Diverse signaling pathways through the SDF-1/CXCR4 chemokine axis in prostate cancer cell lines leads to altered patterns of cytokine secretion and angiogenesis

The establishment of metastatic bone lesions in prostate cancer (CaP) is a process partially dependent on angiogenesis. Previously we demonstrated that the stromal-derived factor-1 (SDF-1 or CXCL12)/CXCR4 chemokine axis is critical for CaP cell metastasis. In this investigation, cell lines were established in which CXCR4 expression was knocked down using siRNA technology. When CaP cells were co-transplanted with human vascular endothelial cells into SCID mice, significantly fewer human blood vessels were observed paralleling the reductions in CXCR4 levels. Likewise, the invasive behaviors of the CaP cells were inhibited in vitro. From these functional observations we explored angiogenic and signaling mechanisms generated following SDF-1 binding to CXCR4. Differential activation of the MEK/ERK and PI3K/AKT pathways that result in differential secretion IL-6, IL-8, TIMP-2 and VEGF were seen contingent on the cell type examined; VEGF and TIMP-2 expression in PC3 cells are dependent on AKT activation and ERK activation in LNCaP and LNCaP C4-2B cells leads to IL-6 or IL-8 secretion. At the same time, expression of angiostatin levels were inversely related to CXCR4 levels, and inhibited by SDF-1 stimulation. These data link the SDF-1/CXCR4 pathway to changes in angiogenic cytokines by different signaling mechanisms and, suggest that the delicate equilibrium between proangiogenic and antiangiogenic factors may be achieved by different signal transduction pathways to regulate the angiogenic phenotype of prostate cancers. Taken together, our results provide new information regarding expression of functional CXCR4 receptor-an essential role and potential mechanism of angiogenesis upon SDF-1 stimulation.     

Highly specific expression of luciferase gene in lungs of naïve nude mice directed by prostate-specific antigen promoter

PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10(9)PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases.     

Gene expression in precursor cells of prostate cancer associated with activin by combination of subtractive hybridization and microarray technologies

Prostatic intraepithelial neoplasia (PIN) is considered the pre-malignant stage of prostate carcinoma, but little is known of its initiation and evolution. The identification of genes associated with these precursors of prostate cancer may elucidate the pathways of the early oncogenesis of this disease. Previously, we have reported that activin, a member of the TGFbeta superfamily, acted as an inhibitory growth factor in prostate cancer. We used laser capture microdissection, mRNA-library amplification (RNA-PCR), subtractive hybridization, and complementary DNA microarray to examine gene expression profiles in activin-positive PIN, compared with activin-negative PIN. Subtractive hybridization showed that 28 genes were differentially expressed (13 and 15 genes were up- and down-regulated, respectively). Microarray analysis identified 29 and 56 more genes (4 times) up- and down-regulated, respectively, suggesting that DNA microarray is a more effective method in screening gene profiles. We have validated the known genes identified by both subtractive hybridization and microarray technologies, using Northern blot analysis in the mRNA libraries generated from cells microdissected from pathological slides. We have successfully showed that at least 13 genes are involved in activin-associated PIN. The evaluation of candidate genes that emerge from these experiments provides a rational approach to investigate those genes significant in evolution from PIN to prostate carcinoma.     

Gangliosides of organ-confined versus metastatic androgen-receptor-negative prostate cancer

Prior development of a unique androgen-receptor (AR)-negative cell line (HH870) from organ-confined (T2b) human prostate cancer (CaP) enabled comparison of the gangliosides associated with normal and neoplastic prostate epithelial cells, organ-confined versus metastatic (DU 145, PC-3), and AR-negative versus AR-positive CaP cell lines. Resorcinol-HCl and specific monoclonal antibodies were used to characterize gangliosides on 2D-chromatograms, and to visualize them on the cell surface with confocal-fluorescence microscopy. AR-negative cells expressed GM1b, GM2, GD2, GD1a, and GM3. GM1a, GD1b, and GT1b were undetectable. GM1b and GD1a were more prominent in AR-negative than in AR-positive cells. PC-3 and HH870 cells were unique in the expression of O-acetylGD2 (O-AcGD2) and two alpha2,3-sialidase-resistant, alkali-susceptible GMR17-reactive gangliosides. Expression of GD1a, GM1b, doublets of GD3, GD2, and O-AcGD2, and the presence of an additional alkali-labile-14.G2a-reactive ganglioside, two alkali-susceptible, and three alkali-resistant GMR17-reactive gangliosides makes HH870 a potential component of a polyvalent-vaccine for active-specific immunotherapy of CaP.     

Physico-chemical and biological characterizations of two human prolactin analogs exhibiting controversial bioactivity, synthesized in Chinese hamster ovary (CHO) cells

The synthesis, purification and characterization of G129R-hPRL and S179D-hPRL, the two better-studied antagonists of human prolactin (hPRL), is described. Both of these have been expressed for the first time, in their authentic form, by a stable CHO cell line, at secretion levels of 7.7 and 4.3 microg/10(6) cells/day, respectively. Previous studies had shown that these hPRL analogs, when produced in bacterial cytoplasm, consistently contained misfolded forms and multimers according to the specific denaturation, refolding and purification conditions. These versions also have an N-terminal extra methionine. An extensive physico-chemical characterization was carried out after a practical two-step purification process and included SDS-PAGE and Western blotting analysis, matrix-assisted laser-desorption ionization time-of-flight mass spectral (MALDI-TOF-MS) analysis, high-performance size-exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). This last technique revealed a considerable difference in hydrophobicity due to a single amino acid substitution, with S179D-hPRL less (t(RR) = 0.85 +/- 0.010) and G129R-hPRL more (t(RR) = 1.10 +/- 0.013) hydrophobic than hPRL, where t(RR) is the relative retention time. The biological characterization was based on further refinement of a sensitive proliferation assay using the pro-B murine cell line (Ba/F3) transfected with the long form hPRL receptor cDNA such that the minimal detectable dose was 0.04 ng of hPRL/mL, the Ba/F3-LLP assay. On the basis of this assay, the relative residual agonistic activity of these two products, determined against a hPRL international standard in four independent assays, was 53 x 10(-3) for S179D-hPRL and 70 x 10(-5) for G129R-hPRL. We believe that the present synthesis and characterization could be extremely helpful for studies of these two proteins, which have been reported to antagonize tumor growth-promoting effects of hPRL in vivo in animal models of breast and prostate cancer.     

Correlation of protein expression, Gleason score and DNA ploidy in prostate cancer

The prognosis of prostate cancer correlates with tumor differentiation. Gleason score and DNA ploidy are two prognostic factors that correlate with prognosis. We analyzed differences in protein expression in prostate cancer of high and low aggressiveness according to these measures. From 35 prostatectomy specimens, 29 cancer samples and 10 benign samples were harvested by scraping cells from cut surfaces. DNA ploidy was assessed by image cytometry. Protein preparations from cell suspensions were examined by 2-DE. Protein spots that differed quantitatively between sample groups were identified by MS fingerprinting of tryptic fragments and MS/MS sequence analysis. We found 39 protein spots with expression levels that were raised or lowered in correlation with Gleason score and/or DNA ploidy pattern (31 overexpressed in high-malignant cancer, 8 underexpressed). Of these, 30 were identified by MS. Among overexpressed proteins were heat-shock, structural and membrane proteins and enzymes involved in gene silencing, protein synthesis/degradation, mitochondrial protein import (metaxin 2), detoxification (GST-pi) and energy metabolism. Stroma-associated proteins were generally underexpressed. The protein expression of prostate cancer correlates with tumor differentiation. Potential prognostic markers may be found among proteins that are differentially expressed and the clinical value of these should be validated.     

VEGF-C、FLt4和结核菌L型感染在前列腺癌中的表达及临床意义

目的探讨血管内皮细胞生长因子-C(VEGF-C)与血管内皮细胞生长因子受体VEGFR-3(Flt4),在前列腺癌中的表达以及结核菌L型感染率及临床意义。方法应用免疫组化和原位杂交和抗酸染色等方法检测65例前列腺癌(carcinoma of prostate,PCa)和30例良性前列腺增生(benign prostatic hyperplasia,BPH)中的VEGF-C、Fit4蛋白及mRNA的表达,以及结核菌L型的检出率。并对前列腺肿瘤主要临床资料和病理分级参数进行比较,用χ2检验进行统计学处理。结果VEGF-C、Flt4蛋白及mRNA阳性表达率和结核菌L型检出率,前列腺癌明显高于前列腺增生(P0.001~0.05)。VEGF-C、Flt4蛋白及mRNA阳性表达率和结核菌L型检出率与前列腺癌的临床分期、病理分级有明显差异(P0.05)。淋巴结转移组中VEGF-C、Flt4蛋白及mRNA的阳性表达率明显高于非转移组(P0.01)。结核菌L型检出率淋巴结转移组明显高于非转移组(P0.05)。结论VEGF-C、Flt4的蛋白及mRNA在前列腺肿瘤中不同程度异常表达以及结核菌L型检出率与肿瘤的临床分期、病理分级和转移呈正相关,提示这2种基因均可作为判断前列腺癌生物学行为及患者预后参考指标;结核菌L型感染可能有协同致瘤作用,与前列腺癌的发生发展可能有一定关系。因此研究VEGF-C、Flt4的阳性表达和结核菌L型感染与前列腺癌的关系,具有重要的临床应用价值。