N-Dihydrogalactochitosan Potentiates the Radiosensitivity of Liver Metastatic Tumor Cells Originated from Murine Breast Tumors

Abstract

Radiation is a widely used therapeutic method for treating breast cancer. N-dihydrogalactochitosan (GC), a biocompatible immunostimulant, is known to enhance the effects of various treatment modalities in different tumor types. However, whether GC can enhance the radiosensitivity of cancer cells remains to be explored. In this study, triple-negative murine 4T1 breast cancer cells transduced with multi-reporter genes were implanted in immunocompetent Balb/C mice to track, dissect, and identify liver-metastatic 4T1 cells. These cells expressed cancer stem cell (CSC) -related characteristics, including the ability to form spheroids, the expression of the CD44 marker, and the increase of protein stability. We then ex vivo investigated the potential effect of GC on the radiosensitivity of the liver-metastatic 4T1 breast cancer cells and compared the results to those of parental 4T1 cells subjected to the same treatment. The cells were irradiated with increased doses of X-rays with or without GC treatment. Colony formation assays were then performed to determine the survival fractions and radiosensitivity of these cells. We found that GC preferably increased the radiosensitivity of liver-metastatic 4T1 breast cancer cells rather than that of the parental cells. Additionally, the single-cell DNA electrophoresis assay (SCDEA) and γ-H2AX foci assay were performed to assess the level of double-stranded DNA breaks (DSBs). Compared to the parental cells, DNA damage was significantly increased in liver-metastatic 4T1 cells after they were treated with GC plus radiation. Further studies on apoptosis showed that this combination treatment increased the sub-G1 population of cells, but not caspase-3 cleavage, in liver-metastatic breast cancer cells. Taken together, the current data suggest that the synergistic effects of GC and irradiation might be used to enhance the efficacy of radiotherapy in treating metastatic tumors.

Keywords: N-dihydrogalactochitosan (GC), metastatic tumors; cancer stem cells; radiosensitivity; triple-negative breast cancer.

Figure 1

Tracking and isolation of liver-metastatic 4T1_3R breast cancer cells in Balb/C mice. (A) Coronal views of bioluminescent signals acquired at different time points after initial subcutaneous (s.c.) injection of 4T1_3R cells. (B) Resection of liver for the visualization of metastatic lesions. A major lesion (big arrowhead) and a minor lesion (thin arrow) were observed in the resected liver. (C) Fluorescence microscopic examination of red fluorescent protein (RFP) expression in isolated liver-metastatic 4T1_3R cells (4T1_L_3R) compared to original 4T1_3R cells. Scale bars = 100 μm.

Figure 2

Characterization of liver-metastatic 4T1 cells for tumor-initiating properties. (A) Comparison of sphere-forming capacity between parental 4T1 cells and 4T1_L_3R cells. RFP expression remained detectable in the latter cells; * p < 0.05. (B) Flow cytometric analysis of CD44 marker expression in parental 4T1 cells and 4T1_L_3R cells. (C) Comparison of the degradation rate of d2GFP between parental 4T1 cells and 4T1_L_3R cells after treatment with cycloheximide (CHX) (50 μg/mL) for 4 h. Scale bar =100 μm.

Figure 3

Analysis of survival fractions in cells exposed to X-rays with or without N-dihydrogalactochitosan (GC) pre-treatment. Cells were treated with 100 μg/mL of GC for 24 h before irradiation at different doses. The colony formation assay was used to compare the survival curves of (A) 4T1 cells and (B) 4T1_L_3R cell; * p < 0.05.

Figure 4

DNA damage analysis in cells exposed to X-rays with or without the GC pre-treatment. (A,B) Comet assay for parental 4T1 cells and 4T1_L_3R cells treated with GC, X-rays (10 Gy) or combined treatment, respectively. Scale bar = 50 μm. (C) Tail lengths determined by the comet assay were compared for untreated control, GC-treated, X-ray-treated, and combined treatment groups, and the results were analyzed using two-way ANOVA; ** p < 0.001. The data were presented as a box-and-whisker plot, where the central box represented the values from the lower to upper quartile (25 to 75 percentile). The middle line represented the median, and the dots in the middle position of the boxes represented central value markers. The far out values were displayed as open or solid circles. (D) Tail lengths were compared in cells subjected to combined treatment and individual GC or X-rays treatments. The results were analyzed using the t-test; * p < 0.05; ** p < 0.001.

Figure 5

Effects of GC combined with different doses of X-rays on the expression of γ-H2AX. (A) Western blot analysis was used to detect the expression of γ-H2AX. The band intensity was quantified using densitometry, and the level of γ-H2AX was normalized to that of GAPDH. Effects of GC + X-rays on γ-H2AX were separately compared for different doses of X-rays. (B) γ-H2AX foci assay. The percentage of γ-H2AX-positive cells corresponds to the number of nuclei with γ-H2AX foci normalized to the total number of nuclei in each experimental group; * p < 0.05. Scale bar = 20 μm.

Figure 6

Effects of GC combined with different doses of X-rays on induction of apoptosis. (A) Analysis of sub-G1 population by flow cytometry. The arrows represent the peaks of sub-G1 population in the DNA histograms. The experiments were duplicated. N.A.: not available. (B) Western blot analysis of caspase-3 expression (~32 kDa) and cleavage (~14 kDa) in cells upon various treatments.