Figure 3

Ectopic expression of MT2A inhibits GC cells growth in vitro and in vivo. MT2A inhibited cell proliferation and colony formation in human GC cells. A, Western blot of MT2A was detected in BGC823 cells transfected with ectopic expression of MT2A plasmid and empty vector. The MT2A protein bands were scanned (normalized to β-actin to control for loading). B, Cell proliferation was analyzed by the MTT assay in re-expressing MT2A of BGC823 cells or not. Data was shown as the mean ± .SD of three independent experiments. C, Growth of BGC823 cells was analyzed in a semisolid soft agar medium. The empty vector colonies formed statistically significantly more colonies (mean = 426 large colonies per plate. 95% CIs: 387-472 colonies) than MT2A clones (mean = 136 colonies. 95% CIs: 100-164 colonies. The numbers represent the mean number of colonies of three independent experiments ± S.D. The relative number was calculated in control and BGC823-MT2A cells, and analyzed using Student’s t-test (P = 0.0038). D, MT2A-inhibited tumorigenicity of BGC823 cells in nude mice. The mean tumor volume for BGC823-MT2A at 3 weeks after injection was 112 mm³ (95% CIs: 93-147 mm³), compared with 352 mm³ (95% CIs: 298-423 mm³) for BGC823-Vector. Data represent the mean ± S.D of tumor volume derived from each group. In each group, tumor sizes were measured using a caliper at the indicated time points. Data were shown as mean ± S.D (t- test detection, P = 0.0027). E, FACS analysis showed that MT2A over-expression led to G2/M arrest, and the ratio of G2/M phase was higher than in empty control and parent cells (P < 0.01). F, Antitumor effect of MT2A was detected by annexin V/FITC-PI staining assay. Percentage of apoptotic cells was 52.9% ± 5.4% in MT2A group compared with that in the parent cells (8.3% ± 2.4% and cells transfected with the empty vector (8.2% ± 3.7%, P < 0.001).