Fig 1.
Cytotoxic effects of 3,5-DMAP in A549 lung cells and HLF cells at different concentrations by using MTS assay.
3,5-DMAP showed higher cytotoxic property in A549 lung cells when compared to HLF cells. # shows p<0.05 compared with 3,5-DMAP-treated A549 samples.
Fig 2.
3,5-DMAP-induced high intracellular O2.- and ROS production caused lipid peroxidation, cytotoxicity and DNA damage in A549 cells.
(A) Intracellular O2.- levels were measured by MitoSOX kit. (B) The ROS level was measured by CM-H2DCFDA ROS detection kit. (C) O2.- and (D) ROS images of A549 cells treated with 3,5-DMAP. Magnification = 400X. Scale bar = 20 μm. Arrow heads point out O2.- spots. (E) Western blot suggests that 3,5-DMAP-induced ROS in turn induces SOD-1 and CAT expressions in A549 cells. (F) Lipid peroxidation was detected using MDA assay kit. (G) MTS assay detected the cytotoxicity in response to 3,5-DMAP exposures. The data was normalized and presented as percentage of the control. (H) Cells were treated with 0, 25 and 50 μM 3,5-DMAP, 50–150 comets were collected and analyzed for each experiment. Fluorescence microscopy magnification = 400X. Scale bar = 20 μm. % tail DNA and tail moment (a function of the distance and intensity of DNA from the center of the comet head) are quantified and significant DNA damage was detected after treatment with two different doses of 3,5-DMAP (25 and 50 μM). # and § shows p<0.05 and p<0.01 compared with 3,5-DMAP-treated samples.
Fig 3.
3,5-DMAP caused both activation of apoptosis and cell cycle arrest.
(A) The expression of apoptotic marker proteins p53, Bax, Bad and cytochrome c, and anti-apoptotic marker Bcl-2 were determined by Western blotting. α–tubulin was used as the internal control. (B) Quantification of p53, Bax, Bad and cytochrome c and Bcl-2 as the relative change of the control. (C) Cell cycle analysis. The values represent the number of cells at various phases of cell cycle as a likely percentage of total cells. (D) Quantification analysis of G1, S, and G2/M cell cycle from flow cytometry. Cell cycle percentages were derived from flow cytometric analysis of total cell number in each group.
Fig 4.
3,5-DMAP induced caspase 3-mediated apoptosis in A549 cells.
(A) Apoptosis detection was performed using the TUNEL assay, which was visualized with immunofluorescence microscopy 100X magnifications (Olympus IX51 Inverted Microscope) with emission at 495–529 nm. Scale bar = 50 μm. Nuclei were stained with DAPI, shown in the upper panel. Green spots in the lower panel present apoptotic cells. The cells treated with cisplatin (25 μM) and H2O2 (100 μM) are the positive controls for apoptosis and necrosis, respectively. (B) Quantitation of TUNEL assay. Values were divided by the amount of nuclei stain in the assessed region and expressed as the number of apoptosis events per 100 cells. (C) Apoptotic cells were identified by flow cytometric analysis using Annexin V-FITC and PI staining. (D) Dose-responses based on all data are plotted. (E) 3,5-DMAP-induced ROS caused caspase 3/7 activity. § shows p<0.01 compared with 3,5-DMAP-treated samples.
Fig 5.
3,5-DMAP exposure decline the carcinogenic property of A549 lung cells.
(A) The migrated cells were visualized with using a light microscope with 200 × magnification. Scale bar = 50 μm. (B) Values were presented as the fold change of the control. (C) Two million of cells were injected subcutaneously into the nude mice. The down panel shows the representative subcutaneous flank tumor in the mice. Scale bar = 1 cm. (D) Body weight is indicated by plotted and it shows no difference between the samples. (E) Plots of tumor volumes (cm3) determined by measurements with a caliper. (F) Plots of Growth Inhibition (GI) were calculated by tumor volumes. (G) The 4 μm tumor sections were examined by H&E staining. Magnification = 200×. Scale bar = 50 μm. # shows p<0.05 compared with 3,5-DMAP-treated cells.
Fig 6.
3,5-DMAP exposure down-regulates the expression of oncogene c-Myc and induces apoptosis.
(A) The 4 μm tumor sections were examined by c-Myc, p53 and cytochrome c immunohistochemistry staining. Magnification = 200×. Scale bar = 25 μm. (B) Quantitative analysis of c-myc expression. (C) Quantitative analysis of p53 expression. (D) Quantitative analysis of cytochrome c expression.
Fig 7.
Pathway for production of ROS by 3,5-DMAP.
3,5-DMA undergoes biotransformation with N–O bond heterolysis and converts to an unstable N-OH-3,5-DMA and highly reactive intermediate nitrenium ion, which is able to react with a DNA base to produce a mutagenic adduct [25]. 3,5-DMAP can be produced by cytochrome P450-catalyzed hydroxylation of 3,5-DMA or by nucleophilic attack of H2O on the appropriate resonance form of the nitrenium ion. 3,5-DMAP readily undergoes redox cycling mechanism and converts to 3,5-dimethylquinoneimine (3,5-DMQI). The process accompanies generation of superoxide ion (O2.-). NAC can attenuate this cycling.