[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.


CONCLUSIONS
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.
Dai
De-jian
DJ
Department of Surgery, Lihuili Hospital of Ningbo Medical Center, Ningbo University Medical School, Ningbo 315040, China.
Lu
Cai-de
CD
Lai
Ri-yong
RY
Guo
Jun-ming
JM
Meng
Hua
H
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016503:Drug Delivery Systems / Q000379:methods; D005260:Female; D015316:Genetic Therapy; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008081:Liposomes / Q000494:pharmacology; D008297:Male; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D010190:Pancreatic Neoplasms / Q000473:pathology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D000077022:Survivin; D014162:Transfection
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008081:Liposomes; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D000077022:Survivin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases