Effect of Melatonin in Epithelial Mesenchymal Transition Markers and Invasive Properties of Breast Cancer Stem Cells of Canine and Human Cell Lines

Cancer stem cells (CSCs) have been associated with metastasis and therapeutic resistance and can be generated via epithelial mesenchymal transition (EMT). Some studies suggest that the hormone melatonin acts in CSCs and may participate in the inhibition of the EMT. The objectives of this study were to evaluate the formation of mammospheres from the canine and human breast cancer cell lines, CMT-U229 and MCF-7, and the effects of melatonin treatment on the modulation of stem cell and EMT molecular markers: OCT4, E-cadherin, N-cadherin and vimentin, as well as on cell viability and invasiveness of the cells from mammospheres. The CMT-U229 and MCF-7 cell lines were subjected to three-dimensional culture in special medium for stem cells. The phenotype of mammospheres was first evaluated by flow cytometry (CD44+/CD24low/- marking). Cell viability was measured by MTT colorimetric assay and the expression of the proteins OCT4, E-cadherin, N-cadherin and vimentin was evaluated by immunofluorescence and quantified by optical densitometry. The analysis of cell migration and invasion was performed in Boyden Chamber. Flow cytometry proved the stem cell phenotype with CD44+/CD24low/- positive marking for both cell lines. Cell viability of CMT-U229 and MCF-7 cells was reduced after treatment with 1mM melatonin for 24 h (P<0.05). Immunofluorescence staining showed increased E-cadherin expression (P<0.05) and decreased expression of OCT4, N-cadherin and vimentin (P<0.05) in both cell lines after treatment with 1 mM melatonin for 24 hours. Moreover, treatment with melatonin was able to reduce cell migration and invasion in both cell lines when compared to control group (P<0.05). Our results demonstrate that melatonin shows an inhibitory role in the viability and invasiveness of breast cancer mammospheres as well as in modulating the expression of proteins related to EMT in breast CSCs, suggesting its potential anti-metastatic role in canine and human breast cancer cell lines.

[1]  S. Hill,et al.  Inhibition of breast cancer cell invasion by melatonin is mediated through regulation of the p38 mitogen-activated protein kinase signaling pathway , 2010, Breast Cancer Research.

[2]  Lin Zhao,et al.  Combined expression of ezrin and E-cadherin is associated with lymph node metastasis and poor prognosis in breast cancer. , 2015, Oncology reports.

[3]  Xuebing Yan,et al.  N-cadherin, a novel prognostic biomarker, drives malignant progression of colorectal cancer. , 2015, Molecular medicine reports.

[4]  Carlos Caldas,et al.  Alpha‐6 integrin is necessary for the tumourigenicity of a stem cell‐like subpopulation within the MCF7 breast cancer cell line , 2008, International journal of cancer.

[5]  L. Mishra,et al.  EMT, CTCs and CSCs in tumor relapse and drug-resistance , 2015, Oncotarget.

[6]  A. Satelli,et al.  Vimentin in cancer and its potential as a molecular target for cancer therapy , 2011, Cellular and Molecular Life Sciences.

[7]  B. Karlan,et al.  Evaluation of MCF10A as a Reliable Model for Normal Human Mammary Epithelial Cells , 2015, PloS one.

[8]  M. Naylor,et al.  Breast cancer stem cells , 2013, Front. Physiol..

[9]  A. C. Terzian,et al.  Immunohistochemical evaluation of Ki-67 and PCNA in canine mammary neoplasias: Correlation with prognostic factors and clinical outcome , 2008 .

[10]  C. Alonso-González,et al.  Melatonin interferes in the desmoplastic reaction in breast cancer by regulating cytokine production , 2012, Journal of pineal research.

[11]  E. Manuali,et al.  CA 15–3 cell lines and tissue expression in canine mammary cancer and the correlation between serum levels and tumour histological grade , 2012, BMC Veterinary Research.

[12]  S. Hill,et al.  Melatonin: an inhibitor of breast cancer. , 2015, Endocrine-related cancer.

[13]  Raghu Kalluri,et al.  The basics of epithelial-mesenchymal transition. , 2009, The Journal of clinical investigation.

[14]  S. Cos,et al.  Influence of melatonin on invasive and metastatic properties of MCF-7 human breast cancer cells. , 1998, Cancer research.

[15]  M. Wicha,et al.  Breast cancer stem cells: current advances and clinical implications. , 2015, Methods in molecular biology.

[16]  E. Hellmén,et al.  Canine mammary tumors contain cancer stem-like cells and form spheroids with an embryonic stem cell signature. , 2011, The International journal of developmental biology.

[17]  G. Dontu,et al.  In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. , 2003, Genes & development.

[18]  Chris Maloney,et al.  PubMed Central , 2017 .

[19]  C. Zhang,et al.  Melatonin Inhibits Endoplasmic Reticulum Stress and Epithelial-Mesenchymal Transition during Bleomycin-Induced Pulmonary Fibrosis in Mice , 2014, PloS one.

[20]  J. Trosko,et al.  Metformin Represses Self-Renewal of the Human Breast Carcinoma Stem Cells via Inhibition of Estrogen Receptor-Mediated OCT4 Expression , 2011, PloS one.

[21]  F. Kelleher,et al.  Circadian molecular clocks and cancer. , 2014, Cancer letters.

[22]  S. Zhang,et al.  Abnormal expression of EMT-related proteins, S100A4, vimentin and E-cadherin, is correlated with clinicopathological features and prognosis in HCC , 2014, Medical Oncology.

[23]  R. Reiter,et al.  Melatonin and cell death: differential actions on apoptosis in normal and cancer cells , 2003, Cellular and Molecular Life Sciences CMLS.

[24]  A. Jemal,et al.  Global cancer statistics , 2011, CA: a cancer journal for clinicians.

[25]  Alexander Gheldof,et al.  Cadherins and epithelial-to-mesenchymal transition. , 2013, Progress in molecular biology and translational science.

[26]  Ali S. Arbab,et al.  Effect of Melatonin on Tumor Growth and Angiogenesis in Xenograft Model of Breast Cancer , 2014, PloS one.

[27]  A. Hao,et al.  Melatonin antagonizes hypoxia‐mediated glioblastoma cell migration and invasion via inhibition of HIF‐1α , 2013, Journal of pineal research.

[28]  F. Mattioli,et al.  In vitro and in vivo antiproliferative activity of metformin on stem-like cells isolated from spontaneous canine mammary carcinomas: translational implications for human tumors , 2015, BMC Cancer.

[29]  Won-Jun Jang,et al.  Melatonin suppresses tumor angiogenesis by inhibiting HIF‐1α stabilization under hypoxia , 2010, Journal of pineal research.

[30]  Kyung-Chul Choi,et al.  Effect of melatonin on mRNA expressions of transcription factors in murine embryonic stem cells , 2011, Brain Research.

[31]  Y. Toiyama,et al.  Correlation of CD133, OCT4, and SOX2 in Rectal Cancer and Their Association with Distant Recurrence After Chemoradiotherapy , 2009, Annals of Surgical Oncology.

[32]  A. Hao,et al.  Expression profile of embryonic stem cell‐associated genes Oct4, Sox2 and Nanog in human gliomas , 2011, Histopathology.

[33]  G. Luvoni,et al.  Isolation of canine mammary cells with stem cell properties and tumour-initiating potential. , 2009, Reproduction in domestic animals = Zuchthygiene.

[34]  George A. R. Wiggins,et al.  E-cadherin loss alters cytoskeletal organization and adhesion in non-malignant breast cells but is insufficient to induce an epithelial-mesenchymal transition , 2014, BMC Cancer.

[35]  J. Foekens,et al.  Loss of E-cadherin is not a necessity for epithelial to mesenchymal transition in human breast cancer , 2013, Breast Cancer Research and Treatment.

[36]  Haibo Zhang,et al.  Epithelial-Mesenchymal Transition Associates with Maintenance of Stemness in Spheroid-Derived Stem-Like Colon Cancer Cells , 2013, PloS one.

[37]  M. Hung,et al.  Dual regulation of Snail by GSK-3β-mediated phosphorylation in control of epithelial–mesenchymal transition , 2004, Nature Cell Biology.

[38]  J. Castle,et al.  Comparative expression pathway analysis of human and canine mammary tumors , 2009, BMC Genomics.

[39]  L. Pang,et al.  Canine Mammary Cancer Stem Cells are Radio- and Chemo-Resistant and Exhibit an Epithelial-Mesenchymal Transition Phenotype , 2011, Cancers.

[40]  D. Zanette,et al.  The melatonin action on stromal stem cells within pericryptal area in colon cancer model under constant light. , 2011, Biochemical and biophysical research communications.

[41]  R. Xiu,et al.  Melatonin modulates the expression of VEGF and HIF‐1α induced by CoCl2 in cultured cancer cells , 2008, Journal of pineal research.

[42]  G. Benítez-King,et al.  ROCK‐regulated cytoskeletal dynamics participate in the inhibitory effect of melatonin on cancer cell migration , 2009, Journal of pineal research.

[43]  Minghua Yu,et al.  Melatonin prevents human pancreatic carcinoma cell PANC‐1‐induced human umbilical vein endothelial cell proliferation and migration by inhibiting vascular endothelial growth factor expression , 2012, Journal of pineal research.

[44]  J. Dick,et al.  Stem cell concepts renew cancer research. , 2008, Blood.

[45]  C. Alonso-González,et al.  Melatonin promotes differentiation of 3T3‐L1 fibroblasts , 2012, Journal of pineal research.

[46]  S. Morrison,et al.  Prospective identification of tumorigenic breast cancer cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[47]  J. Dopazo,et al.  Mammosphere Formation in Breast Carcinoma Cell Lines Depends upon Expression of E-cadherin , 2013, PloS one.

[48]  A. Arbab,et al.  Melatonin decreases breast cancer metastasis by modulating Rho‐associated kinase protein‐1 expression , 2016, Journal of pineal research.

[49]  H. Suemizu,et al.  Aldehyde dehydrogenase activity in cancer stem cells from canine mammary carcinoma cell lines. , 2012, Veterinary journal.

[50]  Yuan Wang,et al.  Melatonin Inhibits the Migration of Human Lung Adenocarcinoma A549 Cell Lines Involving JNK/MAPK Pathway , 2014, PloS one.

[51]  Giuseppe Alberto Di Bella,et al.  Melatonin Anticancer Effects: Review , 2013, International journal of molecular sciences.

[52]  J. Foidart,et al.  TRANSACTIVATION OF VIMENTIN BY BETA-CATENIN IN HUMAN BREAST CANCER CELLS , 2003, International Journal of Gynecologic Cancer.

[53]  G. B. Gelaleti,et al.  Evaluation of melatonin treatment in primary culture of canine mammary tumors. , 2015, Oncology reports.

[54]  S. Hill,et al.  Circadian gating of epithelial-to-mesenchymal transition in breast cancer cells via melatonin-regulation of GSK3β. , 2012, Molecular endocrinology.

[55]  J. Foidart,et al.  Transactivation of Vimentin by β-Catenin in Human Breast Cancer Cells , 2003 .

[56]  Y. Qiu,et al.  A Role for OCT4 in Tumor Initiation of Drug-Resistant Prostate Cancer Cells. , 2010, Genes & cancer.

[57]  S. Chiou,et al.  Coexpression of Oct4 and Nanog enhances malignancy in lung adenocarcinoma by inducing cancer stem cell-like properties and epithelial-mesenchymal transdifferentiation. , 2010, Cancer research.

[58]  Ke Chen,et al.  Understanding and targeting cancer stem cells: therapeutic implications and challenges , 2013, Acta Pharmacologica Sinica.

[59]  Lingling Sun,et al.  Prognostic significance of the epithelial-to-mesenchymal transition markers e-cadherin, vimentin and twist in bladder cancer. , 2014, International braz j urol : official journal of the Brazilian Society of Urology.

[60]  I. Antolín,et al.  Involvement of autophagy in melatonin‐induced cytotoxicity in glioma‐initiating cells , 2014, Journal of pineal research.

[61]  A. C. Alessi,et al.  Immunodetection of cells with a CD44+/CD24- phenotype in canine mammary neoplasms , 2013, BMC Veterinary Research.

[62]  Danila Coradini,et al.  Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. , 2005, Cancer research.

[63]  Araceli Olivares,et al.  Simvastatin exhibits antiproliferative effects on spheres derived from canine mammary carcinoma cells. , 2015, Oncology reports.

[64]  K. Hashimoto,et al.  N-cadherin-based adherens junction regulates the maintenance, proliferation, and differentiation of neural progenitor cells during development , 2015, Cell adhesion & migration.

[65]  T. Deroo,et al.  A Positive Role of Cadherin in Wnt/β-Catenin Signalling during Epithelial-Mesenchymal Transition , 2011, PloS one.

[66]  S. Agelaki,et al.  Epithelial to mesenchymal transition markers expressed in circulating tumour cells of early and metastatic breast cancer patients , 2011, Breast Cancer Research.

[67]  Wenjun Guo,et al.  The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells , 2008, Cell.

[68]  A. del Sol,et al.  Stemness of the hybrid Epithelial/Mesenchymal State in Breast Cancer and Its Association with Poor Survival , 2015, PloS one.

[69]  Shaorong Gao,et al.  Melatonin improves the reprogramming efficiency of murine‐induced pluripotent stem cells using a secondary inducible system , 2013, Journal of pineal research.

[70]  B. Zhou,et al.  Chinese Anti鄄 Cancer a Ssociation , 2022 .

[71]  W. Cui,et al.  Stably Transfected Human Embryonic Stem Cell Clones Express OCT4‐Specific Green Fluorescent Protein and Maintain Self‐Renewal and Pluripotency , 2005, Stem cells.

[72]  R. Ordoñez,et al.  Inhibition of matrix metalloproteinase‐9 and nuclear factor kappa B contribute to melatonin prevention of motility and invasiveness in HepG2 liver cancer cells , 2014, Journal of pineal research.

[73]  B. Colori,et al.  The Di Bella Method (DBM) in the treatment of prostate cancer: a preliminary retrospective study of 16 patients and a review of the literature. , 2013, Neuro endocrinology letters.

[74]  L. Li,et al.  Protective and sensitive effects of melatonin combined with adriamycin on ER+ (estrogen receptor) breast cancer. , 2015, European journal of gynaecological oncology.

[75]  T. Borin,et al.  Melatonin Regulates Angiogenic Factors under Hypoxia in Breast Cancer Cell Lines. , 2016, Anti-cancer agents in medicinal chemistry.

[76]  R. Clarke,et al.  A Detailed Mammosphere Assay Protocol for the Quantification of Breast Stem Cell Activity , 2012, Journal of Mammary Gland Biology and Neoplasia.

[77]  S. Zhang,et al.  The in vitro and in vivo effects of human umbilical cord mesenchymal stem cells on the growth of breast cancer cells , 2012, Breast Cancer Research and Treatment.

[78]  R. Ordoñez,et al.  Inhibition of VEGF expression through blockade of Hif1α and STAT3 signalling mediates the anti-angiogenic effect of melatonin in HepG2 liver cancer cells , 2013, British Journal of Cancer.

[79]  G. Johnson,et al.  Implications of Mesenchymal Cells in Cancer Stem Cell Populations: Relevance to EMT , 2014, Current Pathobiology Reports.

[80]  B. Bao,et al.  Overview of Cancer Stem Cells (CSCs) and Mechanisms of Their Regulation: Implications for Cancer Therapy , 2013, Current protocols in pharmacology.