|dc.description.abstract||Background. Oxytocin (OT) is involved in regulating prostate growth. This study investigated whether OT, which normally inhibits prostatic growth, may stimulate proliferation in prostate cancer and whether this is accompanied by a change in oxytocin receptor (OTR) location. Caveolae are specialized invaginations in the cell membrane involved in the regulation of signal transduction. This study also investigated whether the number of caveolae and the expression of caveolae-associated proteins, caveolin-1 (cav-1) and -2 (cav-2), and polymerase 1 and transcript release factor (PTRF) change in prostate cancer cells.
Methods. Normal human prostate epithelial cells (PrEC) and prostate stromal cells (PrSC) and androgen-dependent lymph node carcinoma of the prostate (LNCaP) and androgen-independent (PC3) cancer cell lines were used. Human prostate tissue was obtained from surgical patients. OTR, cav-1 and -2, PTRF and androgen receptor (AR) expression were identified by immunocytochemistry and quantified by Western blot analysis. The MTS assay measured cell proliferation. Transmission electron microscopy (TEM) determined the number of caveolae per 100μm of cell membrane. Double and triple immunohistochemistry was performed and co-localization was measured using Pearson's correlation coefficient. Total internal reflection fluorescence (TIRF) microscopy was used to determine the movement of OTR in living cells.
Results. The cell lines were characterized and shown to express OTR and androgen receptor (AR). In malignant cell lines and tissue, there was a loss of PTRF and an up regulation of cav-1 and -2. This was accompanied by a loss of caveolae as determined by TEM.
PC3 cells showed increased proliferation in response to an acute or chronic dose of OT or DHT alone. Addition of physiological concentrations of steroids combined with OT decreased proliferation of PC3 cells. Chronic androgen withdrawal therapy (AWT) concentrations of DHT stimulated LNCaP cell proliferation.
Western blot analysis showed that cav-2 expression was increased in PC3 cells compared to PrEC cells. OTR expression was increased when PrEC cells were treated with T, E, and T+E. In LNCaP cells OTR expression increased following treatment with low levels of DHT. In PC3 cells OTR expression was reduced following treatment with OT alone, but increased with DHT. Cav-1 expression was up regulated by DHT and OT together in PrEC and down regulated by OT or DHT alone. Cav-1 was reduced by OT or DHT alone and with T+E in PC3 cells.
In PrEC cells, OT treatment increased co-localization of OTR & lipid rafts, and DHT decreased co-localization of OTR & cav-2. In PC3 cells, there was increased co-localization of OTR & lipid rafts with physiological OT and DHT alone and increased co-localization of OTR & cav-2 with DHT. In PC3, T or R1881 plus OT resulted in decreased co-localization of OTR and lipid rafts.
TIRF analysis using quantum dots showed that in PrEC, the OTR moved and there were immobile periods when OTR was stimulated with the OT ligand. In PC3 cells, OTR appeared to move erratically and more quickly across the cell membrane.
Conclusion. In prostate cancer OT increases cell number, an effect that is lost when androgen is administered with OT. This study demonstrates the presence of caveolae in PrEC and shows that caveolae are lost in malignant epithelial cells. The changes in the cell membrane are accompanied by an up regulation of cav-2 and by a loss of PTRF expression. In PrEC cells, OTR is co-localized with PTRF but in PC3 cells PTRF is lost and increased co-localization with lipid rafts is observed. Thus it appears that in prostate cancer, OTRs move out of caveolae and this may favour cell proliferation. These results suggest that in prostate cancer cells oxytocin, may stimulate proliferation and that this is accompanied by a change in OTR location.||