This study was conducted according to the principles of the Declaration of Helsinki. It was approved by the ethical committee of the Second Affiliated Hospital of Wenzhou Medical University. A written informed consent was granted from all subjects at the time of enrollment.
Study population and data collection
Patients with uterine fibroids and hyperlipidemia at the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, between January 1, 2012, and June 30, 2015, were identified. Uterine fibroids were diagnosed by ultrasonography and the diagnosis of hyperlipidemia was followed guidelines published by the American Heart Association . A total of 120 subjects were enrolled with a median age of 44 years (range 34–51 years). All of these 120 cases were intramural myoma (according to evaluation of ultrasonography) and asymptomatic fibroids, in which 53 patients administrated 20 mg atorvastatin everyday, orally (study group), while 67 cases without statins (control group). All individuals were followed up for 2 years.
None of these 120 patients had received medical treatment that might affect the tumor growth of uterine fibroids during the following-up, such as GnRHa, oral contraceptive, estrogen and progesterone drugs or mifepristone. The tumor size in these patients was measured by ultrasonography both before and after atorvastatin treatment. Ultrasound examination of the uterus was performed at 3 monthly intervals. The length (d1), width (d2) and depth (d3) of each tumor were measured, and the volumes were calculated by the following formula: volume (cm3) = π × d1 × d2 × d3/6. Volume change was calculated using the following formula: post-treatment volume (cm3) − pre-treatment volume (cm3).
Atorvastatin, GGPP and FPP were purchased from Sigma Biochemicals (St. Louis, MO, USA). Atorvastatin was dissolved in dimethylsulfoxide (DMSO) and diluted with medium to reach 0, 1, 5, 10, 20, 40 μM before treatment. GGPP and FPP were used at 10 μM.
Cell line and primary cell cultures
The immortalized human uterine fibroids cell line (HuLM) was a generous gift from William H. Catherino (Uniformed Service University). These HuLM cells were cultured and maintained in DMEM/F12 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 1% antibiotic (penicillin–streptomycin), in 5% CO2 at 37 °C.
Primary human uterine fibroid cells were generated from uterine fibroid specimens collected from the Second Affiliated Hospital of Wenzhou Medical University with an approved protocol. Uterine fibroids tissue samples were collected from 45 Chinese women aged 31–45 years, who underwent hysterectomy to surgically remove confirmed uterine fibroids between Jan 2016 and Jan 2017. The patients were not administered any hormone supplementations including statins for at least 6 months before the hysterectomy was performed. Patients with various complications, such as infections, chronic diseases, uterine malignancy and/or adenomyosis were also excluded.
For preparation of the primary cell population, a portion of the fresh uterine fibroids tissue was washed in cold phosphate-buffered saline (PBS) to remove blood and then chopped into small pieces (1 mm3) under sterile conditions, digested with 0.2% (v/v) collagenase II (Invitrogen, Carlsbad, CA, USA) in Dulbecco’s modified Eagle’s medium (DMEM) for 4 h in a 37 °C with shaking. The dissociated cells were centrifuged at 400×g for 5 min. The resultant cell deposit was suspended with complete culture medium (DMEM, 10% fetal bovine serum, 100 IU/ml of penicillin G and 100 μg/ml streptomycin) and centrifuged at 400×g for 5 min. The resultant cells were cultured at a density of 2 × 105 cells/ml under 5% CO2 at 37 °C. Cells from third passages to the seventh were used for the experiments.
Staining of α-smooth muscle actin by immunocytochemistry
Uterine fibroids cells were identified by the expression of α-smooth muscle actin. Briefly, cells were fixed with 4% paraformaldehyde, permeabilized in PBS containing 0.2% Triton X-100 for 15 min, incubated in a serum-free blocking solution for 15 min at room temperature, and then incubated with mouse monoclonal anti-α-smooth muscle actin antibody (Zhongshan Golden Bridge Biotechnology, Beijing, China) overnight at 4 °C. After extensive washing with PBS, cells were incubated with biotinylated goat anti-mouse IgG as secondary antibody. After incubation the bound antibodies were visualized using 3,3′-diaminobenzidine. Finally, nuclei were stained with hematoxylin. Negative control incubated with PBS instead of primary antibody.
Cell counting kit-8 (CCK-8) assay
To determine the cell proliferation, 1 × 104 cells/well were seeded into 96-well plates and incubated at 37 °C with 5% CO2. After 24 h of incubation, the cells were treated with indicated drugs. By the end of treatments, 10 μl of CCK-8 (Dojindo, Kumamoto, Japan) was added to each well and incubated for an additional 2 h. The reaction product was quantified by spectrophotometry at 450 nm wavelength, and the percentage of viability or number of cells was calculated by formula: (treated cells absorbent/non treated cells absorbent) × 100.
Flow cytometric assay of apoptosis with Annexin-V FITC Staining
Cell apoptosis was assayed by flow cytometry after Annexin-V FITC staining. Cells were plated at 5 × 105 cells/dish into 60 mm dishes. After reaching 70–80% confluence during exponential growth, cells were harvested, washed with cold PBS and resuspended with binding buffer at a concentration of 2 × 106 cell/ml. Cells were analyzed by using the ApoTarget™Annexin-V FITC Apoptosis kit (Invitrogen, Grand Island, NY) according to the manufacture’s protocol.
Western blot analysis
Total proteins were prepared by whole-cell lysis with the buffer (60 mM Tris–HCl, pH 6.8; 5% glycerol; 2% SDS); on ice. The protein from each experimental group was quantified by bicinchoninic acid method (Beyotime, Jiangsu, China). Cellular proteins (30 μg) were solubilized in sample buffer (5× DualColor, ddH2O), and heated at 100 °C for 10 min to denature proteins. The proteins were separated by using electrophoresis on 12% sodium dodecyl sulfate–polyacrylamide gel and then electro-transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 2 h at room temperature in 0.05 M Tris-buffered saline with 0.1% Tween-20 (TBS-T, pH 7.4) containing 5% skimmed milk and then incubated in TBS-T overnight at 4 °C with one of the appropriate primary antibodies at 1:1000 dilutions, unless specified otherwise. The primary antibodies used were PCNA (CST, USA), Bim (CST, USA), Bax (CST, USA), Bcl-2 (CST, USA), caspase 3 (CST, USA), cleaved-caspase 3 (CST, USA), ERK1/2 (CST, USA); p-ERK1/2 (CST, USA); JNK (Abcam, USA), p-JNK (Abcam, USA), α-actin (1:2000; Beyotime, China); and β-tubulin (1:2000; Beyotime, China). After washing with TBS-T, the proteins were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000 for anti-rabbit-IgG or anti-mouse-IgG) for 1 h at room temperature. Blots then were developed by an enhanced chemiluminescence. The expression levels of above proteins were quantified with densitometry and normalized by corresponding levels of β-tubulin or α-actin respectively. Each experiment was repeated at least three times.
Human phospho-mitogen-activated protein kinase (MAPK) antibody array
MAPK protein phosphorylation was screened with 300 μg of cell extracts using Human Phospho-MAPK Array Kit according to the manufacturer’s instructions (Proteome Profiler; R&D Systems,minneapolis, MN, USA), which could be used to detect the relative levels of phosphorylation of 26 kinases (Akt1, Akt2, Akt3, Akt pan, CREB, ERK1, ERK2, GSK-3α/β, GSK-3β, HSP27, JNK1, JNK2, JNK3, JNK pan, MKK3, MKK6, SK2, p38α, p38β, p38δ, p38γ, p53, p70 S6 Kinase, RSK1, RSK2, TOR).
All statistical analyses were performed with SPSS17.0 software. If each group data was of normal distribution and homogeneity of variance, quantitative data was presented as the mean ± standard deviation while skewed variables were reported as median and interquartile range (IQR). Wilcoxon test or t test was used for comparison as appropriate. Differences among multiple groups were analyzed by one-way ANOVA. Qualitative variables were expressed as proportions and were compared with Chi squared or Fisher exact test as appropriate. A 2-tailed P value < 0.05 was considered statistically significant.