Survivin antisense compound inhibits proliferation and promotes apoptosis in liver cancer cells.

AIM: To evaluate the effects of survivin on cell proliferation and apoptosis in liver cancer. METHODS: MTT assay was used to generate and optimize phosphorothioate antisense oligonucleotides (ODNs)-Lipofectamine2000 (LiP) compound by varying ODNs (mug):LiP (muL) ratios from 1:0.5 to 1:5. Then, liver cancer cells (HepG2) were transfected with the compound. By using RT-PCR and Western blot, the expression levels of survivin mRNA and proteins were detected in HepG2 cells treated with antisense compounds (ODNs:LiP = 1:4), and compared with those treated with sense compounds (1:4) as control. MTT assay was applied to the determination of cell proliferation in HepG2 cells. Active caspase-3 was evaluated by flow cytometric analysis. The morphological changes were assessed by electron microscopy. Laser scanning confocal microscopy was performed to detect the subcellular localization of survivin proteins in treated and untreated 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 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.

[Effect of survivin targeting on cell proliferation and apoptosis in pancreatic cancer cells].

OBJECTIVE: To investigate the effects of antisense survivin-Lip compound on pancreatic cancer cell proliferation and apoptosis and its mechanism. METHODS: Pancreatic cancer cells of the line PANC-1 were cultured. Survivin oligonucleotide (ODN) was transfected into the PANC-1 cells mediated by Lip reagent. The expression of survivin mRNA and that of surviving protein were detected by RT-PCR and Western blotting. MTT assay was applied to determine the proliferation of PANC-1 cells. Active caspase-3 and apoptosis rate were evaluated by flow cytometric analysis. The morphological changes were assessed by electron microscope. Lascar scanning confocal microscope immunofluorescence analysis was performed to detect the subcellular localization of survivin protein on treated cells and untreated cells. RESULTS: Antisense compound efficiently down-regulated the survivin expression (mRNA and protein) in dose-dependent manner with an IC50 of 300 nmol/L. Its maximum effect was achieved at the concentration of 500 nM, at which the expression level was down-regulated by 80%. The similar results were found in MTT assay. As revealed by gradually increased caspase-3-like protease activity and apoptosis rate in a time-dependent manner, and the morphological changes of apoptosis such as blebbing and loss of microvilli, vacualization in cytoplasm, condensation of cytoplasm and nucleus, and fragmented chromatin, treatment with antisense compound induced apoptosis and inhibited cell growth. Fluororescein isothiocyanate (FITC)-labeled immunofluorescence staining of survivin clearly showed that survivin was expressed mainly in the formation of a spotted distribution inside the cytoplasm of untreated cells. Survivin protein molecules were clearly seen in the cytoplasm of the untransfected cells and distributed like spots and almost disappeared in the transfected cells with morphological changes conforming to the changes of apoptosis. CONCLUSION: Survivin protein is a key molecular connecting proliferation with apoptosis and antisense oligonucleotides targeting survivin have a bright prospect in therapy of pancreatic cancer cells.