Effects of tetrathiomolybdate in a mouse model of retinal neovascularization.

PURPOSE: To determine the effects of tetrathiomolybdate (TM), a copper-chelating agent, on retinal angiogenesis and vascular endothelial growth factor (VEGF) in a mouse model of retinal neovascularization. METHODS: Postnatal day (P)7 C57BL/6N mice were exposed to 75% +/- 2% oxygen for 5 days (P7-P11) and then returned to room air for 5 days (P12-P17) to induce retinal neovascularization. Beginning on P10 or P12, mice received daily intraperitoneal injections of TM or phosphate-buffered saline (PBS; control) through P17. Retinal neovascularization was examined by fluorescein dextran angiography after 5 days in room air and was quantitated histologically by counting the neovascular endothelial cell nuclei anterior to the inner limiting membrane. TM's effects on VEGF expression were measured by ELISA. RESULTS: TM-treated and control animals demonstrated comparable regions of retinal nonperfusion. Retinas from control mice at P17 contained neovascular tufts at the junction between perfused and nonperfused retina. The tufts contained numerous neovascular nuclei. Retinas from mice treated with TM beginning on P10 (2 days before returning to room air), but not P12, demonstrated a 41% reduction in neovascular cell nuclei compared with control mice (P <0.01). The P10-treated mice also demonstrated a 24% reduction of VEGF compared with control animals (P=0.01). CONCLUSIONS: TM significantly inhibits retinal neovascularization and VEGF production in a mouse model of retinal neovascularization.

A novel role for XIAP in copper homeostasis through regulation of MURR1.

XIAP is a potent suppressor of apoptosis that directly inhibits specific members of the caspase family of cysteine proteases. Here we demonstrate a novel role for XIAP in the control of intracellular copper levels. XIAP was found to interact with MURR1, a factor recently implicated in copper homeostasis. XIAP binds to MURR1 in a manner that is distinct from that utilized by XIAP to bind caspases, and consistent with this, MURR1 did not affect the antiapoptotic properties of XIAP. However, cells and tissues derived from Xiap-deficient mice were found to contain reduced copper levels, while suppression of MURR1 resulted in increased intracellular copper in cultured cells. Consistent with these opposing effects, XIAP was observed to negatively regulate MURR1 protein levels by the formation of K48 polyubiquitin chains on MURR1 that promote its degradation. These findings represent the first described phenotypic alteration in Xiap-deficient mice and demonstrate that XIAP can function through MURR1 to regulate copper homeostasis.

Inhibition of the growth of squamous cell carcinoma by tetrathiomolybdate-induced copper suppression in a murine model.

OBJECTIVE: To determine whether long-term therapy with tetrathiomolybdate suppresses tumor growth in an animal model. DESIGN: In vivo murine model. SUBJECTS: Thirteen 8-week-old C3H/HeJ mice, randomly assigned to a tetrathiomolybdate treatment group (n = 7) or a control group (n = 6). INTERVENTIONS: To render the treatment group mice copper deficient, tetrathiomolybdate (0.7 mg/d per mouse) was added to their drinking water on days 1 through 20. Control group mice received only fresh drinking water. A flank injection of 1.5 x 10(5) SCCVII/SF cells was administrated to all mice on day 21. The treatment group mice continued to receive daily tetrathiomolybdate throughout the remainder of the experiment (70 days). Tumor volume measurements (square of the width x length x 0.52) were taken every other day beginning on day 40. MAIN OUTCOME MEASURES: Mean tumor volume differences. RESULTS: Mean +/- SD tumor volumes on day 40 were 146 +/- 263 mm3 (n = 7) and 274 +/- 331 mm3 (n = 6) for the treatment and control groups, respectively. By day 54, the mean tumor volume for the treatment group was 65 +/- 0 mm3, compared with 1716 +/- 960 mm3 for the control group (P<.001). Treatment was withheld on day 54, resulting in a dramatic increase in tumor growth in the treatment group mice such that by day 60, there was no significant difference in mean tumor volume between groups. CONCLUSION: This study demonstrates the ability of tetrathiomolybdate to maintain a significant and reversible suppression of long-term tumor growth in this murine model of squamous cell carcinoma, suggesting a potential application for the use of tetrathiomolybdate in human squamous cell carcinoma.

Copper deficiency induced by tetrathiomolybdate suppresses tumor growth and angiogenesis.

Copper plays an essential role in promoting angiogenesis. Tumors that become angiogenic acquire the ability to enter a phase of rapid growth and exhibit increased metastatic potential, the major cause of morbidity in cancer patients. We report that copper deficiency induced by tetrathiomolybdate (TM) significantly impairs tumor growth and angiogenesis in two animal models of breast cancer: an inflammatory breast cancer xenograft in nude mice and Her2/neu cancer-prone transgenic mice. In vitro, TM decreases the production of five proangiogenic mediators: (a) vascular endothelial growth factor; (b) fibroblast growth factor 2/basic fibroblast growth factor; (c) interleukin (IL)-1alpha; (d) IL-6; and (e) IL-8. In addition, TM inhibits vessel network formation and suppresses nuclear factor (NF)kappaB levels and transcriptional activity. Our study suggests that a major mechanism of the antiangiogenic effect of copper deficiency induced by TM is suppression of NFkappaB, contributing to a global inhibition of NFkappaB-mediated transcription of proangiogenic factors.

Radiotherapy and antiangiogenic TM in lung cancer.

Tetrathiomolybdate (TM) is a potent nontoxic orally delivered copper complexing agent under development for the last several years for the treatment of Wilson's disease. It has been shown to block angiogenesis in primary and metastatic tumors. Therefore, the combination of cytotoxic radiotherapy (RT) and antiangiogenic TM could target both the existing tumor and the tumor microvasculature in a comprehensive strategy. Using a Lewis lung high metastatic (LLHM) carcinoma mouse tumor model, we demonstrate that the combination of TM and RT is more effective than either used as monotherapy. We also show that their therapeutic effects are additive, with no additional toxicity. We show that TM has no significant cytotoxicity in vitro against LLHM tumor cells, further supporting the antiangiogenic mechanism for its action.