N-Acyl Dopamines Induce Apoptosis in Endometrial Stromal Cells from Patients with Endometriosis

Abstract

Endometriosis is characterized by the formation and development of endometrial tissues outside the uterus, based on an imbalance between proliferation and cell death, leading to the uncontrolled growth of ectopic foci. The potential target for the regulation of these processes is the endocannabinoid system, which was found to be involved in the migration, proliferation, and survival of tumor cells. In this paper, we investigated the effect of endocannabinoid-like compounds from the N-acyl dopamine (NADA) family on the viability of stromal cells from ectopic and eutopic endometrium of patients with ovarian endometriosis. N-arachidonoyldopamine, N-docosahexaenoyldopamine, and N-oleoyldopamine have been shown to have a five-times-more-selective cytotoxic effect on endometrioid stromal cells. To study the mechanisms of the toxic effect, inhibitory analysis, measurements of caspase-3/9 activity, reactive oxygen species, and the mitochondrial membrane potential were performed. It was found that NADA induced apoptosis via an intrinsic pathway through the CB1 receptor and downstream serine palmitoyltransferase, NO synthase activation, increased ROS production, and mitochondrial dysfunction. The higher selectivity of NADA for endometriotic stromal cells and the current lack of effective drug treatment can be considered positive factors for further research of these compounds as possible therapeutic agents against endometriosis.

Keywords: CB1 receptor; N-acyl dopamines; apoptosis; endocannabinoid system; endometrial stromal cells; endometriosis; reactive oxygen species; selective toxicity.

Figure 1

Dose-dependent cytotoxic effect of N-acyl dopamines on stromal cells of eutopic (A) and ectopic (B) endometrium. Cell viability was measured using MTT-test as described in Section 4. Data are presented in percent from vehicle-treated controls as mean ± standard deviation.

Figure 2

Kinetics of cytotoxic action of N-acyl dopamines on stromal cells of ectopic endometrium. Cell viability was measured after indicated time intervals (NADA 10 μM) as described in Section 4. Data are presented as mean ± standard deviation.

Figure 3

The evaluation of the type of NADA-induced ectopic stromal cell death with annexin V-FITC/PI assay. A. Representative plots of fluorescence distribution in ectopic stromal cells incubated with DMSO (1) or AA-DA (2).

Figure 4

Representative plot of AA-DA effect on ROS production by endometriotic stromal cells. ROS production was measured using a 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) in the presence of AA-DA (5 μM) or H₂O₂ (5 μM). *—statistically significant difference, p < 0.05, ANOVA with the Tukey post-hoc test.

Figure 5

Representative plot of AA-DA effect on the mitochondrial membrane potential in ectopic stromal cells. The mitochondrial membrane potential (MMP) was assessed by a fluorometric assay using tetramethylrhodamine ethyl ester (TMRE) in the presence of AADA 5 μM or FCCP 20 μM. *—a statistically significant difference, p < 0.05, ANOVA with the Tukey post-hoc test.