2-Arachidonoylglycerol effects in cytotrophoblasts: metabolic enzymes expression and apoptosis in BeWo cells

Abstract

The major endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is a member of the endocannabinoid system (ECS) that participates in cell proliferation and apoptosis, important events for the homoeostasis of biological systems. The formation of placenta is one of the most important stages of pregnancy and its development requires highly regulated proliferation, differentiation and apoptosis of trophoblasts. Anomalies in these processes are associated with gestational pathologies. In this work, we aimed to study the involvement of 2-AG in cytotrophoblast cell turnover. We found that 2-AG biosynthetic (diacylglycerol lipase A) and degradative (monoacylglycerol lipase) enzymes are expressed in human cytotrophoblasts and in BeWo cells. We also found that 2-AG induces a decrease in cell viability in a time-and concentration-dependent manner and exerts antiproliferative effects. The loss of cell viability induced by a 48-h treatment with 2-AG (10 mM) was accompanied by chromatin fragmentation and condensation, morphological features of apoptosis. Additionally, 2-AG induced an increase in caspase 3/7 and 9 activities, a loss of mitochondrial membrane potential (Djm) and an increase in reactive oxygen species (ROS)/reactive nitrogen species (RNS) generation, suggesting the activation of the mitochondrial pathway. Moreover, whereas Djm loss and ROS/RNS generation were significantly attenuated by the antagonists of both the cannabinoid receptors 1 and 2 (CB1 and CB2), the increase in caspase 3/7 and 9 activities and loss of cell viability were reversed only by the antagonist of CB2 receptor; the blockage of the eCB membrane transporter and the depletion of cholesterol failed to reverse the effects of 2-AG. Therefore, this work supports the importance of cannabinoid signalling during cytotrophoblast cell turnover and that its deregulation may be responsible for altered placental development and poor pregnancy outcomes.

Modulation of cellular redox homeostasis by the endocannabinoid system

“…Conversely, stimulation of the ECS has also been demonstrated to induce the production of ROS in certain cell types [ 69 – 71 ]. For example, 2-AG stimulation has been shown to promote an increase in cellular ROS in BeWo trophoblasts [ 71 ].…”

“…Conversely, stimulation of the ECS has also been demonstrated to induce the production of ROS in certain cell types [ 69 – 71 ]. For example, 2-AG stimulation has been shown to promote an increase in cellular ROS in BeWo trophoblasts [ 71 ]. Moreover, increased ROS and concomitant TNF- α cytokine production have been reported in human macrophages following CB1R activation, with both responses being attenuated by pharmacological inhibition of CB1R [ 69 ].…”

The endocannabinoid 2-arachidonoylglycerol promotes endoplasmic reticulum stress in placental cells

“…We previously described that 2-AG interferes with cytotrophoblast syncytialization through a CB receptor-dependent mechanism ( Costa et al 2015 c ). Moreover, in BeWo cells, we found that 2-AG induces trophoblast apoptosis, through mitochondrial membrane potential loss and increase in ROS generation and caspase -3/-7 and -9 activities, suggesting the activation of the mitochondrial pathway ( Costa et al 2014 a ). The endocannabinoid 2-AG is therefore implicated in normal trophoblast turnover.…”

“…Moreover, we previously demonstrated that AEA and 2-AG impair the synthesis of proteins and hormones by the human syncytiotrophoblast ( Costa et al 2015 a , 2016 ). In addition, AEA and 2-AG are able to modulate trophoblast apoptosis by inducing caspase activation and reactive oxygen species (ROS) generation ( Costa et al 2014 a , b , 2015 b ).…”

Anandamide restricts uterine stromal differentiation and is critical for complete decidualization

“…The functional activity of the decidual cells was evaluated by measuring the alkaline phosphatase (ALP) activity as previously described (Costa et al, 2014). Briefly, cells from 24-well plates were rinsed with buffer and then incubated at 37°C for 1 h with the pALP substrate p-nitrophenilphosphate, 2.5 mM (Sigma Chemical Co, St. Louis, MO, USA), in the presence of 100 mM MgCl2 (Sigma Chemical Co, St. Louis, MO, USA).…”