Luteolin reduces cellular S1P by inhibiting SphK2.

(A) and (B) CC cells were pulsed with 3H-Sph for 2 h in the absence (white bar) or presence of 100 μM luteolin (grey bar). At the end, labeled S1P (A) and water (B) were evaluated in cells and medium, respectively. (C) CC cells were treated with 50–100 μM luteolin for 2h, and equal amounts of proteins were then analyzed for SphK1 and SphK2 proteins by immunoblotting. GAPDH was used as loading control. (D) SphK1 and SphK2 activities were assayed in the absence or presence of luteolin, using CC cell homogenate as enzyme source. Data are mean ± S.D. of three experiments in duplicate. **, p < 0.01 vs. control.</p

Luteolin increases ceramide level in CC cells, and induced elevation of ceramide leads to CC cell toxicity.

<p>(A) DEs and CC cells were treated with luteolin, and, after 24 h, cellular ceramide was quantified. (B) and (C) CC cells were treated with different concentrations of C2-Cer (B, square), C6-Cer (B, triangle) or D609 (C), and after 48 h cell viability was assessed by MTT. All data are the mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01 vs control.</p

Luteolin induces apoptosis in CC cells but not in DEs.

<p>(A) CC cells (CCs) and DEs were treated with different concentrations of luteolin, and after 48 h cell viability was assayed by MTT. Data are the mean ± SD of three independent experiments; *, p < 0.05 and **, p < 0.01 vs. control. (B) Representative microscopic images of DEs and CCs stained with Hoechst 33342 after treatment with 100 μM luteolin for 36 h. (C) Western blot analysis of Pro-caspase-3 and active Caspase-3 intracellular levels of DEs and CCs treated or not with 100 μM luteolin for 36 h.</p

Akt inhibition by luteolin is involved in its effect on ceramide metabolism.

<p>(A) CCs were treated with 50 and 100 μM of luteolin for 2 h and submitted to immunoblotting with anti-pAKT antibodies. β-actin was used as loading control. (B) Cells were submitted to pulse with ³H-Sph in the absence (CT) or presence of LY294002 for 2h. The levels of ceramide (Cer), sphingomyelin (SM) and glycosphingolipids (GSLs) are reported as mean ± S.D. of at least three independent experiments. **, p < 0.01 vs CT cells.</p

S1P protects CC cells from luteolin-induced toxicity by activating Akt.

<p>(A) Upper panel: CC cells were treated with 0.5–1 μM S1P for 2 h. Equal amounts of cell proteins were then analyzed for phosphorylated Akt (pAkt) by immunoblotting. Lower panel: CC cells were treated with different concentrations of S1P in the presence of 50 μM luteolin, and after 48 h cell viability was analyzed by MTT assay. (B) P-Akt/β-actin ratio (mean ± S.D) prior and after treatment of CC cells with 1 μM S1P and/or 50 μM luteolin for 2 h. *, p < 0.05 and **, p < 0.01 vs. untreated cells; #, p < 0.05 vs. luteolin-treated cells. A representative Western blot is reported in the lower part. (C) CC cells were incubated with luteolin alone (Lu) or in the presence of 1 μM S1P and/or 1 μM SEW2871; 5 μM W123; or 100 ng/ml PTX. (D) CC cells were incubated with luteolin in the absence or presence of 1 μM caged S1P without or with 100 ng/ml PTX for 48 hours. Cell viability was measured without (dark grey) or with (light grey) UV irradiation for 30 s. In (C) and (D), cell viability was determined by MTT assay, and the viability of luteolin-treated cells was regarded as 100%. Data are mean ± S.D. of at least two independent experiments. **, p < 0.01.</p

Luteolin inhibits ceramide metabolism to complex sphingolipids.

<p>(A) CC cells were pulsed with 25 nM ³H-Sph (upper panel) or 200 nM ³H-serine (lower panel) in the absence (white bar) or presence (grey bar) of 100 μM luteolin for 2 h. At the end the content of cellular radiolabeled ceramide (Cer), sphingomyelin (SM) and glycosphingolipids (GSLs) was measured. **, p < 0.01. (B) CC cells were incubated in the absence (CT) or presence of luteolin (LU), then with BODIPY-C₅-Cer and analyzed by fluorescence microscopy (bar, 10 μm). (C) CC cells were incubated without (a) or with (b) BFA (1 μg/ml) for 30 min, and then pulsed with ³H-Sph or ³H-serine with luteolin (100 μM) for 2 h. The ceramide/complex sphingolipid ratio is reported. Data are the mean ± S.D. of two independent experiments. **, p < 0.01.</p

Extracellular/intracellular S1P ratio in U87-MG and glioma stem cells.

<p>U87-MG and both GSC types were pulsed with 20 nM [³H]-Sph (0.4 µCi/ml) for 30 or 60 minutes. At the end, S1P was extracted from cells and media and analyzed as described in “Materials and methods”. The extracellular/intracellular ratio of S1P-associated radioactivity is shown, as the mean ± SD of at least three independent experiments.</p

Effect of TMZ on cell survival.

<p>Representative images of (A) U87-MG and U–SC, and (B) L0627 morphology after 48 h treatment with vehicle (0.1% DMSO) or 100 µM TMZ (+ TMZ). Images were viewed on a phase contrast microscope, and digital images were acquired (magnification, 10X; scale bar, 100 µm). (C) U87-MG and U–SC, and (D) L0627 cells were exposed to different concentrations of TMZ or vehicle. Cell viability was assessed after 48 h of treatment by MTT assay. Results are expressed as percentage of cell viability with respect to vehicle-treated cells (100%). Data are the mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01 vs vehicle-treated cells. (E) Cell lysates (60 µg of proteins) were analyzed by immunoblotting with anti-MGMT and anti-β-actin antibodies. 98G human glioblastoma cell lysates were used as control. The immunoblottings are representative of one out of three.</p

Role of S1P in GSC survival properties.

<p>U–SC (A) and L0627 cells (B) were exposed to 50-200 µM of TMZ alone (♦) or in combination with 4 µM SKI (●). After 48 hours of treatment, cell viability was assessed by the MTT assay. Results are expressed as percentage of cell viability with respect to vehicle-treated cells (♦) or cells treated with SKI alone (●) (100%). Data are the mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01 vs vehicle-treated cells.</p

Biosynthesis and fates of S1P in U87-MG and glioma stem cells.

<p>U87-MG, U–SC and L0627 cells were pulsed with [³H]-sphingosine (Sph) for the indicated periods of time. At the end, cells and media were processed and analyzed as described in “Materials and methods”. Panel A, total incorporated radioactivity and intracellular Sph. Panel B, radioactivity associated to total Sph phosphorylation, total S1P and S1P degradation (³H ₂O). Panel C, radioactivity incorporated into intracellular and extracellular S1P. Data are the mean ± SD of at least three independent experiments.</p