Abstract
BackgroundWe have previously shown that galactosylceramide (GalCer) affects the tumourigenic and metastatic properties of breast cancer cells by acting as an anti-apoptotic molecule. Since GalCer is a precursor molecule in the synthesis of sulfatides, the present study was aimed to define the role of sulfatides in apoptosis and breast cancer progression.MethodsExpression of GAL3ST1 in breast cancer cell lines and breast cancer tissue specimens was analysed using real-time PCR, western blotting and immunohistochemistry analysis. The amount of sulfatide, GalCer and ceramide was analysed by thin-layer chromatography binding assay and by the modified hydrophilic interaction liquid chromatography coupled with electrospray mass spectrometry methodology. The tumourigenicity of cancer cells was analysed by an in-vivo tumour growth assay. Apoptotic cells were detected based on caspase-3 activation and the TUNEL assay. The interaction of breast cancer cells with P-selectin or E-selectin was analysed using the flow adhesion assay. The ability of sulfatide-expressing cells to activate and aggregate platelets was studied using the flow-cytometry-based aggregation assay.ResultsUsing two models of breast cancer, T47D cells with blocked synthesis of sulfatide and MDA-MB-231 cells with neosynthesis of this glycosphingolipid, we showed that high sulfatide levels resulted in increased sensitivity of cancer cells to apoptosis induced by hypoxia and doxorubicin in vitro, and decreased their tumourigenicity after transplantation into athymic nu/nu mice. Accordingly, a clinical study on GAL3ST1 expression in invasive ductal carcinoma revealed that its elevated level is associated with better prognosis. Using MDA-MB-231 cells with neosynthesis of sulfatide we also showed that sulfatide is responsible for adhesion of breast cancer cells to P-selectin-expressing cells, including platelets. Sulfatide also acted as an activating molecule, increasing the expression of P-selectin.ConclusionsThis study demonstrates that increased synthesis of sulfatide sensitises cancer cells to microenvironmental stress factors such as hypoxia and anticancer drugs such as doxorubicin. However, sulfatide is probably not directly involved in apoptotic cascades, because its increased synthesis by GAL3ST1 decreased the amounts of its precursor, GalCer, a known anti-apoptotic molecule. On the other hand, our data support the view that sulfatides are malignancy-related adhesive molecules involved in activating and binding P-selectin-expressing platelets to breast cancer cells.
Highlights
We have previously shown that galactosylceramide (GalCer) affects the tumourigenic and metastatic properties of breast cancer cells by acting as an anti-apoptotic molecule
Accumulation of sulfatides in breast cancer cells correlates with increased sensitivity to doxorubicin-induced and hypoxia-induced apoptosis We have previously shown that galactosylceramide (GalCer) increases the resistance of breast cancer MDA-MB-231 cells to doxorubicin-induced apoptosis [14]
We have shown for the first time that sulfatides are involved in adhesion of cancer cells, acting as ligands for P-selectin expressed by endothelial cells and platelets, but they contribute to programmed cell death, acting as āpro-apoptotic moleculesā, making cancer cells more prone to environmental stressors such as hypoxia and anticancer drugs such as doxorubicin
Summary
We have previously shown that galactosylceramide (GalCer) affects the tumourigenic and metastatic properties of breast cancer cells by acting as an anti-apoptotic molecule. Little is known about the biological role of sulfatides in cancer progression It has been proposed, but not experimentally proven, that sulfatides present on the surface of cancer cells are ligands for P-selectin expressed by activated endothelial cells, and such interactions facilitate the formation of aggregates, which in turn increase their metastatic potential [10]. Sulfated galactocerebroside was described as a P-selectin ligand on MC-38 murine colon carcinoma cells [11] These studies revealed that in-vitro adhesion of activated platelets expressing P-selectin to MC-38 cells is mediated solely through sulfatides present on the latter. When mice were transplanted intravenously with MC-38 cells, their aggregates with platelets were observed in the lungs after 30 min
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