Abstract

Although androgen ablative therapy can suppress growth of localized prostate cancer (PC) cells that are androgen receptor positive, skeletal metastases and tumor relapse ultimately dictate PC related morbidity and mortality. Bone metastasis of PC cells results from a complex cascade of events that involves PC cell intravasation into blood or lymphatic vessels, followed by recruitment by bone marrow microvascular endothelial cells and trans-endothelial migration (TEM) in the bone marrow microenvironment. We have tested the hypothesis that enhanced expression of the chemokine receptor, CXCR4 in PC cells plays a crucial role in their adhesion to the endothelium followed by TEM into the bone microenvironment. To test this hypothesis, PC3 (bone metastatic PC cell line) and LnCaP (lymph node metastatic PC cell line) cells were treated with stromal derived factor-1 alpha (SDF-1α, 100 ng/ml) for 6 or 24 hr and RT-PCR amplification of both CXCR4 and GAPDH mRNAs was carried out. Within 6 hr CXCR4 levels of mRNA expression were increased (3–4 fold) in PC3 cells but not in LnCaP cells. By 24 hr the CXCR4 mRNA levels in PC3 cells returned back to normal and remain unchanged in LnCaP cells. The treatment of PC3 cells with SDF-1α (100 ng/ml) for 6 hr showed a significant (p<0.05) increase (200–225 %) in their adhesion to the confluent human umbilical vein endothelial cells (HUVECs) monolayer and increased the TEM of PC3 cells by 80–100 % in trans-well culture chambers. The CXCR4 expression was shown to be a critical determinant in adhesion of PC3 cells to HUVECs since addition of the monoclonal CXCR4 antibody (1 μg/ml) to SDF-1α treated PC3 cells effectively blocked their adhesion and TEM. Furthermore, SDF-1α (100ng/ml for 6 hr) significantly increased NF-kB transactivational activity in PC-3 cells transfected with NF-kB-luciferase reporter plasmid. However, as predicted this activation was not observed in LnCaP cells under these conditions. Suppression of NF-κB activation in SDF-1α (100ng/ml) treated PC3 cells with an adenoviral vector expressing dominant negative mutant I-κB decreased the CXCR4 mRNA expression by 2–3-fold and resulted in inhibition of their adhesion and TEM by 50–80 %. As expected, over-expression of p65 subunit of NF-κB by transient transfection of PC3 cells with pCMV-p65 expression plasmid (3 μg per 5x105 cells) for 48 hr up-regulated the CXCR4 mRNA expression (2–3 fold) and cell surface CXCR4 receptor expression (2–3 fold) as determined by flow cytometric analysis. Introduction of p65 expression plasmid in PC3 cells increased adhesion (50–80 %) to the endothelium and their TEM (80–100 %) in response to SDF-1α (100 ng/ml). In contrast, introduction of pCMV-p50 expression plasmid in PC3 cells did not affect the CXCR4 gene expression. These data clearly implicate that SDF-1α induced CXCR4 expression and activation of NF-κB plays a significant role in adhesion, TEM and ultimately in bone metastasis of PC cells.

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