Figure 1.
Effect of 5-aza-dC and ionizing radiation on colony formation in colon cancer cell lines.
(A) Clonogenic assay of HCT116, SW480, Colo320, and RKO cells treated with 5-aza-dC (0.5 and 1 µM) and/or IR (2 Gy, 5 Gy, and 10 Gy). Clonogenic assay of irradiated (2 Gy, 5 Gy, and 10 Gy) DKO cells. Cells were seeded into 6-well plates (5000 cells/well) and treated with 5-aza-dC/IR. After 2 weeks, cultures were fixed with ethanol and stained with 1.25% crystal violet. Photographs of single colonies are also shown. (B–E) Survival fraction (SF) of HCT116/DKO (B), SW480 (C), Colo320 (D) and RKO (E) cells treated with 5-aza-dC (0.5 and 1 µM) and/or ionizing radiation (IR; 2 Gy, 5 Gy, and 10 Gy). The SF was calculated as mean colonies/seeded cells. The dose enhancement ratio was calculated as the ratio of the SF curve obtained by treatment with a combination of 5-aza-dC and IR to that obtained by treatment with IR alone. Data are expressed as the mean ± standard deviation of three independent experiments. P-values were calculated using Student's t-test. *P<0.05; **P<0.01; ***P<0.001.
Figure 2.
Growth inhibition in colon cancer cell lines in response to treatment with 5-aza-dC and irradiation.
(A) Cell growth curves obtained using 0.5 µM 5-aza-dC and two different radiation doses (2 Gy and 5 Gy) in colon cancer cells (HCT116, DKO and SW480) and (B) MTT assays in colon cancer cells treated with 5-aza-dC (0.5 µM) and/or irradiation (2 Gy and 5 Gy). Data are expressed as the mean ± standard deviation of triplicate experiments. (C) Tumor growth following 5-aza-dC (0.5 µM) treatment and/or irradiation (2 Gy and 5 Gy) in SCID mice. HCT116 cells (5×106 cells) that had been treated with 5-aza-dC (0.5 µM) and irradiated (2 Gy and 5 Gy) were injected subcutaneously into SCID mice (n = 4), and the average tumor size was measured once weekly for 7 weeks. P-values were calculated using Student's t-test. *P<0.05; **P<0.01.
Figure 3.
Cell cycle distributions of colon cancer cell lines in response to 5-aza-dC and irradiation exposure.
Colon cancer cells (HCT116, DKO and SW480) treated with 5-aza-dC (0.5 µM) and/or irradiation (2 Gy and 5 Gy) were stained with propidium iodide and analyzed using a FACS flow cytometer. Columns show the proportion of cells in each cell cycle phase. Black column, sub-G1 phase; bright gray column, G1 phase; dark gray column, S phase; white column, G2-M phase.
Figure 4.
Induction of apoptosis in colon cancer cells by 5-aza-dC or IR, both alone and in combination.
(A) The levels of apoptosis were measured using annexin V and 7-amino-actinomycin and analyzed using a FACS flow cytometer. The levels of apoptosis in HCT116, DKO and SW480 cells treated with 5-aza-dC (0.5 µM) and/or irradiation (2 Gy and 5 Gy) were expressed as percentages of the total cell population at both the early and late stages of apoptosis. (B) The activities of caspases 3 and 7 were determined using the Caspase-Glo assay and were represented as percentages of HCT116, DKO and SW480 cells treated with 5-aza-dC (0.5 µM) and/or irradiation (2 Gy and 5 Gy) compared with untreated cells. The graph represents data (mean ± standard deviation) from three independent experiments. (C) Representative micrographs of fluorescent DNA stain using the comet assay. DNA fragmentation by the comet assay in HCT116, SW480 and DKO cells treated with 0.5 µM 5-aza-dC and/or irradiation (2 Gy and 5 Gy). (D) Quantification of DNA damaged cells represents the mean of three random microscopic fields per sample, and the error bars represent ± standard deviations. NS indicates not significant. P-values were calculated using Student's t-test. *P<0.05; **P<0.01.
Figure 5.
Expression levels of apoptosis-associated proteins in colon cancer cells following treatment with 5-aza-dC or IR, both alone and in combination.
Western blot analysis for the expression of apoptosis-associated proteins (cleaved caspase 3, cleaved caspase 9, cleaved PARP1, survivin, and p53) in HCT116 and SW480 cells treated with 5-aza-dC (0.5 µM) and/or irradiation (2 Gy and 5 Gy), as well as in irradiated DKO cells, using cleaved caspase 3, cleaved caspase 9, cleaved PARP1, survivin, and p53 antibodies.