Meccanismi di azione dell'Ailantone alla base della downregolazione dell'espressione proteica di Nrf2 e YAP in cellule di carcinoma ovarico e della vescica resistenti al cisplatino

Chemoresistance represents one of the main obstacles in the successful treatment of many types of cancers, including ovarian and bladder cancers. One of the strategies adopted by cancer cells to escape to the toxic effects of ROS-generating chemotherapeutic agents such us cisplatin (CDDP) is the development of a redox adaptation based on the upregulation of their antioxidant capacity. Among the several players involved in this adaptive response, an important role is attributed to the transcription factor Nrf2 and to the protein YAP and the search for their inhibitors is an active field of research. Ailanthone (Aila), is a natural active compound extracted from the plant Ailanthus altissima that was recently found to exert an anticancer effect on CDDP-resistant bladder cancer cells by the downregulation of Nrf2 and YAP expression. In this study, we aimed to investigate Aila effect on a CDDP-sensitive (A2780) and CDDP-resistant (A2780/CP70) ovarian cancer cell line. Moreover, we tried to elucidate the mechanisms of action of this compound, extending our analysis also to a CDDP-sensitive (253J B-V) and CDDP-resistant (253J B-V C-r) bladder cancer cell line. Our results demonstrated that Aila had a strong inhibitory effect on viability (MTT assay) and growth (colony forming assay) of A2780 and A2780/CP70 cells, with a higher effectiveness compared to CDDP. Boyden chamber invasion assay also demonstrated Aila ability to affect cell invasion, particularly in the A2780/CP70 cells. Western blot analysis revealed that Aila was able to trigger a strong downregulation of Nrf2 and YAP protein expression and of their transcriptional activities, represented by the expression of GSTA4 and survivin, Nrf2 and YAP targets, respectively. Such effect was particularly important for the resistant cell line, which presented a basal protein expression of Nrf2 and YAP higher than the sensitive counterpart. Moreover, we found that Aila mechanism of action underlying the downregulation of Nrf2 and YAP was not related to a decrease of their transcription, since mRNAs levels, quantified by qRT-PCR, resulted increased. The use of the proteasome inhibitor MG132 demonstrated that the reduction of protein levels of Nrf2 and YAP was instead due to a post-translational mechanism involving protein degradation, common to ovarian and bladder cancer cells. We also demonstrated that Aila-mediated Nrf2 proteasomal degradation was associated to an increase of Keap1, the Nrf2 negative regulator, and a decrease of p62, that is a Nrf2 target but at the same time able to drive Keap1 to autophagic degradation. Regarding instead YAP proteasomal degradation induced by Aila, we found it to be related to an increase of its phosphorylation, verified by the analysis of pYAP expression. In addition, we found that the expression of the deubiquitinase UCHL1, which was increased in CDDP-resistant ovarian and bladder cancer cells, was downregulated upon Aila treatment. This datum suggests that UCHL1 could be involved in chemoresistance and in Aila-mediated mechanism of protein degradation. In conclusion, our study reported Aila anticancer effect in ovarian cancer cells and the existence of a post-translational mechanism underlying Aila downregulation of Nrf2 and YAP, common to ovarian and bladder cancer cells. These findings overall suggest that Aila may be a promising agent for the treatment of chemoresistant cancers characterized by high Nrf2 and YAP expression.