Figure 2.

Treatment of Lovo and HCT116 cells with Genz lead to reduced GSL expression and cell cycle arrest. (A) Formula of the GCS inhibitor Genz-123346 (N-[(1R,2R)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-1-hydroxy-3-pyrrolidin-1-ylpropan-2-yl]nonanamide). (B,C) As shown by thin layer chromatography (TLC), GSL-synthesis was almost completely disrupted by treatment of Lovo (B) and HCT116 cells (C) with 1 µM Genz in the culture medium after 4 and 6 days; x, no GSL-positive band; running solvent for neutral GSLs was CHCl₃/CH₃OH, H₂O, 62.5:30:6 (v/v) and for acidic GSLs CHCl₃/CH₃OH, 0.2% CaCl₂, 60:35:8 (v/v). (D) Anti-Gb₃Cer immune overlay of the neutral GSL-fraction from Lovo and HCT116 cells. Chemical staining with orcinol reagent (left) and immune overlay with anti-Gb₃Cer antibodies. HCT116 cells expressed globosides in addition to hexosylceramides and lactosylceramides, which were not detectable in Lovo cells. (E,F, quantification) Cell cycle arrest of the Lovo cells in S and G2/M phases occurred after treatment with 1 µM Genz (G,H, quantification). A significant influence on the cell cycle of HCT116 cells as shown by FACS analysis occurred at 5 µM Genz and higher concentrations. (I–L) One micrometer of Genz inhibited growth of tumor spheroids from Lovo (I,J, quantification) and HCT116 cells (K,L, quantification). Note: HCT116 microspheres broke apart with elevated Genz concentrations after longer incubation period (K) and could therefore not be included in the calculations (L, nd, not done); each data point on the graphs represents one biological replicate; scale bars, 100 µM; significances are *, p ≤ 0.05; **, p < 0.01; ***, p < 0.001.