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Biomedical subjects

Roberto Benelli

Publications and source records attributed to Roberto Benelli.

At least 19 recordsLinked to original sources
PubMed2006

Biological assays and genomic analysis reveal lipoic acid modulation of endothelial cell behavior and gene expression.

Lipoic acid (LA) is a sulfated antioxidant produced physiologically as a coenzyme of the pyruvate dehydrogenase complex; it is currently used for treatment of non-insulin-dependent diabetes to favor the cellular uptake of glucose. We have previously described the angiopreventive potential of molecules sharing common features with LA: N-acetyl cysteine, epigallocatechin-3-gallate and xanthohumol. To expand these studies, we have tested the capacity of LA to modulate angiogenesis in tumor growth using a Kaposi's sarcoma model. Endothelial cells exposed to LA displayed a dose-dependent reduction of cell migration and a time-dependent modulation of the phosphorylation of key signaling molecules. In vivo, LA efficiently repressed angiogenesis in matrigel plugs and KS-Imm tumor growth. We analyzed modulation of gene expression in endothelial cells treated with LA for 5 h (early response), finding a mild anti-apoptotic, antioxidant and anti-inflammatory response. A group of LA-targeted genes was selected to perform real-time polymerase chain reaction time-lapse experiments. The long-term gene regulation (48 h and 4 days) shows higher rates of modulation as compared with the array data, confirming that LA is able to switch the regulation of several genes linked to cell survival, inflammation and oxidative stress. LA induced the production of tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) in KS-Imm and activin-A in KS-Imm and endothelial cells; these factors show anti-angiogenic activity in vivo contributing to explain the inhibitory effect of LA on neovascularization. According to our data, LA has promising anti-angiogenic properties, though its influence on central metabolic pathways should suggest more caution about its widespread and not prescribed use at pharmacological doses.

Anti-Inflammatory Agents↗
PubMed2005

Tumor inflammatory angiogenesis and its chemoprevention.

The importance of angiogenesis for the growth of tumors is widely recognized. Drugs that successfully target the endothelium, such as antivascular endothelial growth factor antibodies, are beginning to have an effect on the life expectancy of cancer patients. However, the endothelial cell is not the only possible target for antiangiogenic therapy or prevention of vascularization (angioprevention). It is evident from the literature that native immune cells recruited into tumors in turn stimulate the endothelium and are responsible for an indirect pathway of tumor vascularization. Inflammation-dependent angiogenesis seems to be a central force in tumor growth and expansion, a concept supported by the observation that the use of "classic" anti-inflammatory drugs, such as nonsteroidal anti-inflammatory drugs, leads to angiogenesis inhibition. The mechanisms of inflammatory angiogenesis provide new approaches to target, cure, or even better, prevent tumor angiogenesis by treatment with synthetic or natural agents with anti-inflammatory properties. We propose chemoprevention of inflammatory angiogenesis as a way of checking the cancer before it progresses.

Angiogenesis Inhibitors↗
PubMed2005

Cell density-dependent regulation of matrix metalloproteinase and TIMP expression in differently tumorigenic breast cancer cell lines.

Matrix metalloproteinases (MMPs) play a key role in cellular invasion and growth. Recent observations on tumor tissue samples suggest that MMP activity is altered in relation to cell density. Therefore, we examined MMP(-1,-2,-3,-8,-9,-10,-11 and -13) and TIMP-1/-2 expression of well-defined cell densities in breast carcinoma cell lines with differing in vivo tumorigenicity/invasiveness (MCF-7 < MDA-MB-468 < MDA-MB-231 < MDA-MB-435). Chemoinvasion assays were performed to link the in vitro data to the in vivo behavior. In accord with previous in vivo data, expression levels of most MMPs decreased significantly with increasing cell density and correlated well with a lower in vitro invasiveness of confluent cells. Since these data suggested that cell density regulates transcription and the promoter regions of most MMPs have AP-1 transcription factor binding consensus sequences, we tested whether functional AP-1 protein was involved in the mechanism of MMP downregulation by cell density. A role for AP-1 was confirmed by over-expression of c-Jun and c-fos in confluent MDA-MB-231 cells, showing with c-Jun increased MMP-2 (5-fold), MMP-3 (1.6-fold), and MMP-9 (160-fold) expression, as well as enhanced invasive potential, while TIMP-1 expression was down-regulated (2-fold) when compared to vector controls. Our data provide clear evidence that cell density regulates major MMPs and TIMPs which are controlled by AP-1 activity so that ultimately a major regulation pathway for the control of the invasive potential of tumor cells is presented.

Breast Neoplasms↗
PubMed2005

N-i-Propoxy-N-biphenylsulfonylaminobutylhydroxamic acids as potent and selective inhibitors of MMP-2 and MT1-MMP.

Structural manipulation of the pharmacophoric model of type A selective MMP inhibitors (MMPi), obtained by the insertion of some alkyl substituents R2 possessing an appropriate geometry, steric bulkiness and lipophilicity, is able to improve potency, in the subnanomolar range on MMP-2, and to give a good MMP inhibition on MMP-14 (MT1-MMP) in the designed MMPi of type C, while maintaining a good MMP-1/MMP-2 selectivity profile. The simultaneous inhibition of these two enzymes yields type C compounds, which are potent antiangiogenic agents, able to block a chemoinvasion model on HUVEC cells in the micromolar range.

Angiogenesis Inhibitors↗
PubMed2005

The urokinase-type plasminogen activator, its receptor and u-PA inhibitor type-1 affect in vitro growth and invasion of Kaposi's sarcoma and capillary endothelial cells: role of HIV-Tat protein.

The aggressive and malignant nature of AIDS-associated Kaposi's sarcoma (KS) lesions have largely been ascribed to Tat, the HIV-1 transactivator protein. Among other activities, HIV-Tat induces the migration and invasion of KS and endothelial cells. Since cell invasion is strictly correlated to the activity of lytic enzymes, we elucidated the role of the cell-associated plasminogen activation system in Tat-dependent and in constitutive invasion and proliferation of KS and of microvascular endothelial cells (MVEC). We demonstrate that KS cells and MVEC express the u-PA receptor (u-PAR) and release plasminogen activators and plasminogen activator inhibitor type-1 (PAI-1). The urokinase-type plasminogen activator (u-PA) is chemotactic, chemoinvasive and mitogenic for KS cells and for MVEC. Conditioned medium from KS cells induced invasion and proliferation of MVEC through the u-PA/u-PAR system. Tat is motogenic and mitogenic on KS cells and MVEC, and stimulates morphogenesis of MVEC. These activities were inhibited following antagonization of u-PA and u-PAR, which also reduced constitutive proliferation and invasion of KS cells and MVEC. These data indicate that the u-PA/u-PAR/PAI-1 system is involved in KS-induced endothelial cell invasion, proliferation, and differentiation. Further, exogenous Tat protein could up-regulate the fibrinolytic system, increasing its influence on KS and endothelial cell proliferation and migration, potentially promoting KS progression. These observations suggest the potential for application of u-PA/u-PAR system inhibitors for control of AIDS-associated KS, that has a high risk of recurrence with highly active antiretroviral therapy failure, and of other KS forms.

Antibodies, Monoclonal↗
PubMed2004

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.

Animals↗
PubMed2004

New N-arylsulfonyl-N-alkoxyaminoacetohydroxamic acids as selective inhibitors of gelatinase A (MMP-2).

New N-arylsulfonyl-substituted alkoxyaminoaceto hydroxamic acid derivatives of types 8 and 10 designed as oxa-analogues of known sulfonamide-based MMPi of types 2 and 7 were synthesized and tested for their inhibitory activities on some matrix metalloproteinases. The combination of a biphenylsulfonamide group with oxyamino oxygen in the pharmacophoric central skeleton of sulfonamide-based MMPi obtained in the new sulfonamides 10 seems to be able to give selectivity for MMP-2 over MMP-1. The most potent derivative of this type, 10a, shows similar anti-invasive properties to the analogue reference drug CGS27023A, 2, in an in vitro model of invasion on matrigel, carried out on cellular lines of fibrosarcoma HT1080 (tumoural cells over-expressing MMP-2 and MMP-9).

Cell Line, Tumor↗
PubMed2004

Bladder cell culture on small intestinal submucosa as bioscaffold: experimental study on engineered urothelial grafts.

OBJECTIVES: To investigate the feasibility to perform primary urothelial cell culture using porcine small intestinal submucosa as a delivery scaffold both in vitro and after in vivo implantation in a rabbit model. MATERIALS AND METHODS: Bladder mucosa samples were aseptically obtained from a group of eight male rabbits. The mucosa was cut into fragments and placed on small intestinal submucosa matrices for selective urothelial cell culture. After complete in vitro epithelization the matrices were shaped into tubes and placed in the subcutaneous tissue and subdartos of donor rabbits. The pattern of cell growth and delivery was evaluated on retrieved grafts using histology and immunostaining at the end of the in vitro phase; then 5, 10 and 20 days after implantation. RESULTS: Histological and immunohistochemical analysis of the in vitro primary culture showed the acellular matrices covered with a thin uninterrupted monolayer of urothelial cells. The implants examined on the day 5 maintained the epithelial configuration of the cultured grafts in all samples retrieved. On the day 10 the urothelium showed increased thickness taking on a bilayer configuration. On day 20, all grafts presented the transitional cells arranged in a double layer closely resembling the natural urothelium. The immunostaining pattern displayed the maintaining of urothelial cell phenotype. No differences in epithelium growth and delivery were noted between the two sites of implantation. Five days after implantation, the histological analysis of small intestinal submucosa showed a medium degree tissue reaction with the presence of acute inflammatory cells. Angiogenesis was demonstrated by the development of several new vessels inside the matrix. After twenty days, small intestinal submucosa was gradually replaced with host tissue. CONCLUSION: The small intestinal submucosa proved to function as a means of delivering of autologous urothelial cells cultured in vitro. After ectopic in vivo implantation the bioscaffold maintained viability and growth of the surrounding cells until its degradation.

Animals↗
PubMed2004

The "chemoinvasion assay": a tool to study tumor and endothelial cell invasion of basement membranes.

Several genetic and epigenetic factors, both in the cell and in the host, contribute to the progression of tumors towards metastases. The escape of cancer cells from a primary, localized tumor to distant organs transforms a relatively curable pathology to an almost untreatable one. Metastatic lesions are often resistant to cancer therapy because of the progressive phenotypic changes that they have undergone. In this article we will give a bird's eye view on the features of metastatic cells and potential therapeutic targets. In particular, the invasion of basement membranes represents a fundamental step for cell dispersion. Over seventeen years ago we established the Matrigel "chemoinvasion" assay, a useful tool for studying the mechanisms involved in tumor and endothelial cell invasion of basement membranes and for the screening of anti-invasive agents. We will describe the assay and review some of the major results it enabled to obtain.

Animals↗
PubMed2003

Allele-specific patterns of the mouse parathyroid hormone-related protein: influences on cell adhesion and migration.

The mouse parathyroid hormone-like hormone Pthlh(Pro) and Pthlh(Thr) variants are linked with susceptibility and resistance to skin carcinogenesis of Car-S and Car-R mice, respectively, and with in vitro effects (Oncogene, 19: 5324-5328, 2000). We have identified an additional Pthlh variant, consisting of Thr and three amino-acid changes in the C-terminus (Pthlh(SerAspTyr)), carried by an evolutionarily distant Mus spretus (SPRET/Ei) inbred strain. When transfected into NCI-H520 tumor cells, this Pthlh(SerAspTyr) variant did not stimulate tumor growth in nude mice. Analysis of cell adhesion, migration, and invasion patterns of Pthlh(Pro)-, Pthlh(Thr)-, and Pthlh(SerAspTyr)-transfected NCI-H520 cells revealed a 1.5-fold decrease in adhesion efficiency on both collagen type I and Matrigel, and a 5-6-fold increase in migration capability in Pthlh(Pro) transfectants as compared to nontransfected, vector-transfected, Pthlh(Thr)-, or Pthlh(SerAspTyr)-transfected cells. These findings suggest that the cancer modifier effects of the mouse Pthlh gene are mediated by differential cell adhesion and migration effects of PTHrP variants.

Alleles↗
PubMed2003

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.

Apoptosis↗
PubMed2003

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.

Administration, Oral↗
PubMed2003

Proteinase-3 directly activates MMP-2 and degrades gelatin and Matrigel; differential inhibition by (-)epigallocatechin-3-gallate.

Proteinase-3 (PR-3), a serine-proteinase mainly expressed by polymorphonuclear leukocytes (PMNs), can degrade a variety of extracellular matrix proteins and may contribute to a number of inflammation-triggered diseases. Here, we show that in addition to Matrigel(TM) components, PR-3 is also able to degrade denatured collagen and directly activate secreted but not membrane-bound pro-MMP-2, a matrix metallo-proteinase instrumental to cellular invasion. In contrast, following addition of purified PR-3 or PMNs to HT1080 tumor cells, dose-dependent inhibition of in vitro Matrigel(TM) invasion is registered. (-)Epigallocatechin-3-gallate (EGCG), the main flavanol in green tea and known to inhibit inflammation and tumor invasion, exerts dose-dependent inhibition of degradation of gelatin (IC(50)<20 micro M) and casein, which is directly triggered by PR-3. The presence of EGCG does not modify the colocalization of MMP-2 and exogenous PR-3 at the cell surface and does not restrain secreted pro-MMP-2 and pro-MMP-9 activation or degradation of a specific, synthetic peptide by PR-3. These results add new activities to the list of those exerted by PR-3 and indicate a differential inhibition as a result of EGCG.

Catechin↗
PubMed2003

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-gamma-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.

Adult↗
PubMed2003

Angiostatin inhibits extracellular HIV-Tat-induced inflammatory angiogenesis.

The HIV-Tat protein can be released extracellularly where it is able to recruit leukocytes and induce angiogenesis. These activities are mediated by the direct interaction of Tat with VEGFR2 on endothelial cells and chemokine receptors on leukocytes. We have recently shown that angiostatin, an anti-angiogenic peptide fragment of plasminogen, is able to inhibit the recruitment of neutrophils induced by bacterial fMLP and alpha chemokines both in vitro and in vivo. In vivo this was associated with an inhibition of the angiogenic response by angiostatin. These observations suggested that angiostatin could be a suitable inhibitor of Tat-induced angiogenesis, as it acts on both endothelial and neutrophil at the same time. In vitro, chemotaxis assays demonstrated that angiostatin inhibited Tat-induced chemotaxis of neutrophils with an inverse bell shaped profile. In vivo the injection of matrigel plugs containing Tat or its chemokine-like peptide (CysL24-51) caused the infiltration of neutrophils and a strong angiogenic response. Angiostatin completely blocked this inflammatory response, inhibiting the recruitment of inflammatory and endothelial cells inside the implant. Taken together, these results indicate that angiostatin can act as an inhibitor of both endothelial and neutrophil recruitment. As these cell types are also the targets of extracellularly released Tat, angiostatin could be used to contrast Tat-associated vasculopathies.

Angiogenesis Inhibitors↗
PubMed2002

Polyphenolic antioxidant (-)-epigallocatechin-3-gallate from green tea as a candidate anti-HIV agent.

Epigallocatechin-3-gallate (EGCG), one of the components of green tea, has been suggested to have antiviral activity. To determine the effects of EGCG on HIV infection, peripheral blood lymphocytes were incubated with either LAI/IIIB or Bal HIV strains and increasing concentrations of EGCG. EGCG strongly inhibited the replication of both virus strains as determined by reverse transcriptase and p24 assays on the cell supernatants.

Anti-HIV Agents↗
PubMed2002

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), by focussing more specifically to the relation between inflammation and tumor invasion and how this could drive rational therapeutic approaches.

Chemokines↗