Figure 1.
UA induced cell proliferation inhibition and morphology change.
Human colon cancer cell line SW480 and LoVo and normal cell lines CCD841 and LO2 were treated with UA at the indicated doses. At 48 hours after treatment, cell viability was determined by a MTT assay (A, E), and the tumor cell-induced clone formation was analyzed (B, C). The changes in cell morphology, spreading and blebbing in cells treated without or with UA (10 µM and 20 µM) for 48 h were observed and cells were photographed using a microscope fitted with digital camera (D). The percent cell viability or clone formation ratio in each treatment group was calculated relative to cells treated with DMSO vehicle control. The data are presented as mean ± SD of three separate experiments. *, P<0.05, significant differences between treatment groups and DMSO control groups.
Figure 2.
UA inhibited cell migration inhibition by regulating MMP9 and CDH1 expression.
(A), Cell migration was analyzed by a scratch assay. SW480 and LoVo cells were grown to full confluency. The cell monolayers were wounded with a sterile pipette tip, and washed with medium to remove detached cells from the plates. Cells were left either untreated or treated with indicated doses of UA. After 56 h, the wound gap was observed and cells were photographed. (B, C), SW480 cells were treated with UA at the indicated doses. At 56 hours after treatment, the expression of MMP9 (B) or CDH1 (C) was quantitatively analyzed by a real-time qPCR analysis. *, P<0.05, significant differences between the UA-treated groups and the DMSO-treated groups.
Figure 3.
UA inactivated Akt/ERK signaling to inhibit cell proloferation.
(A, B), SW480 cells were treated with UA at 20µM or 40 µM for 48 h. The expression of the phosphorylated or total PI3K, Akt, mTOR, PTEN, JNK, ERK1/2 proteins was detected by Western blot. GAPDH was used as a control for sample loading. (C, D), SW480 cells were treated with a PI3K/Akt-selective inhibitor LY294002 (LY, 5 µM) (C) or with an ERK-selective inhibitor U0126 (20 µM) (D) for 4 hours, and then treated with UA at 20 µM. At 48 hours after treatment, cell viability was determined by MTT analysis. The percent cell viability in each treatment group was calculated relative to cells treated with the vehicle control DMSO. The data are presented as the mean ± SD of three separate experiments. *, P<0.05, significant differences between treatment groups and control groups.
Figure 4.
UA suppressed COX-2/PGE2 signaling to inhibit cell proliferation.
(A–D), SW480 and LoVo cells were treated with UA for 48 h. The expression of COX-2 protein (A) and mRNA (B) were analyzed by Western blotting and RT-PCR, respectively. The densities of COX-2 mRNA and the ratio of COX-2/GAPDH were quantitatively analyzed (C). The production of PGE2 was detected by ELSIA analysis (D). GAPDH was used as controls for sample loading. (E), SW480 cells were treated with a COX-2-selective inhibitor celecoxib (CB, 20 µM) for 4 hours, and then treated with UA at 20 µM. At 48 hours after treatment, cell viability was determined by MTT analysis. The data are presented as the mean ± SD of three separate experiments. *, P<0.05, significant differences between treatment groups and control groups.
Figure 5.
UA induced the translocation of NF-κB and p300 from nuclei to cytoplasm.
Human SW480 and LoVo cells grown on chamber slides were treated with UA. At 48 hours after treatment, the sub-cellular localization of p50, p65 and p300 and the co-localization of p65 with p50 (A) or p300 (B) were examined by confocal microscopy analysis with a confocal microscope. More than 100 cells were inspected per experiment, and cells with typical morphology were presented.
Figure 6.
UA targeted p300 signaling to inhibit NF-κB and CREB2 acetylation and cell proliferation.
(A, B), SW480 cells were transfected with FLAG-p300 for 24 h and then treated with UA for 48 h. The nuclear extracts were prepared and p50, p65 and CREB2 were immunoprecipitated with an acetyl-lysine antibody. The acetylated proteins (A) and the protein expression (B) of p50, p65 or CREB2 were analyzed by Western blot. (C, D), SW480 cells were pretreated with a p300-selective inhibitor roscovitine (Rosc, 20 μM) (C), or transfected with a p300-expressing vector (D) for 8 hours, and then treated with UA. At 40 hours after treatment, the cell viability was determined. Empty vector (EV) was used a transfection control. Each bar represents mean ±SD of three experiments. *, P<0.05, significant differences between treatment groups and control groups.
Figure 7.
UA activated the cytochroem c/caspase-dependent pathway to induce apoptosis.
SW480 and LoVo cells were treated with UA (20 µM and 40 µM). At 48 hours after treatment, the apoptotsis was determined by an AnnexinV-FITC staining-based FACS analysis (A, B). The levels of the cleaved caspase-3, caspase-9 and PARP proteins in SW480 cells were analyzed by Western blot (C). The release of cytochrome c (Cyto-c) from the inter-mitochondrial space into the cytosol was determined by immunofluorescence imaging analysis (D). SW480 cells were transfected with p300 siRNA for 24 hours and followed by the treatment with UA (20 µM). At 48 hours after treatment, the apoptotsis was determined by FACS analysis (E). The apoptosis are represented by relative percentages of apoptotic cells versus that in DMSO-treated cells. *, P<0.05, significant differences between the UA-treated groups and the control groups.
Figure 8.
The proposed mechanisms by which UA suppress proliferation and induce apoptosis in colon cancer cells.
UA inhibited cell proliferation and migration and induced apoptosis in colon cancer cells by simultaneously modulating the MMP9/CDH1, Akt/ERK, COX-2/PGE2, p300/NF-κB/CREB2, and cytochrome c/caspase-dependent signaling pathways.