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Cell growth regulation of ouabain and Methyl-B-Cyclodextrin in Breast Cancer Cells
Leandro A Barbosa1, Lijun Liu2, Amir Askari2
1 - Intituto de Bioquímica Médica, UFRJ, Rio de Janeiro, Brasil
2 - Dpt. of Pharmacology, Medical University of Ohio, Toledo, USA
Objective: Recently, it was demonstrated that when breast cancer cells MDA-MB-435s were treated with ouabain, the activation of Scr/ERK1/2 and increase of expression of p21cip1 occured (Kometiani et al., 2005). However, several studies demonstrated that the cellular content of caveolin-1 was related with cell proliferation (Williams et al., 2005). Breast cancer cell has a restoration of normal phenotype after the transfection of caveolin-1 (Lee et al., 1998). In our work, we treated the cells by a long-term fashion with ouabain and with methyl-beta-cyclodextrin (MBC) to evaluate its effects on caveolae and cell growth.
Methods: The analysis of caveolae content after treatment with ouabain 100nM for 24h and MBC 10mM for 1h was carried out, we used the sucrose gradient fractionation of cell lysates by carbonate method, following an ultracentrifugation step to obtain the plasma membrane fraction (Lijun et al., 2003). We performed immunoblotting experiments using antibodies anti-a1 and anti-b1-Na, K-ATPase, Src kinase and caveolin-1. To proliferation assay, we performed cell counts using a Z2 Coulter Counter (Beckman Coulter). The counts were made after ouabain treatment (100nM for 24h and 48h) or MBC treatment (10mM for only 1h) after MBC treatment a new medium was added or medium containing ouabain (100nM for 24h or 48h) was added.
Results and Conclusions: After ultracentrifugation we obtained 12 fractions of membrane proteins, caveolae was detected in fractions 4-6. After the treatment with ouabain and MBC, we observed a redistribuition of caveolae proteins, with a spread out profile, to heavy fractions. It is important to notice that the protein profile suffered non-significant changes from control to cell-treated conditions. Futhermore, the total expression of proteins after ouabain treatment caused a downregulation of only a1-Na, K-ATPase levels. In the proliferation assays we observed the growth arrest effect of ouabain in these cells, and that MBC alone was sufficient to cause growth arrest. Interestingly, when cells were treated with both, MBC plus ouabain, an additional growth arrest effect occurred. Moreover, when we treated the cells with an inhibitor of Src kinase, PP2, we did not observe any effect in ouabain treated cells. Assuming that MBC was a potent depletor of cholesterol in cell membranes, we can presume similar pathways on cholesterol depletion with long-term treatment with ouabain and MBC.
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