Lactacystin (Synthetic) br Fig Invasion assay at
Fig. 9. Invasion assay at 24 h in 253J B-V and 253J B-V C-r cells untreated (C) or treated with Aila. Data are expressed as percentages of inhibition of cell invasion versus the control invasion measured on untreated cells. The data of each assay was done from 5 independent experiments and shown as the mean ± SD.**p value ≤ 0.01 and *p ≤ 0.05 vs. C.
In the present study, Aila was found to be able to inhibit the pro-liferation of sensitive and CDDP resistant Lactacystin (Synthetic) cancer cells with the same effectiveness, while its effect in reducing the growth of normal kidney cells was lesser. Nrf2, YAP and c-Myc proteins play an important role in controlling proliferative capacity of cells. Besides the canonical Nrf2 role in or-chestrating antioxidant responses, accumulating evidence has estab-lished that Nrf2 sustains cell proliferation and migration (Rojo de la Vega et al., 2018). As a consequence, the down-regulation of Nrf2 ex-pression by Aila, could reduce the growth and the migration of cancer cells. Another naturally occurring quassonoid, brusatol, extracted from the aerial parts of the Brucea javanica plant, has been shown to inhibit Nrf2 and to sensitize cancer cells to several chemotherapeutic drugs (Ren et al., 2011). However, the brusatol-mediated inhibition of Nrf2 was transient, persisting only 8 h from the treatment (Olayanju et al., 2015). On the contrary, we demonstrated that after 48 h the reduction of Nrf2 expression by Aila was still present, as well as the reduction of the Nrf2 target gene GSTA4.
The high antiproliferative effect of Aila in CDDP-resistant cells could be sustained not only by the persistent inhibition of Nrf2 expression, but also by the simultaneous inhibition of YAP and Myc expression. Indeed YAP, through the activation of TEAD transcription factors, has been demonstrated to be implicated in the control of growth, oncogenic transformation, and epithelial-mesenchymal transition (EMT) (Zao et al., 2008). Thus, its inhibition could reduce cell growth and migration. Among the several genes regulated by the YAP-TEAD in-teraction, the binding of YAP with TEADs up-regulates the expression of the well-known oncogene c-myc (Neto-Silva et al., 2010). C-Myc pro-tein has a central role in almost every aspect of the oncogenic process, orchestrating a broad spectrum of cellular genes responsible for en-hancing cell proliferation, cellular metabolism, growth, angiogenesis, metastasis, genomic instability and stem cell self-renewal (Chen et al., 2018). A recent report demonstrated that c-Myc and YAP-TEAD in-tegrate mitogenic and mechanical cues at the transcriptional level to control cell proliferation and cell cycle entry (Croci et al., 2017). Thus, the inhibition of YAP and c-Myc expression could be responsible for the accumulation of cells in the G0/G1 phase of cell cycle after Aila treatment.
Traditionally, the cytotoxic effect of platinum compounds depends on the induction of double-strand breaks, that lead to the G2 arrest, apoptosis induction and generation of oxidative stress (Yu et al., 2018). In this study we demonstrated that Aila affected cell growth through mechanisms other than cisplatin. Indeed, Aila did not induce apoptosis, even after the treatment with the highest concentration. Phytomedicine 56 (2019) 156–164
Another important effect displayed by low concentrations of Aila regarded the inhibition of both random and directional migration. As previously reported, Nrf2 promoted the EMT by down-regulation of E-cadherin, and Nrf2 knock-down greatly impaired migration and inva-sion (Rojo de la Vega et al., 2018). For its part, YAP also is involved in cell invasion, since it induced EMT in cancer cells and YAP knock-down rescued the expression of epithelial markers (Zhao et al., 2008). Pre-viously, we demonstrated that both Nrf2 and YAP silencing reduced the migration of bladder cancer cells and that the silencing of both protein expressions synergistically acted in reducing the migration (Ciamporcero et al., 2018). Accordingly with these observations, we observed a reduction of cell migration after Aila treatment, which could be ascribed to the in-hibition of Nrf2 and YAP expression.
Our results demonstrated, for the first time, that Aila inhibited proliferation and migration of bladder cancer cells, by reducing Nrf2, YAP and c-Myc expression. Importantly, this effect was displayed in CDDP-resistant cancer cells in which the down-regulation of Nrf2 and YAP expressions was required to overcome the resistance. Since CDDP resistance is a common feature in muscle-invasive urothelial carcinoma of the bladder after platinum-based chemotherapy (Shah et al., 2011), the identification of a novel agent which can be employed in treating this resistant disease is of great interest. From this point of view, Aila demonstrated favorable drug-like properties due to its good bioavail-ability, high solubility and low hepatoxicity (He et al., 2016) and a low cytotoxic effect in kidney normal cells.