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  • br Statistical analysis br Data were


    Statistical analysis
    Data were expressed as means ± SD. Significance between experi-mental groups was determined by one-way ANOVA followed by the 
    Bonferroni multiple comparison post-test using GraphPad InStat soft-ware (San Diego, CA, USA). Values of p ≤ 0.05 were considered sta-tistically significant.
    Aila effect on AWD 131-138 cancer cell growth and colony forming
    To analyze the ability of Aila to affect cell growth and colony forming of sensitive and CDDP-resistant bladder cancer cells, 253J B-V and 253J B-V resistant to CDDP (253J B-V C-r), 253J and 253J resistant to CDDP (253J C-r), and T24 (intrinsically CDDP resistant) cells were exposed to different concentrations of Aila. We analyzed the resistance of the T24 cell line, by comparing the growth of this line with the sensitive and the resistant 253J B-V cells exposed to CDDP and to other DNA damaging agents such as the anthracycline antibiotic, doxor-ubicin, and the Podophyllum peltatum toxin, etoposide. T24 cells were more resistant toward cisplatin, doxorubicin, and etoposide with re-spect to 253J BV cells, but less resistant than 253J B-V C-r cells. These experiments confirmed the intrinsic resistance of T24 cells to other DNA damage agents and demonstrated that the resistance of 253J B-V C-r cells was also extended to doxorubicin and etoposide (Fig. 1 supple-mentary data).
    In addition, to analyzing Aila toxicity in normal cells, we exposed HK-2 cells (human kidney-2) to the same Aila concentrations used in treating cancer cells. HK-2 cells were obtained by a primary proximal tubular cell culture from normal adult human renal cortex and exposed to a recombinant retrovirus containing HPV 16 E6/E7 genes. These cells retain a phenotype indicative of well-differentiated primary proximal tubular epithelium and retain functional characteristics of proximal tubular epithelium (Ryan et al., 1994). Results obtained demonstrated that the Aila was more effective than CDDP in reducing cell growth of 253J B-V cells and, in particular, Aila
    Fig. 2. MTT and colony forming assays in 253J and 253J C-r cells. (A) MTT assay. Results are expressed as percent of control values, obtained after 72 h from the treatment with Aila or CDDP. Results are the mean ± SD of four separate experiments. **p ≤ 0.01 vs.sensitive cells. (B): Number of colonies expressed as percent of control number. Results are the mean ± SD of three separate experiments. **p ≤ 0.01 vs. sensitive cells. (C) Representative colony forming assay.
    reduced the growth of 253J B-V C-r cells to the same extent as the sensitive cells (Fig. 1A). Colony forming assay confirmed the effec-tiveness of Aila treatment in sensitive and CDDP resistant 253 J B-V cells (Fig. 1B and C). The cytotoxic and antiproliferative effect of Aila was demonstrated in 253J cells, also (Fig. 2). The reduction of proliferation after 72 h from the treatment with 0.5 and 1 µg/ml of Aila was similar in sensitive and CDDP resistant cells (Fig. 2A). The colony forming assay also confirmed the effectiveness of Aila in 253J C-r cells (Fig. 2B and C). The third cancer cell line employed in our experiments was the T24 cell line. Analogously to that observed in the previous cell lines Aila reduced T24 cell growth and colony forming to a higher extent than CDDP (Fig. 3A, B and C). Interestingly, Aila displayed a very low efficiency in HK-2 cells, indicating that this substance is less toxic for normal cells than for cancer cells (Fig. 3D, E and F). These data are according to that reported by He et al. (2016), indicating a normal kidney morphology after Aila treatment of tumor-bearing mice. On the contrary, CDDP highly re-duced the growth of HK-2 cells.
    Effect of Aila on cell cycle and apoptosis of bladder cancer cells
    To deepen the study of the antiproliferative activity of Aila we performed an analysis of cell cycle and apoptosis in sensitive and CDDP-resistant 253J B-V cells. In both cell lines, Aila (from 0.1 to 1 µg/ml) induced a significant increase in G0/G1 cells (Fig. 4A and B), whereas treatments with the same concentrations of Aila did not induce an in-crease of apoptotic cells (data not shown).
    Aila effects on Nrf2 and Nrf2 target gene expressions
    Since the reduction of proliferation by Aila could depend on the reduction of Nrf2 expression, we analyzed Nrf2 expression in Aila treated cells. The analysis was performed after 24 and 48 h from the treatment. Results demonstrated that Nrf2 expression was inhibited 
    until 48 h in a concentration-dependent way in both sensitive and re-sistant bladder cancer cells (Fig. 5A and B). The reduction of Nrf2 protein was confirmed by the contemporary reduction of GSTA4, a Nrf2 target gene (Fig. 6A and B).