Methods Aila toxicity and proliferation were determined by MTT and

Methods: Aila toxicity and proliferation were determined by MTT and colony forming methods, respectively. Cell cycle was determined by cytofluorimetric analysis through PI staining method. Apoptosis was detected using Annexin V and PI double staining followed by quantitative flow cytometry. Expressions of Nrf2, Yap, c-Myc, and house-keeping genes were determined by western blot with specific antibodies. Cell migration was detected by wound healing and Boyden chamber analysis.
Results: Aila inhibited the growth of sensitive and CDDP-resistant Bafilomycin A1 cancer cells with the same effec-tiveness. On the contrary, the growth of HK-2 cells was only slightly reduced by Aila. Cell cycle analysis revealed an accumulation of Aila-treated bladder cancer cells in the G0/G1 phase. Interestingly, Aila strongly reduced Nrf2 expression in these cell lines. Moreover, Aila significantly reduced YAP, and c-Myc protein expression. The random and the oriented migration of bladder cancer cells were strongly inhibited by Aila treatment, in parti-cular in CDDP-resistant cells.
Conclusion: Aila inhibited proliferation and invasiveness of bladder cancer cells. Its high effectiveness in CDDP resistant cells could be related to the inhibition of Nrf2, YAP, and c-Myc expressions. Aila could represent a new tool to treating CDDP-resistant bladder cancers.
Abbreviation: Aila, ailanthone; CDDP, cisplatin; Nrf2, NF-E2-related factor 2; YAP, yes-associated protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide; Keap1, Kelch-like ECH-associated protein 1; ARE, antioxidant response element; EpRE, electrophile response element; RPMI 1640, Roswell Park Memorial Institute medium; FBS, fetal bovine serum; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulfate; TBS, tris-buffered saline; GAPDH, gly-ceraldehyde 3 phosphate dehydrogenase; GSTA4, glutathione S-transferase A4; PI, propidium iodide; FITC, fluorescein isothiocyanate
Corresponding author.
E-mail address: stefania.pizzimenti@unito.it (S. Pizzimenti).
1 Martina Daga and Stefania Pizzimenti contributed equally to this work. 2 Co-last authors.
Introduction
Ailanthone (Aila) [(1β,11β,12α)-11,20-Epoxy-1,11,12-trihydrox-ypicrasa-3,13(21)-diene-2,16‑dione] is a natural active compound iso-lated from the plant Ailanthus altissima (Bray et al., 1987). Aila has a wide spectrum of biological activities, it is traditionally used to treat ascariasis, diarrhea, spermatorrhea, bleeding and gastrointestinal dis-eases, and it has been found to have anti-inflammatory activity (Kim et al., 2015). Aila has been shown to possess an “in vitro” growth-inhibitory effect against several cancer cell lines (Wang et al., 2016), but the mechanisms involved in the antiproliferative activity of Aila are not completely elucidated and they seem to be related to the cancer cell type. Indeed, in some cell models Aila induced G0/G1-phase cell cycle arrest, and triggered DNA damage and apoptosis pathway (Zhuo et al., 2015), in others, Aila induced G2/M phase cell cycle arrest and apop-tosis through downregulation of Bcl2 and upregulation of Bax (Chen et al., 2017). Ni et al. (2017) found that Aila inhibited the growth of several lung cancer cells through repression of DNA replication via RPA1 down-regulation. He et al. (2016) demonstrated that Aila was a potent inhibitor of androgen receptor and it was able to overcome re-sistance in castration-resistant cancer cells through the binding with the co-chaperone p23 protein.
Urothelial carcinoma of the bladder is a common malignancy in men. At the initial diagnosis, about 30% of tumors have already in-filtrated the bladder muscle wall and are classified as muscle-invasive bladder cancers. Muscle-invasive bladder cancer is associated with poor prognosis. Standard of care for muscle-invasive bladder cancer is cy-stectomy combined with platinum-based chemotherapy regimens (Madersbacher et al., 2003). The clinical benefit of cisplatin-based chemotherapy is limited and the majority of the patients eventually develop the cisplatin-resistant disease (Shah et al., 2011). Thus, the identification of novel agents able to overcome this resistant disease is an urgent and unmet need.