Figure 4

Effects of SM13 on tumour cell growth in vitro. (A) KAT-4 cells were treated with SM13 for 24 and 48 h, and cell proliferation was analyzed. SM13 reduced cell proliferation in a time-dependent manner (*P<0.05 vs control 24H; **P<0.05 vs control 48H). Results are representative of five independent experiments and are presented as mean±s.e.m. (B) To evaluate the mechanism of action of SM13, we analyzed its effect on mitochondrial-dependent apoptotic signalling by western blot. SM13-dependent increase of p53 induces activation of Bax, release of citochrome c from mitochondria and activation of caspase 9 and caspase 3. Images are the mean of three independent experiments. (C) To confirm the effect of SM13 on apoptosis, we evaluated DNA fragmentation through a TUNEL assay. Positive nuclei were counted and results were expressed in graph as mean±s.d. SM13 is able to induce apoptotic events in tumour cells. Images are representative of three independent experiments. (D) The ability of SM13 to induce apoptosis was evaluated in FRO cells, a tumour cell line, which do not express p53. p53 and cleaved caspase 3 levels were evaluated by western blot. SM13 was not able to regulate cleaved caspase 3 levels in FRO cells with respect to KAT-4 cells. Results are representative of three independent experiments. (E) To confirm the p53 transcription-independent effect of SM13, we evaluated gene expression of p53 target genes, p21 and Gadd45. In a p53 WT tumour cell line, MCF7, the treatment with SM13 induced p21 and Gadd45 gene expression, whereas in p53-mutant cell type, KAT-4, such phenomenon was reduced (*P<0.05 vs control). Results are the mean of five independent experiments and are presented as mean±s.e.m.