Inhibitory Effects of 6,8-Diprenylorobol on Endometriosis Progression in Humans by Disrupting Calcium Homeostasis and Mitochondrial Function

Abstract

6,8-Diprenylorobol is a flavonoid compound extracted from Cudrania tricuspidata. It has various biological functions, such as inhibiting melanin synthesis and inducting cell death in cancerous cells. In addition, Cudrania tricuspidata is known to be effective in female diseases, and previous studies have shown anticancer effects in cervical cancer, a female reproductive disease. Outside of that, Cudrania tricuspidata has various physiological effects. However, the effect of 6,8-diprenylorobol is not well known in other benign and chronic diseases, even in endometriosis, which commonly arises in the female reproductive tract. In the present study, we determined the inhibitory effects of 6,8-diprenylorobol on the growth of endometriosis VK2/E6E7 and End1/E6E7 cells. Results indicated that 6,8-diprenylorobol suppressed cellular proliferation and increased the disruption of the cell cycle, mitochondrial membrane potential (MMP), generation of reactive oxygen species, and Ca2+ homeostasis in both endometriosis cells. However, the proliferation of normal stromal cells isolated from endometrial tissue was not altered by 6,8-diprenylorobol. The change in Ca2+ levels was estimated in fluo-4- or rhod-2-stained VK2/E6E7 and End1/E6E7 cells after the treatment of the intracellular calcium regulators 2-aminoethoxydiphenyl borate (2-APB) and ruthenium red (RUR) with 6,8-diprenylorobol. A combination of 6,8-diprenylorobol with each regulator decreased the calcium accumulation in endometriosis cells. Furthermore, Western blot analysis indicated that 6,8-diprenylorobol inactivated AKT pathways, whereas it activated P38 MAPK pathways. In addition, 6,8-diprenylorobol decreased mitochondrial respiration, leading to the reduction in ATP production in VK2/E6E7 and End1/E6E7 cells. Collectively, our results suggested that 6,8-diprenylorobol might be a potential therapeutic agent or adjuvant therapy for the management of endometriosis.