Enes inhibit autophagy [11]. We thus hypothesized that the GC 14 custom synthesis klotho gene could also regulate autophagy in GC. In this study, we investigated the involvement of klotho in GC cell apoptosis and autophagy at the same time as the linked signaling by delivering klotho gene expression vector into two GC cell lines. Our study provided the evidence for klotho’s regulation of signaling involved in cell survival, proliferation, and apoptosis in GC.ResultsDifference in klotho gene expression and promoter methylation amongst gastric cancer and normal cellsThe mRNA expression of klotho gene was detected by RT-PCR and naturally reduce klotho expression was observed in MNK-45, AGS, and GC-7901 gastric cancer cells than in the GES-1 typical gastric epithelial cells (Figure 1A). Western blot also showed decrease klotho protein level in tumor cells than in normal cells (Figure 1B, C). The bisulfate-based PCR strategy was applied to examine the CpG methylation of klotho gene promoter. As shown in Figure 1D, the tested CpG website exhibited virtually complete methylation in GC-7901 cells, partial methylation in MNK-45 and AGS cells, but just about no methylation in GES-1 regular gastric epithelial cells.Restoration of klotho expression by a demethylating agent increases tumor cell apoptosis and authophagyAmong the 3 tested gastric cancer cells, GC-7901 showed the lowest expression of klotho mRNA and protein and highest level in methylation of klotho promoter.GC -7 90 1 GE S250 bp 100 bp 250 bp 100 bpMklothoGAPDHC#DMNK-45 AGS GC-7901 GES-M250Me UM Me UM Me UM Me UMFigure 1 Klotho gene expression and methylation. A) RT-PCR detection of klotho gene mRNA expression in GES-1 typical gastric epithelial cells and, MNK-45, AGS, and GC-7901 gastric cancer cells. B) Western blot of klotho protein expression. C) The relative klotho protein levels in B). D) Methylation of klotho gene promoter. The methylated (Me) and unmethylated (UM) klotho gene promoter fragments have been amplified from bisulfite-treated genomic DNA by PCR. M: DNA ladder.GC -7 90 1 GE SM NK -4 five AG SM NK -4 5 AG SABklotho GAPDHXie et al. Cancer Cell International 2013, 13:18 http://www.cancerci.com/AOH1160 supplier content/13/1/Page 3 ofTherefore, GC-7901 cells were chosen for further tests. As shown in Figure two, DNA demethylating agent 20-deoxy-5azacytidine (5-Aza) enhanced klotho protein level in a dose-dependent manner (Figure 2A, B). The flow cytometry assay revealed that 5-Aza can restore klotho gene expression and substantially induce cell apoptosis in GC-7901 cells (Figure 2C, D). We additional tested irrespective of whether a DNA demethylating agent could affect autophagy and whether or not autophagy inhibitor (3-MA) could block it. GC7901 cells were treated with 5, ten, and 20 mol/L of 5Aza for 12 hrs. 5-Aza dose-dependently decreased LC3-I level and enhanced LC3-II level (Figure 3A) using a substantial increase in LC3-II/LC3-I ratio (Figure 3B), suggesting that a demethylating agent could significantly boost autophagy in gastric cancer cells. To test whether 3-MA could block the effect of 5-Aza in inducing autophagy, GC-7901 cells had been incubated with 10 mol/L of 5-Azaand 10 mM of 3-MA for 8 hrs. Benefits showed that ten mM of 3-MA considerably blocked the function of ten mol/ L of 5-Aza in inducing LC3-II expression and also the ratio of LC3-II/LC3-I (Figure 3C, D).The signaling adjustments in GC-7901 cells subjected to demethylating agent and autophagy inhibitor treatmentThe total protein and phosphorylated protein in demethylating agent and autophagy inhibitor-treated G.
