr1 function. We have made model peptides based on the extracellular copper acquisition domain of hCtr1 that is rich in His residues and Mets motifs. The peptides' Cu(I) and Cu(II) binding properties have been characterized by UV-Vis and mass spectrometry. Our findings have been extended to a mouse cell model and we show that His residues are important for hCtr1 function likely because of their contribution to strong copper-binding sites in the hCtr1 extracellular domain responsible for copper acquisition.

Copper's pro-oxidant property is also medicinally promising if it can be harnessed to induce oxidative stress as a cancer chemotherapy strategy. Our lab has designed a photocleavable caged copper complex that can selectively release redox-active copper in response to light. The thermodynamic copper binding properties of these potential chemotherapeutics have been characterized

Mild hyperthermia (39°C-43°C) has numerous therapeutic benefits as an adjuvant therapy in the treatment of a variety of tumor types. Hyperthermia increases tumor blood flow and vascular permeability, promoting drug delivery and tumor oxygenation. Hyperthermia enhances the uptake and efficacy of numerous chemotherapeutic agents, including cisplatin, resulting in increased cytotoxicity. In addition to these biological responses, hyperthermia can be used as a drug-release trigger for temperature-sensitive nanoparticles, resulting in an improved and more targeted drug delivery system. Cisplatin was chosen because 1) it shows broad spectrum activity against a wide range of heatable cancers (i.e., those in sites such as the pancreas, colon and rectum, cervix and bladder, and 2) the same hyperthermic temperatures that enable temperature-sensitive lipsome-drug release also enhance cisplatin-induced cytotoxicity.

The role of hyperthermia in enhancing cisplatin delivery and cytotoxicity was investigated at both the cellular and tissue levels. While hyperthermia treatment is applicable to a variety of tumor types, the focus of this work was on bladder cancer. The synergistic effects of hyperthermia and cisplatin were investigated, along with the role of copper transport protein 1 (Ctr1) in this process. In addition, cisplatin was encapsulated within temperature-sensitive liposomes, which were used in combination with hyperthermia for targeted drug delivery. These studies demonstrated that the combination of cisplatin and hyperthermia improved drug delivery, and potentially anti-tumor efficacy, and that targeted delivery was enhanced through incorporation of temperature-sensitive liposomes. As many current methods for administering bladder hyperthermia have drawbacks, such as invasiveness and regional heating, the final aim of this study was to develop and test a less-invasive and more focused preclinical bladder heating device in a rat model.

Hyperthermia sensitizes cells to the cytotoxic effects of the commonly used chemotherapeutic agent cisplatin by increasing drug accumulation and subsequent platinum-DNA adduct formation. However, the molecular mechanisms underlying this enhancement remain unclear. Understanding the fundamental mechanisms involved in the synergistic interaction is necessary to increase the therapeutic benefits of this combination in the clinic. The synergism between the anti-cancer benefits of cisplatin and the drug delivery benefits of hyperthermia may offer a novel and more effective treatment for many cancer patients. We hypothesized that hyperthermia increases cisplatin accumulation and efficacy in part by modulating the function of Ctr1, a major regulator of cellular cisplatin uptake. To test this hypothesis, we examined the significance of Ctr1 during combined hyperthermia and cisplatin therapies and assessed the importance of cisplatin- and hyperthermia-induced Ctr1 multimerization in enhancing cisplatin cytotoxicity. We observed increased Ctr1 multimerization following hyperthermia treatment (41°C) in vitro, compared to normothermic controls (37°C), suggesting that this may be a mechanism for increased cisplatin uptake in heat-treated cells. The impact of increased Ctr1 multimerization was evaluated by measuring platinum accumulation in wild-type (WT) and Ctr1-/- cells. WT cells contained greater levels of platinum compared to Ctr1-/- cells. A further increase in platinum was observed following hyperthermia treatment, but only in the WT cells. Hyperthermia enhanced cisplatin-mediated cytotoxicity in WT cells with a dose-modifying factor (DMF) of 1.8 compared to 1.4 in Ctr1-/- cells. Our data suggest that heat increases Ctr1 activity by increasing multimerization, resulting in enhanced drug accumulation. Although we recognize that the effect of heat on cells is multi-factorial, our results support the hypothesis that Ctr1 is, in part, involved in the synergistic interaction observed with cisplatin and hyperthermia treatment.

In addition to assessing cisplatin delivery at the cellular level, we evaluated cisplatin delivery at the tissue level, using novel cisplatin-loaded temperature-sensitive liposomes. We hypothesized that delivering cisplatin encapsulated in liposomes under hyperthermic conditions would improve the pharmacokinetic profiles of cisplatin, increase drug delivery to the tumor, decrease normal tissue toxicity, and enhance the anti-tumor activity of cisplatin. We successfully prepared temperature-sensitive liposomes loaded with cisplatin and demonstrated that heat (42°C) sensitizes cisplatin-resistant cells to the cytotoxic effects of cisplatin in vitro.

Decreased toxicity was observed in animals treated with the cisplatin liposome (± heat) compared to the free drug treatments. A pharmacokinetic study of cisplatin-loaded temperature-sensitive liposomes and free drug was performed in tumor-bearing mice under normothermic and hyperthermic conditions. Cisplatin half-life in plasma was increased following liposome treatment compared to free cisplatin, and cisplatin delivery to the tumors was greatest in mice that received liposomal cisplatin under hyperthermia. These initial in vivo data demonstrate the potential effectiveness of this cisplatin-loaded liposome formulation in the treatment of certain types of cancer. To assess the anti-cancer efficacy of the liposome treatment, a tumor growth delay study was conducted and demonstrated equivalent efficacy for the cisplatin-loaded temperature-sensitive liposome compared to free drug.

In addition to the liposome work, we developed and evaluated a novel heating device for the bladder. Despite the evidence that hyperthermia is an effective adjuvant treatment strategy, current clinical heating devices are inadequate, warranting the development of a new and improved system. We induced hyperthermia using ferromagnetic nanoparticles and an alternating magnetic field device developed by Actium Biosystems. Initial preclinical studies in a rat model demonstrated preferential bladder heating. However, our preliminary studies show severe toxicity with the direct instillation of the nanoparticles in the bladder, and further studies are needed to potentially modify the nanoparticle coating, the catheterization procedure, as well as to develop a different animal model.