Enhanced autophagy in colorectal cancer stem cells does not contribute to radio-resistance

Abstract

Autophagy, an essential catabolic pathway of degrading cellular components within the lysosome, has been found to benefit the growth and therapeutic resistance of cancer cells. In this study, we investigated the role of autophagy in the radio-sensitivity of cancer stem cells. By separating CD44+/CD133+ cancer stem cells from parental HCT8 human colorectal cancer cells, we found a significantly higher level of autophagy in the CD44+/CD133+ cells than in the parental cells. Exposure to 5 Gy of γ-ray significantly damaged both CD44+/CD133+ cells and parental cells, but the radiation-induced damage did not differ between the groups. Unexpectedly, autophagy was not significantly induced by radiation exposure in the CD44+/CD133+ cells and parental cells. The inhibition of autophagy by the silencing of ATG7, a factor required for autophagy at the stage of autophagosome precursor synthesis, did not significantly change the growth and radiation-induced damage in both CD44+/CD133+ cells and parental cells. Although an enhanced basic level of autophagy was found in the CD44+/CD133+ cancer stem cells, our data suggest that the canonical autophagy in cancer cells plays few roles, if any, in radio-sensitivity.

Keywords: autophagy; cancer stem cells; radio-resistance.

Figure 1. Autophagy activity in CD44⁺/CD133⁺ cancer stem cells

A. CD44⁺/CD133⁺ cancer stem cells were purified from the HCT8 human colorectal cancer cell line, and the purity was confirmed by flow cytometry. B. Western blot analysis was used to detect the expression levels of LC3-I and LC3-II, and the LC3-II/LC3-I ratio represents the extent of autophagy. C. Autophagic flux by inferring with LC3-II turnover by Western blotting in the presence and absence of a lysosomal inhibitor. Cells were incubated with 50 μM chloroquine (CQ) for 4 hrs. Parental cells without any treatment were used as the control of relative expression. PBS: Phosphate-buffered saline. The data are represented as the means ± SD from three independent experiments.

Figure 2. Radiation-induced cell damage

A. CD44⁺/CD133⁺ cancer stem cells and parental HCT8 human colorectal cancer cells were exposed to 5 Gy of γ-ray followed by incubation for another 2 days. The growth of cells was observed under a microscope with 40-fold magnification. Scale bar, 200 μm. B. Western blot analysis of the expression level of cleaved PARP1 in cells. C. Cells were labeled with 10 μM H₂DCFDA for 10 min and then exposed to 5 Gy of γ-ray. The ROS level was evaluated by measuring the fluorescence intensity within cells. D. Cells were exposed to 5 Gy of γ-ray and then fixed at the indicated times. The number of γ-H2AX foci in each cell was counted under fluorescence microscopy, and the mean number of γ-H2AX foci per cell was calculated. The data are represented as the means ± SD from three independent experiments.

Figure 3. Western blot analysis of the expression of ATG7 and LC3

A. Cells were treated with ATG7 siRNA and then incubated for 2 days. After autophagy was inhibited, cells were exposed to 5 Gy of γ-ray followed by incubation for another 2 days. The representative western blot shows the expression of ATG7 and LC3 in cells. Quantitative analysis of the LC3-I/LC3-II ratio and relative expression level of ATG7. Parental cells without any treatment were used as the control of relative expression. B. Autophagic flux was measured after ATG7 knockdown. Cells were incubated with 50 μM chloroquine (CQ) for 4 hrs. C. Autophagic flux was measured after irradiation. Parental cells without any treatment were used as the control of relative expression. NC: negative control siRNA. PBS: Phosphate-buffered saline. The data are represented as the means ± SD from three independent experiments.

Figure 4. Cell growth and the cell cycle

Cells were treated with ATG7 siRNA and then incubated for 2 days. After autophagy was inhibited, cells were exposed to 5 Gy of γ-ray followed by incubation for another 2 days. A. Cell growth was observed under a microscope with 40-fold magnification. Scale bar, 200 μm. B. The cell cycle was measured by PI staining.

Figure 5. Apoptosis and clonogenic survival

Autophagy was inhibited by ATG7 siRNA for 2 days or 50 μM chloroquine (CQ) for 4 hrs. A. The cell apoptosis was measured by Annexin V/PI staining. B. Quantitative analysis of the apoptosis rate. C. Quantitative analysis of the necrosis rate. D. Western blot analysis of the expression of cleaved PARP1. Parental cells exposed to 5 Gy of γ-ray were used as the control of relative expression. E. Clonogenic survival assay. NC: negative control siRNA. The data are represented as the means ± SD from three independent experiments.