Kaempferol suppresses cell metastasis via inhibition of the ERK-p38-JNK and AP-1 signaling pathways in U-2 OS human osteosarcoma cells

Chen,Hui-Jye; Lin,Chung-Ming; Lee,Chao-Ying; Shih,Nai-Chen; Peng,Shu-Fen; Tsuzuki,Minoru; Amagaya,Sakae; Huang,Wen-Wen; Yang,Jai-Sing

doi:10.3892/or.2013.2490

Kaempferol suppresses cell metastasis via inhibition of the ERK-p38-JNK and AP-1 signaling pathways in U-2 OS human osteosarcoma cells

Authors:
- Hui-Jye Chen
- Chung-Ming Lin
- Chao-Ying Lee
- Nai-Chen Shih
- Shu-Fen Peng
- Minoru Tsuzuki
- Sakae Amagaya
- Wen-Wen Huang
- Jai-Sing Yang
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Affiliations: Graduate Institute of Molecular Systems Biomedicine, China Medical University, Taichung 40402, Taiwan, R.O.C., Department of Biotechnology, Ming Chuan University, Taoyuan 333, Taiwan, R.O.C., School of Pharmacy, China Medical University, Taichung 40402, Taiwan, R.O.C., Department of Pharmacology, China Medical University, Taichung 40402, Taiwan, R.O.C., Department of Biological Science and Technology, China Medical University, Taichung 40402, Taiwan, R.O.C., Department of Biochemistry, Nihon Pharmaceutical University, Saitama 362-0806, Japan, Department of Kampo Pharmaceutical Sciences, Nihon Pharmaceutical University, Saitama 362-0806, Japan
Published online on: May 23, 2013 https://doi.org/10.3892/or.2013.2490
Pages: 925-932

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Abstract

Kaempferol is a natural flavonoid that possesses anti-proliferative and apoptosis-inducing activities in several cancer cell lines. In the present study, we investigated the anti-metastatic activity of kaempferol and its molecular mechanism(s) of action in human osteosarcoma cells. Kaempferol displayed inhibitory effects on the invasion and adhesion of U-2 osteosarcoma (OS) cells in a concentration-dependent manner by Matrigel Transwell assay and cell adhesion assay. Kaempferol also inhibited the migration of U-2 OS cells in a concentration-dependent manner at different treatment time points by wound-healing assay. Additional experiments showed that kaempferol treatment reduced the enzymatic activities and protein levels of matrix metalloproteinase (MMP)-2, MMP-9 and urokinase plasminogen activator (uPA) by gelatin and casein-plasminogen zymography assays and western blot analyses. Kaempferol also downregulated the mRNA levels of MMP-2 and MMP-9 by quantitative PCR analyses. Furthermore, kaempferol was able to reduce the protein phosphorylation of ERK, p38 and JNK by western blotting. By electrophoretic mobility-shift assay (EMSA), we demonstrated that kaempferol decreased the DNA binding activity of AP-1, an action likely to result in the reduced expression of MMP-2, MMP-9 and uPA. Collectively, our data showed that kaempferol attenuated the MAPK signaling pathways including ERK, JNK and p38 and resulted in the decreased DNA binding ability of AP-1, and hence, the downregulation in the expression and enzymatic activities of MMP-2, MMP-9 and uPA, contributing to the inhibition of metastasis of U-2 OS cells. Our results suggest a potential role of kaempferol in the therapy of tumor metastasis of OS.

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1	Kansara M and Thomas DM: Molecular pathogenesis of osteosarcoma. DNA Cell Biol. 26:1–18. 2007. View Article : Google Scholar
2	Ottaviani G and Jaffe N: The epidemiology of osteosarcoma. Cancer Treat Res. 152:3–13. 2009. View Article : Google Scholar
3	Whelan JS: Osteosarcoma. Eur J Cancer. 33:1611–1618. 1997. View Article : Google Scholar
4	Krishnan K, Khanna C and Helman LJ: The biology of metastases in pediatric sarcomas. Cancer J. 11:306–313. 2005. View Article : Google Scholar : PubMed/NCBI
5	Hayden JB and Hoang BH: Osteosarcoma: basic science and clinical implications. Orthop Clin North Am. 37:1–7. 2006. View Article : Google Scholar : PubMed/NCBI
6	Meyers PA, Schwartz CL, Krailo M, et al: Osteosarcoma: a randomized, prospective trial of the addition of ifosfamide and/or muramyl tripeptide to cisplatin, doxorubicin, and high-dose methotrexate. J Clin Oncol. 23:2004–2011. 2005. View Article : Google Scholar : PubMed/NCBI
7	Meyers PA: Muramyl tripeptide (mifamurtide) for the treatment of osteosarcoma. Expert Rev Anticancer Ther. 9:1035–1049. 2009. View Article : Google Scholar : PubMed/NCBI
8	Hodek P, Trefil P and Stiborová M: Flavonoids-potent and versatile biologically active compounds interacting with cytochromes P450. Chem Biol Interact. 139:1–21. 2002. View Article : Google Scholar : PubMed/NCBI
9	Pietta PG: Flavonoids as antioxidants. J Nat Prod. 63:1035–1042. 2000. View Article : Google Scholar
10	Weng CJ and Yen GC: Flavonoids, a ubiquitous dietary phenolic subclass, exert extensive in vitro anti-invasive and in vivo anti-metastatic activities. Cancer Metastasis Rev. 31:323–351. 2012. View Article : Google Scholar : PubMed/NCBI
11	Middleton E Jr, Kandaswami C and Theoharides TC: The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev. 52:673–751. 2000.PubMed/NCBI
12	Guo Q, Zhao L, You Q, et al: Anti-hepatitis B virus activity of wogonin in vitro and in vivo. Antiviral Res. 74:16–24. 2007. View Article : Google Scholar : PubMed/NCBI
13	Cárdenas M, Marder M, Blank VC and Roguin LP: Antitumor activity of some natural flavonoids and synthetic derivatives on various human and murine cancer cell lines. Bioorg Med Chem. 14:2966–2971. 2006.PubMed/NCBI
14	Burda S and Oleszek W: Antioxidant and antiradical activities of flavonoids. J Agric Food Chem. 49:2774–2779. 2001. View Article : Google Scholar : PubMed/NCBI
15	Crespo I, García-Mediavilla MV, Gutiérrez B, Sánchez-Campos S, Tuñón MJ and González-Gallego J: A comparison of the effects of kaempferol and quercetin on cytokine-induced pro-inflammatory status of cultured human endothelial cells. Br J Nutr. 100:968–976. 2008. View Article : Google Scholar : PubMed/NCBI
16	Yamamoto Y and Gaynor RB: Therapeutic potential of inhibition of the NF-κB pathway in the treatment of inflammation and cancer. J Clin Invest. 107:135–142. 2001.
17	Cushnie TP and Lamb AJ: Antimicrobial activity of flavonoids. Int J Antimicrob Agents. 26:343–356. 2005. View Article : Google Scholar : PubMed/NCBI
18	Yu SF, Shun CT, Chen TM and Chen YH: 3-O-β-D-glucosyl-(1→6)-β-D-glucosyl-kaempferol isolated from Sauropus androgenus reduces body weight gain in Wistar rats. Biol Pharm Bull. 29:2510–2513. 2006.
19	Yao P, Nussler A, Liu L, et al: Quercetin protects human hepatocytes from ethanol-derived oxidative stress by inducing heme oxygenase-1 via the MAPK/Nrf2 pathways. J Hepatol. 47:253–261. 2007. View Article : Google Scholar : PubMed/NCBI
20	Tijburg LB, Mattern T, Folts JD, Weisgerber UM and Katan MB: Tea flavonoids and cardiovascular disease: a review. Crit Rev Food Sci Nutr. 37:771–785. 1997. View Article : Google Scholar : PubMed/NCBI
21	Park JS, Rho HS, Kim DH and Chang IS: Enzymatic preparation of kaempferol from green tea seed and its antioxidant activity. J Agric Food Chem. 54:2951–2956. 2006. View Article : Google Scholar : PubMed/NCBI
22	Stewart AJ, Bozonnet S, Mullen W, Jenkins GI, Lean ME and Crozier A: Occurrence of flavonols in tomatoes and tomato-based products. J Agric Food Chem. 48:2663–2669. 2000. View Article : Google Scholar : PubMed/NCBI
23	Jaganathan SK and Mandal M: Antiproliferative effects of honey and of its polyphenols: a review. J Biomed Biotechnol. 2009:8306162009. View Article : Google Scholar : PubMed/NCBI
24	Zhang Y, Seeram NP, Lee R, Feng L and Heber D: Isolation and identification of strawberry phenolics with antioxidant and human cancer cell antiproliferative properties. J Agric Food Chem. 56:670–675. 2008. View Article : Google Scholar : PubMed/NCBI
25	Kang JW, Kim JH, Song K, Kim SH, Yoon JH and Kim KS: Kaempferol and quercetin, components of Ginkgo biloba extract (EGb 761), induce caspase-3-dependent apoptosis in oral cavity cancer cells. Phytother Res. 24(Suppl 1): S77–S82. 2010.PubMed/NCBI
26	Bajpai M, Pande A, Tewari SK and Prakash D: Phenolic contents and antioxidant activity of some food and medicinal plants. Int J Food Sci Nutr. 56:287–291. 2005. View Article : Google Scholar : PubMed/NCBI
27	Calderón-Montaño JM, Burgos-Morón E, Pérez-Guerrero C and López-Lázaro M: A review on the dietary flavonoid kaempferol. Mini Rev Med Chem. 11:298–344. 2011.PubMed/NCBI
28	Leung HW, Lin CJ, Hour MJ, Yang WH, Wang MY and Lee HZ: Kaempferol induces apoptosis in human lung non-small carcinoma cells accompanied by an induction of antioxidant enzymes. Food Chem Toxicol. 45:2005–2013. 2007. View Article : Google Scholar : PubMed/NCBI
29	Zhang Q, Zhao XH and Wang ZJ: Flavones and flavonols exert cytotoxic effects on a human oesophageal adenocarcinoma cell line (OE33) by causing G2/M arrest and inducing apoptosis. Food Chem Toxicol. 46:2042–2053. 2008. View Article : Google Scholar : PubMed/NCBI
30	Marfe G, Tafani M, Indelicato M, et al: Kaempferol induces apoptosis in two different cell lines via Akt inactivation, Bax and SIRT3 activation, and mitochondrial dysfunction. J Cell Biochem. 106:643–650. 2009. View Article : Google Scholar : PubMed/NCBI
31	Kim KS, Rhee KH, Yoon JH, Lee JG, Lee JH and Yoo JB: Ginkgo biloba extract (EGb 761) induces apoptosis by the activation of caspase-3 in oral cavity cancer cells. Oral Oncol. 41:383–389. 2005. View Article : Google Scholar
32	De Leo M, Braca A, Sanogo R, Cardile V, DeTommasi N and Russo A: Antiproliferative activity of Pteleopsis suberosa leaf extract and its flavonoid components in human prostate carcinoma cells. Planta Med. 72:604–610. 2006.
33	Mutoh M, Takahashi M, Fukuda K, et al: Suppression of cyclooxygenase-2 promoter-dependent transcriptional activity in colon cancer cells by chemopreventive agents with a resorcin-type structure. Carcinogenesis. 21:959–963. 2000. View Article : Google Scholar : PubMed/NCBI
34	Huang WW, Chiu YJ, Fan MJ, et al: Kaempferol induced apoptosis via endoplasmic reticulum stress and mitochondria-dependent pathway in human osteosarcoma U-2 OS cells. Mol Nutr Food Res. 54:1585–1595. 2010. View Article : Google Scholar : PubMed/NCBI
35	Sliva D: Suppression of cancer invasiveness by dietary compounds. Mini Rev Med Chem. 8:677–688. 2008. View Article : Google Scholar : PubMed/NCBI
36	Phromnoi K, Yodkeeree S, Anuchapreeda S and Limtrakul P: Inhibition of MMP-3 activity and invasion of the MDA-MB-231 human invasive breast carcinoma cell line by bioflavonoids. Acta Pharmacol Sin. 30:1169–1176. 2009. View Article : Google Scholar : PubMed/NCBI
37	Labbé D, Provençal M, Lamy S, Boivin D, Gingras D and Béliveau R: The flavonols quercetin, kaempferol, and myricetin inhibit hepatocyte growth factor-induced medulloblastoma cell migration. J Nutr. 139:646–652. 2009.PubMed/NCBI
38	Lin CW, Shen SC, Chien CC, Yang LY, Shia LT and Chen YC: 12-O-tetradecanoylphorbol-13-acetate-induced invasion/migration of glioblastoma cells through activating PKCα/ERK/NF-κB-dependent MMP-9 expression. J Cell Physiol. 225:472–481. 2010.
39	Chiu YJ, Hour MJ, Lu CC, et al: Novel quinazoline HMJ-30 induces U-2 OS human osteogenic sarcoma cell apoptosis through induction of oxidative stress and up-regulation of ATM/p53 signaling pathway. J Orthop Res. 29:1448–1456. 2011. View Article : Google Scholar
40	Chen KT, Hour MJ, Tsai SC, et al: The novel synthesized 6-fluoro-(3-fluorophenyl)-4-(3-methoxyanilino)quinazoline (LJJ-10) compound exhibits anti-metastatic effects in human osteosarcoma U-2 OS cells through targeting insulin-like growth factor-I receptor. Int J Oncol. 39:611–619. 2011.
41	Hour MJ, Tsai SC, Wu HC, et al: Antitumor effects of the novel quinazolinone MJ-33: Inhibition of metastasis through the MAPK, AKT, NF-κB and AP-1 signaling pathways in DU145 human prostate cancer cells. Int J Oncol. 41:1513–1519. 2012.PubMed/NCBI
42	Lai KC, Huang AC, Hsu SC, et al: Benzyl isothiocyanate (BITC) inhibits migration and invasion of human colon cancer HT29 cells by inhibiting matrix metalloproteinase-2/-9 and urokinase plasminogen (uPA) through PKC and MAPK signaling pathway. J Agric Food Chem. 58:2935–2942. 2010. View Article : Google Scholar
43	Kao WT, Lin CY, Lee LT, et al: Investigation of MMP-2 and -9 in a highly invasive A431 tumor cell sub-line selected from a Boyden chamber assay. Anticancer Res. 28:2109–2120. 2008.PubMed/NCBI
44	Mazar AP: The urokinase plasminogen activator receptor (uPAR) as a target for the diagnosis and therapy of cancer. Anticancer Drugs. 12:387–400. 2001. View Article : Google Scholar : PubMed/NCBI
45	Kawabata K, Murakami A and Ohigashi H: Citrus auraptene targets translation of MMP-7 (matrilysin) via ERK1/2-dependent and mTOR-independent mechanism. FEBS Lett. 580:5288–5294. 2006. View Article : Google Scholar : PubMed/NCBI
46	Mook OR, Frederiks WM and Van Noorden CJ: The role of gelatinases in colorectal cancer progression and metastasis. Biochim Biophys Acta. 1705:69–89. 2004.PubMed/NCBI
47	Ried S, Jäger C, Jeffers M, et al: Activation mechanisms of the urokinase-type plasminogen activator promoter by hepatocyte growth factor/scatter factor. J Biol Chem. 274:16377–16386. 1999. View Article : Google Scholar : PubMed/NCBI
48	Lengyel E, Stepp E, Gum R and Boyd D: Involvement of a mitogen-activated protein kinase signaling pathway in the regulation of urokinase promoter activity by c-Ha-ras. J Biol Chem. 270:23007–23012. 1995. View Article : Google Scholar : PubMed/NCBI
49	Peng PL, Hsieh YS, Wang CJ, Hsu JL and Chou FP: Inhibitory effect of berberine on the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2. Toxicol Appl Pharmacol. 214:8–15. 2006. View Article : Google Scholar : PubMed/NCBI
50	Hanahan D and Weinberg RA: The hallmarks of cancer. Cell. 100:57–70. 2000. View Article : Google Scholar
51	Denys H, De Wever O, Nusgens B, et al: Invasion and MMP expression profile in desmoid tumours. Br J Cancer. 90:1443–1449. 2004. View Article : Google Scholar : PubMed/NCBI
52	Masson V, de la Ballina LR, Munaut C, et al: Contribution of host MMP-2 and MMP-9 to promote tumor vascularization and invasion of malignant keratinocytes. FASEB J. 19:234–236. 2005.PubMed/NCBI
53	Duffy MJ, Maguire TM, Hill A, McDermott E and O’Higgins N: Metalloproteinases: role in breast carcinogenesis, invasion and metastasis. Breast Cancer Res. 2:252–257. 2000. View Article : Google Scholar : PubMed/NCBI
54	Hanemaaijer R, Verheijen JH, Maguire TM, et al: Increased gelatinase-A and gelatinase-B activities in malignant vs. benign breast tumors. Int J Cancer. 86:204–207. 2000. View Article : Google Scholar : PubMed/NCBI
55	Chambers AF and Matrisian LM: Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst. 89:1260–1270. 1997. View Article : Google Scholar : PubMed/NCBI
56	Ferrari C, Benassi S, Ponticelli F, et al: Role of MMP-9 and its tissue inhibitor TIMP-1 in human osteosarcoma: findings in 42 patients followed for 1–16 years. Acta Orthop Scand. 75:487–491. 2004.PubMed/NCBI
57	Bjornland K, Flatmark K, Pettersen S, Aaasen AO, Fodstad O and Maelandsmo GM: Matrix metalloproteinases participate in osteosarcoma invasion. J Surg Res. 127:151–156. 2005. View Article : Google Scholar : PubMed/NCBI
58	Uchibori M, Nishida Y, Nagasaka T, Yamada Y, Nakanishi K and Ishiguro N: Increased expression of membrane-type matrix metalloproteinase-1 is correlated with poor prognosis in patients with osteosarcoma. Int J Oncol. 28:33–42. 2006.PubMed/NCBI
59	Foukas AF, Deshmukh NS, Grimer RJ, Mangham DC, Mangos EG and Taylor S: Stage-IIB osteosarcomas around the knee. A study of MMP-9 in surviving tumour cells. J Bone Joint Surg Br. 84:706–711. 2002. View Article : Google Scholar : PubMed/NCBI
60	Sliva D, English D, Lyons D and Lloyd FP Jr: Protein kinase C induces motility of breast cancers by upregulating secretion of urokinase-type plasminogen activator through activation of AP-1 and NF-κB. Biochem Biophys Res Commun. 290:552–557. 2002.PubMed/NCBI
61	Choong PF and Nadesapillai AP: Urokinase plasminogen activator system: a multifunctional role in tumor progression and metastasis. Clin Orthop Relat Res. (Suppl 415): S46–S58. 2003. View Article : Google Scholar : PubMed/NCBI
62	Choong PF, Fernö M, Akerman M, et al: Urokinase-plasminogen-activator levels and prognosis in 69 soft-tissue sarcomas. Int J Cancer. 69:268–272. 1996. View Article : Google Scholar : PubMed/NCBI
63	Dass CR, Nadesapillai AP, Robin D, et al: Downregulation of uPAR confirms link in growth and metastasis of osteosarcoma. Clin Exp Metastasis. 22:643–652. 2005. View Article : Google Scholar : PubMed/NCBI
64	Gondi CS and Rao JS: Therapeutic potential of siRNA-mediated targeting of urokinase plasminogen activator, its receptor, and matrix metalloproteinases. Methods Mol Biol. 487:267–281. 2009.PubMed/NCBI
65	Chen PN, Hsieh YS, Chiou HL and Chu SC: Silibinin inhibits cell invasion through inactivation of both PI3K-Akt and MAPK signaling pathways. Chem Biol Interact. 156:141–150. 2005. View Article : Google Scholar : PubMed/NCBI
66	Westermarck J and Kähäri VM: Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J. 13:781–792. 1999.PubMed/NCBI
67	Aguirre Ghiso JA, Alonso DF, Farías EF, Gomez DE and de Kier Joffè EB: Deregulation of the signaling pathways controlling urokinase production. Its relationship with the invasive phenotype. Eur J Biochem. 263:295–304. 1999.PubMed/NCBI
68	Wu JX, Carpenter PM, Gresens C, et al: The proto-oncogene c-fos is over-expressed in the majority of human osteosarcomas. Oncogene. 5:989–1000. 1990.PubMed/NCBI
69	Franchi A, Calzolari A and Zampi G: Immunohistochemical detection of c-fos and c-jun expression in osseous and cartilaginous tumours of the skeleton. Virchows Arch. 432:515–519. 1998. View Article : Google Scholar : PubMed/NCBI
70	Gamberi G, Benassi MS, Bohling T, et al: C-myc and c-fos in human osteosarcoma: prognostic value of mRNA and protein expression. Oncology. 55:556–563. 1998. View Article : Google Scholar : PubMed/NCBI
71	Leaner VD, Chick JF, Donninger H, et al: Inhibition of AP-1 transcriptional activity blocks the migration, invasion, and experimental metastasis of murine osteosarcoma. Am J Pathol. 174:265–275. 2009. View Article : Google Scholar : PubMed/NCBI
72	Tan ML, Choong PF and Dass CR: Direct anti-metastatic efficacy by the DNA enzyme Dz13 and downregulated MMP-2, MMP-9 and MT1-MMP in tumours. Cancer Cell Int. 10:92010. View Article : Google Scholar : PubMed/NCBI