Alterations of The Stem-Like Properties in The Breast Cancer Cell Line MDA-MB-231 Induced by Single Pulsed Doxorubicin Treatment

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Development of chemoresistance remains a significant limitation for the treatment of cancer and contributes to recurrence of the disease. Both intrinsic and acquired mechanisms of chemoresistance are characteristics of cancer stem cells (CSCs) or stem-like cells (SLCs). The aim of the study was to assess the stem-like properties in the breast cancer cell line MDA-MB-231 during and after pulsed treatment with doxorubicin (DOX) in comparison to the untreated controls.The experimental cultures were exposed to therapeutic concentration of DOX for 48 hours (treatment cultures), and subcultured to post-treatment cultures 24 hours after the removal of DOX. Stem-like properties of the cellular populations in the treatment and post--treatment cultures were assessed by the expression of the stem-cell marker genes (CD24, CD44, ITGA6, ITGB1, POU5F1, NANOG, ALDH1A1), colony-formation efficiency, growth rates, and sensitivity to DOX, 5-fluorouracil (5FU), cisplatin (CIS), and vinblastine (VBL). Exposure to DOX induced formation of giant polyploid cells that persisted in the post-treatment culture. The recovery period was characterised by a decrease in the proliferation rate, viability, and cellular adherence. The post-treatment cultures displayed decreased sensitivity to DOX and increased sensitivities to 5FU, CIS, and VBL. Cells treated with DOX displayed increased expression levels of CD24, CD44, and ALDH1A, while their expression levels at least partially normalised in the post-treatment culture. The post-treatment cultures demonstrated significantly increased colony-formation ability. During treatment with sub-lethal levels of doxorubicin and during the acute recovery period, the survival mechanisms in the breast cancer cell line MDA-MB-231 may be mediated by formation of the cellular population with stem-like properties.

Achuthan, S., Santhoshkumar, T. R., Prabhakar, J., Nair, S. A., Pillai, M. R. (2011). Drug-induced senescence generates chemoresistant stemlike cells with low reactive oxygen species. J. Biol. Chem., 286 (43), 37813–37829.

Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., Clarke, M. F. (2003). Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. USA, 100 (7), 3983–3988.

Anonymous (2018). R: A Language and Environment for Statistical Computing. R foundation for statistical computing, Vienna. Available from: (accessed 20.01.2019).

Brooks, D. L. P., Schwab, L. P., Krutilina, R., Parke, D. N., Sethuraman, A., Hoogewijs, D., Schörg, A., Gotwald, L., Fan, M., Wenger, R. H., Seagroves, T. N. (2016). ITGA6 is directly regulated by hypoxia- inducible factors and enriches for cancer stem cell activity and invasion in metastatic breast cancer models. Mol. Cancer, 15, 26.

Cortós-Funes, H., Coronado, C. (2007). Role of anthracyclines in the era of targeted therapy. Cardiovasc. Toxicol., 7 (2), 56–60.

Cox, J., Weinman, S. (2016). Mechanisms of doxorubicin resistance in hepatocellular carcinoma. Hepat Oncol., 3 (1), 57–59.

Dasari, S., Tchounwou, P. B. (2014). Cisplatin in cancer therapy: Molecular mechanisms of action. Eur. J. Pharmacol., 740, 364–378.

Dean, M., Fojo, T., Bates, S. (2005). Tumour stem cells and drug resistance. Nat. Rev. Cancer, 5 (4), 275–284.

Dean, M., Hamon, Y., Chimini, G. (2001). The human ATP-binding cassette transporter superfamily. J. Lipid Res., 42 (7), 1007–1017.

Deng, X., Apple, S., Zhao, H., Song, J., Lee, M., Luo, W., Wu, X., Chung, D., Pietras, R. J., Chang, H. R. (2017) CD24 expression and differential resistance to chemotherapy in triple-negative breast cancer. Oncotarget, 8 (24), 38294–38308.

El-Badawy, A., Ghoneim, M. A., Gabr, M. M., Salah, R. A., Mohamed, I. K., Amer, M., El-Badri, N. (2017). Cancer cell-soluble factors reprogram mesenchymal stromal cells to slow cycling, chemoresistant cells with a more stem-like state. Stem Cell Res. Ther., 8 (1), 254.

Erenpreisa, J., Ivanov, A., Wheatley, S. P., Kosmacek, E. A., Ianzini, F., Anisimov, A. P., Mackey, M., Davis, P. J., Illidge, T. M. (2008). Endopolyploidy in irradiated p53-deficient tumour cell lines: Persistence of cell division activity in giant cells expressing Aurora-B kinase. Cell Biol. Int., 32 (9), 1044–1056.

Fei, F., Zhang, D., Yang, Z., Wang, S., Wang, X., Wu, Z., Wu, Q., Zhang, S. (2015). The number of polyploid giant cells and epithelial-mesenchymal transition-related proteins are associated with invasion and metastasis un human breast cancer. J. Exp. Clin. Cancer Res., 34, 158.

Freshney, R. I. (2011). Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. Hoboken, NY.: Wiley-Blackwell. 728 pp.

Gewirtz, D. A. (1999). A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem. Pharmacol., 57 (7), 727–741.

Gottesman, M. M., Fojo, T., Bates, S. E. (2002). Multidrug resistance in cancer: Role of ATP-dependent transporters. Nat. Rev. Cancer, 2 (1), 48–58.

Hafner, M., Niepel, M., Chung, M., Sorger, P. K. (2016). Growth rate inhibition metrics correct for confounders in measuring sensitivity to cancer drugs. Nat. Methods, 13 (6), 521–527.

Hafner, M., Niepel, M., Sorger, P. K. (2017). Alternative drug sensitivity metrics improve preclinical cancer pharmacogenomics. Nat. Biotechnol., 35 (6), 52–54.

Holohan, C., Van Schaeybroeck, S., Longley, D. B., Johnston, P. G. (2013). Cancer drug resistance: An evolving paradigm. Nat. Rev. Cancer, 13 (10), 714–726.

Hu, H., Wang, M., Guan, X., Yuan, Z., Liu, Z., Zou, C., Guiyu, W., Gao, X., Wang, X. (2018). Loss of ABCB4 attenuates the caspase-dependent apoptosis regulating resistance to 5-Fu in colorectal cancer. Biosci Rep., 38 (1), BSR20171428.

Huang, Z. J., You, J., Luo, W. Y., Chen, B. S., Feng, Q. Z., Wu, B. L., Jiang, L., Luo, Q. (2015). Reduced tumorigenicity and drug resistance through the downregulation of octamer-binding protein 4 and Nanog transcriptional factor expression in human breast stem cells. Mol. Med. Rep., 11 (3), 1647–1654.

Hwang-Verslues, W. W., Kuo, W. H., Chang, P. H., Pan, C. C., Wang, H. H., Tsai, S. T., Jeng, Y. M., Shew, J. Y., Kung, J. T., Chen, C. H., Lee, E. Y., Chang, K. J., Lee, W. H. (2009). Multiple lineages of human breast cancer stem/progenitor cells identified by profiling with stem cell markers. PLoS ONE, 4 (12), e8377.

Jia, D., Tan, Y., Liu, H., Ooi, S., Li, L., Wright, K., Bennett, S., Addison, C.L., Wang, L. (2016). Cardamonin reduces chemotherapy-enriched breast cancer stem-like cells in vitro and in vivo. Oncotarget, 7 (1), 771–785.

Kibria, G., Hatakeyama, H., Akiyama, K., Hida, K., Harashima, H. (2014). Comparative study of the sensitivities of cancer cells to doxorubicin, and relationships between the effect of the drug-efflux pump P-gp. Biol. Pharm. Bull., 37 (12), 1926–1935.

Kim, W., Ryu, C. J. (2017). Cancer stem cell surface markers on normal stem cells. BMB Rep., 50 (6), 285–298.

Leggett, S. E., Sim, J. Y., Rubins, J. E., Neronha, Z. J., Williams, K., Wong, I. Y. (2016). Morphological single cell profiling of the epithelial-mesenchymal transition. Integr. Biol. (Camb)., 8 (11), 1133–1144.

Liang, Y., Zhong, Z., Huang, Y., Deng, W., Cao, J., Tsao, G., Liu, Q., Pei, D., Kang, T., Zeng, Y.X. (2010). Stem-like cancer cells are inducible by increasing genomic instability in cancer cells. J. Biol. Chem., 285 (7), 4931–4940.

Ling, G. Q., Chen, D. B., Wang, B. Q., Zhang, L. S. (2012). Expression of the pluripotency markers Oct3/4, Nanog and Sox2 in human breast cancer cell lines. Oncol. Lett., 4 (6), 1264–1268.

Liu, P., Kumar, I. S., Brown, S., Kannappan, V., Tawari, P. E., Tang, J. Z., Jiang, W., Armesilla, A. L., Darling, J. L., Wang, W. (2013). Disulfiram targets cancer stem-like cells and reverses resistance and cross-resistance in acquired paclitaxel-resistant triple-negative breast cancer cells. Brit. J. Cancer, 109 (7), 1876–1885.

Livak, K. J., Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods, 25 (4), 402–408.

McDermott, M., Eustace, A. J., Busschots, S., Breen, L., Crown, J., Clynes, M., O’Donovan N., Stordal, B. (2014). In vitro development of chemotherapy and targeted therapy drug-resistant cancer cell lines: A practical guide with case studies. Front. Oncol., 4, 40.

Mckenna, M. T., Weis, J. A., Barnes, S. L., Tyson, D. R., Miga, I., Quaranta, V., Yankeelov, T. E. (2017). Modeling approach for the study of doxorubicin treatment in triple negative breast cancer. Sc. Rep., 7 (1), 5725.

Mirzayans, R., Andrais, B., Murray, D. (2018). Roles of polyploid/multinucleated giant cancer cells in metastasis and disease relapse following anticancer treatment. Cancers, 10, 118.

Moitra, K., Lou, H., Dean, M. (2011). Multidrug efflux pumps and cancer stem cells: Insights into multidrug resistance and therapeutic development. Clin. Pharmacol. Ther., 89 (4), 491–502.

Niu, N., Zhang, J., Zhang, N., Mercado-Uribe, I., Tao, F., Han, Z., Pathak, S., Multani, A. S., Kuang, J., Yao, J., Bast, R.C., Sood, A.K., Hung, M.-C., Liu, J. (2016). Linking genomic reorganization to tumor initiation via the giant cell cycle. Oncogenesis, 5, e281.

Nowell, P. C. (1976). The clonal evolution of tumor cell populations. Science, 194 (4260), 23–28.

Rajaraman, R., Guernsey, D. L., Rajaraman, M. M., Rajaraman, S. R. (2006). Stem cells, senescence, neosis and self-renewal in cancer. Cancer Cell Int., 6, 1–26.

Rivera, E., Gomez, H. (2010). Chemotherapy resistance in metastatic breast cancer: The evolving role of ixabepilone. Breast Cancer Res., 12 (Suppl 2), S2.

Saxena, M., Stephens, M. A., Pathak, H., Rangarajan, A. (2011). Transcription factors that mediate epithelial-mesenchymal transition lead to multidrug resistance by upregulating ABC transporters. Cell Death Dis., 2, e179.

Sheridan, C., Kishimoto, H., Fuchs, R. K., Mehrotra, S., Bhat-Nakshatri, P., Turner, C. H., Goulet, R. Jr., Badve, S., Nakshatri, H. (2006). CD44+/CD24-Breast cancer cells exhibit enhanced invase properties: An early step necessary for metastasis. Breast Cancer Res., 8 (5), R59.

Skehan, P., Storeng, R., Scudiero, D., Monks, A., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S., Boyd, M. R. (1990). New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst., 82 (13), 1107–1112.

Smith, L. Watson, M. B., O’Kane, S. L., Drew, P. J., Lind, M. J., Cawkwell, L. (2006). The analysis of doxorubicin resistance in human breast cancer cells using antibody microarrays. Mol. Cancer Ther., 5 (8), 2115–2120.

Sun, L., Cabarcas, S. M., Farrar, W. L. (2012). Radioresistance and cancer stem cells: Survival of the fittest. J. Carcinogene Mutagenes, s1(01), 1–12.

Sundaram, M., Guernsey, D. L., Rajaraman, M. M., Rajaraman, R. (2004). Neosis: A novel type of cell division in cancer. Cancer Biol. Ther., 3, 2017–2218.

Tegze, B., Szállási, Z., Haltrich, I., Pénzváltó, Z., Tóth, Z., Likó, I., Gyorffy, B. (2012). Parallel evolution under chemotherapy pressure in 29 breast cancer cell lines results in dissimilar mechanisms of resistance. PLoS ONE, 7 (2), 1–9.

Vassilopoulos, A., Chisholm, C., Lahusen, T., Zheng, H., Deng, C. (2013). A critical role of CD29 and CD49f in mediating metastasis for cancer-initiating cells isolated from a Brca1-associated mouse model of breast cancer. Oncogene, 33 (47), 5477–5482.

Vichai, V., Kirtikara, K. (2006). Sulforhodamine B colorimetric assay for cytotoxicity screening. Nature Protocols, 1 (3), 1112–1116.

Vinogradov, S., Wei, X. (2012). Cancer stem cells and drug resistance: The potential of nanomedicine. Nanomedicine (Lond)., 7 (4), 597–615.

Wang, L., Li, P., Hu, W., Xia, Y., Hu, C., Liu, L., Jiang, X. (2017). CD44 + CD24 + subset of PANC-1 cells exhibits radiation resistance via decreased levels of reactive oxygen species. Oncol. Lett., 14 (2), 1341–1346.

Weihua, Z., Lin, Q., Ramoth, A. J., Fan, D., Fidler, I. J. (2011). Formation of solid tumors by a single multinucleated cancer cell. Cancer, 117 (17), 4092–4099.

Xiang, D., Shigdar, S., Bean, A. G., Bruce, M., Yang, W., Mathesh, M., Wang, T., Yin, W., Tran, P. H., Al Shamaileh, H., Barrero, R. A., Zhang, P. Z., Li, Y., Kong, L., Liu, K., Zhou, S. F., Hou, Y., He, A., Duan, W. (2017). Transforming doxorubicin into a cancer stem cell killer via EpCAM aptamer-mediated delivery. Theranostics., 7 (17), 4071–4086.

Zhang, S., Mercado-Uribe, I., Xing, Z., Sun, B., Kuang, J., Liu, J. (2014). Generation of cancer stem-like cells through the formation of polyploid giant cancer cells. Oncogene, 33 (1), 116–128.

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SCImago Journal Rank (SJR) 2017: 0.127
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