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

De-jian Dai, Cai-de Lu, Ri-yong Lai, Jun-ming Guo, Hua Meng
Zhonghua Yi Xue za Zhi [Chinese medical journal] 2004 November 17, 84 (22): 1894-8

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.

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