{"paper_id":"385780af-ac45-4e91-abe9-249805df5472","body_text":"Int. J. Med. Sci. 2022, Vol. 19 \n \n \nhttps://www.medsci.org \n1779 \nInternational Journal of Medical Sciences\n \n2022; 19(12): 1779-1786. doi: 10.7150/ijms.75203 \nResearch Paper \nThe Potential of Transforming Growth Factor-beta \nInhibitor and Vascular Endothelial Growth Factor Inhibitor \nas Therapeutic Agents for Uterine Leiomyoma \nJung Yoon Park, Boah Chae, Mee-Ran Kim \nDepartment of Obstetrics and Gynecology, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea. \n Corresponding author: Professor Mee-Ran Kim, M.D., Ph.D., Division of Reproductive Endocrinology, Department of Obstetrics & Gynecology, Seoul St. \nMary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo -daero, Seocho-gu, Seoul 06591, Korea. Tel .: 82 -2-2258-6170; Fax: \n82-2-595-7549; E-mail: mrkim@catholic.ac.kr. \n© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecom mons.org/licenses/by/4.0/). \nSee http://ivyspring.com/terms for full terms and conditions. \nReceived: 2022.05.16; Accepted: 2022.09.13; Published: 2022.10.03 \nAbstract \nBackground: Uterine leiomyoma is the most common benign tumor in women of reproductive age, and \nit can cause infertility. The growth of uterine leiomyoma is mediated by various steroids and growth \nfactors. The purpose of this study was to evaluate the expression of various growth factors in uterine \nleiomyoma. Additionally, comparing the effects of existing medication and specific growth factor \ninhibitors on leiomyoma and the normal myometrium, we aimed to see the potential of transforming \ngrowth factor -beta (TGF -β) inhibitors and vascular endothelial growth factor (VEGF) inhibitors as \ntherapeutic drugs for uterine leiomyoma. \nMethods: This in vitro study included uterine leiomyoma samples from 12 patients who underwent \nhysterectomy by laparoscopy or laparotomy at Seoul St. Mary’s Hospital between May 2016 and March \n2018. Normal myometrium and uteri ne leiomyoma tissue were obtained from each patient and the \nexpression of growth factors was compared using immunohistochemical staining. After the primary \nculture of normal myometrial and leiomyoma cells, cell viability was evaluated following treatment with \n100 nM ulipristal acetate (UPA) and mifepristone for 48 h. Western blot analysis was performed to \ndetermine the protein expression of  each growth factor. Cell viability was determined following \ntreatment with a 10 -µM TGF-β inhibitor (LY364947) and a 5-µ M VEGF inhibitor (axitinib) for 24 h in \ncultured normal myometrium and leiomyoma cells. \nResults: Immunohistochemical staining revealed the significantly higher intensity of TGF-β and VEGF in \nthe leiomyoma tissue than in the normal myometrium (P < 0.05). M ifepristone treatment decreased \nVEGF expression by 62% in the leiomyoma cells (P < 0.05). According to the cell counting kit-8 (CCK-8) \nassay, cell viability was decreased after UPA, mifepristone, TGF-β1 inhibitor, and VEGF inhibitor \ntreatments in the norma l myometrium and leiomyoma  tissue. The effects of the TGF -β1 inhibitor \nsignificantly differed between normal myometrium and leiomyoma tissue, with a greater decrease in cell \nsurvival in the leiomyoma tissue (P < 0.05).  Post-hoc analysis showed that the TGF -β1 and VEGF \ninhibitors had a greater inhibitory effect on leiomyoma tissue compared with that of UPA. \nConclusion: TGF-β and VEGF inhibitors significantly decreased the viability of uterine leiomyoma cells, \nshowing stronger effects than the conventional dr ug, UPA. TGF-β1 inhibitors affect both  leiomyoma \ntissue and the normal uterus; thus, targeted local treatment rather than systemic treatment should be \nconsidered. \nKey words: leiomyoma; axitinib; infertility; ulipristal acetate; mifepristone \nIntroduction \nUterine leiomyomas are the most common \nbenign tumors in women of childbearing age, causing \nexcessive menstruation and pressure symptoms [1]. \nThis condition can cause infertility, miscarriage, and \ncomplications during pregnancy [2, 3]. The \nprevalence of lei omyomas in infertile women is \n \nIvyspring  \nInternational Publisher \n\nInt. J. Med. Sci. 2022, Vol. 19 \n \nhttps://www.medsci.org \n1780 \n5%-10% [4, 5]. A meta- analysis published in 2009 \nbased on data from 18 studies showed that regardless \nof localization, the presence of leiomyoma results in a \nsignificant decrease in fertility rates and an increase in \nmiscarriage rates [6]. Uterine leiomyomas and \ninfertility are closely related to age [7]; the prevalence \nof leiomyoma increases with age, which is important \nbecause the age at which women bear children has \ngradually increased [7]. Despite the importance of \nfertility and the quality of life of women, treatments \nfor leiomyoma are limited and have not been widely \nexamined. In addition to surgical treatment, only a \nfew me dical treatments  are available [1]. Such \nmedications exert therapeutic effects by regulating \novarian steroid signaling but show limitations, such \nas long -term side effects, resulting in hypo - \nestrogenemia [8]. Thus, new targeted therapies are \nneeded. Recently, studies on the early disease stages \nsuggested that leiomyoma development is a \nmulti-step proces s involving various cytokines and \nvarious growth factors, although the molecular \nmechanisms are not completely understood. The \nprocess is considered to involve insulin- like growth \nfactor-1 (IGF -1), platelet -derived growth factor, \nvascular endothelial growth factor (VEGF), epidermal \ngrowth factor (EGF), and basic fibroblast growth \nfactor [9, 10]. The purpose of this study was to \ncompare the expression of the representative growth \nfactors in patients with uterine leiomyoma and to \ncompare the effect of the cur rently used medications \nwith growth factor inhibitors on myoma tissue and \nthe normal myometrium. \nMaterials and methods \nSubjects and tissue collection \nTwelve uterine leiomyoma tissues were obtained \nfrom women who underwent hysterectomy by \nlaparoscopy or lap arotomy at Seoul St. Mary’s \nHospital from May 2016 to March 2018. The patients \nwere aged between 38 and 52 years, with a mean age \nof 44.5 years. All patients had a menstrual cycle with \nsymptomatic leiomyoma , such as abnormal uterine \nbleeding, dysmenorrhea, and pressure symptoms. \nUterine leiomyoma is diagnosed preoperatively by \nultrasonography and magnetic resonance imaging \nand histologically confirmed after surgery. Eight \npatients had multiple leiomyomas and four had single \nleiomyoma. For all patients, norm al uterine \nmyometrium and leiomyoma tissues were collected \nfrom the uterus parts removed after hysterectomy. \nTwo of the 12 tissue samples obtained from the \npatients were discarded because they did not reach \nsufficient saturation. Informed consent was obtai ned \nfrom each patient before surgery for the use of uterine \ntissues. All tissues were collected in accordance with \nthe guidelines of the Declaration of Helsinki , and \napproval for the use of uterine leiomyoma was \ngranted by the Institutional Review Board of Seoul St. \nMary’s Hospital (No. KC 12TNSI0822). \nCell culture \nUterine leiomyoma tissues were dissected from \nthe uterus and washed with PBS. The tissues were \nminced into small piece s and digested in 30 mg/mL \ncollagenase IV and 4 mg DNase I at 37 °C for 3 h. The \nleiomyoma cells were collected by centrifugation at \n1,000g for 3 min and washed three times with \nphosphate buffered saline (PBS). Isolated leiomyoma \ncells were cultured in 100-mm\n2 culture dishes at 37 °C \nand 5% CO 2 in a humidified atmosphere in phenol \nred-free Dulbecco’s modified Eagle’s medium/F12 \n(Gibco Life Technologies, Grand Island, NY, USA) \nsupplemented with 10% fetal bovine serum and 100 \nU/mL penicillin (Gibco Life Techn ologies, Grand \nIsland, NY, USA). The morphological characteristics \nwere observed daily under an inverted microscope \nwith the medium changed every 2 days. When the \ncells reached saturation, they were isolated using \ntrypsin-EDTA (Gibco Life Technologies, Gra nd \nIsland, NY, USA). \nDrug treatment \nUlipristal acetate (UPA) (100 nM; HRA Pharma, \nParis, France), mifepristone (RU486; Sigma- Aldrich, \nSt. Louis, MO, USA), LY364947 (inhibitor of TGF -β; \nCat. #2718, Tocris Co., Ellisville, MO, USA), and \naxitinib (inhibitor o f VEGF; Cat. #4350, Tocris Co.) \nwere used to treat the cells for 48 h. \nImmunohistochemistry staining \nFor pathological examination of resected \nspecimens, the tissues were fixed with 10% formalin \nfor 24 h at 25 °C and embedded in paraffin. \nFormalin-fixed and  paraffin-embedded specimens \nwere cut into 5 -µm-thick sections. The tissues were \nrehydrated using xylene three times for 10 min and a \nseries of graded ethanol (absolute ethanol twice, 90% \nethanol, 80% ethanol, and 70% ethanol each for 5 min) \nat 25 °C. Afte r washing with water for 5 min, the \nsections were permeabilized using 3% H\n2O2 in \nmethanol for 10 min at 25 °C and washed again with \nwater for 5 min. For antigen retrieval, the sections \nwere immersed in citrate buffer (pH 9.0; DAKO S2367, \nGlostrup, Denmark), boiled for 10 min, cooled to 25 \n°C for 20 min on ice, washed with water, and blocked \nwith protein block serum -free (DAKO X0909) for 30 \nmin at 25 °C. The tissues were incubated with \nanti-EGF (ab9695, Abcam), TGF-β (ab92486, Abcam), \nVEGF-A (ab1316, Abcam), and IGF-1 (ab9572, Abcam, \nCambridge, UK) antibodies (1:500) overnight at 4 °C. \n\nInt. J. Med. Sci. 2022, Vol. 19 \n \nhttps://www.medsci.org \n1781 \nThe sections were washed with wash buffer (DAKO \nS3006) prior to incubation with DAKO REAL TM \nENVISION/HRP, rabbit/mouse (DAKO K5007) for \n30 min at 25 °C. \nWestern blot immunoassay \nProteins were extracted from the cultured \nuterine leiomyoma  cells. After drug treatment, the \ncells were lysed at 4 °C for 30 min  in lysis buffer. The \nlysates were centrifuged at 1,000 g for 30 min at 4 °C, \nand the supernatants were collected. The protein \ncontent in the supernatants was determined using the \nBCA assay (cat 23227). Each 50- µg aliquot of protein \nextracted from cultured cells was separated using a \nNuPAGE Novex 4%–12% Bis-Tris Gel (Invitrogen Life \nTechnologies, Carlsbad, CA, USA) under reducing \nconditions at 200 V for 50 min. The proteins were \nelectrophoretically transferred from the gels onto \npolyvinylidene fluoride transfer membranes \n(Amersham, Piscataway, NJ, USA). The blots were \nexposed overnight at 4 °C to primary antibodies: a \nmouse monoclonal antibody to VEGF (ab1316, \nAbcam) and a rabbit polyclonal antibody to EGF \n(ab9695, Abcam), IGF -1 (ab9572, Abcam ), or TGF -β \n(ab92486, Abcam) at a dilution of 1:200. The blots were \nwashed three times with Tris -buffered saline \ncontaining 1X Tween 20. The membranes were \nincubated for 1 h with anti -mouse or anti -rabbit \nsecondary antibodies (Santa Cruz Biotechnology, \nDallas, TX, USA) diluted to 1:1000 with blocking \nbuffer. Characteristic protein bands were detected  \nusing the ECL western blotting system (Amersham). \nProtein levels on the blots were standardized to the \nlevels of β -actin 1:1000 (sc47778, Santa Cruz \nBiotechnology). The membranes were visualized by \nexposure to an X -OMAT film (Eastman Kodak Co., \nRochester, NY, USA).\n \nCell viability assay \nThe leiomyoma and normal myometrial cells \nwere seeded into a 96 -well plate, incubated for 24 h, \nand then treated with 100 nM UPA and mifepristone \nfor 48 h at 37 °C and 5% CO\n2. The cells were treated \nwith 10 µM LY364947 (inhibitor of TGF-β; Cat. #2718, \nTocris Co., Ellisville, MO, USA) and 5 µM axitinib \n(inhibitor of VEGF; Cat. #4350, Tocris Co.) for 24 h. \nCell counting kit -8 (CCK- 8, Dojindo Molecular \nTechnologies, Inc., Kumamoto, Japan) assay was used \nto determine cell viabili ty according to the \nmanufacturer’s instructions. \nStatistical analysis \nStatistical software ( SPSS 23.0, SPSS, Inc., \nChicago, IL, USA) w as used for data analysis. For \nstatistical analysis, the chi -square test and one -way \nanalysis of variance were performed. Measured data \nwere expressed as the mean ± standard deviation, and \nt-tests were used to analyze data between the two \ngroups. One -way analysis of variance and Scheffe’s \npost-hoc test were used to compare multiple \ntreatment options. Statistical significance was set at P  \n< 0.05. \nResults \nHistological expression of growth factor and \nquantification \nImmunohistochemical staining was performed \nusing leiomyoma cells from 12 patients. Figure 1 \nshows the immunohistochemical staining results of \nEGF, IGF-1, TGF-β, and VEGF in normal myometrial \ncells (Fig. 1A, upper panel) and leiomyoma cells (Fig. \n1B, middle panel). The expression of growth factors \nwas investigated by calculating the relative mask area \n(%) in the normal myometrium layer and leiomyoma \ntissue (Fig. 1C, lowe r panel). Among the various \ngrowth factors, TGF -β and VEGF showed \nsignificantly higher staining intensity in leiomyoma \ntissue than in normal myometrium (P < 0.05). The \nintensity of TGF -β was 3.1-fold higher in leiomyoma \ncells than in normal myometrium cells; VEGF showed \n14.4-fold higher intensity, whereas EGF and IGF -1 \nshowed no significant difference in normal \nmyometrium and leiomyoma cells. \nEvaluation of growth factor expression on \nleiomyoma tissue after conventional drug \ntreatment with ulipristal acetate and \nmifepristone \nFigure 2 shows the results of western blotting, \nwhich was performed to evaluate the protein \nexpression of growth factors in uterine leiomyoma \ntissue in the presence or absence of drug treatment for \n48 h. The most commonly used medications  for \nleiomyoma treatment, namely UPA and mifepristone, \nwere used to treat uterine leiomyoma tissues. The \nprotein expression of growth factors such as EGF, \nIGF-1, TGF -β, and VEGF was quantified (Fig. 2A). \nAfter mifepristone treatment, VEGF expression was \nsignificantly decreased by 62% in uterine leiomyoma \ncells (P < 0.05). The expression levels of EGF, IGF, \nTGF-β, and VEGF increased when UPA was used for \nleiomyoma tissue, but this increase was not \nstatistically significant. In the case of mifepristone, the \nexpression levels of EGF and TGF -β increased and \nthose of IGF and VEGF decreased; however, only the \n62% decrease in the expression level of VEGF was \nstatistically significant (P < 0.05) (Fig. 2B). \n \n\nInt. J. Med. Sci. 2022, Vol. 19 \n \nhttps://www.medsci.org \n1782 \n \nFigure 1. Immunocytochemical staining of IGF-1, 217EGF, TGF-β, and VEGF in the normal myometrium (A) and leiomyoma tissue (B). Leiomyoma cells are cultured for 24 h \nat 25 °C and the tissues are incubated overnight with antibodies diluted by 1:500. Bar = 500 µm. The expression (C) of each growth factor is calculated using the relative mask \narea [rMA  =  (MA/FA) × 100]. FA, overall field area (mm2); MA, overall mask area (mm2), which is the summed area for each detected object in each layer. *P < 0.05 is considered \nas significant. Abbreviations: EGF, epidermal growth factor; IGF -1, insulin-like growth factor -1; TGF-β, transforming growth factor-beta; VEGF, vascular endothelial growth \nfactor. \n \nEffect on cell viability after TGF-β1 inhibitor, \nVEGF inhibitor, UPA, and mifepristone on \nleiomyoma and normal myometrium \nTo examine the survival of cells in the \nleiomyoma tissue and normal myometrium after each \ndrug treatment, a CCK-8 assay was performed. \nBecause TGF-β and VEGF were signifi cantly \nincreased in the leiomyoma tissue, we used inhibitors \nof VEGF and TGF-β1. The cells were also treated with \nUPA and mifepristone for comparison. Drugs were \nadministered to both the normal  myometrium and \nleiomyoma tissues. \nAs illustrated in Fig. 3, co mpared with the \nuntreated group, each drug significantly decreased \ncell viability in both the normal myometrium tissue \nand leiomyoma tissue. The viability of uterine \nleiomyoma cells decreased to 67%, 59%, 27%, and 29% \nafter treatment with UPA, mifepristone , TGF -β1 \ninhibitor, and VEGF inhibitor, respectively, compared \nto the untreated control. In the normal myometrium, \ncell viability decreased to 69%, 57%, 49%, and 35% \ncompared to the control group. Only the TGF -β1 \ninhibitor resulted in significantly differe nt effects on \ncell viability in the normal myometrium and \nleiomyoma tissue, causing a greater decrease in the \nsurvival rate of leiomyoma cells. There was no \nsignificant difference between the two groups  after \ntreatment with the other drugs . Post -hoc analys is \nrevealed that the change in cell viability in leiomyoma \n\n\nInt. J. Med. Sci. 2022, Vol. 19 \n \nhttps://www.medsci.org \n1783 \nwas greater following treatment with the VEGF and \nTGF-β1 inhibitors than following treatment with \nUPA. In the normal myometrium, cell viability was \ndecreased more by the VEGF inhibitor than by UPA \n(Table 1). \n \n \nFigure 2. Expression of growth factors EGF, IGF-1, TGF-β, and VEGF in myoma tissue after treatment with UPA and mifepristone. (A) Western blotting of growth factors after \nUPA and 100 nM mifepristone (10−7 M) treatment for 48 h in myoma tissue. (B) Densitometric analysis of each growth factor expression. All data are presented as the mean ± \nstandard deviation and were analyzed by using a one -way analysis of variance. *P < 0.05 was considered as significant. Abbreviations: EGF: epidermal growth factor; IGF -1: \ninsulin-like growth factor-1; TGF-β: transforming growth factor-beta; VEGF: vascular endothelial growth factor; UPA: ulipristal acetate. \n \nFigure 3. Effect of UPA, mifepristone, TGF-β1 inhibitor, and VEGF inhibitor treatment in normal uterine myometrium and leiomyoma tissue. The normal myometrial tissue and \nleiomyoma tissue are treated with 100 nM of UPA and mifepristone for 48 h and 10 µM TGF -β inhibitor and 5 µM VEGF inhibitor for 24 h.  CCK-8 assays are performed to \nevaluate cell survival. Cell viability after UPA, mifepristone, TGF-β1 inhibitor, and VEGF inhibitor treatment are analyzed as a percentage compared to non-drug tissue. All data \nare presented as the mean ± standard deviation and are analyzed u sing a one-way analysis of variance and Scheffe’s post-hoc test. * P < 0.05 was considered as significant.  \nAbbreviations: EGF: epidermal growth factor; TGF-β: transforming growth factor-beta; VEGF: vascular endothelial growth factor; UPA: ulipristal acetate. \n\n\nInt. J. Med. Sci. 2022, Vol. 19 \n \n \nhttps://www.medsci.org \n1784 \nTable 1. Changes in cell viability of myometrium and leiomyoma \ntissue after UPA,  mifepristone, TGF -β1 inhibitor, and VEGF \ninhibitor treatment and post- hoc analysis of differences between \ntreatments \n Treatment Mean SD P Post-hoc test \nNormal \nmyometrium \nUPA (U) 69.10 17.83 0.03 U>A (Scheffe) \nMifepristone (M) 57.20 15.24 \nTGF-β1 inhibitor (L) 49.09 10.83 \nVEGF inhibitor (A) 34.74 10.14 \nMyoma UPA (U) 67.81 17.53 0.02 U>L,A \n(Scheffe) Mifepristone (M) 58.59 21.31 \nTGF-β1 inhibitor (L) 26.76 1.30 \nVEGF inhibitor (A) 29.24 7.74 \nAll data are presented as the mean and standard deviation (SD) and were analyzed \nby using one-way analysis of variance and Scheffe’s post hoc test. \nAbbreviations: EGF: as epidermal growth factor; TGF-β: transforming growth \nfactor-beta; VEGF: vascular endothelial growth factor; UPA: ulipristal acetate. \n \nDiscussion \nUterine leiomyoma is the most common benign \ntumor i n women of childbearing age and  causes \nsymptoms such as bleeding or pel vic pain infertility \n[1]. A total survey using data from cohorts of one \nmillion patients collected from Korean health \ninsurance data from 2002 to 2013 showed that the \nprevalence of leiomyoma is increasing in women of all \nchildbearing ages, quadrupling to 2.48% from 2002 to \n2013 [11]. In 2018, the average age of women who \ngave birth in Korea was 32 years, and the proportion \nof elderly mothers had increased to 31.8% [12].  The \nprevalence of uterine leiomyoma is expected to \ngradually increase with the developme nt of imaging \nequipment for diagnosis, leading to socioeconomic \nloss as the marriage and childbirth age increases. \nHysterectomy is considered the absolute treatment for \nuterine leiomyoma, but alternative treatments are \nwidely used to preserve fertility and  avoid invasive \nsurgery [13]. \nGonadotropin-releasing hormone agonists, \nwhich are currently widely used for treatment, can  \nreduce the size by 40% and improve symptoms after 3 \nmonths of treatment, but the effect is  temporary (3–6 \nmonths) and used only in lim ited cases because of \ntheir various side effects [14, 15]. Treatment with \ngonadotropin-releasing hormone  analogs leads to a \nrebound in fibroid growth and the loss of bone \nmineral density when the administration is stopped \n[16]. As another option, a selecti ve progesterone \nreceptor modulator with a tissue-specific effect on the \nprogesterone receptor can be used [17]. Mifepristone \nis the first progesterone receptor antagonist and has \nbeen used clinically for more than 25 years [18, 19]. \nCochrane's review of ra ndomized controlled studies \nshowed a decrease in bleeding and increased quality \nof life in patients administered mifepristone; \nhowever, there was no significant decrease in the \nleiomyoma volume [20]. UPA is widely used as a \nselective progesterone receptor modulator and was \napproved by the US FDA in 2010 for treating \nsymptomatic leiomyoma in females of childbearing \nage. However, side effects such as nausea, vomiting, \nbreast tenderness, headache, and malaise can occur ; \ntherefore, this drug can only be used before surgery or \nfor a limited time [21 -23]. Liver injury necessitating \nliver transplantation has recently been reported in \nwomen treated with UPA [22,23]. This has led to the \nsuspension of UPA as a medical therapy for treating \nuterine fibroids  while the Eur opean Medicines \nAgency conducts a review of this liver injury risk [16]. \nThe European Medicine Agency safety committee \nadvised that women should stop taking 5 mg UPA \nand that no new patients should commence treatment \nuntil the ongoing review is completed. \nThe effect of long -term use of current \nmedications is currently being evaluated for PERL 4 \nand VENUS 2, which are not available to women who \nare planning to become pregnant because of ovulation \ninhibition [23, 24]. The fertility outcome has not been \nfully discussed. Growth factor control may prevent \nthe effects of steroid changes to effectively treat \nlong-lasting leiomyomas. Unlike previous studies of \nthe effects of individual growth factors, we compared \nseveral growth factors simultaneously. We also \ncompared the effects on cell viability in both \nleiomyoma tissue and normal myometrium. Our \nresults show that currently used medications decrease \ncell viability not only in leiomyoma tissue, but also in \nnormal tissues. However, among the growth factor \ninhibitors used in this study, the TGF -β1 inhibitor \nshowed more specific action towards uterine \nleiomyoma, demonstrating its potential as a \ntherapeutic agent. Several studies showed that TGF -β \nnot only stimulates smooth muscle cells, but also  \ncontributes to the growth of uterine leiomyoma [9, \n25], and studies of selected cell lines revealed that this \ngrowth factor significantly affects the accumulation of \nextracellular matrix in uterine leiomyoma [26, 27]. \nCellular responses to TGF -β are diver se, and several \nstudies have demonstrated its pre-tumorigenic role in \nvarious stages of cancer; accordingly, many strategies \nto suppress TGF-β are being evaluated [28]. However, \nsystemic therapy, such as targeting TGF -β signaling, \nshows some limitations  such as cardiovascular toxic \nside effects and benign tumor formation (Colak and \nTen Dijke, 2017). We found that the TGF -β inhibitor \naffected not only myoma tissue, but also the normal \nmyometrium; therefore, targeted local treatment \nrather than systemic treat ment should be considered. \nAs we only evaluated one concentration of each drug, \ndifferences in culture conditions and concentrations \nshould be examined to determine the safety margin \n\nInt. J. Med. Sci. 2022, Vol. 19 \n \nhttps://www.medsci.org \n1785 \nfor the normal myometrium while  treating the \nleiomyoma tissue. \nVEGF is an  important growth factor that \nstimulates the proliferation of vascular endothelial \ncells. VEGF promotes cell migration and proliferation \nby binding to associated receptors, thereby increasing \nangiogenesis. Although uterine fibroids are benign \ntumors, angiogenesis is critical for the occurrence and \ndevelopment of these fibroids [29]. The effect of VEGF \non the prognosis of leiomyoma gained attention \nfollowing a study demonstrating a difference in the \nexpression of VEGF and IGF -1 after uterine artery \nembolization treatment. \nInhibitors of the growth factors TGF -β1 and \nVEGF showed greater inhibitory effects on \nleiomyoma cell viability than conventional drugs, \nindicating their potential as therapeutic agents for \nuterine leiomyoma. In addition, as previous studies \nrevealed risks associated with the systemic action of \ngrowth factor inhibitors, their use as local treatments \ncould be considered. \nConclusion \nThe expression of various growth factors (EGF, \nIGF-1, TGF -β, and VEGF) was evaluated in uterine \nleiomyoma tissues.  The expression levels of TGF -β \nand VEGF were 3.1- and 14-fold higher in leiomyoma \nthan in the normal myometrium. Among the \nleiomyoma treatments used in the clinic, UPA did not \nsignificantly alter growth factor expression in \nleiomyoma tissue, whereas mifep ristone significantly \ndecreased the expression of VEGF in leiomyoma \ntissue. \nThe TGF-β1 inhibitor caused a more significant \nchange in cell viability in leiomyoma tissue than in the \nnormal myometrium. In the post -hoc test, TGF -β1 \ninhibitor and VEGF inhibitor s tended to have greater \ninhibitory effects than the conventional drug, UPA. \nThrough this study, the potential of growth-inhibitors \nas a treatment for leiomyoma was examined, and the \npossibility as a local treatment could also be \nconsidered. It is expected  that a safer therapeutic \nconcentration can be found through additional \nexperiments with various concentrations in the future. \nAbbreviations \nEGF: epidermal growth factor; TGF -β: \ntransforming growth factor -beta; VEGF: vascular \nendothelial growth factor; UPA: ulipristal acetate. \nAcknowledgements \nFunding \nThis research was supported by the Basic Science \nResearch Program through the National Research \nFoundation of Korea (NRF) funded by the Ministry of \nEducation (2020R1F1A1063199), Basic Science \nResearch Program through the NRF funded by the \nMinistry of Education (2017R1D1A1B03028045), and \nKorea Research Foundation for Gynecologic Cancer \n(2021-1). \nEthics Committee Approval \nThis study was granted exemption by the \nInstitutional Review Board of The Catholic University \nof Korea, Seoul, St, Mary’s Hospital (IRB No. KC \n12TNSI0822). \nORCID \nJung Yoon Park Http://orcid.org/0000 -0003- \n0756-2439; Mee-Ran Kim Http://orcid.org/0000 - \n0003-4492-0768. \nAuthor Contributions \nJYP and MRK and BC  designed the study. JYP \nand BC performed the experiments. JYP and MRK \nand BC analyzed the data. JYP and BC  drafted the \nmanuscript. All authors contributed to the \ninterpretation and discussion of the results. \nData Availability \nData are available at International Journal of \nMedical Sciences online. \nCompeting Interests \nThe authors have declared that no competing \ninterest exists. \nReferences \n1. Sohn GS, Cho S, Kim YM, et al. Current medical treatment of uterine fibroids. \nObstet Gynecol Sci. 2018; 61: 192-201. \n2. Parazzini F, Tozzi L, Bianchi S. Pregnan cy outcome and uterine fibroids. Best \nPract Res Clin Obstet Gynaecol. 2016; 34: 74-84. \n3. Klatsky PC, Tran ND, Caughey AB, et al. Fibroids and reproductive outcomes: \na systematic literature review from conception to delivery. Am J Obstet \nGynecol. 2008; 198: 357-66. \n4. Guo XC, Segars JH. The impact and management of fibroids for fertility: an \nevidence-based approach. Obstet Gynecol Clin North Am. 2012; 39: 521-33. \n5. 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