Decoy Nuclear Acid Treated In LNCaP Cells And P-Akt Downstream

Objective When prostate cancer treated with maximal androgen blockade (MAB) for 12-24 months, half of the patients turn out to be androgen-independent, in this status the androgen receptor (AR) continue being activated by other signal transductions. AR binds to the androgen-responsive element (ARE) and regulates genes transcription. We synthesized "ARE decoy", to investigate the effect of "ARE decoy" treated in LNCaP cells and the downstream signal pathway.Methods Based on the DNA sequence around-170 of the androgen-responsive element (ARE-Ⅰ) and around -394 of the androgen-responsive element (ARE-Ⅱ) in the promoter region of the human PSA gene, we synthesized ARE-Ⅰ, ARE-Ⅱdecoy, modified them with partially sulfurized or totally sulfurized. All of the decoys were end-labeled with biotin or FITC. We detected 4 kinds of decoy with DNA-protein interactions by electrophoretic mobility shift assay (EMSA), to see which decoy has the strongest affinity with nuclear protein. The nuclear extract was prepared from LNCaP cells, which is most likely AR. Then the anti-androgen effect of the ARE decoy was studied in LNCaP cells, transfecting the ARE decoy by Lipofectin2000. Before transfection, the medium was changed to RPMI-1640 medium with 5% charcoal-stripped FBS (CS-FBS) supplemented with 10-9 M dihydrotestosterone (DHT) for 3 days. After 24-72hr incubation, cell viability was detected by MTS assay. Cellular apoptosis was examined by DNA fragmentation assay. Confocal laser scanning microscopy was used to observe cellular morphological changes, after LNCaP cells transfected with totally sulfurized ARE-II decoy. Western-blot analysis was used to detect p-Akt protein changes in different groups.Results1. The gel shift assay demonstrated: ARE-I, ARE-II decoy with totally sulfurized have more specific affinity to the LNCaP nuclear protein which is most likely AR.2. Transfected with 2.0μmol/L four kinds of ARE decoy for 24-72hr, MTS assay showed:cellular proliferation activities can be inhibited by ARE-II decoy with totally sulfurized (P< 0.01), though not in the groups transfected with the other decoys, comparing with the control decoy.3. Observing by confocal laser sacnning microscopy, the cellular morphology changed a lot in the group transfected with totally sulfurized ARE-Ⅱdecoy, such as nuclear condensation, cytoplasmic shrinkage.4. DNA was extracted from LNCaP cells, transfected with totally sulfurized ARE-Ⅱdecoy, and was examined for an internucleosomal DNA ladder, characteristic of apoptosis.5. Western blot analysis show a significant reduction of p-Akt protein in the cells transfected with totally sulfurized ARE-Ⅱdecoy, comparing with other groups.Conclusions The totally sulfurized ARE-Ⅱdecoy had a stronger antiandrogen effection comparing with other decoys, and induced apoptosis by reducing the p-Akt protein expression, subsequently changed signal transduction in LNCaP. This totally sulfurized ARE-II decoy may become a potential therapeutic tool for prostate cancers.