PMC

At low concentrations, p,p′-DDE promotes colorectal adenocarcinoma cell proliferation through c-Myc and cyclin D1 overexpression resulted from the stimulation of Wnt/β-catenin and Hedgehog/Gli1 signalings, which were mediated by oxidative stress.

After DLD1 or SW620 cells were exposed to 10⁻¹² to 10⁻⁶ M p,p′-DDE for 96 h, cell viability (A) and proliferation rate (B) were determined using MTT and cell number assays, respectively. Values are percent changes above the control (DMSO, 0.1%) as the mean ± SD of three independent experiments. *p<0.05 and **p<0.01 compared to control cells.

After DLD1 or SW620 cells were exposed to p,p′-DDE (10⁻¹⁰, 10⁻⁹, 10⁻⁸ M) for 96 h, the levels of Nox1 (A), p22^phox (B), p40^phox (C), p47^phox (D) and p67^phox (E) mRNA expression were determined by quantitative real-time PCR and normalized to control mRNA. The data were acquired from three biologically independent experiments. Values shown were given as the ± SD. *p<0.05 and **p<0.01 compared to control.

After DLD1 or SW620 cells were treated with p,p′-DDE (10⁻⁹ M) alone or co-treated with NAC (10⁻³ M), SOD (100 U/ml) or CAT (500 U/ml) for 96 h, (A) ROS levels, (B) SOD activity, (C) GSH content, (D) CAT activity, (E) cell viability and (F) proliferation rate were analyzed. The results were the mean ± SD of three independent experiments. **p<0.01 compared to the cells treated with 10⁻⁹ M p,p′-DDE.

After DLD1 cells were treated with p,p′-DDE (10⁻⁹ M) alone or co-treated with NAC (10⁻³ M) (A), SOD (100 U/ml) or CAT (500 U/ml) (B) for 96 h, western blotting was performed to analyzed β-catenin, phospho-β-catenin (Ser33), phospho-GSK3β (Ser9), GSK3β, Gli1, c-Myc and cyclin D1. α-tubulin was used as the loading control. Grayscale scan analysis of western blot bands were shown in right panel of graph. (C) mRNA expression of PTCH1 was quantified by quantitative real-time PCR analysis. Relative mRNA levels were normalized with control mRNA. In (A), (B) and (C), value shown was given as the ± SD and acquired from three biologically independent experiments. **p<0.01 compared to the cells treated with 10⁻⁹ M p,p′-DDE.

After DLD1 cells were transfected with control siRNA or β-catenin siRNA, followed by treating with p,p′-DDE (10⁻⁹ M) for 96 h, (A) western blotting was performed to analyze β-catenin, c-Myc and cyclin D1. α-tubulin was used as a control; Images are representative of three independent experiments that showed similar results. Grayscale scan analysis of western blot bands were shown in right panel of graph. *p<0.05 and **p<0.01 compared to the cells only transfected with control siRNA. ^## p<0.01 compared to the cells transfected with β-catenin siRNA along with p,p′-DDE treatment. (B) Cell viability and (C) proliferation rate were analyzed by MTT and cell number assay. Values are percent as the mean ± SD of three independent experiments. **p<0.01 compared to control.

After DLD1 cells were transfected with control siRNA or Gli1 siRNA, followed by treating with p,p′-DDE (10⁻⁹ M) for 96 h, (A) Protein levels of Gli1, c-Myc and cyclin D1 were examined by western blotting. α-tubulin was used as a control. Grayscale scan analysis of western blot bands from three independent experiments were shown in right panel of graph. *p<0.05 and **p<0.01 compared to the cells only transfected with control siRNA. ^## p<0.01 compared to the cells transfected with Gli1 siRNA along with p,p′-DDE treatment. (B) Cell viability and (C) proliferation rate were analyzed by MTT and cell number assays. The results are percent as the mean ± SD of three independent experiments. **p<0.01 compared to control.

After DLD1 cells were treated with p,p′-DDE (10⁻¹⁰, 10⁻⁹, 10⁻⁸ M) for 96 h, (A) the protein levels of β-catenin, phospho-β-catenin (Ser33), phospho-GSK3β (Ser9), GSK3β, Gli1, c-Myc, cyclin D1 and PCNA were examined by western blotting. α-tubulin was used as the loading control; Grayscale scan analysis of western blot bands from three independent experiments were shown in right panel of graph. **p<0.01 compared to control cells. (B) mRNA expression of PTCH1 was quantified by quantitative real-time PCR analysis. Relative mRNA levels were normalized with control mRNA. The data were acquired from three biologically independent experiments. Value shown was given as the ± SD. *p<0.05 and **p<0.01 compared to control. (C) Western blotting was performed to analyze the amounts of cytoplasmic or nuclear β-catenin and Gli1 proteins. α-tubulin or lamin B1 was used as a control. Grayscale scan analysis of western blot bands from three independent experiments were shown in right panel of graph. **p<0.01 compared to control cells.