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Role of copper in tumour angiogenesis—clinical implications
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Trace Elements in Medicine and Biology Journal of Trace Elements in Medicine and Biology 18 (2004) 1–8 www.elsevier.de/jtemb
REVIEW
Role of copper in tumour angiogenesis—clinical implications Anna Nasulewicza, Andrzej Mazurb, Adam Opolskia,* a
Laboratory of Tumor Immunology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Ul. R. Weigla 12, 53-114 Wroclaw, Poland b CRNH d’Auvergne, Unite! Maladies M!etaboliques et Micronutriments, INRA, Theix, 63122 St. Gen"es Champanelle, France Received 4 August 2003; accepted 26 February 2004
Abstract The formation of new blood vessels is the initial step in progressive tumour development and metastasis. The first stage in tumour angiogenesis is the activation of endothelial cells. Copper ions stimulate proliferation and migration of endothelial cells. It has been shown that serum copper concentration increases as the cancer disease progresses and correlates with tumour incidence and burden. Copper ions also activate several proangiogenic factors, e.g., vascular endothelial growth factor, basic fibroblast growth factor, tumour necrosis factor alpha and interleukin 1. This review concerns a brief introduction into the basics of tumour blood vessel development as well as the regulatory mechanisms of this process. The role of copper ions in tumour angiogenesis is discussed. The new antiangiogenic therapies based on a reduction of copper levels in tumour microenvironment are reviewed. r 2004 Elsevier GmbH. All rights reserved. Keywords: Tumour angiogenesis; Dietary copper; Antiangiogenic therapy; Copper reduction
Introduction
Basic mechanisms of angiogenesis
The concept of antiangiogenic treatment for solid tumours was pioneered by Judah Folkman in the early 1970s [1]. Since that time many studies have supported the rationale and efficacy of antiangiogenic therapy in animal models. Several compounds interfering with critical steps in blood vessel formation have already reached the clinic. Many others are being tested in preclinical studies. It has been demonstrated that copper is required for the angiogenic process and reduction of the copper level in cancer patients has become one of the newest approaches in antiangiogenic treatment.
Every cell of the human organism requires oxygen and nutrients for its survival and development. The efficient supply of substances is sustained by the dense net of blood vessels. Therefore, the cells are located within 100–200 mm of blood vessels—the diffusion limit for nutrients and inorganic substances. Tumours can develop without limit until they reach the size of 1–2 mm3 [2,3]. Further growth depends on the formation of blood vessels [1]. These new vessels develop either from pre-existing ones or from endothelial cell precursors—angioblasts—derived from bone marrow. This process is called angiogenesis [4]. Newly formed blood vessels supply tumour cells with oxygen and nutrients. They are also the gate which makes tumour development and the spread of metastatic cells possible [5]. In adults, under physiological conditions, angiogenesis takes place very rarely and is restricted to very few
*Corresponding author. E-mail address: [email protected] (A. Opolski). 0946-672X/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2004.02.004
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organs (ovaries, Fallopian tubes, uterus, placenta) [6]. This is due to a tight control at the molecular level and to a balance between synthesis of pro- and antiangiogenic factors. As long as the inhibitory molecules override the action of the stimulators, angiogenesis is ‘‘switched off’’ [3]. The antiangiogenic factors may act by regulating: *
*
*
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the transcription of angiogenesis activators produced by tumour as well as by endothelial cells (e.g. interferon alpha and beta), the activity of metalloproteases (e.g. tissue inhibitor of metalloproteases— TIMP), the motility and migration of endothelial cells (e.g. thrombospondin 1 and 2), apoptosis of endothelial cells [5,7].
Tumour angiogenesis—the ‘‘angiogenic switch’’ Most commonly, angiogenesis is associated with pathological lesions, e.g. wound healing, rheumatoid arthritis, psoriasis, retinitis and cancer disease. In cancer, angiogenesis starts when the tumour cells gain angiogenic phenotype, at a size of approximately 2 mm3 (106 cells) [2]. Angiogenesis ‘‘switches on’’ as a result of tipping the balance between the synthesis of activators and inhibitors in favour of proangiogenic molecules [3]. Various signals that are able to trigger angiogenesis have been described. These include mechanical stress, inflammatory response and genetic mutations, but the most important factor is metabolic stress, especially the lack of oxygen within the tumour cells [8]. Low pO2 in tumour cell induces the synthesis of several proteins, among which vascular endothelial growth factor (VEGF) plays the most significant role at the early stages of angiogenesis, as it stimulates endothelial cells migration and proliferation [3–5]. VEGF also prevents endothelial cell apoptosis and increases the permeability of blood vessels [9,10]. The activation of endothelial cells is always the first stage in tumour angiogenesis. The following steps include: (i) release of proteolytic enzymes from activated cells, (ii) degradation of the basal membrane surrounding existing vessels, (iii) migration of endothelial cells to intratissue space, (iv) endothelial cell proliferation, (v) formation of the new blood vessel lumen, (vi) formation of a new basal membrane, (vii) fusion of new vessels, (viii) blood flow initiation [11]. Each of these steps may be a potential target in antiangiogenic cancer treatment. The advantages of angiosuppressive therapy are: low risk of drug resistance (a drug acts on endothelial– cancer cell interactions rather than on tumour cells themselves), relatively high availability of a drug to the cells and relatively high specificity of the drug action (tumour microenvironment).
Copper in cancer Copper is an essential trace element with many physiological functions. It affects activity of many enzymes (Cu/Zn-superoxide dismutase (Cu/Zn-SOD), ceruloplasmin, cytochrome oxidase, tyrosinase, dopamine hydroxylase and lysine oxidase), both as a cofactor and as an allosteric component. These enzymes are essential for cellular respiration, defence against free radicals, melanin synthesis, formation of connective tissue and for iron metabolism. In addition, copperdependent transcription factors play an important role in gene expression [12]. A daily dietary intake of 1–2 mg copper seems necessary to enable copper-dependent metabolic processes in adults [13]. The main sources of dietary copper are dried beans, nuts, shellfish and liver. Copper is absorbed readily from the stomach and small intestine. The liver is the major location of stored copper, containing about 10% of the total body content [14]. In blood serum, copper is transported mainly by ceruloplasmin [15] and, in smaller part, by histidine and albumin [16]. Serum copper level is raised in anaemia, normal pregnancy and during administration of oral contraceptives [17]. Its level is also increased during infections due to the high activity of ceruloplasmin—one of the acute phase proteins [18]. Copper metabolism is profoundly altered in neoplastic disease. It has been found that serum copper concentration correlates with tumour incidence and burden, malignant progression, and recurrence in a variety of human cancers: Hodgkin’s lymphoma, sarcoma, leukaemia, cervix, liver, lung, brain and breast cancers [19–26]. Serum ceruloplasmin level increases 4–8 times as a cancer disease progresses. Furthermore, the cellular deposition of copper is also altered in tumour tissues—from cytoplasm in normal tissue to intranuclear and perinuclear zones in tumours [27]. Copper plays an important role in tumour angiogenesis, especially at its early stages. Copper seems to be necessary for endothelial cell activation as it stimulates their proliferation and migration. Several angiogenic factors, e.g. VEGF, basic fibroblast growth factor (bFGF), tumour necrosis factor alpha (TNF-a) and interleukin (IL) 1 have been found to be copperactivated. Activation factors bind to endothelial cells, ‘‘switch’’ them from G0 into G1 phase and force proliferation. Brem and his co-workers have shown that copper reduction by copper deficient diet or/and penicillamine (copper-chelating molecule) blocks angiogenesis by ‘‘switching’’ endothelial cells back into G0 phase or apoptosis and by down regulating the angiogenic activity of VEGF, bFGF, TNF-a, IL-1 and probably IL-6 and IL-8 as well [28,29]. Probably copper influences angiogenesis in a few ways. Not only does it activate endogenous angiogenic
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factors, but it also binds to several proteins (heparin, ceruloplasmin) which therefore gain angiogenic activity that manifests by stimulation of endothelial cells [15,30]. It has also been shown that additional copper augments, by over four times, the angiogenin binding to calf pulmonary artery endothelial cells [31].
Copper reduction as a therapeutic approach The steps required for successful angiogenesis at the primary and metastatic sites are diverse and they depend on the imbalance between angiogenesis activators and inhibitors. The relative importance of the different angiogenesis-modulating molecules often varies with respect to tissue type. Therefore, it would be very desirable to develop an antiangiogenic therapy that would affect multiple activators of angiogenesis in order to be applicable to various types of tumours [32]. Compounds of copper-reducing activity seem to meet (at least partly) this criterion, since copper is a required cofactor of several key angiogenic factors. In vitro and in vivo studies conducted during the last few years have confirmed the expediency as well as the efficacy of copper-reducing and copper-chelating agents in antiangiogenic treatment. In a series of experiments, Gullino and co-workers showed that application of a low copper diet and penicillamine therapy results in prostaglandin E-stimulated angiogenesis suppression [33]. Experiments conducted by Brem et al. showed that such a treatment leads to tumour growth retardation and angiogenesis inhibition in a rabbit brain tumour model [34]. Several copper-reducing compounds that may be effective in antiangiogenic therapy, including penicillamine, tetrathiomolybdate (TM), captopril, tachpirydine, zinc and trientine, have entered pre- and clinical studies. These agents have been widely used to treat other diseases, e.g. Wilson’s disease (genetic disorder of copper transport resulting in abnormal copper accumulation and toxicity), rheumatoid arthritis or hypertension. Copper-reducing drugs offer advantages to cancer therapy for several reasons, including: *
* *
* *
effectiveness in treatment of various types of tumours, low toxicity risk, possibility of combination with almost every other antitumour treatment strategy, low cost of therapy, may be of a special interest as low risk strategy in a situation of ‘‘Watch and Wait’’ (W&W), when medical procedures are limited to monitoring of the tumour progression before taking a therapeutic decision.
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A serious limitation of using copper-reducing compounds in antitumour treatment is the long time of drug administration required. To provide therapeutic effects, copper level in the patient’s serum needs to be reduced to 20% of the physiological concentration, which takes (dependent on the drug used) from several weeks to 6 months. There is a considerable lag between the initiation of therapy and the reduction of copper level due to copper release from the liver. Effective inhibition of angiogenesis may also lead to a local necrosis, lysis of tumour cells and release of copper into the tumour microenvironment [32]. Moreover, tumour cells are thought to sequester copper [35]. Hence, it is logical that a sufficiently long time is required to deplete copper from the tumour microenvironment. Thus, patients with rapidly progressing tumours may be relatively poor candidates for this antitumour therapy as a single modality.
Copper-reducing agents in experimental and clinical studies Penicillamine d-Penicillamine (sold as Cuprimine or Depen) is a drug commonly used to treat Wilson’s disease and rheumatoid arthritis. It binds copper with sulphydryl groups and this complex is subsequently removed from the organism via the urine. In the presence of copper ions, penicillamine produces H2O2 [36]. This may lead to generation of free radicals, which can inhibit endothelial cell proliferation, as these cells are highly sensitive to reactive oxygen species (ROS) [37]. In addition to copper chelation, penicillamine has multiple functions that may account for its antiangiogenic activity. It is a dose-dependent inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). It inhibits collagen synthesis, gelatinase B activity and both proliferation and migration of endothelial cells. Finally, penicillamine stabilises the activity of TIMP and converts plasminogen to angiostatin, a potent antiangiogenic compound. In a single-drug therapy penicillamine exerts antitumour activity that is enhanced when copper level is strongly reduced [34]. Studies performed on rats inoculated orthotopically [34] or subcutaneously [38] with gliosarcoma cells, as well as experiments conducted in a mouse hepatocellular carcinoma model [39], confirmed the antitumour and antiangiogenic activity of penicillamine when combined with a low copper diet. This combination leads to tumour growth inhibition and decrease in tumour blood vessel density. Penicillamine has entered phase II clinical study for angiosuppressive therapy of glioblastoma. The
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treatment dose starts at 250 mg per day and escalates to 2 g per day by the fifth week. The protocol begins simultaneously with a low copper diet (0.5 mg/day) and a radiation therapy. The reduced copper level probably decreases the antioxidative activity of Cu/Zn-SOD and, as a result, leads to increased sensitivity to oxidative stress. Unfortunately, long-term administration of penicillamine brings several adverse effects, including neurological symptoms (in up to 20% of patients), fever, rash and joint pains (in about half) [14] or even autoagressive diseases [39].
Tetrathiomolybdate Tetrathiomolybdate (TM) is a complex of sulphur and molybdenum, a fast acting copper chelator. TM is administered orally in the form of ammonium salt, which increases its absorption from the digestive tract [40,41]. As a copper chelator TM acts quickly, specifically and, very importantly, seems to be nontoxic. TM forms a tripartite complex with copper and protein and this way prevents absorption of copper from the gastrointestinal tract. Taken orally with meals, TM also complexes with copper in saliva and gastric secretions associated with food intake, thereby preventing copper reabsorption. If TM is given between meals, it is absorbed into the bloodstream, where it binds either free copper or copper loosely bound with serum albumin. Such a complex is no longer available for cellular uptake, possesses no biological activity, and is cleared in bile and urine [32]. Further studies on biological properties of TM revealed the molecular mechanisms of the anticancer effect of TM. Probably, the major mechanism used by TM to inhibit angiogenesis and metastasis is a suppression of the NFkB signalling pathway. TM-treated SUM149 inflammatory breast cells secret lower amounts of VEGF, IL-8, IL-1a and exhibit a less invasive phenotype [42]. A phase I/II clinical trial with TM therapy in a variety of advanced and metastatic cancers was initiated at the University of Michigan, USA in the late 1990s. A total number of 40 patients have been enrolled, 18 of which (representing 11 types of malignancies) have met the criteria of TM treatment, which requires copper depletion to 20% of baseline for at least 2 months. The rest of the patients enrolled had disease progression before the therapeutic level of copper was achieved. Brewer and his colleagues found that 120 mg of TM per day was the most effective of the three doses tested, bringing copper concentrations to the target level without causing sideeffects. All 18 patients achieved disease stabilisation for longer than 2 months, with the average being 9.5 months, two (with metastatic breast cancer and metastatic chondrosarcoma) for about 3 years. Six patients were still on the study in May of 2001. Brewer et al. also
found that even long-term administration of TM leads to toxicity that is limited to easily reversible anaemia, sometimes with leucopoenia [32,43]. Parallel studies in a murine model of head and neck squamous cell carcinoma suggest a potential application of TM in the therapy of this disease in humans. TM treatment led to the reduction of total body copper by 28% from baseline levels in mice and to 79% inhibition of tumour growth as compared to the control, untreated group. In addition, microvessel density was reduced by 50% in the TM-treated group [44,45]. There are also some data suggesting that application of classic cytotoxic chemotherapy (e.g. with doxorubicin) or radiotherapy combined with TM may result in additive effect, with no additional toxicity [46,47]. Studies conducted by Brewer and his colleagues showed that the time required to achieve copper therapeutic levels induced by TM treatment in humans is a minimum 2–3 months. For this reason TM (in a single-drug therapy) may not be effective enough in the treatment of patients with rapidly progressing disease.
Captopril Captopril is an orally administered drug already in widespread use to treat hypertension, acting as an inhibitor of the angiotensin converting enzyme (ACE). Captopril antagonises the angiogenic process via several distinct pathways by [48,49]: * * *
copper chelation, inhibition of metalloproteases activity, converting plasminogen to angiostatin.
Captopril treatment may provoke side effects, including anaemia, rash or low blood pressure.
Trientine This drug (sold as Syprine) is also used to treat patients with Wilson’s disease. It is administered as an alternative agent for the patients with penicillamine intolerance due to severe side effects, such as bonemarrow suppression and induction of auto-immune diseases. In vitro studies revealed that trientine is not cytotoxic for tumour cells, but stimulates them to undergo apoptosis [39]. Its antiangiogenic effect may occur through suppression of interleukin 8 production [50]. Oral administration of trientine (with food or water) led to statistically significant inhibition of tumour growth in mice inoculated subcutaneously with hepatocellular carcinoma cells (HCC). In addition, suppression of tumour blood vessel formation, increased apoptotic index and inhibition of endothelial cell proliferation and migration after treatment with trientine were found [39]. It has been also suggested that trientine might exert
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chemopreventive effect against HCC due to the suppression of angiogenesis [51].
Zinc Zinc is an essential trace element for every living organism. More than 200 enzymes and transcription factors require zinc as a functional component. Therefore, zinc affects major metabolic processes, as well as regulation of the cell cycle and cell division. The first symptom of zinc deficiency is an inhibition of cell growth and proliferation [52]. Since the late 1970s zinc has been used to treat Wilson’s disease. Its ‘anti-copper’ activity is not due to copper chelation. Zinc acts as copper antagonist by inducing synthesis of metallothioneins in the cells of intestinal tract. These proteins are able to bind copper ions with very high affinity and therefore to prevent reabsorption of copper into the bloodstream. These complexes are excreted from an organism with the stool [53]. In addition, antiproliferative and pro-apoptotic effects of zinc on human prostate cancer cell lines (LNCaP and PC-3) have been shown in in vitro studies [54]. Zinc is well tolerated by patients. It is administered orally in the form of acetate or gluconate for better absorption. When taken in doses even three times higher than the daily requirement (15 mg), zinc does not cause side effects. Surplus zinc is efficiently removed from the human body with faecal matter, urine or sweat. Only doses over 2 g per day can provoke gastrointestinal irritation and vomiting [54]. Contrary to TM, zinc is a stable compound, easy to store, but as a copper reducing agent, it acts much slower than other drugs—up to 6 months is required to decrease copper levels down to 20% of baseline values. Nevertheless, zinc may be used as a complementary drug in combination with other agents, for instance with TM [40].
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tumour growth and angiogenesis require a copper optimum. Therefore, we cannot exclude the possibility that supplementation with high doses of copper may cause, similarly to copper deficiency, an inhibitory effect on tumour growth. On the other hand, convincing data suggests that copper depletion causes a significant inhibition of activity of cupric enzymes (up to 50%) without exerting any effect on their levels. This may lead to overproduction of free radicals, abnormalities in the structure of connecting tissue and immune system dysfunction. The early manifestations of copper deficiency include: neutropenia, impairment of iron absorption and mobilisation leading to inadequate erythropoiesis, decrease in interleukin 2 production and inhibition of T cell proliferation [55,56]. In addition, bone abnormalities, including osteoporosis and fractures have been found in infants with copper deficiency [57]. Observations coming from the studies carried out on patients with Menkes’ syndrome imply a possible relation between copper deficiency and neurodegenerative disease. There is some concern that copper deficiency may contribute to the initiation of tumour development. It has been shown that multiple intestinal neoplasia (Min) mice fed copper-deficient diet have a significantly higher incidence and burden of spontaneous intestine tumours as compared to animals fed copper-balanced diet [58]. Moreover, it was also reported that ingestion of copper-deficient diet significantly increased the formation of chemically induced aberrant crypt foci in Fisher-344 rats [59]. There are also data showing a correlation between copper deficiency and atherosclerosis. An increased concentration of total cholesterol and LDL and a reduction of HDL are observed in the subjects fed an experimental diet low in copper [60]. It has been demonstrated that experimental copper deficiency significantly increases the susceptibility of lipoproteins and cardiovascular tissues to lipid peroxidation, thus increasing the risk of cardiovascular disease [61].
Side effects of copper depletion Data concerning clinical effects of copper deficiency in humans are not consistent. The results of clinical studies with TM suggest that even long-term (up to 17 months) copper depletion to 10–20% of the baseline does not induce significant side effects. These are limited to mild anaemia, reversible after adjustment of TM doses. Low concentrations of copper seem to be sufficient for the activity of copper-dependent enzymes, but at the same time are not high enough to sustain angiogenesis. The underlying hypothesis is that the level of copper required for blood vessel formation is higher than that required for essential copper-dependent cellular processes [32]. One may also suspect that
Conclusions Tumours develop in a complex, step-wise manner and many factors can influence this process. One of the crucial stages in tumour development is the initiation of blood vessel formation. Many studies point at the undeniable influence of microelements, particularly copper, on angiogenesis. It has been found that copper metabolism is profoundly altered during the development of human cancer, and copper level in plasma correlates positively with tumour incidence, burden, malignant progression and recurrence in many human and animal cancers. Copper ions seem to play an
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important role in the stimulation of angiogenesis, especially at the earliest stages of this process. Therefore, copper became one of the targets in antiangiogenic cancer treatment. One of the advantages of antiangiogenic therapy based on reduction of copper level is its limited toxicity and a low risk of drug resistance. Several agents of copper-reducing activity have entered clinical trials, e.g. TM, penicillamine, captopril. Based on the experimental data coming from in vitro and in vivo studies, it seems reasonable to use copper chelators or copper-reducing agents in antiangiogenic treatment alone or in combination with other therapeutic approaches, like surgery or classic chemotherapy. Another interesting approach may be the combination of several antiangiogenic drugs with different mechanisms of action. There are, however, certain limitations of using copper-reducing compounds in antitumour treatment. One of them is the long time of drug administration required to produce therapeutic effects. Thus, patients with rapidly progressing tumours may be relatively poor candidates for ‘anti-copper’ therapy as a single modality. In addition, more work has to be done to explore the exact mechanisms of action of copper-reducing agents and, even more important, to define the effects of a long-term copper deficiency.
Acknowledgements We are very grateful to Prof. Jack T. Saari from USDA, ARS, Grand Forks Human Nutrition Research Centre, Grand Forks, USA for help in the preparation of the manuscript.
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of the NFkB signaling cascade. Mol Cancer Res 2003;1:701–6. Brewer GJ, Madawell BR, Kent M, Dick R. Antiangiogenic cancer therapy in mice, humans and dogs with the anticopper drug tetrathiomolybdate, 2001. URL: http:// www.ivis.org Cox C, Teknos TN, Barrios M, Brewer G, Dick RD, Merajver SD. The role of copper suppression as an antiangiogenic strategy in head and neck squamous cell carcinoma. Laryngoscope 2001;111:696–701. Cox C, Merajver SD, Yoo S, Dick RD, Brewer GJ, Lee JS, Teknos TN. Inhibition of the growth of squamous cell carcinoma by tetrathiomolybdate-induced copper suppression in a murine model. Arch Otolaryngol Head Neck Surg 2003;129:781–5. Khan MK, Miller MW, Taylor J, Gill NK, Dick RD, Van Golen K, Brewer GJ, Merajver SD. Radiotherapy and antiangiogenic TM in lung cancer. Neoplasia 2002;4: 164–70. Pan Q, Bao LW, Kleer CG, Brewer GJ, Merajver SD. Antiangiogenic tetrathiomolybdate enhances the efficacy of doxorubicin against breast carcinoma. Mol Cancer Ther 2003;2:617–22. Kotsaki-Kovatsi VP, Koehler-Samouilidis G, Kovatsis A, Rozos G. Fluctuation of zinc, copper, magnesium and calcium concentrations in guinea pig tissues after administration of captopril (SQ 14225). J Trace Elem Med Biol 1997;11:32–6. Mueller SA, Driscoll WJ, Mueller GP. Captopril inhibits peptidylglycine-alpha-hydroxylating monoxygenase: implication for therapeutic effects. Pharmacology 1999;58: 270–80. Moriguchi M, Nakjima T, Kimura H, Watanabe T, Takashima H, Mitsumoto Y, Katagishi T, Okanoue T, Kagawa K. The copper chelator trientine has an antiangiogenic effect against hepatocellular carcinoma, possibly through inhibition of interleukin-8 production. Int J Cancer 2002;102:445–52. Yoshiji H, Kuriyama S, Yoshii J, Ikenaka Y, Noguchi R, Yanase K, Namisaki T, Yamazaki M, Tsujinoue H, Imazu H, Fukui H. The copper-chelating agent, trientine, attenuates liver enzyme-altered preneoplastic lesions in rats by angiogenesis suppression. Oncol Rep 2003;10: 1369–73. MacDonald RS. The role of zinc in growth and cell proliferation. J Nutr 2000;130(5S):1500S–8S. DeJong K. Zinc/copper—will it work? Anticopper/zinc therapy, 2000. URL: http://www.dnai.com/Bdcooley/ cancer/cancer1/cazinco.htm#cazinco Liang JY, Liu YY, Zou J, Franklin RB, Costello LC, Feng P. Inhibitory effect of zinc on human prostatic carcinoma cell growth. Prostate 1999;40:200–7. Cordano A. Clinical manifestations of nutritional copper deficiency in infants and children. Am J Clin Nutr 1998;67(S):1012S–6S. Percival SS. Copper and immunity. Am J Clin Nutr 1998;67:1064S–8S. Heller RM, Kirchner SG, O’Neill Jr JA, Hough Jr AJ, Howard L, Kramer SS, Green HL. Skeletal changes of copper deficiency in infants receiving
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prolonged total parenteral nutrition. J Pediatr 1978;92: 947–9. [58] Davis CD, Newman S. Inadequate dietary copper increases tumorigenesis in the Min mouse. Cancer Lett 2000;159:57–62. [59] Davis CD, Feng Y. Dietary copper, manganese and iron affect the formation of aberrant crypts in colon of rats administered 3,2-dimethyl-4-aminobiphenyl. J Nutr 1999;129:1060–7.
[60] Klevay LM, Inman L, Johnson LK, Lawler M, Mahalko JR, Milne DB, Lukaski HC, Bolonchuk W, Sandstead HH. Increased cholesterol in plasma in a young man during experimental copper depletion. Metabolism 1984;33:1112–8. [61] Rayssiguier Y, Gueux E, Bussiere L, Mazur A. Copper deficiency increases the susceptibility of lipoproteins and tissues to peroxidation in rats. J Nutr 1993;123: 1343–8.
Trace Elements in Medicine and Biology Journal of Trace Elements in Medicine and Biology 18 (2004) 1–8 www.elsevier.de/jtemb
REVIEW
Role of copper in tumour angiogenesis—clinical implications Anna Nasulewicza, Andrzej Mazurb, Adam Opolskia,* a
Laboratory of Tumor Immunology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Ul. R. Weigla 12, 53-114 Wroclaw, Poland b CRNH d’Auvergne, Unite! Maladies M!etaboliques et Micronutriments, INRA, Theix, 63122 St. Gen"es Champanelle, France Received 4 August 2003; accepted 26 February 2004
Abstract The formation of new blood vessels is the initial step in progressive tumour development and metastasis. The first stage in tumour angiogenesis is the activation of endothelial cells. Copper ions stimulate proliferation and migration of endothelial cells. It has been shown that serum copper concentration increases as the cancer disease progresses and correlates with tumour incidence and burden. Copper ions also activate several proangiogenic factors, e.g., vascular endothelial growth factor, basic fibroblast growth factor, tumour necrosis factor alpha and interleukin 1. This review concerns a brief introduction into the basics of tumour blood vessel development as well as the regulatory mechanisms of this process. The role of copper ions in tumour angiogenesis is discussed. The new antiangiogenic therapies based on a reduction of copper levels in tumour microenvironment are reviewed. r 2004 Elsevier GmbH. All rights reserved. Keywords: Tumour angiogenesis; Dietary copper; Antiangiogenic therapy; Copper reduction
Introduction
Basic mechanisms of angiogenesis
The concept of antiangiogenic treatment for solid tumours was pioneered by Judah Folkman in the early 1970s [1]. Since that time many studies have supported the rationale and efficacy of antiangiogenic therapy in animal models. Several compounds interfering with critical steps in blood vessel formation have already reached the clinic. Many others are being tested in preclinical studies. It has been demonstrated that copper is required for the angiogenic process and reduction of the copper level in cancer patients has become one of the newest approaches in antiangiogenic treatment.
Every cell of the human organism requires oxygen and nutrients for its survival and development. The efficient supply of substances is sustained by the dense net of blood vessels. Therefore, the cells are located within 100–200 mm of blood vessels—the diffusion limit for nutrients and inorganic substances. Tumours can develop without limit until they reach the size of 1–2 mm3 [2,3]. Further growth depends on the formation of blood vessels [1]. These new vessels develop either from pre-existing ones or from endothelial cell precursors—angioblasts—derived from bone marrow. This process is called angiogenesis [4]. Newly formed blood vessels supply tumour cells with oxygen and nutrients. They are also the gate which makes tumour development and the spread of metastatic cells possible [5]. In adults, under physiological conditions, angiogenesis takes place very rarely and is restricted to very few
*Corresponding author. E-mail address: [email protected] (A. Opolski). 0946-672X/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2004.02.004
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organs (ovaries, Fallopian tubes, uterus, placenta) [6]. This is due to a tight control at the molecular level and to a balance between synthesis of pro- and antiangiogenic factors. As long as the inhibitory molecules override the action of the stimulators, angiogenesis is ‘‘switched off’’ [3]. The antiangiogenic factors may act by regulating: *
*
*
*
the transcription of angiogenesis activators produced by tumour as well as by endothelial cells (e.g. interferon alpha and beta), the activity of metalloproteases (e.g. tissue inhibitor of metalloproteases— TIMP), the motility and migration of endothelial cells (e.g. thrombospondin 1 and 2), apoptosis of endothelial cells [5,7].
Tumour angiogenesis—the ‘‘angiogenic switch’’ Most commonly, angiogenesis is associated with pathological lesions, e.g. wound healing, rheumatoid arthritis, psoriasis, retinitis and cancer disease. In cancer, angiogenesis starts when the tumour cells gain angiogenic phenotype, at a size of approximately 2 mm3 (106 cells) [2]. Angiogenesis ‘‘switches on’’ as a result of tipping the balance between the synthesis of activators and inhibitors in favour of proangiogenic molecules [3]. Various signals that are able to trigger angiogenesis have been described. These include mechanical stress, inflammatory response and genetic mutations, but the most important factor is metabolic stress, especially the lack of oxygen within the tumour cells [8]. Low pO2 in tumour cell induces the synthesis of several proteins, among which vascular endothelial growth factor (VEGF) plays the most significant role at the early stages of angiogenesis, as it stimulates endothelial cells migration and proliferation [3–5]. VEGF also prevents endothelial cell apoptosis and increases the permeability of blood vessels [9,10]. The activation of endothelial cells is always the first stage in tumour angiogenesis. The following steps include: (i) release of proteolytic enzymes from activated cells, (ii) degradation of the basal membrane surrounding existing vessels, (iii) migration of endothelial cells to intratissue space, (iv) endothelial cell proliferation, (v) formation of the new blood vessel lumen, (vi) formation of a new basal membrane, (vii) fusion of new vessels, (viii) blood flow initiation [11]. Each of these steps may be a potential target in antiangiogenic cancer treatment. The advantages of angiosuppressive therapy are: low risk of drug resistance (a drug acts on endothelial– cancer cell interactions rather than on tumour cells themselves), relatively high availability of a drug to the cells and relatively high specificity of the drug action (tumour microenvironment).
Copper in cancer Copper is an essential trace element with many physiological functions. It affects activity of many enzymes (Cu/Zn-superoxide dismutase (Cu/Zn-SOD), ceruloplasmin, cytochrome oxidase, tyrosinase, dopamine hydroxylase and lysine oxidase), both as a cofactor and as an allosteric component. These enzymes are essential for cellular respiration, defence against free radicals, melanin synthesis, formation of connective tissue and for iron metabolism. In addition, copperdependent transcription factors play an important role in gene expression [12]. A daily dietary intake of 1–2 mg copper seems necessary to enable copper-dependent metabolic processes in adults [13]. The main sources of dietary copper are dried beans, nuts, shellfish and liver. Copper is absorbed readily from the stomach and small intestine. The liver is the major location of stored copper, containing about 10% of the total body content [14]. In blood serum, copper is transported mainly by ceruloplasmin [15] and, in smaller part, by histidine and albumin [16]. Serum copper level is raised in anaemia, normal pregnancy and during administration of oral contraceptives [17]. Its level is also increased during infections due to the high activity of ceruloplasmin—one of the acute phase proteins [18]. Copper metabolism is profoundly altered in neoplastic disease. It has been found that serum copper concentration correlates with tumour incidence and burden, malignant progression, and recurrence in a variety of human cancers: Hodgkin’s lymphoma, sarcoma, leukaemia, cervix, liver, lung, brain and breast cancers [19–26]. Serum ceruloplasmin level increases 4–8 times as a cancer disease progresses. Furthermore, the cellular deposition of copper is also altered in tumour tissues—from cytoplasm in normal tissue to intranuclear and perinuclear zones in tumours [27]. Copper plays an important role in tumour angiogenesis, especially at its early stages. Copper seems to be necessary for endothelial cell activation as it stimulates their proliferation and migration. Several angiogenic factors, e.g. VEGF, basic fibroblast growth factor (bFGF), tumour necrosis factor alpha (TNF-a) and interleukin (IL) 1 have been found to be copperactivated. Activation factors bind to endothelial cells, ‘‘switch’’ them from G0 into G1 phase and force proliferation. Brem and his co-workers have shown that copper reduction by copper deficient diet or/and penicillamine (copper-chelating molecule) blocks angiogenesis by ‘‘switching’’ endothelial cells back into G0 phase or apoptosis and by down regulating the angiogenic activity of VEGF, bFGF, TNF-a, IL-1 and probably IL-6 and IL-8 as well [28,29]. Probably copper influences angiogenesis in a few ways. Not only does it activate endogenous angiogenic
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factors, but it also binds to several proteins (heparin, ceruloplasmin) which therefore gain angiogenic activity that manifests by stimulation of endothelial cells [15,30]. It has also been shown that additional copper augments, by over four times, the angiogenin binding to calf pulmonary artery endothelial cells [31].
Copper reduction as a therapeutic approach The steps required for successful angiogenesis at the primary and metastatic sites are diverse and they depend on the imbalance between angiogenesis activators and inhibitors. The relative importance of the different angiogenesis-modulating molecules often varies with respect to tissue type. Therefore, it would be very desirable to develop an antiangiogenic therapy that would affect multiple activators of angiogenesis in order to be applicable to various types of tumours [32]. Compounds of copper-reducing activity seem to meet (at least partly) this criterion, since copper is a required cofactor of several key angiogenic factors. In vitro and in vivo studies conducted during the last few years have confirmed the expediency as well as the efficacy of copper-reducing and copper-chelating agents in antiangiogenic treatment. In a series of experiments, Gullino and co-workers showed that application of a low copper diet and penicillamine therapy results in prostaglandin E-stimulated angiogenesis suppression [33]. Experiments conducted by Brem et al. showed that such a treatment leads to tumour growth retardation and angiogenesis inhibition in a rabbit brain tumour model [34]. Several copper-reducing compounds that may be effective in antiangiogenic therapy, including penicillamine, tetrathiomolybdate (TM), captopril, tachpirydine, zinc and trientine, have entered pre- and clinical studies. These agents have been widely used to treat other diseases, e.g. Wilson’s disease (genetic disorder of copper transport resulting in abnormal copper accumulation and toxicity), rheumatoid arthritis or hypertension. Copper-reducing drugs offer advantages to cancer therapy for several reasons, including: *
* *
* *
effectiveness in treatment of various types of tumours, low toxicity risk, possibility of combination with almost every other antitumour treatment strategy, low cost of therapy, may be of a special interest as low risk strategy in a situation of ‘‘Watch and Wait’’ (W&W), when medical procedures are limited to monitoring of the tumour progression before taking a therapeutic decision.
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A serious limitation of using copper-reducing compounds in antitumour treatment is the long time of drug administration required. To provide therapeutic effects, copper level in the patient’s serum needs to be reduced to 20% of the physiological concentration, which takes (dependent on the drug used) from several weeks to 6 months. There is a considerable lag between the initiation of therapy and the reduction of copper level due to copper release from the liver. Effective inhibition of angiogenesis may also lead to a local necrosis, lysis of tumour cells and release of copper into the tumour microenvironment [32]. Moreover, tumour cells are thought to sequester copper [35]. Hence, it is logical that a sufficiently long time is required to deplete copper from the tumour microenvironment. Thus, patients with rapidly progressing tumours may be relatively poor candidates for this antitumour therapy as a single modality.
Copper-reducing agents in experimental and clinical studies Penicillamine d-Penicillamine (sold as Cuprimine or Depen) is a drug commonly used to treat Wilson’s disease and rheumatoid arthritis. It binds copper with sulphydryl groups and this complex is subsequently removed from the organism via the urine. In the presence of copper ions, penicillamine produces H2O2 [36]. This may lead to generation of free radicals, which can inhibit endothelial cell proliferation, as these cells are highly sensitive to reactive oxygen species (ROS) [37]. In addition to copper chelation, penicillamine has multiple functions that may account for its antiangiogenic activity. It is a dose-dependent inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). It inhibits collagen synthesis, gelatinase B activity and both proliferation and migration of endothelial cells. Finally, penicillamine stabilises the activity of TIMP and converts plasminogen to angiostatin, a potent antiangiogenic compound. In a single-drug therapy penicillamine exerts antitumour activity that is enhanced when copper level is strongly reduced [34]. Studies performed on rats inoculated orthotopically [34] or subcutaneously [38] with gliosarcoma cells, as well as experiments conducted in a mouse hepatocellular carcinoma model [39], confirmed the antitumour and antiangiogenic activity of penicillamine when combined with a low copper diet. This combination leads to tumour growth inhibition and decrease in tumour blood vessel density. Penicillamine has entered phase II clinical study for angiosuppressive therapy of glioblastoma. The
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treatment dose starts at 250 mg per day and escalates to 2 g per day by the fifth week. The protocol begins simultaneously with a low copper diet (0.5 mg/day) and a radiation therapy. The reduced copper level probably decreases the antioxidative activity of Cu/Zn-SOD and, as a result, leads to increased sensitivity to oxidative stress. Unfortunately, long-term administration of penicillamine brings several adverse effects, including neurological symptoms (in up to 20% of patients), fever, rash and joint pains (in about half) [14] or even autoagressive diseases [39].
Tetrathiomolybdate Tetrathiomolybdate (TM) is a complex of sulphur and molybdenum, a fast acting copper chelator. TM is administered orally in the form of ammonium salt, which increases its absorption from the digestive tract [40,41]. As a copper chelator TM acts quickly, specifically and, very importantly, seems to be nontoxic. TM forms a tripartite complex with copper and protein and this way prevents absorption of copper from the gastrointestinal tract. Taken orally with meals, TM also complexes with copper in saliva and gastric secretions associated with food intake, thereby preventing copper reabsorption. If TM is given between meals, it is absorbed into the bloodstream, where it binds either free copper or copper loosely bound with serum albumin. Such a complex is no longer available for cellular uptake, possesses no biological activity, and is cleared in bile and urine [32]. Further studies on biological properties of TM revealed the molecular mechanisms of the anticancer effect of TM. Probably, the major mechanism used by TM to inhibit angiogenesis and metastasis is a suppression of the NFkB signalling pathway. TM-treated SUM149 inflammatory breast cells secret lower amounts of VEGF, IL-8, IL-1a and exhibit a less invasive phenotype [42]. A phase I/II clinical trial with TM therapy in a variety of advanced and metastatic cancers was initiated at the University of Michigan, USA in the late 1990s. A total number of 40 patients have been enrolled, 18 of which (representing 11 types of malignancies) have met the criteria of TM treatment, which requires copper depletion to 20% of baseline for at least 2 months. The rest of the patients enrolled had disease progression before the therapeutic level of copper was achieved. Brewer and his colleagues found that 120 mg of TM per day was the most effective of the three doses tested, bringing copper concentrations to the target level without causing sideeffects. All 18 patients achieved disease stabilisation for longer than 2 months, with the average being 9.5 months, two (with metastatic breast cancer and metastatic chondrosarcoma) for about 3 years. Six patients were still on the study in May of 2001. Brewer et al. also
found that even long-term administration of TM leads to toxicity that is limited to easily reversible anaemia, sometimes with leucopoenia [32,43]. Parallel studies in a murine model of head and neck squamous cell carcinoma suggest a potential application of TM in the therapy of this disease in humans. TM treatment led to the reduction of total body copper by 28% from baseline levels in mice and to 79% inhibition of tumour growth as compared to the control, untreated group. In addition, microvessel density was reduced by 50% in the TM-treated group [44,45]. There are also some data suggesting that application of classic cytotoxic chemotherapy (e.g. with doxorubicin) or radiotherapy combined with TM may result in additive effect, with no additional toxicity [46,47]. Studies conducted by Brewer and his colleagues showed that the time required to achieve copper therapeutic levels induced by TM treatment in humans is a minimum 2–3 months. For this reason TM (in a single-drug therapy) may not be effective enough in the treatment of patients with rapidly progressing disease.
Captopril Captopril is an orally administered drug already in widespread use to treat hypertension, acting as an inhibitor of the angiotensin converting enzyme (ACE). Captopril antagonises the angiogenic process via several distinct pathways by [48,49]: * * *
copper chelation, inhibition of metalloproteases activity, converting plasminogen to angiostatin.
Captopril treatment may provoke side effects, including anaemia, rash or low blood pressure.
Trientine This drug (sold as Syprine) is also used to treat patients with Wilson’s disease. It is administered as an alternative agent for the patients with penicillamine intolerance due to severe side effects, such as bonemarrow suppression and induction of auto-immune diseases. In vitro studies revealed that trientine is not cytotoxic for tumour cells, but stimulates them to undergo apoptosis [39]. Its antiangiogenic effect may occur through suppression of interleukin 8 production [50]. Oral administration of trientine (with food or water) led to statistically significant inhibition of tumour growth in mice inoculated subcutaneously with hepatocellular carcinoma cells (HCC). In addition, suppression of tumour blood vessel formation, increased apoptotic index and inhibition of endothelial cell proliferation and migration after treatment with trientine were found [39]. It has been also suggested that trientine might exert
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chemopreventive effect against HCC due to the suppression of angiogenesis [51].
Zinc Zinc is an essential trace element for every living organism. More than 200 enzymes and transcription factors require zinc as a functional component. Therefore, zinc affects major metabolic processes, as well as regulation of the cell cycle and cell division. The first symptom of zinc deficiency is an inhibition of cell growth and proliferation [52]. Since the late 1970s zinc has been used to treat Wilson’s disease. Its ‘anti-copper’ activity is not due to copper chelation. Zinc acts as copper antagonist by inducing synthesis of metallothioneins in the cells of intestinal tract. These proteins are able to bind copper ions with very high affinity and therefore to prevent reabsorption of copper into the bloodstream. These complexes are excreted from an organism with the stool [53]. In addition, antiproliferative and pro-apoptotic effects of zinc on human prostate cancer cell lines (LNCaP and PC-3) have been shown in in vitro studies [54]. Zinc is well tolerated by patients. It is administered orally in the form of acetate or gluconate for better absorption. When taken in doses even three times higher than the daily requirement (15 mg), zinc does not cause side effects. Surplus zinc is efficiently removed from the human body with faecal matter, urine or sweat. Only doses over 2 g per day can provoke gastrointestinal irritation and vomiting [54]. Contrary to TM, zinc is a stable compound, easy to store, but as a copper reducing agent, it acts much slower than other drugs—up to 6 months is required to decrease copper levels down to 20% of baseline values. Nevertheless, zinc may be used as a complementary drug in combination with other agents, for instance with TM [40].
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tumour growth and angiogenesis require a copper optimum. Therefore, we cannot exclude the possibility that supplementation with high doses of copper may cause, similarly to copper deficiency, an inhibitory effect on tumour growth. On the other hand, convincing data suggests that copper depletion causes a significant inhibition of activity of cupric enzymes (up to 50%) without exerting any effect on their levels. This may lead to overproduction of free radicals, abnormalities in the structure of connecting tissue and immune system dysfunction. The early manifestations of copper deficiency include: neutropenia, impairment of iron absorption and mobilisation leading to inadequate erythropoiesis, decrease in interleukin 2 production and inhibition of T cell proliferation [55,56]. In addition, bone abnormalities, including osteoporosis and fractures have been found in infants with copper deficiency [57]. Observations coming from the studies carried out on patients with Menkes’ syndrome imply a possible relation between copper deficiency and neurodegenerative disease. There is some concern that copper deficiency may contribute to the initiation of tumour development. It has been shown that multiple intestinal neoplasia (Min) mice fed copper-deficient diet have a significantly higher incidence and burden of spontaneous intestine tumours as compared to animals fed copper-balanced diet [58]. Moreover, it was also reported that ingestion of copper-deficient diet significantly increased the formation of chemically induced aberrant crypt foci in Fisher-344 rats [59]. There are also data showing a correlation between copper deficiency and atherosclerosis. An increased concentration of total cholesterol and LDL and a reduction of HDL are observed in the subjects fed an experimental diet low in copper [60]. It has been demonstrated that experimental copper deficiency significantly increases the susceptibility of lipoproteins and cardiovascular tissues to lipid peroxidation, thus increasing the risk of cardiovascular disease [61].
Side effects of copper depletion Data concerning clinical effects of copper deficiency in humans are not consistent. The results of clinical studies with TM suggest that even long-term (up to 17 months) copper depletion to 10–20% of the baseline does not induce significant side effects. These are limited to mild anaemia, reversible after adjustment of TM doses. Low concentrations of copper seem to be sufficient for the activity of copper-dependent enzymes, but at the same time are not high enough to sustain angiogenesis. The underlying hypothesis is that the level of copper required for blood vessel formation is higher than that required for essential copper-dependent cellular processes [32]. One may also suspect that
Conclusions Tumours develop in a complex, step-wise manner and many factors can influence this process. One of the crucial stages in tumour development is the initiation of blood vessel formation. Many studies point at the undeniable influence of microelements, particularly copper, on angiogenesis. It has been found that copper metabolism is profoundly altered during the development of human cancer, and copper level in plasma correlates positively with tumour incidence, burden, malignant progression and recurrence in many human and animal cancers. Copper ions seem to play an
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important role in the stimulation of angiogenesis, especially at the earliest stages of this process. Therefore, copper became one of the targets in antiangiogenic cancer treatment. One of the advantages of antiangiogenic therapy based on reduction of copper level is its limited toxicity and a low risk of drug resistance. Several agents of copper-reducing activity have entered clinical trials, e.g. TM, penicillamine, captopril. Based on the experimental data coming from in vitro and in vivo studies, it seems reasonable to use copper chelators or copper-reducing agents in antiangiogenic treatment alone or in combination with other therapeutic approaches, like surgery or classic chemotherapy. Another interesting approach may be the combination of several antiangiogenic drugs with different mechanisms of action. There are, however, certain limitations of using copper-reducing compounds in antitumour treatment. One of them is the long time of drug administration required to produce therapeutic effects. Thus, patients with rapidly progressing tumours may be relatively poor candidates for ‘anti-copper’ therapy as a single modality. In addition, more work has to be done to explore the exact mechanisms of action of copper-reducing agents and, even more important, to define the effects of a long-term copper deficiency.
Acknowledgements We are very grateful to Prof. Jack T. Saari from USDA, ARS, Grand Forks Human Nutrition Research Centre, Grand Forks, USA for help in the preparation of the manuscript.
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