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Abstract
Sphingosine 1-phosphate (S1P) is a mitogenic lipid molecule formed by the enzymes, sphingosine kinase (SphK) 1 and 2. SphK1, which is commonly overexpressed in malignant tumours, is recognised as a significant contributor to cancer cell survival and tumour angiogenesis, and is accordingly implicated in the pathogenesis of various types of cancer. We investigated the roles of SphK1 and 2 in glioblastoma multiforme (GBM), the most common and most aggressive malignant primary brain cancer. Using RNA interference, our research confirmed that both SphKs are vital for the proliferation of GBM cell lines, T98G and U87MG. Interestingly, suppressing the gene expression of SphK2 produced a more potent inhibition of cell proliferation, reduction of viability, and G1 cell cycle arrest than suppressing SphK1. We subsequently tested if a reduced intracellular S1P level was responsible for such anti-proliferative effects. However, intracellular lipid measurements by mass spectrometry and in situ SphK activity measurements revealed that the total S1P generation by each SphK isoform was independent of the effect on cell growth, and was determined by the particular cell line used and the presence or absence of serum. Furthermore, supplying the cells with a high concentration of exogenous S1P could not rescue the anti-proliferative effect of SphK suppression, and overexpressing SphK1 could not compensate the loss of SphK2. Thus, our data highlight the significance of specifically localised or compartmentalised S1P signalling in cell proliferation and survival.
Potential targets of S1P required for the growth of GBM cells include the S1P receptors, which are expressed on the cell surface. Once generated, S1P is thought to be secreted through efflux transporters such as ATP-binding cassette transporters, leading to autocrine or paracrine stimulation of S1P receptors. The regulation of extracellular S1P levels by cells is not thoroughly understood despite abundant literature emphasising the importance of S1P receptors. Therefore, we investigated the regulation of extracellular S1P on GBM cells using mass spectrometry and thin layer chromatography. We present data indicating that U87MG cells actively import S1P across the cell membrane through an as-yet unidentified transporter. These findings will contribute to our deeper understanding of the mechanisms of S1P signalling.