Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines

Although the rictor-mTOR complex (mTORC2) has been shown to act as phosphoinositide-dependent kinase (PDK)2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK) versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial-mesenchymal transition in MDA-MB-468 and PC-3 cells. Thus, we hypothesize that ILK might bestow growth advantage and metastatic potential in the course of tumor progression.     

Bone morphogenetic protein signaling in prostate cancer cell lines

Prostate cancer is the most commonly diagnosed malignancy in men and is often associated with bone metastases. Prostate cancer bone lesions can be lytic or schlerotic, with the latter predominating. Bone morphogenetic proteins (BMPs) are a family of growth factors, which may play a role in the formation of prostate cancer osteoblastic bone metastases. This study evaluated the effects of BMPs on prostate cancer cell lines. We observed growth inhibitory effects of BMP-2 and -4 on LNCaP, while PC-3 was unaffected. Flow cytometric analysis determined that LNCaP cell growth was arrested in G(1) after bone morphogenetic protein-2 treatment. Treatment of LNCaP and PC-3 with BMP-2 and -4 activated downstream signaling pathways involving SMAD-1, up-regulation of p21(CIP1/WAF1) and changes in retinoblastoma (Rb) phosphorylation. Interestingly, bone morphogenetic protein-2 treatment stimulated a 2.7-fold increase in osteoprotegerin (OPG), a molecule, which inhibits osteoclastogenesis, production in PC-3.     

Combination of Quercetin and 2-Methoxyestradiol Enhances Inhibition of Human Prostate Cancer LNCaP and PC-3 Cells Xenograft Tumor Growth

Quercetin and 2-Methoxyestradiol (2-ME) are promising anti-cancer substances. Our previous in vitro study showed that quercetin synergized with 2-Methoxyestradiol exhibiting increased antiproliferative and proapoptotic activity in both androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cell lines. In the present study, we determined whether their combination could inhibit LNCaP and PC-3 xenograft tumor growth in vivo and explored the underlying mechanism. Human prostate cancer LNCaP and PC-3 cells were inoculated subcutaneously in male BALB/c nude mice. When xenograft tumors reached about 100 mm³, mice were randomly allocated to vehicle control, quercetin or 2-Methoxyestradiol singly treated and combination treatment groups. After therapeutic intervention for 4 weeks, combination treatment of quercetin and 2-ME i) significantly inhibited prostate cancer xenograft tumor growth by 46.8% for LNCaP and 51.3% for PC-3 as compared to vehicle control group, more effective than quercetin (28.4% for LNCaP, 24.8% for PC3) or 2-ME (32.1% for LNCaP, 28.9% for PC3) alone; ii) was well tolerated by BALB/c mice and no obvious toxic reactions were observed; iii) led to higher Bax/Bcl-2 ratio, cleaved caspase-3 protein expression and apoptosis rate; and iv) resulted in lower phosphorylated AKT (pAKT) protein level, vascular endothelial growth factor protein and mRNA expression, microvascular density and proliferation rate than single drug treatment. These effects were more remarkable compared to vehicle group. Therefore, combination of quercetin and 2-ME can serve as a novel clinical treatment regimen owning the potential of enhancing antitumor effect on prostate cancer in vivo and lessening the dose and side effects of either quercetin or 2-ME alone. These in vivo results will lay a further solid basis for subsequent researches on this novel therapeutic regimen in human prostate cancer.     

Anti-proliferative effect of a putative endocannabinoid, 2-arachidonylglyceryl ether in prostate carcinoma cells

Endocannabinoids (ECs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), inhibit proliferation of carcinoma cells. Several enzymes hydrolyze ECs to reduce endogenous EC concentrations and produce eicosanoids that promote cell growth. In this study, we determined the effects of EC hydrolysis inhibitors and a putative EC, 2-arachidonylglyceryl ether (noladin ether, NE) on proliferation of prostate carcinoma (PC-3, DU-145, and LNCaP) cells. PC-3 cells had the least specific hydrolysis activity for AEA and administration of AEA effectively inhibited cell proliferation. The proliferation inhibition was blocked by SR141716A (a selective CB1R antagonist) but not SR144528 (a selective CB2R antagonist), suggesting a CB1R-mediated inhibition mechanism. On the other hand, specific hydrolysis activity for 2-AG was high and 2-AG inhibited proliferation only in the presence of EC hydrolysis inhibitors. NE inhibited proliferation in a concentration-dependent manner; however, SR141716A, SR144528 and pertussis toxin did not block the NE-inhibited proliferation, suggesting a CBR-independent mechanism of NE. A peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662 did not block the NE-inhibited proliferation, suggesting that PPARγ was not involved. NE also induced cell cycle arrest in G(0)/G(1) phase in PC-3 cells. NE inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB p65) and down-regulated the expression of cyclin D1 and cyclin E in PC-3 cells, suggesting the NF-κB/cyclin D and cyclin E pathways are involved in the arrest of G1 cell cycle and inhibition of cell growth. These results indicate therapeutic potentials of EC hydrolysis inhibitors and the enzymatically stable NE in prostate cancer.     

Secretion of endogenous kallikreins 2 and 3 by androgen receptor-transfected PC-3 prostate cancer cells

Androgen independent PC-3 cells lack androgen receptor (AR) expression and do not produce kallikrein 2 (hK2) or 3 (prostate-specific antigen, PSA). In this paper, we examined the ability of androgens to stimulate PSA and hK2 production in AR transfected PC-3 cells (PC-3(AR)) and compared this to LNCaP cells. PSA and hK2 were measured in the culture medium and cell lysates using an ELISA-based immunofluorometric assay. Only androgens were able to induce PSA and hK2 secretion in PC-3(AR) cells in a dose- and time-dependent manner depending on the level of AR present. The level of androgen-induced PSA and hK2 secretion in PC-3(AR) cells was approximately 1.5 and 0.9% that induced in LNCaP cells, respectively. Insulin-like growth factor-I (IGF-I), which has been shown to activate AR in the absence of ligand, did not activate PSA secretion in the absence of androgen, but further increased the dihydrotestosterone-induced PSA secretion in PC-3(AR) cells. The lack of PSA and hK2 production in parental PC-3 cells is thus a result of their lack of AR expression. PSA and/or hK2 production in PC-3(AR) cells can thus serve as an endogenous reporter system to investigate AR action or to screen putative endocrine disrupters.     

Difference in Protein Expression Profile and Chemotherapy Drugs Response of Different Progression Stages of LNCaP Sublines and Other Human Prostate Cancer Cells

Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, 80-90% of the patients who receive androgen ablation therapy ultimately develop recurrent tumors in 12-33 months after treatment with a median overall survival time of 1-2 years after relapse. LNCaP is a commonly used cell line established from a human lymph node metastatic lesion of prostatic adenocarcinoma. We previously established two relapsed androgen receptor (AR)-rich androgen-independent LNCaP sublines 104-R1 (androgen depleted for 12 months) and 104-R2 cells (androgen depleted for 24 months) from AR-positive androgen-dependent LNCaP 104-S cells. LNCaP 104-R1 and 104-R2 mimics the AR-positive hormone-refractory relapsed tumors in patients receiving androgen ablation therapy. Androgen treatment stimulates proliferation of 104-S cells, but causes growth inhibition and G1 cell cycle arrest in 104-R1 and 104-R2 cells. We investigated the protein expression profile difference between LNCaP 104-S vs. LNCaP 104-R1, 104-R2, PC-3, and DU-145 cells as well as examined the sensitivity of these prostate cancer cells to different chemotherapy drugs and small molecule inhibitors. Compared to 104-S cells, 104-R1 and 104-R2 cells express higher protein levels of AR, PSA, c-Myc, Skp2, BCL-2, P53, p-MDM2 S166, Rb, and p-Rb S807/811. The 104-R1 and 104-R2 cells express higher ratio of p-Akt S473/Akt, p-EGFR/EGFR, and p-Src/Src, but lower ratio of p-ERK/ERK than 104-S cells. PC-3 and DU-145 cells express higher c-Myc, Skp2, Akt, Akt1, and phospho-EGFR but less phospho-Akt and phospho-ERK. Overexpression of Skp2 increased resistance of LNCaP cells to chemotherapy drugs. Paclitaxel, androgen, and inhibitors for PI3K/Akt, EGFR, Src, or Bcl-2 seem to be potential choices for treatment of advanced prostate cancers. Our study provides rationale for targeting Akt, EGFR, Src, Bcl-2, and AR signaling as a treatment for AR-positive relapsed prostate tumors after hormone therapy.     

Phenethyl Isothiocyanate Inhibits Oxidative Phosphorylation to Trigger Reactive Oxygen Species-mediated Death of Human Prostate Cancer Cells

Phenethyl isothiocyanate (PEITC), a constituent of edible cruciferous vegetables such as watercress, not only affords significant protection against chemically induced cancer in experimental rodents but also inhibits growth of human cancer cells by causing apoptotic and autophagic cell death. However, the underlying mechanism of PEITC-induced cell death is not fully understood. Using LNCaP and PC-3 human prostate cancer cells as a model, we demonstrate that the PEITC-induced cell death is initiated by production of reactive oxygen species (ROS) resulting from inhibition of oxidative phosphorylation (OXPHOS). Exposure of LNCaP and PC-3 cells to pharmacologic concentrations of PEITC resulted in ROS production, which correlated with inhibition of complex III activity, suppression of OXPHOS, and ATP depletion. These effects were not observed in a representative normal human prostate epithelial cell line (PrEC). The ROS production by PEITC treatment was not influenced by cyclosporin A. The Rho-0 variants of LNCaP and PC-3 cells were more resistant to PEITC-mediated ROS generation, apoptotic DNA fragmentation, and collapse of mitochondrial membrane potential compared with respective wild-type cells. The PEITC treatment resulted in activation of Bax in wild-type LNCaP and PC-3 cells, but not in their respective Rho-0 variants. Furthermore, RNA interference of Bax and Bak conferred significant protection against PEITC-induced apoptosis. The Rho-0 variants of LNCaP and PC-3 cells also resisted PEITC-mediated autophagy. In conclusion, the present study provides novel insight into the molecular circuitry of PEITC-induced cell death involving ROS production due to inhibition of complex III and OXPHOS.     

Remnant lipoproteins induced proliferation of human prostate cancer cell,PC-3 but not LNCaP,via low density lipoprotein receptor

Background: Hypertriglyceridemia has been shown to be one of the risk factors for prostate cancer. In this study, we investigated the effect of remnant lipoproteins on cell growth in prostate cancer cell lines. Methods: Remnant lipoproteins were isolated as remnant like particles (RLP) from human plasma. We used RLP for TG-rich lipoproteins and low density lipoproteins (LDL) for cholesterol-rich lipoproteins respectively and examined the effect of lipoproteins on proliferation of PC-3 and LNCaP cells using MTS assays. Moreover, we studied the effect of RLP and LDL treatment on the regulation of lipoprotein receptors in prostate cancer cells to investigate the relationship between lipoprotein-induced cell proliferation and lipoprotein receptor expression using real-time PCR, Western blotting assays and siRNA. Results: RLP effectively induced PC-3 cell proliferation more than LDL, whereas both RLP and LDL could not induce LNCaP cell proliferation except at a higher concentration of RLP. LDL receptor (LDLr) was expressed in both prostate cancer cells but there was a sharp difference of sterol regulation between two cells. In PC-3 cells, LDL decreased the LDLr expression in some degree, but RLP did not. Meanwhile LDLr expression in LNCaP was easily downregulated by RLP and LDL. Blocking LDLr function significantly inhibited both RLP- and LDL-induced PC-3 cell proliferation. Conclusions: This study demonstrated that RLP-induced PC-3 cell proliferation more than LDL; however, both RLP and LDL hardly induced LNCaP cell proliferation. The differences of proliferation by lipoproteins might be involved in the regulation of LDLr expression.     

Expression of prostate specific membrane antigen in androgen-independent prostate cancer cell line PC-3

During the progression of prostate cancer from androgen-dependence or sensitivity to androgen-independence, the overall expression of prostate specific membrane antigen (PSMA) increases with its appearance in plasma membrane. However, surprisingly some androgen-independent metastatic prostate cancer cell lines do not express this protein. Estradiol (E2) and basic fibroblast growth factor (bFGF) due to their recognized and strong involvement in prostate growth, development, and pathology were selected with the aim of restoring the expression of PSMA in markedly dedifferentiated prostate cancer PC-3 cells and in Du 145. E2 (10(-7)-10(-11)M) and bFGF (10ng/ml) stimulated the expression of mRNAs for PSMA (2- to 4-fold increase) that apparently were further translated and processed to its membrane form in LNCaP, PC-3, and Du 145 cells. The values of interaction force between the same anti-PSMA antibodies and all studied cells were almost identical (45-64pN), indicating antigenic similarity of the membrane form of PSMA expressed in LNCaP, PC-3, and Du 145 cells.     

Adrenomedullin inhibits prostate cancer cell proliferation through a cAMP-independent autocrine mechanism

Adrenomedullin (AM) is a multifunctional peptide expressed in the normal and malignant prostate, and in prostate cancer cells. To elucidate the potential role of AM in prostate cancer, we have transfected the human AM gene into PC-3, DU 145, and LNCaP prostate cancer cells. Northern blot, Western blot, and radioimmunoassay techniques confirmed an increase in the synthesis and secretion of the 6kDa mature peptide, in the AM-transfected clones. Proliferation and cell cycle assays demonstrated that AM overexpression inhibited cell proliferation in PC-3 and LNCaP cells through a G0/G1 cell cycle arrest, but not in DU 145 cells. In vivo growth assays also confirmed that, at least in PC-3, AM produced a very significant reduction of tumor volume. In addition, the three cell lines expressed the CL/RCP/RAMP-2 receptor complex by RT-PCR, which suggests that AM peptide acts through an autocrine loop in prostate cancer cells. Although cAMP elevation is the most common pathway involved in AM signalling, stimulation of PC-3, DU 145, and LNCaP with synthetic AM did not increase intracellular cAMP. However, short-term stimulation of PC-3 cells with synthetic AM increased ERK1/2 activation. On the contrary, long-term stimulation, or AM overexpression, caused a reduction in the basal activation of ERK1/2. In summary, our results demonstrate that AM (either overexpressed or exogenously added) causes an inhibition of prostate cancer cell growth. This inhibition does not depend on changes in intracellular cAMP levels, but may be related to ERK1/2 activation.     

人同源盒基因NKX3.1对前列腺癌细胞的诱导凋亡作用

构建人同源盒基因NKX3.1 cDNA真核表达载体,研究其在前列腺癌细胞PC-3、LNCaP 中的表达及对细胞的促凋亡作用.以人前列腺癌细胞LNCaP细胞中的总RNA为模板,RT-PCR扩增NKX3.1基因全长编码片段,将NKX3.1 cDNA重组到真核表达载体pcDNA3.1（+）中; 将pcDNA3.1-NKX3.1表达载体瞬时转染前列腺癌细胞PC-3和LNCaP 细胞,用RT-PCR和Western印迹检测NKX3.1 cDNA在转录水平和蛋白水平的表达;绘制细胞生长曲线,观察NKX3.1对前列腺癌细胞增殖的抑制作用;用DNA/ladder和流式细胞术检测NKX3.1对前列腺癌细胞凋亡的影响,进一步用RT PCR检测凋亡相关基因caspase3、caspase8、caspase9、Apaf1、survivin和Bcl2表达的变化.人同源盒基因NKX3.1 cDNA真核表达载体pcDNA3.1-NKX3.1经酶切及测序鉴定正确. pcDNA3.1-NKX3.1转染PC-3和LNCaP细胞后,经RT-PCR和Western印迹证明能有效表达NKX3.1.生长曲线显示,前列腺癌细胞转染NKX3.1 cDNA后细胞增殖受到抑制;前列腺癌细胞转染NKX3.1 cDNA 48 h后,DNA电泳呈现具有凋亡特征的DNA ladder;流式细胞术检测出现明显凋亡峰;RT-PCR检测凋亡相关基因.结果显示,caspase3、caspase8、caspase9基因表达明显增加,Bcl2基因表达明显减少.本研究成功构建了真核表达载体pcDNA3.1 NKX3.1, 转染PC3和LNCaP细胞后能有效表达,并对细胞具有诱导凋亡作用     

Adrenomedullin prevents apoptosis in prostate cancer cells

The 52-aminoacid peptide adrenomedullin (AM) is expressed in the normal and malignant prostate. We have previously shown that prostate cancer cells produce and secrete AM, which acts as an autocrine growth inhibitory factor. We have evaluated in the present study the role of AM in prostate cancer cell apoptosis, induced either by serum deprivation or treatment with the chemotherapeutic agent etoposide (which acts as an inhibitor of topoisomerase II). For this purpose we over-expressed AM in PC-3, DU 145 and LNCaP cells, which were transfected with an expression vector carrying AM. We also treated the parental cell lines with synthetic AM in normal culture conditions and in conditions of induced-apoptosis. After serum removal, AM prevented apoptosis in DU 145 and PC-3 cells, but not in LNCaP cells. When treated with etoposide, AM prevented apoptosis in PC-3 and LNCaP cells, but not in DU 145 cells. Cell cycle analysis demonstrated a significant decrease in the percentage of AM-overexpressing PC-3 cells in the subG0/G1 phase after treatment with etoposide, as compared to the percentage of mock-transfected PC-3 treated cells. Western blot showed that protein levels of phosphorylated ERK1/2 increased in parental PC-3 cells after treatment with etoposide. In PC-3 cells overexpressing AM, phosphorylated ERK1/2 basal levels were lower than basal levels of parental PC-3 cells, and treatment with etoposide did not result in such an increase. Etoposide produced a significant increase in cleaved PARP in parental PC-3 cells. However, PC-3 clones overexpressing AM that were treated with etoposide only showed a mild increase in fragmented PARP. The ratio Bcl-2/Bax was reduced in parental or mock-transfected PC-3 cells after treatment with etoposide. On the contrary, this ratio was not reduced in PC-3 clones with AM overexpression that were treated with etoposide. All these data demonstrate that AM plays a protective role against induced apoptosis in prostate cancer cells. These results may have important implications in prostate cancer resistance to chemotherapeutic agents.     

Different effect of beta-carotene on proliferation of prostate cancer cells

It was shown that high doses of beta-carotene (>30 microM) decrease proliferation of prostate cancer cells in vitro. However, it is rather doubtful whether such concentration of beta-carotene is really accessible at cellular level. We studied the effect of 3 and 10 microM beta-carotene on proliferation and gene expression in LNCaP and PC-3 prostate cancer cell lines. Beta-carotene--more efficiently absorbed from medium by androgen-sensitive LNCaP cells--increased proliferation of LNCaP cells whereas it had weaker effect on PC-3 cells. Initial global analysis of expression of genes in both cell lines treated with 10 microM beta-carotene (Affymetrix HG-U133A) showed remarkable differences in number of responsive genes. Their recognition allows for conclusion that differences between prostate cancer cell lines in response to beta-carotene treatment are due to various androgen sensitivities of LNCaP and PC-3 cells. Detailed analysis of expression of selected genes in beta-carotene treated LNCaP cells at the level of mRNA and protein indicated that the observed increase of proliferation could have been the result of slight induction of a few genes affecting proliferation (c-myc, c-jun) and apoptosis (bcl-2) with no significant effect on major cell cycle control genes (cdk2, RB, E2F-1).     

Inhibition of cell survival signal protein kinase B/Akt by curcumin in human prostate cancer cells

Although curcumin has been shown to inhibit prostate tumor growth in animal models, its mechanism of action is not clear. To better understand the anti-cancer effects of curcumin, we investigated the effects of curcumin on cell survival factor Akt in human prostate cancer cell lines, LNCaP, PC-3, and DU-145. Our results demonstrated differential activation of Akt. Akt was constitutively activated in LNCaP and PC-3 cells. Curcumin inhibited completely Akt activation in both LNCaP and PC-3 cells. The presence of 10% serum decreased the inhibitory effect of curcumin in PC-3 cells whereas complete inhibition was observed in 0.5% serum. Very little or no activation of Akt was observed in serum starved DU-145 cells (0.5% serum). The presence of 10% serum activated Akt in DU-145 cells and was not inhibited by curcumin. Results suggest that one of the mechanisms of curcumin inhibition of prostate cancer may be via inhibition of Akt. To our knowledge this is the first report on the curcumin inhibition of Akt activation in LNCaP and PC-3 but not in DU-145 cells.     

Vav3 oncogene is overexpressed and regulates cell growth and androgen receptor activity in human prostate cancer

The purpose of this research was to investigate the role of Vav3 oncogene in human prostate cancer. We found that expression of Vav3 was significantly elevated in androgen-independent LNCaP-AI cells in comparison with that in their androgen-dependent counterparts, LNCaP cells. Vav3 expression was also detected in other human prostate cancer cell lines (PC-3, DU145, and 22Rv1) and, by immunohistochemistry analysis, was detected in 32% (26 of 82) of surgical specimens of human prostate cancer. Knockdown expression of Vav3 by small interfering RNA inhibited growth of both androgen-dependent LNCaP and androgen-independent LNCaP-AI cells. In contrast, overexpression of Vav3 promoted androgen-independent growth of LNCaP cells induced by epidermal growth factor. Overexpression of Vav3 enhanced androgen receptor (AR) activity regardless of the presence or absence of androgen and stimulated the promoters of AR target genes. These effects of Vav3 could be attenuated by either phosphatidylinositol 3-kinase (PI3K) inhibitors or dominant-negative Akt and were enhanced by cotransfection of PI3K. Moreover, phosphorylation of Akt was elevated in LNCaP cells overexpressing Vav3, which could be blocked by PI3K inhibitors. Finally, we ascertained that the DH domain of Vav3 was responsible for activation of AR. Taken together, our data show that overexpression of Vav3, through the PI3K-Akt pathway, inappropriately activates AR signaling axis and stimulates cell growth in prostate cancer cells. These findings suggest that Vav3 overexpression may be involved in prostate cancer development and progression.     

Prostate tumor CXC-chemokine profile correlates with cell adhesion to endothelium and extracellular matrix

Though chemokines of the CXC family are thought to play key roles in neoplastic transformation and tumor invasion, information about CXC chemokines in prostate cancer is sparse. To evaluate the involvement of CXC chemokines in prostate cancer, we analyzed the CXC coding mRNA of both chemokine ligands (CXCL) and chemokine receptors (CXCR), using the prostate carcinoma cell lines PC-3, DU-145 and LNCaP. CXCR proteins were further evaluated by Western blot, CXCR surface expression by flow cytometry and confocal microscopy. The expression pattern was correlated to adherence of the tumor cells to an endothelial cell monolayer or to extracellular matrix components. Based on growth and adhesion capacity, PC-3 and DU-145 were identified to be highly aggressive tumor cells (PC-3>DU-145), whereas LNCaP belonged to the low aggressive phenotype. CXCL1, CXCL3, CXCL5 and CXCL6 mRNA, chemokines with pro-angiogenic activity, were strongly expressed in DU-145 and PC-3, but not in LNCaP. CXCR3 and CXCR4 surface level differed in the following order: LNCaP>DU-145>PC-3. The differentiation factor, fatty acid valproic acid, induced intracellular CXCR accumulation. Therefore, prostate tumor malignancy might be accompanied by enhanced synthesis of angiogenesis stimulating CXC chemokines. Further, shifting CXCR3 and CXCR4 from the cell surface to the cytoplasm might activate pro-tumoral signalling events and indicate progression from a low to a highly aggressive phenotype.