Inhibition of tumor growth and microvascular angiogenesis by the potent angiogenesis inhibitor, TNP-470, in rats

OBJECTIVE: To analyse the longitudinal relationships between body mass index (BMI)/sum of skinfolds (SSF) and biological and lifestyle risk factors for coronary heart disease (CHD). DESIGN: An observational longitudinal study; that is, the Amsterdam Growth and Health Study. SUBJECTS: 181 males and females, initially aged 13 y. Over a period of 15 y, six repeated measurements were carried out. MEASUREMENTS: BMI and SSF, biological CHD risk factors; that is, total cholesterol (TC), high density lipoprotein (HDL), TC:HDL ratio, systolic/diastolic blood pressure (SBP/DBP) and cardiopulmonary fitness (VO2-max) and lifestyle CHD risk factors (that is, daily physical activity, dietary parameters, smoking, and alcohol consumption). The longitudinal relationships were analysed by an autoregressive model, in which the value of the outcome variable at time-point t is not only related to the value of the predictor variable at t, but also to the value of the outcome variable at t-1. RESULTS: Both BMI and SSF were positively related to TC and the TC:HDL ratio. Only BMI was positively related to SBP and only SSF was negatively related to VO2-max. Physical activity was negatively related to SSF. None of the other lifestyle parameters were related to SSF and/or BMI. CONCLUSIONS: Both BMI and SSF were related to a high risk profile regarding CHD. Different relationships for SSF and BMI are found, because BMI not only reflects body fatness, but also lean body mass. Analyses with BMI as an indicator for body fatness should therefore be interpreted cautiously.     

Inhibition of LDL oxidation by melatonin requires supraphysiologic concentrations

Melatonin has been suggested as a potent antioxidant that may protect against development of atherosclerosis and cancer; however, these effects are unproven and controversial. The antioxidant capacity of melatonin was tested in comparison with alpha-tocopherol, ascorbic acid, and the melatonin precursors tryptophan and serotonin, by measuring inhibition of metal ion-mediated and human macrophage-mediated oxidation of LDL. Melatonin had weak antioxidant activity that was detectable only at concentrations 10000- to 100000-fold higher than physiologic concentrations. These results were comparable with published data showing that the radical scavenging activity of melatonin requires markedly supraphysiologic concentrations. In contrast, alpha-tocopherol was 50- to 100-fold more potent and was efficacious at physiologic concentrations. Ascorbic acid and tryptophan also were active at physiologic concentrations and were significantly more potent than melatonin. In summary, extremely supraphysiologic concentrations of melatonin had only weak antioxidant activity, which was surpassed by alpha-tocopherol, ascorbic acid, and tryptophan.     

Hydrogen peroxide is involved in lymphocyte activation mechanisms to induce angiogenesis

Ligands of the benzodiazepine binding site allosterically modulate gamma-aminobutyric acidA receptors. Their binding pocket is made up of amino acid residues located on both alpha and gamma subunits. We transiently expressed wild-type alpha1beta2gamma2 and mutant GABAA receptors in human embryonic kidney 293 cells and determined their binding properties. Receptors containing the mutant alphaY209A showed approximately 40-fold decrease in affinity for [3H]Ro 15-1788 and diazepam, whereas zolpidem displayed no measurable affinity. Receptors containing the mutant alphaY209F showed a small-to-moderate decrease in affinity for [3H]Ro 15-1788, diazepam, zolpidem, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, and Cl 218872, amounting to 2-8-fold. Receptors containing the mutant alphaY209Q appeared in the surface membrane of transfected cells, bound [3H]muscimol with wild-type affinity, but failed to bind [3H]Ro 15-1788 or [3H]flunitrazepam with detectable affinity. If these mutant receptors were expressed in Xenopus laevis oocytes, the apparent affinity for GABA was only slightly decreased, whereas the ability of the currents to be stimulated by low concentrations of flunitrazepam was abolished. Receptors containing a point mutant of another amino acid residue, alphaT206A, surprisingly showed an increase in affinity of 5- and 16-fold, for the negative allosteric modulator methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate and the partial positive allosteric modulator Cl 218872, respectively, whereas there was only a small decrease in affinity for Ro 15-1788, diazepam, and zolpidem, amounting to 2-, 4-, and 5-fold. Both alpha206 and alpha209 are thus both important in determining the binding affinities for ligands of the benzodiazepine binding site. The residues are spaced at an interval of three amino acids and may be part of an alpha helix.     

Clotrimazole is an inhibitor of inflammatory angiogenesis and the metabolic activity in sponge granuloma

BACKGROUND: Recently, the automated cardiac output method (ACM) was introduced for the calculation of blood flow at the left ventricular outflow tract (LVOT). This study was performed to examine the possibility of using ACM for flow calculation at the level of the mitral valve and for the quantification of mitral regurgitation (MR) in vitro and in vivo. METHODS AND RESULTS: In a computer-controlled in vitro model of the human heart, aortic and mitral normal bioprosthetic valves were inserted. ACM and electromagnetic probe flow measurements correlated well at the LVOT and at the mitral level (r2 = 0.79 and 0.77, respectively). For stroke volumes ranging from 30 to 100 ml/beat, there was no statistically significant bias between ACM and electromagnetic flow probe (-1.5 and 1.3 ml for LVOT and mitral level, respectively). Limits of agreement were [-14; +11] ml and [-18; +16] ml, respectively. We evaluated 68 patients in our in vivo study. They were divided into three groups according to the results of "standard" echocardiographic Doppler methods for the semiquantification of MR: echocardiographic color Doppler cartography, intensity of the continuous wave Doppler spectra, and in some patients, pulmonary venous flow, conventional Doppler, and proximal isovelocity surface area quantitative data. Group 1 consisted of 35 patients without MR or a physiologic one; the 17 patients in group 2 had a mild MR (1-2/4) and in group 3, 16 patients with MR 3-4/4 were included. Regurgitant volume (RV) was calculated as the difference between ACM mitral flow and ACM aortic flow, and regurgitant fraction (RF) was defined as the ratio between RV and ACM mitral flow. When mitral flow was measured only from the four-chamber view, we found in group 1, RV = -0.57 (0.67) L/min and RF = -16% (19%); in group 2, RV = -0.31 (1.06) L/min and RF = -8% (19%); and in group 3, RV = 1.53 (0.94) L/min and RF = 23% (13%). RV and RF were statistically higher in group 3 compared with group 2 or group 1 (p < 0.0005), but no significant difference was found between groups 1 and 2. When mitral flow was measured by the mean value of ACM four-chamber and two-chamber views, this resulted in group 1, RV = -0.26 (0.63) L/min and RF = -8% (15%); in group 2, RV = 0.01 (1.04) L/min and RF = -2% (18%); and in group 3, RV = 2.07 (1.21) L/min and RF = 34% (19%). RV and RF were again significantly higher in group 3 (p < 0.0001). There was no significant difference between group 1 and group 2, but in group 1 RF was no longer statistically different from 0%. CONCLUSIONS: (1) In our in vitro setting, ACM is reliable both at the LVOT and at the mitral valve. (2) In the in vivo situation, some overlapping does exist between the three groups of MR. However, ACM is a very easy, rapid, and objective method to differentiate hemodynamic nonsignificant (<3/4) from significant (> or =3/4) MR. Together with other well-known methods for the quantification of MR, it should facilitate the gradation of MR in the clinical setting. The absence of significant differences between group 1 and group 2 proves that the accuracy of ACM measurements at the mitral valve needs to be ameliorated before ACM can be used as a gold standard for the noninvasive measurement of RV and RF.     

Inhibition of angiogenesis and growth of human nerve-sheath tumors by AGM-1470

The effectiveness of AGM-1470, a potent, fungal-derived inhibitor of angiogenesis, in suppressing the neovascularization and growth of human Schwann cell tumors was tested in six schwannomas, seven neurofibromas, and one neurofibrosarcoma. Tumor fragments from surgical specimens were implanted into the subrenal capsule of 348 nude mice (nu/nu). Seven days after implantation, the tumors were measured and vascularity was graded. The animals were then randomly assigned to one of two groups, to receive either saline (control group) or systemic AGM-1470 treatment. After 2 to 6 weeks of treatment, tumor size and degree of vascularity were recorded. In the six different schwannomas implanted into 138 mice, the average vascular grade in the control group after 2 weeks of treatment increased from 2.2 to 3.2 (+1.0), while in the AGM-1470-treated group it decreased from 2.2 to 1.7 (-0.5) (p < 0.01). In the seven different neurofibromas implanted into 158 mice, the change in the average vascular grade in control and AGM-1470-treated animals was +0.5 and -1.0, respectively (p < 0.01). In the one neurofibrosarcoma implanted into 52 mice, the change in average vascular grade in each group during the 6-week treatment period was +1.9 and -1.0, respectively (p < 0.01). Neurofibrosarcoma growth after 6 weeks of AGM-1470 treatment was only 8.5% of the growth found in the control animals (p < 0.01). This study determined that AGM-1470 is effective in inhibiting angiogenesis and the growth of human nerve-sheath tumors.     

Inhibition of protein synthesis induced by adenine nucleotides requires their metabolism into adenosine

Adverse reactions to food may be mediated by immunological or non immunological mechanisms. The term "food allergy" describes an event in which a definite immunopathological process can be demonstrated and a cause and effect relationship must be present. Symptoms and signs of food allergy may appear in any organ system, depending in part on the age of the subject and on the allergen involved. At present it is generally agreed that the only effective therapy for food allergy is strict elimination of the offending food antigen. Institution of a food elimination diet should be considered comparable to prescribing a medication, which carries along definite risk-benefit ratio. Consequently, appropriate diagnostic measures base on history, skin test, or radioallergosorbent test (Rast) and blind food challenges, must be utilized before implementing special diets. The allergist and other health care professionals must recognize the advantages of elimination diets (improvement of symptoms) as well as disadvantages (increase of the time required to purchase food and prepare meals, impossibility to eat at restaurants, at friends' houses or at school with consequent possible social isolation, nutritional disorders) and choose the most appropriate elimination diet.     

Inhibition of collagen-induced platelet activation by arachidonyl trifluoromethyl ketone

Collagen-induced platelet activation is associated with, and markedly potentiated by, the release of arachidonic acid and its subsequent conversion to thromboxane A2. The precise mechanism of arachidonic acid release is unknown. An inhibitor of isolated cytosolic phospholipase A2 (cPLA2), arachidonyl trifluoromethyl ketone (AACOCF3), was used to examine the role that cPLA2 plays in this process. AACOCF3 inhibited platelet aggregation in response to collagen and arachidonic acid but not to thrombin, calcium ionophore, phorbol ester, or a thromboxane mimetic. Thromboxane formation stimulated by thrombin or collagen was inhibited by AACOCF3. However, AACOCF3 did not inhibit collagen-induced [14C]arachidonic acid release. These data are consistent with the inhibitory effects of AACOCF3 on collagen-induced aggregation involving an action on the conversion of arachidonic acid to thromboxane.     

Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis

Soy isoflavones exhibit a number of biological effects, suggesting that they may have a role in cancer prevention. Our objectives are to determine whether components of soy products or purified soy isoflavones can inhibit the progression of bladder cancer. We compared the in vitro effects of pure soy isoflavones and soy phytochemical concentrate on growth curves, cell cycle progression, and apoptosis in murine and human bladder cancer cell lines. Pure soy isoflavones (genistein, genistin, daidzein, and biochanin A) and soy phytochemical concentrate exhibit dose-dependent growth inhibition of murine (MB49 and MBT-2) and human (HT-1376, UM-UC-3, RT-4, J82, and TCCSUP) bladder cancer cell lines, although the degree of inhibition varies among lines. Soy isoflavones induce a G2-M cell cycle arrest in all human and murine lines evaluated by flow cytometry. In addition, some bladder cancer lines show DNA fragmentation consistent with apoptosis. We next evaluated the ability of genistein, soy phytochemical concentrate, and soy protein isolate, respectively, to inhibit the growth of transplantable murine bladder cancer in vivo. C57BL/6 mice were randomly assigned to treatment groups (n = 12/group): (a) AIN-76A diet; (b) AIN-76A diet plus genistein, i.p., 50 mg/kg body weight/day; (c) AIN-76 diet with soy phytochemical concentrate at 0.2% of the diet; (d) AIN-76 diet with soy phytochemical concentrate at 1.0% of the diet; and (e) AIN-76A diet with soy protein isolate, 20% by weight. Mice were inoculated s.c. with 5 x 10(4) syngeneic MB49 bladder carcinoma cells, and tumor growth was quantitated. Neither genistein nor soy products reduced body weight gain. Tumor volumes from mice treated with genistein, dietary soy phytochemical concentrate at 1%, or dietary soy protein isolate were reduced by 40% (P < 0.007), 48% (P < 0.001), or 37% (P < 0.01), respectively, compared with controls. We characterized the effects of treatment on several biomarkers in tumor tissue: proliferation index by proliferating cell nuclear antigen staining, apoptotic index by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining, and angiogenesis by microvessel quantitation. Soy products reduced angiogenesis, increased apoptosis, and slightly reduced proliferation while showing no histopathological effects on the normal bladder mucosa. Our data suggest that soy isoflavones can inhibit bladder tumor growth through a combination of direct effects on tumor cells and indirect effects on the tumor neovasculature. Soy products warrant further investigation in bladder cancer prevention and treatment programs or as antiangiogenic agents.     

Inhibition of NNK-induced lung tumorigenesis by modulators of NNK activation

Xenografts originated from human tumours offer the most appropriate research material for in vivo experimental research. However, primary human breast carcinomas are difficult to grow when transplanted in athymic mice: tumour take is less than 15%. Recently, we have achieved 60% tumour take by injecting tumour cell suspensions mixed with Matrigel. Human breast xenografts originated from primary breast carcinoma also frequently show the potential to metastasize spontaneously. In the present study, we generated a human breast carcinoma xenograft line (UISO-BCA-NMT-18) that shows 100% tumorigenicity and 80-100% lung metastasis when transplanted s.c. in athymic mice. We have studied in detail the characteristics of the xenograft and the patient's tumour from which the xenograft line originated. Both the xenograft and the patient's tumour showed intense staining for mutant p53 nuclear protein, and high expression of U-PA, PAI and u-PAR. In vivo growth of the xenograft is stimulated by exogenous supplementation of oestrogen. This xenograft is continuously growing in mice and has shown 80-100% metastasis for the last three successive in vivo passages. This well-characterized, oestrogen-responsive, metastatic breast carcinoma xenograft line will provide excellent research material for metastasis-related research.     

Inhibition in verbal working memory revealed by brain activation

There are many occasions in which humans and other animals must inhibit the production of some behavior or inhibit the processing of some internal representation. Success in inhibitory processing under normal circumstances can be revealed by the fact that certain brain pathologies render inhibitory processing ineffective. These pathologies often have been associated with damage to frontal cortex, including lateral and inferior aspects. We provide behavioral evidence of a verbal working memory task that, by hypothesis, engaged inhibitory processing, and we show (by using positron emission tomograpny) that the inhibitory processing is associated with a lateral portion of the left prefrontal cortex. The task in which subjects engaged was item-recognition: Four target letters were presented for storage followed, after a brief interval, by a probe letter that could match a target letter or not. On some trials, when the probe did not match a target letter and therefore required a "no" response, the probe had matched a target letter of the previous trial, so on these trials a "yes" response was prepotent and had to be inhibited, by hypothesis. Compared with a condition in which no prepotent response was created, this condition yielded brain activation in left inferior frontal gyrus, in the region of Brodmann's area 45.     

Clotrimazole, an imidazole antimycotic, is a potent inhibitor of angiogenesis

PURPOSE: To evaluate the roles of fibroblast proteins in the remodeling of the subconjunctival connective tissue, we immunohistochemically assessed the expression of matrix metalloproteinases (MMP)-1 and -2, and the tissue inhibitors of matrix metalloproteinases (TIMP)-1 and -2 in cultured human subconjunctival fibroblasts and in normal and healing human subconjunctival connective tissue. METHODS: Cultured fibroblasts derived from human subconjunctival connective tissue and surgical specimens of normal and healing conjunctiva were immunostained with monoclonal antibodies directed against human MMPs and TIMPs and examined by light and electron microscopy. RESULTS: In the cultured fibroblasts, MMP-1 and TIMP-1 antibodies stained the cytoplasm in a fine granular pattern, suggesting localization of those proteins in the endoplasmic reticulum (ER) and Golgi apparatus. Antibodies to MMP-2 and TIMP-2 reacted with fibroblast cytoplasm in a granular pattern. Electron microscopy of those fibroblasts revealed MMP-1 and TIMP-1 immunoreactivity in the ER cisternae or on the membrane of the ER. In surgical samples, MMP-1 and TIMP-1 were immunohistochemically detected in healing subconjunctival tissue, but not in conjunctival epithelium or normal subconjunctival tissue. CONCLUSIONS: MMPs and TIMPs may be involved in remodeling of subconjunctival connective tissue and in fibroblast population after surgical interventions. These proteins may play a crucial role in the post-operative fibrotic process occurring during scar formation in subconjunctival tissue.     

Inhibition of angiogenesis by antitumor antibiotic C1027 and its effect on tumor metastasis

Seven genes were regionally localized on rat Chromosome (Chr) 1, from 1p11 to 1q42, and two of these genes were also included in a linkage map. This mapping work integrates the genetic linkage map and the cytogenetic map, and allows us to orient the linkage map with respect to the centromere, and to deduce the approximate position of the centromere in the linkage map. These mapping data also indicate that the Slc9a3 gene, encoding the Na+/H+ exchanger 3, is an unlikely candidate for the blood pressure loci assigned to rat Chr 1. These new localizations expand comparative mapping between rat Chr 1 and mouse or human chromosomes.