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


CONCLUSIONS
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.
Dai
De-Jian
DJ
Department of Surgery, Second Affiliated Hospital of Zhejiang University Medical School, Hangzhou, Zhejiang Province, China.
Lu
Cai-De
CD
Lai
Ri-Yong
RY
Guo
Jun-Ming
JM
Meng
Hua
H
Chen
Wei-Sheng
WS
Gu
Jun
J
D000951:Antigens, Neoplasm / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects*; D045744:Cell Line, Tumor; D017931:DNA Primers; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008113:Liver Neoplasms / Q000473:pathology*; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D009363:Neoplasm Proteins; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077022:Survivin
D000951:Antigens, Neoplasm; C107489:BIRC5 protein, human; D017931:DNA Primers; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D000077022:Survivin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases