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. 2010 Oct;22(10):1536-42.
doi: 10.1016/j.cellsig.2010.05.022. Epub 2010 Jun 4.

FTY720 and (S)-FTY720 Vinylphosphonate Inhibit Sphingosine Kinase 1 and Promote Its Proteasomal Degradation in Human Pulmonary Artery Smooth Muscle, Breast Cancer and Androgen-Independent Prostate Cancer Cells

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Free PMC article

FTY720 and (S)-FTY720 Vinylphosphonate Inhibit Sphingosine Kinase 1 and Promote Its Proteasomal Degradation in Human Pulmonary Artery Smooth Muscle, Breast Cancer and Androgen-Independent Prostate Cancer Cells

Francesca Tonelli et al. Cell Signal. .
Free PMC article

Abstract

Sphingosine kinase 1 (SK1) is an enzyme that catalyses the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that FTY720 (Fingolimod) and (S)-FTY720 vinylphosphonate are novel inhibitors of SK1 catalytic activity and induce the proteasomal degradation of this enzyme in human pulmonary artery smooth muscle cells, MCF-7 breast cancer cells and androgen-independent LNCaP-AI prostate cancer cells. Proteasomal degradation of SK1 in response to FTY720 and (S)-FTY720 vinylphosphonate is associated with the down-regulation of the androgen receptor in LNCaP-AI cells. (S)-FTY720 vinylphosphonate also induces the apoptosis of these cells. These findings indicate that SK1 is involved in protecting LNCaP-AI from apoptosis. This protection might be mediated by so-called 'inside-out' signalling by S1P, as LNCaP-AI cells exhibit increased expression of S1P(2/3) receptors and reduced lipid phosphate phosphatase expression (compared with androgen-sensitive LNCaP cells) thereby potentially increasing the bioavailability of S1P at S1P(2/3) receptors.

Figures

Fig. 1
Fig. 1
Structures of FTY720, (S)-FTY720 vinylphosphonate, (R)-FTY720 vinylphosphonate, (S)-FTY720 phosphonate, (R)-FTY720 phosphonate and (S)-FTY720 phosphate.
Fig. 2
Fig. 2
Assessment of FTY720 analogues on SK1 activity. (a) Effect of DMS, SKi and various FTY720 analogues on purified SK1 activity (assayed using 10 μM sphingosine and [32P]ATP (250 μM) as substrates). Results, expressed as percentage of vehicle control are means and standard deviations of 3–4 independent experiments performed in duplicate samples. All inhibitors were screened at 50 μM; (b) Concentration response of the inhibitory effect of (S)-FTY720 vinylphosphonate on SK1 activity (using 10 μM sphingosine and 250 μM [32P]ATP as the substrates) in lysates of HEK 293 cells in which SK1 has been ectopically over-expressed.
Fig. 3
Fig. 3
Effect of FTY720 and (S)-FTY720 vinylphosphonate on proteasomal degradation of SK1a in hPASMC. Western blots showing the effect of: (a) FTY720 and (S)-FTY720 vinylphosphonate (both 10 μM, 24 h) on SK1a, ERK-2 and phosphorylated ERK-1/2 levels in hPASMC; (b) MG132 (10 μM, 24 h) on the FTY720- and (S)-FTY720 vinylphosphonate-induced down-regulation of SK1a; (c) FTY720 and (S)-FTY720 vinylphosphonate (both 10 μM, 24 h) on caspase-3 activation in hPASMC. C = control. In each case, results are representative of 2–4 separate experiments.
Fig. 4
Fig. 4
Effect of SKi, FTY720 and (S)-FTY720 vinylphosphonate on proteasomal degradation of SK1a in MCF-7 cells. Western blot showing the effect of MG132 (10 μM, 24 h) on SKi-, FTY720- and (S)-FTY720 vinylphosphonate-induced degradation of SK1a in MCF-7 cells. The cells were treated for 24 h with the inhibitors at 10 μM. Results are representative of 3 separate experiments.
Fig. 5
Fig. 5
Effect of FTY720 and (S)-FTY720 vinylphosphonate on proteasomal degradation of SK1a and SK1b in LNCaP-AI cells. Western blots showing the effect of MG132 (10 μM, 48 h) on the FTY720- and (S)-FTY720 vinylphosphonate-induced degradation of (a) SK1a and (b) SK1b in LNCaP-AI cells. The cells were treated for 48 h with 10 μM of each inhibitor. Western blots were reprobed with anti-actin antibody to ensure comparable protein loading. In each case, results are representative of 3 separate experiments.
Fig. 6
Fig. 6
Effect of FTY720 and (S)-FTY720 vinylphosphonate on PARP cleavage and AR expression in LNCaP-AI cells. Western blots showing (a) the effect of FTY720 and (S)-FTY720 vinylphosphonate (both 10 μM, 48 h) on PARP cleavage in LNCaP-AI cells; (b) the effect of SKi (1 and 10 μM, 48 h), FTY720 (10 μM, 48 h) or (S)-FTY720 vinylphosphonate (10 μM, 48 h) on AR expression in LNCaP-AI cells. Western blots were re-probed with anti-actin antibody to ensure comparable protein loading. In each case, results are representative of 3 separate experiments.
Fig. 7
Fig. 7
Comparison of mRNA transcript levels of S1P2 and S1P3 in LNCaP and LNCaP-AI cells. RT-PCR was conducted using S1P2 or S1P3 specific primers using 0.15 μg of RNA isolated from either LNCaP or LNCaP-AI cells. An RT-PCR using GAPDH primers was performed to confirm that similar amounts of RNA were used between cell types. In each case, results are representative of 3 separate experiments.
Fig. 8
Fig. 8
Comparison of mRNA transcript levels of LPP1-3 in LNCaP and LNCaP-AI cells. RT-PCR was conducted using LPP1 or LPP2 or LPP3 specific primers using 0.15 μg of RNA isolated from either LNCaP or LNCaP-AI cells. An RT-PCR using GAPDH primers was performed to confirm that similar amounts of RNA were used between cell types. In each case, results are representative of 3 separate experiments.

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