Icaritin synergistically enhances the radiosensitivity of 4T1 breast cancer cells

Abstract

Icaritin (ICT) is a hydrolytic form of icariin isolated from plants of the genus Epimedium. This study was to investigate the radiosensitization effect of icaritin and its possible underlying mechanism using murine 4T1 breast cancer cells. The combination of Icaritin at 3 µM or 6 µM with 6 or 8 Gy of ionizing radiation (IR) in the clonogenic assay yielded an ER (enhancement ratio) of 1.18 or 1.28, CI (combination index) of 0.38 or 0.19 and DRI (dose reducing index) of 2.51 or 5.07, respectively. These strongly suggest that Icaritin exerted a synergistic killing (?) effect with radiation on the tumor cells. This effect might relate with bioactivities of ICT: 1) exert an anti-proliferative effect in a dose- and time-dependent manner, which is different from IR killing effect but likely work together with the IR effect; 2) suppress the IR-induced activation of two survival paths, ERK1/2 and AKT; 3) induce the G2/M blockage, enhancing IR killing effect; and 4) synergize with IR to enhance cell apoptosis. In addition, ICT suppressed angiogenesis in chick embryo chorioallantoic membrane (CAM) assay. Taken together, ICT is a new radiosensitizer and can enhance anti-cancer effect of IR or other therapies.

Figure 1. ICT inhibits the growth of 4T1 breast cancer cells in a dose- and time-dependent manner.

The 4T1 cells were seeded into 96-well plates at a density of 5–10×10³ cells per well, treated with vehicle alone or ICT at the indicated different doses for 24, 48 or 72 h, and then the cell viability was determined by MTT assay. (A) ICT inhibits the growth of 4T1 breast cancer cells in a dose- and time-dependent manner. (B) Representative images of 4T1 cells treated with the indicated concentrations of ICT for 24 h, 48 h or 72 h.

Figure 2. ICT acts synergistically with IR in clonogenic assay.

The 4T1 breast cancer cells were treated with vehicle alone, ICT alone (0, 1.5, 3, or 6 µmol/L), radiation alone (0, 2, 4, 6 or 8 Gy) or combination of ICT with radiation. The cells were plated in 60 mm dishes at different densities, based on the stringency of the treatments. After 14 days, the colonies were stained with crystal violet, and the numbers of colonies (containing ≥50 cells) were counted using Image-Pro® Plus. The images represent the synergistic killing effects of ICT and IR on 4T1 cells.

Figure 3. Icaritin inhibits irradiation-induced phosphorylation of ERK1/2 and AKT.

(A) p-ERK1/2 assay. 4T1 cells were treated with vehicle alone or Icaritin at13 uM for 4 hr, and then irradiated with 0, 1, 4, or 6 Gy. Ten minutes later, the cells were fixed with 4% formalin for 5 min and subjected to an in-plate ELISA-like test with rabbit anti-phosphorylated ERK followed by horseradish peroxidase-anti-rabbit and a TMB substrate and A₄₅₀ reading. (B) p-AKT assay: 4T1 cells were treated with vehicle alone or Icaritin at 3 uM for 24 hr, and then irradiated with 0, 1, 4, or 6 Gy; One hour later, the cells were fixed with 4% formalin and subjected to an in-plate ELISA-like assay using rabbit anti-phosphorylated AKT as first antibody followed by the same method as detection for phosphorylated ERK1/2. ∗ represents p<0.05; ∗∗ represents p<0.01; NS means not significant.

Figure 4. Accumulation of G2-M cell population after combination of ICT and IR.

4T1 cells were treated with ICT (0, 12.5, 25 µM), IR (0, 4, or 6 Gy), or ICT combined with IR. 72 hours after treatment, the cells were harvested and subjected to flow cytometry. (A) FCM images represent the fraction of cells in G0G1 phase (M1), S phase (M2) and G2-M phase (M3) after different single or combined treatment. (B) Bar graphs represent the percentage of cells in three different phases.

Figure 5. Combination of ICT and Radiation Increased Cell Apoptosis.

4T1 cells were treated with ICT (0, 12.5, 25 µM), IR (0, 4, or 6 Gy), or ICT combined with IR. 72 hours after treatment, the cells were harvested and stained with Annexin V and PI. Combination of ICT and IR enhanced apoptosis.

Figure 6. Icaritin inhibits angiogenesis in the chick embryo chorioallantoic membrane (CAM) assay.

The fertilized eggs were incubated at 37°C for eight days and then divided into groups (8 eggs/group). 100 µl of 0, 10, 20 or 40 µM ICT was added to the top of CAM. After incubation for 3 more days, the CAM of each alive egg was harvested and placed individually in 6 well plate. (A) Images of the blood vessels of each CAM after treatment with various concentrations of ICT for 8–10 days were captured using Motic Images Plus®. (B) Mean vessel area as a percentage of the total area, (C) mean vessel length and (D) mean number of branch points were obtained by using Image Pro® analysis software.