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 microg 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 alpha(v)beta3, 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 alpha(v)beta3, and perhaps other integrins, and thus inhibits in vivo progression.

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 ·vß3 negative, but ·vß5 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.

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.

Newborn male mice (45 control and 45 experimental) were tested after 6 weeks of 0.3 ppm nitrogen dioxide (NO,) 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. NO, 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 NO,-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 NO, 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.

Colonies 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 (Nod, ozone (03, 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 0, exceeded the 7985 California state 0, 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 ratio of the alveolar wall perimeter to the alveolar wall area was decreased for the LA animals (p < .02). No definitive conclusions can be made since the study is the first of its kind. The trends observed were expected in that Type 2 cell numbers and size, and also alveolar wall thickness, were increased relative to the SB animals. Cell and wall increases are well recognized as early responses to injury of both the human and animal lung.An independent indoor test at a separate vivaria1 facility was carried out at the same time with measurements of 0, and NO2 only There were two groups in the testing, one exposed to room air (RA) and the other in the same room but within a chamber that excluded air pollutants (C). The restricted monitoring, and technical problems with monitoring, obviated comparisons of all four groups and independent conclusions as well. The lung data have been included for the suggestion that the effects of indoor air may be intermediate between those of the two outdoor environments. Supported by contract A4-160-33, California Air Resources Board, and the Hastings Foundation. The investigators wish to acknowledge the assistance provided to us for completion of this project: The California Air Resources Board provided the ozone and nitrogen dioxide detectors; Lee Lewis and John lung, El Monte Division of the Air ResourcesBoard, standardized and maintained the instruments. Steven Horvath, Ph.D., was instrumental in our obtaining facilities and personnel assistance at the vivarium of the University of California at Santa Barbara, and arranged for the assistance of Linda Flegel, Supervisor of the Santa Barbara Vivaria. Fernando Aluzzi, USC Vivaria, afforded temperature 37 Inhalation Toxicology, 3:37-47, 1991 Copyright 0 1991 by Hemisphere Publishing Corporation Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 01/05/15 For personal use only. and humidity recordings as well as animal transportation; and helped with animal census, maintenance of supplies, and temperaturelhumi...