Discovery of phosphatidylcholines and sphingomyelins as biomarkers for ovarian endometriosis.

BACKGROUND: Current non-invasive diagnostic methods for endometriosis lack sensitivity and specificity. In search for new diagnostic biomarkers for ovarian endometriosis, we used a hypothesis-generating targeted metabolomics approach. METHODS: In a case-control study, we collected plasma of study participants and analysed their metabolic profiles. We selected a group of 40 patients with ovarian endometriosis who underwent laparoscopic surgery and a control group of 52 healthy women who underwent sterilization at the University Clinical Centre Ljubljana, Slovenia. Over 140 targeted analytes included glycerophospholipids, sphingolipids and acylcarnitines. The analytes were quantified by electrospray ionization tandem mass spectrometry. For assessing the strength of association between the metabolite or metabolite ratios and the disease, we used crude and adjusted odds ratios. A stepwise logistic regression procedure was used for selecting the best combination of biomarkers. RESULTS: Eight lipid metabolites were identified as endometriosis-associated biomarkers due to elevated levels in patients compared with controls. A model containing hydroxysphingomyelin SMOH C16:1 and the ratio between phosphatidylcholine PCaa C36:2 to ether-phospholipid PCae C34:2, adjusted for the effect of age and the BMI, resulted in a sensitivity of 90.0%, a specificity of 84.3% and a ratio of the positive likelihood ratio to the negative likelihood ratio of 48.3. CONCLUSIONS: Our results suggest that endometriosis is associated with elevated levels of sphingomyelins and phosphatidylcholines, which might contribute to the suppression of apoptosis and affect lipid-associated signalling pathways. Our findings suggest novel potential routes for therapy by specifically blocking highly up-regulated isoforms of phosphpolipase A2 and lysophosphatidylcholine acyltransferase 4.

Expression of estrogen and progesterone receptors and estrogen metabolizing enzymes in different breast cancer cell lines.

Prolonged exposure to estrogens is a significant risk factor for the development of breast cancer. Estrogens exert carcinogenic effects by stimulating cell proliferation or through oxidative metabolism that forms DNA-damaging species. In the present study, we aimed to provide a better understanding of estrogen metabolism and actions in breast cancer, and to characterize model breast cancer cell lines. We determined the expression profiles of the genes for the estrogen and progesterone receptors, and for 18 estrogen-metabolizing enzymes in eight cell lines: MCF-7, MCF-10A, T47D, SKBR3, MDA-MB-231, MDA-MB-361, Hs-578T and Hs-578Bst cells. Similar gene expression profiles of these receptors and enzymes for the formation of estradiol via the aromatase and sulfatase pathways were observed in the MCF-7 and T47D metastatic cell lines. The MDA-MB-361 cells expressed ESR1, ESR2 and PGR as well, but differed in expression of the estrogen-metabolizing enzymes. In the MDA-MB-231 and SKBR3 cells, all of these estrogen-forming enzymes were expressed, although the lack of ESR1 and the low levels of ESR2 expression suggested that the estrogens can only act via non-ER mediated pathways. In the non-tumorigenic MCF-10A cell line, the key enzymes of the aromatase pathway were not expressed, and the sulfatase pathway also had a marginal role. The comparison between gene expression profiles of the non-tumorigenic Hs-578Bst cells and the cancerous Hs-578T cells revealed that they can both form estrogens via the sulfatase pathway, while the aromatase pathway is less important in the Hs-578Bst cells. The Hs-578T cells showed low levels of ESR1, ESR2 and PGR expression, while only ESR1 and ESR2 expression was detected in the Hs-578Bst cells. Our data show that the cell lines examined provide the full range of model systems and should further be compared with the expression profiles of breast cancer specimens.

Aldo-keto reductases AKR1C1, AKR1C2 and AKR1C3 may enhance progesterone metabolism in ovarian endometriosis.

Endometriosis is a very common disease that is characterized by increased formation of estradiol and disturbed progesterone action. This latter is usually explained by a lack of progesterone receptor B (PR-B) expression, while the role of pre-receptor metabolism of progesterone is not yet fully understood. In normal endometrium, progesterone is metabolized by reductive 20α-hydroxysteroid dehydrogenases (20α-HSDs), 3α/ß-HSDs and 5α/ß-reductases. The aldo-keto reductases 1C1 and 1C3 (AKR1C1 and AKR1C3) are the major reductive 20α-HSDs, while the oxidative reaction is catalyzed by 17ß-HSD type 2 (HSD17B2). Also, 3α-HSD and 3ß-HSD activities have been associated with the AKR1C isozymes. Additionally, 5α-reductase types 1 and 2 (SRD5A1, SRD5A2) and 5ß-reductase (AKR1D1) are responsible for the formation of 5α- and 5ß-reduced pregnanes. In this study, we examined the expression of PR-AB and the progesterone metabolizing enzymes in 31 specimens of ovarian endometriosis and 28 specimens of normal endometrium. Real-time PCR analysis revealed significantly decreased mRNA levels of PR-AB, HSD17B2 and SRD5A2, significantly increased mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1, and negligible mRNA levels of AKR1D1. Immunohistochemistry staining of endometriotic tissue compared to control endometrium showed significantly lower PR-B levels in epithelial cells and no significant differences in stromal cells, there were no significant differences in the expression of AKR1C3 and significantly higher AKR1C2 levels were seen only in stromal cells. Our expression analysis data at the mRNA level and partially at the cellular level thus suggest enhanced metabolism of progesterone by SRD5A1 and the 20α-HSD and 3α/ß-HSD activities of AKR1C1, AKR1C2 and AKR1C3.