Roberto Benelli

Tumor gelatinases and invasion inhibited by the green tea flavanol epigallocatechin‐3‐gallate

Given the association of consumption of green tea with prevention of cancer development, metastasis, and angiogenesis, the effect of the main flavanol present, epigallocatechin‐3‐gallate (EGCG), on two gelatinases most frequently overexpressed in cancer and angiogenesis (MMP‐2 and MMP‐9) and on tumor cell invasion and chemotaxis were examined.

Neutrophil restraint by green tea: Inhibition of inflammation, associated angiogenesis, and pulmonary fibrosis

Neutrophils play an essential role in host defense and inflammation, but the latter may trigger and sustain the pathogenesis of a range of acute and chronic diseases. Green tea has been claimed to exert anti-inflammatory properties through unknown molecular mechanisms. We have previously shown that the most abundant catechin of green tea, (−)epigallocatechin-3-gallate (EGCG), strongly inhibits neutrophil elastase. Here we show that 1) micromolar EGCG represses reactive oxygen species activity and inhibits apoptosis of activated neutrophils, and 2) dramatically inhibits chemokine-induced neutrophil chemotaxis in vitro; 3) both oral EGCG and green tea extract block neutrophil-mediated angiogenesis in vivo in an inflammatory angiogenesis model, and 4) oral administration of green tea extract enhances resolution in a pulmonary inflammation model, significantly reducing consequent fibrosis. These results provide molecular and cellular insights into the claimed beneficial properties of green tea and indicate that EGCG is a potent anti-inflammatory compound with therapeutic potential.

Tumors and inflammatory infiltrates: Friends or foes?

The recognition of a role for inflammation in the natural history of a tumor has a long record, stretching from the mid-19th century. From the times of Virkow, who postulated that cancer originates from inflamed tissues, to Metchnikoff and many others, this field has continued to excite (and divide) the scientific community. The question as to whether the inflammatory infiltrate helps or hinders tumors is still open. In a sense, modern molecular biology has, if anything, worsened this dualism, and the literature on this issue shows a plethora of conflicting reports. We would like to provide another contribution to this topic, which was the subject of a recent brilliant review (Balkwill F and Mantovani A. Lancet 2001; 357: 539–45 [1]), by focussing more specifically to the relation between inflammation and tumor invasion and how this could drive rational therapeutic approaches.

Tumor invasion: molecular shears blunted by green tea.

To the editor–The recent press, both popular and scientific, has given wide coverage of the beneficial properties of green tea, most commonly used in Asian countries. Consumption has been associated with prevention of cancer development and metastasis. The main flavonol of green tea, epigallocatechin-3-gallate (EGCG), inhibits urokinase, one of the hydrolases implicated in tumor invasion. Moreover, green tea consumption by mice significantly limits angiogenesis, crucial for the growth of all solid tumors.

Neutrophils as a key cellular target for angiostatin: implications for regulation of angiogenesis and inflammation

Angiostatin effectively blocks tumor angiogenesis through still poorly understood mechanisms. Given the close association between immune and vascular regulation, we investigated the effects of angiostatin on angiogenesis‐associated leukocytes. Angiostatin inhibited the migration of monocytes and, even more markedly, neutrophils. Angiostatin blocked chemotaxis of neutrophils to CXCR2 chemokine receptor agonists (IL‐8, MIP‐2, and GROα), formyl‐Met‐Leu‐Phe (fMLP), and 12‐O‐tetradecanoylphorbol 13‐acetate, and repressed fMLP‐induced mitochondrial activity. Two different angiostatin forms (kringles 1–4 and 1–3) were effective, whereas whole plasminogen had no effect. IL‐8, MIP‐2, and GROα induced intense angiogenic reactions in vivo, but no angiogenic response to these factors was observed in neutropenic mice, demonstrating an essential role for neutrophils. Angiostatin potently inhibited chemokine‐induced angiogenesis in vivo, and consistent with in vitro observations, both angiostatin forms were active and whole plasminogen had little effect. Angiostatin inhibition of angiogenesis in vivo was accompanied by a striking reduction in the number of recruited leukocytes. In vivo, the inflammatory agent lipopolysaccharide also induced extensive leukocyte infiltration and angiogenesis that were blocked by angiostatin. Neutrophils expressed mRNAs for ATP synthase and angiomotin, two known angiostatin receptors. These data show that angiostatin directly inhibits neutrophil migration and neutrophil‐mediated angiogenesis and indicate that angiostatin might inhibit inflammation.

Mechanisms of Inhibition of Tumor Angiogenesis and Vascular Tumor Growth by Epigallocatechin-3-Gallate

Purpose: Green tea consumption has been linked to a reduced occurrence of some tumor types. Current data indicate that the principal mediator of this chemopreventive effect is epigallocatechin-3-gallate (EGCG), the most abundant polyphenol found in dried tea leaves. Here, we examined the effects of this compound on the two key cell populations typically involved in tumor growth: tumor cells and endothelial cells. Experimental Design: The effects of green tea and EGCG were tested in a highly vascular Kaposi’s sarcoma (KS) tumor model and on endothelial cells in a panel of in vivo and in vitro assays. Results: EGCG inhibited KS-IMM cell growth and endothelial cell growth, chemotaxis, and invasion over a range of doses; high concentrations also induced tumor cell apoptosis. EGCG inhibited the metalloprotease-mediated gelatinolytic activity produced by endothelial cell supernatants and the formation of new capillary-like structures in vitro. Green tea or purified EGCG when administered to mice in the drinking water inhibited angiogenesis in vivo in the Matrigel sponge model and restrained KS tumor growth. Histological analysis of the tumors were consistent with an anti-angiogenic activity of EGCG and green tea. Conclusions: These data suggest that the green tea gallate or its derivatives may find use in the prevention and treatment of vascular tumors in a chemoprevention or adjuvant setting.

The chemoinvasion assay: a method to assess tumor and endothelial cell invasion and its modulation

Invasive and metastatic cells, as well as endothelial cells, must cross basement membranes (BMs) in order to disseminate or to form new blood vessels. The chemoinvasion assay using the reconstituted BM Matrigel in Boyden blind-well chambers is a very rapid, easy, inexpensive and flexible test that can be used to quantify the invasive potential of most cell types; it can be applied to detect the migratory activity associated with matrix degradation and can also be adapted to study the selective degrading activity on different matrix substrates. Transwell inserts can also be used. Once the optimal experimental conditions are empirically determined for specific cellular models, the chemoinvasion assay can be used for the screening of inhibitors of invasiveness and angiogenesis, or to select for invasive cellular populations. This protocol can be completed in 9 h.

Melanoma Cells Inhibit Natural Killer Cell Function by Modulating the Expression of Activating Receptors and Cytolytic Activity

Natural killer (NK) cells play a key role in tumor immune surveillance. However, adoptive immunotherapy protocols using NK cells have shown limited clinical efficacy to date, possibly due to tumor escape mechanisms that inhibit NK cell function. In this study, we analyzed the effect of coculturing melanoma cells and NK cells on their phenotype and function. We found that melanoma cells inhibited the expression of major NK receptors that trigger their immune function, including NKp30, NKp44, and NKG2D, with consequent impairment of NK cell-mediated cytolytic activity against various melanoma cell lines. This inhibitory effect was primarily mediated by indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2). Together, our findings suggest that immunosuppressive barriers erected by tumors greatly hamper the antitumor activity of human NK cells, thereby favoring tumor outgrowth and progression.

Phenotypic and functional analysis of T cells homing into the CSF of subjects with inflammatory diseases of the CNS.

The recruitment of lymphocytes across the blood brain barrier (BBB) is mediated by adhesion molecules and chemokines. The expression of activation markers and of chemokine receptors on T cells homing to the nervous system (NS) may help define their functional state. In the cerebrospinal fluid (CSF) of subjects with inflammatory neurological diseases (IND), including multiple sclerosis, we observed an increased number of T cells coexpressing CXCR3 and CCR5 as well as T cells with a CD45RO+ CCR7+ CD27+ memory phenotype. A subset of CCR7+ T cells coexpressed CXCR3 and CCR5. We also detected an increased number of interferon‐γ‐producing T cells in the CSF compared with peripheral blood, mostly but not exclusively in the CD45RO+ CCR7− CD27− compartment. T helper 1 (Th1) clones, established from the CSF of individuals with IND and from a healthy subject, similarly migrated to CXCL10, CXCL12, and CCL5. CXCL10, CXCL12, and CCL19 were increased in the CSF of individuals with neuroinflammation. These findings suggest that CSF is enriched in Th1‐polarized memory T cells capable of differentiating into effector cells upon antigen encounter. These cells are recruited into the CSF by inducible chemokines. Thus, CSF represents a transitional station for T cells trafficking to and from the NS.

The guanylate binding protein-1 GTPase controls the invasive and angiogenic capability of endothelial cells through inhibition of MMP-1 expression

Expression of the large GTPase guanylate binding protein‐1 (GBP‐1) is induced by inflammatory cytokines (ICs) in endothelial cells (ECs), and the helical domain of the molecule mediates the repression of EC proliferation by ICs. Here we show that the expression of GBP‐1 and of the matrix metalloproteinase‐1 (MMP‐1) are inversely related in vitro and in vivo, and that GBP‐1 selectively inhibits the expression of MMP‐1 in ECs, but not the expression of other proteases. The GTPase activity of GBP‐1 was necessary for this effect, which inhibited invasiveness and tube‐forming capability of ECs in three‐dimensional collagen‐I matrices. A GTPase‐deficient mutant (D184N‐GBP‐1) operated as a transdominant inhibitor of wild‐type GBP‐1 and rescued MMP‐1 expression in the presence of ICs. Expression of D184N‐GBP‐1, as well as paracrine supplementation of MMP‐1, restored the tube‐forming capability of ECs in the presence of wild‐type GBP‐1. The latter finding indicated that the inhibition of capillary formation is specifically due to the repression of MMP‐1 expression by GBP‐1, and is not affected by the anti‐proliferative activity of the helical domain of GBP‐1. These findings substantiate the role of GBP‐1 as a major regulator of the anti‐angiogenic response of ECs to ICs.