Heparin increases the adhesion of murine mammary adenocarcinoma cells (LM3). Correlation with the presence of heparin receptors on cell surface.

Mastocytosis is a common feature around solid tumors. Due to mast cell (MC) degranulation, heparin and other chemical mediators are released to surrounding tissues. The aim of this paper is to investigate the role of heparin and chemically modified heparins, on a murine mammary adenocarcinoma cell line adhesion properties, and the relationship with the presence of heparin binding sites in tumor cells. We show that heparin increases tumor cell adhesion in a dose-dependent manner. When the number of heparin binding sites was regulated, by culturing the cells with different FCS concentration for 24 hours, a correlation between binding capacity and heparin effect on cell adhesion was observed. The increment on cell adhesion by heparin was lower on cells with less heparin binding sites. Moreover, only heparin and a chemically modified heparin (partially N-desulfated N-acetylated), which bound to heparin-receptor, retained the ability to stimulate cell adhesion, while other modified heparins lost both effects. The increase in cell adhesion was observed on plastic dishes, albumin, as well as on fibronectin pre-coated ones suggesting that heparin effect is substratum independent. Our results show a direct relation between heparin binding to specific cell receptors and increase in cell attachment.

Nitric oxide synthase-cyclooxygenase interactions are involved in tumor cell angiogenesis and migration.

Nitric oxide (NO), produced by distinct nitric oxide synthase (NOS) isoforms, and prostaglandins generated by expression of cyclooxygenases are important mediators in tumor progression. Previous studies have shown that NO can influence the formation of prostaglandin E2 (PGE2). We provide evidence that NO, derived from iNOS and eNOS activity in LMM3 murine mammary adenocarcinoma cell line, is involved in tumor angiogenesis and in tumor cell migration. LMM3 cells that also stimulate their neovascularization activity and migration liberate high basal amounts of PGE2. There is large amount of evidence that postulates positive regulatory interactions between NOS and cyclooxygenase (COX) isoforms. We here show that, in the LMM3 cell line, while PGE2 exerts a positive modulation on NOS activity, NO closes the loop with a negative feed back on COX activity. We also provide evidence of a positive regulatory effect of protein tyrosine kinases on NOS as well as on COX enzymatic functions affecting tumor induced angiogenesis and cell migration.

Heparin receptors in two murine mammary adenocarcinomas with different metastatic ability: relationship with growth inhibition.

Binding of heparin to primary cultured cells of two murine mammary adenocarcinomas with low (M3) and high (MM3) lung, metastatic capacity was determined. Heparin binding was rapid, specific and saturable. MM3 cells grown for 24 h in fetal calf serum (FCS)-free medium exhibited a higher number of binding sites for 3H-heparin [(11 +/- 1) x 10(5) sites per cell than M3 cells [(6.9 +/- 0.6) x 10(5) sites per cell]. However, when M3 cells were grown in the presence of 2% FCS, they showed less heparin binding sites [(3.5 +/- 0.4) x 10(5) sites per cell]. In contrast, dissociation constants were very similar for MM3 and M3 cells grown with or without FCS (Kd = 2-4 x 10(-9) M). Furthermore, heparin inhibited MM3 and M3 cell growth both in the absence or presence of FCS. Competition studies showed that chemically modified heparins lacking antiproliferative effect (O-desulfated; O/N-desulfated N-acetylated and N-desulfated heparins) were not able to inhibit 3H-heparin binding. N-desulfated N-acetylated heparin, which had partial antiproliferative effect, partially inhibited 3H-heparin binding, while heparin with a high antiproliferative activity inhibited more than 90% 3H-heparin binding. The antiproliferative effect of heparin and chemically modified heparins seems to be related to their binding ability to the cell membrane.

Growth inhibition in vitro of murine mammary adenocarcinoma cells by heparin and chemically modified heparins.

Heparin, a highly sulfated polysaccharide used as an antithrombotic and anticoagulant, inhibits proliferation of several cell types. We have investigated the effect of heparin and chemically modified heparins on the growth of a cell culture of a murine mammary adenocarcinoma (M3). We found that heparin inhibited the proliferation of M3 cells growing either with or without 2% fetal calf serum (FCS) in a dose-dependent and reversible fashion. Several heparins with different anticoagulant properties showed a similar antiproliferative effect. Histological assays showed that heparin was internalized and appeared in cytoplasmic vesicules. O-desulfated, O/N-desulfated N-acetylated and N-desulfated heparins lost their antiproliferative activity, while N-desulfated N-acetylated heparin significantly inhibited cell proliferation with or without FCS. The finding of an antiproliferative action of N-desulfated N-acetylated heparin which does not show anticoagulant activity suggests a possible therapeutic role for this compound as an antineoplastic drug.

Mast cell kinetics during tumor growth.

The behavior of mast cells was studied during tumor growth. Sarcoma 13, and normal syngeneic kidney, as control, were implanted subcutaneously in BALBc mice. The number of mast cells in the hypodermis and peritumoral tissue increased 3-fold, 20 days after implantation. In the peritumoral tissue, mast cell degranulation increased together with tumor growth, while in the dermis and hypodermis, it first decreased and only became evident on day 20. Mitosis and mast cell degranulation, more conspicuous in tissues near the tumor, seem to indicate the existence of tumoral factors which spread slowly from the tumor to distant zones. The role of mast cells in peritumoral tissues will be evaluated in the near future.