Resveratrol induces the production of reactive oxygen species, interferes with the cell cycle, and inhibits the cell migration of bladder tumour cells with different TP53 status

Abstract Resveratrol is a polyphenolic compound whose antitumor activity has been demonstrated in several types of cancer. However, there are few studies on its molecular mechanisms of action in bladder cancer. Therefore, we aimed to evaluate resveratrol activity in bladder tumour cells with different TP53 gene status. Cytotoxicity, cell proliferation, reactive oxygen species (ROS) production, cell migration, mutagenicity, and CDH1, CTNNBIP1, HAT1, HDAC1, MYC, and SMAD4 gene expression were evaluated. An increase in ROS after resveratrol treatment was accompanied by reduced cell viability and proliferation in all cell lines. In TP53 wild-type cells, the inhibition of cell migration was accompanied by CDH1 and SMAD4 modulation. In TP53 mutated cells, cell migration inhibition with CDH1 and CTNNB1P1 upregulation was observed. In conclusion, resveratrol has antiproliferative effect in bladder tumour cells and its mechanism of action occurred through ROS production, interference with cell cycle, and inhibition of cell migration, independent of TP53 status. Graphical Abstract


Introduction
Bladder cancer is the ninth most common type of cancer worldwide (Ferlay et al. 2021).Incidence and mortality rates are elevated in North America and Europe and low in South and Central America, although the number of new cases in some countries, such as Brazil and Cuba, has increased (Antoni et al. 2017).Bladder tumours can be classified as superficial or muscle-invasive, depending on their localisation.In general, superficial bladder cancers are treated with resection and intravesical therapy, while muscle-invasive types are treated with partial or total cystectomy plus chemotherapy (Sanli et al. 2017).Non-muscle invasive bladder tumours present high rates of recurrence and progression (approximately 60-70%).Furthermore, there has been no improvement in survival rates for bladder cancer in the last three decades (Berdik 2017).
The TP53 gene plays an essential role in the cell cycle and apoptosis regulation, contributing to cellular transformation and malignancy (Kastenhuber and Lowe 2017).Mutations in the TP53 gene commonly occur in muscle-invasive bladder cancer and are associated with increased angiogenesis, invasiveness, metastasis, recurrence, and poor prognosis (Mitra et al. 2006).However, little is known about the differential gene expression patterns in human tumours that present with the wild-type (wt) or mutated TP53 gene and the chemotherapy response (Stadler et al. 2011).
Natural products are a relevant source of compounds in new drug development (Yin et al. 2019).Regarding antitumor drugs, more than 60% are natural products or derived from natural products (Newman et al. 2003).Resveratrol is a polyphenolic compound found in grapes, berries, and peanuts (Wang et al. 2013).Plants produce resveratrol in response to environmental stress, such as exposure to sunlight and heavy metals, climate change, and pathogenic microorganism infection (Athar et al. 2009).Several studies demonstrated its antitumor effects on different types of cancer (Rauf et al. 2018), including bladder cancer (Almeida and Silva 2021).Besides, non-tumour cells were more resistant to resveratrol treatment than bladder tumour cells, indicating selectivity (Zhou et al. 2014).Several studies demonstrated the indirect mechanism for p53 activation by resveratrol through activation of MAP kinases (Lin et al. 2006;Liao et al. 2010) and post-translational modifications, such as acetylation (Zhang et al. 2004).Furthermore, it was demonstrated in breast cancer that resveratrol interacts directly with mutated p53, inhibiting its aggregation and reducing cell proliferation and migration (Ferraz da Costa et al. 2018).Despite all these findings, few studies have been performed to understand the molecular effects of resveratrol on bladder cancer cells with different TP53 status.
Considering the evidence of the antineoplastic potential of resveratrol and the importance of TP53 mutations in bladder cancer, this study aimed to investigate the effects of this natural product on cellular responses and gene expression in bladder cancer cell lines with wild-type (wt) or mutated TP53 gene.

Results and discussion
The influence of TP53 mutations on cellular responses to therapy is poorly understood because it depends on a complex signalling cascade.In bladder cancer, the interaction between the TP53 status of a primary tumour and the response to therapy using natural products is poorly defined.Firstly, we demonstrated that resveratrol treatment was cytotoxic for RT4 TP53 wild type cells at the concentrations of 50 mM, 100 mM, 150 mM, and 200 mM.For 5637 and T24 TP53 mutated cells, the cytotoxic effect was observed at 100 mM, 150 mM, and 200 mM of resveratrol (Figure S1-a).The IC 50 values were estimated to be 97.11mM for RT4 cells, 220.08 mM for 5637 cells, and 198.38 mM for T24 cells.We can observe that TP53 mutated cells were approximately two times more resistant to resveratrol cytotoxic effects than wt TP53 cells.Mutations in TP53 are often connected to resistance to standard medications, including cisplatin, doxorubicin, and gemcitabine (Hientz et al. 2017).Since p53 serves as a central inducer of apoptosis, TP53 mutations can protect, at least partially, from this death pathway induced by drugs.Additionally, the functions of p53 extend to several non-apoptotic forms of cell death and other processes such as DNA repair (Kruiswijk et al. 2015), which are all known to affect anticancer drug responses.
We also observed a significant decrease in cell proliferation in all cell lines after resveratrol treatment (Figure S1-b).This result provided evidence that the resveratrol led to the loss of reproductive integrity, probably due to its sustained lethal damage.The significant decrease in NDI values after resveratrol treatment (Table S1) also confirmed its antiproliferative effects since this parameter measures the proliferative situation of the viable cell fraction (Fenech 2007).These findings agree with our previous observation of RT4, 5637, and T24 cell colonies decrease in clonogenic survival assay after resveratrol treatment (Almeida et al. 2019).
The increase in the production of reactive oxygen species (ROS) observed in all cell lines after resveratrol treatment (Figure S2) may be at least partially responsible for the reduction in cell proliferation.It seems contradictory that resveratrol, a compound widely known for its antioxidant effect, produces oxidative stress.However, it is important to note that resveratrol is a polyphenol, a compound that can act as both antioxidant and pro-oxidant, depending on its concentration, its redox potential, the presence of metal ions and, the pH of the medium (Biasutto et al. 2008).Other studies have confirmed that high resveratrol concentrations (12.5 mM, 25 mM, 50 mM, 100 mM, and 200 mM) induce excessive production of reactive oxygen species in tumour cells (Lin et al. 2012;Stocco et al. 2012;Ji et al. 2018;Rodr ıguez-Enr ıquez et al. 2019).
Although previously reported as genotoxic (Almeida et al. 2019), resveratrol did not cause mutagenic effects (Table S2), suggesting that it is not aneugenic or clastogenic to mutated or wt TP53 cells in the evaluated conditions.
While cell migration is crucial in the development and tissue repair, it also mediates diseases such as cancer (Friedl and Gilmour 2009).We demonstrated that resveratrol treatment reduced cell migration rates in all studied cell lines (Figure S3).This modulation was accompanied by CDH1 upregulation in all cell lines (2.49-upregulation at 150 mM for RT4, 1.98-and 3.65-upregulation at 100 mM and 150 mM, respectively, for 5637 and 1.83-upregulation for T24) (Figure S4; Table S3).This gene encodes a protein (cadherin 1) that mediates cell-cell adhesion.Loss of CDH1 expression is one of the characteristics observed in epithelial-mesenchymal transition, a process often observed in carcinogenesis in which an epithelial cell loses cell-cell polarity and adhesion, acquiring migratory and invasive properties (Schneider and Kolligs 2015).
In wt TP53 cells, the reduction of SMAD4 expression (4.54-, 6.67-and 4.35-fold downregulation at 50 mM, 100 mM and 150 mM, respectively) (Figure S4; Table S3) probably also contributed to cell migration reduction.As previously reported, Nodal/ ALK/Smad4 signalling pathway is related to cell migration in RT4 cells.NODAL (Nodal Growth Differentiation Factor) functions as an oncogene by regulating migration and invasion through the ALK/Smad signalling pathway.NODAL knockdown inhibits SMAD4 expression, resulting in inhibited migration and invasion (Li et al. 2018).
In TP53 mutated cells, the reduction of cell migration caused by resveratrol was also accompanied by CTNNB1P1 upregulation (4.81-fold upregulation for T24 at 150 mM and 1.58-, 2.13-and 2.65-fold upregulation for 5637 at 50 mM, 100 mM and 150 mM, respectively) (Figure S4; Table S3).The CTNNB1P1 encoded protein (catenin beta interacting protein 1) is a negative regulator of the Wnt/b-catenin, a crucial signalling pathway in cell proliferation and migration in bladder cancer (Chen et al. 2018).
The MYC oncogene expression is related to the transcription of genes involved in processes such as angiogenesis, metastasis, and cell migration (Walz et al. 2014).However, we did not detect any changes in MYC expression in either wt or mutated TP53 cells (Figure S4), suggesting that this gene is not involved in resveratrol effects in bladder cancer cells.
Several natural products have been studied as possible acetylation modulators for cancer treatment (Merarchi et al. 2019).Acetylation is the most common post-translational epigenetic modification.The balance between acetylation and deacetylation through the enzymes histone acetyltransferases and deacetylases regulates the general level of acetylation (Huang et al. 2019).We did not observe modulation of HDAC1 expression, however, there was a decrease of HAT1 expression in RT4 and T24 cells after resveratrol treatment (1.49-fold downregulation for RT4 at 150 mM and 2.56-, 4.17-and 3.70-fold downregulation for T24 at 50 mM, 100 mM and 150 mM, respectively) (Figure S4; Table S3).High expression of HAT1 is related to several kinds of cancer and could reflect its role in chromatin assembly (Parthun 2007).Depletion of HAT1 sensitised cells to DNA damage compromised the global chromatin structure, inhibited cell proliferation, and induced cell death (Yang et al. 2013).

Experimental
See Supplementary Material.

Conclusion
In conclusion, although the TP53 mutated cells seem to be more resistant, resveratrol presented antiproliferative activity independent of TP53 status in bladder cancer cells, interfering with the cell cycle, inducing the production of reactive oxygen species, and inhibiting the cell migration.However, the molecular targets of resveratrol appear to differ.For example, while resveratrol acts decreasing SMAD4 expression in TP53 wild type cells, altered expression of CTNNB1P1 was detected in TP53 mutated cells.