Adipocyte differentiation influences proliferation and migration of normal and tumoral epithelial breast cells

LUCIANO VIDAL ,VIRGINIA PISTONE CREYDT, PAULA ALEJANDRA SACCA, AMELIA JULIETA TESONE , JUAN CARLOS CALVO

Daegu, República de Korea

Congreso; 7th Annual Meeting of the International Federation for Adipose Therapeutics and Science; 2009

International Federation for Adipose Therapeutics and Science

Breast epithelial cells are in constant development and differentiation throughout a woman’s life. Stromal tissue plays a key role in the regulation of those changes. Adipocytes are the main stromal cell type in breast tissue and can be found as preadipocytes, mature adipocytes and in intermediate differentiation states. Heparanase degrades heparan sulphate and the importance of this enzyme has been described in relation to tumoral metastasis. In addition, metalloproteinases (MMPs) cleave proteoglycans and therefore degrade the extracellular matrix. The aim of this work was to evaluate the effect of soluble and non-soluble factors obtained from distinct adipocyte differentiation states on proliferation and migration as well as the activity of heparanase and MMP-9 of normal (NMuMG) and tumoral (LM3) murine epithelial cells. NMuMG and LM3 were grown on plastic or on 3T3-L1 irradiated cells at various degrees of differentiation (stromal support –SS-, preadipocytes –preA-, poorly differentiated adipocytes –pDA-, mature adipocytes –MA-), and in the presence or absence of conditioned medium (CM) from preA, pDA or MA 3T3-L1 cells. Proliferation was quantified by MTS and cell migration by wound healing. Heparanase activity was determined by heparin cleavage in native polyacrylamide gels dyed with Rubipy (a cationic dya), while MMP-9 expression was quantified by Western blot. NMuMG and LM3 proliferation increased on all three SSs after 24 and 48 h of culture compared to controls (p<0.05). When NMuMG were cultured on the SS in the presence of CM from preA, pDA or MA 3T3-L1 cells for 24 h, proliferation was enhanced with respect to control (p<0.05). In the same growth conditions LM3 proliferation was not significantly modified. Initial NMuMG and LM3 adhesion to SSs did not show significant differences compared to plastic. All three CM from 3T3-L1 increased LM3 migration since wound healing occurred in 75±3% (preA); 53±4% (pDA) and 39±4% (MA) after 6h incubation (p<0.05). When LM3 were cultured on the three SSs migration was observed only on the preA SS (33±3%, p<0.05). NMuMG migration was not observed when cultured on the SSs or with CM. In order to evaluate the role of heparanase enzyme on NMuMG and LM3 cell proliferation and migration, we measured this enzyme’s activity by means of heparin cleavage, in both cell lines previously incubated with the CM of preA, pDA and MA 3T3-L1 cells. We observed a significant decrease of heparin band intensity, corresponding to an increase of heparanase activity, in NMuMG cells previously incubated with the three CM. On the other hand, LM3 cells presented enzyme activity when previously incubated with the CM from pDA and MA 3T3-L1 cells. None of the cell lines presented a detectable basal heparanase activity. Finally, we evaluated the expression of MMP-9 in NMuMG and LM3 cells previously incubated with CM from 3T3-L1 cells at various degrees of differentiation. Both cell lines presented basal expression of MMP-9. We observed a significant increase of MMP-9 expression in LM3 cells previously incubated with CM from preA; pDA and MA 3T3-L1 cells, with a significantly higher expression in presence of preA 3T3-L1 CM. We conclude that adipocyte differentiation influences normal and tumoral epithelial breast cell proliferation and migration. Heparanase and MMP-9 seem to be involved in this regulation. We present an experimental model that allows maintaining the characteristics of the physiological environment of epithelial breast cells, regarding both factors and stromal structure per se.