Figure 5. Effect of StSPL on S1P-stimulated MAPK phosphorylation, proliferation and migration and VEGF synthesis in MCF-7 breast carcinoma cells.

(A) Quiescent MCF-7 cells were treated for 10 min with either vehicle (DMEM, -) or S1P (1 µM) in the absence or presence of WT StSPL (StSPL; 10 µg/ml) or the K311A mutant (10 µg/ml). Cell lysates were prepared and separated by SDS-PAGE, transferred to nitrocellulose and subjected to Western blotting using antibodies against phospho-p42/p44 (dilution of 1∶1000, upper panel) and total p42/p44-MAPK (dilution each 1∶6000, lower panel). (B) Quiescent MCF-7 cells were treated for 24 h with either vehicle (Co) or S1P (1 µM), which had been pretreated for 30 min at 37°C with either vehicle (-), WT StSPL (StSPL; 10 µg/ml) or the K311A mutant (10 µg/ml), in the presence of [³H]thymidine. Incorporated radioactivity was measured as described in the Materials and Methods section. Results are expressed as cpm/well of incorporated [³H]thymidine and are means ±S.D. (n = 4). (C) Quiescent MCF-7 cells were treated for 24 h with DMEM (Co) or S1P (1 µM), which had been pretreated for 30 min at 37°C with either vehicle (-), WT StSPL (StSPL) or the K311A mutant. Thereafter, migrated cells were analysed as described in the Materials and Methods section. Results are expressed as migrated cells per counted field and are means ±S.D. (n = 3). (D) Quiescent MCF-7 cells were treated for 24 h with DMEM (Co) or S1P (1 µM) which had been pretreated for 30 min at 37°C with either vehicle (-), WT StSPL (StSPL; 10 µg/ml), or the K311A mutant (10 µg/ml). Thereafter, supernatants were taken for a VEGF ELISA. Results are expressed as pg/ml of VEGF and are means ±S.D. (n = 4). *p<0.05, ***p<0.001 considered statistically significant when compared to the vehicle treated control values; ^#p<0.05, ^###p<0.001 when compared to the S1P-treated values.