EWS Knockdown and Taxifolin Treatment Induced Differentiation and Removed DNA Methylation from p53 Promoter to Promote Expression of Puma and Noxa for Apoptosis in Ewing’s Sarcoma


Ewing’s sarcoma is a pediatric tumor that mainly occurs in soft tissues and bones. Malignant characteristics of Ewing’s sarcoma are correlated with expression of EWS oncogene. We achieved knockdown of EWS expression using a plasmid vector encoding EWS short hairpin RNA (shRNA) to increase anti-tumor mechanisms of taxifolin (TFL), a new flavonoid, in human Ewing’s sarcoma cells in culture and animal models. Immunofluorescence microscopy and flow cytometric analysis showed high expression of EWS in human Ewing’s sarcoma SK-N-MC and RD-ES cell lines. EWS shRNA plus TFL inhibited 80% cell viability and caused the highest decreases in EWS expression at mRNA and protein levels in both cell lines. Knockdown of EWS expression induced morphological features of differentiation. EWS shRNA plus TFL caused more alterations in molecular markers of differentiation than either agent alone. EWS shRNA plus TFL caused the highest decreases in cell migration with inhibition of survival, angiogenic and invasive factors. Knockdown of EWS expression was associated with removal of DNA methylation from p53 promoter, promoting expression of p53, Puma, and Noxa. EWS shRNA plus TFL induced the highest amounts of apoptosis with activation of extrinsic and intrinsic pathways in both cell lines in culture. EWS shRNA plus TFL also inhibited growth of Ewing’s sarcoma tumors in animal models due to inhibition of differentiation inhibitors and angiogenic and invasive factors and also induction of activation of caspase-3 for apoptosis. Collectively, knockdown of EWS expression increased various anti-tumor mechanisms of TFL in human Ewing’s sarcoma in cell culture and animal models.

Share and Cite:

Hossain, M. and Ray, S. (2014) EWS Knockdown and Taxifolin Treatment Induced Differentiation and Removed DNA Methylation from p53 Promoter to Promote Expression of Puma and Noxa for Apoptosis in Ewing’s Sarcoma. Journal of Cancer Therapy, 5, 1092-1113. doi: 10.4236/jct.2014.512114.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Khoury, J.D. (2005) Ewing Sarcoma Family of Tumors. Advances in Anatomic Pathology, 12, 212-220. http://dx.doi.org/10.1097/01.pap.0000175114.55541.52
[2] Ma, C., Bower, K.A., Chen, G., Shi, X., Ke, Z.J. and Luo, J. (2008) Interaction between ERK and GSK3β Mediates Basic Fibroblast Growth Factor-Induced Apoptosis in SK-N-MC Neuroblastoma Cells. Journal of Biological Chemistry, 283, 9248-9256. http://dx.doi.org/10.1074/jbc.M707316200
[3] Ohno, T., Ouchida, M., Lee, L., Gatalica, Z., Rao, V.N. and Reddy, E.S. (1994) The EWS Gene, Involved in Ewing Family of Tumors, Malignant Melanoma of Soft Parts and Desmoplastic Small Round Cell Tumors, Codes for an RNA Binding Protein with Novel Regulatory Domains. Oncogene, 9, 3087-3097.
[4] Ohno, T., Rao, V.N. and Reddy, E.S. (1993) EWS/Fli-1 Chimeric Protein Is a Transcriptional Activator. Cancer Research, 53, 5859-5863.
[5] Kovar, H., Aryee, D.N., Jug, G., Henockl, C., Schemper, M., Delattre, O., Thomas, G. and Gadner, H. (1996) EWS/ FLI-1 Antagonists Induce Growth Inhibition of Ewing Tumor Cells in Vitro. Cell Growth and Differentiation, 7, 429-437.
[6] Uren, A. and Toretsky, J.A. (2005) Ewing’s Sarcoma Oncoprotein EWS-FLI1: The Perfect Target without a Therapeutic Agent. Future Oncology, 1, 521-528.
[7] Maksimenko, A. and Malvy, C. (2005) Oncogene-Targeted Antisense Oligonucleotides for the Treatment of Ewing Sarcoma. Expert Opinion on Therapeutic Targets, 9, 825-830.
[8] Spahn, L., Siligan, C., Bachmaier, R., Schmid, J.A., Aryee, D.N. and Kovar, H. (2003) Homotypic and Heterotypic Interactions of EWS, FLI1 and Their Oncogenic Fusion Protein. Oncogene, 22, 6819-6829. http://dx.doi.org/10.1038/sj.onc.1206810
[9] Embree, L.J., Azuma, M. and Hickstein, D.D. (2009) Ewing Sarcoma Fusion Protein EWSR1/FLI1 Interacts with EWSR1 Leading to Mitotic Defects in Zebrafish Embryos and Human Cell Lines. Cancer Research, 69, 4363-4371. http://dx.doi.org/10.1158/0008-5472.CAN-08-3229
[10] Paronetto, M.P., Micana, B. and Valcárcel, J. (2011) The Ewing Sarcoma Protein Regulates DNA Damage-Induced Alternative Splicing. Molecular Cell, 43, 353-368.
[11] Li, H., Watford, W., Li, C., Parmelee, A., Bryant, M.A., Deng, C., O’Shea, J. and Lee, S.B. (2007) Ewing Sarcoma Gene EWS Is Essential for Meiosis and B Lymphocyte Development. Journal of Clinical Investigation, 117, 1314-1323. http://dx.doi.org/10.1172/JCI31222
[12] Sankar, S., Gomez, N.C., Bell, R., Patel, M., Davis, I.J., Lessnick, S.L. and Luo, W. (2013) EWS and RE1-Silencing Transcription Factor Inhibit Neuronal Phenotype Development and Oncogenic Transformation in Ewing Sarcoma. Genes & Cancer, 4, 213-223.
[13] Chakrabarti, M., Banik, N.L. and Ray, S.K. (2013) MiR-138 Overexpression Is More Powerful than hTERT Knockdown to Potentiate Apigenin for Apoptosis in Neuroblastoma in Vitro and in Vivo. Experimental Cell Research, 319, 1575-1585. http://dx.doi.org/10.1016/j.yexcr.2013.02.025
[14] Das, A., Banik, N.L. and Ray, S.K. (2006) Mechanism of Apoptosis with the Involvement of Calpain and Caspase Cascades in Human Malignant Neuroblastoma SH-SY5Y Cells Exposed to Flavonoids. International Journal of Cancer, 119, 2575-2585. http://dx.doi.org/10.1002/ijc.22228
[15] Wallace, S.N., Carrier, D.J. and Clausen, E.C. (2005) Batch Solvent Extraction of Flavanolignans from Milk Thistle (Silybum marianum L. Gaertner). Phytochemical Analysis, 16, 7-16.
[16] Slimestad, R., Fossen, T. and V?gen, I.M. (2007) Onions: A Source of Unique Dietary Flavonoids. Journal of Agricultural and Food Chemistry, 55, 10067-10080. http://dx.doi.org/10.1021/jf0712503
[17] Makena, P.S., Pierce, S.C., Chung, K.T. and Sinclair, S.E. (2009) Comparative Mutagenic Effects of Structurally Similar Flavonoids Quercetin and Taxifolin on Tester Strains Salmonella typhimurium TA102 and Escherichia coli WP-2 uvrA. Environmental and Molecular Mutagenesis, 50, 451-459. http://dx.doi.org/10.1002/em.20487
[18] Lee, S.B., Cha, K.H., Selenge, D., Solongo, A. and Nho, C.W. (2007) The Chemopreventive Effect of Taxifolin Is Exerted Through ARE-Dependent Gene Regulation. Biological and Pharmaceutical Bulletin, 6, 074-1079.
[19] Luo, H., Jiang, B.H., King, S.M. and Chen, Y.C. (2008) Inhibition of Cell Growth and VEGF Expression in Ovarian Cancer Cells by Flavonoids. Nutrition and Cancer, 60, 800-809.
[20] Oi, N., Chen, H., Ok Kim, M., Lubet, R.A., Bode, A.M. and Dong, Z.G. (2012) Taxifolin Suppresses UV-Induced Skin Carcinogenesis by Targeting EGFR and PI3K. Cancer Prevention Research, 5, 1103-1114. http://dx.doi.org/10.1158/1940-6207.CAPR-11-0397
[21] Brusselmans, K., Vrolix, R., Verhoeven, G. and Swinnen, J.V. (2005) Induction of Cancer Cell Apoptosis by Flavonoids Is Associated with Their Ability to Inhibit Fatty Acid Synthase Activity. Journal of Biological Chemistry, 280, 5636-5645. http://dx.doi.org/10.1074/jbc.M408177200
[22] Zhang, Z.R., Al Zaharna, M., Wong, M.M., Chiu, S.K. and Cheung, H.Y. (2013) Taxifolin Enhances Andrographolide-Induced Mitotic Arrest and Apoptosis in Human Prostate Cancer Cells via Spindle Assembly Checkpoint Activation. PLoS ONE, 8, e54577.
[23] Hossain, M.M., Banik, N.L. and Ray, S.K. (2012) Survivin Knockdown Increased Anti-Cancer Effects of (-)-Epigallocatechin-3-Gallate in Human Malignant Neuroblastoma SK-N-BE2 and SH-SY5Y Cells. Experimental Cell Research, 318, 1597-1610. http://dx.doi.org/10.1016/j.yexcr.2012.03.033
[24] van Meerloo, J., Kaspers, G.J. and Cloos, J. (2011) Cell Sensitivity Assays: The MTT Assay. Methods in Molecular Biology, 731, 237-245. http://dx.doi.org/10.1007/978-1-61779-080-5_20
[25] Chakrabarti, M., Banik, N.L. and Ray, S.K. (2014) MiR-7-1 Potentiated Estrogen Receptor Agonists for Functional Neuroprotection in VSC4.1 Motoneurons. Neuroscience, 256, 322-333.
[26] Mohan, N., Ai, W., Chakrabarti, M., Banik, N.L. and Ray, S.K. (2013) KLF4 Overexpression and Apigenin Treatment down Regulated Anti-Apoptotic Bcl-2 Proteins and Matrix Metalloproteinases to Control Growth of Human Malignant Neuroblastoma SK-N-DZ and IMR-32 Cells. Molecular Oncology, 7, 464-474. http://dx.doi.org/10.1016/j.molonc.2012.12.002
[27] Herman, J.G., Graff, J.R., My?h?nen, S., Nelkin, B.D. and Baylin, S.B. (1996) Methylation-Specific PCR: A Novel PCR Assay for Methylation Status of CpG Islands. Proceedings of the National Academy of Sciences of the United States of America, 93, 9821-9826. http://dx.doi.org/10.1073/pnas.93.18.9821
[28] Yang, D., Thangaraju, M., Greeneltch, K., Browning, D.D., Schoenlein, P.V., Tamura, T., Ozato, K., Ganapathy, V., Abrams, S.I. and Liu, K. (2007) Repression of IFN Regulatory Factor 8 by DNA Methylation Is a Molecular Determinant of Apoptotic Resistance and Metastatic Phenotype in Metastatic Tumor Cells. Cancer Research, 67, 3301-3309. http://dx.doi.org/10.1158/0008-5472.CAN-06-4068
[29] Carr, J., Bell, E., Pearson, A.D., Kees, U.R., Beris, H., Lunec, J. and Tweddle, D.A. (2006) Increased Frequency of Aberrations in the p53/MDM2/p14(ARF) Pathway in Neuroblastoma Cell Lines Established at Relapse. Cancer Research, 66, 2138-2145. http://dx.doi.org/10.1158/0008-5472.CAN-05-2623
[30] Wachsberger, P.R., Burd, R., Marero, N., Daskalakis, C., Ryan, A., McCue, P. and Dicker, A.P. (2005) Effect of the Tumor Vascular-Damaging Agent, ZD6126, on the Radioresponse of U87 Glioblastoma. Clinical Cancer Research, 11, 835-842.
[31] Dauphinot, L., De Oliveira, C., Melot, T., Sevenet, N., Thomas, V., Weissman, B.E. and Delattre, O. (2001) Analysis of the Expression of Cell Cycle Regulators in Ewing Cell Lines: EWS-FLI-1 Modulates p57KIP2 and c-Myc Expression. Oncogene, 20, 3258-3265. http://dx.doi.org/10.1038/sj.onc.1204437
[32] Matsumoto, Y., Tanaka, K., Nakatani, F., Matsunobu, T., Matsuda, S. and Iwamoto, Y. (2001) Downregulation and Forced Expression of EWS-Fli1 Fusion Gene Results in Changes in the Expression of G(1) Regulatory Genes. British Journal of Cancer, 84, 768-775.
[33] Kim, S., Schein, A.J. and Nadel, J.A. (2005) E-Cadherin Promotes EGFR-Mediated Cell Differentiation and MUC5AC Mucin Expression in Cultured Human Airway Epithelial Cells. American Journal of Physiology-Lung Cellular and Molecular Physiology, 289, L1049-L1060.
[34] J?gi, A., ?ra, I., Nilsson, H., Lindeheim, H., Makino, Y., Poellinger, L., Axelson, H. and P?hlman, S. (2002) Hypoxia Alters Gene Expression in Human Neuroblastoma Cells toward an Immature and Neural Crest-Like Phenotype. Proceedings of the National Academy of Sciences of the United States of America, 99, 7021-7026. http://dx.doi.org/10.1073/pnas.102660199
[35] Janardhanan, R., Banik, N.L. and Ray, S.K. (2009) N-Myc Down Regulation Induced Differentiation, Early Cell Cycle Exit, and Apoptosis in Human Malignant Neuroblastoma Cells Having Wild Type or Mutant p53. Biochemical Pharmacology, 78, 1105-1114. http://dx.doi.org/10.1016/j.bcp.2009.06.009
[36] Pelloski, C.E., Lin, E., Zhang, L., Yung, W.K., Colman, H., Liu, J.L., Woo, S.Y., Heimberger, A.B., Suki, D., Prados, M., Chang, S., Barke III, F.G., Fuller, G.N. and Aldape, K.D. (2006) Prognostic Associations of Activated Mitogen-Activated Protein Kinase and Akt Pathways in Glioblastoma. Clinical Cancer Research, 12, 3935-3941. http://dx.doi.org/10.1158/1078-0432.CCR-05-2202
[37] Maddika, S., Ande, S.R., Panigrahi, S., Paranjothy, T., Weglarczyk, K., Zuse, A., Eshraghi, M., Manda, K.D., Wiechec, E. and Los, M. (2007) Cell Survival, Cell Death and Cell Cycle Pathways Are Interconnected: Implications for Cancer Therapy. Drug Resistance Updates, 10, 13-29.
[38] Plate, K.H., Breier, G., Weich, H.A., Mennel, H.D. and Risau, W. (1994) Vascular Endothelial Growth Factor and Glioma Angiogenesis: Coordinate Induction of VEGF Receptors, Distribution of VEGF Protein and Possible in Vivo Regulatory Mechanisms. International Journal of Cancer, 59, 520-529. http://dx.doi.org/10.1002/ijc.2910590415
[39] Argyriou, A.A., Giannopoulou, E. and Kalofonos, H.P. (2009) Angiogenesis and Anti-Angiogenic Molecularly Targeted Therapies in Malignant Gliomas. Oncology, 77, 1-11.
[40] Oren, M. (1999) Regulation of the p53 Tumor Suppressor Protein. Journal of Biological Chemistry, 274, 36031-36034. http://dx.doi.org/10.1074/jbc.274.51.36031
[41] Schroeder, M. and Mass, M.J. (1997) CpG Methylation Inactivates the Transcriptional Activity of the Promoter of the Human P53 Tumor Suppressor Gene. Biochemical and Biophysical Research Communications, 235, 403-406. http://dx.doi.org/10.1006/bbrc.1997.6796
[42] Labi, V. and Villunger, A. (2010) PUMA-Mediated Tumor Suppression: A Tale of Two Stories. Cell Cycle, 9, 4269-4275. http://dx.doi.org/10.4161/cc.9.21.13666
[43] Roberts, C.G., Millar, E.K.A., O’Toole, S.A., McNei, C.M., Lehrbach, G.M., Pinese, M., Tobelmann, P., McCloy, R.A., Musgrove, E.A., Sutherland, R.L. and Butt, A.J. (2011) Identification of PUMA as an Estrogen Target Gene that Mediates the Apoptotic Response to Tamoxifen in Human Breast Cancer Cells and Predicts Patient Outcome and Tamoxifen Responsiveness in Breast Cancer. Oncogene, 30, 3186-3197. http://dx.doi.org/10.1038/onc.2011.36
[44] Ming, L., Wang, P., Bank, A., Yu, J. and Zhang, L. (2006) PUMA Dissociates Bax and Bcl-XL to Induce Apoptosis in Colon Cancer Cells. Journal of Biological Chemistry, 281, 16034-16042.
[45] Lopez, H., Zhang, L., George, N.M., Liu, X., Pang, X., Evans, J.J., Targy, N.M. and Luo, X. (2010) Perturbation of the Bcl-2 Network and an Induced Noxa/Bcl-XL Interaction Trigger Mitochondrial Dysfunction after DNA Damage. Journal of Biological Chemistry, 285, 15016-15026.
[46] Luo, X., Budihardjo, I., Zou, H., Slaughter, C. and Wang, X.D. (1998) Bid, a Bcl2 Interacting Protein, Mediates Cytochrome c Release from Mitochondria in Response to Activation of Cell Surface Death Receptors. Cell, 94, 481-490. http://dx.doi.org/10.1016/S0092-8674(00)81589-5
[47] Fulda, S. and Debatin, K.M. (2006) Extrinsic versus Intrinsic Apoptosis Pathways in Anticancer Chemotherapy. Oncogene, 25, 4798-4811. http://dx.doi.org/10.1038/sj.onc.1209608
[48] Karmakar, S., Banik, N.L., Patel, S.J. and Ray, S.K. (2006) Curcumin Activated both Receptor-Mediated and Mitochondria-Mediated Proteolytic Pathways for Apoptosis in Human Glioblastoma T98G Cells. Neuroscience Letters, 407, 53-58. http://dx.doi.org/10.1016/j.neulet.2006.08.013
[49] Sun, X.M., MacFarlane, M., Zhuang, J., Wolf, B.B., Green, D.R. and Cohen, G.M. (1999) Distinct Caspase Cascades Are Initiated in Receptor-Mediated and Chemical-Induced Apoptosis. Journal of Biological Chemistry, 274, 5053-5060. http://dx.doi.org/10.1074/jbc.274.8.5053
[50] Choudhury, S.R., Karmakar, S., Banik, N.L. and Ray, S.K. (2011) Valproic Acid Induced Differentiation and Potentiated Efficacy of Taxol and Nanotaxol for Controlling Growth of Human Glioblastoma LN18 and T98G Cells. Neurochemical Research, 36, 2292-2305. http://dx.doi.org/10.1007/s11064-011-0554-7

Copyright © 2020 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.