The observed increase in oxidative stress
The observed increase in oxidative stress was also associated with decreased migration of cells as well as estriol disturbances. The migration of PNT1A cells was significantly decreased after both ZEA and ZEA+PHTPP treatments in two highest concentrations and correlated with the decreased ZEB2 expression. As we showed that ZEA affected cell cycle regulation, similarly, a cell cycle arrest effect of ZEA was observed in Sertoli cells, where ZEA caused a statistically significant increase in the number of cells in the G2/M cell cycle phase (Zheng et al., 2018a, Zheng et al., 2018b). The G2/M cell cycle arrest is also associated with the lowered expression of CDK2 and CDK4. The addition of PHTPP caused significant decrease in the number of G2/M cells as compared to ZEA treatment, visible also in the expression of CDC2, the increase in the number of cells in the G0/G1 cell cycle phase was also apparent, but it was still lower as compared to control cells. Interestingly, we observed that simultaneous treatment of cells with ZEA and PHTPP caused an increase in the number of cells in the sub-G0 cell cycle phase visible on the representative results, indicating that lack of ERβ might trigger a different effect of ZEA-induced changes in cell cycle progression. Due to the fact that chronic inflammation in prostate is associated with a higher cancer risk as well as the fact that ROS generation might be also associated with reactive nitric species (RNS) generation (Ying and Hofseth, 2007), we evaluated the effect of ZEA and involvement of ERβ on the expression of iNOS (NOS2) and eNOS (NOS3). We observed that ZEA in a dose-dependent manner increases the expression of iNOS, a similar effect was observed for addition of PHTPP, however, it was presented to a lower extent. The induction of oxidative stress by ZEA is also visible in the NOS3 expression, which was increased after ZEA treatment and interestingly this effect was not visible in cells treated with ZEA and PHTPP. NOS3 was previously reported to be potentially modulated by estrogens via ERβ in prostate cancer (Re et al., 2018). In this study, we observed that the lack of active ERβ increases the expression of NOS3 for control and E2 treated cells. Our results suggest that the observed ZEA-induced ROS generation might be associated with RNS species too. As demonstrated by Mak and colleagues, ERβ is known to be a part of the NFκB-HIF-1α signaling pathway during hypoxia as well as chronic inflammation of prostate (Mak et al., 2015), therefore in the next part of the study, we evaluated whether ZEA-induced ROS production is also associated with NFκB-HIF-1α signaling. As previously observed, a decrease in the HIF-1α expression is associated with RelA, RelB and p52 expression (van Uden et al., 2008). We demonstrated that ZEA caused a significant decrease in the HIF-1α as well as RelA expression, whereas the addition of PHTPP caused an increase as compared to ZEA treatment alone. Similar effect was observed for the expression of FOXO3 known to be involved in immunity of cancer cells (Deng et al., 2018). The results suggest that NFκB-HIF-1α signaling pathways might be involved in ZEA-induced oxidative stress, however, this statement needs a confirmation in further studies.