All-trans retinoic acid inhibits proliferation, migration, invasion and induces differentiation of hepa1-6 cells through reversing EMT in vitro

Cui,Jiejie; Gong,Mengjia; He,Yun; Li,Qilin; He,Tongchuan; Bi,Yang

doi:10.3892/ijo.2015.3235

All-trans retinoic acid inhibits proliferation, migration, invasion and induces differentiation of hepa1-6 cells through reversing EMT in vitro

Authors:
- Jiejie Cui
- Mengjia Gong
- Yun He
- Qilin Li
- Tongchuan He
- Yang Bi
View Affiliations

Affiliations: Department of Pediatric Surgery, The Children's Hospital of Chongqing Medical University, Chongqing 400014, P.R. China, Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Stem Cell Therapy Engineering Technical Center, The Children's Hospital of Chongqing Medical University, Chongqing 400014, P.R. China, Department of Ultrasound, The Third People's Hospital of Chongqing, Chongqing 400014, P.R. China
Published online on: November 6, 2015 https://doi.org/10.3892/ijo.2015.3235
Pages: 349-357

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Hepatocellular carcinoma (HCC) has the characristics of tumor invasiveness, frequent intrahepatic spread and extra hepatic metastases, which affects the therapy efficiency and prognosis. Epithelial-mesenchymal transition (EMT) is now recognized as a key process in tumor invasion, metastasis and the generation of cancer initiating cells. All-trans retinoic acid (ATRA) is currently used as a potential chemo-therapeutic or chemo-preventive agent because of its anti-proliferative, pro-apoptotic and antioxidant properties. This study investigated the effects of ATRA at different concentrations on the proliferation, migration, invasion, differentiation and functions of the mouse hepa1-6 hepatocarcinoma cell line and explored whether ATRA regulates EMT in the antitumor process. Trypan blue staining and colony formation assay were used to detect cell proliferation. Wound-healing assay and Transwell Matrigel assay were performed to examine migration. Invasion was assessed by using Transwell invasion assay. In the present study, ATRA significantly inhibited the cell growth, colony formation, migration, and invasion capability of hepa1-6 cells in a dose-dependent manner. Furthermore, ATRA at low concentration (0.1 µmol/l) could generate these influences. After treated in the ATRA medium, the expression of mature hepatic markers ALB (albumin), CK18 (cytokeratin 18), TAT (tyrosine aminotransferase), ApoB (apolipoprotein B) decreased and that of hepatocarcinoma marker AFP (α fetoprotein) increased. At day 7 after ATRA induction, hepa1-6 cells showed comparable indocyanine green (ICG) uptake and glycogen storage function to the blank control. The mRNA expression of mesenchymal markers N-cadherin, vimentin, snail and twist decreased, while expression of epithelial marker E-cadherin increased in hepa1-6 cells after treated with ATRA. Therefore, this study demonstrates that ATRA remarkably suppressed the proliferation, migration, invasion of hepa1-6 hepatocarcinoma cell line and effectively induced its differentiation and liver functions in vitro through the reversal of EMT. HCC may be more sensitive to ATRA than other cancers, suggesting the prospective usefulness of ATRA in the treatment of HCC.

View Figures

View References

1	Forner A, Llovet JM and Bruix J: Hepatocellular carcinoma. Lancet. 379:1245–1255. 2012. View Article : Google Scholar : PubMed/NCBI
2	Llovet JM and Bruix J: Molecular targeted therapies in hepatocellular carcinoma. Hepatology. 48:1312–1327. 2008. View Article : Google Scholar : PubMed/NCBI
3	Bogachek MV, De Andrade JP and Weigel RJ: Regulation of epithelial-mesenchymal transition through SUMOylation of transcription factors. Cancer Res. 75:11–15. 2015. View Article : Google Scholar
4	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
5	Tsai JH and Yang J: Epithelial-mesenchymal plasticity in carcinoma metastasis. Genes Dev. 27:2192–2206. 2013. View Article : Google Scholar : PubMed/NCBI
6	Trimboli AJ, Fukino K, de Bruin A, Wei G, Shen L, Tanner SM, Creasap N, Rosol TJ, Robinson ML, Eng C, et al: Direct evidence for epithelial-mesenchymal transitions in breast cancer. Cancer Res. 68:937–945. 2008. View Article : Google Scholar : PubMed/NCBI
7	Ross SA, McCaffery PJ, Drager UC and De Luca LM: Retinoids in embryonal development. Physiol Rev. 80:1021–1054. 2000.PubMed/NCBI
8	Feldman DR, Patil S, Trinos MJ, Carousso M, Ginsberg MS, Sheinfeld J, Bajorin DF, Bosl GJ and Motzer RJ: Progression-free and overall survival in patients with relapsed/refractory germ cell tumors treated with single-agent chemotherapy: Endpoints for clinical trial design. Cancer. 118:981–986. 2012. View Article : Google Scholar
9	Li M, Sun Y, Guan X, Shu X and Li C: Advanced progress on the relationship between RA and its receptors and malignant tumors. Crit Rev Oncol Hematol. 91:271–282. 2014. View Article : Google Scholar : PubMed/NCBI
10	Woo YJ and Jang KL: All-trans retinoic acid activates E-cadherin expression via promoter hypomethylation in the human colon carcinoma HCT116 cells. Biochem Biophys Res Commun. 425:944–949. 2012. View Article : Google Scholar : PubMed/NCBI
11	Zanetti A, Affatato R, Centritto F, Fratelli M, Kurosaki M, Barzago MM, Bolis M, Terao M, Garattini E and Paroni G: All-trans-retinoic acid modulates the plasticity and inhibits the motility of breast cancer cells: Role of NOTCH1 and transforming growth factor (TGFβ). J Biol Chem. 290:17690–17709. 2015. View Article : Google Scholar : PubMed/NCBI
12	Chansri N, Kawakami S, Yamashita F and Hashida M: Inhibition of liver metastasis by all-trans retinoic acid incorporated into O/W emulsions in mice. Int J Pharm. 321:42–49. 2006. View Article : Google Scholar : PubMed/NCBI
13	Zhang Y, Guan DX, Shi J, Gao H, Li JJ, Zhao JS, Qiu L, Liu J, Li N, Guo WX, et al: All-trans retinoic acid potentiates the chemotherapeutic effect of cisplatin by inducing differentiation of tumor initiating cells in liver cancer. J Hepatol. 59:1255–1263. 2013. View Article : Google Scholar : PubMed/NCBI
14	Rodriguez LG, Wu X and Guan JL: Wound-healing assay. Methods Mol Biol. 294:23–29. 2005.
15	Bi Y, Huang J, He Y, Zhu GH, Su Y, He BC, Luo J, Wang Y, Kang Q, Luo Q, et al: Wnt antagonist SFRP3 inhibits the differentiation of mouse hepatic progenitor cells. J Cell Biochem. 108:295–303. 2009. View Article : Google Scholar : PubMed/NCBI
16	Yang MH, Chen CL, Chau GY, Chiou SH, Su CW, Chou TY, Peng WL and Wu JC: Comprehensive analysis of the independent effect of twist and snail in promoting metastasis of hepatocellular carcinoma. Hepatology. 50:1464–1474. 2009. View Article : Google Scholar : PubMed/NCBI
17	Mima K, Hayashi H, Kuroki H, Nakagawa S, Okabe H, Chikamoto A, Watanabe M, Beppu T and Baba H: Epithelial-mesenchymal transition expression profiles as a prognostic factor for disease-free survival in hepatocellular carcinoma: Clinical significance of transforming growth factor-β signaling. Oncol Lett. 5:149–154. 2013.
18	Yamada T, Yoshikawa M, Kanda S, Kato Y, Nakajima Y, Ishizaka S and Tsunoda Y: In vitro differentiation of embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green. Stem Cells. 20:146–154. 2002. View Article : Google Scholar : PubMed/NCBI
19	Kamo N, Yasuchika K, Fujii H, Hoppo T, Machimoto T, Ishii T, Fujita N, Tsuruo T, Yamashita JK, Kubo H, et al: Two populations of Thy1-positive mesenchymal cells regulate in vitro maturation of hepatic progenitor cells. Am J Physiol Gastrointest Liver Physiol. 292:G526–G534. 2007. View Article : Google Scholar
20	Zeisberg M and Neilson EG: Biomarkers for epithelial-mesenchymal transitions. J Clin Invest. 119:1429–1437. 2009. View Article : Google Scholar : PubMed/NCBI
21	Mongan NP and Gudas LJ: Diverse actions of retinoid receptors in cancer prevention and treatment. Differentiation. 75:853–870. 2007. View Article : Google Scholar : PubMed/NCBI
22	Connolly RM, Nguyen NK and Sukumar S: Molecular pathways: Current role and future directions of the retinoic acid pathway in cancer prevention and treatment. Clin Cancer Res. 19:1651–1659. 2013. View Article : Google Scholar : PubMed/NCBI
23	Lo-Coco F, Ammatuna E, Montesinos P and Sanz MA: Acute promyelocytic leukemia: Recent advances in diagnosis and management. Semin Oncol. 35:401–409. 2008. View Article : Google Scholar : PubMed/NCBI
24	Arisi MF, Starker RA, Addya S, Huang Y and Fernandez SV: All trans-retinoic acid (ATRA) induces re-differentiation of early transformed breast epithelial cells. Int J Oncol. 44:1831–1842. 2014.PubMed/NCBI
25	de Almeida Vasconcelos Fonseca EM, Chagas CE, Mazzantini RP, Heidor R, Ong TP and Moreno FS: All-trans and 9-cis retinoic acids, retinol and beta-carotene chemopreventive activities during the initial phases of hepatocarcinogenesis involve distinct actions on glutathione S-transferase positive preneoplastic lesions remodeling and DNA damage. Carcinogenesis. 26:1940–1946. 2005. View Article : Google Scholar : PubMed/NCBI
26	Song K, Li W, Wang H, Wang H, Liu T, Ning R and Wang L: Investigation of coculture of human adipose-derived stem cells and mature adipocytes. Appl Biochem Biotechnol. 167:2381–2387. 2012. View Article : Google Scholar : PubMed/NCBI
27	Zanatta G, Steffens D, Braghirolli DI, Fernandes RA, Netto CA and Pranke P: Viability of mesenchymal stem cells during electrospinning. Braz J Med Biol Res. 45:125–130. 2012. View Article : Google Scholar
28	Fan TT, Cheng Y, Wang YF, Gui SY, Chen FH, Zhou Q and Wang Y: A novel all-trans retinoid acid derivative N-(3-trifluoromethyl-phenyl)-retinamide inhibits lung adenocarcinoma A549 cell migration through down-regulating expression of myosin light chain kinase. Asian Pac J Cancer Prev. 15:7687–7692. 2014. View Article : Google Scholar
29	Zhou RJ, Liao WG, Yang ZZ, Min J and Xiao Y: Effects and mechanisms of ATRA on proliferation, cell cycle of lung carcinoma cellline A549. Acta Academiae Medicinae Militaris Tertiae. 14:1399–1401. 2007.
30	Nitto T and Sawaki K: Molecular mechanisms of the antileukemia activities of retinoid and arsenic. J Pharmacol Sci. 126:179–185. 2014. View Article : Google Scholar : PubMed/NCBI
31	Okuno M, Kojima S, Matsushima-Nishiwaki R, Tsurumi H, Muto Y, Friedman SL and Moriwaki H: Retinoids in cancer chemoprevention. Curr Cancer Drug Targets. 4:285–298. 2004. View Article : Google Scholar : PubMed/NCBI
32	Tania M, Khan MA and Fu J: Epithelial to mesenchymal transition inducing transcription factors and metastatic cancer. Tumour Biol. 35:7335–7342. 2014. View Article : Google Scholar : PubMed/NCBI
33	Liu Z, Jin ZY, Liu CH, Xie F, Lin XS and Huang Q: MicroRNA-21 regulates biological behavior by inducing EMT in human cholangiocarcinoma. Int J Clin Exp Pathol. 8:4684–4694. 2015.PubMed/NCBI
34	Ye Z, Zhou M, Tian B, Wu B and Li J: Expression of lncRNA-CCAT1, E-cadherin and N-cadherin in colorectal cancer and its clinical significance. Int J Clin Exp Med. 8:3707–3715. 2015.PubMed/NCBI
35	Zhou J, Tao D, Xu Q, Gao Z and Tang D: Expression of E-cadherin and vimentin in oral squamous cell carcinoma. Int J Clin Exp Pathol. 8:3150–3154. 2015.PubMed/NCBI
36	Ting HJ, Deep G, Jain AK, Cimic A, Sirintrapun J, Romero LM, Cramer SD, Agarwal C and Agarwal R: Silibinin prevents prostate cancer cell-mediated differentiation of naive fibroblasts into cancer-associated fibroblast phenotype by targeting TGF beta2. Mol Carcinog. 54:730–741. 2015. View Article : Google Scholar
37	Thaiparambil JT, Bender L, Ganesh T, Kline E, Patel P, Liu Y, Tighiouart M, Vertino PM, Harvey RD, Garcia A, et al: Withaferin A inhibits breast cancer invasion and metastasis at sub-cytotoxic doses by inducing vimentin disassembly and serine 56 phosphorylation. Int J Cancer. 129:2744–2755. 2011. View Article : Google Scholar : PubMed/NCBI
38	Lee J, Hahm ER, Marcus AI and Singh SV: Withaferin A inhibits experimental epithelial-mesenchymal transition in MCF-10A cells and suppresses vimentin protein level in vivo in breast tumors. Mol Carcinog. 54:417–429. 2015. View Article : Google Scholar
39	Tian Q, Xue Y, Zheng W, Sun R, Ji W, Wang X and An R: Overexpression of hypoxia-inducible factor 1α induces migration and invasion through Notch signaling. Int J Oncol. 47:728–738. 2015.PubMed/NCBI
40	Wang YP, Yu GR, Lee MJ, Lee SY, Chu IS, Leem SH and Kim DG: Lipocalin-2 negatively modulates the epithelial-to-mesenchymal transition in hepatocellular carcinoma through the epidermal growth factor (TGF-beta1)/Lcn2/Twist1 pathway. Hepatology. 58:1349–1361. 2013. View Article : Google Scholar : PubMed/NCBI