Fig 1.
Cell viability following 5-AzaD treatment.
Cell viability was detected by XTT following treatment of FaDu cells with increasing concentrations of 5-AzaD (0, 0.1, 0.5,1,2.5,5 and 10 μM) for 48 h (A). FaDu and normal fibroblast cells (NDHF) were treated with 5 μM of AzaD for a period of 24, 48 and 72 h (B). Untreated control cells, FaDu and NDHF, were exposed to 0.01% DMSO. Data were presented as mean ± SD of three independent experiments in which each treatment was performed in eight replicates, and are expressed as percentages of the respective untreated control cells that was defined as 100%. Statistical significance was determined by two-tailed Student’s t-test. Significance is indicated as **P<0.01, ***P<0.001.
Fig 2.
Cell cycle analysis of FaDu cells following 5-AzaD treatment.
FaDu cells were treated with 5 μM of 5-AzaD for 24, 48 and 72 h; Untreated FaDu cells (control) were exposed to 0.01% DMSO. Cells were subjected to cell cycle analysis by flow cytometry as described under “Materials and methods”. The graph summarize percentages of cells in sub-G1 and G2/M phases following treatment. The results are presented as mean ± SD of four experiments each conducted in duplicates and are expressed as percentages from total of 10,000 analysed cells. Statistical significance was determined by two-tailed student’s t-test (treatment VS. control) and is indicated as *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 3.
Measurement of apoptotic cells using an Annexin V binding.
FaDu cells were treated with 5 μM of 5-AzaD for 24, 48, 72 h. Untreated FaDu cells were exposed to 0.01% DMSO. Analysis of Annexin V-FITC/PI double-stained was performed using flow cytometry. In a representative plot (A), the power left quadrant (Q3) represents viable cells, the upper left quadrant (Q1) indicates necrotic cells, the lower right quadrant (Q4) denotes early apoptotic cells and the upper right quadrant (Q2) represents late apoptotic cells. The percentage of Annexin V-FITC positive cells from the early and late stage of apoptosis following treatment for 24, 48 and 72h is shown. Data are presented as mean ± SE of three independent experiments, each conducted in duplicates [mean (B)]. Statistical significance was determined by two-tailed student’s t-test [treatment vs. control (untreated cells)] and is indicated as **p < 0.01, ***p < 0.001.
Fig 4.
Activation of caspases following 5-AzaD treatment.
FaDu cells were treated with 5 μM of 5-AzaD for 48 h. Untreated FaDu cells were exposed to 0.01% DMSO. Total protein was extracted and 60-μg was separated on SDS-PAGE and transferred to nitrocellulose membranes. Caspase 3 (A), Caspase 8 (B), Caspase 9 (C), Cleaved PARP (D), and Cytochrome C (E) were detected by using specific primary antibodies. For equalization of protein amounts, mouse anti-human β-actin monoclonal antibody was used. The figure shown is representative of three independent experiments. Results were analysed with Amersham Imager 600.
Fig 5.
The effect of 5-AzaD on tumour development using xenograft mice.
Athymic nude male mice were SC implanted with 1X106 FaDu cells. When the tumours reached a volume of ~200 mm3 the mice were divided into 2 groups (n = 8). Mice were intraperitoneal (IP) injected three times a week either with PBS X 1 to the control group, or with 2.5 mg/kg of 5-AzaD for 3 weeks. During the experiment, tumour volumes were measured twice a week using calibre meter (A). On day 21 at the end of the experiments, mice were sacrificed, and the tumour tissues were dissected and measured for weight (B) and volume (C). The results were presented as the mean± SD (n = 8). Statistical significance was determined by two tailed student’s–t–test and assigned as **P < 0.01, ***P < 0.001. In order to better understand how 5-AzaD treatment affects tumour growth in vivo, tumours were fixed and sectioned for histological staining. H&E staining indicated that 5-AzaD treatment induced apoptosis of tumour cells and inhibited mitosis when comparing to the control group (Fig 6A). Morphological criteria for diagnosis of mitosis, using the H&E staining, included reporting of increased mitosis above or below the normal background levels, of what is usually seen in control animals [22].
Fig 6.
H&E stained images and Ki-67 and active caspase 3 immunostaining of tumour sections from control and treated animals.
Nude mice bearing FaDu tumour cells were treated with 2.5 mg/kg of AzaD or PBS 3 times a week for 3 weeks. At the end of the experiment, mice were sacrificed and the tumour tissues were dissected. (A) Representative H&E stained images of isolated tumours are shown. Mitotic cells in the control and apoptotic cells in the treated groups are marked with arrows. (B) Proliferation profiles of the cells were detected by Ki-67 immunostaining as described under “Material and Methods”. The images are representatives of the results obtained from control and treatment groups (Magnification, 400X). (C) Apoptotic cells were detected by caspase 3 immunostaining positivity; criteria are explained in the materials and methods section. The images are representatives of the results obtained from control and treatment groups.
Table 1.
Qquantitative analysis of Ki67 and caspase 3 on tumor sections.
Fig 7.
The effect of 5-AzaD on body weight.
Body weight of control and treated animals was measure twice a week through the experiment. Data are presented as Mean ± SE.