The Effect of Curcumin on Angiogenic and Proliferative Factors inHuman Endometriotic Cells

Endometriosis is a chronic inflammatory disease and one of the most common gynaecological disorders in women of childbearing age. It is characterised by the growth of endometrial-like tissue outside the uterus ( 1 ). An estimated 6 to 10% of reproductive-age women are affected by endometriosis, and most suffer from pelvic pain and infertility ( 2 ). The pathogenesis of endometriosis has been widely studied, and various hypotheses have been proposed. The most commonly accepted theory is Sampson’s transplantation theory, which describes retrograde menstruation. This phenomenon can cause the transfer of endometrial cells to the peritoneal cavity through the fallopian tubes and result in their Placement in the peritoneal cavity ( 1 ). However, endometriosis does not occur in all women with recurrent retrograde menstrual bleeding, and this indicates the involvement of genetic, epigenetic, and environmental factors in this disease ( 3 ). According to Sampson’s theory, adhesion and proliferation of endometrial tissue, cellular invasion, and neoangiogenes are key factors in the pathogenesis of endometriosis. Therefore, growth and angiogenesis factors such as insulin-like growth factors (IGF1, IGF2) and vascular endothelial growth factor (VEGF) play a critical role in ectopic endometrial cell proliferation ( 4 ). Long non-coding RNA (lncRNA) H19 is one of the first identified imprinted lncRNAs expressed from the maternal allele. H19 is involved in the regulation of cell proliferation and differentiation and serves a critical role in various biological processes of different diseases ( 5 ). Recent research shows that decreased expression of H19 alters stromal cell growth through IGF signalling in the endometrium of patients with endometriosis ( 6 ). New agents can effectively improve endometriosis in patients. Considering that diet is a potential risk factor for this disease, food compounds have recently been considered as therapeutic and preventive agents ( 7 , 8 ). One of these nutrients is curcumin, which is produced by the Curcuma longa plant. Curcumin is a potent anti-inflammatory agent. Several studies have shown that curcumin has antiinflammatory, antioxidant, anti-cancer, and anti-angiogenic effects ( 9 , 10 ).Curcumin can inhibit angiogenesis, proliferation, invasion, and metastasis of different cancers through targeting signalling pathways ( 11 ). In addition, the anti-inflammatory, anti-angiogenic, anti-proliferative, and anti-invasive effects of curcumin on endometriosis have been reported ( 12 - 15 ). In our previous study, we analyzed the expression levels of H19 lncRNA along with genes involved in angiogenesis (VEGF) and proliferation (IGF1, IGF2) , in endometrial tissues from patients with endometriosis in comparison with healthy women. Increased VEGF levels along with decreased H19 lncRNA, IGF1 , and IGF2 expressions were observed in the eutopic endometria of women with endometriosis ( 16 ). For the current study, we took into consideration the role of an giogenesis, proliferation factors, and migration in endometriosis and the anti-angiogenesis and anti-proliferation effects of curcumin. We intend to evaluate the impact of curcumin on VEGF, IGF1, IGF2, and H19 expressions, in addition to cell migration and proliferation in eutopic endometrial stromal cells (EU-ESCs) from women with endometriosis compared to normal endometrial tissues.

ESCs incubated with various concentrations of curcumin (0-100 mM) for 72 hours showed that curcumin inhibited cell proliferation and migration in a time- and dose-dependent manner. The IC50 value for curcumin at 72 hours was 42.20 mM for the E 50 U-ESCs from endometriosis patients ( Fig .1 ). The wound healing assay was performed to evaluate the effects of curcumin on proliferation and migration of EU-ESCs. Curcumin significantly inhibited cell migration in cultured EU-ESCs compared to untreated EU-ESCs ( Fig .2A ). The inhibitory effect of curcumin was time- and dose-dependent ( Fig .2B ). Based on the cell viability results and IC 50 at 72 hours, we selected the 30 mM dose of curcumin and an incubation time of 72 hours for further assessment. Curcumin effect on cell proliferation. Effect of different doses of curcumin (0-100 mM) on human EU-ESCs cultured in vitro after 72 hours of incubation. Curcumin inhibited the growth of EU-ESCs after 72 hours of treatment at different concentrations. The IC 50 value for curcumin at 72 hours was 42.20 mM for EU-ESCs. IC 50 ; Half maximal inhibitory concentration, EU-ESCs; Eutopic endometrial stromal cells, and OD; Optical density. Gene expression analysis showed a decrease in VEGFA expression in EU-ESCs following treatment with curcumin (P=0.250, Fig .3A ). IGF1 expression increased in N-ESCs and EU-ESCs treated with curcumin compared to non-treated cells, but these increases were not significant (P=0.250, Fig .3B ). IGF2 expression increased in curcumin-treated EU-ESCs compared to those cultured without curcumin; however, it decreased in N-ESCs treated with curcumin compared to non-treated cells. These differences were not significant (P=0.250, Fig .3C ). Although N-ESCs and EU-ESCs had higher H19 expression after curcumin treatment compared to the untreated conditions, this increase in the curcumin-treated EU-ESCs and EU-ESCs was not significant (P=0.250, Fig .3D ). Table 2 shows the descriptive statistics obtained from the non-parametric test (Wilcoxon). Evaluation of migration of EU-ESCs by the scratch test. A, B. Cell migration of EU-ESCs was decreased by curcumin treatment in a time-and concen tration-dependent manner (scale bar: 500 μm). EU-ESCs; Eutopic endometrial stromal cells. Gene expression patterns following treatment with curcumin. Relative mRNA expression levels of: A. VEGF , B. IGF1 , C. IGF2 , and D. H19 treated with 30 mM curcumin for 72 hours were evaluated in cultured N-ESCs and EU-ESCs. Gene expression was analysed by qRT-PCR. GAPDH was used as a reference gene. The results are expressed as median and interquartile range. ns; P>0.05, N-ESCs; Normal endometrial stromal cells, EU-ESCs; Eutopic endometrial stromal cells, and qRT-PCR; Reverse transcription quantitative polymerase chain reaction. Descriptive statistics of the data The descriptive statistics of data obtained from non-parametric test (Wilcoxon). N-ESCs; Normal endometrial stromal cells, EU-ESCs; Eutopic endometrial stromal cells, and IQR; Interquartile range.

Endometriosis is a gynaecological disorder characterised by abnormal cell adhesion, invasion, growth, and proliferation of endometrial cells, along with neoangiogenesis, which leads to implantation in ectopic sites ( 19 ). In our previous study, overexpression of VEGF in the eutopic endometrium of women with endometriosis was detected compared to the control endometrium. Expression of H19 was lower in eutopic endometrial samples compared with the control endometrium. The expression levels of IGF1 and IGF2 were also decreased in the eutopic samples compared to the control group ( 16 ). These altered patterns of expression suggest an impaired regulation of cellular growth and differentiation in endometriotic tissues. The aim of the present experimental study was to determine if curcumin, as an anti-angiogenic and anti-proliferative agent, could affect the expression of these genes. Curcumin (diferuloylmethane) is the main active poly phenol in turmeric, and has a low molecular weight. The chemical formula of curcumin is C 21 H 20 O 6 ; it contains 2-8% turmeric and was first identified in 1910 for its chemical properties ( 20 , 21 ). Several studies have shown the effects of curcumin on inflammation, invasion, angiogenesis, cell proliferation, and apoptosis ( 22 , 23 ). In the present study, we found that curcumin decreased cellular migration and proliferation of EU-ESCs in a time- and dose-dependent manner. Consistent with the present results, Zhang et al. ( 24 ) observed a dose-dependent, antiproliferative effect of curcumin in ESCs from patients with endometriosis. Curcumin inhibited cell proliferation in ovarian and endometrial cancer cells ( 25 ). It blocked the increase in size and weight of endometriosis lesions in endometriotic rats in a time- and dose-dependent manner ( 26 ). These studies suggest that treatment with curcumin is associated with decreased cell proliferation in endometriosis. In the present study, curcumin decreased VEGF expression, which is consistent with previous studies. Zhang et al. ( 27 ) showed that curcumin reduced VEGF protein expression in ectopic tissues of a rat model with endometriosis. In another study, curcumin reduced the survival of endometriotic stroma cells in vitro by reducing VEGF protein expression ( 28 ). Also, the anti-angiogenic effect of curcumin has been reported in ovarian cancer ( 29 ). Downregulation of VEGF was attributed to curcumin treatment, and might lead to a decrease in the ability of endometrial cells to implant at the ectopic sites. The IGFs play a main role in regulating endometrial cell growth and differentiation ( 30 ). lncRNA H19 is involved in the regulation of cell proliferation and differentiation ( 31 ). The current study results showed that curcumin increased the expression levels of IGF1, IGF2, and H19. However, the effect of curcumin on the expressions of these genes in endometriosis has not been studied. Other studies have shown that curcumin reduces IGF1 and IGF2 expression in various diseases and cancers under in vivo and in vitro conditions ( 32 - 34 ). Similar to our results, only one study showed an increase in IGF1 gene expression in curcumin-treated diabetic rats ( 35 ). Although there are limited studies on the effect of curcumin on H19 gene expression, these studies have shown that curcumin reduces its expression in tumour cells, and this was inconsistent with our results ( 36 , 37 ). This discrepancy may be related to the diverse biological functions of H19 in cancer biology, which is known both as an oncogene and a tumour suppressor gene ( 38 ). In addition, we previously observed decreased expression of H19 in endometriosis ( 16 ), and this finding suggests that H19 may have a suppressive role in endometriosis. In the present study, the increased H19 gene expression after curcumin treatment might indicate the therapeutic effect of curcumin in endometriosis. However, in vivo investigations are needed, especially in animal models. The role of H19 in regulating cell proliferation and differentiation may be due to its association with IGF1 and IGF2 ( 6 , 38 ). We previously reported that the reduction in H19 in endometriosis lesions possibly caused decreased IGF1 and IGF2 expressions. This pattern implies that the endometriotic tissue may undergo a disturbance in cellular growth regulation and differentiation ( 16 ). In support of this hypothesis, the present findings showed that although curcumin treatment increases H19, IGF1, and IGF2 expressions in endometriotic tissue (EU-ESC), this pattern was not completely detected in normal endometrium (NESC). It implied that the EU-ESC of endometriosis has different behaviour than N-ESC of normal endometrium in response to curcumin treatment. Thus, the increased expressions of IGF1, IGF2, and H19 after curcumin treatment could be considered a new finding of the present study. However, due to the limited studies in this field and the inconsistent results of the effect of curcumin on other diseases, further investigations, especially in vivo studies, are recommended to determine the role of curcumin in endometriosis.

Our data demonstrated that curcumin decreases cell migration and proliferation of endometriotic stromal cells in a dose - and time-dependent manner. The present study also showed that curcumin reduces the expression of genes involved in angiogenesis. Another finding of this study was the increase in IGFs and H19 expressions in the presence of curcumin, which suggests that curcumin can be an effective treatment for endometriosis.

DNase I, Dispase II, collagenase types І and IV, β-mercaptoethanol, and curcumin were purchased from Sigma Corporation, USA. DMED/F-12 (1:1), foetal bovine serum (FBS), GlutaMAX, non-essential amino acids (NEAA), penicillin-streptomycin, and phosphate-buffered saline were purchased from Gibco, USA. This experimental study was approved by the Ethics Committee of Royan Institute (IR.ACECR.ROYAN. REC.1398.95) and written informed consent was obtained from participants before the endometrial biopsies. Based on our previous study ( 17 ), we enrolled three women with endometriosis (case group) and three women without endometriosis (control group) in the current study. The age range of the participants was 30 to 40 years. All women had regular menstrual cycles and had not received any hormonal therapy for at least three months before endometrial sampling. Endometriosis was diagnosed by the existence of endometriotic lesions during laparoscopy and after pathological examination. These endometriosis patients had stage ІІІ or ІV disease according to the revised classification of the American Fertility Society. Endometrial specimens were collected from both groups during the proliferative phase of their menstrual cycles. The samples were obtained under sterile conditions using a pipelle by an expert gynaecologist. The endometrial specimens were placed in sterile medium and transferred to the laboratory. Tissues were washed using washing medium with gentle stirring to remove blood cells and mucous. Then, the tissues were dissected into small pieces and incubated in DMEM/F-12 that included 10% FBS, collagenase type I (1 mg/ml), collagenase type IV (1 mg/ml), DNase I (1 mg/ml), and Dis pase II (4 mg/ml) for 30 minutes at 37°C. The epithelial cells were removed by serial filtration of the stromal cells through 70 and 40 µm sieves. The ESCs were obtained by centrifuging the cell suspension at 500 ×g for 5 minutes. The resultant pellet was resuspended and cultured in a 25 cm 2 flask in DMEM/F‐12 medium that contained 10% FBS, 1% GlutaMAX, 1% penicillin/streptomycin, and 1% NEAA, then incubated at 37°C in a humidified 5% CO2 incubator. The culture medium was replaced every 2 -3 days. All cultures were passaged three times, and when they reached 70-80% confluency, the cells were used for treatment with or without curcumin. Purity of the stromal cells was assessed by flow cytometry and an antibody panel against CD29, CD31, CD45, CD73, and CD90 (all from Becton Dickinson Biosciences, USA), and immunofluorescent staining for vimentin ( 17 ). ESC proliferation was assessed by the methylthiazole tetrazolium (MTT) test. The cells were seeded in 96-well culture plates at 5×10 3 cells/well. After the cells attached, they were treated with various concentrations of curcumin (0-100 mM) for 72 hours. This time was selected based on the doubling time of ESCs. MTT was performed as previously reported ( 18 ). Curcumin was dissolved in dimethyl sulphoxide (DMSO) and diluted in culture media to the desired concentrations. The final concentration of DMSO was less than 0.1%. The wound-healing migration assay was used to measure cell migration. For this purpose, a line was scratched using a pipette tip after the cells reached confluency. Then, the detached cells were removed by washing them. Fresh media without curcumin or with different concentrations of curcumin were added to the plates. Representative photographs were taken under an inverted light microscope (Olympus, Japan) at various times (0, 24, 48, and 72 hours). RNA was extracted from the cells using an RNeasy Micro kit (Qiagen, Germany) according to the manufacturer’s instructions. DNase I (Takara, USA) was used to remove any DNA contamination. Both the concentration and purity of the RNA samples were evaluated using a Nanodrop 2000 spectrophotometer (Thermo Scientific, USA). Complementary DNA synthesis was performed using a TaqMan reverse transcription kit (Takara, USA), according to the manufacturer’s instructions. Subsequently, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed using a Step-One RT-PCR (AB Applied Biosystems, USA) with primers designed for VEGF, IGF1, IGF2, and H19 ( Table 1 ). The mean fold changes of these genes were calculated using the 2 −ΔΔCT algorithm, and their expressions were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal standard. Primer pairs used in this study All experiments were performed in triplicate. The data were analysed using GraphPad Prism software, version 8 (GraphPad, San Diego, CA, USA) and the non-parametric Wilcoxon test. Data are presented as medians and interquartile ranges. P<0.05 indicated statistical significance. The distance between the edges of the lines was measured with ImageJ software, and Prism software was used to determine the half maximal inhibitory concentration (IC 50 ).