Figure 1.
Inhibition of the interaction between Bcl-2 or Bcl-XL and fluorescent Puma BH3 peptide measured by the decrease of fluorescence polarisation as a function of S44563-2 concentration.
Three independent experiments are presented. FP data are presented in millipolarization units (mP). For each experiment, measures are assessed in triplicate.
Figure 2.
Bcl-2, Bcl-XL, and Mcl-1 Immunohistochemistry.
Bcl-2, Bcl-XL, and Mcl-1 expression determined by immunohistochemical analyses of the 4 human UM xenografts (between 3 to 5 tumors have been studied per condition).
Figure 3.
In vitro apoptosis induction by S44563.
A and B. Apoptosis induction by S44563. The MP41, MM26, and MM66 cell lines were incubated with 17 µM and 34 µM S44563 for 24 hours, after which the samples were labeled with DAPI/annexin V-FITC (A) or JC1 (B). The proportion of annexin V-positive cells and low Δψm cells was indicated in C and E, respectively. A two-way ANOVA with Bonferroni post-test was then performed (* p<0.05). C. Cell cycle analyses after S44563 exposure (17 or 34 µM for 24 h) in the 3 UM cell lines. Cell cycle analysis was determined by labeling the DNA with propidium iodide. Each of the three lines was treated by 17 µM or 34 µM of S44563. The proportion of cells (%) in different cell cycle phases was compared with the control (untreated). Two-way ANOVA with Bonferroni post-test was then performed (* means a p<0.05). D. Cell cycle analyses of the xMP41 UM cell line. I and J. Measurement of autophagy on the 3 UM cell lines after S44563 exposure (2 or 4 µM for 24 h). After S44563 treatment, the amount of active and inactive protein LC3 was determined and reported to ß-actine. The quantity of total LC3 protein was calculated to study the activation of LC3. Results were presented as percentage and total amount of active cleaved LC3, relative to β-actin. A two-way ANOVA with Bonferroni post-test was then performed (* means a p<0.05).
Figure 4.
In vivo responses of UM PDXs to S44563 administered alone or in combination with fotemustine.
A. MP41 xenograft was treated either by S44563 at 50/kg (▪) or 100 mg/kg (□) 5 days a week for 5 weeks. B. Overall survival of mice bearing MP41 tumors treated by S44563 (O), fotemustine (▪), and concomitant fotemustine+S44563 (⧫). C. MP77 xenograft was treated by S44563 at 100 mg/kg (□), fotemustine 15 mg/kg (Δ), or both (▴). D. MM66 xenograft was treated by S44563 at 50 mg/kg (□), fotemustine 30 mg/kg (Δ), or both (▴). Mice in the control group (•) received 0,3 ml of the drug-formulating vehicle with the same schedule as the treated animals. Tumor growth was evaluated by plotting the mean of the RTV (relative tumor volume) ± SD per group. Between 8 to 12 mice per group were included in in vivo experiments.
Table 1.
TGI and complete remission induced by a combination of S44563 and fotemustine.
Table 2.
Overall and median survival after combination of S44563 and fotemustine.
Figure 5.
Immunohistochemical analyses under S44563 administration.
Immunohistochemical analyses of the MM66 UM xenograft after S44563, fotemustine, or S44563+fotemustine.