Figure 1.
U2OS cells are sensitive to genetic and pharmacological Wnt pathway modulation.
(A) U2OS cells infected with LVTCF-Luciferase and LVTA-Renilla were exposed to different amounts of SEN461 and the Wnt transcriptional activity was measured 24 h later. (B) The effect on Wnt transcriptional activity after inducible (10 ng/ml of doxycyclin) lentiviral infection with TCF4dn was measured by reporter activity. (C) Representative images for U2OS cells expressing Axin1-GFP (green) stained for P-β-catenin S33/S37/T41 (red) and their co-localization (yellow). Scale bar = 10 µm. U2OS cells, transiently transfected with GFP-tagged AXIN1 and stimulated with Wnt3a CM were either treated overnight with 10 µmol/L of SEN461 or with the inactive analogue SEN973 at the same concentration for the same length of time. (D) The bar-graph showed the result of three independent experiments where the number of Axin1/P-β-catenin co-localization puncta was counted in 5 different fields and normalized vs. untreated-Wnt3a-CM control. Data represent means ± SEM. ***, P<0.05 relative to control cells (Student t test). (E) Quantitative RT-PCR assay to measure the effect of SEN461 treatment after Wnt3a CM stimulation on the mRNA level of the Wnt target gene AXIN2. Data represent means ± SEM. ***, P<0.0001 relative to Wnt3a stimulated cells (Student t test).
Figure 2.
Effects of SEN461 on growth and on Wnt pathway components in sarcoma cells.
(A) Summary of the half-maximal inhibitory concentration (IC50) for the four sarcoma lines examined in soft agar is shown, and ranked from lowest to highest. (B, C) IC50 for U2OS and HT-1080 cancer cells after XAV939 and SEN973 is shown, determined from the soft agar assay. Soft agar data (from two independent experiments) represent means ± SEM. (D, E) Western blotting analysis of Axin1 and β-catenin levels after SEN461 treatment in 143B and G292 osteosarcoma cells.
Figure 3.
SEN461 effects on Wnt molecular components in sarcoma cells.
(A, D) Effect of SEN461 or XAV939 on Wnt target genes mRNA expression measured by quantitative RT-PCR in U2OS and HT-1080 cells after 10 µmol/L treatment. Data (collected from three independent experiments) represent means ± SEM. **, P<0.05 ***, P<0.005 relative to control cells (Student t test). (B, E) Western blotting analysis of U2OS and HT-1080 cell lines treated with SEN461 or XAV939 overnight. Cytoplasmic cell lysates were probed with anti-Axin1, anti-TNKS1/2 and anti-Tubulin as loading control. (C, F, G) Western blotting analysis of U2OS and HT-1080 treated with SEN461 or XAV939 overnight. Cytoplasmic and nuclear cell lysates were then probed with anti-c-Myc, anti-p21 and anti-β-actin as loading control. The asterisk represents a background band migrating below the p21 band.
Figure 4.
Axin1 over-expression phenocopies SEN461 activity at molecular and phenotypic level.
(A) U2OS and HT-1080 cells were transiently transfected with AXIN1 cDNA. Cell lysates were then analyzed by Western blotting and probed with anti-Axin1, anti-c-Myc and anti-β-actin as loading control. (B, C) Ectopic expression of AXIN1 in U2OS and HT-1080 cells produced a strong reduction in their ability to grow in anchorage-independent fashion. Soft agar data (from two independent experiments) represent means ± SEM. *, P<0.05 ***, P<0.0005 relative to control cells (Student t test).
Figure 5.
In vivo effects of SEN461 in HT-1080 xenograft model.
(A) Pharmacokinetics and pharmacodynamics of SEN461 in mice. Concentration of SEN461 in tumors (black line) and relative c-MYC human mRNA values (columns) in HT-1080 xenograft tumors at 1, 4 and 8 hours after 30 mpk BID oral administration of SEN461. The data are presented as mean ± SEM (n = 5) and T0 represents c-MYC human mRNA value at 1 hour after vehicle administration in the control group. (B) mRNA level for the human VEGFA gene in HT-1080 xenograft tumors at 1 hour after 30 mpk BID of SEN461. The data are presented as means ± SEM (n = 5). (C) Antitumor activity of SEN461 in a HT-1080 xenograft tumor model. Treatment groups (5 mice per group) received 30 mg/kg twice a day for seven consecutive days.
Table 1.
Plasma and tumor exposure of SEN461 in mice.