L'Ailanthone influisce sull'espressione di Nrf2 e YAP nelle cellule tumorali di ovaio e vescica, sensibili e resistenti al trattamento con CDDP.

Ailanthone (Aila), is a quassonoid extracted from the plant Ailanthus Altissima, which has been shown to possess an in vitro growth-inhibitory effect against several cancer cell lines. Advanced bladder cancer and ovarian cancers are diseases characterized by a frequent onset of resistance to platinum salts-based therapy. The cisplatin (CDDP) resistance is accompanied by an increase in Nrf2 and YAP protein expressions, which contributes to conferring resistance. Recently, it has been demonstrated a cross-talk between Nrf2 and YAP. In this thesis we analyzed the antitumor effect of Aila in sensitive and CDDP-resistant bladder and ovarian cancer cells and the molecular mechanisms involved in Aila activity. Sensitive and CDDP-resistant 253J B-V and 253J bladder cancer cells, CDDP-resistant T24 bladder cancer cells, A2780 sensitive, and CP70 CDDP-resistant ovarian cancer cells, and HK-2 human renal cortex cells were used. Cells were treated with diverse concentrations of Aila and proliferation, cell cycle, mRNA and protein expressions were determined. Moreover, MG132, a proteasomal inhibitor, was added to control and Aila-treated 253J B-V CDDP-resistant cells to verify the Aila effect on proteasomal degradation. Aila toxicity and proliferation were determined by MTT and colony forming methods, respectively. Cell cycle was determined by cytofluorimetric analysis through PI staining method. Expressions of Nrf2, Yap, c-Myc, Keap1, P-YAP, and house-keeping genes were determined by western blot with specific antibodies. Nrf2 and YAP mRNA expression was determined in CDDP-resistant 253J and 253J B-V cells by RT-PCR. Nrf2 and YAP protein expression in MG 132-treated CDDP-resistant 253J B-V cells was determined by western blot. Bladder cancer cell migration was detected by wound healing and Boyden chamber analysis. Aila inhibited growth of sensitive and CDDP-resistant bladder and ovarian cancer cells with the same effectiveness. 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 of cell cycle. Aila strongly reduced Nrf2 and YAP protein expression in all cancer cell lines. Moreover, Aila significantly reduced c-Myc protein expression in bladder cancer cells. On the contrary, mRNA expression of Nrf2 and YAP genes was increased in bladder cancer cells after Aila treatment, indicating that Aila inhibitory effect was not dependent on mRNA transcription. To deepen the mechanism involved in Aila inhibition of Nrf2 and YAP protein expression, we treated 253J B-V CDDP-resistant cells with MG132, a proteasomal inhibitor. Results demonstrated that in presence of MG132 the Nrf2 and YAP expression was strongly increased in Aila-treated cells, thus confirming that Aila treatment increased Nrf2 and YAP proteasomal degradation. According to this results we found an increase of protein expression of Keap1, the Nrf2 inhibitor, and P-YAP, the inactive form of YAP which is driven to proteasomal degradation. The random and the oriented migration of bladder cancer cells were strongly inhibited by Aila treatment, in particular in CDDP-resistant cells. Our results demonstrated that Aila, is able to inhibit proliferation and invasiveness of cancer cells through the inhibition of Nrf2 ,YAP and c-Myc expressions. The down-regulation of Nrf2 and YAP expression can be ascribed to the increase of protein degradation. In conclusion Aila demonstrated favourable drug-like properties due to low toxicity, also demonstrated by the low cytotoxic effect in kindey normal cells.