Briefly, the cells were incubated for 1 h at the end of treatment with 20 ng/ml Hydroethidine stock solution

(2,5 mg/ml). At the time of processing the cells were RO4929097 order scraped, washed twice with PBS and the pellet was resuspended in 1 ml PBS. The dye accumulation was analysed by FACScan flow cytometer (FACScan, Becton Dickinson) SGC-CBP30 mw by the CellQuest software. For each sample, 2 × 104 events were acquired. Analysis was carried out by triplicate determination on at least three separate experiments. Statistical analysis All data are expressed as mean + SD. Statistical analysis was performed by analysis of variance (ANOVA) with Neumann-Keul’s multiple comparison test or Kolmogorov-Smirnov where appropriate. Results Effects of DOXO and 5-FU on H9c2 and HT-29 cell proliferation and apoptosis We studied the effect of increasing concentrations of DOXO and 5-FU in presence or not of LF on growth inhibition of HT-29 and H9c2 cells by MTT assay as described in “Materials and Methods”. We have found a dose and time-dependent growth inhibition in both cell mTOR inhibitor lines. In details, the IC50 (50% inhibitory concentration) value of 5-FU was 4 μM and 400 μM in HT29 and H9c2, respectively (Figure 1 and Table 1). Moreover, LF potentiated growth inhibition induced by 5-FU. In fact, IC50 of HT-29 and H9c2 cells was 2 μM and 43 μM, respectively. These results suggest, as expected, that the

colon cancer cell line HT29 was more sensitive to 5-FU than H9c2 normal cells (Table 1). Interestingly, these concentrations of 5-FU can be reached in vivo after the routinely used ways of administration of this agent in the clinical practice [34]. Figure 1 Effects of DOXO and 5-FU on H9c2 and HT-29 cell proliferation. Growth inhibition of H9c2 (A-C) and HT-29 (D-F) cells treated with 5-FU alone (A and D) or combined with LF (B and E) or DOXO alone (C and F) for 24, 48 and 72 h, evaluated by MTT assay and expressed as a percentage of untreated cells. Data are reported as mean of three independent experiments ± SD. The experiments were repeated at least three times and gave always similar results. Table 1 IC 50 s of

the different drugs in cardiocytes and colon cancer cells Drugs IC 50 H9c2 IC 50 HT-29 5-FU 400 μM ± 0.06 4 μM ± 0.01 5-FU + 10 −4 M LF 43 μM ± 0.01 2 μM ± 0.009 DOXO 0.12 μM ± 0.001 0.31 μM ± 0.002 On mafosfamide the other hand, H9c2 cells appeared to be more sensitive to DOXO than HT-29. In fact, the IC50 of DOXO was 0.12 μM and 0.31 μM on HT-29 and H9c2, respectively (Figure 1). Thereafter, we have evaluated the effects of the different treatments in inducing apoptosis, assessed by FACS analysis after double labelling with Annexin V and PI. We have found that the treatment with DOXO induced apoptosis in only about 8% of H9c2 cell population (Figure 2 and Table 2), while the treatment with 5-FU alone induced apoptosis in about 38% of H9c2 cell population compared to 5% of untreated cells as demonstrated with FACS analysis.