Cyanidin-3-O-glucoside inhibits epithelial-to-mesenchymal transition, and migration and invasion of breast cancer cells by upregulating KLF4

  • Dahu Chen Shandong University of Technology
  • Mei Yuan
  • Qin Ye
  • Xing Wang
  • Jing Xu
  • Guangyi Shi
  • Zhaodi Hu
Keywords: anthocyanin, cell migration, cell invasion, KLF4, FBXO32

Abstract

Background: Anthocyanins (ACNs) are capable of suppressing breast cancer growth; however, investigation on the effect and mechanism of ACNs on epithelial-to-mesenchymal transition (EMT), and cell migration and invasion in breast cancer cells is limited. A complete understanding of those properties may provide useful information on of how to use these natural compounds for cancer prevention and treatment.

Objectives: The aim of this work was to investigate the role of cyanidin-3-O-glucoside (Cy3G), one of the most widely distributed ACNs in edible fruits, in the EMT process, and cell migration and invasion of breast cancer cells, and its underlying molecular mechanisms of how Cy3G establishes these functional roles in these cells.

Methods: MDA-MB-231 and MDA-MB-468 breast cancer cells were treated with Cy3G (20 μM) for 24 h, and then the cells were used for cell migration and invasion assay. Western blotting, luciferase assay, ubiquitination assay, gene knockdown, and cycloheximide chase assay were performed to analyze the molecular mechanisms of Cy3G in suppressing EMT, and cell migration and invasion.

Results: Cy3G inhibited the EMT process in these cells and significantly suppressed the migration and invasion of breast cancer cells (P ≤ 0.05) by upregulating Krüppel-like factor 4 (KLF4) expression at protein level. KLF4 knockdown in MDA-MB-231 cells did not reveal any change in EMT marker expression, and cell migration and invasion upon treatment with Cy3G (P ≥ 0.05), which strongly indicated that the effects of Cy3G were mediated by KLF4. Furthermore, we determined that Cy3G indirectly upregulated KLF4 expression by downregulating FBXO32, which is the E3 ligase of KLF4.

Conclusion: Cy3G is a potential anticancer reagent as it can inhibit EMT and breast cancer cell migration and invasion by upregulating KLF4.

Downloads

Download data is not yet available.

References


  1. Matamala N, Vargas MT, Gonzalez-Campora R, Minambres R, Arias JI, Menendez P, et al. Tumor microRNA expression profiling identifies circulating microRNAs for early breast cancer detection. Clin Chem 2015; 61(8): 1098–106. doi: 10.1373/clinchem.2015.238691

  2. Weigelt B, Peterse JL, van’t Veer LJ. Breast cancer metastasis: markers and models. Nat Rev Cancer 2005; 5(8): 591–602. doi: 10.1038/nrc1670

  3. Gallo S, Sangiolo D, Carnevale Schianca F, Aglietta M, Montemurro F. Treating breast cancer with cell-based approaches: an overview. Expert Opin Biol Ther 2017; 17(10): 1255–64. doi: 10.1080/14712598.2017.1356816

  4. Chaffer CL, San Juan BP, Lim E, Weinberg RA. EMT, cell plasticity and metastasis. Cancer Metastasis Rev 2016; 35(4): 645–54. doi: 10.1007/s10555-016-9648-7

  5. Lambert AW, Pattabiraman DR, Weinberg RA. Emerging biological principles of metastasis. Cell 2017; 168(4): 670–91. doi: 10.1016/j.cell.2016.11.037

  6. Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2002; 2(6): 442–54. doi: 10.1038/nrc822

  7. Yang J, Weinberg RA. Epithelial-mesenchymal transition: at the crossroads of development and tumor. Dev Cell 2008; 14(6): 818–29. doi: 10.1016/j.devcel.2008.05.009

  8. Scheel C, Onder T, Karnoub A, Weinberg RA. Adaptation versus selection: the origins of metastatic behavior. Cancer Res 2007; 67(24): 11476–9. doi: 10.1158/0008-5472.CAN-07-1653

  9. Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 2008; 10(5): 593–601. doi: 10.1038/ncb1722

  10. Wellner U, Schubert J, Burk UC, Schmalhofer O, Zhu F, Sonntag A, et al. The EMT-activator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs. Nat Cell Biol 2009; 11(12): 1487–95. doi: 10.1038/ncb1998

  11. Shimono Y, Zabala M, Cho RW, Lobo N, Dalerba P, Qian D, et al. Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells. Cell 2009; 138(3): 592–603. doi: 10.1016/j.cell.2009.07.011

  12. Ma L, Young J, Prabhala H, Pan E, Mestdagh P, Muth D, et al. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis. Nat Cell Biol 2010; 12(3): 247–56. doi: 10.1038/ncb2024

  13. Song X, Xing YM, Wu W, Cheng GH, Xiao F, Jin G, et al. Expression of Kruppel-like factor 4 in breast cancer tissues and its effects on the proliferation of breast cancer MDA-MB-231 cells. Exp Ther Med 2017; 13(5): 2463–67. doi: 10.3892/etm.2017.4262

  14. Rowland BD, Peeper DS. KLF4, p21 and context-dependent opposing forces in cancer. Nat Rev Cancer 2006; 6(1): 11–23. doi: 10.1038/nrc1780

  15. Tiwari N, Meyer-Schaller N, Arnold P, Antoniadis H, Pachkov M, van Nimwegen E, et al. Klf4 is a transcriptional regulator of genes critical for EMT, including Jnk1 (Mapk8). PLoS One 2013; 8(2): e57329. doi: 10.1371/journal.pone.0057329

  16. Yori JL, Johnson E, Zhou G, Jain MK, Keri RA. Kruppel-like factor 4 inhibits epithelial-to-mesenchymal transition through regulation of E-cadherin expression. J Biol Chem 2010; 285(22): 16854–63. doi: 10.1074/jbc.M110.114546

  17. Yori JL, Seachrist DD, Johnson E, Lozada KL, Abdul-Karim FW, Chodosh LA, et al. Kruppel-like factor 4 inhibits tumorigenic progression and metastasis in a mouse. Neoplasia 2011; 13(7): 601–10. doi: 10.1593/neo.11260

  18. Wu X, Beecher GR, Holden JM, Haytowitz DB, Gebhardt SE, Prior RL. Concentrations of anthocyanins in common foods in the United States and estimation of normal consumption. J Agric Food Chem 2006; 54(11): 4069–75. doi: 10.1021/jf060300l

  19. Higgins JA, Zainol M, Brown K, Jones GD. Anthocyans as tertiary chemopreventive agents in bladder cancer: anti-oxidant mechanisms and interaction with mitomycin C. Mutagenesis 2014; 29(4): 227–35. doi: 10.1093/mutage/geu009.

  20. Chen PN, Chu SC, Chiou HL, Chiang CL, Yang SF, Hsieh YS. Cyanidin 3-glucoside and peonidin 3-glucoside inhibit tumor cell growth and induce apoptosis in vitro and suppress tumor growth in vivo. Nutr Cancer 2005; 53(2): 232–43. doi: 10.1207/s15327914nc5302_12

  21. Zhang H, Yousef H, Renaud J, Liu R, Yang C, Sun Y, et al. Bioaccessibility, bioavailability and anti-inflammatory effects of anthocyanins from purple root vegetable using mono and coculture cell models. Mol Nutr Food Res 2017; 61(10). doi: 10.1002/mnfr.201600928

  22. Wu X, Prior RL. Systematic identification and characterization of anthocyanins by HPLC-ESI-MS/MS. In common foods in the United States: fruits and berries. J Agric Food Chem 2005; 53(7): 2589–99. doi: 10.1021/jf048068b

  23. Serra D, Paixao J, Nunes C, Dinis TCP, Almeida LM. Cyanidin-3-glucoside suppresses cytokine-induced inflammatory response in human intestinal cells: comparison with 5aminosaliccylic acid. PLoS One 2013; 8(9): e73001. doi: 10.1371/journal.pone.0073001

  24. Xu M, Bower KA, Wang S, Frank JA, Chen G, Ding M, et al. Cyanidin-3-glucoside inhibits ethanol-induced invasion of breast cancer cells overexpressing ErbB2. Mol Cancer 2010; 9: 285. doi: 10.1186/1476-4598-9-285

  25. Wang L, Li H, Yang S, Ma W, Liu M, Guo S, et al. Cyanidin-3-o-glucoside directly binds to ERalpha36 and inhibits EGFR-positive triple negative breast cancer. Oncotarget 2016; 7(42): 68864–82. doi: 10.18632/oncotarget.12025

  26. Liu M, Du Y, Li H, Wang L, Ponikwicka-Tyszko D, Lebiedzinska W, et al. Cyanidin-3-o-glucoside pharmacologically inhibits tumorigenesis via estrogen receptor β in melanoma mice. Front Oncol 2019; 9: 1110. doi: 10.3389/fonc.2019.01110

  27. Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS, et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 2003; 9(4): 493–501. doi: 10.1261/rna.2192803

  28. Ma L, Teruya-Feldstein J, Weinberg RA. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 2007; 449(7163): 682–8. doi: 10.1038/nature06174

  29. Khoo HE, Azlan A, Tang ST, Lim SM. Anthocyanidins and anthocyanins: colored pigments as food, pharmaceutical ingredients, and the potential health benefits. Food Nutr Res 2017; 61(1): 1361779. doi: 10.1080/16546628.2017.1361779

  30. Kristo AS, Klimis-Zacas D, Sikalidis AK. Protective role of dietary berries in cancer. Antioxidants (Basel) 2016; 5(4): E37. doi: 10.3390/antiox5040037

  31. Grimes KL, Stuart CM, McCarthy JJ, Kaur B, Cantu EJ, Forester SC. Enhancing the cancer cell growth inhibitory effects of table grape anthocyanins. J Food Sci 2018; 83(9): 2369–74. doi: 10.1111/1750-3841.14294

  32. Kimble R, Keane KM, Lodge JK, Howatson G. Dietary intake of anthocyanins and risk of cardiovascular disease: a systematic review and meta-analysis of prospective cohort studies. Crit Rev Food Sci Nutr 2019; 59(18): 3032–3043. doi: 10.1080/10408398.2018.1509835

  33. Liobikas J, Skemiene K, Trumbeckaite S, Borutaite V. Anthocyanins in cardioprotection: a path through mitochondria. Pharmacol Res 2016; 113(Pt. B): 808–15. doi: 10.1016/j.phrs.2016.03.036

  34. Wang Y, Lin J, Tian J, Si X, Jiao X, Zhang W, et al. Blueberry Malvidin-3-galactoside suppresses hepatocellular carcinoma by regulating apoptosis, proliferation, and metastasis pathways in vivo and in vitro. J Agric Food Chem 2019; 67(2): 625–36. doi: 10.1021/acs.jafc.8b06209

  35. Han B, Peng X, Cheng D, Zhu Y, Du J, Li J, et al. Delphinidin suppresses breast carcinogenesis through the HOTAIR/microRNA-34a axis. Cancer Sci 2019; 110(10): 3089–97. doi: 10.1111/cas.14133

  36. Diaconeasa Z, Ayvaz H, Ruginǎ D, Leopold L, Stǎnilǎ A, Socaciu C, et al. Melanoma inhibition by anthocyanins is associated with the reduction of oxidative stress biomarkers and changes in mitochondrial membrane potential. Plant Foods Hum Nutr 2017; 72(4): 404–10. doi: 10.1007/s11130-017-0638-x

  37. Liu W, Xu J, Liu Y, Yu X, Tang X, Wang Z, et al. Anthocyanins potentiate the activity of trastuzumab in human epidermal growth factor receptor 2-positive breast cancer cells in vitro and in vivo. Mol Med Rep 2014; 10(4): 1921–26. doi: 10.3892/mmr.2014.2414

  38. Sousa A, Araújo P, Azevedo J, Cruz L, Fernandes I, Mateus N, et al. Antioxidant and antiproliferative properties of 3deoxyanthocyanidins. Food Chem 2016; 192: 142–8. doi: 10.1016/j.foodchem.2015.06.108

  39. Pacheco SM, Soares MSP, Gutierres JM, Gerzson MFB, Carvalho FB, Azambuja JH, et al. Anthocyanins as a potential pharmacological agent to manage memory deficit, oxidative stress and alterations in ion pump activity induced by experimental sporadic dementia of Alzheimer’s type. J Nutr Biochem 2018; 56: 193–204. doi: 10.1016/j.jnutbio.2018.02.014

  40. Min J, Yu SW, Baek SH, Nair KM, Bae ON, Bhatt A, et al. Neuroprotective effect of cyanidin-3-O-glucoside anthocyanin in mice with focal cerebral ischemia. Neurosci Lett 2011; 500(3): 157–61. doi: 10.1016/j.neulet.2011.05.048

  41. Qin Y, Zhai Q, Li Y, Cao M, Xu Y, Zhao K, et al. Cyanidin-3-O-glucoside ameliorates diabetic nephropathy through regulation of glutathione pool. Biomed Pharmacother 2018; 103: 1223–30. doi: 10.1016/j.biopha.2018.04.137

  42. Ma B, Wu Y, Chen B, Yao Y, Wang Y, Bai H, et al. Cyanidin-3-O-β-glucoside attenuates allergic airway inflammation by modulating the IL-4Rα-STAT6 signaling pathway in a murine asthma model. Int Immunopharmacol 2019; 69: 1–10. doi: 10.1016/j.intimp.2019.01.008

  43. Bhaswant M, Fanning K, Netzel M, Mathai ML, Panchal SK, Brown L. Cyanidin 3-glucoside improves diet-induced metabolic syndrome in rats. Pharmacol Res 2015; 102: 208–217. doi: 10.1016/j.phrs.2015.10.006

  44. Adams LS, Kanaya N, Phung S, Liu Z, Chen S. Whole blueberry powder modulates the growth and metastasis of MDA-MB-231 triple negative breast tumor in nude mice. J Nutr 2011; 141(10): 1805–12. doi: 10.3945/jn.111.140178

  45. Zhou H, Liu Y, Zhu R, Ding F, Wan Y, Li Y, et al. FBXO32 suppresses breast cancer tumorigenesis through targeting KLF4 to proteasomal degradation. Oncogene 2017; 36(23): 3312–21. doi: 10.1038/onc.2016.479

  46. Celià-Terrassa T, Meca-Cortés O, Mateo F, Martínez de Paz A, Rubio N, Arnal-Estapé A, et al. Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells. J Clin Invest 2012; 122(5): 1849–68. doi: 10.1172/JCI59218

  47. Chen D, Sun Y, Yuan Y, Han Z, Zhang P, Zhang J, et al. miR-100 induces epithelial-mesenchymal transition but suppresses tumorigenesis, migration and invasion. PLoS Genet 2014; 10(2): e1004177. doi: 10.1371/journal.pgen.1004177

Published
2020-09-28
How to Cite
Chen, D., Yuan, M., Ye, Q., Wang, X., Xu, J., Shi, G., & Hu, Z. (2020). Cyanidin-3-O-glucoside inhibits epithelial-to-mesenchymal transition, and migration and invasion of breast cancer cells by upregulating KLF4. Food & Nutrition Research, 64. https://doi.org/10.29219/fnr.v64.4240
Section
Original Articles