Endometriosis is a common gynecological disease characterized by endometrial tissue growth outside the uterus, owing to increased cellular proliferation or decreased apoptosis in response to appropriate stimuli. Globally, endometriosis reportedly affects 5%-20% of women of reproductive age, with patients generally experiencing non-cyclical pelvic pain, dyspareunia, dysmenorrhea, and subfertility. Based on accumulated evidence, apoptosis helps maintain cellular homeostasis by promoting the removal of senescent cells from the functional layer of the endometrial lining during menstrual and late secretory phases of the cycle (Herington
Luteolin (3,4,5,7-tetrahydroxyflavone) is a natural flavonoid abundant in several food sources, including fruits, vegetables, nuts, and herbs (Lopez-Lazaro, 2009; Bhagwat
Luteolin was isolated from the ethyl acetate (EtOAc) fraction of the 70% EtOH extract flower buds of
Human endometriotic 12Z cells were generously gifted by Dr. Starzinski-Powitz (Johann-Wolfgang-Goethe-Universitaet, Frankfurt, Germany) and were maintained at 37°C in a containing 5% CO2 and cultured in DMEM containing 5% (v/v) fetal bovine serum, 100 U/ml penicillin, and 100 μg/mL streptomycin sulfate. The cells were treated with vehicle (DMSO) or luteolin. The human monocyte THP-1 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI 1640 containing FBS 10% (v/v), streptomycin sulfate (100 μg/mL), penicillin (100 U/mL), and 2-mercaptoethanol (0.05 mM). THP-1 cells were differentiated into macrophages with 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h. To prepare the endometriosis-associated macrophages (EAMs), the macrophages were stimulated with conditioned medium (CM) containing various soluble factors derived from endometriotic 12Z cells. The 12Z cells (1×106) with 3 mL complete medium were seeded and cultured in 60 mm dish. After 48 h, the medium of the cultures was harvested and used for stimulation of macrophages.
12Z cells (1×105) were seeded into each well of a 96-well plates and allowed for incubation overnight. After 48 h treatment, 25 μL of a 0.5 mg/mL MTT solution was added into each well and the cells were further incubated at 37°C for 4 h. Thereafter the media was aspirated and dimethyl sulfoxide (DMSO) was added to dissolve the formazan blue. Absorbance of the samples was determined at a wavelength of 540 nm by Spectra Max (Molecular Devices, Sunnyvale, CA, USA).
Cells were treated with luteolin for 24 h. After collection, the cells were washed in ice-cold PBS. Subsequently, the cells were centrifuged at 1,000 rpm for 10 min and washed twice with PBS. The cells were resuspended with a binding buffer (0.1 M Hepes/NaOH pH 7.4, 25 mM CaCl2, 1.4 M NaCl), containing PI and Annexin V- FITC, and stored in the dark at room temperature for 20 min. Apoptosis of cells analyzed by Guava easyCyte flow cytometry system (Millipore Corporation, Billerica, MA, USA).
Lysates from cells were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to the polyvinylidene fluoride (PVDF) membranes at 300 mA for 1 h. The membranes were blocked in 5% skimmed milk/TBST (Tris-buffered saline buffer containing 0.1% Tween-20) for 30 min, followed by incubated with primary antibodies using TBST with 3% skimmed milk. Antibodies used include: caspase-3 (1:1,000), caspase-8 (1:1,000), caspase-9 (1:1,000), and β-actin (1:1,000). The washed membranes were probed with horseradish peroxidase-conjugated secondary antibody (1:1,000) in TBST with 3% skimmed milk. The membranes were washed again three times in TBST buffer and incubated with ECL solution for 1 min. Immunoreactivity for the transferred proteins was visualized and analyzed by Image Quant LAS-4000 (Fujifilm Life science, Tokyo, Japan).
According to the manufacture’s protocol, total RNA was isolated from 12Z cells and macrophages using Easy Blue reagent (Invitrogen, San Diego, CA, USA). After total RNA was reverse transcribed into the cDNA using first-strand cDNA synthesis kit (Amersham Pharmacia Biotech), SYBR Premix Ex Taq™ Kit and Thermal Cycler Dice Real-Time PCR System (TaKaRa) were used for the reverse-transcription (RT)-PCR. The primers pairs used for real-time RT-PCR reactions (Bioneer, Seoul, Korea) are as follow (forward and reverse): human CCL5, 5’-CCTCATTGCTAGGCCCTCT-3’ and 5’-GGTGTGGTGTCCCGAGGAAT-3’; human CCL2, 5’-GCTCATAGCAGCCACCTTCA-3’ and 5’-GGACACTTGCTGCTGGTGAT-3’; human CD206, 5’-ACCTCACAAGTATCCACACCATC-3’ and 5’-CTTTCATCACCACACAATCCTC-3’; human Trem-2, 5’-TTGCCCCTATGACTCCATGA-3’ and 5’-CGCAGCGTAATGGTGAGAGT-3’; human MMP-9, 5’-CGATGACGAGTTGTGGTCCC-3’ and 5’-TCGTAGTTGGCCGTGGTACT-3’; human MMP (matrix metalloproteinase)-2, 5’-ACCGCGACAAGAAGTATGGC-3’ and 5’-CCACTTGCGGTCATCATCGT-3’; human IL-10, 5’-GACCAGCTGGACAACATACTGCTAA-3’ and 5’-GATAAGGCTTGGCAACCCAAGTAA-3’; human VEGF (vascular endothelial growth factor), 5’-ATGGCAGAAGGAGGAGGGCA-3’ and 5’-ATCGCATCAGGGGCACACAG-3’; human GAPDH, 5’-GAGTCAACGGATTTGGTCGT-3’ and 5’-TTGATTTTGGAGGGATCTCG-3’. To analyze the relative mRNA levels of target gene, the average cycle threshold (Ct) value of each triplicate reaction was normalized relative to that of an internal control, GAPDH. Melting curve analysis was performed to verify the presence of gene-specific peak and the absence of primer dimer.
Statistical analysis for comparison was performed using the One-way ANOVA or Student’s
Endometriosis is characterized by the ectopic growth of endometrial tissues. In the present study, we investigated the effect of luteolin (Fig. 1A) on the growth of 12Z human endometriotic cells using the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Luteolin significantly inhibited cell growth in 12Z cells in a dose-dependent manner (Fig. 1B), prompting us to further explore the molecular mechanism underlying this effect. To investigate whether luteolin-induced cell death is associated with the induction of apoptotic cell death, Annexin V-FITC staining was performed. As shown in Fig. 2, luteolin markedly enhanced the population of apoptotic Annexin V-FITC-positive cells in a dose-dependent manner, indicating that luteolin induces apoptotic cell death in human endometriotic cells.
Next, we investigated whether luteolin-induced apoptosis was caspase-dependent. Western blotting was performed to evaluate the activation of initiator caspases (caspase-8 and caspase-9) and an effector caspase (caspase-3) indicated by the density of pro-caspases. As shown in Fig. 3A, luteolin dose-dependently and markedly decreased the levels of pro-caspase -3, -8, and -9 in 12Z cells. Various caspase inhibitors were then employed to further confirm the involvement of caspases in luteolin-induced cell death (Fig. 3B). Pretreatment with Z-VAD-FMK (a broad caspase inhibitor), Z-DEVD-FMK (caspase-3 inhibitor), Z-IEVD-FMK (caspase-8 inhibitor), and Z-LEHD-FMK (caspase-9 inhibitor) markedly reduced the inhibitory effect of luteolin on cell growth. These findings indicate the involvement of the caspase-dependent pathway in luteolin-induced apoptosis in human endometriotic cells.
The increased production of chemokines, such as C-C motif chemokine ligand 2 (CCL2) and CCL5, by endometriotic tissues, has been postulated to promote a pro-inflammatory environment that fuels disease pathogenesis by inducing macrophage recruitment to endometriotic lesions (Hornung
Reportedly, macrophages are recruited to endometriotic lesions, where they undergo alternative activation (M2 polarization) (Capobianco and Rovere-Querini, 2013). Therefore, we investigated the effect of luteolin on the M2 polarization of macrophages. Macrophages were stimulated by conditioned medium containing various soluble factors (cytokines, chemokines, and growth factors) derived from endometriotic 12Z cells and the so-called endometriosis-associated macrophages (EAMs), revealing increased intracellular expression of the CD206 and Trem-2 M2 phenotype markers when compared with that in control macrophages (Fig. 5). Treatment with luteolin significantly reduced the mRNA expression of CD206 and Trem-2 in EAMs. Notably, EAMs are known to produce various endometriosis-promoting factors, such as matrix metalloproteinase (MMP)-2/9, interleukin (IL)-10, and vascular endothelial growth factor (VEGF) (Woo
In recent years, natural compounds commonly found to occur in food have been widely researched to prevent and treat various gynecological disorders, including endometriosis (Varma
The growth of endometriotic cells plays a central role in the development of endometriotic lesions (Di Paola
The levels of CCL2 and CCL5, which play vital roles in macrophage recruitment, are known to be significantly increased in the peritoneal fluid of women with endometriosis (Arici
Overall, these data indicate that luteolin stimulates apoptosis in human endometriotic cells and suppresses the activation of EAMs, implying that the anti-inflammatory properties of luteolin are associated with its inhibitory effect on EAMs and endometriotic cells.
This work was supported by a grant the National Research Foundation of Korea (NRF) (No. NRF-2017R1A6A3A11034320 and NRF-2020R111A1A01075620).
Authors have nothing to declare.