Bisphenol S and F affect cell cycle distribution and steroidogenic activity of human ovarian granulosa cells, but not primary granulosa tumour cells.

Bisphenol S (BPS) and F (BPF), a new generation of bisphenols (BPs), are the main substitutes for bisphenol A (BPA). Both have been detected in human body fluids. Importantly, bisphenols are structurally similar to oestrogen, the main sex hormone in females. Because bisphenols bind to nuclear oestrogen receptors (ESR1 and ESR2) and to membrane G-coupled receptor 30 (GPR30), they can disrupt ovarian function. Here, we reveal the molecular mechanism underlying the effects of BPS and BPF on the cell cycle and steroidogenesis in the human ovarian granulosa cell (GC) line HGrC1. We show that BPS and BPF arrest GCs at the G0/G1 phase by inducing expression of cyclin D2, an important event that triggers maximal steroid synthesis in response to the BPS and BPF. We used pharmacological inhibitors to show that BPS and BPF, despite acting via already described pathways, also stimulate steroid secretion via IGF1R pathways in HGrC1 cells. Moreover, we identified differences critical to bisphenols response between normal (HGrC1) and primary tumour granulosa (COV434) cells, that enable COV434 cells to be more resistant to bisphenols. Overall, the data suggest that BPS and BPF drive steroidogenesis in human ovarian GCs by affecting the cell cycle. Furthermore, the results indicate that BPS and BPF act not only via the classical and non-classical ESR pathways, but also via the IGF1R pathway.

Orotic acid induces apoptotic death in ovarian adult granulosa tumour cells and increases mitochondrial activity in normal ovarian granulosa cells.

Orotic acid (OA) is a natural product that acts as a precursor in the pyrimidine nucleotide biosynthesis pathway. Most studies concerning administration of OA focus on its therapeutic effects; however, its effect on tumours is unclear. We aimed to determine whether treatment with OA influences the viability and apoptosis of normal (HGrC1) and tumour-derived (KGN) human ovarian granulosa cells. The effects of OA (10-250 µM) on viability and apoptosis of both cell lines were determined by using alamarBlue and assessing caspase-3/7 activity, respectively. Annexin V binding and loss of membrane integrity were evaluated in KGN cells. The cell cycle and proliferation of HGrC1 cells were assessed by performing flow cytometric and DNA content analyses, respectively. The influence of OA (10 and 100 µM) on cell cycle- and apoptosis-related gene expression was assessed by RT-qPCR in both cell lines. Mitochondrial activity was analysed by JC-1 staining in HGrC1 cells. In KGN cells, OA reduced viability and increased caspase-3/7 activity, but did not affect mRNA expression of Caspase 3, BAX, and BCL2. OA enhanced proliferation and mitochondrial activity in HGrC1 cells without activating apoptosis. This study demonstrates that the anti-cancer properties of OA in ovarian granulosa tumour cells are not related to changes in apoptosis-associated gene expression, but to increased caspase-3/7 activity. Thus, OA is a promising therapeutic agent for ovarian granulosa tumours. Further, our results suggest that differences in basal expression of cell cycle- and apoptosis-related genes between the two cell lines are responsible for their different responses to OA.