Liver Cancer
Copyright ©2005 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Jan 14, 2005; 11(2): 193-199
Published online Jan 14, 2005. doi: 10.3748/wjg.v11.i2.193
Survivin antisense compound inhibits proliferation and promotes apoptosis in liver cancer cells
De-Jian Dai, Cai-De Lu, Ri-Yong Lai, Jun-Ming Guo, Hua Meng, Wei-Sheng Chen, Jun Gu
De-Jian Dai, Hua Meng, Department of Surgery, Second Affiliated Hospital of Zhejiang University Medical School, Hangzhou 310009, Zhejiang Province, China
Cai-De Lu, Department of Surgery, Lihuili Hospital of Ningbo Medical Center, Ningbo University Medical School, Ningbo 315040, Zhejiang Province, China
Ri-Yong Lai, Department of Biochemistry and Molecular Biology, Gannan Medical College, Ganzhou 341000, Jiangxi Province, China
Jun-Ming Guo, Medical School, Ningbo University, Ningbo 315211, Zhejiang Province, China
Wei-Sheng Chen, Jun Gu, Ningbo Institute of Microcirculation and Henbane, Ningbo 315040, Zhejiang Province, China
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China, No.30171059
Correspondence to: Cai-De Lu, M.D., Department of Surgery, Lihuili Hospital of Ningbo Medical Center, Ningbo University Medical School, Ningbo 315040, Zhejiang Province, China. lucd71@hotmail.com
Telephone: +86-574-87392290-7707 Fax: +86-574-87392232
Received: April 7, 2004
Revised: April 8, 2004
Accepted: May 24, 2004
Published online: January 14, 2005
Abstract

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)-LipofectamineTM2000 (LiP) compound by varying ODNs (μg):LiP (μL) 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.

Keywords: Liver cancer; Survivin; Cell proliferation; Apoptosis