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Apigenin inhibits tumor necrosis factor α–induced cell proliferation and prostaglandin E2 synthesis by inactivating NFκB in endometriotic stromal cells

2010; Elsevier BV; Volume: 95; Issue: 4 Linguagem: Inglês

10.1016/j.fertnstert.2010.09.046

1556-5653

Kana Suou, Fuminori Taniguchi, Yukiko Tagashira, Tomoiki Kiyama, Naoki Terakawa, Tasuku Harada,

Reproductive System and Pregnancy

Apigenin suppressed tumor necrosis factor α–induced cell proliferation and prostaglandin E2 expression via the attenuation of nuclear factor κB pathway in endometriotic stromal cells in vitro. Apigenin reduced the mitogenic activity and inflammatory reaction in endometriotic stromal cells. Apigenin suppressed tumor necrosis factor α–induced cell proliferation and prostaglandin E2 expression via the attenuation of nuclear factor κB pathway in endometriotic stromal cells in vitro. Apigenin reduced the mitogenic activity and inflammatory reaction in endometriotic stromal cells. Local inflammatory reaction in the peritoneal environment is considered one of the contributing factors for pathogenesis of endometriosis. We demonstrated that the levels of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α are elevated in the peritoneal fluid of women with endometriosis (1Harada T. Yoshioka H. Yoshida S. Iwabe T. Onohara Y. Tanikawa M. et al.Increased interleukin-6 levels in peritoneal fluid of infertile patients with active endometriosis.Am J Obstet Gynecol. 1997; 176: 593-597Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 2Iwabe T. Harada T. Tsudo T. Tanikawa M. Onohara Y. Terakawa N. Pathogenetic significance of increased levels of interleukin-8 in the peritoneal fluid of patients with endometriosis.Fertil Steril. 1998; 69: 924-930Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). We also showed that both TNF-α and IL-8 enhances mitogenic activity, and TNF-α upregulates IL-8 expression through nuclear factor (NF) κB activation in endometriotic stromal cells (ESCs) (3Sakamoto Y. Harada T. Horie S. Iba Y. Taniguchi F. Yoshida S. et al.Tumor necrosis factor-alpha-induced interleukin-8 (IL-8) expression in endometriotic stromal cells, probably through nuclear factor-kappa B activation: gonadotropin-releasing hormone agonist treatment reduced IL-8 expression.J Clin Endocrinol Metab. 2003; 88: 730-735Crossref PubMed Scopus (142) Google Scholar). Increased prostaglandin E2 (PGE2) levels are present in the peritoneal fluid of women with endometriosis (4Karck U. Reister F. Schafer W. Zahradnik H.P. Breckwoldt M. PGE2 and PGF2 alpha release by human peritoneal macrophages in endometriosis.Prostaglandins. 1996; 51: 49-60Crossref PubMed Scopus (77) Google Scholar). Cyclooxygenase (COX)-2 is more abundantly expressed in ectopic endometria than in eutopic endometria (5Chishima F. Hayakawa S. Sugita K. Kinukawa N. Aleemuzzaman S. Nemoto N. et al.Increased expression of cyclooxygenase-2 in local lesions of endometriosis patients.Am J Reprod Immunol. 2002; 48: 50-56Crossref PubMed Scopus (144) Google Scholar), and it can regulate survival and invasion of endometriotic cells (6Banu S.K. Lee J. Speights Jr., V.O. Starzinski-Powitz A. Arosh J.A. Cyclooxygenase-2 regulates survival, migration, and invasion of human endometriotic cells through multiple mechanisms.Endocrinology. 2008; 149: 1180-1189Crossref PubMed Scopus (126) Google Scholar). It is therefore rational to attempt to identify new therapeutic agents that can inhibit the inflammatory reaction in the peritoneal fluid of women with endometriosis. Apigenin is a dietary flavonoid in various fruits and vegetables such as parsely, celery, onion, orange, and herbs, and it is considered to possess multiple biologic activities such as antiproliferative, antiinflammatory, and free radical scavenging properties. Recently, there has been interest in using the flavonoid derivatives as anticancer drugs. Apigenin has been studied in several cancer cell lines, including breast, colon, skin, and leukemia cells (7Losi G. Puia G. Garzon G. de Vuono M.C. Baraldi M. Apigenin modulates GABAergic and glutamatergic transmission in cultured cortical neurons.Eur J Pharmacol. 2004; 502: 41-46Crossref PubMed Scopus (67) Google Scholar, 8Takahashi T. Kobori M. Shinmoto H. Tsushida T. Structure-activity relationships of flavonoids and the induction of granulocytic- or monocytic-differentiation in HL60 human myeloid leukemia cells.Biosci Biotechnol Biochem. 1998; 62: 2199-2204Crossref PubMed Scopus (76) Google Scholar, 9Van Dross R.T. Hong X. Pelling J.C. Inhibition of TPA-induced cyclooxygenase-2 (COX-2) expression by apigenin through downregulation of Akt signal transduction in human keratinocytes.Mol Carcinog. 2005; 44: 83-91Crossref PubMed Scopus (64) Google Scholar, 10Panes J. Gerritsen M.E. Anderson D.C. Miyasaka M. Granger D.N. Apigenin inhibits tumor necrosis factor-induced intercellular adhesion molecule-1 upregulation in vivo.Microcirculation. 1996; 3: 279-286Crossref PubMed Scopus (72) Google Scholar). In skin cancer cells, apigenin decreases COX-2 transcription and expression (9Van Dross R.T. Hong X. Pelling J.C. Inhibition of TPA-induced cyclooxygenase-2 (COX-2) expression by apigenin through downregulation of Akt signal transduction in human keratinocytes.Mol Carcinog. 2005; 44: 83-91Crossref PubMed Scopus (64) Google Scholar). To identify a new candidate agent for the alleviation of endometriosis, we evaluated the inhibitory activity of apigenin on TNF-α–induced cell growth and COX-2/PGE2 synthesis of ESCs. We hypothesized that apigenin possesses the favorable effects to control the propagation of endometriosis. With their informed consent, we recruited 35 women with endometriomas who had regular menstrual cycles (follicular phase [n = 15] and luteal phase [n = 20]) and who had not received any hormonal treatment before the surgery. The institutional review boards of Tottori University Faculty of Medicine approved this project. The women underwent laparoscopic surgery for ovarian endometriomas (more than 4 cm in diameter) at Tottori University Hospital. The patients' ages (mean ± SD) were 33.8 ± 0.91 years; their values of serum FSH and E2 were within normal limits. For the indication of surgery, infertility and/or severe dysmenorrhea mainly due to endometrioma were included. The chocolate cysts lining the ovaries of patients with endometriosis were the source of endometriotic tissues. ESCs were isolated from endometriotic tissues and used in a monolayer culture after the first passage (11Osteen K.G. Yeaman G.R. Bruner-Tran K.L. Matrix metalloproteinases and endometriosis.Semin Reprod Med. 2003; 21: 155-164Crossref PubMed Scopus (127) Google Scholar). The apoptosis rates in the ESCs of proliferative and secretory phase were similar (12Izawa M. Harada T. Deura I. Taniguchi F. Iwabe T. Terakawa N. Drug-induced apoptosis was markedly attenuated in endometriotic stromal cells.Hum Reprod. 2006; 21: 600-604Crossref PubMed Scopus (40) Google Scholar), and samples derived from endometriomas were not classified according to menstrual phase for analysis. ESCs were pretreated with apigenin (4', 5, 7-trihydroxyflavone; Sigma-Aldrich, St. Louis, MO) in the medium. Next, TNF-α (0.1 ng/mL; R&D Systems, Minneapolis, MN) was exposed to evaluate IL-8 and PGE2 production in the supernatant. We chose TNF-α simply because it is a potent proinflammatory cytokine. The concentration of IL-8 and PGE2 was determined using IL-8 ELISA kits (R&D Systems). Total RNA was extracted from cultured ESCs using an RNeasy Mini Kit (Qiagen, Tokyo, Japan). Reverse transcription of RNA (1 μg) from ESCs into complementary DNA was performed. The mRNA levels were quantified using the ABI 7900 HT real-time PCR system (Applied Biosystems, Tokyo, Japan). All primers and probes were designed using the PRIMER express program (Applied Biosystems). The absolute values for IL-8, COX-1, and COX-2 were normalized to that for glyceraldehyde-3-phosphate dehydrogenase. To examine DNA synthesis in proliferating cells, bromodeoxyuridine incorporation was assessed with the cell proliferation ELISA (Amersham Biosciences, Tokyo, Japan). Total cell lysates (30 μg) were applied on the polyacrylamide gel and transferred to a membrane. These transferred proteins reacted with antibodies recognizing the phosphorylation of extracellular signal-regulated kinase (p-ERK1/2), p38 MAPK (p-p38MAPK), c-Jun N-terminal protein kinase (p-JNK1/2), and inhibitor NFκB (p-IκB). The concentration of intranuclear p65 protein was measured using a NFκB Transcription Factor Assay ELISA Kit (Active Motif, Carlsbad, CA). The effect of a NFκB inhibitor, TPCK (Calbiochem, San Diego, CA) on intranuclear p65 levels in ESCs was evaluated. Results were analyzed using one-way ANOVA, followed by Fisher's protected least significant differences post hoc test. All data sets are presented as means with SEMs; P<0.05 was accepted as indicating statistical significance. As demonstrated previously (13Iwabe T. Harada T. Tsudo T. Nagano Y. Yoshida S. Tanikawa M. et al.Tumor necrosis factor-alpha promotes proliferation of endometriotic stromal cells by inducing interleukin-8 gene and protein expression.J Clin Endocrinol Metab. 2000; 85: 824-829Crossref PubMed Scopus (191) Google Scholar, 14Taniguchi F. Harada T. Miyakoda H. Iwabe T. Deura I. Tagashira Y. et al.TAK1 activation for cytokine synthesis and proliferation of endometriotic cells.Mol Cell Endocrinol. 2009; 307: 196-204Crossref PubMed Scopus (38) Google Scholar), TNF-α enhanced bromodeoxyuridine incorporation of ESCs (130% vs. the untreated group). Apigenin (50 and 100 μM) repressed TNF-α–induced ESC proliferation by 50 % compared with only TNF-α addition (Fig. 1A ). Similarly, TNF-α markedly enhanced IL-8 gene and protein expression. By apigenin pretreatment, TNF-α–induced IL-8 expression was repressed (Fig. 1B). To evaluate the cytotoxicity of apigenin, the lactate dehydrogenase release, as a marker of cell cycle arrest and apoptosis from ESCs, was analyzed. High concentration (50–100 μM) of apigenin, regardless of TNF-α addition, led to the increase of lactate dehydrogenase release (data not shown). TNF-α induced an approximately fourfold increase of COX-2 gene expression of ESCs (Fig. 1C). COX-2 gene expression was repressed by apigenin treatment. In contrast, COX-1 gene expression was not enhanced by TNF-α, and apigenin treatment did not show any discernible effect (data not shown). As anticipated, TNF-α enhanced PGE2 synthesis of ESCs, whereas pretreatment of apigenin suppressed PGE2 synthesis (Fig. 1D). On the other hand, we evaluated the effect of apigenin on IL-8 and COX-2 gene expression in eutopic endometrial stromal cells obtained from patients with uterine myomas. TNF-α–induced IL-8 and COX-2 gene expression was significantly suppressed by apigenin treatment (10–100 μM) in eutopic endometrial stromal cells (data not shown) as shown in ESCs. As we showed previously (14Taniguchi F. Harada T. Miyakoda H. Iwabe T. Deura I. Tagashira Y. et al.TAK1 activation for cytokine synthesis and proliferation of endometriotic cells.Mol Cell Endocrinol. 2009; 307: 196-204Crossref PubMed Scopus (38) Google Scholar), TNF-α addition induced all the phosphorylated protein expression, IκB, JNK1/2, p38MAPK, and ERK1/2. Pretreatment with apigenin prior to TNF-α addition obviously attenuated only IκB phosphorylation. JNK1/2, p38MAPK, and ERK1/2 phosphorylation was not altered (Fig. 1E). To provide further support for our proposal that apigenin can inhibit TNF-α–induced IκB phosphorylation, we assessed the level of intranuclear p65 protein. In addition to the pretreatment with TPCK, a NFκB inhibitor, apigenin treatment reduced TNF-α–induced translocation of p65 protein (Fig. 1F). Flavonoids are distributed widely in foods and have been suggested to play a role in preventing disease (15Gates M.A. Vitonis A.F. Tworoger S.S. Rosner B. Titus-Ernstoff L. Hankinson S.E. et al.Flavonoid intake and ovarian cancer risk in a population-based case-control study.Int J Cancer. 2009; 124: 1918-1925Crossref PubMed Scopus (94) Google Scholar, 16Wang L. Lee I.M. Zhang S.M. Blumberg J.B. Buring J.E. Sesso H.D. Dietary intake of selected flavonols, flavones, and flavonoid-rich foods and risk of cancer in middle-aged and older women.Am J Clin Nutr. 2009; 89: 905-912Crossref PubMed Scopus (233) Google Scholar). A great deal of attention has been focused on how flavonoids function in relation to their biologic properties. Several reports have shown that apigenin has anti-inflammatory and anti-tumor properties (7Losi G. Puia G. Garzon G. de Vuono M.C. Baraldi M. Apigenin modulates GABAergic and glutamatergic transmission in cultured cortical neurons.Eur J Pharmacol. 2004; 502: 41-46Crossref PubMed Scopus (67) Google Scholar, 9Van Dross R.T. Hong X. Pelling J.C. Inhibition of TPA-induced cyclooxygenase-2 (COX-2) expression by apigenin through downregulation of Akt signal transduction in human keratinocytes.Mol Carcinog. 2005; 44: 83-91Crossref PubMed Scopus (64) Google Scholar, 10Panes J. Gerritsen M.E. Anderson D.C. Miyasaka M. Granger D.N. Apigenin inhibits tumor necrosis factor-induced intercellular adhesion molecule-1 upregulation in vivo.Microcirculation. 1996; 3: 279-286Crossref PubMed Scopus (72) Google Scholar, 17Choi E.J. Kim G.H. Apigenin causes G(2)/M arrest associated with the modulation of p21(Cip1) and Cdc2 and activates p53-dependent apoptosis pathway in human breast cancer SK-BR-3 cells.J Nutr Biochem. 2009; 20: 285-290Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). It is expected that apigenin has specific inhibitory activity for the cell growth; however, the mechanisms underlying the effects of apigenin have been still unknown in endometriotic cells. We ascertained that TNF-α significantly promoted COX-2 gene expression and PGE2 production of ESCs. We previously provided evidences that lipopolysaccharide stimulated TNF-α and IL-8 expression in ESCs through NFκB activation (18Iba Y. Harada T. Horie S. Deura I. Iwabe T. Terakawa N. Lipopolysaccharide-promoted proliferation of endometriotic stromal cells via induction of tumor necrosis factor alpha and interleukin-8 expression.Fertil Steril. 2004; 82: 1036-1042Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Lipopolysaccharide promotes also the proliferation and invasion of ESCs by upregulation of the COX-2–PGE2 pathway, indicating that pelvic inflammation may promote the progression of endometriosis (19Takenaka Y. Taniguchi F. Miyakoda H. Takai E. Terakawa N. Harada T. Lipopolysaccharide promoted proliferation and invasion of endometriotic stromal cells via induction of cyclooxygenase-2 expression.Fertil Steril. 2010; 93: 325-327Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Apigenin is noted to inhibit COX-2 expression in lipopolysaccharide-treated macrophages (20Liang Y.C. Huang Y.T. Tsai S.H. Lin-Shiau S.Y. Chen C.F. Lin J.K. Suppression of inducible cyclooxygenase and inducible nitric oxide synthase by apigenin and related flavonoids in mouse macrophages.Carcinogenesis. 1999; 20: 1945-1952Crossref PubMed Scopus (512) Google Scholar, 21Raso G.M. Meli R. Di Carlo G. Pacilio M. Di Carlo R. Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in macrophage J774A.1.Life Sci. 2001; 68: 921-931Crossref PubMed Scopus (393) Google Scholar). In accordance with our data, apigenin decreased the proliferation of human breast cancer SK-BR-3 cells only at 50 and 100 μM (17Choi E.J. Kim G.H. Apigenin causes G(2)/M arrest associated with the modulation of p21(Cip1) and Cdc2 and activates p53-dependent apoptosis pathway in human breast cancer SK-BR-3 cells.J Nutr Biochem. 2009; 20: 285-290Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Signal transduction kinases, such as IκB, JNK, p38MAPK, and ERK were phosphorylated by TNF-α addition. After phosphorylation, the transcription factors may bind the cis-acting elements in the COX-2 promoter, thereby resulting in positive regulation of COX-2 expression (22Hunot S. Vila M. Teismann P. Davis R.J. Hirsch E.C. Przedborski S. et al.JNK-mediated induction of cyclooxygenase 2 is required for neurodegeneration in a mouse model of Parkinson's disease.Proc Natl Acad Sci U S A. 2004; 101: 665-670Crossref PubMed Scopus (367) Google Scholar). Because COX-2 expression may be primarily controlled by transcriptional regulation, apigenin may inhibit directly the transcriptional factors in the COX-2 promoter via the IκB phosphorylation. NFκB, is a key component for the inducible expression of a wide variety of genes mediating immune and inflammatory responses. TNF-α induces IκB polyubiquitination and degradation resulting in nuclear translocation of the active p50/p65 NFκB that eventually regulates the expression of target genes, such as IL-8 and COX-2. Our current data indicate that apigenin inhibits TNF-α–inducible IκB phosphorylation and the intranuclear level of p65 protein in ESCs, suggesting that apigenin possesses antiinflammatory effects through the NFκB pathway. As with many flavonoids, several investigators reported that apigenin may possess the special interest for the development of a novel chemopreventive or chemotherapeutic agent. Gates et al. (15Gates M.A. Vitonis A.F. Tworoger S.S. Rosner B. Titus-Ernstoff L. Hankinson S.E. et al.Flavonoid intake and ovarian cancer risk in a population-based case-control study.Int J Cancer. 2009; 124: 1918-1925Crossref PubMed Scopus (94) Google Scholar) reviewed the data in regard to the foods commonly eaten over a 1-week period by women with or without ovarian cancer. Interestingly, within five common flavonoids (myricetin, kaempferol, quercetin, luteolin, and apigenin), only apigenin intake was associated with a suggestive decrease in ovarian cancer risk. In contrast, a recent study showed that, among 38,408 women identified a total of 3,234 cancer cases, no significant associations were observed between intake of flavonoid-rich foods and the incidence of cancers, including ovarian cancer and endometrial cancer (16Wang L. Lee I.M. Zhang S.M. Blumberg J.B. Buring J.E. Sesso H.D. Dietary intake of selected flavonols, flavones, and flavonoid-rich foods and risk of cancer in middle-aged and older women.Am J Clin Nutr. 2009; 89: 905-912Crossref PubMed Scopus (233) Google Scholar). Therefore, to verify the findings of previous studies and the biologic plausibility of this association, additional studies about the effects of apigenin treatment will be required. Our findings indicate that apigenin attenuates TNF-α–induced cell proliferation and COX-2/PGE2 synthesis via the inactivation of NFκB pathway in ESCs. Further study will be needed to test the potential of this agent in the prevention or treatment of endometriosis. Collectively, these results suggest that apigenin is a novel therapeutic agent in the treatment of endometriosis. The authors thank Ms. Hiroko Miyakoda (Tottori University Faculty of Medicine) for providing technical assistance and Dr. Apostolos Kaponis for reviewing the manuscript (University of Patras, Patras, Greece).