Moreover, we identified that the compounds (1:4) were the most potent

Moreover, we identified that the compounds (1:4) were the most potent. to detect the subcellular localization of survivin proteins in treated and untreated RG7800 cells. RESULTS: Antisense compounds (1:4) down-regulated survivin expression (mRNA and protein) in a dose-dependent manner with an IC50 of 250 nmol/L. Its maximum effect was achieved at a concentration of 500 nmol/L, at which mRNA and protein levels were down-regulated by 80%. The similar results were found in MTT assay. Antisense compound (1:4)-treated cells revealed increased caspase-3-like protease activity compared with untreated cells. Untreated cells as control were primarily negative for the presence of active-caspase-3. As shown by transmission electron microscopy, treated cells with RG7800 antisense compounds (1:4) resulted in morphological changes such as blebbing and loss of microvilli, vacuolization in the cytoplasm, condensation of the cytoplasm and nuclei, and fragmented chromatin. Immunofluorescence analysis confirmed the presence of survivin protein pool inside the cytoplasm in untreated cells. Labeled-FITC immunofluorescence staining of survivin clearly showed that survivin was distributed mainly in a spotted form inside the cytoplasm. Whereas cells treated with antisense compounds were rare and weak inside the cytoplasm. CONCLUSION: Down-regulation of survivin expression induced by the antisense compounds reduces tumor growth potential, promotes apoptosis and affects the localization of survivin proteins in HepG2 cells. Furthermore, survivin protein is a key molecule associated with proliferation and apoptosis, and antisense oligonucleotides targeting survivin have a bright prospect in the therapy of liver cancer. test was used to compare the distribution of individual variables. A two-tailed value less than 0.05 was considered statistically significant. RESULTS Generation and optimization of antisense compound With the aim of obtaining the highest transfection efficiency and low non-specific effects, the compounds were generated using the antisense phosphorothioate oligonucleotides (ODNs) and highly effective Lipofectamine 2000 (LiP) by varying ODNs (g):LiP (L) ratios from 1:0.5 to 1 1:5. The ODNs designed by Olie et al[22], which targeted nucleotides (232-251) revealed the strongest effect. MTT was performed to test the effect of those compounds in surviving-HepG2 high-expression cells. As shown in Figure ?Figure1,1, the antisense compounds (1:4, 1:5) were identified as the most potent compounds. There was no significant difference in the effect between the compounds (1:4) and (1:5). Because the antisense compounds (1:0.5-1:3) could not achieve the best effect, and LiP had slight cytotoxicity, we only used the DP2.5 antisense compounds (1:4) in the following experiments. Open in a separate window Figure 1 Generation and optimization of antisense compound. Each value represents the meanSD of three independent experiments. Down-regulatory effects of antisense oligonucleotides on survivin expression levels To further characterize the potency of antisense compounds (1:4) and their dose dependent effect on survivin expression levels (mRNA and protein) in HepG2 cells, the cells highly expressing survivin were transfected with different concentrations of antisense compounds (100-600 nmol/L), whereas the cells transfected with sense compound (600 nmol/L), LiP (600 nmol/L) and untreated cells were used as controls. Forty-eight hours after the start of transfection, the cells were examined by RT-PCR and Western blot. As shown in Figure ?Figure2,2, antisense compounds (1:4) down-regulated the survivin expression level in a dose-dependent manner with an IC50 of about 250 nmol/L. At a concentration of 500 nmol/L, a maximum down-regulation to 20% of the initial mRNA level was achieved. A further increase in oligonucleotide concentration did not result in increased antisense efficacy. The sense RG7800 compound (600 nmol/L), LiP (600 nmol/L) and antisense compound controls without LiP (600 nmol/L) did not down-regulate the survivin expression level. Open in a separate window Figure 2 Survivin expression level (mRNA and protein) in HepG2 cells treated with antisense compound (1:4). A: Sensitivity of survivin mRNA in RT-PCR assay; B: Level of survivin mRNA expression in HePG2; C and D: Western blot analysis of survivin protein expression in HePG2 cells. Inhibition of cell growth by antisense compound (1:4) To analyze the biological effect associated with the down-regulation of survivin expression, the growth of HepG2 cells treated with antisense compounds was investigated by MTT assay. As shown in Figure ?Figure3A,3A, 48 h after the start of transfection, antisense compounds reduced the growth of HepG2 cells dose-dependently, with an IC50 of 300 nmol/L. The unspecific growth-inhibitory.