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EGCG inhibits properties of glioma stem-like cells and synergizes with temozolomide through downregulation of P-glycoprotein inhibition

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Abstract

Rational: Combination therapy to inhibit cancer stem cells may have important clinical implications. Here, we examine the molecular mechanisms by which epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, inhibits the stem cell characteristics of glioma stem-like cells (GSLCs) and synergizes with temozolomide (TMZ), a DNA-methylating agent commonly used as first-line chemotherapy in gliomas. GSLCs were enriched from the human glioblastoma cell line U87 using neurosphere culture. Cells were analyzed using flow cytometry, quantitative PCR, and western blotting. Compared to U87 cells, a higher percentage of U87 GSLCs remained in the G0/G1 phase, with downregulation of the cell-cycle protein CylinD1 and overexpression of stem cell markers CD133 and ALDH1. The drug-resistance gene ABCB1 (but not ABCG2 or MGMT) also showed high mRNA and protein expression. The resistance index of U87 GSLCs against TMZ and carmustine (BCNU) was 3.0 and 16.8, respectively. These results indicate that U87 GSLCs possess neural stem cell and drug-resistance properties. Interestingly, EGCG treatment inhibited cell viability, neurosphere formation, and migration in this cell model. EGCG also induced apoptosis, downregulation of p-Akt and Bcl-2, and cleaving PARP in a dose-dependent manner. Importantly, EGCG treatment significantly downregulated P-glycoprotein expression but not that of ABCG2 or MGMT and simultaneously enhanced sensitivity to TMZ. Our study demonstrates that the use of EGCG alone or in combination with TMZ may be an effective therapeutic strategy for glioma.

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References

  1. Sarkaria JN, Kitange GJ, James CD, Plummer R, Calvert H, Weller M, Wick W (2008) Mechanisms of chemoresistance to alkylating agents in malignant glioma. Clin Cancer Res 14:2900–2908. doi:10.1158/1078-0432.CCR-07-1719

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Sai K, Yang QY, Shen D, Chen ZP (2013) Chemotherapy for gliomas in mainland China: an overview. Oncol Lett 5:1448–1452. doi:10.3892/ol.2013.1264

    CAS  PubMed Central  PubMed  Google Scholar 

  3. Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S, Fiocco R, Foroni C, Dimeco F, Vescovi A (2004) Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 64:7011–7021. doi:10.1158/0008-5472.CAN-04-1364

    Article  CAS  PubMed  Google Scholar 

  4. Christmann M, Verbeek B, Roos WP, Kaina B (2011) O(6)-Methylguanine-DNA methyltransferase (MGMT) in normal tissues and tumors: enzyme activity, promoter methylation and immunohistochemistry. Biochim Biophys Acta 1816:179–190. doi:10.1016/j.bbcan.2011.06.002

    CAS  PubMed  Google Scholar 

  5. Nakai E, Park K, Yawata T, Chihara T, Kumazawa A, Nakabayashi H, Shimizu K (2009) Enhanced MDR1 expression and chemoresistance of cancer stem cells derived from glioblastoma. Cancer Invest 27:901–908. doi:10.3109/07357900801946679

    Article  CAS  PubMed  Google Scholar 

  6. Yin BB, Wu SJ, Zong HJ, Ma BJ, Cai D (2011) Preliminary screening and identification of stem cell-like sphere clones in a gallbladder cancer cell line GBC-SD. J Zhejiang Univ Sci B 12:256–263. doi:10.1631/jzus.B1000303

    Article  PubMed Central  PubMed  Google Scholar 

  7. Imai Y, Tsukahara S, Asada S, Sugimoto Y (2004) Phytoestrogens/flavonoids reverse breast cancer resistance protein/ABCG2-mediated multidrug resistance. Cancer Res 64:4346–4352. doi:10.1158/0008-5472.CAN-04-0078

    Article  CAS  PubMed  Google Scholar 

  8. Shi Z, Liang YJ, Chen ZS, Wang XW, Wang XH, Ding Y, Chen LM, Yang XP, Fu LW (2006) Reversal of MDR1/P-glycoprotein-mediated multidrug resistance by vector-based RNA interference in vitro and in vivo. Cancer Biol Ther 5:39–47

    Article  CAS  PubMed  Google Scholar 

  9. Vlachostergios PJ, Hatzidaki E, Befani CD, Liakos P, Papandreou CN (2013) Bortezomib overcomes MGMT-related resistance of glioblastoma cell lines to temozolomide in a schedule-dependent manner. Invest New Drugs. doi:10.1007/s10637-013-9968-1

    PubMed  Google Scholar 

  10. Singh BN, Shankar S, Srivastava RK (2011) Green tea catechin, epigallocatechin-3-gallate (EGCG): mechanisms, perspectives and clinical applications. Biochem Pharmacol 82:1807–1821. doi:10.1016/j.bcp.2011.07.093

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Shankar S, Ganapathy S, Srivastava RK (2007) Green tea polyphenols: biology and therapeutic implications in cancer. Front Biosci 12:4881–4899

    Article  CAS  PubMed  Google Scholar 

  12. Chen H, Landen CN, Li Y, Alvarez RD, Tollefsbol TO (2013) Epigallocatechin gallate and sulforaphane combination treatment induce apoptosis in paclitaxel-resistant ovarian cancer cells through hTERT and Bcl-2 down-regulation. Exp Cell Res 319:697–706. doi:10.1016/j.yexcr.2012.12.026

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Sadava D, Whitlock E, Kane SE (2007) The green tea polyphenol, epigallocatechin-3-gallate inhibits telomerase and induces apoptosis in drug-resistant lung cancer cells. Biochem Biophys Res Commun 360:233–237. doi:10.1016/j.bbrc.2007.06.030

    Article  CAS  PubMed  Google Scholar 

  14. Farabegoli F, Papi A, Bartolini G, Ostan R, Orlandi M (2010) (-)-Epigallocatechin-3-gallate downregulates Pg-P and BCRP in a tamoxifen resistant MCF-7 cell line. Phytomedicine 17:356–362. doi:10.1016/j.phymed.2010.01.001

    Article  CAS  PubMed  Google Scholar 

  15. Huang HY, Niu JL, Zhao LM, Lu YH (2011) Reversal effect of 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone on multi-drug resistance in resistant human hepatocellular carcinoma cell line BEL-7402/5-FU. Phytomedicine 18:1086–1092. doi:10.1016/j.phymed.2011.04.001

    Article  CAS  PubMed  Google Scholar 

  16. Burnette WN (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112:195–203

    Article  CAS  PubMed  Google Scholar 

  17. Wan F, Zhang S, Xie R, Gao B, Campos B, Herold-Mende C, Lei T (2010) The utility and limitations of neurosphere assay, CD133 immunophenotyping and side population assay in glioma stem cell research. Brain Pathol 20:877–889. doi:10.1111/j.1750-3639.2010.00379.x

    CAS  PubMed  Google Scholar 

  18. Chaichana K, Zamora-Berridi G, Camara-Quintana J, Quinones-Hinojosa A (2006) Neurosphere assays: growth factors and hormone differences in tumor and nontumor studies. Stem Cells 24:2851–2857. doi:10.1634/stemcells.2006-0399

    Article  CAS  PubMed  Google Scholar 

  19. Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG (2005) Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2− cancer cells are similarly tumorigenic. Cancer Res 65:6207–6219. doi:10.1158/0008-5472.CAN-05-0592

    Article  CAS  PubMed  Google Scholar 

  20. Venezia TA, Merchant AA, Ramos CA, Whitehouse NL, Young AS, Shaw CA, Goodell MA (2004) Molecular signatures of proliferation and quiescence in hematopoietic stem cells. PLoS Biol 2:e301. doi:10.1371/journal.pbio.0020301

    Article  PubMed Central  PubMed  Google Scholar 

  21. Persano L, Rampazzo E, Basso G, Viola G (2013) Glioblastoma cancer stem cells: role of the microenvironment and therapeutic targeting. Biochem Pharmacol 85:612–622. doi:10.1016/j.bcp.2012.10.001

    Article  CAS  PubMed  Google Scholar 

  22. Mori H, Ninomiya K, Kino-oka M, Shofuda T, Islam MO, Yamasaki M, Okano H, Taya M, Kanemura Y (2006) Effect of neurosphere size on the growth rate of human neural stem/progenitor cells. J Neurosci Res 84:1682–1691. doi:10.1002/jnr.21082

    Article  CAS  PubMed  Google Scholar 

  23. Tabatabai G, Weller M (2011) Glioblastoma stem cells. Cell Tissue Res 343:459–465. doi:10.1007/s00441-010-1123-0

    Article  PubMed  Google Scholar 

  24. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63:5821–5828

    CAS  PubMed  Google Scholar 

  25. Deng S, Yang X, Lassus H, Liang S, Kaur S, Ye Q, Li C, Wang LP, Roby KF, Orsulic S, Connolly DC, Zhang Y, Montone K, Butzow R, Coukos G, Zhang L (2010) Distinct expression levels and patterns of stem cell marker, aldehyde dehydrogenase isoform 1 (ALDH1), in human epithelial cancers. PLoS One 5:e10277. doi:10.1371/journal.pone.0010277

    Article  PubMed Central  PubMed  Google Scholar 

  26. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1:555–567. doi:10.1016/j.stem.2007.08.014

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Li T, Su Y, Mei Y, Leng Q, Leng B, Liu Z, Stass SA, Jiang F (2010) ALDH1A1 is a marker for malignant prostate stem cells and predictor of prostate cancer patients’ outcome. Lab Invest 90:234–244. doi:10.1038/labinvest.2009.127

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Huang EH, Hynes MJ, Zhang T, Ginestier C, Dontu G, Appelman H, Fields JZ, Wicha MS, Boman BM (2009) Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res 69:3382–3389. doi:10.1158/0008-5472.CAN-08-4418

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Zang Y, Yu LF, Nan FJ, Feng LY, Li J (2009) AMP-activated protein kinase is involved in neural stem cell growth suppression and cell cycle arrest by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside and glucose deprivation by down-regulating phospho-retinoblastoma protein and cyclin D. J Biol Chem 284:6175–6184. doi:10.1074/jbc.M806887200

    Article  CAS  PubMed  Google Scholar 

  30. Zhao S, Liu H, Liu Y, Wu J, Wang C, Hou X, Chen X, Yang G, Zhao L, Che H, Bi Y, Wang H, Peng F, Ai J (2013) miR-143 inhibits glycolysis and depletes stemness of glioblastoma stem-like cells. Cancer Lett 333:253–260. doi:10.1016/j.canlet.2013.01.039

    Article  CAS  PubMed  Google Scholar 

  31. Lin CH, Shen YA, Hung PH, Yu YB, Chen YJ (2012) Epigallocathechin gallate, polyphenol present in green tea, inhibits stem-like characteristics and epithelial-mesenchymal transition in nasopharyngeal cancer cell lines. BMC Complement Altern Med 12:201. doi:10.1186/1472-6882-12-201

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Lee SH, Nam HJ, Kang HJ, Kwon HW, Lim YC (2013) Epigallocatechin-3-gallate attenuates head and neck cancer stem cell traits through suppression of Notch pathway. Eur J Cancer. doi:10.1016/j.ejca.2013.06.025

    Google Scholar 

  33. Chen D, Pamu S, Cui Q, Chan TH, Dou QP (2012) Novel epigallocatechin gallate (EGCG) analogs activate AMP-activated protein kinase pathway and target cancer stem cells. Bioorg Med Chem 20:3031–3037. doi:10.1016/j.bmc.2012.03.002

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Tang SN, Fu J, Nall D, Rodova M, Shankar S, Srivastava RK (2012) Inhibition of sonic hedgehog pathway and pluripotency maintaining factors regulate human pancreatic cancer stem cell characteristics. Int J Cancer 131:30–40. doi:10.1002/ijc.26323

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Tang SN, Singh C, Nall D, Meeker D, Shankar S, Srivastava RK (2010) The dietary bioflavonoid quercetin synergizes with epigallocathechin gallate (EGCG) to inhibit prostate cancer stem cell characteristics, invasion, migration and epithelial-mesenchymal transition. J Mol Signal 5:14. doi:10.1186/1750-2187-5-14

    Article  PubMed Central  PubMed  Google Scholar 

  36. Van Aller GS, Carson JD, Tang W, Peng H, Zhao L, Copeland RA, Tummino PJ, Luo L (2011) Epigallocatechin gallate (EGCG), a major component of green tea, is a dual phosphoinositide-3-kinase/mTOR inhibitor. Biochem Biophys Res Commun 406:194–199. doi:10.1016/j.bbrc.2011.02.010

    Article  PubMed  Google Scholar 

  37. Premkumar DR, Jane EP, DiDomenico JD, Vukmer NA, Agostino NR, Pollack IF (2012) ABT-737 synergizes with bortezomib to induce apoptosis, mediated by Bid cleavage, Bax activation, and mitochondrial dysfunction in an Akt-dependent context in malignant human glioma cell lines. J Pharmacol Exp Ther 341:859–872. doi:10.1124/jpet.112.191536

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Chen NG, Lu CC, Lin YH, Shen WC, Lai CH, Ho YJ, Chung JG, Lin TH, Lin YC, Yang JS (2011) Proteomic approaches to study epigallocatechin gallate-provoked apoptosis of TSGH-8301 human urinary bladder carcinoma cells: roles of AKT and heat shock protein 27-modulated intrinsic apoptotic pathways. Oncol Rep 26:939–947. doi:10.3892/or.2011.1377

    CAS  PubMed  Google Scholar 

  39. Liu D, Li P, Song S, Liu Y, Wang Q, Chang Y, Wu Y, Chen J, Zhao W, Zhang L, Wei W (2012) Pro-apoptotic effect of epigallo-catechin-3-gallate on B lymphocytes through regulating BAFF/PI3 K/Akt/mTOR signaling in rats with collagen-induced arthritis. Eur J Pharmacol 690:214–225. doi:10.1016/j.ejphar.2012.06.026

    Article  CAS  PubMed  Google Scholar 

  40. Li C, Zhou C, Wang S, Feng Y, Lin W, Lin S, Wang Y, Huang H, Liu P, Mu YG, Shen X (2011) Sensitization of glioma cells to tamoxifen-induced apoptosis by Pl3-kinase inhibitor through the GSK-3beta/beta-catenin signaling pathway. PLoS One 6:e27053. doi:10.1371/journal.pone.0027053

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. Manero F, Gautier F, Gallenne T, Cauquil N, Gree D, Cartron PF, Geneste O, Gree R, Vallette FM, Juin P (2006) The small organic compound HA14-1 prevents Bcl-2 interaction with Bax to sensitize malignant glioma cells to induction of cell death. Cancer Res 66:2757–2764. doi:10.1158/0008-5472.CAN-05-2097

    Article  CAS  PubMed  Google Scholar 

  42. Bleau AM, Huse JT, Holland EC (2009) The ABCG2 resistance network of glioblastoma. Cell Cycle 8:2936–2944

    Article  PubMed  Google Scholar 

  43. Liu L, Gerson SL (2006) Targeted modulation of MGMT: clinical implications. Clin Cancer Res 12:328–331. doi:10.1158/1078-0432.CCR-05-2543

    Article  CAS  PubMed  Google Scholar 

  44. Chahal M, Xu Y, Lesniak D, Graham K, Famulski K, Christensen JG, Aghi M, Jacques A, Murray D, Sabri S, Abdulkarim B (2010) MGMT modulates glioblastoma angiogenesis and response to the tyrosine kinase inhibitor sunitinib. Neuro Oncol 12:822–833. doi:10.1093/neuonc/noq017

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Ryu CH, Yoon WS, Park KY, Kim SM, Lim JY, Woo JS, Jeong CH, Hou Y, Jeun SS (2012) Valproic acid downregulates the expression of MGMT and sensitizes temozolomide-resistant glioma cells. J Biomed Biotechnol 2012:987495. doi:10.1155/2012/987495

    Article  PubMed Central  PubMed  Google Scholar 

  46. Chen TC, Wang W, Golden EB, Thomas S, Sivakumar W, Hofman FM, Louie SG, Schonthal AH (2011) Green tea epigallocatechin gallate enhances therapeutic efficacy of temozolomide in orthotopic mouse glioblastoma models. Cancer Lett 302:100–108. doi:10.1016/j.canlet.2010.11.008

    Article  CAS  PubMed  Google Scholar 

  47. Qian F, Wei D, Zhang Q, Yang S (2005) Modulation of P-glycoprotein function and reversal of multidrug resistance by (−)-epigallocatechin gallate in human cancer cells. Biomed Pharmacother 59:64–69. doi:10.1016/j.biopha.2005.01.002

    Article  CAS  PubMed  Google Scholar 

  48. Weksler BB, Subileau EA, Perriere N, Charneau P, Holloway K, Leveque M, Tricoire-Leignel H, Nicotra A, Bourdoulous S, Turowski P, Male DK, Roux F, Greenwood J, Romero IA, Couraud PO (2005) Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19:1872–1874. doi:10.1096/fj.04-3458fje

    CAS  PubMed  Google Scholar 

  49. Jodoin J, Demeule M, Beliveau R (2002) Inhibition of the multidrug resistance P-glycoprotein activity by green tea polyphenols. Biochim Biophys Acta 1542:149–159

    Article  CAS  PubMed  Google Scholar 

  50. Riganti C, Salaroglio IC, Caldera V, Campia I, Kopecka J, Mellai M, Annovazzi L, Bosia A, Ghigo D, Schiffer D (2013) Temozolomide downregulates P-glycoprotein expression in glioblastoma stem cells by interfering with the Wnt3a/glycogen synthase-3 kinase/beta-catenin pathway. Neuro Oncol. doi:10.1093/neuonc/not104

    PubMed Central  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 81072059, 31201028, 81201727), the Science and Technology Innovation Key Project of Guangdong Higher Education Institutes (No. CXZD1110).

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The authors declare that no competing interests exist.

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Authors and Affiliations

  1. Department of Pathology, Medical School of Jinan University, Guangzhou, 510632, People’s Republic of China

    Yong Zhang, Shao-Xiang Wang, Ji-Wei Ma, Hai-Ying Li, Jie-Cheng Ye, Si-Ming Xie, Bin Du & Xue-Yun Zhong

  2. Guangdong Province Key Laboratory of Molecule Immunology and Antibody Engineering, Jinan University, Guangzhou, 510632, China

    Yong Zhang, Shao-Xiang Wang, Ji-Wei Ma, Hai-Ying Li, Jie-Cheng Ye, Si-Ming Xie, Bin Du & Xue-Yun Zhong

  3. School of Medicine, Shenzhen University, Shenzhen, 518060, China

    Shao-Xiang Wang

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  1. Yong Zhang
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Correspondence to Bin Du or Xue-Yun Zhong.

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Yong Zhang and Shao-Xiang Wang contributed equally to this study.

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Zhang, Y., Wang, SX., Ma, JW. et al. EGCG inhibits properties of glioma stem-like cells and synergizes with temozolomide through downregulation of P-glycoprotein inhibition. J Neurooncol 121, 41–52 (2015). https://doi.org/10.1007/s11060-014-1604-1

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