Tetrathiomolybdate Research Articles

Antiangiogenic Tetrathiomolybdate Protects against Her2/neu-Induced Breast Carcinoma by Hypoplastic Remodeling of the Mammary Gland

The objective of the present study was to delineate the efficacy of tetrathiomolybdate (TM), a novel antiangiogenic anticancer agent, as a chemopreventative agent. Nulliparous Her2/neu transgenic mice were treated with water or TM for 180 days and observed for tumor development during treatment and for 180 days after treatment. Mammary gland composition and architecture were also observed following TM treatment of Her2/neu transgenic and normal FVB mice. At the 1-year follow-up, 86.7% of control and 40% of TM-treated Her2/neu mice had palpable mammary tumors with a median time to tumor development of 234 days (95% confidence interval, 202-279 days) for control and >460 days for TM-treated mice (P < 0.0005, n = 15). The mammary glands from TM-treated Her2/neu and FVB mice showed a blunted epithelial ductal branching system due to a significant decrease in the number of secondary branches and total number of differentiated mammary epithelial cells. Microvessel density in Her2/neu and FVB mammary glands was lowered by 65.6 +/- 6.2% and 50.9 +/- 4.5% (P < 0.005), respectively, following TM therapy, consistent with the antiangiogenic effect of TM. Lastly, TM treatment resulted in a 2-fold increase in the absolute number of aldehyde dehydrogenase-positive mammary stem cells in Her2/neu and FVB mammary glands. Taken together, these results strongly support that TM is a potent chemopreventative agent as a consequence of hypoplastic remodeling of the mammary gland through modulation of the mammary stem cell compartment.

Preoperative chemoradiation and postoperative adjuvant tetrathiomolybdate for patients with resectable esophageal cancer

e15504 Background: Tetrathiomolybdate (TM) is an oral copper chelator. Copper is necessary for blood vessel growth, and so TM functions as an antiangiogenic agent. Methods: Pts with resectable esophageal cancer were treated at the University of Michigan with chemoradiation followed by surgery, and then 2 years of TM. Chemoradiation was cisplatin 60 mg/m2 on days #1 and 22, paclitaxel 60 mg/m2 on days #1, 8, 15, and 22, and radiation 1.5 Gy fractions b.i.d. x 3 weeks to a total of 45 Gy. Transhiatal esophagectomy was performed on approximately day #50. TM 20 mg p.o. q.d. was started approximately 4 weeks post-op, and continued for 2 years. Doses were adjusted to maintain the ceruloplasmin level between 5 and 15. Results: Between 1/23/02 and 1/24/06, 69 pts were enrolled. Pt characteristics: males - 62 (90%), females - 7 (10%); median age - 60 (range 42–74); adenocarcinoma - 56 (81%), squamous cell carcinoma 13 (19%). The chemoradiation was well tolerated: grade 3 leukopenia in 15 pts (22%), and 17 pts (25%) required feeding tubes. 66 pts went to surgery. 61 pts had a complete resection; 4 pts had metastases discovered at surgery, and 1 pt had extensive positive margins. Histologic complete response rate was 10%. 21 pts never received TM because of metastases noted in the peri-operative period, long post-op recovery time, or pt refusal. 48 pts started TM after surgery and it was well tolerated. Grade 3 toxicities were diarrhea -2 (3%), nausea - 2 (3%), dizziness - 2 (3%). 13 pts completed the full 24 mos of treatment, 12 completed 10–23 mos, 15 completed 2–8 mos, and 8 completed only 1 month or less. 27 pts have had disease recurrence, the majority (23 of the 27) of which was distant. Current status of pts with median follow-up time of 55 months: 25 alive and disease-free, 1 alive with disease, and 43 have died. 3-year survival probability is 47% (95% CI 35%-58.6%). 3-year recurrence-free probability is 51% (95% CI 38%- 62.7%). Conclusions: TM is an antiangiogenic agent which is fairly well tolerated. Prolonged adjuvant treatment over 2 years is difficult but not impossible to complete. Disease-free survival and overall survival are promising when compared to historical controls treated with a very similar chemoradiation regimen without TM in the past at the University of Michigan. [Table: see text]

Copper chelation in cancer therapy using tetrathiomolybdate: an evolving paradigm

Background: Tetrathiomolybdate (TM) is a novel anticancer and anti-angiogenic agent, which acts through copper chelation and NF-κB inhibition. Objective: This review summarizes the scientific rationale for the use of TM as an anticancer agent in human studies. Methods: A systematic review of the literature was conducted for the use of TM in cancer including preclinical, animal and human studies. The results of this search are summarized in this review. Results/conclusions: Copper chelation using TM has demonstrated efficacy in preclinical and animal models as an alternative and novel anti-angiogenic agent. Phase I and II clinical trials conducted in solid tumors using TM have demonstrated efficacy with favorable toxicity profile. The use of copper lowering as an anti-angiogenic strategy in the cancer chemopreventative setting remains to be investigated.

The effect of tetrathiomolybdate on circulating endothelial progenitor cells in patients with breast cancer at high risk of recurrence.

Abstract Abstract #1036 Background: Endothelial progenitor cells are critical to tumor angiogenesis and are increased in breast cancer patients. Copper is required for angiogenesis, and pre-clinical data suggest that tetrathiomolybdate (TM), a copper-depleting compound, inhibits angiogenesis and maintains tumor dormancy. We sought to measure circulating endothelial progenitor cells (CEPCs) in patients at high risk of breast cancer recurrence and to evaluate the effect of copper depletion on CEPCs.&amp;#x2028; Methods: This analysis is part of an ongoing phase II study of TM in breast cancer patients at high risk of recurrence defined as Stage III or IV with no evidence of disease. All therapy other than hormonal was completed at least 6 weeks prior to study. Treatment: TM 180 mg daily to achieve a target ceruloplasmin (Cp) level of 5-15 mg/dL (copper depletion), and then 100 mg daily. We monitored levels of CEPCs (CD45dim, CD133+, VEGFR2+), CEA, CA15-3, and Cp at baseline and monthly. CEPCs were also measured in 6 healthy controls.&amp;#x2028; Results: To date we have enrolled 16 patients with a median age of 51 years (range: 29-64). 14 had a history of Stage III disease, while 2 were considered to be Stage IV with no evidence of disease. The median number of positive lymph nodes among Stage III patients was 7 (1-42), with 2 patients having received neoadjuvant therapy. The median baseline Cp level was 28 mg/dL (21-41). Among 12 patients who have reached target Cp, the median time to target was 1 month (1-3 months). The median follow-up of the 4 patients who have not yet achieved target is 2.5 months. 1 of these discontinued treatment before reaching target. The median baseline CEPCs was lower in patients than healthy controls: 0.022 cells/μL (0.000-0.286) vs. 0.123 cells/μL (0.058-0.418); p=0.03. There was no statistically significant change in CEPCs from baseline over time.&amp;#x2028; One patient was diagnosed with recurrent breast cancer at month 10. A rise in her CEPCs preceded a rise in a CEA and overt relapse by 1 and 5 months, respectively.&amp;#x2028; Toxicity: Grade 3/4 neutropenia occurred in 3 patients. TM was held, and this resolved 5-13 days later, after which TM was resumed. No other grade 3/4 toxicity was observed. One patient discontinued TM due to diarrhea attributed to the lactose used in the compounding of TM.&amp;#x2028; Conclusions: TM is well tolerated in breast cancer patients. We postulate that the increased CEPCs noted in one patient at month 4, 6 months prior to overt relapse, could represent the “turning on” of an angiogenic switch, resulting in an outpouring of CEPCs to the new site of metastasis. The trial is ongoing, and with additional follow-up other trends might emerge.&amp;#x2028; Supported by Komen for the Cure Foundation, Anbinder Foundation, NY Community Trust and Breast Cancer Alliance of Greenwich. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1036.

The use of copper-lowering therapy with tetrathiomolybdate in medicine

Background: Tetrathiomolybdate (TM), an anticopper drug, has been developed for the neurologic presentation of Wilson's disease. In animal models, lowering copper levels with TM produces antifibrotic, anti-inflammatory, antiautoimmune, and anticancer effects, thought to be due to inhibition of many cytokines that are dependent on available copper for their activity. Clinical testing has been done relatively extensively in Wilson's disease and advanced cancers, but remains in its infancy in other diseases. Objectives: To review current preclinical and clinical studies done with TM, and our current knowledge of TM efficacy and toxicity. Methods: We have reviewed the last 10 years of literature on TM therapy. Results/conclusion: TM has excellent efficacy and acceptable toxicity for the initial treatment of neurologically presenting Wilson's disease. TM has excellent efficacy in animal models of fibrotic, inflammatory, autoimmune, and neoplastic diseases, as well as Alzheimer's disease models.

Efficacy of tetrathiomolybdate in a mouse model of multiple sclerosis

Tetrathiomolybdate (TM) is a potent anticopper drug developed for Wilson's disease. We have found multiple efficacious results from decreasing copper levels with TM in mouse models of disease, using serum Cp as a surrogate marker of copper status and targeting Cp values of 20% to 50% of baseline. We have found efficacious results of TM therapy in mouse models of fibrosis; inflammation; damage from exogenous agents, such as acetaminophen and doxorubicin; and immune-modulated diseases, such as concanavalin A hepatitis, collagen II-induced arthritis, and the non-obese diabetic (NOD) mouse model of type I diabetes. In the current study, we examine TM efficacy in the EAE mouse model of multiple sclerosis (MS). We find that clinical scores of neurologic damage are significantly inhibited by TM therapy, whether therapy is started before MS-inducing antigen administration or after symptoms from antigen administration develop. Furthermore, we find that experimental autoimmune encephalomyelitis (EAE) treatment produces a marked increase of oxidant damage, as measured by urine isoprostane levels, and TM suppresses these isoprostane increases strongly and significantly. Finally, we find marked increases of inflammatory and immune-related cytokines in this model, and we find that TM strongly and significantly suppresses these increases.

A pilot trial of the anti-angiogenic copper lowering agent tetrathiomolybdate in combination with irinotecan, 5-flurouracil, and leucovorin for metastatic colorectal cancer

Tetrathiomolybdate (TM) is an oral copper chelator under development as an anti-angiogenic agent. We evaluated TM in combination with irinotecan, 5-fluorouracil, and leucovorin (IFL). Serum vascular endothelial growth factor (VEGF), basic fibroblast growth factor, interleukin 6 (IL-6), and IL-8 were measured to evaluate the anti-angiogenic effect. Twenty-four patients with metastatic colorectal cancer were treated. The combination with IFL was well tolerated and dose intensity of IFL was maintained during combination therapy with TM. By intention to treat analysis, the overall response rate (RR) was 25% (95% CI 9.8-46.7) and the median time to progression (TTP) was 5.6 months (95% CI 2.7-7.7). VEGF levels were correlated with TTP, as were changes in VEGF, IL-8, and IL-6. TM can be safely added to IFL without compromising dose intensity or diminishing the expected RR. Changes in serum VEGF, IL-8, and IL-6 after treatment may directly reflect changes in CRC tissue angiogenesis.

Tetrathiomolybdate Is Partially Protective Against Hyperglycemia in Rodent Models of Diabetes

The objective was to evaluate whether copper lowering therapy with tetrathiomolybdate (TM) affected blood sugar levels in three rodent models of diabetes, streptozotocin (STZ) treated rats and mice, and the db/db mouse model. STZ was administered to rats and mice, and blood sugar levels were followed over a protracted time in these and non-STZ control animals. TM was administered by oral gavage (rats) or in the drinking water (mice) to a portion of the rats and mice to observe effects on blood sugar. Mice with genetically determined diabetes (db/db) were studied by giving half the mice TM in the drinking water and following blood sugar. The results show that TM caused a significant reduction in blood glucose in both STZ treated rats and mice, but no effect on blood glucose in db/db mice. However, TM caused a significant reduction in proteinuria in db/db animals. The results are discussed around the likelihood that TM is inhibiting ongoing inflammatory damage in the pancreas from STZ. A metabolic effect of TM on blood glucose is possible but seems less likely. TM is also likely inhibiting inflammatory and/or fibrogenic effects in the kidneys of db/db mice.

A Phase II Trial of Tetrathiomolybdate After Surgery for Malignant Mesothelioma: Final Results

Tetrathiomolybdate (TM) is an oral copper-depleting agent that has been shown to inhibit angiogenesis, and angiogenesis is a predictor of poor prognosis in malignant pleural mesothelioma. We hypothesized that cytoreduction of malignant pleural mesothelioma followed by TM will delay time to progression. Between November 2000 and August 2003, 30 patients with malignant pleural mesothelioma received postoperative TM beginning 4 to 6 weeks after surgery at a dose adjusted to keep ceruloplasmin between 5 and 15 mg/dL). Time to progression was compared with the 55 stage I and II patients and 109 stage III patients previously treated with cytoreduction by one of us (H.P.). The 30 patients (25 men, 5 women; 13 stage I and II, 17 stage III), median age 67 years (range, 49-81 years), remained on TM a median of 14.9 months (range, 2 to 57 months). All patients reached target ceruloplasmin levels at a mean of 34 +/- 2 days (95% confidence interval, 30 to 39 days), and vascular endothelial growth factor levels at baseline (ceruloplasmin = 45.2 +/- 2 mg/dL) decreased from 2,086 +/- 390 pg/mL to 1,250 +/- 712 pg/mL (p < 0.002) at target ceruloplasmin (13 +/- 2 mg/dL; p < 0.0001 from baseline). The time to progression for all stage I or II TM patients was 20 months whereas that of 55 stage I or II non-TM-treated patients was 10 months (p = 0.046 versus TM). No differences in time to progression for the stage III TM patents from surgery were seen (7 months). Tetrathiomolybdate has antiangiogenic effects in malignant pleural mesothelioma patients after resection of gross disease, and exhibits minimal toxicity and comparable efficacy to previous multimodality trials. Tetrathiomolybdate should be evaluated for efficacy in combination with standard malignant pleural mesothelioma regimens, as well as for postsurgical maintenance therapy.

A phase II trial of tetrathiomolybdate (TM) in metastatic head &amp; neck cancer (MHNC) patients (pts) after treatment with platinum &amp; 5-fluorouracil (P&amp;5FU)

6080 Background: The use of antiangiogenesis agents to promote disease stabilization following maximal response to chemotherapy in HNC is not well studied. TM is an oral copper (Cu) depleting agent that inhibits angiogenesis & decreases levels of VEGF when ceruloplasmin (Cp) is reduced to 5–15 mg/dl. We hypothesize that treatment of MHNC with P&5FU followed by continuous TM can delay disease progression by > 2 months (mo) by keeping maximally treated disease in a dormant state. Methods: Eligible pts were treated with 2–4 (21 day) cycles of cisplatin (100 mg/m2) or carboplatin (AUC 6) + 5FU (1000 mg/m2/day over 5 days). Pts who attained complete response (CR), partial response (PR), or stable disease (SD) were given TM (180 mg/day). Cp levels were drawn weekly & TM doses were adjusted to maintain a Cp level of 5–15 mg/dl. Neck/chest CTs were performed every 8 weeks. TM was continued until disease progression (PD). Results: From November 2002 to March 2004, 24 pts were enrolled; 23 were eligible (19 men; 4 ...

Tetrathiomolybdate Protects against Bile Duct Ligation-Induced Cholestatic Liver Injury and Fibrosis

Tetrathiomolybdate (TM), a potent copper-chelating drug, was initially developed for the treatment of Wilson's disease. Our working hypothesis is that the fibrotic pathway is copper-dependent. Because biliary excretion is the major pathway for copper elimination, a bile duct ligation (BDL) mouse model was used to test the potential protective effects of TM. TM was given in a daily dose of 0.9 mg/mouse by means of intragastric gavage 5 days before BDL. All the animals were killed 5 days after surgery. Plasma liver enzymes and total bilirubin were markedly decreased in TM-treated BDL mice. TM also inhibited the increase in plasma levels of tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1 seen in BDL mice. Cholestatic liver injury was markedly attenuated by TM treatment as shown by histology. Hepatic collagen deposition was significantly decreased, and it was paralleled by a significant suppression of hepatic smooth muscle alpha-actin and fibrogenic gene expression in TM-treated BDL mice. Although the endogenous antioxidant ability was enhanced, oxidative stress as shown by malondialdehyde and 4-hydroxyalkenals, hepatic glutathione/oxidized glutathione ratio, was not attenuated by TM treatment, suggesting the protective mechanism of TM may be independent of oxidative stress. In summary, TM attenuated BDL-induced cholestatic liver injury and fibrosis in mice, in part by inhibiting TNF-alpha and TGF-beta1 secretion. The protective mechanism seems to be independent of oxidative stress. Our data provide further evidence that TM might be a potential therapy for hepatic fibrosis.

Early Biomarkers for the Anti-Angiogenic and Anti-Tumor Activity of the Superoxide Dismutase 1 (SOD1) Inhibitor Tetrathiomolybdate.

Background: Identifying biomarkers that monitor clinical response to cancer therapies is crucial. Tetrathiomolybdate (TM) is a highly specific, high-affinity, orally available copper-binding compound that inhibits CuZn SOD1, leading to robust anti-angiogenic and anti-tumor effects. TM is currently being evaluated in Phase II cancer trials including for relapsed and refractory multiple myeloma (MM). TM's inhibition of angiogenesis has been attributed to depletion of systemic Cu, which is known to affect multiple key angiogenesis regulators; however, in mice, inhibition of angiogenesis by TM occurred before a measurable decrease in systemic Cu. We have recently shown that in MM, circulating endothelial progenitor cell (EPC) levels serve as a reliable biomarker of disease severity which covaries with tumor load and disease progression. Furthermore, recent evidence from our laboratory and by others indicates that EPCs display evidence of clonality and are genetically related to MM cells. In the present study, the relationship between anti-angiogenic and anti-SOD1 activities of TM were compared by determining its effects on:1.circulating endothelial progenitor cells (EPCs) in bonnet macaques (M. radiata);2.on angiogenesis and SOD1 activity in blood cells from mice and humans.Methods: TM was manufactured with >99% purity using a proprietary process (choline salt: ATN-224). For animal studies, three female and three male young adult monkeys were treated with TM (0.5 mg/kg, s.c.) daily for 22 days, followed by a 26 day drug holiday, and a second course of drug. Blood was drawn from the cephalic vein, separated by Ficoll, and red cell lysis. EPCs were identified as CD31+/CD133+/CD45− weak cells using 3-color flow cytometry. For SOD activity, blood from normal control subjects was incubated with TM for 5.5 hr at 37°C. SOD activity was measured in 30 μg of protein from treated blood pellets using an SOD Activity Kit.Results: Administration of TM to the monkeys resulted in a rapid, profound, and reversible decrease in circulating EPCs without significant toxicity. The levels of EPCs increased initially at day 6 in 5 of the 6 subjects, and then decreased to 7% of baseline at day 21. The EPC levels rebounded to normal levels once treatment was discontinued and dropped again after treatment was reinstated. Other hematopoietic parameters (RBC, WBC, platelet counts, and hemoglobin) were not affected by treatment. Although we did not measure blood cell SOD in the macaque study, substantial inhibition of blood cell SOD is associated with hematologic toxicities which did not occur in these animals, suggesting that RBC SOD activity is inhibited at higher doses than those needed for anti-angiogenic activity. TM treatment inhibited SOD activity in blood cell pellets obtained from human controls in a dose dependent manner, with an IC50 3 μM.Conclusion: These results show, for the first time, that in vivo effects of TM include suppression of circulating EPC levels. Since circulating EPCs are genetically related to tumor cells in MM, at least the anti-MM effects of TM may depend on its suppressive effects on EPCs as well as on angiogenesis in general. Furthermore, EPCs are a sensitive indicator of TM actions since their reversible modulation correlated with drug therapy. Lastly, macaques may represent a reliable model of human response to determine drug effects on EPCs and angiogenesis.

Phase II Trial of Copper Depletion with Tetrathiomolybdate as an Antiangiogenesis Strategy in Patients with Hormone-Refractory Prostate Cancer

Objective: Preclinical studies suggest antiangiogenesis strategies may be effective in the treatment of prostate cancer. In tumor models, the copper-chelating agent tetrathiomolybdate (TM) has been shown to be antiangiogenic. We evaluated the antitumor activity of TM in patients with hormone-refractory prostate cancer (HRPC). Methods: Nineteen patients with asymptomatic HRPC enrolled. Copper depletion was monitored using serum ceruloplasmin levels. Once the target ceruloplasmin level of 5–15 mg/dl was attained, patients underwent staging evaluation. Patients were reassessed every 12 weeks, and TM was continued until they developed evidence of disease progression or intolerable toxicity. Prostate-specific antigen and levels of vascular endothelial growth factor, basic fibroblast growth factor, interleukin (IL)-6 and IL-8 were measured at study entry, at the time of copper depletion, and monthly while on therapy. Results: Seventeen of 19 patients achieved copper deficiency on TM therapy. Of the 16 evaluable patients, 14 developed progressive disease, 1 discontinued therapy because of toxicity and 1 patient opted to discontinue therapy because of rising prostate-specific antigen level without objective evidence of progressive disease. Levels of vascular endothelial growth factor, IL-6 and IL-8, but not basic fibroblast growth factor, were elevated when compared to normal controls prior to TM therapy, but there was no significant change during therapy. There was no correlation between prostate-specific antigen and levels of angiogenesis factors. Conclusions: Copper depletion with TM did not delay disease progression in patients with asymptomatic metastatic HRPC.

Tetrathiomolybdate versus Trientine in the Initial Treatment of Neurologic Wilson's Disease

ABSTRACTBackground: The initial treatment of the neurologic presentation of Wilson's disease is problematic. Penicillamine, used for years on most patients, causes neurologic worsening in up to half of such patients, and half of those who worsen never recover. Zinc, ideal for maintenance therapy, is too slow for these acutely ill patients. We have developed tetrathiomolybdate (TM) for this type of patient, and it has worked well in open label studies. Trientine, another anticopper drug on the market approved for penicillamine intolerant patients, had not been tried in this type of patient. Here, we report on a double blind trial of TM versus trientine in the neurologically presenting Wilson's disease patient. Design and Methods: The study was a double blind design in which patients received either TM plus zinc, or trientine plus zinc, for 8 weeksThis study was originally published in reference 1. Patients were accepted if they presented with neurologic symptoms from Wilson's disease, if they had not been treated longer than 4 weeks with penicillamine or trientine. Patients were followed in the hospital for the 8 weeks of treatment with weekly semiquantitative neurologic and speech examinations, to evaluate possible neurologic worsening. They also had blood and urine studies done weekly. At discharge from hospital they were continued on zinc maintenance therapy, and returned at yearly intervals for 3 years for further evaluation. Results: Twenty-three patients were entered into the trientine arm and 6 reached criteria for neurologic deterioration, while 25 patients were entered into the TM arm and only 1 deteriorated (p < 0.05). One patient on trientine had an adverse event while 7 on TM had adverse events. All adverse events were mild. Four patients in the trientine arm died during follow-up, 3 having shown initial neurologic deterioration, 2 patients in the TM arm died. In those patients who did not deteriorate or die, neurologic and speech recovery over 3 years was good. Interpretation: TM is a superior choice to trientine for the initial therapy of neurologic Wilson's disease.

Antitumor and antiinflammatory effects of tetrathiotungstate in comparison with tetrathiomolybdate

Tetrathiomolybdate (TM) is an anticopper drug under development for treating Wilson's disease. Its mechanism of action involves forming a tight tripartite complex in the blood with serum albumin and available copper. When available copper levels are lowered in animals with TM, strong antiangiogenic and antitumor effects are observed. Similarly, TM has excellent efficacy in animal models of fibrotic, inflammatory, and autoimmune diseases, and it protects against heart damage from doxorubicin (DXR) and liver damage from acetaminophen, carbon tetrachloride, and concanavalin A. Tetrathiotungstate (TT) also forms a similar tripartite complex in the blood and has similar effects to TM on copper. In this article, whether TT had similar antitumor effects, and similar effects in protecting the heart against DXR toxicity, as TM was evaluated. It was found that the 2 drugs were comparable in their effects when doses were used that lowered copper availability to the same extent.

Comparison of lowering copper levels with tetrathiomolybdate and zinc on mouse tumor and doxorubicin models

Tetrathiomolybdate (TM), presumably by lowering copper levels and availability, has shown excellent efficacy in animal models of cancer and models of injury that produce fibrotic or inflammatory damage in lung, heart, and liver. Trials in human patients are underway. If the efficacy of TM is indeed through lowering copper levels, other anticopper drugs should be equally efficacious. Zinc is an anticopper drug, with proven efficacy in Wilson's disease, a disease of copper toxicity. In this study, the efficacy of zinc is compared with TM on a mouse tumor model and on the doxorubicin model of heart damage, and it is hypothesized that when copper availability is lowered to an equivalent extent, the 2 drugs would show equivalent efficacy. No effect is found of zinc on inhibiting growth of a tumor that is markedly inhibited by TM, and zinc is found to be less effective than TM in inhibiting cardiac damage from doxorubicin. This study shows that TM's mechanism of action in protecting against doxorubicin toxicity is because of its anticopper effects, as copper supplementation eliminated the protective effect of TM. It is also hypothesized that the differences between TM and zinc may be caused by TM's mechanism of action in which it binds copper already in the body, whereas zinc does not.

Multifocal Angiostatic Therapy: An Update

Multifocal angiostatic therapy (MAT) is a strategy that seeks to impede cancer-induced angiogenesis by addressing multiple targets that regulate the angiogenic capacity of a cancer and/or the angiogenic responsiveness of endothelial cells, using measures that are preferentially, but not exclusively, nutraceutical. A prototype of such a regimen has been proposed previously, composed of green tea polyphenols, fish oil, selenium, and high-dose glycine, complementing a low-fat vegan diet, exercise training, and the copper-sequestering drug tetrathiomolybdate (TM). A review of more recent evidence suggests additional agents that could appropriately be included in this regimen and clarifies to some extent the mechanisms of action of its constituents. Diindolylmethane, a widely available crucifera-derived nutraceutical, has inhibited cancer growth in several mouse xenograft models; this effect may be largely attributable to an angiostatic action, as concentrations as low as 5 to 10 muM inhibit proliferation, migration, and tube-forming capacity of human endothelial cells in vitro, and a parenteral dose of 5 mg/kg markedly impairs matrigel angiogenesis in mice. Silymarin/silbinin, which has slowed the growth of human xenografts in a number of studies, suppresses the proliferation, migration, and tube-forming capacity of endothelial cells and inhibits vascular endothelial growth factor (VEGF) secretion by a range of human cancer cell lines, in concentrations that should be clinically feasible. The angiostatic activity of orally administered green tea now appears likely to reflect inhibition of the kinase activity of VEGFR-2. Glycine's angiostatic activity may be attributable to a hyperpolarizing effect on endothelial cells that decreases the activity of NADPH oxidase, now known to promote tyrosine kinase signaling in endothelial cells. The ability of TM to suppress cancer cell production of a range of angiogenic factors results at least in part from a down regulation of NF-kappaB activation. Dual-purpose molecular targets, whose inhibition could be expected to decrease the aggressiveness and chemoresistance of cancer cells while simultaneously impeding angiogenesis, include NF-kappaB, cox-2, c-Src, Stat3, and hsp90; drugs that can address these targets are now in development, and salicylates are notable for the fact that they can simultaneously inhibit NF-kappaB and cox-2. The potential complementary of the components of MAT should be assessed in nude mouse xenograft models.

Control of Copper Status for Cancer Therapy

Copper is a trace element which is tightly regulated in mammals and lower animals. Disruptions of copper homeostasis in humans are rare and they cause serious disorders such as Wilson's disease and Menke's disease. Copper plays an important role in promoting physiological and malignant angiogenesis. Formation of new blood vessels by a tumor enables tumor growth, invasion, and metastasis are copper requiring processes. The copper chelator tetrathiomolybdate (TM), which quickly and effectively depletes copper stores, is under investigation as an anti-angiogenic agent. Promising results from in vitro experiments, in pre-clinical animal models, and in a phase I clinical trial have led to several phase II trials of TM in patients with advanced cancers.

Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells

IntroductionA physiological feature of many tumor tissues and cells is the tendency to accumulate high concentrations of copper. While the precise role of copper in tumors is cryptic, copper, but not other trace metals, is required for angiogenesis. We have recently reported that organic copper-containing compounds, including 8-hydroxyquinoline-copper(II) and 5,7-dichloro-8-hydroxyquinoline-copper(II), comprise a novel class of proteasome inhibitors and tumor cell apoptosis inducers. In the current study, we investigate whether clioquinol (CQ), an analog of 8-hydroxyquinoline and an Alzheimer's disease drug, and pyrrolidine dithiocarbamate (PDTC), a known copper-binding compound and antioxidant, can interact with copper to form cancer-specific proteasome inhibitors and apoptosis inducers in human breast cancer cells. Tetrathiomolybdate (TM), a strong copper chelator currently being tested in clinical trials, is used as a comparison.MethodsBreast cell lines, normal, immortalized MCF-10A, premalignant MCF10AT1K.cl2, and malignant MCF10DCIS.com and MDA-MB-231, were treated with CQ or PDTC with or without prior interaction with copper, followed by measurement of proteasome inhibition and cell death. Inhibition of the proteasome was determined by levels of the proteasomal chymotrypsin-like activity and ubiquitinated proteins in protein extracts of the treated cells. Apoptotic cell death was measured by morphological changes, Hoechst staining, and poly(ADP-ribose) polymerase cleavage.ResultsWhen in complex with copper, both CQ and PDTC, but not TM, can inhibit the proteasome chymotrypsin-like activity, block proliferation, and induce apoptotic cell death preferentially in breast cancer cells, less in premalignant breast cells, but are non-toxic to normal/non-transformed breast cells at the concentrations tested. In contrast, CQ, PDTC, TM or copper alone had no effects on any of the cells. Breast premalignant or cancer cells that contain copper at concentrations similar to those found in patients, when treated with just CQ or PDTC alone, but not TM, undergo proteasome inhibition and apoptosis.ConclusionThe feature of breast cancer cells and tissues to accumulate copper can be used as a targeting method for anticancer therapy through treatment with novel compounds such as CQ and PDTC that become active proteasome inhibitors and breast cancer cell killers in the presence of copper.

Copper Lowering Therapy With Tetrathiomolybdate as an Antiangiogenic Strategy in Cancer

Tetrathiomolybdate (TM) is a novel anticopper agent under development for use in Wilson's disease. It acts by forming a stable tripartite complex with serum albumin and copper, rendering the complexed copper unavailable for cellular uptake. TM is a very potent anticopper agent and has an excellent safety profile. It has been shown that normal copper levels are required for optimal angiogenesis. Based on this background, we decided to evaluate TM as an anticancer agent. TM treatment of Her/2neu mice, genetically programmed to develop breast cancer, completely prevented the development of visible mammary cancers, although avascular microscopic clusters of cancer cells were present in the breasts of TM treated animals. Controls developed grossly visible tumors. TM was able to strongly inhibit tumor growth in six other rodent models. In a phase 1/2 clinical trial of advanced and metastatic cancers, freedom from progression averaged 11 months, and some individual results were quite dramatic. Eight phase 2 studies of specific cancers have been launched. TM's hypothesized mechanism of action is inhibition of angiogenic cytokines. Unlike other current approaches to antiangiogenic therapy which target single agents, we hypothesize that TM inhibits multiple angiogenic cytokines. Part of this effect appears to stem from inhibition of nuclear factor kappa B (NF(K)B), which in turn controls transcription of many angiogenic and other cytokines. However, there are probably multiple mechanisms, in that some angiogenic cytokines appear to have separate mechanisms of copper dependence. The inhibition of multiple angiogenic cytokines gives TM the potential to be a more global inhibitor of angiogenesis.