Ann Surg Treat Res.  2019 Nov;97(5):230-238. 10.4174/astr.2019.97.5.230.

Inhibition of tamoxifen's therapeutic effects by emodin in estrogen receptor-positive breast cancer cell lines

  • 1Department of Surgery, Bundang Jesang General Hospital, Seongnam, Korea.
  • 2Clinical Science, Department of Medicine, The Graduate School of Konkuk University, Seoul, Korea.
  • 3Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul, Korea.
  • 4Ewha Womans University Mokdong Hospital/Cancer Center for Women, Breast and Thyroid Cancer Center, Seoul, Korea.
  • 5Department of Surgery, Kyung Hee University School of Medicine, Seoul, Korea.
  • 6Department of Surgery, Konkuk University School of Medicine, Seoul, Korea.
  • 7Research Institute of Medical Science, Konkuk University School of Medicine, Seoul, Korea.


This study was aimed to investigate the combination effect of endoxifen and emodin on estrogen receptor (ER) positive breast cancer cell lines and to explain the mechanism of the combination effect.
We conducted this study on MCF-7 (ER+/human epidermal growth factor receptor-2 [HER2]−), T47D (ER+/HER2−), ZR-75-1 (ER+/HER2+), and BT474 (ER+/HER2+) cell lines, which confirmed combination effect of endoxifen and emodin. Optimal concentrations for combination were determined to study the effects on proliferation of MCF-7 and ZR-75-1 cells. Analysis of the combination effect was carried out in the CompuSyn software. The combination of downstream mechanisms, and combined effects of other similar compounds were tested on the MCF-7 and ZR 75-1 cell lines. Protein expression was confirmed by western blot.
The combination of endoxifen and emodin had antagonistic effects on MCF-7 and ZR-75-1cell lines (combination index > 1). We validated the antagonistic effect in T47D and BT474 cell lines. During the combined treatment, the results showed elevated amounts of cyclin D1 and phosphorylated extracellular signal-regulated kinase (pERK). Analysis of drug interactions showed antagonistic effect between endoxifen and chemical compounds similar to emodin, such as chrysophanol or rhein, in MCF-7 and ZR-75-1 cells.
Addition of emodin attenuated tamoxifen's treatment effect via cyclin D1 and pERK up-regulation in ER-positive breast cancer cell lines.


Breast neoplasm; Cyclin D1; Emodin; Phytoestrogens; Tamoxifen

MeSH Terms

Blotting, Western
Breast Neoplasms*
Cell Line*
Cyclin D1
Drug Interactions
Epidermal Growth Factor
Therapeutic Uses*
Cyclin D1
Epidermal Growth Factor
Therapeutic Uses


  • Fig. 1 Analysis of cell viability in MCF-7 and ZR75-1 breast cancer cell lines. Cell viability was analyzed using the EZ-Cytox cell viability assay kit. The cell lines were treated with 0–16 µM endoxifen for 48 hours (A) and 0–120 µM emodin for 48 hours (B). After analysis using EZ-Cytox, the cytotoxic concentrations of endoxifen (4 µM) and emodin (60 µM) were obtained.

  • Fig. 2 Cell viability and microscopic feature after combined drug combination. After treatment with the drug combination, cell viability was analyzed using the EZ-Cytox assay. Combinations emodin (0, 15, 30, 60 µM) and endoxifen (0, 2, 4 µM) were applied for 48 hours. Cell viability decreased gradually with increasing drug dose in the MCF-7 breast cancer cell line (A) and the ZR 75-1 breast cancer cell line (B). Cell morphology was observed after 48 hours of incubation with the drug combinations (×250; A, MCF-7; B, ZR-75-1). *P < 0.05, **P < 0.01.

  • Fig. 3 Isobologram analysis using CompuSyn software. Isobologram analysis was based on the results of cell viability after treatment using the drug combination. The MCF-7 breast cancer cell line (A) and the ZR 75-1 breast cancer cell line (B) had combination index (CI) values > 1. These results indicated an antagonistic effect in the 2 cell lines.

  • Fig. 4 Validation of the antagonistic effects on other breast cancer cell lines. Cell viability was analyzed using the EZ-Cytox assay. Isobologram analysis was performed using the CompuSyn program. The T47D breast cancer cell line (A) and the BT-474 breast cancer cell line (B) also showed the antagonistic effect between endoxifen and emodin. CI, combination index. *P < 0.05, **P < 0.01.

  • Fig. 5 Western blotting analysis of cells in response to the combination of endoxifen and emodin. The breast cancer cell lines were treated endoxifen (0, 2, 4 µM) and emodin (0, 60 µM). After 48 hours, protein levels in harvested cells were analyzed using western blotting analysis. β-actin was used as a loading control – EGFR; EGFR, epidermal growth factor receptor; ERα, estrogen receptor alpha; p-ERK, phosphorylated extracellular signal-regulated kinase.

  • Fig. 6 Cell viability and isobologram analysis of MCF-7 breast cancer cells treated with combinations of endoxifen and other compounds. Cell viability was analyzed using the EZ-Cytox assay. Isobologram analysis was performed using the CompuSyn program. The MCF-7 breast cancer cell line showed an antagonistic effect toward endoxifen with chrysophanol or rhein. CI, combination index. *P < 0.05, **P < 0.01.

  • Fig. 7 Cell viability and isobologram analysis with drug combination between endoxifen and other compounds in ZR75-1 breast cancer cell line Cell viability analyzed by EZ-Cytox assay. Isobologram analysis was used to Compusyn program. ZR 75-1 breast cancer cell line showed antagonistic effect between endoxifen and chysophanol or rhein. CI, combination index. *P < 0.05, **P < 0.01.


1. Ko BS, Noh WC, Kang SS, Park BW, Kang EY, Paik NS, et al. Changing patterns in the clinical characteristics of Korean breast cancer from 1996–2010 using an online nationwide breast cancer database. J Breast Cancer. 2012; 15:393–400.
2. Osborne CK. Tamoxifen in the treatment of breast cancer. N Engl J Med. 1998; 339:1609–1618.
3. Early Breast Cancer Trialists' Collaborative Group. Tamoxifen for early breast cancer: an overview of the randomised trials. Lancet. 1998; 351:1451–1467.
4. Burstein HJ, Temin S, Anderson H, Buchholz TA, Davidson NE, Gelmon KE, et al. Adjuvant endocrine therapy for women with hormone receptor-positive breast cancer: american society of clinical oncology clinical practice guideline focused update. J Clin Oncol. 2014; 32:2255–2269.
5. Aiello Bowles EJ, Boudreau DM, Chubak J, Yu O, Fujii M, Chestnut J, et al. Patient-reported discontinuation of endocrine therapy and related adverse effects among women with early-stage breast cancer. J Oncol Pract. 2012; 8:e149–e157.
6. Jiao Y, Zuo Y. Ultrasonic extraction and HPLC determination of anthraquinones, aloe-emodine, emodine, rheine, chrysophanol and physcione, in roots of Polygoni multiflori. Phytochem Anal. 2009; 20:272–278.
7. Lin SY, Lai WW, Ho CC, Yu FS, Chen GW, Yang JS, et al. Emodin induces apoptosis of human tongue squamous cancer SCC-4 cells through reactive oxygen species and mitochondria-dependent pathways. Anticancer Res. 2009; 29:327–335.
8. Basu S, Ghosh A, Hazra B. Evaluation of the antibacterial activity of Ventilago madraspatana Gaertn., Rubia cordifolia Linn. and Lantana camara Linn.: isolation of emodin and physcion as active antibacterial agents. Phytother Res. 2005; 19:888–894.
9. Shrimali D, Shanmugam MK, Kumar AP, Zhang J, Tan BK, Ahn KS, et al. Targeted abrogation of diverse signal transduction cascades by emodin for the treatment of inflammatory disorders and cancer. Cancer Lett. 2013; 341:139–149.
10. Wang XD, Gu LQ, Wu JY. Apoptosis-inducing activity of new pyrazole emodin derivatives in human hepatocellular carcinoma HepG2 cells. Biol Pharm Bull. 2007; 30:1113–1116.
11. Meng G, Liu Y, Lou C, Yang H. Emodin suppresses lipopolysaccharide-induced pro-inflammatory responses and NF-κB activation by disrupting lipid rafts in CD14-negative endothelial cells. Br J Pharmacol. 2010; 161:1628–1644.
12. Wei WT, Lin SZ, Liu DL, Wang ZH. The distinct mechanisms of the antitumor activity of emodin in different types of cancer (Review). Oncol Rep. 2013; 30:2555–2562.
13. Holliday DL, Speirs V. Choosing the right cell line for breast cancer research. Breast Cancer Res. 2011; 13:215.
14. Lim YC, Li L, Desta Z, Zhao Q, Rae JM, Flockhart DA, et al. Endoxifen, a secondary metabolite of tamoxifen, and 4-OH-tamoxifen induce similar changes in global gene expression patterns in MCF-7 breast cancer cells. J Pharmacol Exp Ther. 2006; 318:503–512.
15. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev. 2006; 58:621–681.
16. Chung YS, Cho S, Ryou HJ, Jee HG, Choi JY, Yoon K, et al. Is there a treatment advantage when paclitaxel and lovastatin are combined to dose anaplastic thyroid carcinoma cell lines? Thyroid. 2011; 21:735–744.
17. Levenson AS, Jordan VC. MCF-7: the first hormone-responsive breast cancer cell line. Cancer Res. 1997; 57:3071–3078.
18. Burdall SE, Hanby AM, Lansdown MR, Speirs V. Breast cancer cell lines: friend or foe? Breast Cancer Res. 2003; 5:89–95.
19. Sartorius CA, Groshong SD, Miller LA, Powell RL, Tung L, Takimoto GS, et al. New T47D breast cancer cell lines for the independent study of progesterone B- and A-receptors: only antiprogestin-occupied B-receptors are switched to transcriptional agonists by cAMP. Cancer Res. 1994; 54:3868–3877.
20. Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, et al. A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell. 2006; 10:515–527.
21. Pontano LL, Diehl JA. Speeding through cell cycle roadblocks: nuclear cyclin D1-dependent kinase and neoplastic transformation. Cell Div. 2008; 3:12.
22. Zhang X, Gureasko J, Shen K, Cole PA, Kuriyan J. An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell. 2006; 125:1137–1149.
23. Lahusen T, Fereshteh M, Oh A, Wellstein A, Riegel AT. Epidermal growth factor receptor tyrosine phosphorylation and signaling controlled by a nuclear receptor coactivator, amplified in breast cancer 1. Cancer Res. 2007; 67:7256–7265.
24. Saini KS, Loi S, de Azambuja E, Metzger-Filho O, Saini ML, Ignatiadis M, et al. Targeting the PI3K/AKT/mTOR and Raf/MEK/ERK pathways in the treatment of breast cancer. Cancer Treat Rev. 2013; 39:935–946.
25. Mohammend MM. Structure antimutagenicity relationship of anthraquinones. Nat Prod Chem Res. 2016; 4:1000228.
26. Jones JL, Daley BJ, Enderson BL, Zhou JR, Karlstad MD. Genistein inhibits tamoxifen effects on cell proliferation and cell cycle arrest in T47D breast cancer cells. Am Surg. 2002; 68:575–577.
27. Ju YH, Doerge DR, Allred KF, Allred CD, Helferich WG. Dietary genistein negates the inhibitory effect of tamoxifen on growth of estrogen-dependent human breast cancer (MCF-7) cells implanted in athymic mice. Cancer Res. 2002; 62:2474–2477.
Full Text Links
  • ASTR
export Copy
  • Twitter
  • Facebook
Similar articles
Copyright © 2023 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: