A key molecule in obesity is leptin, a 16 kDa peptide hormone predominantly produced by white adipose tissue 10. Circulating leptin is actively transported through the blood-brain barrier and acts on the hypothalamic satiety center to decrease food intake. The main function of leptin in the human body is the regulation of energy expenditure and control of appetite. Indeed, lack of leptin in mice with a mutation in the gene encoding leptin, or absence of functional leptin receptor (db/db mice) results in obesity and many associated metabolic complications such as insulin resistance 11. Serum level of leptin reflects the amount of energy stored in the adipose tissue and is in proportion to body fat mass 12, i.e. increased in obese and decreased after several months of pronounced weight loss 13.

Leptin acts via transmembrane receptors (OB-R), which belong to the class I cytokine receptor family, such as the receptors of interleukin-2 (IL-2), IL-3, IL-4, IL-6, IL-11, IL-12, granulocyte colony-stimulating factor (G-CSF) or leukemia inhibitory factor (LIF). The OB-R has at least six isoforms, termed OB-R(a-f), which are generated primarily by alternative splicing of the ob gene. They all share an identical extracellular ligand-binding domain but differ in their C-terminal regions 14. Two main leptin receptor isoforms dominate: the short leptin receptor isoform (OB-Ra) and the long leptin receptor isoform (OB-Rb). OB-Rb contains the full-length intracellular domain and is believed to be the main leptin signaling receptor. OB-Rb has full signaling capabilities and is able to activate the JAK/STAT pathway, the major pathway used by leptin to exert its effects 15. High levels of this isoform exist in the hypothalamus, but is represented on many other cell types as well, such as adipocytes, osteoclasts, endothelial cells, lung and kidney cells, mononuclear blood cells, muscle, endometrial and liver cells 14. OB-Ra and OB-Rc are highly expressed in choroids plexus and microvessels, where they may play a role in leptin uptake or efflux from the cerebrospinal fluid as well as in receptor mediated transport of leptin across the blood-brain barrier 16; OB-Re, which lacks the intracellular domain, may encode a soluble receptor. Consistent with leptin's role in controlling appetite and energy metabolism, OB-Rs have been found in the hypothalamus and adjacent brain regions. However, the almost universal distribution of OB-Ra and OB-Rb reflects the multiplicity of biological effects in extraneural tissues, providing evidence for the extreme functional pleiotropy of leptin 12.

Leptin plays important roles in both adaptive and innate immunity, and humans lacking leptin function exhibit impaired immunity. Accordingly, the leptin receptor is found to be expressed on a variety of immune cells 17. With regard to innate immunity, leptin is a direct potent chemoattractant for monocytes and macrophages, whereas the presence of full-length receptors on migrating cells is required. In addition, leptin increases the recruitment of blood monocytes via adipose-tissue derived endothelial cells (EC) by stimulating the upregulation of EC adhesion molecules necessary for the diapedesis of the monocytes 18. Acting on monocytes leptin induces the release of other cytokines such as tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6) as well as CC-chemokine ligand 2 (CCL2) and vascular endothelial growth factor (VEGF) 19. Leptin is also able to stimulate the chemokinesis of eosinophils 20, and the chemotaxis of neutrophils 2122. However, whereas leptin alone induces the migration of neutrophils and exerts by itself a chemoattractive effect comparable to that of well-known formyl-methionyl-leucyl-phenylalanin 21, neutrophil locomotion in response to classical chemoattractants is inhibited by simultaneous treatment with leptin 22. Most of these effects are mediated through the OB-Rb, which is expressed mainly by endothelial cells and various leukocytes. In adaptive immunity, leptin enhances T cell proliferation and Th 1 proinflammatory cytokine production in vitro, whereas nothing is known about the effect of this adipocytokine on the migratory behaviour of T cells. Acting on dendritic cells leptin activates them, licenses them for Th 1 priming, and increases migratory performance 23.

Epidemiological studies have shown that obesity is a risk factor for postmenopausal breast cancer, cancers of the endometrium, colon and kidney, and malignant adenomas of the oesophagus 3. Obese individuals have approximately a 1.5-3.5-fold increased risk of developing these cancers compared with normal-weight individuals, and it is estimated that 15 to 45% of these cancers are attributable to overweight (BMI 25.0-29.9 kg/m2) and obesity in Europe 24. Moreover, in high income countries the attributable fraction of all cancers due to obesity was estimated as 3% 25.

Functional leptin receptors are found to be expressed on diverse cancer cells derived from different tissues such as breast, colon or prostate 2627282930. The breast cancer cell lines HTB-26 and ZR75-1 27, the prostate cancer cell lines DU145 and PC-3 30, and various colon carcinoma cell lines such as LS174T and HM7 29 as well as SW480, SW620 and HCT116 26 all express OB-Ra and OB-Rb. In breast cancer cell lines and in human primary breast carcinoma leptin receptor has been demonstrated to occur in combination with leptin. Therein, leptin is able to induce the growth of these cells via different pathways, can mediate angiogenesis by inducing the expression of VEGF, and promotes invasion and migration by transactivation of the epidermal growth factor receptor (EGFR) 15. A bidirectional crosstalk between leptin and insulin-like growth factor-I (IGF-I) signaling was also shown to stimulate invasion and migration of breast cancer cells 31. Thereby, IGF-I induced phosphorylation of Ob-Rb and leptin induced phosphorylation of IGF-I receptor, whereas cotreatment induced synergistic phosphorylation and association of Ob-Rb and IGF-IR along with activation of downstream effectors, Akt and extracellular signal-regulated kinase; in parallel this cotreatment synergistically transactivated EGFR. In 92% of breast carcinomas examined leptin was found to be overexpressed, but in none of the cases of normal breast epithelium. Remarkably, distant metastasis was present in 34% of OB-R-positive tumors with leptin overexpression, but in none of the cases where the tumors lacked OB-R expression or leptin overexpression 32. A multitude of other studies have demonstrated that leptin mediates a significant increase of proliferation in breast, colon, esophagal and prostate cancer cells, too 32730. For example, Somasundar et al. 30 showed that leptin induced in vitro proliferation and inhibited apoptosis in DU145 and PC-3 prostate cancer cell lines. In a murine model of preneoplastic Apc(Min/+) colon epithelial cells leptin treatment was demonstrated to promote cell proliferation by an autocrine IL-6 production and trans-IL-6 signaling 33.

Moreover, leptin promotes the migration and invasion of cells derived from glioma 34, chondrosarcoma 35, colon carcinoma 2936, hepatocellular and endometrial carcinoma cells 3738, as well as prostate cancer 28. In DU145 and PC-3 prostate cancer leptin significantly enhanced cell migration and induced expression of VEGF, transforming growth factor-beta1 (TGF-beta1), and basic fibroblast growth factor (bFGF), and thus overall likely contributes to the progression of prostate cancer 28. Interestingly, Deo et al 39 observed differential effects of leptin on the invasive potential of prostate carcinoma cells depending on their androgen sensitivity. Androgen-sensitive LNCaP cells showed a significant increase in cellular proliferation upon treatment with leptin, whereas no effect was observable on androgen-insensitive PC-3 and DU145 cells. In contrast, leptin caused a significant dose-dependent decrease in migration and invasion solely of PC-3 and DU145 prostate carcinoma cell lines. These results are further supported by our own experiments demonstrating divergent effects of leptin on the proliferation and migration of carcinoma cells derived from different tissues (Table 1). Whereas leptin enhanced the proliferation of various breast carcinoma cell lines, including MDA-MB-468 and MDA-MB-231, it did not have any impact on the migratory activity of these cells. However, in various human colon carcinoma cells leptin significantly stimulated the locomotory behaviour of the cells 2629. These contradictory leptin effects on the migration and proliferation, especially of prostate and breast carcinoma cells, might be ascribable to the hormone-sensitivity of the cells. This conclusion is confirmed by results which demonstrate an influence of leptin on breast cancer development in relation to estrogen receptor status 40, and illuminate the growth-inducing effect of leptin in estrogen receptor-positive breast cancer cells by its stimulation of aromatase expression and the accompanied increase of estrogen levels through the aromatization of androgens 15. In summary, all these data clearly support a direct functional role of leptin in processes related to cancer initiation and/or progression by promoting the migration and proliferation of carcinoma cells, thus resulting in metastatic development.

Leptin acts through its receptor OB-R, which is a member of the cytokine receptor family. Accordingly, leptin signaling is thought to be transmitted predominantly by the intracellular Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway with the activation of STAT-3 and extracellular signal-regulated kinase (ERK)1/2 3. The engagement of OB-R by leptin leads to JAK-2 phosphorylation, which can then recruit STAT-3 tyrosine phosphorylation, finally leading to a nuclear translocation and stimulation of transcription. In addition, the leptin receptor is also known to intracellularly activate MAPK pathway after leptin binding 41. In human endometrial and hepatocellular carcinoma cells leptin was shown to promote proliferation and invasion by rapidly stimulating the JAK/STAT-pathway and inducing the phosphorylation of ERK and AKT, thus activating these key signal transduction pathways associated with cell growth and cell migration 373842. Recent studies have shown that the ERK pathway is an attractive target for anticancer therapies due to its central role in the regulation of proliferation, invasiveness, and survival of tumors 43. AKT provides a survival signal protecting cells from apoptosis induced by various stresses by multiple mechanisms, such as the phosphorylation of Bad, glycogen synthase-3, and caspase-9 44. The PI3K/AKT pathway is frequently altered in human cancers, and an activation of AKT is correlated e.g with invasive and metastasizing breast tumors 45. In leptin-treated human chondrosarcoma, hepatocellular and endometrial carcinoma cells AKT phosphorylation was found to be increased, and inhibition of PI3K with specific inhibitors abolished leptin-induced proliferation, migration and invasion 35373842. Other studies explain the leptin stimulating effect on migration and invasion of various carcinoma cells through the increased expression of various matrix metalloproteinase or integrins via an activation of the nuclear factor κB (NFκB) pathway 3435. Moreover, in colon carcinoma cells leptin-induced promotion of motility and invasion was mediated by an activation of PI3K and src kinase pathways resulting in a stimulation of the Rho GTPases rac1, cdc42 and rhoA, proteins known to regulate cell migration by affecting the reorganization of the actin cytoskeleton 29.

Adiponectin is a 30 kDa protein secreted exclusively by white adipocytes. Adiponectin circulates as several multimeric species, including a high molecular weight form thought to be the most clinically relevant. Serum levels of adiponectin are markedly decreased in individuals with visceral obesity and states of insulin resistance, such as type 2 diabetes mellitus and artherosclerosis. In contrast to leptin, adiponectin seems to have several beneficial and protective effects. Although its role has not been definitely established, adiponectin was shown to have anti-inflammatory, vasculoprotective, and anti-diabetic effects. Adiponectin is highly abundant in the circulation, with plasma concentrations in healthy humans around 3-30 μg/ml 46, and has a broad spectrum of biological activities. Adiponectin increases the body's sensitivity to insulin including stimulation of glucose uptake in skeletal muscle and suppression of glucose production in liver, and affects the lipid metabolism by decreasing tissue fatty acid content and serum lipids. Interestingly, levels of adiponectin in obese individuals have shown to be decreased even though it comes primarily from adipose tissue. Adiponectin acts through its two receptors, AdipoR1 and AdipoR2, which have unique distributions and affinities for the different forms of circulating adiponectin. AdipoR1 is expressed widely in various tissues, including breast tissue, with the highest level of expression in skeletal muscle, while AdipoR2 is most abundantly expressed in the liver 46.

With regard to the immune system, Pang et al. 47 investigated that AdipoR1 is present approximately on 1% of T cells, 93% of monocytes, 47% of B cells, and 21% of NK cells, and the distribution of AdipoR2 was found to be similar. Acting on adaptive immunity, adiponectin inhibits T cell activation and proliferation, although data regarding adiponectin effects on adaptive immune response are relatively spare. In adiponectin-deficient mice, absence of adiponectin was associated with a 2-fold increase in leukocyte-rolling and a 5-fold increase in leukocyte adhesion in the microcirculation 48. Here, we show for the first time that adiponectin induced the migratory activity of human neutrophil granulocytes within a three-dimensional collagen matrix (from 13% locomoting cells in the control to 39% locomoting cells after stimulation with 1 μg/ml adiponectin; Fig. 1A), whereas it did not have any effect on the migratory activity of human CD8 T lymphocytes. This contradictory results of adiponectin on neutrophil granulocytes could be a consequence of the experimental setup. Whereas in the adiponectin-deficient mice the behaviour of leukocytes during the extravasation process was examined, we investigated in our study the migration of neutrophils, a process subsequently following extravasation. This is of major importance for the interpretation of the results, because participating molecules and molecular mechanisms are different in this course of action. Whereas the extravasation of neutrophils is primarily regulated by the interaction of leukocytes with the endothelium via various adhesion molecules such as selectins or N-cadherin 49, the migratory process is mediated e.g. by the interaction of the cells with the surrounding extracellular matrix via integrins or stimuli-induced rearrangement of the cytoskeleton 50. In addition, adiponectin inhibits NF-κB activation in endothelial cells and interferes with the function of macrophages; treatment of cultured macrophages with adiponectin markedly inhibit their phagocytotic activity and their production of TNFα in response to lipopolysaccharide stimulation 51. It suppresses IL-2 enhanced cytotoxic activity of natural killer (NK) cells without affecting basal NK cell cytotoxicity 52. The exact roles of adiponectin in inflammation and immunity, and especially the underlying molecular mechanism, remain to be defined.

Low serum levels of adiponectin are associated with several metabolic diseases, including obesity and insulin resistance in type-2 diabetes 53. Moreover, evidence from several studies indicates that serum levels of adiponectin in vivo are inversely associated with the risk for multiple cancers, including colon in men 54, prostate 55 as well as breast and endometrium 5657. For example, women with a high body mass index and low plasma adiponectin have a risk of endometrial cancer that is 6.5-fold that in women with normal body mass index and higher adiponectin concentrations 58. Interestingly, the inverse association of adiponectin with breast cancer risk was found solely among postmenopausal women, and not among premenopausal women 56. In postmenopausal women obesity has been shown to increase rates of breast cancer consistently by 30-50% 59. Recently, elevated serum adiponectin levels were demonstrated to be independently associated with childhood non-hodgkin's lymphoma (NHL) and poor prognosis. Moreover NHL specimens expressed the adiponectin receptors, suggesting that adiponectin may represent not only a potential clinically significant diagnostic and prognostic marker but also a molecule that may be implicated in NHL pathogenesis 60. Adiponectin receptors are primarily found to be expressed solely in the malignant and not in the normal control tissue, and the expression can differ by disease stage. For example, in cancerous lung tissue expression of adiponectin receptors was apparent only in the cancerous lung tissue (64.2% AdipoR1 and 61.9% AdipoR2 in cancerous vs. 0% among noncancerous tissue). Specifically, AdipoR1 was expressed in all disease types, but no difference was noted with disease stage, whereas AdipoR2 was mainly expressed in the non-small cell carcinomas and more prominently in the advanced disease stage (80%) 61. Thus obese individuals with low levels of adiponectin could be at higher risk of developing tumors.

Adiponectin may influence cancer risk through its well recognized effects on insulin resistance, but it is also plausible that it acts on tumor cells directly, because several tumor cell lines express AdipoR1 and AdipoR2. Hence, adiponectin receptors are found to be expressed on breast (6263, Table 1), colon 64, and prostate 55 carcinoma cells. Furthermore, adiponectin has been shown to suppress tumor growth in mice, most likely due to inhibition of neovascularization through suppression of endothelial cell proliferation, migration and survival 65. Several recent studies found that adiponectin decreased cell growth in various breast cancer cell lines such as MCF-7, MDA-MB-231, SK-BR-3 6263, inhibited proliferation in prostate cancer cell lines 66, and SW480 colon carcinoma cells (Table 1). In contrast with findings in breast and prostate cancer cell lines, adiponectin stimulated colonic epithelial cell proliferation 67. This is in accordance with the results in our study, where we observed an enhanced proliferation of various breast carcinoma cell lines, including MDA-MB-468 and MDA-MB-231 upon treatment with adiponectin (Table 1). Also, study of the effects of adiponectin on the apoptosis of cancer cells in vitro, which may well differ from the in vivo condition, remains inconclusive. In some studies 6869 apoptosis was increased upon incubation with adiponectin, but this was not observed in other studies 6266. These divergent results might be caused by variations in experimental conditions, because especially in vitro proliferation studies have limitations; one is the fact that all these studies were performed under serum-free conditions, which may favor an inhibition of cell growth, whereas e.g. in our proliferation assay cells were grown with serum.

To date only two studies have investigated the physiological relevance of the above data by examining the effects of in vivo adiponectin administration on tumor growth 6568. Animal studies showed that adiponectin supplement therapy could inhibit the development of breast tumors in nude mice, and mice treated with adiponectin displayed a much lower degree of breast tumor metastasis 68. Although it was not possible to discriminate whether the decreased metastasis was caused by the direct suppressive effects of adiponectin on cell migration and invasion or is simply secondary to the reduced sizes of primary tumors 68, these data clearly support a role of adiponectin in breast cancer progression.

With regard to cell migration we observed differences for the impact of adiponectin on the migratory behaviour of tumor cells. Herein, we show for the first time that adiponectin significantly stimulated the migration of human SW480 colon carcinoma cells, whereas it did not have any effect on the locomotion of various breast carcinoma cells (Table 1). In accordance with the pro-migratory effect on colon carcinoma cells demonstrated herein, very recently two studies report that adiponectin increased the motility of human prostate cancer cells 70 and chondrosarcoma cells 71, too. Although adiponectin research with regard to metastasis is at the beginning, current results on its effects on the migratory and proliferative activity of carcinoma cells indicate a distinct role of this adipocytokine in cancer pathogenesis.