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The mitochondriotropic compound 7-O-(4-triphenylphosphoniumbutyl)quercetin iodide (Q-7BTPI) in the μM concentration range caused necrotic death of cultured cells by acting as a prooxidant, with generation of superoxide anion in the mitochondria. Externally added membrane-permeating superoxide dismutase or catalase largely prevented death. Rescue by permeant catalase indicates that the toxicant is H2O2, or reactive species derived from it. Rescue by permeant dismutase suggests the possibility of a chain mechanism of H2O2 production, in which dismutation of superoxide constitutes a termination step. Oxidative stress was due to the presence of free phenolic hydroxyls and to accumulation in mitochondria, since the analogous mitochondriotropic per-O-methylated compound -3,3′,4′,5-tetra-O- methyl,7-O-(4-triphenylphosphoniumbutyl) quercetin iodide (QTM-7BTPI) - or Quercetin itself induced no or little superoxide production and cell death. Q-7BTPI did not cause a significant perturbation of the mitochondrial transmembrane potential or of respiration in cells. On the other hand its presence led to inhibition of glutathione peroxidase, an effect expected to accentuate oxidative stress by interfering with the elimination of H 2O2. An exogenous permeable glutathione precursor determined a strong increase of cellular glutathione levels but did not rescue the cells. Death induction was selective for fast-growing C-26 tumoral cells and mouse embryonic fibroblasts (MEFs) while sparing slow-growing MEFs. This suggests a possible use of Q-7BTPI as a chemotherapeutic agent. © 2012 Elsevier B.V.

Protocols and results of case-control studies, pooled- and meta-analyses on the relationship between mobile phone use and risk of head tumours are critically analysed, and for each study strict elements necessary for evaluating its reliability are identified and applied (see Methods). In studies funded by public bodies, blind protocols give positive results revealing cause-effect relationships between long-term latency or use of mobile phones (cellulars and cordless) and statistically significant increases of ipsilateral risk of brain gliomas and acoustic neuromas, with biological plausibility. In studies funded or co-funded by the cellphone companies non-blind protocols give overall negative results with systematic underestimation of risk; however, also in these studies a statistically significant increase in risk of ipsilateral brain gliomas, acoustic neuromas, and parotid gland tumours is quite common when only subjects with at least 10 years of latency or exposure to mobile phones (only cellulars) are considered. Informed precautionary measures are therefore recommended as a step towards reducing risk of head tumours, especially for young people and those making intense use of cellular and cordless phones.

Mitochondria-targeted redox-active polyphenol derivatives are being developed to affect redox processes in the organelles and as tools either to protect cells from oxidative damage or to precipitate their death. The properties of such compounds may be altered by substituents introduced to enhance mitochondrial delivery. Accordingly, we have characterized the redox behaviour of two isomeric mitochondriotropic quercetin-based compounds, 3O- and 7O-[4-triphenylphosphonio)butyl]quercetin iodide. In both cyclic voltammetric determinations of the oxidation potential and in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assays, the 7-substituted isomer behaved much like quercetin itself, whereas the 3-substituted isomer was less reactive. Low μM concentrations of either compound increased superoxide radical anion formation in cultured cells; both compounds were also cytotoxic. The 7-substituted derivative proved to be more active in both types of assays, thus emerging as the isomer of choice for further bioactivity studies and in efforts to understand the link between redox properties, pro-oxidant behaviour and cytotoxicity.

Mitochondria-targeted polyphenols are being developed with the intent to intervene on the levels of reactive oxygen species (ROS) in mitochondria. Polyphenols being more than just anti-oxidants, the interaction of these derivatives with the organelles needs to be characterised. We have studied the effects of two quercetin derivatives, 3-(4-O-triphenylphosphoniumbutyl)quercetin iodide (Q3BTPI) and its tetracetylated analogue (QTA3BTPI), on the inner membrane aspecific permeability, transmembrane voltage difference and respiration of isolated rat liver mitochondria. While the effects of low concentrations were too small to be reliably defined, when used in the 5–20 μM range these compounds acted as inducers of the mitochondrial permeability transition (MPT), an effect due to pro-oxidant activity. Furthermore, Q3BTPI behaved as an uncoupler of isolated mitochondria, causing depolarisation and stimulating oxygen consumption. When applied to tetramethylrhodamine methyl ester (TMRM)-loaded HepG2 or Jurkat cells uptake of the compounds was predictably associated with a loss of TMRM fluorescence, but there was no indication of MPT induction. A production of superoxide could be detected in some cells upon prolonged incubation of MitoSOX®-loaded cells with QTA3BTPI. The overall effects of these model mitochondriotropic polyphenols may thus differ considerably depending on whether their hydroxyls are protected or not and on the experimental system. In vivo assays will be needed for a definitive assessment of their bioactivities.

Glycogen synthase kinase (GSK)-3 modulates the production of inflammatory cytokines. Because bleomycin (BLM) causes lung injury, which is characterized by an inflammatory response followed by a fibrotic degeneration, we postulated that blocking GSK-3 activity with a specific inhibitor could affect the inflammatory and profibrotic cytokine network generated in the BLM-induced process of pulmonary inflammation and fibrosis. Thus, here we investigated the effects of the GSK-3 inhibitor 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) on a BLM-induced lung fibrosis model in mice. SB216763 prevented lung inflammation and the subsequent fibrosis when coadministered with BLM. Bronchoalveolar lavage fluid analysis of mice treated with BLM plus SB216763 revealed a significant reduction in BLM-induced alveolitis. Furthermore, SB216763 treatment was associated with a significantly lower production of inflammatory cytokines by macrophages. BLM-treated mice that received SB216763 developed alveolar epithelial cell damage and pulmonary fibrosis to a significantly lower extent compared with BLM-treated controls. These findings suggest that GSK-3 inhibition has a protective effect on lung fibrosis induced by BLM and candidate GSK-3 as a potential therapeutic target for preventing pulmonary fibrosis.

We report the development of a chemical modification method of general applicability to polyphenols, which increases solubility to influence absorption. Glucosyl groups were added to the resveratrol kernel via a succinate linker, yielding 3,4′,5-tri-(α- d -glucose-3- O -succinyl) resveratrol. The construct was only slowly hydrolyzed in acid and at pH 6.8, but it was destroyed by blood esterases in less than 1 h. In rats its administration resulted in a blood concentration versus time curve shifted to longer times in comparison to resveratrol, a useful modulation of pharmacokinetics. The area-under-curve parameter and the metabolite mix were similar to those of resveratrol. The method may be advantageously employed to solubilize other polyphenols and to make them more palatable.

We previously documented a clear-cut antihypertensive effect of green teat extract (GTE), which was associated with correction of endothelial dysfunction and prevention of left ventricular hypertrophy in an angiotensin II (Ang II)-dependent model of hypertension, but the molecular mechanisms remain to be defined. As several effects of Ang II involve production of reactive oxygen species (ROS) and activation of 2nd messengers, such as mitogen-activated protein kinase (MAPK) and Akt, we investigated the effect of GTE on these signal transduction pathways in Ang II-treated rats. Rats were treated for 2 wk with Ang II infusion (700 mug.kg(-1).d(-1); n = 6, via osmotic minipumps), Ang II plus GTE (6 g/L) dissolved in the drinking water; n = 6), or vehicle (n = 6) to serve as controls. Blood pressure was monitored by telemetry throughout the study. The activation and expression of NAD(P)H oxidase subunits, protein kinase C isoforms, Src, epidermal growth factor receptor (EGFR), Akt, and MAPK were determined in the heart in vitro through immunoprecipitation and western blot analysis with specific antibodies. NAD(P)H oxidase enzymatic activity was measured by cytochrome c reduction assay. GTE blunted Ang II-induced blood pressure increase and cardiac hypertrophy. In Ang II-treated rats, GTE decreased the expression of the NAD(P)H oxidase subunit gp91(phox) and the translocation of Rac-1, as well as NAD(P)H oxidase enzymatic activity. Furthermore, it specifically reduced Ang II-induced Src, EGFR, and Akt phosphorylation. These results show that GTE blunts Ang II-induced cardiac hypertrophy specifically by regulating ROS production and the Src/EGFR/Akt signaling pathway activated by Ang II.

Aim: Tetrahydrocurcumin (THC) is an active metabolite of curcumin. It has been reported to have similar pharmacological activity to curcumin. The proteases that participate in extracellular matrix (ECM) degradation are involved in cancer cell metastasis. The present study investigates the effect of an ultimate metabolite of curcumin, THC, on the invasion and motility of highly-metastatic HT1080 human fibrosarcoma cells. Methods: The effect of THC on HT1080 cell invasion and migration was determined using Boyden chamber assay. Cell-adhesion assay was used for examining the binding of cells to ECM molecules. Zymography assay was used to analyze the effect of THC on matrix metalloproteinase (MMP)-2, MMP-9, and urokinase plasminogen activator (uPA) secretion from HT1080 cells. Tissue inhibitor of metalloproteinase (TIMP)-2 and membrane-type 1 matrix metalloproteinase (MT1-MMP) proteins levels were analyzed by Western blotting. Results: Treatment with THC reduced HT1080 cell invasion and migration in a dose-dependent manner. THC also decreased the cell adhesion to Matrigel and laminin-coated plates. Analysis by zymography demonstrated that treatment with THC reduced the levels of MMP-2, MMP-9, and uPA. THC also inhibited the levels of MT1-MMP and TIMP-2 proteins detected by Western blot analysis. Conclusion: Our findings revealed that THC reduced HT1080 cell invasion and migration. The inhibition of cancer cell invasion is associated with the downregulation of ECM degradation enzymes and the inhibition of cell adhesion to ECM proteins.

Prostate cancer is the most common cancer in men and one of the leading causes of cancer-related deaths in Western countries. The extraordinary biological heterogeneity, the increasing incidence of this disease, and the presence of putative premalignant conditions make prostate cancer a crucial pathology to study and test pharmacological or nutritional chemopreventive strategies. It has been demonstrated that the incidence of prostate cancer is lower in Asian people, and that it increases in Asian men living in Western countries; these data point to a pivotal role of diet in the onset of prostate cancer. A large amount of work has been done in investigating chemopreventive properties of dietary compounds widely used in Asian countries (i.e. soy, soybeans, green tea, fish) in respect of the oxidants- and meatrich diet typical of Western people, particularly of central and northern Europe. Some dietary products appear promising as chemopreventive agents for prostate cancer, because they display both anti-oxidant and anti-inflammatory activity – and inflammation is crucial for the aetiology of adeno-carcinoma of the prostate. There is increasing evidence for close correlation between inflammation, the microenvironment and tumour-associated neo-angiogenesis causing the adverse outcomes of prostate cancer. It may thus be useful to develop new strategies to couple the treatment of inflammation-related prostate cancer and the generation of angiopreventive or antiinflammatory molecules to prevent this disease. The search for compounds with few or no adverse effects – particularly cardiovascular – as compared with the agents currently in use is therefore of greatest relevance. This paper reviews the beneficial effects in this context of the most promising compounds: β-carotene, capsaicin, curcumin, daidzein, EGCG, genistein, hyperforin, lycopene, N-acetyl-L-cysteine, reductase inhibitors, resveratrol, selenium, silybinin, quercetin, vitamin-D and vitamin-E.

Background We investigated the effect of green tea extract (GTE) in arterial hypertension with high oxidative stress. Angiotensin (Ang) II induces endothelial dysfunction (ED) that is crucial for the development of atherosclerosis and hypertension. Methods Male Sprague-Dawley rats, 13 weeks old, randomly assigned to drinking water with or without GTE (6 mg/mL) received a vehicle, a high (700 μg/kg/d) or a low (350 μg/kg/d) Ang II dose for 13 days, by osmotic mini-pumps. Blood pressure (BP) was measured with telemetry. After sacrifice, left ventricular (LV) mass index, small mesenteric artery media-to-lumen ratio, and concentration–response curves of phenylephrine-precontracted arteries to acetylcholine were evaluated. The effect of the superoxide dismutase (SOD-1) analog tempol on artery responses to acetylcholine was assessed. Oxidative stress was measured by plasma hydroperoxides and nitrotyrosine levels. The mRNA of heme oxygenase 1 (HO-1), NADPH oxidase endothelial p22phox subunit, and SOD-1 was also measured in the aorta. Results Compared with vehicle high Ang II increased BP, LV mass index, media-to-lumen ratio, and hydroperoxide radicals. The GTE blunted these increases, prevented the increase in HO-1, p22phox, and SOD-1 mRNA in aorta caused by Ang II, and reduced them below baseline levels. Low Ang II dose increased BP values and plasma hydroperoxides only during the first week. Both Ang II doses shifted rightward the curves to acetylcholine; this was prevented in vivo by GTE and abolished in vitro by tempol. Conclusions The GTE prevented hypertension and target organ damage induced by a high Ang II dose, likely by prevention or scavenging of superoxide anion generation.

Hyperforin (Hyp), a polyphenol-derivative of St. John9s wort ( Hypericum perforatum ), has emerged as key player not only in the antidepressant activity of the plant but also as an inhibitor of bacteria lymphocyte and tumor cell proliferation, and matrix proteinases. We tested whether as well as inhibiting leukocyte elastase (LE) activity, Hyp might be effective in containing both polymorphonuclear neutrophil (PMN) leukocyte recruitment and unfavorable eventual tissue responses. The results show that, without affecting in vitro human PMN viability and chemokine-receptor expression, Hyp (as stable dicyclohexylammonium salt) was able to inhibit in a dose-dependent manner their chemotaxis and chemoinvasion (IC 50 = 1 μM for both); this effect was associated with a reduced expression of the adhesion molecule CD11b by formyl-Met-Leu-Phe-stimulated neutrophils and block of LE-triggered activation of the gelatinase matrix metalloproteinase-9. PMN-triggered angiogenesis is also blocked by both local injection and daily i.p. administration of the Hyp salt in an interleukin-8-induced murine model. Furthermore, i.p. treatment with Hyp reduces acute PMN recruitment and enhances resolution in a pulmonary bleomycin-induced inflammation model, significantly reducing consequent fibrosis. These results indicate that Hyp is a powerful anti-inflammatory compound with therapeutic potential, and they elucidate mechanistic keys.

beta-Lactams, a well known class of antibiotics, have been investigated as inhibitors of the disruptive protease released by inflammatory cells, leukocyte elastase (LE). We have synthesized a new beta-lactam with an N-linked galloyl moiety, the latter identified as strategic in conferring anti-LE properties to some flavonols. This N-galloyl-derivative beta-lactam inhibits the LE activity with a K(i) of 0.7 microM, whereas it exerts weak activity against cathepsin G and protease-3 (IC(50)100 microM), and matrix metalloproteinase (MMP)-2 and MMP-9. Without affecting chemotactic response and viability of polymorphonuclear (PMN) leukocytes, the compound efficiently restrains their chemoinvasion (IC(50) of 1-2 microM) blocking the LE-triggered activation of pro-MMP-9, instrumental to extravasation. Daily i.p. injection of compound enhances resolution in a pulmonary inflammation model, significantly reducing consequent fibrosis. These results indicate that the new beta-lactam is a potent anti-inflammatory compound with therapeutic potential.

Cultured fibroblasts from the dermis of normal subjects and of Pseudoxanthoma elasticum (PXE) patients were analysed for enzyme activity, protein and mRNA expression of metalloproteases (MMP-2, MMP-3, MMP-9, MT1-MMP) and of their specific inhibitors (TIMP-1, TIMP-2 and TIMP-3). MMP-3, MMP-9 and TIMP-3 mRNAs and proteins failed to be detected in both the medium and the cell layer of both controls and PXE patients. MMP-2 mRNA was significantly more expressed in PXE than in control cell lines, whereas MT1-MMP, TIMP-1 and TIMP-2 mRNAs appeared unchanged. MMP-2 was significantly higher in the cell extracts from PXE fibroblasts than in control cells, whereas differences were negligible in the cell medium. Data suggest that PXE fibroblasts have an increased proteolytic potential, and that MMP-2 may actively contribute to connective tissue alterations in this genetic disorder.

Green tea infusion has been shown to inhibit metastatic spreading of the transgenic adenocarcinoma of mouse prostate (TRAMP). Investigation on the molecular mechanisms triggered by the main green tea flavonoid, (-)epigallocatechin-3-gallate (EGCG), shows that EGCG restrains TRAMP-C1 cell proliferation in a dose-dependent manner, at concentrations (IC(50) 50%). In vivo, s.c. injection of TRAMP-C1 cells dispersed in Matrigel gives origin to a tumor mass, whose growth is not inhibited by green-tea regimen. This growth is contained greater than two-thirds by LPS-triggered polymorpho-nuclear phagocyte (PMN) recruitment but this effect is abolished by green tea. Nevertheless, while tumor-released pro-MMP-2 is activated by co-incubation of TRAMP-C1 cells with PMNs, in the presence of 10 microM EGCG the activation is almost abolished. These results suggest that inflammatory involvement of prostate carcinoma could be efficaciously prevented by green tea with a concomitant lowering of the invasive potential.

In addition to their antibiotic potency, beta-lactams have recently been investigated as inhibitors of serine proteinase such as leukocyte elastase (LE), released by inflammatory cells. We describe the synthesis of a series of 4-alkylidene-beta-lactams, and investigate how substitutions on C-3, C-4, and N-1 of the beta-lactam ring affect the activity of human LE and gelatinases MMP-2 and MMP-9. LE activity was measured using a chromogenic substrate, while gelatin-zymography assay was used to evaluate gelatinase activity. We demonstrate that C-4 unsaturation on the beta-lactam ring determines the degree of biological activity, with a selectivity over LE by 3-[1-(tert-butyldimethylsilyloxy)-ethyl] derivatives (lowest IC(50) was 4 microM), and over gelatinase MMP-2 by C-3-unsubstituted 4-[1-ethoxycarbonyl]-ethylidene-beta-lactams (lowest IC(50) was 60 microM). (3S)-3-[(1R)-1-hydroxyethyl]-4-(1-ethoxycarbonyl)-ethylidene-azetidin-2-one inhibits gelatinase MMP-9. The compounds tested showed no cytotoxicity against NIH-3T3 murine fibroblasts. This is the first example of beta-lactams inhibiting metallo-proteinases instrumental in cancer invasion and angiogenesis. These molecules are good candidates for prototype drugs showing selective antibiotic, anti-inflammatory, and anti-invasion properties.

The objective of this paper is to assess the gelatinase production by some ocular pathogenic bacterial strains, and evaluate the ability of (-)epigallocatechin-3-gallate (EGCg) to inhibit this gelatinase activity and thus limit bacterial invasion. The effect of EGCg on bacterial gelatinase activity was tested by classic zymography methods, while its effect on bacterial invasion was evaluated through the ability of growing bacteria to liquefy and thus penetrate a semisolid gelatine substrate. It was found that EGCg inhibits bacterial gelatinases with an IC(50) of about 0.2 mM, and limits invasion of gelatinase-positive bacteria at concentrations above 2 mM. These results show for the first time that EGCg, as well as having direct antibacterial activity, can also inhibit bacterial gelatinases, thus limiting their invasion on gelatine. Possible use of EGCg is thus suggested as an adjuvant in antibacterial chemotherapy.

Uncertainty about the association between health risks and exposure to radiofrequency radiation emitted by cellular and cordless mobile phones can be addressed by a critical analysis of the methodology used in studies assessing this relationship. Studies funded by cellphone companies give reassuring conclusions but are affected by biases and flaws, whereas public-funded studies are without these errors and show acute and chronic effects, including head tumors, findings supported by biological evidence. ____________________ *A.P.P.L.E., Associazione Per la Protezione e la Lotta all’Elettrosmog [association for action and awareness-raising on non-ionizing electromagnetic fields (EMF) health risks], www.applelettrosmog.it, Italy **AIE, Associazione Italiana Elettrosensibili (association for electromagnetic hypersensitivity), www.elettrosensibili.it, Italy HEALTH EFFECTS OF MOBILE PHONE USAGE 2 INTRODUCTION We provide an overview of the relationship between exposure to cellular and cordless phone radiofrequencies and possible health effects. One key concern is the large disparity between the results from publicand from private-funded research. Drawing on Tomatis (2005), founder and long-serving (1969-1993) head of International Agency for Research on Cancer (IARC), the method used in many private-funded studies is such as to raise background noise, increasing confusion, thereby making assessment of risk more difficult. It is thus important to critically assess the scientific validity of these studies: a crucial point we raise is the conflicts of interest often affecting the parties involved, whether individual scientists, international journals, or safety agencies. The implications are far reaching, going beyond the lack of awareness in mobile phone (MP) users regarding health risks, and limited insight on potential improvements due to failures in scientific research. The prime consequence is that collective decisions concerning how these devices should be produced and made available are precluded by the fallacious scientific knowledge that vested interests explicitly pursue. A pioneer study exposing carcinogenic risk arising from MP use was published by Hardell (Hardell et al., 2002) of the Dept Oncology, University Hospital, Orebro, Sweden. This seminal work has now been extended by a number of notable authors: Davis (Davis, 2010) of the Environmental Health Trust, Tetom Village, WY USA; Gee (Gee, 2009) at the European Environment Agency, Copenhagen, Denmark; Lloyd Morgan (Lloyd Morgan, 2009) in Albany, CA USA; Kundi (Kundi, 2009) at the Environmental Health Institute, University of Vienna, Austria. In addition, one of the present authors, Levis (Levis et al., 2011), has worked in this field for more than 10 years, and has frequently been called as an expert witness to give evidence in the context of MP court cases. Investigators in newer areas of research, tackling the increasing challenges of acute and chronic effects of electrohypersensitivity, include Johansson (Johansson, 2006) at the Karolinska Institute and Royal Institute of Technology, Stockholm, Sweden; Khurana (Khurana, 2010) at the Australian National University Medical School, Garran, Australia; HEALTH EFFECTS OF MOBILE PHONE USAGE 3 and Grigoriev (Grigoriev 2011) at the Federal Medical Biophysical Center, Moscow, Russia.

Flavanols—a class of plant polyphenols abundant in tea leaves and grape seeds and skins—have been found to inhibit some matrix-proteases instrumental in inflammation and cancer invasion, such as leukocyte elastase (LE) and gelatinases. In order to establish the relationship between chemical structure and activity, 27 different flavonoids (antocyanidins, dihydrochalcones, dihydroflavonols, flavanolignans, flavanols, flavones, flavonols and isoflavones) and other compounds with anti-oxidant properties were evaluated for their potential in blocking LE and gelatinase activities. LE activity was measured using a chromogenic substrate: from comparison of the different levels of inhibition, it was deduced that a crucial role in inhibition might be played by a galloyl moiety or hydroxyl group at C3, three hydroxyl groups at B ring, one hydroxyl group at C4′, and a 2,3-double bond. Gelatinase activity was measured using the gelatin-zymography assay, and its inhibition showed that three hydroxyl groups at the A or B ring, or, for non-planar molecules, a galloyl moiety at C3 could be determinant. This comparative study is proposed as a basis for designing new molecules with enhanced anti-proteolytic activities, and no or reduced side-effects, for use in hindering inflammation, cancer invasion and angiogenesis.

The effect of CNTF and BDNF on a proteolytic complement instrumental to invasion and on differentiation was studied in two murine neuroblastoma clones, N1 and N7. At the membrane level, gelatinase MMP-2--mainly the activated form--was restrained by CNTF and BDNF to a residual 34% with both factors; membrane-type 1 MMP was down-regulated to 50% (10 h) and 34% (24 h) with both factors; and urokinase-type plasminogen activator was restrained mainly by BDNF to 70%. In the medium, the two gelatinases MMP-2 and MMP-9 were mainly in zymogen form: only MMP-2 was restrained in N1 cells, while only MMP-9 was restrained in N7 cells by both factors, single or in combination. These effects were paralleled by the induction of neurite outgrowth, which was more stimulated in the less differentiated clone. These dose-dependent and transient effects make CNTF and BDNF ideal candidates for constraining the potentially invasive behavior of nervous system tumors.

BACKGROUND Monocytes bind to human mesangial cells (HMC) in a co-culture model of leukocyte/ glomerular cell interactions. Since monocytic infiltration has been demonstrated in the early stages of diabetic glomerulopathy, we examined whether co-culture with myelomonocytes of the U937 cell line in media mimicking the diabetic microenvironment modulated phenotype, growth, and extracellular matrix production patterns of HMC. METHODS HMC monolayers grown for 5 days in 5.5 mmol/l (NG) or 30 mmol/l (HG) glucose media were examined 3, 24 and 48 h after addition of U937 cells by computer-assisted image analysis/fluorescence microscopy following fixation, staining for cell adhesion, and TUNEL/propidium iodide labelling for apoptosis. As matrix components may be relevant to both phenotype of cultured HMC and monocyte adhesion, reverse transcription-polymerase chain reaction, zymography, and ELISA were used to detect urokinase-plasminogen activator (uPa), collagen type IV (COL IV), transforming growth factor beta1 (TGF-beta1), matrix metalloproteinases (MMP), and relative inhibitors (tissue inhibitor of MMP (TIMP)) expression in co-cultures in NG/HG. RESULTS U937 adhesion at 1-3 h was increased in HG (from 54.9+/-6.6 to 87.1+/-5.8% U937/HMC). Control HMC proliferating in NG supplemented with 10% fetal bovine serum had an average cross-sectional area of 9993+/-505 micro(2) with 1.2+/-0.1 hillocks/high-power field, which increased to 13 651+/- 1114 micro(2) with 0.5+/-0.2 hillocks/high-power field in HG (P

The phototoxicity of liposome-incorporated Zn(II)-phthalocyanine (ZnPc) and its water-soluble tetrasulphonated derivative (ZnPcTS) was studied in the tumorigenic but nonmetastatic (RE4) and the highly metastatic (4R) transformed rat embryo fibroblasts. Upon irradiation with 585–605 nm light in the presence of ZnPc, the cell survival drastically decreased, while it was unaffected by ZnPcTS. Enzymatic assays showed that ZnPc induced about a 60% decrease in the activity of the mitochondrial enzymes NADH and succinate dehydrogenase after 3 min of irradiation, while no significant reduction in the activity of lactate dehydrogenase and lysosomal N-acetyl-β-glucosaminidase was observed. The transport of thymidine, deoxyglucose and α-aminoisobutyric acid through the plasma membrane was strongly inhibited after irradiation. Similarly, the intracellular ATP content was significantly reduced. The reduction of DNA biosynthesis showed a time dependence quite similar to the photo-induced decrease in cell survival. No repair of cellular functions affected by ZnPc was observed in the 2 cell lines. These results indicate that, under our experimental conditions, hydrophobic ZnPc exerts its cytotoxic activity mainly by impairing those functions localized in the plasma membrane of the cells. Int. J. Cancer 75:412–417, 1998. © 1998 Wiley-Liss, Inc.

Treatment of mouse Lewis lung carcinoma with razoxane or dacarbazine was protracted for 10 transplant generations. While the capacity of the treated tumors to grow locally in immuno-competent or in immuno-depressed hosts was retained and not significantly modified, the metastatic phenotype was eliminated when the treated tumor cells were transplanted into immuno-competent hosts. The reduction in metastatic potential was slightly less pronounced, in terms of both number and volume of metastases, when the treated tumor cells were transplanted into immuno-depressed hosts. These properties were retained after 3 transplant generations without treatment. Northern blotting and zymography of primary-tumor crude extracts revealed that treatment with either razoxane or dacarbazine for one generation approximately doubled the expression of MMP-2 and MMP-9, while lacking any effect on that of 1.0 and of 3.5 kb TIMP-2. When the treatment was maintained for 10 generations, the expression of MMP-2 and MMP-9 for both drugs showed up-regulation of approximately 10- and 2-fold respectively. TIMP-2 mRNA of 1.0 kb doubled its expression, while that of 3.5 kb registered just above the control. Dacarbazine doubled the expression of uPA after 10 generations, while razoxane boosted it approximately 3-fold after either 1 or 10 generations. The permanent loss of metastatic phenotype induced in Lewis lung carcinoma by dacarbazine and razoxane is thus attributable to biological mechanisms independent of downregulation of expression and/or activation of the 2 gelatinases. Int. J. Cancer 72:1056‐1061, 1997. r 1997 Wiley-Liss, Inc.

Matrix metalloproteinases are inhibited by a growing family of specific tissue inhibitors, TIMPs. The cDNA of the third member of the family, TIMP-3, was obtained by using a reverse transcription-polymerase chain reaction (RT-PCR) to amplify the corresponding mRNA from human placenta. Cloning and expression of the TIMP-3 were performed in Escherichia coli as a fusion protein with a 36 amino acid N-tail containing a His cluster. In the host vector system, rhTIMP-3 was stored intracellularly in its denatured, insoluble form in inclusion bodies. Slow dilution of denaturing and reducing agents, from rhTIMP-3 His bound to a metal affinity solid phase, was followed by partial acid removal of the N-tail, which leaves a residue of four amino acids. Circular dichroism, fluorescence and second-derivative UV spectroscopic analyses supported correct refolding of the recombinant and zymography showed inhibition of both MMP-2 and MMP-9 gelatinolytic activities. The role of the C-terminus, which has closer homology with TIMP-2 than TIMP-1, was also investigated: a C-truncated mutant, similarly cloned and expressed in E. coli, shows complete lack of inhibitory activity on MMP-9, still retaining some on MMP-2. The described protein engineering shows high yield of active inhibitor, unglycosylated as in the native form.

The anti-metastatic ruthenium complex NaCtransRuCI4(DMSO)lm] was given i.p. at 22 and 44 mg/kg/day, on days 8-13 after tumour implantation, to mice carrying S.C. implants of MCa mammary carcinoma. The aim ofthe study was to compare the effects on lung metastasis formation with those on primary tumour cells. This investigation was based on flow cytometry analysis after propidium iodide and acridine orange staining, histology of tumour parenchyma and RT-PCR analysis for the type-IV collagenases MMP-9 and MMP-2 and their respective inhibitors TIMP-I and TIMP-2 mRNAs. NactransRuCl,(DMSO)lm] is not cytotoxic for tumour cells but has the capacity of interacting with nucleic acids, giving a general reduction of nucleic acid content as shown by a marked reduction of acridine orange staining and a tendency to a reduction of DNA polyploidy with marked reduction of 8n and 4n cell populations. Na[trans-RuCI~(DMSO)lm] also influences a proteolytic system which has the potential of degrading the basement membrane and has been related to metastatic aggressiveness: it markedly reduces, in a dose-dependent manner, MMP-2/TIMP-2 balance, but not that of MMP-9/TIMP-I. The different enzyrne/inhibitor mRNA levels between untreated and treated tumours seem to be unaffected by tumourinfiltrating lymphocytes and are paralleled by the maintenance of connective tissue around blood vessels in the tumour mass. Correspondingly, lung metastasis formation is markedly reduced, to less than 10% of that seen in conbols. o 1996 Wiley-Liss, Inc. Basic research on synthetic drugs, effective against tumour metastases, has recently highlighted the effects of a ruthenium complex, namely sodium trans-rutheniumtetrachloridedimethylsulphoxideimidazole (hereafter indicated by Na[transRuCI4(DMSO)Im]) (Sava er al., 1992a, b, 1993, 1994). The effects of this new generation ruthenium(II1) complex on solid metastasizing tumour are particularly evident on the formation of spontaneous metastases. The selectivity of Na[transRuCI4(DMSO)Im] on lung metastases is also marked on advanced metastases and accounts for a significant prolongation of the host's survival time; combined with surgical removal of primary tumour, Na[trans-RuC14(DMSO)Im] prevents the formation of metastases and inhibits the growth of those already formed (Sava et al., 1994). The histological analysis of tumour growth and of healthy host tissues such as lung and kidney epithelia, muscle and liver cells, splenocytes and bone-marrow cells, by light microscopy and by SEM, shows a lack of significant cytotoxicity (Gagliardi et al., 1994). It thus appears that the selective anti-metastatic effects do not result directly from histological modification of the primary tumour structure.

A highly metastatic (4R) and a nonmetastatic (RE4) transformed rat embryo fibroblast cell line were incubated with lipid-soluble Zn(II)-phthalocyanine (ZnPc) and its water-soluble tetrasulphonated derivative (ZnPcTS) and compared for phthalocyanine uptake. The hydrophobic liposome-delivered ZnPc showed a significantly greater uptake by both cell lines than did ZnPcTS. Moreover, the two phthalocyanines appear to interact with cells according to different pathways, as suggested by the different temperature-dependence of the binding process and the different inhibitory action exerted by selected serum proteins, such as lipoproteins and heavy proteins. Under all experimental conditions, the two cell lines exhibited similar interactions with ZnPc and ZnPcTS, suggesting that heterogeneity of the tumor cell population has a minor influence on the accumulation of photosensitizers.

The levels of mRNA of both gelatinases A and B were dramatically decreased in HIV-infected cells, when compared to uninfected cells. The expression of gelatinase A in HIV-infected cells was selectively increased by tumor necrosis factor (TNFα) while the expression of gelatinase B was not affected. In contrast, in uninfected cells TNFα down regulated gelatinase B mRNA level, without affecting the gelatinase A. N-acethylcysteine (NAC) increased the levels of mRNA of both gelatinases. The conditioned media from HIV-infected and uninfected cells had comparable level of secreted gelatinase A protein. These data suggest that in monocytic cells different regulatory pathways control gelatinases A and B and that HIV could modulate in vivo the expression of these proteolytic enzymes, critically involved in regulation of invasion of basement membrane.

The metastatic potential of cancer cells has been associated to their ability to elaborate and secrete basement membrane degradative enzymes. In this process a major role appears to be played by a protease known as gelatinase A (72 kDa type IV collagenase, MMP-2). In an effort to assess the significance of these findings to breast cancer progression, we have evaluated the gelatinase A/MMP-2 serum levels in a cohort of 80 breast cancer patients, 27 subjects affected by benign breast disease and 27 healthy controls. Although differences between the three groups were observed, with the highest values monitored in benign breast disease, they were not statistically significant. On the contrary, within the breast cancer cohort, the patients presenting clinical evidence of distant metastases (M+, n=40) had statistically elevated enzyme serum levels (p

Background Malignant canine mammary tumors represent 50% of all neoplasms in female dogs. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in tumor progression, and they are also associated with the reactive stroma, which provides structural and vascular support for tumor growth. Results MMP-2, MMP-9 and MT1-MMP were expressed at both the mRNA and protein levels in tumor samples. MMP-2 and MMP-9 immunohistochemical reactions were evident both in the epithelial tumor cells and in the stromal compartment to varying degrees; in particular, the intensity of the MMP-2 staining was stronger in the stromal fibroblasts close to epithelial tumor cells in simple carcinomas than in adenomas. These data were supported by gelatin-zymography; bands for the active form of MMP-2 were found in 94% of carcinoma samples, compared with 17% of benign tumor samples. The gene expression and immunohistochemical results for MT1-MMP were comparable to those for MMP-2. The immunoreactivity for MMP-13 and TIMP-2 was lower in carcinomas than in adenomas, confirming the mRNA data for MMP-13 and the other MMP inhibitors that were evaluated. The active form of MMP-9, but not the active form of MMP-2, was identified in the plasma of all of the tested dogs. Conclusions Our findings suggest that MMP-9, MMP-2 and MT1-MMP, which are synthesized by epithelial cancer cells and cancer-associated fibroblasts, play an important role in malignant canine mammary tumors. The reduction of MMP-13 and TIMP-2 could also be a significant step in malignant transformation. MMP-2 and MT1-MMP could be further evaluated as future biomarkers for predicting the progression and prognosis of canine mammary tumors.

Background Whether or not there is a relationship between use of mobile phones (analogue and digital cellulars, and cordless) and head tumour risk (brain tumours, acoustic neuromas, and salivary gland tumours) is still a matter of debate; progress requires a critical analysis of the methodological elements necessary for an impartial evaluation of contradictory studies. Methods A close examination of the protocols and results from all case-control and cohort studies, pooled- and meta-analyses on head tumour risk for mobile phone users was carried out, and for each study the elements necessary for evaluating its reliability were identified. In addition, new meta-analyses of the literature data were undertaken. These were limited to subjects with mobile phone latency time compatible with the progression of the examined tumours, and with analysis of the laterality of head tumour localisation corresponding to the habitual laterality of mobile phone use. Results Blind protocols, free from errors, bias, and financial conditioning factors, give positive results that reveal a cause-effect relationship between long-term mobile phone use or latency and statistically significant increase of ipsilateral head tumour risk, with biological plausibility. Non-blind protocols, which instead are affected by errors, bias, and financial conditioning factors, give negative results with systematic underestimate of such risk. However, also in these studies a statistically significant increase in risk of ipsilateral head tumours is quite common after more than 10 years of mobile phone use or latency. The meta-analyses, our included, examining only data on ipsilateral tumours in subjects using mobile phones since or for at least 10 years, show large and statistically significant increases in risk of ipsilateral brain gliomas and acoustic neuromas. Conclusions Our analysis of the literature studies and of the results from meta-analyses of the significant data alone shows an almost doubling of the risk of head tumours induced by long-term mobile phone use or latency.

The matrix metalloproteinase enzymes have been implicated in tumor invasion and metastasis by a series of correlative immunohistochemical studies. In addition, direct evidence for the role of these enzymes in this pathologic process comes from studies using specific metalloproteinase inhibitors to block tumor invasion and metastasis formation, both in vitro and in vivo. Synthetic oligonucleotide primers for four metalloproteinases (MMP-1, MMP-2, MMP-9, MMP-10) and their tissue inhibitors (TIMP-1, TIMP-2) were selected, synthesized, and optimized in the reverse transcriptase-polymerase chain reaction (RT-PCR) to study the qualitative profile of these enzymes and inhibitors in cultured human tumor cells and tumor tissues. These primers are specific and generate unique amplification products for each appropriate enzyme and inhibitor. Slight enhancement in the amplification of cDNA products was achieved by adding dimethylsulfoxide to the reaction mixture, but commercial enhancement reagents were ineffective. Using this RT-PCR method, cDNA amplification was successful with RNA from as few as 20 cultured tumor cells. The RT-PCR analysis was done on three invasive human colon adenocarcinomas and their paired adjacent normal mucosa. The results show MMP-1 and MMP-2 products in all three tumors, and MMP-2 detected in one of the three normal mucosa samples; TIMP-2 expression was present in two of three patients and awaits quantitative assessment of RT-PCR products.

Resveratrol (trans-3,4',5-trihydroxystilbene) and quercetin (3,3’,4’,5,7-pentahydroxyflavone) are two naturally occurring polyphenols with the potential to exert beneficial health effects. Since their low bioavailability is a major obstacle to biomedical applications, efforts are being made to improve their absorption and slow down phase II metabolism. An accurate evaluation of the corresponding levels in the bloodstream is important to assess delivery strategies, as well as to verify claims of efficacy based on in vitro results. In the present work we have optimized a simple method ensuring complete stabilization and extraction of resveratrol and quercetin from whole blood. The suitability of different protocols was evaluated by measuring the recovery of polyphenol and internal standard from spiked blood samples via HPLC/UV analysis. The optimized procedure ensured a satisfactory recovery of both internal standards and compounds. Comparing plasma and whole blood, up to 76% of the analyte, being associated with the cellular fraction, was unaccounted for when examining only plasma. This indicates the importance of analysing whole blood rather than plasma to avoid underestimating polyphenol absorption in bioavailability studies.

The regioselective synthesis of several quercetin (3,3’,4’,5,7-pentahydroxy flavone) tetraesters bearing a single free OH on 5-C was achieved in good yield by proper choice of reaction conditions using common esterification procedures. Tetracetylated quercetin with the free OH on 7-C was selectively obtained instead via imidazole-promoted deacylation of the corresponding pentaester. Unambiguous structural characterization of the two isomeric tetraacetyl quercetin derivatives was obtained by combined HSQC and HMBC 2D-NMR analysis. These molecules can be used as starting materials for the regioselective synthesis of other derivatives. High yield syntheses of the natural polyphenol rhamnetin (7-O-methylquercetin) and of the new mitochondriotropic compound 7-(4-triphenylphosphoniumbutyl) quercetin iodide are reported as examples.

Vascular endothelial growth factor (VEGF) is a key player in neo-angiogenesis; it sustains the progression of solid neoplasias, brain tumours included. It has recently been demonstrated that the use of antidepressants correlates with increasing VEGF levels in the central nervous system (CNS). In order to elucidate whether the most used natural antidepressant [St. John's wort (SJW) extract] modulates VEGF expression, possible relationship between ≤ μM hyperforin (Hyp, the bioactive component in SJW) and VEGF in CNS tumours has been now examined in medulloblastoma and glioblastoma cells. Real-time PCR and ELISA revealed that under Hyp VEGF expression increased more than three fold in DAOY medulloblastoma cells; while, U87 glioblastoma cells – constitutively expressing high VEGF levels – showed no significant differences. Moreover, Hyp induced endothelial pro-angiogenic behaviour in a multi-parametric Matrigel colonisation assay, and down-modulation of pro-MMP-2 and pro-MMP-9 activities as measured by gelatin zymography. Should these results be confirmed in vivo for this and other types of CNS tumour, the antidepressant use of SJW extracts must be carefully re-considered, in particular for brain tumour patients.

Ductal infiltration carcinomas (d.i.c.) of the breast are potentially highly metastatic tumours, associated with drastic alterations of the architecture and molecular composition of the extracellular matrix at the tumour-host interface. 8701-BC, a recently characterized cell line, isolated from primary d.i.c., was used to study different aspects of tumor cell-substratum interactions. Since type V collagen deposition is augmented in d.i.c. we have examined the ability of 8701-BC cells to interact with this collagen species. We have found that cell binding to type V collagen was mediated by protein homologous to the 67 kDa laminin receptor (67-R). This conclusion is substantiated by the following observations: (a) a major band having an apparent molecular mass of 67 kDa and immunoreactive to the anti-67 R antibody was detectable by SDS-PAGE of the membrane proteins; (b) the antibody inhibited cellular adhesion to type V collagen in a dose-dependent way; (c) membrane proteins purified by affinity chromatography on type V collagen were immunoreactive to anti-67 R antibody, but not to anti-VLA1, VLA2 and VLA3 integrin antibodies. This receptor appears to have prominent carbohydrate-binding properties, since lactose competes with cell adhesion to type V collagen.

A morphometric analysis was carried out on electron micrographs of cells of the F1 (low metastatic capacity) and F10 (high metastatic capacity) variant sublines of the murine B16 melanoma, both in in-vitro cultures and in lung-metastatic nodules developed after the intravenous injection of neoplastic cells in syngeneic C57 black male mice. A group of 28 morphometric parameters was derived to describe quantitatively each neoplastic cell profile. No qualitative difference was observed between the two cell lines. The quantitative expression of subcellular organelles was dissimilar in the two sublines, being consistently characterized, both in in-vitro cultured cells and in lung-metastatic colonies, by a significant decrease in the mean values of parameters related to melanosomes in the high metastatic capacity cell line (B16-F10). Moreover, in in-vitro cultured cells, indices describing heterochromatin masses and cytoplasmic membranous compartments displayed statistically significant differences between the two sublines. In this experimental system, an inverse relationship between metastatic capacity and differentiation is detected, since cells with a more aggressive metastatic behavior exhibit a decreased degree of differentiation.

Model prodrugs of resveratrol carrying protecting substituents at the hydroxyls have been synthesised and tested. Resveratrol triacetate and resveratrol-tri-mPEG(1900) were formed by linking methyl groups or poly(ethylene glycol) chains, respectively, via carboxyester bonds. Resveratrol trimesylate, a molecule less susceptible to hydrolytic attack, was synthesised as well. This latter compound proved to be stable in vitro, while the carboxyester derivatives were slowly hydrolysed in solutions mimicking the gastric or intestinal environment, and rapidly converted to resveratrol in blood. In ex vivo permeation experiments with explanted intestinal segments, resveratrol and its triacetate derivative appeared in the basolateral compartment essentially as a mixture of Phase II metabolites. When the PEGylated derivative was provided on the apical side, unconjugated resveratrol accounted for about 50% of the compounds in the basolateral-side chamber. The same result was obtained by providing an equivalent physical mixture of resveratrol and PEG polymer, indicating that this behaviour is likely due to an adjuvating effect of PEG rather than to the covalent polymer conjugation. These observations suggest that the ester derivatives are rapidly hydrolysed at the intestinal surface or inside enterocytes, and are then processed as resveratrol. On the other hand, the mesylate was transported from the apical to the basolateral side without modification. It may thus be possible to enhance absorption and hinder metabolism of natural polyphenols by constructing pro-drugs incorporating bonds with appropriate resistance to enzymatic hydrolysis.

Caco-2 cells are widely used for transepithelial transport and metabolism studies. We analysed the metabolites produced from quercetin (Q) during transport of this flavonoid across Caco-2 monolayers and by plastic-adhering cells. We found that the pattern of Phase II metabolic activity varies markedly depending on the particular cell clone, age of the cell culture, and stressful treatment such as freezing/thawing. Prolonged culturing and stress cause a decrease of "detoxifying" conjugating activity. This can be re-established by growing the cells with a low concentration of the transport/metabolism substrate for a few days. We suggest this metabolism-activating procedure be used to make studies with these cells more readily comparable.

Curcumin (Cur), a component of turmeric ( Curcuma longa ), has been reported to exhibit antimetastatic activities, but the mechanisms remain unclear. Other curcuminoids present in turmeric, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) have not been investigated whether they exhibit antimetastatic activity to the same extent as curcumin. The regulation of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) play important role in cancer cell invasion by cleavage of extracellular matrix (ECM). In this line, we comparatively examined the influence of Cur, DMC and BDMC on the expressions of uPA, MMP-2, MMP-9, membrane Type 1 MMP (MT1-MMP), tissue inhibitor of metalloproteinases (TIMP-2), and in vitro invasiveness of human fibrosarcoma cells. The results indicate that the differential potency for inhibition of cancer cell invasion was BDMC≥DMC>Cur, whereas the cell migration was not affected. Zymography analysis exhibited that curcumin, DMC and BDMC significantly decreased uPA, active-MMP-2 and MMP-9 but not pro-MMP-2 secretion from the cells in a dose-dependent manner, in which BDMC and DMC show higher potency than curcumin. The suppression of active MMP-2 level correlated with inhibition of MT1-MMP and TIMP-2 protein levels involved in pro-MMP-2 activation. Importantly, BDMC and DMC at 10 μM reduced MT1-MMP and TIMP-2 protein expression, but curcumin slightly reduced only MT1-MMP but not TIMP-2. In addition, three forms of curcuminoids significantly inhibited collagenase, MMP-2, and MMP-9 but not uPA activity. In summary, these data demonstrated that DMC and BDMC show higher antimetastasis potency than curcumin by the differentially down-regulation of ECM degradation enzymes.

The Ca(2+)- and oxidative stress-induced mitochondrial permeability transition (MPT) plays an important role in phenomena ranging from tissue damage upon infarction to muscle wasting in some forms of dystrophy. The process is due to the activation of a large pore in the inner mitochondrial membrane. Anti-oxidants are considered a preventive and remedial tool, and mitochondria-targeted redox-active compounds have been developed. Plant polyphenols are generally considered as anti-oxidants, and thus candidates to the role of mitochondria-protecting agents. In patch-clamp experiments, easily oxidizable polyphenols induced closure of the MPT channel. In swelling experiments with suspensions of mitochondria, high (20-50 microM) concentrations of quercetin, the most efficient inhibitor, promoted instead the onset of the MPT. Chelators of Fe(2+/3+) and Cu(+/2+) ions counteracted this effect. Fluorescent indicators of superoxide production confirmed that quercetin potentiates O(2)(*-) generation by isolated mitochondria and cultured cells. Since this was not affected by chelating Fe and Cu ions, the MPT-inducing effect can be ascribed to a "secondary", metal ion-catalyzed production of ROS. These results are a direct demonstration of the ambivalent redox character of polyphenols. Their mode of action in vivo cannot be taken for granted, but needs to be experimentally verified.

In 187 node-negative breast cancers, the expression of laminin receptors and collagenase IV was directly related in 52% of cases, independently of pathological (tumor size and histology) and biological (estrogen receptors and proliferative activity) features. Moreover, the presence of laminin receptors and collagenase IV did not appear to influence tumor proliferative activity, evaluated as 3H-thymidine labelling index. In this case series, relapse-free survival and overall survival at 6 years were significantly affected by tumor size, hormone receptor status and proliferative activity. Conversely, high levels of laminin receptors and collagenase IV failed to influence relapse-free or overall survival, whereas they were strong indicators of local-regional diffusion of the disease.

It has been proposed that among the various cell-surface proteins capable of interacting with laminin, the 67-kd high-affinity laminin receptor plays a crucial role during tumor invasion and metastasis. In this study, the expression of laminin-receptor-precursor messenger RNA (mRNA) and 67-kd protein was analyzed in human colon adenocarcinoma. In 22 of 23 patients with colon cancer, we found a 2- to 23-fold increase in levels of laminin-receptor-precursor mRNA in the cancer tissues compared with those in matched normal adjacent colonic mucosa. In 10 of 11 cases studied, the level of 67-kd laminin receptor, detected by affinity-purified anti-laminin-receptor synthetic peptide antibodies on immunoblots of matched tumor and normal tissue extracts, was higher in the colon carcinoma tissue. Immunodetection of laminin receptor in tissue sections using anti-laminin-receptor-peptide antibodies confirmed that the increased expression of laminin receptor was specifically associated with the cancer cells. In a series of 72 paraffin sections of colon lesions, we observed a correlation between the expression of the laminin receptor and the Dukes' classification. Our observations indicate that increased expression of laminin-receptor-precursor mRNA is associated with enhanced levels of the 67-kd laminin receptor as well as with the invasive phenotype of colon carcinoma. Detection of this metastasis-associated gene product may be a valuable adjunct in the evaluation of human colon cancer.

Hyperforin, the major lipophilic compound contained in extracts of Hypericum perforatum, is responsible for the antidepressant activity associated with the extract. Recently, several other biological properties of Hyperforin have been unveiled including inhibition of tumour invasion and angiogenesis. The mechanism of the anti-angiogenic activity of Hyperforin remains to be fully elucidated. We show that treatment with non-cytotoxic concentrations of Hyperforin restrains, in a dose-dependent manner, the capacity of endothelial cells to migrate towards relevant chemotactic stimuli. Hyperforin inhibits the organisation of HUVE endothelial cells in capillary-like structures in vitro, and potently represses angiogenesis in vivo in the Matrigel sponge assay in response to diverse angiogenic agents. Immunofluorescent staining shows that in cytokine-activated endothelial HUVE cells Hyperforin prevents translocation to the nucleus of NF-κB, a transcription factor regulating numerous genes involved in cell growth, survival, angiogenesis and invasion. Under Hyperforin treatment in vivo, the growth of Kaposi's sarcoma - a highly angiogenic tumour - is strongly inhibited, with the resultant tumours remarkably reduced in size and in vascularisation as compared with controls. Hyperforin has also been reported to have anti-inflammatory properties. Here we show that Hyperforin inhibits neutrophil and monocyte chemotaxis in vitro and angiogenesis in vivo induced by angiogenic chemokines (CXCL8 or CCL2). These results highlight the potential for Hyperforin as an anti-inflammatory angioprevention agent, acting as a strong inhibitor of inflammation- or tumour-triggered angiogenesis, and provide new therapeutic approaches to halting pathology-associated angiogenesis. © 2009 Elsevier Ltd. All rights reserved.

Mitochondria-targeted compounds are needed to act on a variety of processes that take place in these subcellular organelles and that have great pathophysiological relevance. In particular, redox-active molecules that are capable of homing in on mitochondria provide a tool to intervene on a major cellular source of reactive oxygen species and on the processes they induce, notably the mitochondrial permeability transition and cell death. We have linked the 3-OH of quercetin (3,3',4',5,7-pentahydroxy flavone), a model polyphenol, and the triphenylphosphonium moiety, a membrane-permeant cationic group, to produce proof-of-principle mitochondriotropic quercetin derivatives. The remaining hydroxyls were sometimes acetylated to hinder metabolism and improve solubility. The new compounds accumulate in mitochondria in a transmembrane potential-driven process and are only slowly metabolised by cultured human colon cells. They inhibit mitochondrial ATPase activity much as quercetin does, and are toxic for fast-growing cells.

We previously documented a clear-cut antihypertensive effect of green teat extract (GTE), which was associated with correction of endothelial dysfunction and prevention of left ventricular hypertrophy in an angiotensin II (Ang II)-dependent model of hypertension, but the molecular mechanisms remain to be defined. As several effects of Ang II involve production of reactive oxygen species (ROS) and activation of 2nd messengers, such as mitogen-activated protein kinase (MAPK) and Akt, we investigated the effect of GTE on these signal transduction pathways in Ang II-treated rats. Rats were treated for 2 wk with Ang II infusion (700 mug.kg(-1).d(-1); n = 6, via osmotic minipumps), Ang II plus GTE (6 g/L) dissolved in the drinking water; n = 6), or vehicle (n = 6) to serve as controls. Blood pressure was monitored by telemetry throughout the study. The activation and expression of NAD(P)H oxidase subunits, protein kinase C isoforms, Src, epidermal growth factor receptor (EGFR), Akt, and MAPK were determined in the heart in vitro through immunoprecipitation and western blot analysis with specific antibodies. NAD(P)H oxidase enzymatic activity was measured by cytochrome c reduction assay. GTE blunted Ang II-induced blood pressure increase and cardiac hypertrophy. In Ang II-treated rats, GTE decreased the expression of the NAD(P)H oxidase subunit gp91(phox) and the translocation of Rac-1, as well as NAD(P)H oxidase enzymatic activity. Furthermore, it specifically reduced Ang II-induced Src, EGFR, and Akt phosphorylation. These results show that GTE blunts Ang II-induced cardiac hypertrophy specifically by regulating ROS production and the Src/EGFR/Akt signaling pathway activated by Ang II.

To target natural polyphenols to the subcellular site where their redox properties might be exploited at best, that is, mitochondria, we have synthesised new proof-of-principle derivatives by linking resveratrol (3,4',5-trihydroxy-trans-stilbene) to the membrane-permeable lipophilic triphenylphosphonium cation. The new compounds, (4-triphenylphosphoniumbutyl)-4'-O-resveratrol iodide and its bis-acetylated derivative, the latter intended to provide transient protection against metabolic conjugation, accumulate into energized mitochondria as expected and are cytotoxic for fast-growing but not for slower-growing cells. They provide a powerful potential tool to intervene on mitochondrial and cellular redox processes of pathophysiological relevance.

Hyperforin (Hyp) is an active compound contained in the extract of Hypericum perforatum, well known for its antidepressant activity. However, Hyp has been found to possess several other biological properties, including inhibitory effects on tumor invasion, angiogenesis, and inflammation. In this paper, we show that treatment with Hyp inhibited IFN-γ production, with down-regulation of T-box (T-bet; marker of Th1 gene expression) and up-regulation of GATA-3 (marker gene of Th2) on IL-2/PHA-activated T cells. In parallel, we showed a strong down-regulation of the chemokine receptor CXCR3 expression on activated T cells. The latter effect and the down-modulation of matrix metalloproteinase 9 expression may eventually lead to the inhibition of migratory capability and matrix traversal toward the chemoattractant CXCL10 by activated lymphocytes that we observed in vitro. The effect of Hyp was thus evaluated on an animal model of experimental allergic encephalomyelitis (EAE), a classic, Th1-mediated autoimmune disease of the CNS, and we observed that Hyp attenuates the severity of the disease symptoms significantly. Together, these properties qualify Hyp as a putative, therapeutic molecule for the treatment of autoimmune inflammatory disease sustained by Th1 cells, including EAE.