Steroidogenic activity of liposomal methylated resveratrol analog 3,4,5,4′-tetramethoxystilbene (DMU-212) in human luteinized granulosa cells in a primary three-dimensional in vitro model

PURPOSE: Steroid hormone secretion is one of the key functions of granulosa cells (GCs). Resveratrol is a natural polyphenol, known for its beneficial health effects, such as improving reproductive health. However, its application is limited due to poor bioavailability. The methoxy derivative of resveratrol (DMU-212) was demonstrated to be more lipophilic, and therefore of greater bioavailability. However, since the addition of methoxy groups to the stilbene scaffold was found to make the molecule insoluble in water, DMU-212 was loaded into liposomes. This study aimed to evaluate how the liposomal formulation of DMU-212 (lipDMU-212) alters estradiol and progesterone secretion of human ovarian GCs in a primary three-dimensional cell culture model. METHODS: DMU-212-loaded liposomes were prepared by thin film hydration followed by extrusion. Cell viability was measured after exposure of GCs spheroids to the liposomal formulation of DMU-212 using CellTiter-Glo(®) 3D Cell Viability Assay. The secretion of estradiol and progesterone was determined using commercial ELISA kits. RT-qPCR was conducted to analyze the expression of steroidogenesis-related genes. Finally, the western blot technique was used to analyze the effect of lipDMU-212 and FSH treatments on CYP11A1 and HSD3B1 protein levels. RESULTS: lipDMU-212 was found to significantly increase estradiol and progesterone secretion in a dose-dependent manner by enhancing the expression of CYP11A1, HSD3B1, StAR, CYP17A1, CYP19A1, and HSD17B1 genes. We have also shown that lipDMU-212, used alone and in combination with FSH, significantly increased the expression of the HSD3B1 and CYP11A1 proteins in GCs. Furthermore, our study suggests that lipDMU-212 increases FSH activity. CONCLUSIONS: This is the first study to describe the steroidogenic activity of liposomal formulation of DMU-212, possibly through increasing the StAR and CYP19A1 expression. These findings suggest that lipDMU-212 might have a beneficial effect in the treatment of disorders related to estrogen deficiency and hyperandrogenism, such as PCOS.