Figure 1.
IR dose–response survival curves and cytotoxic effects resulting from SAHA and/or IR treatment in breast cancer cells.
(A) Concentration-dependent effects of SAHA on histone and nonhistone proteins for 24 hrs in 4T1 cells. (B) Concentration-dependent effects of SAHA on the viability of breast cancer cells. Cells were treated with 1, 3, 5 or 7 µM SAHA for 24 hrs. *, p<0.05, SAHA versus control. (C) Dose-dependent effects of IR on the viability of breast cancer cells. Cells were treated with 2, 4, 6 or 8 Gy IR for 24 hrs. *, p<0.05, IR versus control. (D) Cytotoxic effects in cells treated with IR (4 Gy) and/or SAHA (3 µM). #, p<0.05, IR versus combined treatment. *, p<0.05, SAHA versus combined treatment. (E) Clonogenic assay in 4T1 cells treated with IR (4 Gy) and/or SAHA (3 µM). (F) The radiation dose-response survival curves of 4T1 cells with or without SAHA. Data are presented as the mean ± standard deviation of three independent experiments.
Figure 2.
DNA damage, DNA repair and ER stress induced by IR and/or SAHA in 4T1 cells.
(A) Time-dependent effects of IR on the expression of γH2AX. Cells were treated with IR (4 Gy). (B) Western blotting for γH2AX, Rad51 and DNA-PK in 4T1 cells. (C) Comet assay induced by IR and/or SAHA. The tails indicate DNA damage. The effect of SAHA (3 µM) or IR (4 Gy) alone or in combination for 24 hrs on average tail DNA. #, p<0.05, IR versus combined treatment. *, p<0.05, SAHA versus combined treatment. (D) ER staining enhanced by IR and/or SAHA. Cells were treated with IR (4 Gy) and SAHA (3 µM) for 24 hrs and treated with ER Tracker Blue-White DPX for ER staining. (E) Western blotting for IRE1α, phospho-eIF2α and eIF2 in 4T1 cells. Cells were treated with IR (4 Gy) and SAHA (3 µM) for 24 hrs.
Figure 3.
Measurement of apoptosis and autophagy in breast cancer cells that received various treatments.
(A) Early apoptosis, detected using an Annexin V apoptosis detection kit, was measured using flow cytometry. Cells were treated with IR (4 Gy) and SAHA (3 µM) for 6, 12 or 24 hrs. (B) Microphotograph of AVOs in 4T1 cells. Detection of green and red fluorescence in acridine orange (AO)-stained cells was performed using a fluorescence microscope. Cells were treated with IR (4 Gy) and SAHA (3 µM) for 24 hrs. (C) Quantification of AVOs with AO-stained cells treated with IR (4 Gy) or SAHA (3 µM) alone or in combination using flow cytometry. #, p<0.05, IR versus combined treatment. *, p<0.05, SAHA versus combined treatment. (D) Western blotting for LC3-I, LC3-II and Beclin 1 in 4T1 cells. Cells were treated with IR (4 Gy) and SAHA (3 µM) for 12 hrs. (E) Immunofluorescence staining of LC3 protein in 4T1 cells treated with IR (4 Gy) and SAHA (3 µM) for 12 hrs. #, p<0.05, IR versus combined treatment. *, p<0.05, SAHA versus combined treatment. (F) EM microphotographs of 4T1 cells treated with IR (4 Gy) and SAHA (3 µM) for 24 hrs. The black arrows point to autophagic vacuoles and autolysosomes. (G) Cytotoxic effects in the absence or presence of 3-MA. Cells were pretreated with 3-MA for 1 hr before combined treatment. *, p<0.05, combined treatment versus combined treatment +3-MA. Data are presented as the mean ± standard deviation of three independent experiments.
Figure 4.
Tumor growth and body weight of the orthotopic breast cancer model mice treated with IR (4 Gy) or SAHA (25 mg/kg×9) alone or in combination.
(A) Body weight in Balb/c mice measured once per week. (B) The 4T1-luc cells were injected into mammary fat pads of Balb/c mice, observed for luciferase signals and photographed using IVIS 200. (C) Quantification of the luciferase signals. *, p<0.05, versus control. (D) IHC staining of the mouse orthotopic tumor tissues. IHC was used to determine the expression levels of LC3 and γH2AX (×100 objective magnification). The percentage of LC3 and γH2AX-positive cells was determined using HistoQuest software (TissueGnostics).
Table 1.
Biochemistry tests including GOT, GPT, albumin, BUN, and creatinine.
Figure 5.
Effects of SAHA on cell viability, migration and invasion of 4T1 cells.
(A) Concentration-dependent effects of SAHA on the viability of 4T1 cells. SAHA inhibits 4T1 cell migration in a wound healing assay (B) and Trans-well migration assay (C). (D) The invasive ability of 4T1 cells was determined using a Matrigel invasion assay. MMP-9 enzyme activity was determined using a gelatin zymography assay (E) and western blot analysis (F) of conditioned medium. Cells were treated with SAHA for 24 hrs. Data are presented as the mean ± standard deviation of three independent experiments.
Figure 6.
SAHA inhibits experimental metastasis in Balb/c mice.
(A) Body weight in Balb/c mice measured once per week. (B) Lung weights of mice. *, p<0.05, versus control. (C) The 4T1-luc cells were observed for the luciferase signals and photographed using IVIS 200. (D) Histological characteristics of mouse lungs stained with H&E. Tumor metastasis is indicated by arrows.
Table 2.
Biochemistry tests including GOT, GPT, albumin, BUN, and creatinine.