The aims of our study were to: (1) identify and quantify progesterone metabolites formed in the HEC-1A and Ishikawa model cell lines of endometrial cancer; and (2) pinpoint the enzymes involved in progesterone metabolism, and delineate their roles. Progesterone metabolism studies combined with liquid chromatography-tandem mass spectrometry enabled identification and quantification of the metabolites formed in these cells. Further quantitative PCR analysis and small-interfering-RNA-mediated gene silencing identified individual progesterone metabolizing enzymes and their relevant roles. In Ishikawa and HEC-1A cells, progesterone was metabolized mainly to 20α-hydroxy-pregn-4-ene-3-one, 20α-hydroxy-5α-pregnane-3-one, and 5α-pregnane-3α/β,20α-diol. The major difference between these cell lines was rate of progesterone metabolism, which was faster in HEC-1A cells. In the Ishikawa and HEC-1A cells, expression of AKR1C2 was 110-fold and 6800-fold greater, respectively, than expression of AKR1C1, which suggests that 20-ketosteroid reduction of 5α-pregnanes and 4-pregnenes is catalyzed mainly by AKR1C2. AKR1C1/AKR1C2 gene silencing showed decreased progesterone metabolism in both cell lines, thus further supporting the significant role of AKR1C2. SRD5A1 was also expressed in these cells, and its silencing confirmed that 5α-reduction is catalyzed by 5α-reductase type 1. Silencing of SRD5A1 also had the most pronounced effects, with decreased rate of progesterone metabolism, and consequently higher concentrations of unmetabolized progesterone. Our data confirm that in model cell lines of endometrial cancer, AKR1C2 and SRD5A1 have crucial roles in progesterone metabolism, and may represent novel targets for treatment.
COBISS.SI-ID: 31704793
We have developed a combined liquid chromatography—tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line.
COBISS.SI-ID: 31822297
Human aldoketo reductases AKR1C1-AKR1C4 and AKR1D1 play essential roles in the metabolism of all steroid hormones, the biosynthesis of neurosteroids and bile acids, the metabolism of conjugated steroids, and synthetic therapeutic steroids. These enzymes catalyze NADPH dependent reductions at the C3, C5, C17 and C20 positions on the steroid nucleus and side-chain. AKR1C1-AKR1C4 act as 3-keto, 17-keto and 20-ketosteroid reductases to varying extents, while AKR1D1 acts as the sole delta4-3-ketosteroid-5beta-reductase (steroid 5beta-reductase) in humans. AKR1 enzymes control the concentrations of active ligands for nuclear receptors and control their ligand occupancy and trans-activation, they also regulate the amount of neurosteroids that can modulate the activity of GABA[sub]A and NMDA receptors. As such they are involved in the pre-receptor regulation of nuclear and membrane bound receptors. Altered expression of individual AKR1C genes is related to development of prostate, breast, and endometrial cancer. Mutations in AKR1C1 and AKR1C4 are responsible for sexual development dysgenesis and mutations in AKR1D1 are causative in bile-acid deficiency.
COBISS.SI-ID: 30940121