{"paper_id":"e20158b1-fdb2-442d-b2fd-392e2d22028a","body_text":"1Scientific  RepoRtS  |         (2020) 10:7993  | https://doi.org/10.1038/s41598-020-64871-y\nwww.nature.com/scientificreports\nfisetin induces apoptosis in uterine \nleiomyomas through multiple \npathways\nJin-Woo Lee1, Hyuck Jai ch oi2, eun-Jin Kim2, Woo Yeon Hwang3, Min-Hyung Jung  3,5 ✉ & \nKyung Sook Kim4,5 ✉\nAlthough uterine leiomyomas are the most common benign uterine tumors in women, there is no \neffective therapy that can also preserve the uterus and maintain fertility. The work aimed to work was \nto discover a potential natural agent that has pharmacological activities on uterine leiomyomas with \nfewer adverse effects. We chose Rhus verniciflua Stokes (RVS) as a candidate after primary cytotoxicity \ntesting, and analyzed the RVS components that showed pharmacological activity. Leiomyoma cells \nand myometrium cells were cultured from uterine tissues obtained from patients, and were treated \nwith RVS at varying concentrations. RVS was cytotoxic in both leiomyoma and myometrium cells; \nhowever, the effects were more prominent in the leiomyoma cells. Among the bioactive components of \nRVS, fisetin showed significant pharmacological effects on leiomyoma cells. Fisetin showed excellent \nleiomyoma cell cytotoxicity and induced apoptotic cell death with cell cycle arrest. The apoptotic cell \ndeath appeared to involve not one specific pathway but multichannel pathways (intrinsic, extrinsic, \nMARK, and p53-mediated pathways), and autophagy. The multichannel apoptosis pathways were \nactivated with a low concentration of fisetin (<ic\n20) and were more vigorously activated at high \nconcentrations (>ic 50). This is the first demonstration to show the pharmacological activities of fisetin \non leiomyoma cells. These findings suggest that fisetin may be used for the prevention and treatment of \nuterine leiomyomas. Since fisetin can be obtained from plants, it may be a safe and effective alternative \ntreatment for uterine leiomyomas.\nUterine leiomyomas are the most common benign uterine tumors in women, especially during their reproductive \nyears1,2. They are monoclonal tumors of the uterine smooth muscle cells. Leiomyomas can be classified by their \nlocation relative to the layers of the uterus, such as subserous, intramural or submucous, and can be single or mul-\ntiple\n3,4. Although they can occur elsewhere in the body, leiomyomas most frequently occur in the myometrium. \nThe uterine leiomyomas do not cause pain or other symptoms in most cases. However, they very occasionally are \nassociated with symptoms such as abnormal bleeding, pain, and pressure depending on their size and location \nwithin the uterus\n5. The exact cause of leiomyomas remains unclear. Several factors such as genetic abnormalities, \nalterations in growth factor expression, and abnormalities in the blood vessels have been suggested to play an \nimportant role in the development of leiomyomas\n6.\nThere are several uterine leiomyoma treatment options, including medications, surgery, high-intensity \nfocused ultrasound, and uterine artery embolization7,8. Although the choice of treatment depends on the symp-\ntoms and leiomyoma size, surgery is currently the most common treatment. Several drugs have been used for \nconservative treatment, but they have been used to treat the symptoms rather than to cure the disease. Selective \nprogesterone-receptor modulators including asoprisnil, ulipristal, and telapristone have been suggested as ther-\napeutic drugs for uterine leiomyomas\n9. Among them, ulipristal acetate showed promising results in the treat-\nment of leiomyomas and the control of leiomyoma-associated bleeding 10. However, the European Medicines \nAgency recently reported that 5 mg daily administration has been linked with liver injury 11. Subsequently, the \nPharmacovigilance Risk Assessment Committee recommended against the prescribing of ulipristal12. Therefore, \n1Medical Science Research Institute, Kyung Hee University Medical Center, Seoul, 02447, Korea. 2East-West Medical \nResearch Institute, Kyung Hee University Medical Center, Seoul, 02447, Korea. 3Department of Obstetrics & \nGynecology, School of Medicine, Kyung Hee University, Kyung Hee University Medical Center, Seoul, 02447, Korea. \n4Department of Biomedical Engineering, College of Medicine, Kyung Hee University, Seoul, 02447, Korea. 5These \nauthors contributed equally: Min-Hyung Jung and Kyung Sook Kim. ✉e-mail: webhospital@naver.com; moosou94@\nkhu.ac.kr\nopen\n\n2Scientific  RepoRtS  |         (2020) 10:7993  | https://doi.org/10.1038/s41598-020-64871-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nit is necessary to develop safer alternative methods or medications for the treatment of leiomyomas that can also \npreserve the uterus and maintain fertility.\nIn this study, we investigated a natural plant, Rhus verniciflua Stokes (RVS), and analyzed which of its compo-\nnents has a pharmacological activity for leiomyomas. RVS is an herbal medicine possessing various pharmacolog-\nical effects including antioxidant, antiproliferative, anti-inflammatory, antitumor, and antimutagenic effects\n13,14. \nLeiomyoma cells and normal myometrium cells were cultured from tissues obtained from patients, and then \ntreated with RVS. After confirmation that RVS was cytotoxic specifically in leiomyomas, three components of RVS \nincluding fustin, fisetin, and sulfuretin were selected as candidates for cytotoxicity studies. The apoptotic effect \nof fisetin on the leiomyoma cells was confirmed and the underlying mechanism of apoptosis was investigated.\nResults\nSelection of the natural product and its extracts. The purpose of this work was to discover new agents \nexerting anti-uterine leiomyoma activities. A natural product that appeared to be pharmacologically active \nagainst leiomyomas was chosen, and the component showing effective pharmacological effects was confirmed. \nThen, the underlying mechanism of apoptosis induced by the selected component was confirmed (Fig. 1).\nFirstly, three kinds of plants—Curcuma longa , Orostachys japonicas, and RVS—were selected for antileiomy-\noma activity assessment. Curcuma longa, which belongs to the family Zingiberaceae, is a well-known herbal med-\nicine used in Asian countries in the treatment of many diseases such as helminthic infections, asthma, gonorrhea \nand urinary infections\n15. Its diverse pharmacological activities, such as anti-inflammatory, antioxidant, antibac-\nterial, and antitumor activities, have been well documented16–19. O. japonicas also have shown various pharma-\ncological activities, including anti-inflammatory, neuroprotective, anti-ulcerative, and anti-oxidant activities20–22. \nIn particular, it has shown excellent anti-cancer effects on hepatic stellate cells, leukemia cells, colon cancer cells, \nand prostate cancer cells\n23–25. RVS is a well-known traditional medicinal plant that possesses a variety of phar -\nmacological activities. It has been widely used for treating various stomach diseases and cancers26–28. With RVS \ntreatment, cell growth was inhibited and apoptosis was induced in human lymphoma cells and human chronic \nmyelogenous leukemia K562 cells\n26,27. RVS also induced apoptosis in paclitaxel-resistant ovarian cancer cells28.\nAs the first step, the cytotoxicity of the three natural plants on uterine leiomyoma cells was examined. \nUnexpectedly, C. longa showed no significant cytotoxic effects (Fig. S1A), whereas the cytotoxic effects of O. \njaponicas were prominent (Fig. S1B,C). The main components of O. japonicas having cytotoxic activities were \nquercetin, kaempferol, and epicatechin gallate, which are also among the main components of green tea29. We \nfound several studies that demonstrated the cytotoxic effects of these components on leiomyomas30,31. Therefore, \nwe focused on RVS in this work. Even though the apoptotic activities of RVS on various types of cancer cells have \nbeen well documented, no studies have investigated the pharmacological effects of RVS or of its single compo-\nnents such as fustin, sulfuretin, and fisetin on leiomyoma cells.\nCytotoxic effects of RVS on leiomyoma cells and myometrium cells.  To evaluate the cytotoxic \neffect of RVS, leiomyoma cells and myometrium cells were inoculated into 60 mm culture dishes at a density of 2.5 \n× 105 cells/dish. The cells were treated with RVS at varying concentrations (20–1,000 µg/mL), and then cultured \nfor 24, 48, and 72 h, respectively. As shown in Fig. 2A, the viability of the leiomyoma cells decreased with increas-\ning concentrations of RVS after 24 h treatment, and similar cytotoxic effects were observed in the groups treated \nfor 48 and 72 h (Fig. S2A,B). The viability of the myometrium cells also decreased with RVS treatment and showed \na dependency on the RVS concentrations in all groups at 24, 48, and 72 h of treatment (Figs. 2A, S2A,B). In both \ncell types, the cytotoxicity of RVS was significant above 400 μg/mL. In leiomyoma cells, the IC 50 values were \nestimated to be 414.0 μg/mL (24 h), 278.1 μg/mL (48 h), and 249.2 μg/mL (72 h), respectively. The IC50 values in \nmyometrium cells were estimated to be 370.1 μg/mL (24 h), 257 μg/mL (48 h), and 220 μg/mL (72 h), respectively.\nTo investigate the mechanism of cell death induced by RVS, both leiomyoma cells and myometrium cells were \ntreated with RVS at concentrations of 50, 100, 200, 400, and 600 μg/mL and then cultured for 24, 48, and 72 h, \nrespectively. The total percentage of apoptotic cells induced by RVS was determined with an annexin V-FITC/\nFigure 1. Flow chart showing the process of discovering natural products that have pharmacological effects on \nuterine leiomyoma cells.\n\n3Scientific  RepoRtS  |         (2020) 10:7993  | https://doi.org/10.1038/s41598-020-64871-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nPI apoptosis assay. Figure 2B,D show the results of the apoptosis assay after 24  h RVS treatment in leiomyoma \ncells and myometrium cells, respectively. Similar proportions of cells underwent apoptosis when comparing the \ncell types at low concentrations (≤ 200 μg/mL). However, the apoptosis rate of the normal cells was remarkably \nincreased at high concentrations: 400 and 600 μg/mL. This is an undesirable result. In cell-based experiments, \ntreatment with higher concentrations than 400 μg/mL of a drug is considered a very harsh condition. The effect at \na lower concentration, of 200 μg/mL or less, is generally considered to be clinically significant. Therefore, we think \nthat the high sensitivity of myometrium to concentrations of RVS over 400 μg/mL is not a meaningful result. We \nobserved a similar dependence of apoptosis on the RVS concentrations after 48- and 72-h treatment (Fig. S2C,D).\nThe flow cytometry analysis was performed to determine the cell cycle phase at which RVS exerts its growth \ninhibitory effects. The population of sub-G1 cells among the leiomyoma cells after 24 h of RVS treatment increased \neven at a low concentration of 50 μg/mL, and the rate of increase was significant at high concentrations (≥400 μg/\nmL) (Fig. 2C,E). In the case of myometrium cells, the population of cells in sub-G1 slightly increased with RVS, \nbut the rate of increase was not significant and no concentration dependency was observed. The changes in the \nproportion of sub-G\n1 cells after 48 and 72 h showed a similar dependency on the RVS concentration after 24 h of \ntreatment (Fig. S2E,F).\nThese results indicated that the cytotoxic activity of RVS is not only generated in leiomyoma cells but also in \nnormal myometrium cells. Therefore, we compared the cytotoxic effects of leiomyoma cells and myometrium \ncells at each concentration of RVS and statistically analyzed the difference. Statistical analysis was performed \nFigure 2. Effects of RVS on proliferation and apoptosis of leiomyoma cells and myometrium cells. (A) Both \ncells were treated with RVS at varying concentrations (0–1000 μg/mL), and cell viability was measured by the \nMTT assay. (B) The populations of cells in early and late apoptosis were counted. (C) Alteration of cell cycle \npopulations (sub-G1) induced by RVS treatment. (D) Analysis of apoptosis by annexin V and PI staining in \nmyometrium cells (left) and leiomyoma cells (right). The apoptotic status of cells is presented as dot-plots. (E) \nCell cycle analysis in myometrium cells (left) and leiomyoma cells (right). The cells were treated with RVS (0, 20, \n100, 200, 400, 600 μg/mL). Each value represents the mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).\n\n4Scientific  RepoRtS  |         (2020) 10:7993  | https://doi.org/10.1038/s41598-020-64871-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nusing repeated measures ANOV A and Bonferroni post-test. The analysis confirmed that the cell death rate in \nleiomyoma cells was statistically greater than that in myometrium cells under certain conditions. Compared to \nmyometrium cells, RVS effectively induced cell cycle arrest and inhibited the cell proliferation of leiomyoma cells \nat concentrations of 200 μg/mL (24 h), 50 μg/mL (48 h), and 50 μg/mL and 200 μg/mL (72 h), respectively.\nCytotoxic effects of fisetin on leiomyoma cells and myometrium cells. To identify causative \nfactors of cytotoxicity in leiomyoma cells, the principal components of RVS were analyzed via HPLC. Among \nthe chemical components of RVS, fustin, sultretin, and fisetin are the main components with anticancer and \nanti-inflammatory effects\n29. The RVS components we used in this study were confirmed by comparison with the \nHPLC results of the reference standards of fustin, sulfuretin, and fisetin. We confirmed that RVS contained 0.05% \n(w/w) fustin, 0.16% (w/w) sulfuretin, and 0.60% (w/w) fisetin, respectively (Fig. 3A). Since the amounts of fustin, \nsulfuretin, and fisetin in RVS were too small to use in our studies on viability, apoptosis, and cell cycle measure-\nments, we purchased the three components to analyze their effects. Because the purchased fustin, sulfuretin, and \nfisetin each consisted of a single chemical component, there should have been no difference in composition or \neffect between the components directly extracted from RVS and those that were commercially obtained. Using the \nlatter, we could exclude the possibility of involvement of foreign substances in the extracts from the laboratory.\nTo evaluate the cytotoxic effects of the RVS components, leiomyoma cells and myometrium cells were seeded \ninto 96-well plates at a density of 1.0 × 10\n4 cells/well. The cells were treated with fustin, sulfuretin, and fisetin at \nvarying concentrations and then cultured. The cytotoxic activity of fustin on myometrium cells was not signifi-\ncant (Fig. S3A). After 24 h of treatment, no change in cell viability was observed at concentrations up to 40 µM, \nand then the viability decreased by only 20% at 60 µM. The cytotoxic effect of fustin on leiomyoma cells was also \ninsignificant (Fig. S3B). The slight decrease in cell viability depending on the concentrations was observed in all \nFigure 3. Effects of fisetin on proliferation and apoptosis of leiomyoma cells and myometrium cells. (A) HPLC \nchromatograms of RVS showed three main components: fustin, sulfuretin, and fisetin. (B) Myometrium cells \nwere treated with fisetin at varying concentrations (0–100 mM), and cell viability was measured by the MTT \nassay. (C) Leiomyoma cells were also treated with fisetin, and their viability was analyzed. (D) The fold changes \nin cell numbers in both early and late apoptosis were counted. (E) Alteration of cell cycle populations (sub-\nG\n1) after fisetin treatment. (F) Analysis of apoptosis by annexin V and PI staining in myometrium cells (left) \nand leiomyoma cells (right). The apoptotic status of cells is presented as dot-plots. (G) Cell cycle analysis in \nmyometrium cells (left) and leiomyoma cells (right). The cells were treated with fisetin (0, 50, 100, 200, 400, \n600 mM). Each value represents mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).\n\n5Scientific  RepoRtS  |         (2020) 10:7993  | https://doi.org/10.1038/s41598-020-64871-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nconditions after 24, 48, and 72 h treatments. Sulfuretin, however, showed more pronounced cytotoxicity. In both \ncell types, the cell viability decreased by more than 50% at 60 µM (Fig. S3C,D). However, the results of Annexin \nV-FITC/PI apoptosis assay showed that necrosis occurs as well as apoptosis by sulfuretin in leiomoyma cells. \nBoth increased with the concentrations of sulfuretin (Fig. S4A,B). Therefore, based on the results of the MTT and \napoptosis analyses, we decided not to investigate fustin and sulfuretin further.\nThe myometrium cells showed no changes in viability at low concentrations (≤ 10 µM) of fisetin. However, \ntheir viability significantly decreased at higher concentrations (≥ 20 µM) (Fig.  3B). The cytotoxic activities of \nfisetin on leiomyoma cells were very effective (Fig. 3C). The decreasing of viability was apparent from 10 µM, and \nthe viability decreased rapidly and statistically at higher concentrations (≥20 µM).\nTo investigate how fisetin induced cell death, both myometrium cells and leiomyoma cells were treated at \nvarying concentrations. The proportion of apoptotic cells increased among the myometrium cells; however, the \nrate of increase was lower than that of the leiomyoma cells (Fig. 3D). Among the leiomyoma cells, the population \nof apoptotic cells increased up to 6.4–7.7-fold after 24 h of treatment compared to the non-treated cells, respec-\ntively. However, there was no significant change in the number of necrotic cells. The difference in the proportions \nof apoptotic cells between the leiomyoma cells and the myometrium cells was significant at concentrations ≥ \n20 µM. Figure 3F shows the detailed results of the apoptosis assays in myometrium cells (left) and leiomyoma \ncells (right).\nIn the cell cycle analysis, the proportion of myometrium cells in sub-G\n1 phase increased with fisetin treatment. \nHowever, the increase in the sub-G1 population was more significant in leiomyoma cells, and it was statistically \nsignificant at the fisetin concentrations of 20, 40, 60, and 80 µM. The proportion of leiomyoma cells in sub-G 1 \nphase after treatment with fisetin for 24 h increased by up to 36-fold (Fig. 3E,G). These results suggest that inhi-\nbition of cell cycle progression is one of the molecular events associated with apoptotic activities of fisetin in \nleiomyoma cells. All measurements of cell apoptosis and cell cycle were conducted after 48 h treatment, and 24 h \ntreatment yielded similar results (Figs. S5 and S6).\nIntrinsic & extrinsic apoptosis pathways. Generally, the cell death process can be classified into apop-\ntosis, necrosis, autophagy, and mitotic catastrophe30. Among them, apoptosis, which is programmed cell death, is \nprocessed in mainly 4 different pathways: the intrinsic, extrinsic, p53-mediated, and activated MAPK pathways.\nThe intrinsic pathway involves mitochondrial outer membrane permeabilization (MOMP) and diverse pro-\nteins causing apoptosis induction and signal transduction 31,32. In particular, apoptosis machinery components \nincluding the death receptor, the bcl-2 family, and caspases play major roles in the intrinsic pathway. Death recep-\ntors are cell surface receptors belonging to the tumor necrosis factor receptor gene superfamily; the death recep-\ntors trigger apoptosis upon ligand binding. The bcl-2 family of proteins, which comprises several proapoptotic \nmultidomain proteins, initiates MOMP by forming pores at the mitochondrial outer membrane. The caspases are \na family of protease enzymes that play essential roles in the initiation and progression of apoptosis. The extrinsic \npathway is triggered by death receptors such as Fas and tumor necrosis factor-related apoptosis-inducing ligand \nreceptors.\nFigure 4 shows the effects of fisetin on the intrinsic and extrinsic apoptosis pathways of leiomyoma cells as \na function of concentration. All concentrations of fisetin except for 20 µM decreased the protein expression of \nbcl-2, and all decreases were statistically significant. Expression of the bax and cytochrome C proteins increased \nwith fisetin treatment in a concentration-dependent manner. The expression levels of both apaf-1 and caspase-9 \nshowed no change at the lowest concentration (10  µM), but significantly increased at higher concentrations \n(≥ 20 µM). Caspase-3 and caspase-6 showed increases in expression with increasing fisetin concentration, but the \nchanges at 10 and 20 µM were not statistically significant. However, when treated at higher concentrations (30 and \n40 µM), their expression levels increased rapidly. This result indicates that fisetin inhibits the activity of bcl-2 in \nleiomyoma cells, which results in the expression of bax and cytochrome C in mitochondria. Then, the cytochrome \nC is released into the cytoplasm and binds to apaf-1 and caspase-9 to form a protein complex that activates \ncaspase-3 and caspase-6 to break down important proteins, which leads to a typical intrinsic pathway leading to \ndeath. Fisetin increased the expression of both the 18-kDa (active) and 43-kDa (intermediate) forms of caspase 8. \nFisetin also increased the expression of PARP , and the increase was significant at high concentrations (≥40 µM). \nThis result indicates that fisetin induces the death of leiomyoma cells through caspase-dependent apoptosis.\nActivation of p53-mediated apoptosis by fisetin. The tumor suppressor gene p53 plays a central role \nin the regulation of the cell cycle, apoptosis, and DNA repair33,34. p53 regulates the expression of various genes in \nresponse to DNA damage. The expression of p53 rapidly increases when DNA is damaged, which results in the \ninduction of p21, one of the target genes of p53. The p21 inhibits the activity of cyclin-CDK (cyclin-dependent \nkinase) complexes. In normal cells, the G\n2/M transition is regulated by the cyclin B-CDK1 complex. At the end \nof the G2 phase, it is phosphorylated by the CDK-activating kinase, which leads the cell into the mitotic phase. \nHowever, p53 in damaged cells inhibits the G2/M transition by inhibiting the activity of the cyclin B-CDK1 com-\nplex. Fisetin increased the expression of p53 in leiomyoma cells, and the changes were evident when the fisetin \nconcentration exceeded 20 µM (Fig. 5A). At the same time, the expression level of cyclin B decreased to less than \n50% of that in untreated cells. These results are consistent with the changes in the proportions of the leiomyoma \ncells in the sub-G\n1 (Fig. 3E) and G2/M phases (Fig. S6D,E). Therefore, it is thought that apoptosis of leiomyoma \ncells is also caused by p53-induced cell cycle arrest.\nActivation of the MAPK pathway by fisetin.  The mitogen-activated protein kinase (MAPK) family \nmembers form a crucial signaling pathway for the maintenance of cells against external stress 35,36. The MAPK \nfamily consists of three main subfamilies: the extracellular signal-regulated protein kinases (ERKs), the c-Jun \nN-terminal kinases (JNKs), and the p38-MAPKs. JNKs and p38-MAPKs are activated by proinflammatory \n\n6Scientific  RepoRtS  |         (2020) 10:7993  | https://doi.org/10.1038/s41598-020-64871-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\ncytokines, UV irradiation, heat, osmotic shock, hydrogen peroxide, and DNA damage, and help to regulate \ngrowth inhibition or apoptosis induction. ERKs are activated by mitogenic stimuli such as growth factors, \ncytokines, and phorbol esters, and help to regulate cell growth and differentiation.\nWith fisetin, there were no changes in total ERK, JNK, and p38-MAPK protein expression (Fig. 5B). However, \nthe expression levels of their phosphorylated forms showed definite changes induced by fisetin. The expression \nlevels of phospho-p38 and phospho-JNK decreased significantly in a concentration-dependent manner, but the \ndecrease of phospho-ERK showed no concentration dependency. These results suggest that the MAPK pathway \nwas activated by fisetin and played a crucial role in the apoptosis of leiomyoma cells.\nActivation of autophagy signaling pathway by fisetin. Autophagy is a self-degradative process \nby which dysfunctional cellular components are degraded inside the cell and delivered to the lysosome 37–39. \nAutophagy has several stages, including induction, vesicle nucleation, vesicle elongation, retrieval, docking/\nfusion, and vesicle breakdown/degradation. Autophagy can be initiated by inhibition of mTOR activity, during \nwhich phosphorylation of Atg13 is suppressed and a complex with Atg1 and Atg17 is formed. Class III PI3K \n(phosphatidylinositol 3-kinase) plays an important role in the early stages of vesicle nucleation. Its activity is \ndetermined by the formation of a multiprotein complex of Beclin-1 (Atg6), UV irradiation resistance-associated \ntumor suppressor gene (UVRAG), and myristylated kinase (Vps15 or p150). The autophagosome formation is \nregulated by Atg proteins, such as the Atg12-Atg5 and LC3-II (Atg8-II) complexes. Atg12 is conjugated to Atg5 \nin a ubiquitin-like reaction, which requires Atg7 and Atg10 (E1- and E2-like enzymes, respectively), in the ves-\nicle elongation stage. The Atg12-Atg5 conjugate interacts non-covalently with Atg16 to form a large complex. \nLC3-I is generated by cleaved LC3/Atg8 and conjugated to phosphatidylethanolamine (PE) by Atg7 and Atg3 \n(E2-like enzymes) in a ubiquitin-like reaction. The binding of PE results in the formation of LC3-II (autophagic \nvesicle-associated form), which is a lipidated form of LC3. LC3-II is considered a marker of autophagosome \nformation.\nFigure 4. Effects of fisetin treatment on expression of Bcl-2, Bax, Cytochrome c, Apaf-1, caspase 3, 6, 8, 9 and \nPARP in leiomyoma cells. Leiomyoma cells were treated with fisetin for 24 h, and the expression of intrinsic and \nextrinsic apoptosis-regulating proteins was analyzed by western blot analysis. β-actin was used as an internal \ncontrol. Results were expressed as the mean ± S.D. from three independent experiments. **p < 0.01 and \n***p < 0.001 were used to indicate statistical significance compared to the untreated control cells.\n\n7Scientific  RepoRtS  |         (2020) 10:7993  | https://doi.org/10.1038/s41598-020-64871-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nFigure  5(C) shows the changes in autophagy-related signals induced by fisetin. In the case of Beclin-1, \nthe change with the lowest concentration of fisetin was not clear, but at concentrations of 20 μM or more, the \nexpression level of Beclin-1 was significantly higher compared to the control group. The expression of Atg7 was \nincreased in a concentration-dependent manner, while phospho-mTOR was decreased. The LC3-II expression \nlevel sharply increased with 10 μM fisetin and slightly decreased with a higher concentration of fisetin, but the \nexpression level of LC3-II in all treated groups was higher than in the control group. In the case of phospho-Akt, \nthe expression level slightly increased when the concentration of fisetin was 20 µM, but decreased sharply over \n40 µM. This result suggests that autophagy occurred in the leiomyoma cells treated with fisetin. The multi-channel \napoptosis pathways induced by fisetin are delineated in a schematic diagram in Fig. 6.\nDiscussion\nThere have been few studies on the treatment of uterine leiomyomas, other than surgical treatment, over the past \n100 years. Because it is a benign tumor, unlike cancer, no widely recognized cell lines and no animal study models \nhave been developed. Therefore, it is very difficult to carry out preliminary studies including ex vivo experiments, \nwhich are necessary before human studies can be performed. However, when natural products are employed, \nthere is an advantage in that human subjects can be relatively easily tested under safe conditions. In this study, \nwe have identified a natural component showing therapeutic effects specifically in leiomyoma cells compared \nwith normal myometrium cells. Both leiomyoma cells and myometrium cells were cultured from uterine tissues \nobtained from patients.\nTo discover agents in natural plants that have pharmacological activities targeting leiomyomas, we screened \nCurcuma longa, O. japonicas, and RVS, which are well-known herbal medicines in Asian countries, using an \nMTT assay. Based on the results, we chose RVS as a candidate for further study. RVS is a tree that belongs to the \nAnacardiaceae family, also commonly known as the lacquer  tree. RVS has been used as a folk herbal medicine \nin Asian countries for a long time, and its various pharmacological activities have been revealed in recent stud-\nies\n13,14. RVS possesses several bioactive compounds including fustin, fisetin, gallic acid, butein, butin, sulfuretin, \nquercetin, coumaric acid, kaempferol-3-O-glucoside, and kaempferol, which are mediators of the pharmacolog-\nical activities of RVS\n40.\nAmong them, fisetin (3,7,3,4-tetrahydroxyflavone) is a naturally occurring flavonoid found not only in \nRVS but also in various fruits and vegetables such as strawberries, apples, and persimmons. 41. Fisetin has been \nreported to induce apoptosis in cells from various cancers such as human non-small cell lung cancer, liver cancer, \nFigure 5. Effect of fisetin on cell cycle, MAPK phosphorylation, and autophagy flux-related protein levels \nin leiomyoma cells. Leiomyoma cells were treated with fisetin at 0, 10, 20, 40, and 80 μM concentrations for \n24 h, and then the levels of proteins related to the apoptosis pathways were evaluated by western blot analysis. \n(A) The expression of p53 and cyclin B1 proteins changed after the fisetin treatment. (B) The protein levels of \nphosphorylated p38, JNK, and ERK were measured to understand the effects of fisetin on the pattern of MAPK \nphosphorylation. (C) To understand the effects of fisetin on autophagy, Beclin-1, Atg7, LC3 I, II, total mTOR, \nAkt, and the phosphorylated forms of mTOR (p-mTOR) and Akt (p-Akt) were analyzed. β-actin was used as \nan internal control. Results are representative of three independent experiments. *p < 0.1, **p < 0.01, and \n***p < 0.001 were used to indicate statistical significance compared to the untreated control cells.\n\n8Scientific  RepoRtS  |         (2020) 10:7993  | https://doi.org/10.1038/s41598-020-64871-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nprostate cancer, and laryngeal cancer, all through apoptosis signaling pathways42. In addition, fisetin can atten-\nuate isoproterenol-induced cardiac ischemic injury, activate anti-inflammatory activity by inhibition of c-Jun \nN-terminal kinase and nuclear factor κ B pathways, and induce the expression of heme oxygenase-1, which is a \nmajor component of cellular antioxidant enzymes43. However, to date, no study has been conducted to identify \nthe pharmacological activities of fisetin in uterine leiomyoma cells.\nWe firstly demonstrated that fisetin was cytotoxic for uterine leiomyoma cells. It induced apoptotic cell death \nand cell cycle arrest. The fisetin-induced apoptosis was not mediated specifically by a single pathway, but by all \nknown apoptosis pathways including intrinsic, extrinsic, MARK, p53-mediated pathways, and autophagy. This is \nin strong contrast with apoptosis caused by a specific drug, which usually occurs along one or two pathways. The \nmultichannel apoptosis pathways were activated even at low concentrations of fisetin. Most of the expression lev-\nels of proteins associated with intrinsic and extrinsic pathways, including Bax, Bcl-2, caspase 8 and 9, Apaf-1, and \ncytochrome C, increased with fisetin treatment at the concentration of 20 µM, which is even lower than the IC\n20 \n(26.0 µM). The activation of p53 and deactivation of cyclin B were remarkable with 20 µM fisetin. The proteins \nassociated with p53-mediated apoptosis and the MRAR pathway also increased with 20 µM fisetin. The expres-\nsion of proteins associated with autophagy, except Akt, was also significant at 20 µM. As expected, the activation \nor deactivation of proteins related to apoptosis was more prominent at higher concentrations, 40 and 60 µM. Note \nthat the IC50 of fisetin for leiomyoma cells is 64.52 µM.\nUterine leiomyoma is the most common tumor in women. It is found in approximately 25%–35% of women \nof childbearing age and 40–50% of women over 35 years old. The cause of uterine leiomyomas is not yet known \nexactly, but it is thought that one of the cells forming the uterine smooth muscle abnormally proliferates to form \nthe leiomyoma. A leiomyoma is a type of hormone-dependent tumor, which is especially affected by follicular \nhormones and estrogen. Previously identified molecular biologic abnormalities of uterine leiomyoma include \nincreased estrogen and progesterone receptors, bcl-2, and aromatase cytochrome P450\n44. The overexpression \nFigure 6. Schematic diagram of the proposed molecular mechanisms of fisetin-induced G2/M arrest, \napoptosis, and autophagy in uterine leiomyomas. The schematic diagram delineates the extrinsic (death \nreceptor) and intrinsic (mitochondrial) pathways of fisetin-induced apoptosis. Fisetin may induce apoptosis \nthrough both pathways. Mitochondria act as major control points involving the regulation of apoptosis. Uterine \nleiomyomas cells were exposed to fisetin, and the phagosomes were converted to double-layered membranes of \nautophagosomes through increasing expression levels of Atg proteins including Beclin-1and Atg-7, while LC3-I \nis converted to LC3-II. The inhibition of the Akt/mTOR signaling pathway contributes to the accumulation of \nLC3-II, which suggests that the pathway is upstream of fisetin-induced autophagy. Once the autophagosome \ndevelops, its maturation is complete upon fusion with a lysosome to form an autophagolysosome. Eventually, \nfisetin induces programmed cell death. In addition, phosphorylation of p53 stimulates its activation of p21, \nresulting in cell cycle arrest in G2/M through inhibiting cyclin B1 (CCNB1) expression and activity.\n\n9Scientific  RepoRtS  |         (2020) 10:7993  | https://doi.org/10.1038/s41598-020-64871-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nof transforming growth factor beta, heparin-binding growth factor, insulin-like growth factor, and basic \nfibroblast growth factor has been reported to play a key role in the development of uterine leiomyomas 45. A \ngonadotropin-releasing hormone agonist is widely used as typical drug therapy for uterine leiomyomas, but its \neffect is temporary and there may be adverse effects due to estrogen deficiency\n46. Other potential medications \ninclude anti-progesterone agents, interferon, estrogen receptor modulators, and anti-fibrosis agents, but they \nare still in the research stage. Fisetin can be considered as a potential therapeutic agent for uterine leiomyomas \nbecause it showed cytotoxicity through multi-channel apoptosis pathways. Also, a lack of significant side effects \nis expected because it is being used as a dietary supplement.\nIn conclusion, we investigated which natural products showed pharmacological effects on uterine leiomyoma \ncells, and chose to study RVS after cytotoxicity analysis. Among the bioactive components of RVS, fisetin showed \nsignificant cytotoxicity in leiomyoma cells. Leiomyoma cells treated with fisetin underwent apoptotic cell death \nthrough a multi-channel pathway. This study is the first to demonstrate the pharmacological activity of fisetin on \nuterine leiomyoma cells.\nMaterials and methods\nRVS extracts and treatment conditions.  RVS was purchased from Kyung Hee Herb Pharm (Wonju, \nGangwon Province, South Korea). A sample of 700 g of RVS was precisely weighed and heated at 180 °C for 1 h, 10 \ntimes the volume of distilled water was added, and then the mixture was extracted at 100 °C for 2 h. The obtained \nextracts were filtered under reduced pressure (EYELA A-1000S, EYELA, NY , USA) and concentrated using a \nrotary vacuum concentrator (BUCHI Rotavapor R-220, BUCHI Labortechnik, Switzerland) at 60 °C for 1 h. The \nresulting concentrates were lyophilized using a lyophilizer (Ilshin Biobase, Gyeonggi-do, South Korea) for 3 days \nat − 80 °C, and 24 g of dried RVS was obtained. For the experiment, the dried RVS was added to the distilled water \nto yield a concentration of 1 mg/mL and dissolved in a mixing agitator for 4 h. The supernatant was transferred \nand the pellet was centrifuged at 12,000 rpm using a microcentrifuge three times, and then it was filtered using a \n0.22-μM filter.\nHigh-performance liquid chromatography (HPLC).  HPLC analysis was performed with an Alliance \n2690 Separations Module with a Waters 996 Photodiode Array Detector and Millennium 32 Chromatography \nManager Version 3.2 (Waters, MA, USA). For preparative HPLC, a Nucleosil C18 column was used (5 μ m, \n4.0 mm × 250 mm I.D.; Macherey-Nagel, Germany). Acetonitrile, methanol, and water (J.T. Baker, USA) were \nused for separation. For simultaneous analysis of fustin, fisetin, and sulfuretin, the mobile phase consisted of 2% \nacetic acid (A) and methanol (B). The flow rate was fixed at 1.0  ml/min and the wavelength was set at 254  nm. \nThe gradient elution was as follows: 5% B for 0 min, 20% B for 10 min, 60% B for 40 min, and 80% B for 50 min. \nFor analysis of the sample, 10 ml of methanol was added to 100 mg of the extract, and the resulting mixture was \nsonicated for 30 min and then filtered through a 0.45 μm membrane filter. Fisetin used as a reference standard \nwas purchased from Sigma (MO, USA), and fustin and sulfuretin were purchased from the company (Indofine \nChemical Co Inc, NJ, USA).\nIsolation and expansion of leiomyoma cells and normal myometrium cells. Uterine leiomyomas \nand normal myometrium tissues were obtained from patients who underwent a hysterectomy, under approval \nof the institutional review board of Kyung Hee University Hospital (IRB No. KUH 2017-11-064). Written \ninformed consent was obtained from all participants before using their tissues. All experimental methods were \nperformed following the guidelines of the IRB. Tissue was immersed in phosphate-buffered saline (PBS, pH 7.4) \nsupplemented with 1% antibiotics/antimycotic (Gibco BRL, Grand Island, NY , USA). The tissue was cut into \n1–2 mm pieces, and enzymatically digested for 3–4 h at 37 °C in sterile Hanks’ Balanced Salt Solution (HBSS) \nsupplemented with 2 mg/mL collagenase type I (Sigma-Aldrich, St. Louis, MO, USA) and 0.2 mg/mL DNase \n(Sigma-Aldrich, St. Louis, MO, USA). Next, 20% fetal bovine serum (FBS) was added to the fully digested tissue \nto stop the enzyme action. The tissue digest was then filtered through a 70 µm cell strainer (SPL, Gyeonggi-do \nKOREA) and centrifuged at 2,500 rpm for 15 min. The pellets were collected, washed with PBS, and centrifuged \nagain. The fully filtered samples were incubated in Dulbecco’s modified Eagle medium/nutrient mixture F-12 \nmedia (Pan Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum for 48 h at 37 °C and \n5% CO\n2. The media was changed every 2 days. Cultured cells were immunohistochemically stained to visualize \nsmooth muscle characteristics, and 99% or more pure muscle cells were identified. Only the first to third passages \nof cells were used in the experiment.\nMTT assay. Both leiomyoma cells and myometrium cells were incubated on 96-well plates (1 ×  104 cells/\nwell) for 24 h. After the incubation, the cells were treated with RVS, fustin, fisetin and sulfuretin, separately. The \nconcentrations of RVS were 0, 50, 100, 200, 400, 500, and 1000 µg/mL. The concentrations of fustin, fisetin and \nsulfuretin were 0, 10, 20, 40, 60, 80, and 100 µM. Cell viability was analyzed using a methylthiazol tetrazolium \n(MTT) assay after 24, 48, and 72 h of treatment. Briefly, an MTT solution (2 mg/mL) was added to each well, and \nthe cells were incubated at 37 °C and 5% CO2 for 2 h. After the incubation, the MTT was aspirated and 200 μl per \nwell of DMSO was added to each well, and then the plates were shaken for 5 min. Subsequently, the cell viability \nwas assessed by measuring the absorbance at 570 nm using a spectrophotometric microplate reader (Molecular \nDevices, Sunnyvale, CA, USA).\nAnnexin V-FITC/PI apoptosis assay. Cells were seeded onto 60-mm culture dishes at a density of 2.5 × \n105 cells/dish and incubated for 48 h at 37 °C and 5% CO 2. After the incubation, the cells were harvested with \ntrypsin-EDTA solution (JBI, Seoul, Korea), immersed in 5% FBS to stop the enzyme reaction, and then washed \ntwice with PBS buffer. The apoptotic effects of RVS, fustin, fisetin, and sulfuretin were analyzed by using an \nannexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (BD Pharmingen, San Diego, CA, USA), \n\n10Scientific  RepoRtS  |         (2020) 10:7993  | https://doi.org/10.1038/s41598-020-64871-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nwhich contains a binding buffer, annexin V-FITC, and PI staining buffer. Briefly, leiomyoma cells and myome-\ntrium cells were suspended in 100  μl binding buffer and sequentially mixed with 5  μl annexin V-FITC and 5 μl \nPI. The mixture was incubated for 15 min at room temperature in the dark. Cell apoptosis was quantitated with \nflow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA) and CellQuest 6.0 software (Becton \nDickinson, Franklin Lakes, NJ, USA).\nCell cycle analysis. For analysis of the sub-G1 DNA content, cells were collected by trypsinization and fixed \nwith ethanol (70%) overnight at 20 °C. The cells were resuspended in PBS buffer containing 10 μg/ml RNase \nand incubated for 30 min at 37 °C. PI at 50 μg/ml was used to stain cells in the dark at room temperature for \n30 min. The cell cycle phase was identified based on DNA content using a FACSCalibur flow cytometer (Becton \nDickinson, New Jersey, USA) and CellQuest 6.0 software.\nWestern blot analysis. Uterine leiomyoma cells were seeded in 6-well plates and incubated with fisetin \nat varying concentrations. The cells were collected and lysed with ice-cold lysis buffer (20 mM Tris-HCl pH \n7.5, 2.5 mM sodium pyrophosphate, 1 mM EGTA, 150 mM NaCl, 1 mM Na2 EDTA, 1 mM β-glycerophosphate, \n1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin, 1% Triton) and placed on ice for 5 min. \nSubsequently, the lysates were centrifuged for 10 min at 12,000 rpm. The supernatants were collected and the pro-\ntein concentrations in the cell lysates were determined using BCA assay kit (Thermo Fisher Scientific, Waltham, \nMA, USA). Protein (20 μg) was mixed with loading buffer, boiled for 5 min, and loaded onto 8%–15% poly-\nacrylamide gels. Electrophoresis was then carried out, and the proteins were transferred to polyvinylidene diflu-\noride membranes. The membranes were blocked with skimmed milk at room temperature for 1 h, and incubated \nwith the antibodies at 4 °C overnight. The antibodies were listed in Table 1. Membranes were subsequently incu-\nbated with secondary antibody at room temperature for 1 h. After a second wash with TBS-T, target protein \nbands were visualized using an Enhanced Chemiluminescence kit (Thermo Scientific, Rockford, IL, USA). The \nAntibody\nCatalog \nnumber Dilution\nPrimary antibodies\nanti-Akt #4085 1:1,000\nanti-phospho Akt #4060 1:1,000\nanti-Apaf-1 #8723 1:1,000\nanti-Bax #2772 1:1,000\nanti-Bcl2 #2876 1:1,000\nanti-caspase 3 #9665 1:1,000\nanti-cleaved caspase 3 #9661 1:1,000\nanti-caspase 6 #9762 1:1,000\nanti-cleaved caspase 6 #97611 1:1,000\nanti-caspase 8, anti-cleaved caspase 8 #9746 1:1,000\nanti-caspase 9 #9508 1:1,000\nanti-cleaved caspase 9 #52873 1:1,000\nanti-PARP #9542 1:1,000\nanti-cleaved PARP #5625 1:1,000\nanti-Erk1/2 #4695 1:1,000\nanti-phospho Erk1/2 #4370 1:1,000\nanti-p38 #8690 1:1,000\nanti-phospho p38 #4511 1:1,000\nanti-JNK #9665 1:1,000\nanti-phospho JNK #9661 1:1,000\nanti-phospho cyclin B1 #4133 1:1,000\nanti-cytochrome c #4272 1:1,000\nanti-p53 #9282 1:1,000\nanti-phospho p53 #9284 1:1,000\nanti-Beclin-1 #3495 1:1,000\nanti-Apaf-1 #8723 1:1,000\nanti-mTOR #2972 1:1,000\nanti-Atg7 #8558 1:1,000\nanti-LC3A/B I and II #12741 1:1,000\nanti-rabbit β actin #4970 1:1,000\nSecondary antibody horseradish peroxidase-conjugated \ngoat anti-rabbit immunoglobulin G #7074 1:2,500\nTable 1. List of the antibodies, catalog number, and dilution used in the analysis of apoptosis pathways. All \nantibodies were purchased by Cell Signaling Technology (Danvers, CO, USA).\n\n11Scientific  RepoRtS  |         (2020) 10:7993  | https://doi.org/10.1038/s41598-020-64871-y\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\namount of protein expression was quantified with an Amersham Imager 600 chemiluminescence imaging system \n(Davinch-K, Seoul, Korea).\nStatistics. All data are expressed as means ± standard deviations and were statistically analyzed by GraphPad \nPrism (version 5.01; GraphPad Software, San Diego, CA). The statistical significance of the difference between the \ncontrol and experimental groups was determined by one-way Analysis of Variance. P-values less than 0.01 were \nregarded as statistically significant.\nReceived: 17 July 2019; Accepted: 20 April 2020;\nPublished: xx xx xxxx\nReferences\n 1. Ryan, G. L. & Syrop, C. H. & Van, Voorhis B. J. 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Sci. \n7(3), 170–174 (2014).\nAcknowledgements\nThis work was supported by the Korea Health Technology R&D Project through the Korea Health Industry \nDevelopment Institute, funded by the Ministry of Health & Welfare, Republic of Korea (grant number, \nHI19C0113).\nAuthor contributions\nThe authorship credited based on substantial contributions as follows. Conception and design of the study - Jin-\nWoo Lee, Min-Hyung Jung, Kyung Sook Kim. Acquisition of data, or analysis and interpretation of data - Jin-\nWoo Lee, Hyuck Jai Choi, Eun-Jin Kim. Drafting of the article or revising it critically for important intellectual \ncontent - Jin-Woo Lee, Woo Y eon Hwang, Min-Hyung Jung, Kyung Sook Kim. Final approval of the version to be \npublished - Jin-Woo Lee, Min-Hyung Jung, Kyung Sook Kim.\ncompeting interests\nThe authors declare no competing interests.\nAdditional information\nSupplementary information is available for this paper at https://doi.org/10.1038/s41598-020-64871-y.\nCorrespondence and requests for materials should be addressed to M.-H.J. or K.S.K.\nReprints and permissions information is available at www.nature.com/reprints.\nPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and \ninstitutional affiliations.\nOpen Access This article is licensed under a Creative Commons Attribution 4.0 International \nLicense, which permits use, sharing, adaptation, distribution and reproduction in any medium or \nformat, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-\native Commons license, and indicate if changes were made. The images or other third party material in this \narticle are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the \nmaterial. If material is not included in the article’s Creative Commons license and your intended use is not per-\nmitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the \ncopyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.\n \n© The Author(s) 2020","source_license":"CC0","license_restricted":false}