Cell lines and cell culture
Human endometrial cancer cell lines (Ishikawa and ECC1) were maintained in our laboratory. Ishikawa cells are derived from a well-differentiated endometrioid adenocarcinoma, so they were used to illuminate some of the molecular mechanisms underlying progestin resistance as an in vitro model of hyperplasia. HEK-293 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured in DMEM:F12 medium (1:1, GIBCO) containing 10% fetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA), 100 U/ml penicillin G and 100 µg/ml streptomycin (Life Technologies, Inc., Rockville, MD) and placed in a 37°C incubator with a humidified atmosphere containing 5% CO2.
Establishment of stable cell lines, transient transfection, small interfering RNA transfection and progestin treatment
To investigate the roles of Nrf2 and AKR1C1 in progestin resistance, stable cell lines with Nrf2/AKR1C1 overexpression or AKR1C1 depletion were established using a retrovirus system as described previously 9, 10. Transient transfection of the indicated plasmids or siRNA was performed with LipofectamineTM 3000 (Invitrogen, Carlsbad, CA, USA). The cells were treated with progestin or brusatol either alone or in combination for 48 hours, and proliferation was measured with a CCK8 assay.
Drug treatment and cell proliferation assay
Endometrial cancer cells were treated with MPA, brusatol, or tBHQ for the indicated times. Cell proliferation was measured with a CCK8 assay.
Cells were harvested after various treatments and lysed with RIPA buffer to extract total protein. After the protein concentration was quantified, 50 µg of protein per lane was loaded onto an SDS-polyacrylamide gel, electrophoresed and transferred to PVDF membranes, which were incubated overnight with primary antibodies against Nrf2 and Tet1 (Sigma Aldrich); α-tubulin (Abcam); and AKR1C1, AKR1B10, GCLM, NQO1, GAPDH and HO-1 (Santa Cruz Biotechnology). After the membranes were washed and treated with the indicated secondary antibodies, detection of the protein bands was carried out using a chemiluminescence detection system (ECL detection kit; Pierce, Rockford, IL). Each experiment was performed at least three times.
Dot blot and HMeDIP assay
A dot blot assay was carried out as previously described. After conducting the indicated treatments, we extracted total DNA and performed a gradient dilution of the samples. The dilutions of total DNA were dropped on nitrocellulose membranes, which were baked at 80°C for 10 min. The membrane was blocked with 10% skim milk for 1 hour before it was incubated with 5hmC primary antibody (1:500 dilution, Active Motif) overnight at 4°C. After the membrane was washed and treated with HRP-conjugated secondary antibody, it was subjected to ECL and scanned to visualized bound antibodies. Methylene blue (MB) staining served as a loading control. Quantification of the dot blots from three independent assays was calculated with Gel-Pro analyzer software (Media Cybernetics). The gray intensity of dots sampled with 100 ng DNA was analyzed by one-way analysis of variance (ANOVA). Endometrial cancer cells were transfected with pPB-Tet1 plasmid and subjected to brusatol treatment, after which total DNA was extracted and sheared via ultrasonication. The DNA fragments were incubated with 5hmC antibody (Active Motif) and pulled down to amplify the AKR1C1 gene promoter via real-time PCR. The primers used are listed in Table 1.
ARE constructs and Dual luciferase reporter assay
The wild type or mutant Tet1 AREs were amplified and cloned into pGL4.27 plasmids (Promega) as previously described 18. Ishikawa cells were cotransfected with pGL4.27-Tet1-ARE plasmids, pRL-SV40-Renilla plasmid (Promega) and Nrf2 plasmid, and the relative luciferase activity was determined by a Dual Luciferase Assay Kit (Promega).
20α-dihydroxyprogesterone concentration detection
Endometrial cancer cells were treated with various drugs were harvested and lysed to determine the 20α-dihydroxyprogesterone concentration with analysis kits (XQ-EN15767, Xinquan Company, Shanghai, China).
Selection of matched cases, tissue processing and immunohistochemical (IHC) analysis
Thirty-four pairs of endometrial samples before and after progestin treatment were assessed in this study. The patients’ clinical information is listed in Table 2. The pathological diagnosis of endometrial hyperplasia or well-differentiated carcinoma was reviewed and confirmed by gynecological pathologists (YJ and WZ) on the basis of the WHO classification. Specifically, the different pathological statuses based on progestin response were defined as follows: no response or residual disease, any architectural abnormalities such as clusters of crowded glands, papillary structures, and complex types of glands with or without cytologic atypia either alone or in combination; partial response, no residual hyperplasia/endometrial intraepithelial neoplasia (EIN) but an incomplete response or abnormal glands or any residual architectural abnormalities that do not reach the level of residual disease or nonresponsive disease; and complete response, attenuated endometrial glands with decidualized stroma. The IHC assay was performed as previously described 6, 9, 18.
Human endometrial organoids culture
Human endometrial organoids culture was carried out as previous report 29. Briefly, fresh hyperplasia endometrial biopsies or endometrial cancer tissues were collected, followed by enzymatically digested and centrifugated. The pellets were resuspended in diluted ice-cold Matrigel medium mix. Fifty-microlitre drops of Matrigel–cell suspension were plated into 24-well plate and overlaid with organoid Expansion Medium (ExM) . The medium was changed every 2–3 d. Cultures were passaged by manual pipetting every 7–10 d. After various treatments with drugs, the organoids were collected and fixed with 4% paraformaldehyde on ice, followed by resuspending and embedding the organiods with 3% low melting point agarose for H&E and IHC analysis.
In vivo xenograft mouse model
Nude mice were subcutaneously injected with 1×106 endometrial cancer cells. After the mice were treated with MPA and brusatol either alone or in combination for 30 days, they were sacrificed. The tumors were harvested for IHC analysis as previously described 22.
SPSS 19.0 was used for data analysis. Comparisons of proliferation, the dot blot assay, the dual luciferase reporter assay and the western blot results after various treatments among multiple groups were made with one-way ANOVA followed by Dunnett’s t-test. P < 0.05 was considered a significant difference when compared with the control group.