Valda Richters

Contortrostatin, a dimeric disintegrin from Agkistrodon contortrix contortrix, inhibits breast cancer progression

We report the results of a multidisciplinary study on the inhibitory effect of a snake venom disintegrin, contortrostatin, a 13.5 kDa homodimeric protein isolated from Agkistrodon contortrix contortrix (southern copperhead) venom, on breast cancer progression. We demonstrate that contortrostatin binds to integrins and blocks the adhesion of human breast cancer cells (MDA-MB-435) to extracellular matrix (ECM) proteins including fibronectin and vitronectin, but it has no effect on adhesion of the cells to laminin and Matrigel. Contortrostatin also prevents invasion of MDA-MB-435 cells through an artificial Matrigel basement membrane. Daily local injection of contortrostatin (5 μg per mouse per day) into MDA-MB-435 tumor masses in an orthotopic xenograft nude mouse model inhibits growth of the tumor by 74% (p = 0.0164). More importantly, it reduces the number of pulmonary macro-metastasis of the breast cancer by 68% (p < 0.001), and micro-metastasis by 62.4% (p < 0.001). Contortrostatin is not cytotoxic to cancer cells, and does not inhibit proliferation of the breast cancer cells in vitro. However, contortrostatin inhibits angiogenesis induced by the breast cancer, as shown by immunohistochemical quantitation of the vascular endothelial cells in tumor tissue removed from the nude mice. We have identified αvβ3, an important integrin mediating cell motility and tumor invasion, as one of the binding sites of contortrostatin on MDA-MB-435 cells. We conclude that contortrostatin blocks αvβ3, and perhaps other integrins, and thus inhibits in vivo progression.

A Novel Snake Venom Disintegrin That Inhibits Human Ovarian Cancer Dissemination and Angiogenesis in an Orthotopic Nude Mouse Model

OVCAR-5 is a human epithelial carcinoma cell line of the ovary, established from the ascitic fluid of a patient with progressive ovarian adenocarcinoma without prior cytotoxic treatment. The unique growth pattern of ovarian carcinoma makes it an ideal model for examining the anticancer activity of contortrostatin (CN), a homodimeric disintegrin from southern copperhead venom. FACS analysis revealed that OVCAR-5 is integrin αvβ3 negative, but αvβ5 positive. CN effectively blocks the adhesion of OVCAR-5 cells to several extracellular matrix proteins and inhibits tumor cell invasion through an artificial basement membrane. In a xenograft nude mouse model with intraperitoneal introduction of OVCAR-5 cells, intraperitoneal injection of CN was used for therapy. Tumor dissemination in CN-treated versus control groups was studied by gross examination, and antiangiogenic potential was examined by factor VIII immunohistochemistry and image analysis. CN not only significantly inhibited ovarian cancer dissemination in the nude mouse model, but it also dramatically prevented the recruitment of blood vessels to tumors at secondary sites.

A new relationship between air pollutant inhalation and cancer.

Many studies have been conducted to investigate the effects of different air pollutants on health. Our studies have focused on the effects of nitrogen dioxide (NO2), and recently we reported that inhalation of low levels of NO2 can facilitate cancer cell metastasis. The study described herein utilized the same B16 mouse melanoma metastasis model of previous investigations, but under different NO2 exposure conditions. The results provide further evidence that inhalation of ambient level NO2 (0.4 ppm) or polluted urban ambient air play a role in facilitation of blood-borne cancer cell metastasis. In addition, results show different patterns of melanoma cell distribution in the lungs of NO2- and ambient-air exposed animals. They also indicate that extended periods of clean air between NO2 exposures may diminish the severity of the insult in the less sensitive animals. It is our conclusion that the results provide strong support for the need of improved air quality and for reduction of noxious pollutants in urban ambient air.

Behavior of cancers of the human lung in short-term tissue cultures.

Forty six human lung cancers have been studied in vitro by phase microscopy, cinemicrography and cytologic staining of monolayers. In addition the in vitro findings have been correlated with histologic examinations of whole lung sections and cancer tissue explants sacrificed at specific intervals. The in vitro study was restricted to short‐term cultures to provide an optimal reflection of in vivo cancer properties. The use of a multidiscipline methodology assured the identification of the in vitro cancer cell and the validity of the findings. Eight distinctive types of in vitro cancer behavior have been found, cellular interactions described and new cytostructural insights gained. Also, in vitro parameters of potential clinical value are suggested, particularly in view of the high yield of lung cancer cells obtainable in short‐term cultures.

Effect of 0.3 ppm ozone exposure on type II cells and alveolar walls of newborn mice: an image-analysis quantitation.

Newborn Swiss-Webster mice were exposed to intermittent 0.3 ppm ozone (O3) for 6 wk, and the lungs of 137 exposed and 138 control animals (275 of the 301 in the colony) were suitable for computer-assisted image-analysis quantitation. Data were obtained on numbers and areas of lactate dehydrogenase stained type II cells, and also area, perimeters, and linear intercepts of the alveolar wall. As noted in an earlier study of adult mice, ozone exposure increased all cell and wall measurements. In contrast to the adult animal findings, there was a greater increase in mean type II cell area (p = 0.07) than in numbers of type II cells, with the latter increase falling short of statistical significance. The increase in cell area at the expense of cell number may largely be due to cell pairing or multicell clustering, events that would mask type II cell hyperplasia. Ozone effects on the type II cell population implicate damage to the type I alveolar lining cells. Moreover, the increases in alveolar wall measurements that were found in both the adult and developing mouse lung imply an alteration of the lung scaffolding, and this raises the question of impaired regeneration of the epithelial lining.

Centriacinar region inflammatory disease in young individuals: a comparative study of Miami and Los Angeles residents

Abstract Semiquantitative measurements of chronic inflammation of the centriacinar region (proximal acinus of lung) were compared between 20 Miami and 18 Los Angeles residents (ages 11–30 years) for whom smoking histories were available. Mean extent and severity scores of four lung sites were higher for Los Angeles than Miami residents, with effect of city statistically significant for extent (P=0.02). Also, maximum scores for extent and severity by city were significantly greater for Los Angeles residents (P=0.02, each), but not by smoking history. Smokers did have higher scores for mean extent and severity (by lung site and smoking history), but neither this nor inclusion of smoking and city in the model reached significance. With respect to maximum extent and maximum severity scores, a stratified comparison of cities by smoking history showed a trend (not significant) toward higher scores for Los Angeles residents. Mean extent and severity scores for the lower lobe were higher for basilar sections than for apical sections (each P<0.001). Cumulative data indicate that expanded pathologic studies are essential for efforts to complete a convergence of epidemiological and experimental data implicating exceedences of the Federal ozone standard as a contributor to human lung injury.

Serum and Lung Clearance of Exogenous Horseradish Peroxidase: Influence of Low Levels of Nitrogen Dioxide

Serum and tissue homogenates of lung and kidney from 264 mice, half of which had been exposed to continuous or intermittent nitrogen dioxide (NO3 at levels of 0.5 ppm, 0.6 ppm, and 0.8 ppm in three independent experiments, were assayed for intravenously introduced horseradish peroxidase (HRP), a molecular probe for protein leakage. Disc gel elec-trophoresis and enzyme kinetic assays were used independently to quantitate HRP content after 3 and 6 weeks of NO2 exposure, and at 5.5 hr after HRP injection. Of 6 test periods, 5 showed a greater lung HRP content for the NO2, exposed animals by gel scan analysis, and 3 of the 5 increases were statistically significant (p >.05, p >.025, and p >.0025). Similar trends were noted with the HRP kinetic assay. Serum and kidney comparisons showed no consistent differences; 1 of 6 test periods for each was statistically significant. The findings implicate an NO2, induced overload of the bidirectional protein transport system of the lung.

Influence of 0.5 ppm nitrogen dioxide exposure of mice on macrophage congregation in the lungs

SummaryThe lungs of 12 mice, half of which were exposed to continuous 0.5 ppm nitrogen dioxide for 3 weeks, were explanted in culture, and the instances of macrophage congregation were quantitated according to numbers of target cells involved, categories of congregation from three to 11 or more, numbers of macrophages participating in each category for the total cultures, and the influence of delaying explantation for 24 and 96 hr. A total of 9042 macrophages and 2140 epithelial and spindle target cells were counted in the outgrowths from 306 explants. The incidence of macrophage congregation (or numbers of target cells) was greater for the cultures from the NO2-exposed animals, both with respect to total incidences between groups (p→0), and the 0-hr (p<0.001) and 24-hr (p<0.01) culture subgroups. In addition, the values for T3 to T6 macrophage congregation were individually and consistently greater for the exposed animal group. Postmortem interval stress at 96 hr appeared to result in large colonies, but they were reduced greatly in number. Also the incidence of macrophage congregation fell by 28% as compared to 0-hr and 24-hr subgroups.

Image Analysis Quantitation of Type 2 Cells and Alveolar Walls Part II: Influence of 0.3 ppm Nitrogen Dioxide Exposure on the Developing Mouse Lung

Newborn male mice (45 control and 45 experimental) were tested after 6 weeks of 0.3 ppm nitrogen dioxide (NO2) exposure and at postexposure periods of 4 and 10 weeks. After 6 weeks of exposure, there was a 12.9% increase in type 2 cell number that was statistically significant (P < 0.025) and an 11% increase in mean type 2 cell area that fell short of statistical significance. The ratio of cell area to alveolar wall area was also statistically significant (P = 0.05). The latter ratio, dividing a combined measurement of numbers and area of type 2 cells (type 2 cell field area) by the alveolar wall area, serves to control for lung volume. Although increases in type 2 cell number were not statistically significant for the 2 postexposure test periods, a significant increase (P < 0.05) was found by analysis of variance for all 3 test periods. NO2 exposure also resulted in a significant interaction (group x time; P < 0.05) of the type 2 cell field area:alveolar wall area ratio, i.e., a progressive fall for the exposed animals vs. a progressive rise for the control group. The interaction is believed to be the result of NO2-induced type 2 cell swelling followed by impairment of normal type 2 cell growth. The increase in type 2 cell numbers most likely reflects damage to the type 1 cell population since type 2 cell hyperplasia is a common denominator for diverse kinds of human lung disease where damaged type 1 cells are replaced by type 2 cells. The finding of a statistically significant interaction of the type 2 cell field area:wall area ratio and the persistence of increased alveolar wall area imply that the effects of NO2 on type 2 cells and alveolar walls were not completely reversed 10 weeks after exposure. Should the effects be irreversible, there would be the further implication of a commensurate depletion of structural and functional reserves of the lung.

Influence of Community Air Pollution on the Lungs of Mice. Part I: Hyperplasia and Hypertrophy of Type 2 Cells, and Increase in Alveolar Wall Area

AbstractColonies of 115 mice each were exposed for 43 days to the ambient outdoor atmosphere of Los Angeles (LA) and Santa Barbara (SB), cities that frequently and infrequently exceed air quality standards, respectively The air monitoring stations closest to the LA and SB vivarial sites provided data on nitrogen dioxide (NO2), ozone (O3, sulfur dioxide, carbon monoxide, hydrocarbons, and particulates. All Los Angeles air pollutants were higher than those in Santa Barbara. In particular, the 1-h average of O3 exceeded the 1985 California state O3 standard of 0.10 ppm on 21 of the 43 days, versus just 2 for Santa Barbara. The NO2 mean for LA was 4 times higher than the SB mean, 0.10 versus 0.03 ppm, respectively Image analysis measurements of Type 2 cells and alveolar walls showed the following greater measurements for the LA animals: (a) numbers of Type 2 cells (p - .05); (b) mean area of Type 2 cells (p -.06); (c) alveolar wall area (p -.001); and (d) alveolar wall perimeter (p - .001). In addition, the r...