Aldoxorubicin and Temozolomide combination in a xenograft mice model of human glioblastoma

Figure 1. Cell viability of U87MG (A), A172 (B) and T98G (C) cells treated with Aldox, TMZ and their combination at the indicated concentrations for 72 h by MTT assay. Results shown are the mean % viability ± S.D. of three independent experiments. *P=<0.01; **=P<0.001 ***=P<0.0001 vs untreated cells; ###=P<0.0001 vs TMZ 100 μM-treated cells; ΩΩP< 0.001; ΩΩΩP< 0.0001 vs TMZ 200 μM-treated cells; °°°P<0.0001 vs Aldox-treated cells; ANOVA one-way followed by Bonferroni post-hoc test.

Figure 2. Pro-apoptotic effect of Aldox, TMZ and their combination (30 h) on U87MG (A), A172 (B) and T98G (C) cells. Upper panel: qualitative representation of TUNEL positivity; lower panel: quantitative representation of TUNEL positivity (mean % TUNEL positivity ± S.D) **P=<0.001; ***=P<0.0001 vs untreated cells; ##=P<0.001; ###=P<0.0001 vs TMZ 100 μM-treated cells; ΩΩP< 0.001; ΩΩΩP< 0.0001 vs TMZ 200 μM-treated cells; ANOVA one-way followed by Bonferroni post-hoc test. Experiments performed two times in triplicate.

Figure 3. (A) Drug stimulated P-gp ATPase activity was estimated by Pgp-GLOassay system and measured in terms of both change in luminescence, that reflects the ATP concentration (upper panel), and ATP consumption (nmol ATP consumed/mg P-gp/minute) (lower panel), that reflects the P-gp ATPase activity. Data expressed as mean ± SD. Experiments performed two times in triplicate. (B) The uptake of Aldox, with or without TMZ, was evaluated in MDCKII parental and P-gp transfected cells by flow cytometry, using the FL2-H channel. Upper panel: qualitative representation of flow cytometric detection of Aldox uptake; lower panel: quantitative representation of Aldox uptake (Mean Aldox Fluorescence ± S.D), ***=P<0.0001 vs untreated samples; ANOVA one-way followed by Bonferroni post-hoc test. Results representative of two independent experiments in triplicate.

Figure 4. (A) BLI signals throughout the experimental period, distinctly for group of treatment and day from injection (from day 7 to day 42). (B) Average IVIS values between experimental groups at day +42, +49 and +56. Data expressed as mean ± SD. Outliers were removed by the ROUT test (Q=1%). Statistical analysis was performed with Mann-Whitney U-Test (**=P<0.01; *=P<0.05). (C) Images of BLI acquisition of brain tumor on day 7 (start of treatments), day 42 (end of treatments) and day 56 (15 days after the end of treatments). The colorimetric scale represents the range of radiance values (red=highest value; blu=lowest value) which translates to tumor growth.

Figure 5. (A) Survival curves were compared between mice bearing GBM tumors, treated with vehicle, TMZ, Aldox or Aldox plus TMZ. Analysis was performed with Kaplan–Meier test. (B) BW measurements were used to evaluate the drug-related side effects. No statistical significant differences were reported between all experimental groups. Data expressed as mean ± SD.