J Breast Cancer.  2016 Mar;19(1):18-25. 10.4048/jbc.2016.19.1.18.

Caffeic Acid Phenethyl Ester Increases Radiosensitivity of Estrogen Receptor-Positive and -Negative Breast Cancer Cells by Prolonging Radiation-Induced DNA Damage

• Khoram NM
• Bigdeli B
• Nikoofar A
• Goliaei B

• KMID: 2176317
• DOI: http://doi.org/10.4048/jbc.2016.19.1.18

Abstract

PURPOSE
Breast cancer is an important cause of death among women. The development of radioresistance in breast cancer leads to recurrence after radiotherapy. Caffeic acid phenethyl ester (CAPE), a polyphenolic compound of honeybee propolis, is known to have anticancer properties. In this study, we examined whether CAPE enhanced the radiation sensitivity of MDA-MB-231 (estrogen receptor-negative) and T47D (estrogen receptor-positive) cell lines.
METHODS
The cytotoxic effect of CAPE on MDA-MB-231 and T47D breast cancer cells was evaluated by performing an 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. To assess clonogenic ability, MDA-MB-231 and T47D cells were treated with CAPE (1 µM) for 72 hours before irradiation, and then, a colony assay was performed. A comet assay was used to determine the number of DNA strand breaks at four different times.
RESULTS
CAPE decreased the viability of both cell lines in a dose- and time-dependent manner. In the clonogenic assay, pretreatment of cells with CAPE before irradiation significantly reduced the surviving fraction of MDA-MB-231 cells at doses of 6 and 8 Gy. A reduction in the surviving fraction of T47D cells was observed relative to MDA-MB-231 at lower doses of radiation. Additionally, CAPE maintained radiation-induced DNA damage in T47D cells for a longer period than in MDA-MB-231 cells.
CONCLUSION
Our results indicate that CAPE impairs DNA damage repair immediately after irradiation. The induction of radiosensitivity by CAPE in radioresistant breast cancer cells may be caused by prolonged DNA damage.

Keyword

Breast neoplasms; Caffeic acid phenethyl ester; DNA damage; Radiation-sensitizing agents

MeSH Terms

Breast Neoplasms*
Breast*
Cause of Death
Cell Line
Comet Assay
DNA Damage*
DNA*
Estrogens*
Female
Humans
Propolis
Radiation Tolerance*
Radiation-Sensitizing Agents
Radiotherapy
Recurrence
DNA
Estrogens
Propolis
Radiation-Sensitizing Agents

Figure

• 

  Figure 1 Effect of CAPE on the viability of breast cancer cell lines. The cells were treated with 1–100 µM CAPE for 24, 48, and 72 hours and cell viability was evaluated using the MTT assay. (A) MDA-MB-231 cells. (B) T47D cells. CAPE reduced the cell viability in time- and dose-dependent manners. The data represent the mean±SD of three independent experiments. DMSO=dimethyl sulfoxide; CAPE=caffeic acid phenethyl ester; MTT=3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide.

• 

  Figure 2 Clonogenic ability of MDA-MB-231 and T47D cells in the presence of different concentrations of CAPE. The cells were treated with 1, 5, and 10 µM CAPE for 72 hours, replated, and incubated for the indicated number of days to form colonies. (A) MDA-MB-231 cells. (B) T47D cells. (C) Representative colony formation assay of MDA-MB-231 (up) and T47D (down) cells at different concentrations of CAPE. (D) Images of single colonies of MDA-MB-231 (up) and T47D (down) cells after crystal violet staining, acquired using an inverted microscope (×5). Data represent the mean±standard deviation of two independent experiments. DMSO=dimethyl sulfoxide; CAPE=caffeic acid phenethyl ester. *p<0.05; †p<0.001.

• 

  Figure 3 CAPE effects on breast cancer cell line radiosensitivity. (A) Survival curve of MDA-MB-231. (B) Survival curve of T47D cells. A colony-forming assay was performed using control cells and cells treated with 1 µM CAPE for 72 hours after exposure to radiation (2, 4, 6, and 8 Gy). The surviving fraction of group treated with CAPE plus radiation was compared to that of the irradiated group. IR=irradiation; CAPE=caffeic acid phenethyl ester. *p<0.05; †p<0.001 represent statistical significance.

• 

  Figure 4 Level of radiation induced breaks in the presence of CAPE. MDA-MB-231 and T47D cells were treated with 1 µM CAPE for 72 hours and then exposed to radiation (6 Gy); the amount of DNA damage was determined by the comet assay immediately after irradiation until 2 hours. (A) %DNA in tail in MDA-MB-231. (B) %DNA in tail in T47D. (C) Olive tail moment in MDA-MB-231. (D) Olive tail moment in T47D. The mean±SD value of each parameter was measured from two independent experiments. CAPE=caffeic acid phenethyl ester; IR=irradiation. *p<0.05.

• 

  Figure 5 Representative comet assay images of MDA-MB-231 and T47D cells show changes in DNA damage levels. CAPE pretreated with 1 µM CAPE for 72 hours and then irradiated. The amount of DNA damage was analyzed immediately after radiation exposure (6 Gy) up to 2 hours. (A) MDA-MB-231 cells. (B) T47D cells. Four different groups of cells were compared at different time points (control, CAPE treated, irradiated, and combination of radiation and CAPE) at 0, 30, 60, and 120 minutes after irradiation. Images of cells were acquired using a fluorescence microscope (×20) followed by staining with ethidium bromide. CAPE=caffeic acid phenethyl ester; IR=irradiation.

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