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  • br Western blotting br T


    2.7. Western blotting
    T24 or 253 J cells (5 × 105 cells) were seeded in 10 cm petri dishes in complete medium and allowed to attach for 24 h. Cells were treated with 20 μM of compounds, TSA (0.16 μM) or 1% DMSO for 24 h. At the end of the incubation period, cells were washed twice with PBS 1x and harvested by scrapping with Laemmli buffer plus protease inhibitors (NaF, PMSF and protease inhibitor cocktail), followed by denaturation at 95 °C for 5 min and frozen at −20 °C. Each sample were resolved on a 15% SDS PAGE followed by transfer to a PVDF membrane. The samples were probed with the primary antibody rabbit anti-histone H4 lysine 8 acetylated (1/6000) and the following day a secondary antibody goat-anti-rabbit HRP (1/2000) was employed. The blots were developed using an enhanced chemiluminescence ECL kit. Loading control was performed incubating the membranes overnight with an anti-H4 total histone made in Rabbit (1/500) and developed in the same way as for the anti-acetylated H4 histone antibody.
    2.8. Image processing and data analysis
    Image processing was performed with ImageJ software using the indicated plugins. Data analyses and statistical calculations were per-formed using One-way ANOVA followed by Dunnett’s multiple com-parisons test and Two-way ANOVA followed by Bonferroni’s multiple comparisons test.
    3. Results
    3.1. Anti-proliferative activity
    Anti-proliferative activity by MTT of the nine studied compounds and TSA was assayed in the normal cell line V79-4 and in two Fulvestrant cancer cell lines T24 and 253 J in the absence or presence of cisplatin. A dose response curve to cisplatin was used to determine the IC50 values in V79-4, T24 and 253 J cell lines. As shown in the Fig. 3a) the IC50 of cisplatin in V79-4 cell line was > 160.0 μM, the T24 cell line is  Environmental Toxicology and Pharmacology 69 (2019) 9–15
    sensitive to cisplatin and presents a IC50 of 20.5 ± 1.1 μM while the 253 J cell line, that presents a IC50 of 97.8 ± 1.1 μM, is resistant against cisplatin. Considering the IC50 values, a subtoxic dose of cis-platin, 10 μM for V79-4 and T24 and 20 μM for 253 J, was chosen to determine the sensitizer activity of the phenazine dioxides. In this sense, the anti-proliferative activity of the phenazine dioxides at 5 μM in absence (black bars) or presence (grey bars) of the subtoxic doses of cisplatin was evaluated in normal and bladder cancer cells (Fig. 3b) and c)).
    Phenazine dioxides were evaluated in V79-4 cells at 5 μM in absence and presence of cisplatin to know their toxicity toward normal cells. In this regard, we observed that only compound 1c and the co-treatments of 3a and 3b with cisplatin presented a slight toxicity as outlined in the Fig. 3b).
    As Fig. 3c) indicates, compounds that showed better anti-pro-liferative activities towards T24 cells in absence of cisplatin were 1a–d. However, when these compounds were co-incubated with cisplatin, they were not able to increase their cancer cell growth inhibition. On the other hand, the anti-proliferative activity of the more complex phenazine dioxide derivatives (2a-c and 3a-b) was very poor, but, when these compounds were co-incubated with cisplatin at the subtoxic dose 10 μM, the anti-proliferative activity increased considerably. This results indicate a sensitizer effect of the more complex phenazine di-oxides to the cisplatin treatment in T24 cells.
    In the case of 253 J cell line, none of the phenazine dioxide deri-vatives present anti-proliferative activity when incubated alone. However, when the compounds were co-incubated with subtoxic dose of cisplatin 20 μM the anti-proliferative activity was increased. In par-ticular, compounds 1a, 1b, 2c and 3a showed a significant increase of the anti-proliferative activity respect to the control treated with cis-platin alone, indicating a sensitizer effect to the cisplatin treatment.