[Molecular mechanism of anti-apoptotic action of survivin in NCI-H446 lung cancer cells].
OBJECTIVE
To investigate cell apoptosis induced by survivin ASODN and clarify the precise mechanism of anti-apoptotic action of survivin.


METHODS
Cells of lung cancer cell line NCI-H446 were treated with survivin ASODN at different concentrations. The changes of survivin mRNA and protein expression were assessed by RT-PCR and Western blot assay. The apoptosis index (AI) and proliferation index (PI) were determined by flow cytometry (FCM). After 500 mmol/L survivin ASODN treatment, cells were stained with Rh123 to detect changes of mitochondrial membrane potential (deltapsim) by FCM. The concentration of cytoplasmic cytochrome c (cyt-c) was continuously determined by ELISA. Relative activities of caspase-9 and caspase-3 were assessed by colorimetric assay. The expression of caspase-8 protein was measured by Western blot assay. The apoptotic rates of lung cancer cells induced by survivin ASODN with or without mitochondrial permeability transition pole (MPTP) inhibitor CsA treatment were assessed by FCM.


RESULTS
Down-regulated survivin mRNA was shown to be in dose-dependent and time-dependent manners. Its maximal effect was achieved at a concentration of 500 nmol/L for 72 h, at which mRNA was down-regulated by 62.7%, the expression of survivin protein in NCI-H446 cells was also obviously decreased. After treatment with survivin ASODN at concentration of 500 mmol/L for 72 h, AI was 48.35%, higher than that of control, lipofectin, NSODN, survivin ASODN 100 mmol/L and 300 mmol/L groups (3.75%, 3.41%, 4.69%, 19.85% and 34.39%, respectively). PI was 24.38%, lower than that of control, lipofectin, NSODN, survivin ASODN100 and 300 mmol/L groups (75.74%, 73.12%, 71.76%, 51.03% and 38.94%, respectively). Deltapsim was decreased in 9.54% of NCI-H446 cells treated with survivin ASODN for 3 h and 97.06% for 24 h. Following it, release of cyt-c from mitochondria to cytosol and activation of caspase-9 and caspase-3 increased significantly. The above mentioned indicators changed with a time-dependent and time diversity relationship. In the presence of CsA, the apoptotic rate of lung cancer cells induced by survivin ASODN was decreased significantly. No up-regrulation and activation in caspase-8 protein was observed.


CONCLUSIONS
Survivin inhibits apoptosis via regulation of mitochondrial-dependent pathway. survivin ASODN can not only induce apoptosis but also inhibit cell proliferation through blocking the expression of survivin mRNA and protein.
Chen
Yu- Qing
Y
Department of Respiratory Medicine, Affiliated Hospital of Bengbu Medical College, Bengbu 233004, China. bbmccyq@126.com
Li
Wei
W
Zhou
Ji-Hong
JH
Li
Dian-Ming
DM
Xia
Xue-Mei
XM
Huang
Li-Nian
LN
Li
Bai-Qing
BQ
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016572:Cyclosporine / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D015536:Down-Regulation; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D051034:Inhibitor of Apoptosis Proteins; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D000077022:Survivin; D014162:Transfection
C107489:BIRC5 protein, human; D007166:Immunosuppressive Agents; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D000077022:Survivin; D016572:Cyclosporine; D045304:Cytochromes c; D053453:Caspase 9