Figure 7. Downregulation of NDRG2 is associated with enhanced phosphorylation of PTEN-Ser380/Thr382/Thr383 and enhanced activation of PI3K-AKT in various cancers.

(a) Bisulfite genomic sequencing of the NDRG2 promoter region in the KLM1 (pancreatic cancer), SKOV3 (ovarian cancer), HeLa (cervical cancer), HepG2 (hepatic cancer) and KatoIII (gastric cancer) cell lines. PCR products amplified from bisulfite-treated genomic DNA were subcloned, and ten clones in each cell line were sequenced. Open circles indicate unmethylated CpGs (Thy) and filled circles indicate methylated CpGs (Cyt). The region sequenced spans from −396 bp to −133 bp. (b) SKOV3, HepG2 and KLM1 cells were cultured with 10 μM 5-aza-dC for 72 h, with 1.2 μM TSA for 48 h, or with 1.2 μM of TSA for 48 h, followed by 10 μM of 5-aza-dC for 24 h. After treatments, total RNA was extracted and quantitative RT–PCR was performed with NDRG2 and β-actin. The relative amounts of mRNA were normalized against β-actin mRNA and expressed relative to the mRNA abundance in untreated cells. The mean±s.d. is shown; *P<0.05 compared with the untreated control (Student’s t-test). The data are representative of two experiments. (c) Western blot analyses of NDRG2, PTEN, p-PTEN (Ser380/Thr382/Thr383), AKT and p-AKT (Ser473) were performed in SAS (OSCC), HSC3 (OSCC), KLM1, SKOV3, HeLa, MT2 (ATLL), KOB (ATLL) and MOLT4 (T-ALL) cell lines. The data are representative of two experiments. (d) HeLa and SAS cells stably transfected with Mock or FLAG-NDRG2 expression vector were subjected to western blotting. The data are representative of three experiments.