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

• Kim YG
• Park YH
• Yang EY
• Park WS
• Park KS

• KMID: 2461898
• DOI: http://doi.org/10.4174/astr.2019.97.5.230

Abstract

PURPOSE
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.
METHODS
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.
RESULTS
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.
CONCLUSION
Addition of emodin attenuated tamoxifen's treatment effect via cyclin D1 and pERK up-regulation in ER-positive breast cancer cell lines.

Keyword

Breast neoplasm; Cyclin D1; Emodin; Phytoestrogens; Tamoxifen

MeSH Terms

Blotting, Western
Breast Neoplasms*
Breast*
Cell Line*
Cyclin D1
Drug Interactions
Emodin*
Epidermal Growth Factor
Estrogens*
Phosphotransferases
Phytoestrogens
Tamoxifen
Therapeutic Uses*
Up-Regulation
Cyclin D1
Emodin
Epidermal Growth Factor
Estrogens
Phosphotransferases
Phytoestrogens
Tamoxifen
Therapeutic Uses

Figure

• 

  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.

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