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Neoangiogenesis is essential for tumor and metastasis growth, but this complex process does not follow the same activation pathway, at least in tumor cell lines originated from different murine mammary adenocarcinomas. LMM3 cells were the most potent to stimulate new blood vessel formation. This response was significantly reduced by preincubating cells with indomethacin and NS-398, non-selective cyclooxygenase (COX) and COX-2 selective inhibitors, respectively. COX-1 and COX-2 isoenzymes were both highly expressed in LMM3 cells, and we observed that indomethacin was more effective than NS-398 to inhibit prostaglandin E2(PGE2) synthesis. In addition, nitric oxide synthase (NOS) inhibitors, N ωmonomethyl l-arginine and aminoguanidine, also reduced LMM3-induced angiogenesis and nitric oxide (NO) synthesis as well. NOS2 > NOS3 proteins and arginase II isoform were detected in LMM3 cells by Western blot. The latter enzyme was also involved in the LMM3 neovascular response, since the arginase inhibitor, N ω hydroxy l-arginine reduced the angiogenic cascade. On the other hand, parental LM3 cells were able to stimulate neovascularization via COX-1 and arginase products since only indomethacin and N ω hydroxy l-arginine, which diminished PGE2 and urea synthesis, respectively, also reduced angiogenesis. In turn, LM2 cells angiogenic response could be due in fact to PGE2-induced VEGF liberation that stimulated neoangiogenesis at very low levels of NO. Fil: Davel, Lilia E.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Rimmaudo, Laura Elizabeth. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Español, Alejandro Javier. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: de la Torre, Eulalia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Jasnis, María Adela. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Ribeiro, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Gotoh, Tomomi. Kumamoto University; Japón Fil: Sacerdote de Lustig, Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Sales, María Elena. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Unidad documental simple

T-lymphocytes from tumour-bearing mice are able to trigger the angiogenic cascade. Since it is known that tumour growth produces reactive oxygen species (ROS), the aim of this study was to evaluate the role of hydrogen peroxide (H202) on the activation of lymphocytes and their induction of this vascular response. Studies on lymphocytes, stimulated in vitro by ROS to induce angiogen- esis, showed that only the enzyme catalase (CAT) could block the activation. The incubation of nor- mal lymphocytes with H202 stimulated these cells to induce angiogenesis. The administration of H202 or an oxidative stress-producing drug (doxorubicin) to normal mice activated in viva angio- genesis. In tumour-bearing mice, high levels of lipid peroxidation products were observed in the spleen, but not in the liver or kidney. Moreover, when the ROS scavenger enzyme activities (super- oxide dismutase (SDM) and CAT) were determined, we observed low CAT activity in normal spleens, reflected in a high SDM/CAT ratio, when compared to liver or kidney values. We also showed an increasing value of the SDM/CAT ratio with tumour growth. These results strongty suggest that H202 could be involved in the mechanisms of lymphocyte activation and their induction of angiogenesis during tumour growth. Fil: Monte, M.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Davel, L. E.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Sacerdote de Lustig, Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Unidad documental simple

Urokinase-type plasminogen activator (uPA) initiates an extracellular proteolytic cascade with which invasive cells eliminate barriers to movement. We have evaluated the antiinvasive and antimetastatic properties of two recently developed synthetic uPA inhibitors, B428 and B623, in a BALB/c mouse mammary carcinoma model. We used the F3II and M3 tumor cell lines, previously described by our laboratory. In vitro, noncytotoxic concentrations of B428 or B623 inhibited secreted and cell-associated uPA activity produced by tumor cells and blocked uPA-mediated whole tumor cell degradation of fibronectin, allowing deposition of extracellular fibronectin fibrils. In vivo, administration of compounds was not associated with overt toxic effects. Daily i.p. treatment with B428 (20 mg/kg/day) or B623 (7.5 mg/kg/day) for 2 weeks, beginning after tumor take, markedly blocked the invasion of the muscle and adipose layers of the subcutis and dermis in mice bearing highly invasive F3II tumors. However, these compounds neither inhibited tumor-induced angiogenesis nor reduced the incidence of spontaneous lung metastasis. Moreover, B623 enhanced the formation of experimental lung metastasis. Our results suggest that synthetic uPA inhibitors act as potent antiinvasiveness agents in vivo but may be unable to control progression of the metastatic disease in the present mammary tumor model.

It is known that tumor cells activate spleen cells to induce an angiogenic response. In this report we studied whether different antigenic stimuli, other than tumor cells, were able to activate spleen lymphocytes to induce angiogenesis in syngeneic combination (SLIA). For this purpose, mice were inoculated with sheep red blood cells (SRBC), allogeneic kidney and syngeneic fetal tissues. The effect of pregnancy (syngeneic or allogeneic) on the ability of spleen cells to induce a neovascular response was also assessed. None of the different stimuli were able to induce spleen lymphocytes to evoke angiogenesis. Although allogeneic lymphocytes from virgin females induced a strong neovascular response, the same population, but from allogeneic pregnant mice, did not evoke this response. We conclude that tumor cells seem to be the only antigenic stimuli able to activate spleen lymphocytes to induce SLIA.

The delayed-type hypersensitivity (DTH) response and lymphocyte-mediated angiogenesis were determined in mice bearing in vivo cultures of mammary tumor cells in diffusion chambers (DCs). Soluble tumor products which diffuse from the DCs were able to stimulate the immune system for both the DTH reaction and angiogenic activity by spleen cells.

Investigations on the influence of the parasympathetic nervous system via muscarinic signaling in tumor progression have produced contradictory evidence. We investigated the expression of muscarinic acetylcholine receptors (mAchR) and their intracellular transduction pathways, in two murine mammary adenocarcinoma cell lines, LM3 and LM2 in comparison with the normal murine mammary epithelial cell line: NMuMG. Saturation binding assays with the tritiated muscarinic antagonist quinuclidinyl benzilate ([3H]-QNB) indicate that LM3 cells express higher amounts of mAchR than LM2 cells. Muscarinic receptor activation with carbachol (CARB) enhanced basal production of citrulline to a greater extent in LM3 cells than in LM2 cells. The nitric oxide synthase (NOS) inhibitor, NGmono-methyl-L-arginine (L-NMMA), blunted this effect only in LM3 cells while in LM2 cells the action of CARB was blocked by N? hydroxy-L-arginine (L-OH-Arg), which is known to inhibit the arginase pathway. Atropine blocks the action of CARB in both cell lines. Additionally, mAchR activation stimulates prostaglandin E2 (PGE2) synthesis only in LM2 cells. NMuMG cells show detectable basal amounts of nitric oxide and PGE2, but they did not respond to CARB. Binding experiments confirm the absence of mAchR in these cells. The findings indicate that mAchR expression in tumor cells, and its control on arginine metabolism, via NOS/arginase, and on PGE2 synthesis by COX activation, could be a switch on mechanism that might lead mammary cells from normal to malignant phenotype. Moreover, mAchR coupling to distinct effectors might be associated with differences in aggressiveness of tumor cells. Fil: Español, Alejandro Javier. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Eiján, Ana M.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mazzoni, Esteban. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Davel, Lilia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Jasnis, Maria Adela. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Sacerdote de Lustig, Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Sales, Maria E.. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Unidad documental simple

Regional lymph nodes are important in the generation of tumor-directed immune responses. The relationship between nitric oxide synthase (NOS) expression and the biological behavior of tumor-draining lymph node (TDLNs) cells in vivo was determined using a spontaneously arising BALB/c mammary adenocarcinoma S13. We first demonstrated a reduction of tumor size and tumor-induced angiogenesis by blocking NOS activity in vivo. TDLNs harvested from tumor-bearing mice (TBM) on day 16 after tumor implant, showed enhanced NOS activity and NOS expression compared to control nodes. Identification of the NOS isoforms present in TDLNs resulted in expression of neuronal NOS (nNOS), endothelial NOS (eNOS) and absence of inducible NOS (iNOS). TDLN cells admixed with tumor cells and inoculated into normal mice (Winn assay) induced a reduction of tumor growth although, when inoculated alone, were able to induce the formation of new blood vessels (angiogenesis). Our data indicate that the in vivo antitumor activity of TDLN cells is modulated by a balance between angiogenesis and antitumor effectors. In our model, when trafficking of leukocytes is obviated, the control of tumor growth by TDLN cells can be explained in part by an antitumor activity great enough to exceed the angiogenic component elicited by the same cells, leading to a reduction of tumor size.

The design of more effective therapies for metastatic disease involves development of new compounds able to specifically block the malignant process. We demonstrated previously that a new synthetic nitrogenated compound 3'-1-chloroethyl-2,3-dihydro-1H-imidazo-(2, 1-i)-purine-4-ium-7-yl-3'-deoxy-1',5', 6'-tri-O-(methylsulfonyl)-muco-inositol chloride (DIC) had an anti-proliferative activity on tumor cells in vitro. In the present work we demonstrate that DIC induces apoptosis on the LM3 murine mammary adenocarcinoma cell line in vitro and has anti-angiogenic activity in vivo. We also evaluated toxicity, biodistribution and anti-neoplastic properties of DIC in vivo. Toxicity studies allowed us to establish the LD50 (750 mg/kg body weight). Administration of 250 mg/kg/day (LD10) for 6 days did not cause overt toxic effects. Biodistribution assays revealed that DIC was rapidly eliminated (60% at t=10 min), although it accumulated in tumor tissue at higher concentrations than in other tissues. Daily s.c. treatment with DIC (LD10) for 24 days significantly reduced the number of spontaneous lung metastases. These results suggest that DIC has the ability of impairing the metastatic development by inhibiting angiogenesis and inducing apoptosis on tumor cells.

The purpose of this study was to determine whether nitric oxide (NO) production by different mammary tumor cell lines correlated with their sensitivity to NO mediated injury. Three mammary tumor cell lines LM2, LM3 and LMM3 syngeneic to BALB/c mice were cultured in vitro with IFNgamma + LPS. Different levels of NO production among the three lines were detected in culture supernatants. The only tumor cell line which did not produce NO (LM2) showed the highest sensitivity to SNP-derived NO cytotoxicity (87%), while LM3 and LMM3 which both produced higher levels of NO than LM2, showed lower cytotoxicity by SNP (39% and 22% respectively). Spleen cells (SC) from M2 tumor bearing mice (TBM) were able to lyse LM2 cells by NO-dependent mechanisms. SC from M3-TBM exerted cytotoxicity against LM3 cells mainly by NO-independent mechanisms. Thus, we postulate an inverse correlation between NO production and NO mediated cytotoxicity in the three mammary tumor cell lines. It is possible that tumor cells producing NO develop mechanisms to resist NO injury. Fil: Eijan, Ana Maria. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Davel, L. E.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Rueda, H. A.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Rozenberg, G.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Sacerdote de Lustig, Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Jasnis, Maria Adela. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Unidad documental simple

Solid tumors induce an angiogenic response by the host blood vessels to form a new vascular network for the supply of fresh nutrients and oxygen responsible for tumor growth. Furthermore, tumor growth and metastatic spread is abrogated or markedly reduced in the absence of neovascularization. Spleen Y lymphocytes from tumor-bearing mice elicit a strong neovascular response. It is well known that certain T cell responses require the presence of active oxygen radicals. Because these metabolites are produced during tumor growth, we studied whether oxygen free radicals play a role in the angionesis induction by lymphocytes. In this study, we demonstrated that the administration of a free radical scavenger (EGb-761) to tumor-bearing mice, blocked the angiogenic response and decrease the lung metastatic incidence. On the other hand, when normal lymphocytes were incubated with the xanthine-xanthine oxidase system (X-XO), a known superoxide anion generator, this elicited a dose-response positive angiogenic reaction in normal recipient mice. No angiogenic response was observed in the absence of X-XO, or when EGb-761 or superoxide dismutase (SOD) plus catalase (CAT) were added to the incubation medium. These results suggest that free radicals are involved in some step of the angiogenic process, and that the EGb-761 treatments block this response due to the free radical scavenging activity of this compound. Fil: Monte, Martin. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Davel, Lilia E.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Sacerdote de Lustig, Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Unidad documental simple

Tumor growth mainly depend on formation of new blood vessels. DFMO (α-difluoromethylornithine), an inhibitor of polyamine biosynthesis, inhibits tumor growth in many animal tumors. Our investigation was to evaluate the requirement of polyamines for induction of angiogenesis by tumor cells and spleen lymphocytes from tumor-bearing mice. In this regard, we have added DFMO to cell cultures. The neovascular response induced either by tumor cells or spleen lymphocytes was completely abrogated. This inhibition could be reversed by the addition of exogenous putrescine. These findings suggest that the effect of DFMO on angiogenesis is, in part, mediated by the inhibition of polyamine biosynthesis. Fil: Jasnis, Maria Adela. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Klein, Slobodanka Mariana. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Monte, Martin. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Davel, Lilia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Sacerdote de Lustig, Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Algranati, Israel David. Instituto de Investigaciones Bioquímicas "Fundación Campomar"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Unidad documental simple

Fil: Davel, Lilia E.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Miguez, Marta M.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Sacerdote de Lustig, Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Unidad documental simple

Supernatants of cultured spleen cells (SCS) from small tumor-bearing mice (STBM) and large tumor-bearing mice (LTBM) are able to enhance tumor growth as well as accelerate tumor takes in vivo when inoculated 24 h before tumor implant. After tumor resection enhancing activity disappears at a rate depending on the size of the resected tumor. Spleen cells from tumor-bearing mice also evoke a complex vascular response, lymphocyte-induced angiogenesis (LIA) elicited via the release of lymphokines from activated T cells. As angiogenic factors promote tumor development we investigated if SCS from tumor-bearing and tumor-resected mice contain factors capable of evoking a LIA response. Angiogenic activity was detected by SCS of small and large tumor-bearing mice as well as in large tumor-resected mice. On the other hand, SCS from small tumor-resected mice are not able to evoke an angiogenic response. Possibly, the large antigenic burden in LTRM stimulate the immune system in a different way than in STRM. We can suggest that the different behavior of spleen cells after small and large tumor removal can be explained by quantitative or qualitative changes in the spleen subpopulations.

Introduction The role of macrophages in tumor progression has generated contradictory evidence. We had previously demonstrated the ability of peritoneal macrophages from LMM3 murine mammary adenocarcinoma-bearing mice (TMps) to increase the angiogenicity of LMM3 tumor cells, mainly through polyamine synthesis. Here we investigate the ability of the parasympathetic nervous system to modulate angiogenesis induced by TMps through the activation of the muscarinic acetylcholine receptor (mAchR). Methods Peritoneal macrophages from female BALB/c mice bearing a 7-day LMM3 tumor were inoculated intradermally (3 × 105 cells per site) into syngeneic mice. Before inoculation, TMps were stimulated with the muscarinic agonist carbachol in the absence or presence of different muscarinic antagonists or enzyme inhibitors. Angiogenesis was evaluated by counting vessels per square millimeter of skin. The expression of mAchR, arginase and cyclo-oxygenase (COX) isoforms was analyzed by Western blotting. Arginase and COX activities were evaluated by urea and prostaglandin E2 (PGE2) production, respectively. Results TMps, which stimulate neovascularization, express functional mAchR, because carbachol-treated TMps potently increased new blood vessels formation. This response was completely blocked by preincubating TMps with pirenzepine and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), M1 and M3 receptor antagonists, and partly by the M2 receptor antagonist methoctramine. M1 receptor activation by carbachol in TMps triggers neovascularization through arginase products because N ω-hydroxy-L-arginine reversed the agonist action. Preincubation of TMps with methoctramine partly prevented carbachol-stimulated urea formation. In addition, COX-derived liberation of PGE2 is responsible for the promotion of TMps angiogenic activity by M3 receptor. We also detected a higher expression of vascular endothelial growth factor (VEGF) in TMps than in macrophages from normal mice. Carbachol significantly increased VEGF expression in TMps, and this effect was totally reversed by methoctramine and pirenzepine. Arginase and COX inhibitors partly decreased VEGF derived from TMps. Conclusion TMps themselves induce a potent angiogenic response that is augmented by carbachol action. mAchR activation triggers arginine metabolism, PGE2 synthesis and VEGF production, promoting neovascularization. Fil: de la Torre, Eulalia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Davel, Lilia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Jasnis, María A.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Gotoh, Tomomi. Kumamoto University; Japón Fil: Sacerdote de Lustig, Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Sales, María Elena. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Unidad documental simple