The medium from cells grown in serum-free medium was analyzed by HPLC-MS; however, the levels of extracellular S1P were below the detection limit of the assay (data not shown)

The medium from cells grown in serum-free medium was analyzed by HPLC-MS; however, the levels of extracellular S1P were below the detection limit of the assay (data not shown). anti-proliferative or anti-migratory effects of the siRNAs. Consistent with these results, differential affects of SK1- and SK2-selective siRNAs on signaling proteins including p53, p21, ERK1, ERK2, FAK and VCAM1 indicate that SK1 and SK2 have only partially overlapping functions in tumor cells. Overall, these data indicate that loss of SK2 has stronger anticancer effects than does suppression of SK1. Consequently, selective inhibitors of SK2 may provide optimal targeting of this pathway in cancer chemotherapy. Relative expression of SK mRNAs in 3 TRi-1 cancer cell lines. Data are presented as percentage of SK1 in A498 cells. and 2Expression above was quantified by imaging pixel intensity/cell, and the relative protein expression levels of Mouse monoclonal to IgM Isotype Control.This can be used as a mouse IgM isotype control in flow cytometry and other applications SK1 (open TRi-1 bars) and SK2 (filled bars) were calculated relative to control (siNC). The enzymatic activity of SK1 was decided relative to control (siNC-treated) cells. Data represent the mean SEM of three impartial experiments. *p 0.05, **p 0.01, TRi-1 ***p 0.001 versus control. Effects of SK Knockdown on Sphingolipids SK convert sphingosine to S1P; however, it is well established that the metabolism of sphingolipids is usually a dynamic process (32), so that an altered flux through SK may modulate several sphingolipids. As shown in Physique 3, SK1-selective knockdown resulted in elevated levels of total ceramides (total Cer, approximately 70% increase from control) and all of the individual ceramide species measured, i.e. dihydro-C16-ceramide (DHC16-Cer), C24-ceramide (C24-Cer), C20:1-ceramide (C20:1-Cer), C20-ceramide (C20-Cer), C18-ceramide (C18-Cer), C14-ceramide (C14-Cer). Also, the level of S1P was decreased as expected, but there was a small decrease in sphingosine, indicating that the sphingosine was likely being converted to ceramides by ceramide synthase. In marked contrast, the selective knockdown of SK2 did not substantially affect the levels of most ceramide species, causing only a slight increase in total ceramide levels. Interestingly, selective knockdown of SK2 resulted in significantly elevated S1P levels, and this was largely abrogated when SK1 was simultaneously suppressed in the double knockdown cells. The medium from cells produced in serum-free medium was analyzed by HPLC-MS; however, the levels of extracellular S1P were below the detection limit of the assay (data not shown). Combined with the qPCR results described above, it seems likely that the elevated S1P level arises from the increased expression of SK1 TRi-1 in the SK2-selectively-depleted cells. These data indicate that SK1 is the dominant isoenzyme in A498 cells for the synthesis of S1P (on a total S1P mass basis). Open in a separate window Physique 3 Effects of SK siRNA TRi-1 transfection on sphingolipid profilesA498 cells were transfected with siRNAs as indicated, and sphingolipid analyses were performed 72 hr later. Bars indicate the ratio of the lipid mass relative to control (siNC). Data are mean SEM of three impartial experiments. *p 0.05, **p 0.01 versus control. Knockdown of SK Suppresses Cell Proliferation Because SK catalyze the production of mitogenic S1P, we evaluated the effects of their knockdown on cell cycle progression and proliferation. As shown in Physique 4and 4or MDA-MB-231 cells were harvested 72 hr after siRNA transfection (and 4Cells were harvested and immunoblotting was conducted with the indicated antibodies and quantified by densitometry. The expression of the indicated proteins is usually normalized to beta-actin. qPCR was performed to determine the expression of mRNA for ERK1 (open bars) or ERK2 (filled bars) relative to control (siNC) after normalization to GAPDH. Cell cycle histogram of siERK1 and siSK co-transfected cells. Data are mean SEM of three impartial experiments, except that represents a single experiment. *p 0.05, **p 0.01, ***p 0.001 versus control. As markers of cell survival and proliferation, AKT and ERK were also analyzed. Levels of pAKT were decreased dramatically by depletion of either SK1 or SK2 (Physique 5indicate that ablation of ERK1 did not alter responses to either SK siRNA. Taken together, the data suggest that SK2 has.