Chrysin inhibits the cell viability, induces apoptosis and modulates expression of genes related to epigenetic events in bladder cancer cells

Abstract This study was conducted with the aim of exploring the molecular and cellular mechanisms of action of the chrysin (natural flavonoid compound) on bladder tumour cell lines with different status of TP53 (RT4, 5637 and T24). The cells were treated with different concentrations of chrysin (20, 40, 60, 80 and 100 µM) to analyze the cell viability, nuclear division index, mutagenicity, apoptosis rates and expression of genes related to epigenetic events (DNMT1, HAT1 and HDAC1). Results showed that the treatment with chrysin reduced the cell viability and caused apoptosis, regardless TP53. Moreover, in the TP53-mutated cell lines, chrysin modulated the expression of the DNMT1, HAT1 and HDAC1 epigenetic genes, which might be a plus to the death observed in the cells with p53 mutation. Graphical Abstract


Introduction
In 2020, 0.573 million new cases and almost 0.212 million deaths worldwide due to bladder cancer were estimated (Ferlay et al. 2021). The therapeutic approach depends on the biologic phenotypes of the tumour which might be influenced by TP53 gene status. Somatic mutations of TP53 are common to be found in muscle-invasive bladder cancer, changing the expression of diverse cell proliferation genes (Rentsch et al. 2017). Moreover, the pleiotropic action of p53 mutated protein might modulate the expression of genes which present important role on genome-wide histone methylation and acetylation (Nasr et al. 2003;Kong et al. 2020). In addition, it is important to note that the high toxicity and failure rates caused by the standard treatment for bladder cancer justifies the constant search for natural products as potential therapeutic agent (Lin et al. 2020).
Chrysin (5,7-dihydroxy-2-phenyl-4H-chromene-4-one or 5,7-dihydroxyflavone) is a natural flavonoid with antitumor effect (Kasala et al. 2015). Although there are only three studies demonstrating the effect of the chrysin on bladder cancer viability (Orsolic et al. 2009;Xu et al. 2018;Lima et al. 2020), only our research group studied the biological effects of the compound based on TP53 status and showed the mechanisms of action are dependent on wild type or mutated status (Lima et al. 2020).
Considering the selectivity of the chrysin for tumour cells (Xu et al. 2018) and our previous evidence of the antineoplastic potential based on TP53 status, the present study aimed to elucidate other mechanisms of action of chrysin (apoptosis rates, cytostatic and mutagenic effects and the expression of genes that regulate histone methylation and acetylation) on bladder cancer cell lines with different TP53 gene status. Our results will complete the mechanisms of action of chrysin on bladder tumours.

Cell viability
Trypan blue exclusion test is based on the principle that live cells possess intact cell membranes that exclude certain dyes. It is important to note that different cellular endpoints should be used to determine the cell viability, since different factors can influence the apparent potency of a compound on viability and toxicity (Riss and Moravec 2004). The results indicated that chrysin decreased the cell viability ( Figure  S1A) for all bladder cell lines tested (for RT4 cells, treatment with all concentrations; for 5637 cells, at 40, 60, 80 and 100 mM, and for T24 cells, at 60, 80 and 100 mM). The IC 50 values (88 mM for RT4, 65 mM for 5637 and 55 mM for T24) revelated that there is no significant difference between cytotoxicity rates in wild-type or mutated TP53 cells, demonstrating effect independent of TP53 status. In addition, the Figures S1B and S1C showed after 24 h or 48 h of recuperation (compound free cells), there was a drastic decrease of cell viability for all cell lines. These results provide evidence that the lost of the cell viability caused by chrysin remain leading to the loss of reproductive integrity.

Cytokinesis-block micronucleus assay and nuclear division index (NDI)
Significant decrease of the NDI in all cell lines confirmed that chrysin caused a cytostatic effect independent of TP53 status (Table SI). NDI is a cell viability marker which indicate cells with greater chromosomal damage will either die before cell division or may be less likely to enter this phase (Ionescu et al. 2011). These findings are in agreement with our previous observation of the genotoxic damage in RT4, 5637 and T24 cells after chrysin treatment (Lima et al. 2020). Nevertheless, it was not detected any mutagenic effect in the micronucleus assay (data not showed), demonstrating that chrysin, although genotoxic, is not aneugenic or clastogenic for the tested cells.

Apoptosis and DNMT1, HAT1, HDAC1 gene expression
The results of apoptosis assay ( Figure S2) demonstrated that chrysin caused different types of death in the cell lines studied. The increase of early (20, 40 and 60 mM) or late (60 mM) apoptosis rates observed in the RT4 cells may be explained by status of TP53 gene. Cells with wild-type TP53 can activate the expression of pro-apoptotic genes, through the activation of p53 protein, and to coordinate cell death signaling pathways after exposition to a harmful compound (Liu et al. 2003;Almeida et al. 2019). On the other hand, the increase of necrosis rates (60 mM) and early (40 and 60 mM) and late (20, 40 and 60 mM) apoptosis in the T24 cells may be explained by pathways that are activated by mutated p53 activity. In the literature, there are studies demonstrating that the epigenetic changes (DNA methylation, histones acetylation) caused by mutated p53 might lead a deconfiguration of the bladder tumour cells DNA (Lee and Song 2017). Here, we observed a significant decrease of the expression of the genes related to epigenetic events in the T24 cells after treatment with chrysin ( Figure S3). The downregulation of DNMT1 (1.56-, 1.93-, 2.89-fold downregulation for 20, 40 and 60 mM), HAT1 (1.97-, 2.51-, 5.02-fold downregulation for 20, 40 and 60 mM) and HDAC1 (2.56-, 2.13-, 3.51-fold downregulation for 20, 40 and 60 mM) observed might cause aberrant DNA methylation and histones acetylation, changing chromatin remodeling and deregulating the whole genomic chain. These alterations can lead to a global instability of the T24 cell lines DNA by compromising the DNA protection mechanisms, such as repair mechanisms, and driving the tumour cells to apoptotic death, as was observed after chrysin treatment (Lin et al. 2016). As for 5637 cells there was no significant change in apoptosis or necrosis rates after chrysin treatment, it is supposed to affirm that the balance between the upregulation of HAT1 (1.28-, 1.59-, 1.67-fold upregulation for 20, 40 and 60 mM) and HDAC1 (1.59-, 1.49-fold upregulation for 40 and 60 mM) genes could not be involved with the apoptosis in these cell line, suggesting that the upregulation of these genes is responsible for another cell death mechanism in the 5637 cells.

Conclusion
Our study supports the cytotoxic potential of chrysin on bladder tumour cells and gives an important complement for the molecular activities of the chrysin on cells with different status of the TP53 gene. Besides the cytostatic and pro-apoptotic effects of the chrysin be regardless TP53 status, our study also demonstrated that the downregulation of the DNMT1, HDAC1 and HAT1 genes is an important issue to be considered in relation to the cell death seen at bladder tumour cells with TP53 mutation. These results work as a strong complement for our previous study, where we demonstrated that the cell death caused by chrysin was correlated to its molecular mechanisms in downregulating oncogenes and inducing genotoxic damage to DNA of these cells. Therefore, now that the importance of chrysin in killing bladder cancer cells is deeply recognized, it is reasonable to assume that more researches in this area must be matured over the years.

Author contribution
APBL carried out the experiments, contributed to the experimental design and data interpretation and wrote the manuscript. ASM supported the experiments. GMF supported the experiments. GNS was the advisor, contributed to the experimental design, as well as critically read the manuscript Compliance with ethical standards

Disclosure statement
No potential conflict of interest was reported by the authors.