Contributed equally
The aim of the present study was to evaluate the effects of baicalein on human endometrial stromal cells
Endometriosis is a benign, hormone-dependent disease that affects at least 10–15% of women of reproductive age (
Although endometriosis is a benign disease, previous results have suggested that it represents the initial stages of neoplastic processes (
Baicalein (5,6,7-trihydroxyflavone) is a component derived from the root of
As baicalein exhibits a wide range of anti-tumor effects, is easily obtained, produces little toxicity, causes few side effects, and has demonstrated potential in treating cancer (
The procedures for the collection of tissue samples were approved by the Ethics Committee of the Obstetrics and Gynecology Hospital of Fudan University (Shanghai, China). All patients signed an informed consent form in compliance with the code of ethics of the World Medical Association (The Declaration of Helsinki). All ectopic endometrium samples were collected from 6 female patients with ovarian endometriosis who underwent laparoscopic surgical procedures at the Obstetrics and Gynecology Hospital of Fudan University from July to September 2015. The patients were 25–42 years old, had regular menstrual cycles. Patients who had received hormonal therapy or antibiotics for at least three months prior to surgery were excluded. After collection, the tissue samples were immediately used for the isolation and culture of human endometrial stromal cells.
Endometrial stromal cells were separated from the isolated endometrial tissues as described previously (
Following 72 h of subculture, cells were fixed with 4% paraformaldehyde for 30 min at room temperature. After blocking with rabbit serum (1:200; Beyotime Institute of Biotechnology, Haimen, China), the cells were incubated with rabbit anti-human vimentin (ab92547; 1:200; Abcam, Cambridge, UK) or rabbit anti-human cytokeratin 7 (CK7) antibodies (ab68459; 1:200; Abcam, Cambridge, UK) overnight at 4°C. After three 15 min washes in PBS (pH 7.4), the cells were incubated with a mouse anti-rabbit antibody at room temperature (sc-2357; 1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 60 min. The slides were subsequently washed in PBS and incubated with 0.01% 3,3-diaminobenzidine tetrahydrochloride (Beyotime Institute of Biotechnology) for 2 min. The cells were then stained with chromogen substrates (Beyotime Institute of Biotechnology) for 3 min and counterstained with hematoxylin. Analysis was carried out under a light microscope at ×400 magnification and an estimation based on microscopic observations. When >98% of cells were positive for vimentin staining and <2% of cells were negative for CK7 staining, it was considered that a homogeneous population of endometrial stromal cells had been isolated.
Cell viability was estimated using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Cells were seeded into 96-well plates at a density of 10,000 cells/well in DMEM/F-12 containing 10% FBS at 37°C. After 24 h of culture, the medium was replaced with DMEM/F-12 without FBS and cultured at 37°C for 12 h. Baicalein was added to DMEM/F-12 containing 10% FBS to obtain concentrations of 0, 5, 10, 20, 40, 80, 160 µM and each concentration was added to separate wells (3 wells for each concentration). At 24, 48 or 72 h after the addition of baicalein, the media was removed and CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 90 min. Absorbance at 450 nm was then measured on a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A culture solution containing CCK-8 reagent without cells served as a blank control (BC). Cell viability rates were expressed as the ratio of the absorbance values from the experimental group to those of the negative control (0 µM) group.
Cells were lysed in ice-cold radioimmunoprecipitation protein lysis buffer (Beyotime Institute of Biotechnology) and protein concentration was measured using a bicinchoninic assay kit (Beyotime Institute of Biotechnology). Equal amounts of total protein (40 µg per lane) were solubilized in sample buffer by boiling for 5 min, then subjected to electrophoresis on 12% SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc.). The membranes were incubated with a blocking buffer [1X Tris-buffered-saline (TBS) containing 5% non-fat, dried milk and 0.1% Tween-20] for 1 h at room temperature. Membranes were then incubated with 1:1,000 dilutions of rabbit anti-human B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), proliferating cell nuclear antigen (PCNA), cyclin D1, β-actin and β-tubulin (cat. no. 4223, 14796, 2586, 2978, 4970 and 2128, respectively, all from Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. The next day, the membranes were washed three times in TBS containing 0.1% Tween-20, then incubated for 60 min with a horseradish peroxidase-conjugated secondary antibody at room temperature (cat. no. 14708; 1:1,000; Cell Signaling Technology, Inc.). The protein bands were visualized using an enhanced chemiluminescence kit (Beijing Transgen Biotech Co., Ltd., Beijing, China). The densities of the bands were quantified using Quantity One Analysis software v4.62 (Bio-Rad Laboratories, Inc.) and normalized against the intensities of β-actin and β-tubulin.
Approximately 10,000 cells were treated with baicalein (0 or 40 µM) for 48 h at 37°C. The cells were then collected, fixed with cold 70% ethanol and stored at −20°C. The cells were washed, resuspended in cold PBS and incubated with 1 mg/ml propidium iodide (Sigma-Aldrich; Merck KGaA) at 37°C for 30 min. DNA content was measured by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). The percentages of cells in different phases of the cell cycle were evaluated using CellQuest acquisition software version 5.1 (BD Biosciences).
Approximately 10,000 endometrial stromal cells were incubated with or without WP1066 [signal transducer and activator of transcription 3 (STAT3) inhibitor; 10 µM], N'-(4-Oxo-4H-chromen-3-yl) methylene) nicotinohydrazide (STAT5 inhibitor; 10 µM), LY294002 (Akt signal pathway inhibitor; 10 µM), SP600125 [c-Jun N-terminal kinase (JNK) inhibitor; 10 µM], SB203580 [p38/mitogen-activated protein kinase (MAPK) inhibitor; 10 µM], U0126 [extracellular signal-regulated kinases 1/2 (ERK1/2) inhibitor; 10 µM], BAY11-7080 [nuclear factor (NF)-κB inhibitor; 10 µM] (all inhibitors were from Santa Cruz Biotechnology, Inc.) for 6 h at 37°C, then treated with or without baicalein (40 µM; Sigma-Aldrich; Merck KGaA) for 48 h at 37°C. Cell viability was evaluated using a CCK-8 assay, as described above.
All statistical analyses were performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Statistical comparisons were performed using a Kruskal-Wallis test for three or more groups and the Wilcoxon signed-rank test for two groups. Data are presented as the median ± interquartile range of three independent experiments. P<0.05 was considered to indicate a statistically significant difference.
The purity and homogeneity of the stromal cell preparation were verified by immunocytochemistry using an antibody against vimentin, as a specific marker of stromal cells, and an antibody against CK7, as a specific marker of epithelial cells (
The effect of baicalein on the growth of human endometrial stromal cells
To determine the effects of baicalein on cell cycle progression, flow cytometry analysis was performed. As depicted in
The effect of baicalein on the expression of apoptotic proteins was evaluated. As depicted in
To investigate the signaling pathways related to baicalein activity in endometrial stromal cells, the viability of endothelial stromal cells was evaluated following treatment with baicalein and inhibitors of different signaling pathways. As depicted in
Endometriosis is among the most prevalent types of benign gynecological diseases, and shares a number of characteristics with tumors (
The effects of baicalein have been studied in numerous types of malignancy, including osteosarcoma, gallbladder cancer, breast cancer and gynecologic tumors such as ovarian cancer (
Due to the reported anti-tumor properties of baicalein and the relationship between endometriosis and cancer, the current study aimed to evaluate the therapeutic potential of baicalein in endometriosis. The effect of baicalein on endometrial stromal cell viability was assessed
In conclusion, the present study observed that baicalein significantly reduced the viability of endometrial stromal cells
The present study was supported by the Science and Technology Commission of Shanghai Municipality, China (grant no. 12401902200).
Verification of endometrial stromal cell purity and homogeneity. Immunocytochemistry was used to assess the levels of (A) vimentin (brown staining) and (B) cytokeratin 7 (no brown staining) expression in cells isolated from 6 female patients presenting with ovarian endometriosis. Magnification, ×400.
Effect of baicalein on endometrial stromal cell viability. Following treatment with (A) 0, (B) 10 or (C) 40 µM baicalein, the morphology of endometrial stromal cells was evaluated under a light microscope. Magnification, ×400. (D) Cell viability was evaluated following treatment with the indicated concentrations of baicalein for 24, 48 and 72 h. Cell viability was expressed relative to the control (0 µM). Statistical comparisons were made using the Kruskal-Wallis test and results are presented as the median and interquartile range (25–75th percentile). *P<0.05 vs. control.
Effect of baicalein on the expression of Bcl-2, Bax, PCNA and cyclin D1 in endometrial stromal cells. Cells were treated with 0 µM (control) or 40 µM baicalein for 48 h, and western blot analysis was used to evaluate the expression of Bcl-2, Bax, PCNA and cyclin D1. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; PCNA, proliferating cell nuclear antigen.
Effect of baicalein on endometrial stromal cell viability.
Cell viability (relative to control) | ||||||
---|---|---|---|---|---|---|
24 h | 48 h | 72 h | ||||
Baicalein (µM) | Median | P25-P75 | Median | P25-P75 | Median | P25-P75 |
0 | 1.000 | 1.000–1.000 | 1.000 | 1.000–1.000 | 1.000 | 1.000–1.000 |
5 | 0.939 |
0.831–0.985 | 0.978 |
0.922–0.991 | 0.937 |
0.902–0.987 |
10 | 0.897 |
0.801–0.957 | 0.856 |
0.728–0.933 | 0.904 |
0.763–0.928 |
20 | 0.814 |
0.686–0.913 | 0.739 |
0.618–0.843 | 0.841 |
0.571–0.914 |
40 | 0.730 |
0.632–0.905 | 0.654 |
0.575–0.764 | 0.776 |
0.538–0.844 |
80 | 0.603 |
0.511–0.874 | 0.543 |
0.417–0.740 | 0.590 |
0.431–0.798 |
160 | 0.541 |
0.424–0.692 | 0.402 |
0.295–0.597 | 0.454 |
0.284–0.511 |
Statistical comparisons were made using the Kruskal-Wallis test.
P<0.05 vs. control group (0 µM baicalein). P25, 25th percentile; P75, 75th percentile.
Effect of baicalein on endometrial stromal cell cycle progression measured by flow cytometry analysis.
Baicalein 0 µM (% cells) | Baicalein 40 µM (% cells) | |||
---|---|---|---|---|
Cell cycle stage | Median | P25-P75 | Median | P25-P75 |
G1 | 61.52 | 60.23–63.62 | 82.54 |
80.45–83.61 |
S | 21.82 | 20.16–22.53 | 13.69 |
12.28–14.57 |
G2/M | 16.17 | 15.21–17.36 | 4.12 |
3.98–5.21 |
Statistical comparisons were made using the Wilcoxon test.
P<0.05 vs. control group (0 µM baicalein). P25, 25th percentile; P75, 75th percentile.
Effect of baicalein on the expression of Bcl-2, Bax, PCNA and cyclin D1.
Relative expression | ||||
---|---|---|---|---|
Baicalein 0 µM | Baicalein 40 µM | |||
Protein | Median | P25-P75 | Median | P25-P75 |
Bcl-2 | 1.000 | 1.000–1.000 | 0.661 |
0.629–0.762 |
Bax | 1.000 | 1.000–1.000 | 1.061 | 0.993–1.141 |
PCN |
1.000 | 1.000–1.000 | 0.683 |
0.652–0.713 |
Cyclin D1 | 1.000 | 1.000–1.000 | 0.698 |
0.659–0.701 |
Statistical comparisons were made using the Wilcoxon test.
P<0.05 vs. control group (0 µM baicalein). P25, 25th percentile; P75, 75th percentile; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; PCNA, proliferating cell nuclear antigen.
Effect of signal pathway inhibition on endometrial stromal cell viability.
Cell viability (relative to control) | |||
---|---|---|---|
Treatment | Inhibited signal | Median | P25-P75 |
Baicalein 0 µM | None | 1.000 | 1.000–1.000 |
Baicalein 40 µM | None | 0.730 |
0.632–0.905 |
LY294002 + baicalein 40 µM | Akt pathway | 0.694 |
0.524–0.753 |
SB203580 + baicalein 40 µM | MAPK pathway | 0.715 |
0.685–0.812 |
WP1066 + baicalein 40 µM | STAT3 | 0.702 |
0.498–0.792 |
Nicotinohydrazide + baicalein 40 µM | STAT5 | 0.683 |
0.576–0.853 |
SP600125 + baicalein 40 µM | JNK pathway | 0.741 |
0.669–0.821 |
U0126 + baicalein 40 µM | ERK 1/2 pathway | 0.659 |
0.518–0.781 |
BAY11-7080 + baicalein 40 µM | NF-κB | 1.100 | 0.978–1.135 |
Statistical comparisons were made using the Kruskal-Wallis test.
P<0.05 vs. control group (0 µM baicalein). P25, 25th percentile; P75, 75th percentile; MAPK, mitogen-activated protein kinase; STAT, signal transducer and activator of transcription; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; NF-κB, nuclear factor-κB.