Casticin Induces DNA Damage and Affects DNA Repair Associated Protein Expression in Human Lung Cancer A549 Cells (Running Title: Casticin Induces DNA Damage in Lung Cancer Cells)

Abstract

Casticin was obtained from natural plants, and it has been shown to exert biological functions; however, no report concerns the induction of DNA damage and repair in human lung cancer cells. The objective of this study was to investigate the effects and molecular mechanism of casticin on DNA damage and repair in human lung cancer A549 cells. Cell viability was determined by flow cytometric assay. The DNA damage was evaluated by 4',6-diamidino-2-phenylindole (DAPI) staining and electrophoresis which included comet assay and DNA gel electrophoresis. The protein levels associated with DNA damage and repair were analyzed by western blotting. The expression and translocation of p-H2A.X were observed by confocal laser microscopy. Casticin reduced total viable cell number and induced DNA condensation, fragmentation, and damage in A549 cells. Furthermore, casticin increased p-ATM at 6 h and increased p-ATR and BRCA1 at 6-24 h treatment but decreased p-ATM at 24-48 h, as well as decreased p-ATR and BRCA1 at 48 h. Furthermore, casticin decreased p-p53 at 6-24 h but increased at 48 h. Casticin increased p-H2A.X and MDC1 at 6-48 h treatment. In addition, casticin increased PARP (cleavage) at 6, 24, and 48 h treatment, DNA-PKcs and MGMT at 48 h in A549 cells. Casticin induced the expressions and nuclear translocation of p-H2AX in A549 cells by confocal laser microscopy. Casticin reduced cell number through DNA damage and condensation in human lung cancer A549 cells.

Keywords: DNA condensation and repair; DNA damage; casticin; human lung cancer A549 cells.

Figure 1

Casticin decreased the percentage of viable cells in A549 cells. Cells were incubated with 0, 10, 20, 30, 40, and 50 μM of casticin for 48 h (A) or treated with 20 μM of casticin for 0, 6, 12, 24, and 48 h (B) and then were collected for measuring the percentage of viable cells by flow cytometry. Experiments were performed in triplicate as described in Materials and Methods. Data represent mean ± S.D. * p < 0.05 was significant difference between casticin-treated and control groups.

Figure 2

Casticin affected DNA condensation in A549 cells. Cells (1 × 10⁵ cells/well) were grown in 12-well plates for 24 h and incubated with 20 μM of casticin for 0, 6, 12, 24, and 48 h. Cells were fixed with 3.7% paraformaldehyde (v/v) in phosphate buffered saline (PBS) for 15 min, permeablized with 0.1% Triton X-100 in PBS for 5 min. Nuclei were stained with 2 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. All samples were examined and photographed using a fluorescence microscope at 200× (A) and were measured the intensity of fluorescence (B) as described in Materials and Methods. Data represent mean ± S.D. * p < 0.05 was significant difference between casticin-treated and control groups.

Figure 3

Casticin induced DNA damage in A549 cells. Cells were incubated with 20 μM of casticin for 24 and 48 h and analyzed by Comet assay (A) and then calculated the fluorescence intensity of comet (B) as described in Materials and Methods. Data represent mean ± S.D. * p < 0.05 was significant difference between casticin-treated and control groups.

Figure 4

Casticin induced DNA fragmentation in A549 cells. Cells were incubated with 20 μM of casticin for 0, 6, 12, 24, and 48 h. Then cells were collected and lysed and individual DNA was extracted for DNA gel electrophoresis as described in Materials and Methods.

Figure 5

Casticin affects the DNA damage and repair associated protein expressions in A549 cells. Cells were incubated with 20 μM casticin for 0, 6, 12, 24, and 48 h, the cells were collected for western blotting, and the resultant membranes were used to probe to anti-p-ATM, -p-ATR, -BRCA1, -p-p53, -p-H2A.X, -MDC1 (A) -PARP, -DNA-PKcs, and -MGMT (B) as described in Materials and Methods. β-actin was used as an internal control.

Figure 6

Casticin affected the translocation of p-H2A.X in A549 cells. Cells (5 × 10⁴ cells/well) were plated on 4-well chamber slides and incubated with 0 and 20 μM of casticin for 48 h, and cells were stained by anti-p-H2A.X (green fluorescence) and then stained with secondary antibody fluorescein isothiocyanate (FITC-conjugated goat anti-mouse IgG). All cells were counterstained by propidium iodide (PI) (red fluorescence) for nucleus examination and were photomicrographed under a Leica TCS SP2 Confocal Spectral Microscope as described in Materials and Methods.

Figure 7

The possible mechanism of casticin-induced DNA repair and cell death in A549 cells.