Curcumin-Induced DNA Demethylation in Human Gastric Cancer Cells Is Mediated by the DNA-Damage Response Pathway

Figure 4

Curcumin-induced cell cycle arrest and DNA demethylation are mediated by DDR. hGCCs were treated with 20 μM curcumin (or combined with 10 μM PFTα, a p53 specific inhibitor) for 24 or 48 h, and the amount of proteins related to DDR or its downstream signaling pathways, the cell cycle, and DNA methylation was determined. (a) PFTα treatment alleviated apoptosis of hGCCs caused by curcumin treatment (exhibited by the decrease in the number of round-shape, apoptosis cells). (b and c) The amount of phosphorylated γH2AX, phosphorylated p53, phosphorylated Rb, DNMT1, and β-actin (endogenous control protein) was determined by western blot, and the band density was analyzed by the ImageJ software. (d and e) The number of cells in different phases of the cell cycle was determined by flow cytometry. (f) One of the CGIs in L1Hs, which contained 370 bp and 19 of CpG sites, was amplified in methylation-specific PCR. (g) The methylation of this CGI was determined by bisulfite sequencing. Methylated and nonmethylated CpG sites are shown as black dots and cycle dots, respectively. (h) The percentage of methylated CpG sites was calculated as follows: . All experiments were carried out in triplicates; bisulfite sequencing was repeated five times. The data are shown as the . The single factor variance analysis was used to compare the significance between groups as: , , and . Difference with was considered statistically significant.

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