Antitumor activity of NF-kB decoy oligodeoxynucleotides in a prostate cancer cell line.
BACKGROUND
Nuclear factor-kappaB (NF-kB), a transcription factor, is abundantly expressed in prostate cancer and regulates many tumor-related genes. Given the important roles of these genes in tumor control, the present study was conducted to test the hypothesis that there was different expression of NF-kB in androgen- dependent or androgen-independent prostate cancer cells. In addition NF-kB decoy oligodeoxynucleotides (ODNs) were transfected into two prostate cancer cells to determine affects on growth and apoptosis.


METHODS
First, NF-kB decoy ODNs were designed according to the NF-κB elements in the promoter region of c-myc gene. Then, NF-kB and control decoy ODNs were transfected with lipofectamine. Their influence on prostate cancer cell line proliferative activity was detected by MTT assay. Cell apoptosis was determined by flow cytometric(FCM) analysis and AO/EB study. Thirdly, nuclear extracts were prepared from PC-3M cells and DNA-protein interactions were examined by electrophoretic mobility shift assay (EMSA). Lastly, to confirm mechanisms of action, a pGL3-C-MYC luciferase expression vector containing a fragment of the c-myc promoter was constructed and co-transfected with NF-kB decoy ODNs into PC-3M cells with lipofectamineTM2000. Expression levels of related endogenous genes were assessed by western blotting.


RESULTS
We found overexpression of NF-kB in the androgen-independent prostate cancer cell line PC-3M compared to the androgen-independent LNCaP. Treatment with NF-kB decoy ODNs resulted in strong suppression of proliferation, especially in the PC-3M case. Induction of apoptosis of PC-3M was observed in FCM and AO/EB studies. Activity of luciferase was significantly reduced in the NF-kB decoy-transfected cells, but not in cells transfected with a control decoy. Furthermore, we found that expression of some endogenous genes was reduced, while other genes transcripts were induced. EMSA demonstrated specific binding of the NF-kB decoy to NF-kB protein.


CONCLUSIONS
These findings indicate that NF-kB activation plays an important role in evolution of androgen-independent prostate cancer via manipulating expression of target genes. Inhibitors of NF-kB may thus offer promise as a therapeutic approach for the treatment of androgen-independent prostate cancer. NF-kB decoy ODNs may allow development of therapeutic and investigative tools for human malignancies.
School of Basic Medicine, Taishan Medical University, China.
Yongqi
Y
Fang
Hongli
H
Sun
Jing
J
Zhai
Yuanying
Y
Zhang
Shuying
S
Yi
Gangping
G
Hao
Tao
T
Wang
D000728:Androgens; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D019938:Cyclin D1 / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D051858:Forkhead Transcription Factors / Q000378:metabolism; D016259:Genes, myc / Q000235:genetics; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D008055:Lipids / Q000235:genetics; D008297:Male; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*
D000728:Androgens; C530104:CCND1 protein, human; C090173:FOXG1 protein, human; D051858:Forkhead Transcription Factors; D051034:Inhibitor of Apoptosis Proteins; D008055:Lipids; C086724:Lipofectamine; D016328:NF-kappa B; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides; D019938:Cyclin D1; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9