Nobiletin inhibits epithelial-mesenchymal transition of human non-small cell lung cancer cells by antagonizing the TGF-β1/Smad3 signaling pathway

Da,Chunli; Liu,Yuting; Zhan,Yiyi; Liu,Kai; Wang,Ruozheng

doi:10.3892/or.2016.4661

Nobiletin inhibits epithelial-mesenchymal transition of human non-small cell lung cancer cells by antagonizing the TGF-β1/Smad3 signaling pathway

Authors:
- Chunli Da
- Yuting Liu
- Yiyi Zhan
- Kai Liu
- Ruozheng Wang
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Affiliations: Department of Intensive Care, Tumor Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830000, P.R. China, Department of Anesthesiology, Tumor Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830000, P.R. China, Department of Chemotherapy for Lung Cancer, Tumor Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830000, P.R. China, Department of Radiotherapy for the Head and Neck, Tumor Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830000, P.R. China
Published online on: March 7, 2016 https://doi.org/10.3892/or.2016.4661
Pages: 2767-2774

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Abstract

Epithelial-mesenchymal transition (EMT) is a critical cellular process in cancer metastasis, during which epithelial polarized cells become motile mesenchymal cells. Since transforming growth factor-β (TGF-β) is a potent inducer of EMT, blocking of TGF-β/Smad signaling has become a promising cancer therapy. Nobiletin, a polymethoxy flavonoid from Citrus depressa, has been shown to be valuable for cancer treatment, yet the mechanism remains unclear. In the present study, lung adenocarcinoma A549 and H1299 cells were used to evaluate the effect of nobiletin on EMT induced by TGF-β1. Nobiletin successfully inhibited TGF-β1-induced EMT, migration, invasion and adhesion in vitro, accompanied by attenuation of MMP-2, MMP-9, p-Src, p-FAK, p-paxillin, Snail, Slug, Twist and ZEB1 expression. Nobiletin inhibited the transcriptional activity of Smads without changing the phosphorylation status or translocation of Smads induced by TGF-β1. Moreover, Smad3 is requisite in TGF-β1-stimulated EMT. Smad3 overexpression meaningfully impaired the ability of nobiletin to reverse TGF-β1-induced EMT. In vivo, nobiletin prohibited the growth of metastatic nodules in the lungs of nude mice. Moreover, nobiletin inhibited tumor growth and reversed EMT in mice bearing A549-Luc xenografts, as revealed by IVIS imaging and immunohistochemical analysis. Collectively, the data suggest that nobiletin prevents EMT by inactivating TGF-β1/Smad3 signaling.

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1	Barton MK: Patients of all ages with advanced non-small cell lung cancer are not receiving chemotherapy. CA Cancer J Clin. 65:337–338. 2015. View Article : Google Scholar : PubMed/NCBI
2	Thiery JP, Acloque H, Huang RY and Nieto MA: Epithelial-mesenchymal transitions in development and disease. Cell. 139:871–890. 2009. View Article : Google Scholar : PubMed/NCBI
3	Hou M, Cheng Z, Shen H, He S, Li Y, Pan Y, Feng C, Chen X, Zhang Y, Lin M, et al: High expression of CTHRC1 promotes EMT of epithelial ovarian cancer (EOC) and is associated with poor prognosis. Oncotarget. 6:35813–35829. 2015.PubMed/NCBI
4	Micalizzi DS, Farabaugh SM and Ford HL: Epithelial-mesenchymal transition in cancer: Parallels between normal development and tumor progression. J Mammary Gland Biol Neoplasia. 15:117–134. 2010. View Article : Google Scholar : PubMed/NCBI
5	Ma L, Teruya-Feldstein J and Weinberg RA: Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature. 449:682–688. 2007. View Article : Google Scholar : PubMed/NCBI
6	Iwatsuki M, Mimori K, Yokobori T, Ishi H, Beppu T, Nakamori S, Baba H and Mori M: Epithelial-mesenchymal transition in cancer development and its clinical significance. Cancer Sci. 101:293–299. 2010. View Article : Google Scholar
7	Kim YJ, Choi WI, Jeon BN, Choi KC, Kim K, Kim TJ, Ham J, Jang HJ, Kang KS and Ko H: Stereospecific effects of ginsenoside 20-Rg3 inhibits TGF-β1-induced epithelial-mesenchymal transition and suppresses lung cancer migration, invasion and anoikis resistance. Toxicology. 322:23–33. 2014. View Article : Google Scholar : PubMed/NCBI
8	Cichon MA and Radisky DC: ROS-induced epithelial-mesenchymal transition in mammary epithelial cells is mediated by NF-κB-dependent activation of Snail. Oncotarget. 5:2827–2838. 2014. View Article : Google Scholar : PubMed/NCBI
9	Kim J, Moon SH, Kim BT, Chae CH, Lee JY and Kim SH: A novel aminothiazole KY-05009 with potential to inhibit Traf2-and Nck-interacting kinase (TNIK) attenuates TGF-β1-mediated epithelial-to-mesenchymal transition in human lung adenocarcinoma A549 cells. PLoS One. 9:e1101802014. View Article : Google Scholar
10	Fong YC, Hsu SF, Wu CL, Li TM, Kao ST, Tsai FJ, Chen WC, Liu SC, Wu CM and Tang CH: Transforming growth factor-beta1 increases cell migration and beta1 integrin up-regulation in human lung cancer cells. Lung Cancer. 64:13–21. 2009. View Article : Google Scholar
11	Ko H: Geraniin inhibits TGF-β1-induced epithelial-mesenchymal transition and suppresses A549 lung cancer migration, invasion and anoikis resistance. Bioorg Med Chem Lett. 25:3529–3534. 2015. View Article : Google Scholar : PubMed/NCBI
12	Nogata Y, Sakamoto K, Shiratsuchi H, Ishii T, Yano M and Ohta H: Flavonoid composition of fruit tissues of citrus species. Biosci Biotechnol Biochem. 70:178–192. 2006. View Article : Google Scholar : PubMed/NCBI
13	Lee YC, Cheng TH, Lee JS, Chen JH, Liao YC, Fong Y, Wu CH and Shih YW: Nobiletin, a citrus flavonoid, suppresses invasion and migration involving FAK/PI3K/Akt and small GTPase signals in human gastric adenocarcinoma AGS cells. Mol Cell Biochem. 347:103–115. 2011. View Article : Google Scholar
14	Baek SH, Kim SM, Nam D, Lee JH and Ahn KS, Choi SH, Kim SH, Shim BS, Chang IM and Ahn KS: Antimetastatic effect of nobiletin through the down-regulation of CXC chemokine receptor type 4 and matrix metallopeptidase-9. Pharm Biol. 50:1210–1218. 2012. View Article : Google Scholar : PubMed/NCBI
15	Gao XJ, Liu JW, Zhang QG, Zhang JJ, Xu HT and Liu HJ: Nobiletin inhibited hypoxia-induced epithelial-mesenchymal transition of lung cancer cells by inactivating of Notch-1 signaling and switching on miR-200b. Pharmazie. 70:256–262. 2015.PubMed/NCBI
16	Song J, Shu L, Zhang Z, Tan X, Sun E, Jin X, Chen Y and Jia X: Reactive oxygen species-mediated mitochondrial pathway is involved in Baohuoside I-induced apoptosis in human non-small cell lung cancer. Chem Biol Interact. 199:9–17. 2012. View Article : Google Scholar : PubMed/NCBI
17	Yang M, Xie X and Ding Y: SALL4 is a marker of poor prognosis in serous ovarian carcinoma promoting invasion and metastasis. Oncol Rep. 35:1796–1806. 2016.PubMed/NCBI
18	Smith AL, Robin TP and Ford HL: Molecular pathways: Targeting the TGF-β pathway for cancer therapy. Clin Cancer Res. 18:4514–4521. 2012. View Article : Google Scholar : PubMed/NCBI
19	Moustakas A and Heldin CH: Signaling networks guiding epithelial-mesenchymal transitions during embryogenesis and cancer progression. Cancer Sci. 98:1512–1520. 2007. View Article : Google Scholar : PubMed/NCBI
20	Puisieux A, Brabletz T and Caramel J: Oncogenic roles of EMT-inducing transcription factors. Nat Cell Biol. 16:488–494. 2014. View Article : Google Scholar : PubMed/NCBI
21	Shi Y, Wu H, Zhang M, Ding L, Meng F and Fan X: Expression of the epithelial-mesenchymal transition-related proteins and their clinical significance in lung adenocarcinoma. Diagn Pathol. 8:892013. View Article : Google Scholar : PubMed/NCBI
22	Bolós V, Peinado H, Pérez-Moreno MA, Fraga MF, Esteller M and Cano A: The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: A comparison with Snail and E47 repressors. J Cell Sci. 116:499–511. 2003. View Article : Google Scholar : PubMed/NCBI
23	Kim J and Hwan Kim S: CK2 inhibitor CX-4945 blocks TGF-β1-induced epithelial-to-mesenchymal transition in A549 human lung adenocarcinoma cells. PLoS One. 8:e743422013. View Article : Google Scholar
24	Hoot KE, Lighthall J, Han G, Lu SL, Li A, Ju W, Kulesz-Martin M, Bottinger E and Wang XJ: Keratinocyte-specific Smad2 ablation results in increased epithelial-mesenchymal transition during skin cancer formation and progression. J Clin Invest. 118:2722–2732. 2008.PubMed/NCBI
25	Chien SY, Hsieh MJ, Chen CJ, Yang SF and Chen MK: Nobiletin inhibits invasion and migration of human nasopharyngeal carcinoma cell lines by involving ERK1/2 and transcriptional inhibition of MMP-2. Expert Opin Ther Targets. 19:307–320. 2015. View Article : Google Scholar : PubMed/NCBI
26	Sato T, Koike L, Miyata Y, Hirata M, Mimaki Y, Sashida Y, Yano M and Ito A: Inhibition of activator protein-1 binding activity and phosphatidylinositol 3-kinase pathway by nobiletin, a polymethoxy flavonoid, results in augmentation of tissue inhibitor of metalloproteinases-1 production and suppression of production of matrix metalloproteinases-1 and -9 in human fibrosarcoma HT-1080 cells. Cancer Res. 62:1025–1029. 2002.PubMed/NCBI
27	De Craene B and Berx G: Regulatory networks defining EMT during cancer initiation and progression. Nat Rev Cancer. 13:97–110. 2013. View Article : Google Scholar : PubMed/NCBI
28	Su BH, Tseng YL, Shieh GS, Chen YC, Wu P, Shiau AL and Wu C: Overexpression of prothymosin α antagonizes TGF-β signalling to promote the development of emphysema. J Pathol. 238:412–422. 2015. View Article : Google Scholar
29	Dayer C and Stamenkovic I: Recruitment of matrix metal-loproteinase-9 (MMP-9) to the fibroblast cell surface by lysyl hydroxylase 3 (LH3) triggers transforming growth factor-β (TGF-β) activation and fibroblast differentiation. J Biol Chem. 290:13763–13778. 2015. View Article : Google Scholar : PubMed/NCBI
30	Cicchini C, Laudadio I, Citarella F, Corazzari M, Steindler C, Conigliaro A, Fantoni A, Amicone L and Tripodi M: TGFbeta-induced EMT requires focal adhesion kinase (FAK) signaling. Exp Cell Res. 314:143–152. 2008. View Article : Google Scholar
31	Baquero P, Jiménez-Mora E, Santos A, Lasa M and Chiloeches A: TGFβ induces epithelial-mesenchymal transition of thyroid cancer cells by both the BRAF/MEK/ERK and Src/FAK pathways. Mol Carcinog. Sep 21–2015.Epub ahead of print. View Article : Google Scholar
32	Guarino M: Src signaling in cancer invasion. J Cell Physiol. 223:14–26. 2010.PubMed/NCBI
33	Frame MC and Inman GJ: NCAM is at the heart of reciprocal regulation of E-cadherin- and integrin-mediated adhesions via signaling modulation. Dev Cell. 15:494–496. 2008. View Article : Google Scholar : PubMed/NCBI
34	Jagadeeswaran R, Surawska H, Krishnaswamy S, Janamanchi V, Mackinnon AC, Seiwert TY, Loganathan S, Kanteti R, Reichman T, Nallasura V, et al: Paxillin is a target for somatic mutations in lung cancer: Implications for cell growth and invasion. Cancer Res. 68:132–142. 2008. View Article : Google Scholar : PubMed/NCBI
35	Nagathihalli NS and Merchant NB: Src-mediated regulation of E-cadherin and EMT in pancreatic cancer. Front Biosci. 17:2059–2069. 2012. View Article : Google Scholar
36	Wilson C, Nicholes K, Bustos D, Lin E, Song Q, Stephan JP, Kirkpatrick DS and Settleman J: Overcoming EMT-associated resistance to anti-cancer drugs via Src/FAK pathway inhibition. Oncotarget. 5:7328–7341. 2014. View Article : Google Scholar : PubMed/NCBI