Meccanismi coinvolti nel crosstalk tra Nrf2/YAP ed effetto dell'inibizione Nrf2 nelle cellule del cancro della vescica sensibili e resistenti al trattamento con cisplatino

Many anticancer therapies, including the use of antineoplastic drugs such as cisplatin (CDDP), are able to induce oxidative stress. Indeed, cancers display a higher level of oxidative stress than the normal counterpart, thus they are more sensitive to apoptosis induced by a further increase of oxidative stress, with respect to normal tissues. However, a continuous oxidative stimulation provokes an adaptive response in cancer cells that, over time, increases the synthesis of antioxidant molecules, which contribute to the onset of drug-resistance. The high levels of antioxidant molecules are due to an increase of Nrf2 transcription factor, the master regulator of cellular antioxidant response. Indeed, it has been demonstrated that the Nrf2 expression was increased in chemo-resistant and radio-resistant cancer cells. YAP protein is another important player involved in chemoresistance. Analogously to that observed for Nrf2 protein, YAP protein was also increased in bladder and ovary CDDP-resistant cancer cells. The aim of this work is to clarify whether a cross-talk exists between Nrf2 and YAP, and if it can contribute to the maintenance of the chemoresistance and antioxidant potential in CDDP-resistant bladder cancer cells. For this study, 253J and 253J CDDP-resistant (C-r) cells were used. In resistant cells, which expressed high YAP and Nrf2 expression levels, YAP was knocked down by using pGIPZ lentiviral vector encoding non-silencing control shRNA or YAP shRNAs, and Nrf2 was silenced with Nrf2-specific siRNA. FOXM1 specific siRNA was used to silenced FOXM1. GSH/GSSG ratio was determined by the Owens and Belcher method. Cell growth was tested by MTT method and Colony Forming assay. Protein expression was detected through Western Blot analysis and RNA level through qRT-PCR. Apoptosis was detected by PARP and cleaved PARP expressions and cytofluorimetric analysis. 253J C-r cells displayed higher GSH/GSSG ratio, and Nrf2 and YAP expressions than 253J cells. YAP knockdown in 253J C-r caused a reduction of GSH/GSSG ratio and Nrf2 mRNA and protein. Furthermore, a down-regulation of FOXM1 protein was observed. The silencing of FOXM1 reduced the expression of Nrf2 protein. On the other hand, the silencing of Nrf2 in 253J C-r caused a reduction of YAP mRNA and protein. A similar result was also achieved by performing a depletion of GSH by treatment with BSO, indicating that YAP expression is negatively regulated by GSH depletion. Taken together, results suggested a possible crosstalk between YAP and Nrf2 proteins. To test a new compound able to inhibit Nrf2 expression and to reduce the growth of resistant cells, we treated 253J and 253J C-r cells with ailanthone, a natural compound extracted from Ailanthus altissima tree. We demonstrated that ailanthone was an effective inhibitor of Nrf2 expression, and that it strongly reduced the growth of CDDP-resistant cells. These results confirm the importance of Nrf2 reduction in overcoming resistance in cancer cells, and sustain the indication of Nrf2 and YAP protein as suitable targets to overcome the CDDP resistance in bladder cancer cells.