Enhanced invasion of hormone refractory prostate cancer cells through hepatocyte growth factor (HGF) induction of urokinase-type plasminogen activator (u-PA)

BACKGROUND: Increased expression of the hepatocyte growth factor (HGF) receptor (MET) is associated with high-grade prostatic adenocarcinoma and metastasis. However, the mechanism through which MET signaling contributes to prostate cancer (CaP) metastasis remains unclear. METHODS: Human PC-3 CaP cells and in vivo selected, isogeneic variant cells of increasing metastatic potential (PC-3M, PC-3M-Pro4, and PC-3M-LN4) were used to investigate the effect of HGF on CaP cell growth, protease production, and invasion. Cell-free urokinase-type plasminogen activator (u-PA) expression and function following HGF treatment were analyzed by Western blot, ELISA, and casein/plasminogen zymography. In vitro invasion stimulated by HGF was measured using Matrigel-coated invasion chambers. RESULTS: Both mRNA and functional protein for MET were detected in each of the CaP cell lines. HGF treatment (0-40 ng/ml) weakly increase proliferation, however, HGF induced soluble u-PA protein and activity 3-fold in the metastatic variant cells. HGF significantly stimulated the invasion of highly metastatic PC-3M-LN4 cells through Matrigel and treatment with specific urokinase receptor inhibitors diminished the HGF-stimulated invasion in a dose-dependent manner. CONCLUSIONS: These results demonstrate the biological significance of u-PA up-regulation in response to HGF in highly metastatic hormone refractory CaP cells.     

Increased levels of urokinase plasminogen activator receptor in prostate cancer cells derived from repeated metastasis

To understand alterations to the urokinase system that may occur in progressively metastatic prostate cancer cells, we assessed urokinase plasminogen activator receptor (uPAR) expression, in vitro motility towards vitronectin, urokinase plasminogen activator (uPA)-induced growth and growth factor regulation of uPAR expression in three cell lines—PC-3 and two derivatives from secondary metastases, PC-3M and PC-3MM2. DU-145 and Tsu-Pr1 cells were included for comparative purposes. uPAR expression increases with metastatic passage in these cell lines and accompanies increased growth and motility responses in the presence of uPA. Growth factors TGF1 and IGF-1 induce uPAR in all three prostate cancer lines; however, PC-3M and PC-3MM2 cells also respond to bFGF. Of the cell lines tested, PC-3MM2 most uniformly respond to added TGF1, IGF-1 and bFGF. These results show that in two progressive derivatives from repeated metastasis of PC-3 cells, constitutive and growth factor-induced uPAR expression is enhanced. This increased uPAR facilitates the properties of growth and motility.     

Plasminogen activators in human prostate cancer cell lines and tumors: correlation with the aggressive phenotype

The levels of several tumor associated proteases, including plasminogen activators (PA), are elevated in many malignant tumors compared to their benign tumor counterparts. Extracellular matrix degradation mediated by PA may facilitate tumor cell invasion and metastasis. To assess whether PA content correlates with the aggressive phenotype in prostate cancer, we studied these activators in the PC-3 human prostate cell line and PC-3CALN, an aggressive in vivo derived variant cell line. Enzymatic assays using H-D-val-leu-lys-pNA (S-2251) as substrate and peroxidase-anti-peroxidase immunohistochemical techniques were used. In an in vitro chemoinvasion assay, the PC-3CALN variant cell line demonstrated significantly greater invasive behavior than the unselected, parental PC-3 line. The activity of PA secreted by PC-3CALN cells was 3.5 times greater than that of PC-3 cells (p less than 0.01). PC-3 metastases obtained following intrasplenic injection of PC-3 cells had greater PA activities than the corresponding primary tumors. Immunohistochemical studies of PC-3 tumors demonstrated preferential localization of urokinase-type PA to areas of apparent tumor cell invasion. These data suggest a correlation between PA and the aggressive phenotype in this model of human prostate cancer. PA, in particular u-PA, may play a role in the migration and invasion of prostate cancer cells and provide a marker of the aggressive phenotype.     

不同胃癌细胞系尿激酶型纤溶酶原激活物表达与腹膜转移潜能相关的体外研究

目的比较不同胃癌细胞系尿激酶型纤溶酶原激活物(uPA)表达及其活性与腹膜转移潜能的关系。方法采用ELISA和Western印迹方法,比较不同胃癌细胞系uPA表达的差异,并测定其活性。在24孔培养板或Boyden小室中与生长良好的间皮细胞共同培养不同时间,在显微镜下直接计数与间皮细胞黏附的胃癌细胞,而用MTT法评估胃癌细胞在间皮细胞间的迁移和侵袭情况。结果4株胃癌细胞(AGS、SGC7901、MKN45和MKN28)的uPA表达以SGC7901最高,uPA活性以MKN45最高,AGS两者皆最低。MKN45的黏附能力明显强于MKN28(P<0.05)、SGC7901(P<0.05)和AGS(P<0.01),但其迁移和侵袭能力与SGC7901和MKN28相比差异无统计学意义;而AGS在3方面均明显弱于其他3株细胞。结论4株胃癌细胞uPA表达量及其活性差异较大,并且与其腹膜转移潜能呈正相关。     

Differential expression of uPA in an aggressive (DU 145) and a nonaggressive (1013L) human prostate cancer xenograft

Prostate cancer has a slow growing noninvasive phase, but, in general, is invasive on diagnosis. An initial step in the invasion of surrounding normal tissue is the activity of proteolytic enzymes such as components of the plasminogen activator system (PA). In cell culture, the primary human prostate cancer cell line 1013L expressed no urokinase type-PA (uPA), while DU 145, a cell line derived from a metastatic lesion, expressed high levels of uPA. The DU 145 cells grew easily as xenografts but the establishment of 1013L in the SCID mice was possible only with the aid of a gelatin sponge (Spongostan). The latency period was 42–64 days, followed by a slow growth phase before a fast growth phase occurred. This fast growth phase was characterized by rapid degeneration of tumor tissue, while high proliferation occurred around the blood vessels. On serial transplantation of tumor material, the growth pattern was similar. Furthermore, the 1013L tumor was encapsulated by connective tissue and no invasiveness could be detected. We found that 1013L tumor homogenates had hardly detectable levels of uPA, i.e., 300-fold lower than we found in the invasive prostate xenograft DU 145. In addition, no expression of uPA was found in the plasma of 1013L tumor-bearing mice whilst uPA antigen was detected in the plasma of DU 145 tumor-bearing mice. In conclusion, the 1013L cell line, which exhibits a nonaggressive pattern, could be a good model for studying progression of prostate cancer to a more aggressive phenotype in vivo and in vitro.     

环氧化酶2在不同前列腺癌细胞系中的表达

目的:研究环氧化酶2(COX-2)在不同前列腺癌细胞系中的表达,探讨COX-2在前列腺癌侵袭进展及转移潜能获得机制中的可能作用。方法:应用Western印迹及RT-PCR鉴定LNCaP及其亚细胞系C4-2和AR-CaP亚细胞系IF11、IA8,以及PC-3细胞中COX-2的表达情况,并初步分析其在不同特性前列腺癌细胞系转移侵袭过程中的作用。结果:Western印迹结果显示:COX-2蛋白在PC-3细胞中表达相对较高,在IF11、IA8、LNCaP和C4-2细胞中表达缺失,差异具有统计学意义(P<0.05)。COX-2mRNA表达结果同蛋白一致。结论:不同来源、不同转移潜能的前列腺癌细胞株中COX-2表达存在差异。高表达COX-2可能在PC-3细胞高侵袭转移潜能获得方面起着一定作用,而与其他细胞系转移作用无关。     

Thrombospondin-1 up-regulates tumor cell invasion through the urokinase plasminogen activator receptor in head and neck cancer cells

BACKGROUND: We have previously demonstrated that thrombospondin-1 (TSP-1) is expressed in squamous cell carcinomas of the head and neck. We have also shown that TSP-1 promotes tumor cell invasion through up-regulation of the urokinase plasminogen activator receptor (uPAR), in adenocarcinoma models. We now determined the role of TSP-1 in the regulation of uPAR expression and tumor cell invasion in squamous cell carcinoma of the head and neck cells. MATERIALS AND METHODS: KB squamous cell carcinoma of the head and neck cells were used. The effect of TSP-1 on uPAR and its ligand, urokinase plasminogen activator (uPA), expression were determined by ELISA. The effect of TSP-1 on KB tumor cell invasion was determined in a modified Boyden chamber collagen invasion assay. To determine the role of uPAR on TSP-1-mediated KB tumor cell invasion, we used the three following different strategies: (a). blocking uPAR or its ligand, uPA, with neutralizing antibodies; (b). enzymatic cleavage of uPAR with glycosylphosphatidylinositol (GPI)-specific phospholipase C; and (c). inhibition of plasminogen binding by using epsilon-aminocaproic acid. RESULTS: TSP-I up-regulated uPAR and uPA expression 3- and 4-fold, respectively. TSP-1 up-regulated KB tumor cell invasion 5-fold. Inhibition of uPAR blocked the TSP-1-mediated up-regulation of KB tumor cell invasion. CONCLUSIONS: Our data support a central role for TSP-1 in the regulation of uPAR and tumor cell invasion in squamous cell carcinomas of the head and neck cells. Furthermore, uPAR seems to play a crucial role in TSP-1-mediated squamous cell carcinoma of the head and neck tumor cell invasion.     

Induction of urinary plasminogen activator by retinoic acid results in increased invasiveness of human prostate cancer cells PC-3

Overproduction of uPA by prostate cancer cells in vivo results in tumor invasiveness and osteoblastic skeletal metastasis due to its mitogenic actions in osteoblasts. In the present study we have examined the effect of several growth factors and steroid hormones on regulating uPA gene expression in the human prostate cancer cell line (PC-3). Treatment of these cells with dexamethasone (Dex) caused a decrease, whereas epidermal growth factor (EGF) and fetal bovine serum (FBS) increased uPA expression in a dosedependent manner. Trans retinoic acid (RA) also induced uPA mRNA and protein production in a dose-dependent manner (10^?6 to 10^?9 M). This increase was seen as early as 2 hr of treatment until 48 hr. Dex treatment resulted in decreased tumor cells invasiveness, whereas exposure to EGF and RA caused an increase in the invasive capacity of PC-3 cells. These studies should help to better understand the control mechanism of uPA expression in prostate cancer, where uPA has been implicated as a major pathogenetic factor. © 1995 Wiley-Liss, Inc.     

E-cadherin和CAR在人膀胱癌细胞中的表达及其意义

目的检测上皮型钙黏素（E—cadherin）和柯萨奇-腺病毒受体（coxsackie and adenovirus receptor，CAR）在多种人膀胱癌细胞中的表达情况，初步讨论E-cadherin与CAR在人膀胱癌细胞中表达的意义及两者间的关联。方法用Western印迹法测定人膀胱癌细胞中E—cadherin和CAR的表达情况。结果E—cadherin，CAR在RT4、5637细胞中表达较高，在253J细胞中表达较低；E—cadherin在J82、T24细胞中不表达，CAR在J82细胞中微弱表达，在T24中不表达。结论E-cadherin和CAR在人膀胱癌细胞中的表达趋势一致，两者可能相互协作，共同参与膀胱癌的侵袭转移过程。     

Prostacyclin (PGI2) and plasminogen activator (PA) activity in umbilical vein grafts. An experimental study

Glutaraldehyde-tanned human umbilical cord vein grafts (Dardik Biograft, Meadox) (HUV) measuring 6 cm in length and 5-6 mm in diameter were placed in the carotid arteries (CA) or jugular veins (JV) of sheep and the anastomoses were made end-to-end. Sections from the anastomotic sites and mid-graft were submitted to prostacyclin (PGI2) (radioimmunoassay) and plasminogen activator, PA, (histochemical) assays. Anastomotic PGI2 production from 6 grafts placed in the CA and removed 10 days later was similar to that of control arteries. The midgraft PGI2 synthesis tended to be less but the difference was not statistically significant. Anastomotic PGI2 from 11 grafts placed in the CA and removed 90 days later was also similar but the midgraft production was significantly less (p less than 0.025). From the distal anastomoses and mid portion of grafts placed in the jugular veins and removed 10 days later PGI2 synthesis was significantly less than that of control veins (p less than 0.025 and p less than 0.005 respectively) but no difference between the proximal anastomosis and control veins was observed. In none of 11 HUV removed on the 10th postoperative day was there any PA in the neointima; however, in 6 out of the 9 HUV removed on the 90th postoperative day, PA was demonstrated in the neointima. In conclusion, PGI2 synthesis similar to that of control vessels but less from the mid-graft regions. Neointima gained the ability to produce plasminogen activator.     

Stimulation of human prostate cancer cell lines by factors present in human osteoblast-like cells but not in bone marrow

Secondary deposits of prostate tumours are frequently found in the skeleton where they produce osteoblastic lesions. In this study both osteoblast-like cells and bone marrow from the proximal femur have been cultured to determine whether or not they can release factors which could support the growth of secondary prostate tumours. Media conditioned by both osteoblast-like cells (OBCM) and bone marrow were examined for their potential to stimulate prostate carcinoma cell lines. Whilst the results obtained demonstrated that OBCM could enhance the growth of both the hormone sensitive (LNCaP) and hormone unresponsive (PC-3 and DU-145) prostate carcinoma cell lines, no proliferative effect could be shown on cell lines derived from cancers of the breast, bladder, and liver. Significantly, media conditioned by either bone marrow or human skin fibroblasts also had no effect on the growth of prostate carcinoma cell lines. This study supports the possibility that the proliferation of prostate cancer cells at secondary skeletal sites, in vivo, may be due to osteoblast derived factors. © 1995 Wiley-Liss, Inc.     

Endopeptidase 24.11 activity in the human prostate cancer cell lines LNCaP and PPC-1

Human endopeptidase 24.11 (EP) occurs in greatest abundance on terminally differentiated prostate cells; thus, loss of EP could mark dedifferentiation of prostate epithelium. To identify laboratory models that would permit continuous work on the biochemistry and hormonal regulation of EP, we examined the well-differentiated LNCaP and poorly differentiated PPC-1 human prostate cancer cell lines. Ultrastructural analysis revealed that LNCaP secretes electron-dense material that resembles the particulate matter of seminal plasma, which is associated with endopeptidase activity. LNCaP medium contained EP activity while PPC-1 medium did not. Whether the apparent deletion of EP from the PPC-1 cell line is characteristic of poorly differentiated prostate adenocarcinoma is not yet clear. However, it may be relevant to the carcinogenic process that EP can limit growth of lung small carcinomas by inactivating cell growth-promoting bombesin-like peptides. Because bombesin has been identified in aggressive human prostate cancers, loss of EP in PPC-1 could represent a necessary step in transformation to aggressive phenotype. The combination of LNCaP and PPC-1, which offers well-differentiated and poorly differentiated cancer phenotypes, appears well suited to studying the relevance of EP in prostate cancer biology.     

Immunological characterization and activity of transglutaminases in human normal and malignant prostate and in prostate cancer cell lines

Using biochemical assays, we compared enzyme activities with the immunoreactivity of antibodies against rat seminal transglutaminase (TGase), human erythrocyte TGase and guinea pig liver TGase in human normal prostate, primary prostatic carcinomas and prostatic carcinoma cell lines. Glandular cells of the epithelium were only exceptionally positive with the antibody against (rat) secretory TGase. Using the antibodies against tissue-type TGase, most immunoreactive cells were found in the basal cell layer of prostatic epithelium as well as in stroma (fibroblasts, endothelial cells), whereas immunoreactive glandular cells were sparse. In the case of benign prostatic hyperplasia, few, irregularly distributed secretory cells along with a small number of stromal cells were also immunoreactive with the tissue-type TGase antibody. In dedifferentiated carcinomas, immunoreactive cells were nearly completely absent. Of the prostate cancer cell lines, the LNCaP line showed neither TGase enzyme activity nor immunoreactivity, whereas the PC-3 cell line displayed significant enzyme activity and immunoreactivity. No hormone-dependent changes in either enzyme activity or immunoreactivity were recorded after in vitro treatment of the respective cell lines with estrogens, androgens and antiandrogens. As there is no correlation between androgen deprivation and TGase expression in nonmalignant and malignant human prostatic epithelial cells, TGase activity more likely indicates cellular lesions and consecutive repair mechanisms.     

Proteomic analysis of dunning prostate cancer cell lines with variable metastatic potential using SELDI-TOF

BACKGROUND: Surface enhanced laser desorption and ionization-time-of-flight (SELDI-TOF) is an evolving proteomic technology for improving biomarker discovery that allows for rapid and sensitive analysis of complex protein mixtures generated from body fluids, cells, and/or tissues. SELDI--based profiling identifies unique, differentially expressed proteins relating to specific cancer-related disease states. We utilized SELDI-TOF following pre-processing with molecular separation and chemical fractionation of cell membrane extracts from three Dunning rat prostate cancer cell lines of varying metastatic potential to search novel proteins that are differentially expressed. METHODS: Dunning rat cell sublines of variable (%) metastatic potential; G (0%), AT-1 (20%), and Mat-Ly-Lu (100%) were cultured in two different laboratories. Cell lysis was performed in a homogenation buffer (320 mM sucrose/50 mM Tris/0.5 mM PSMF) using Dounce homogenation. After centrifugation, the membrane pellet was washed 2x and then solublized in 2% CHAPS/8 M urea. This sample was further processed using positive pressure molecular ultrafiltration at 30 kDa or precipitation with 50% ammonium sulfate. Next, each sample was applied to an IMAC3-Ni ProteinChip (Ciphergen Biosystems, Freemont, CA) and analyzed using Ciphergen's Protein Biology System with protein peak analysis software. RESULTS: SELDI-TOF analysis differentiated the three Dunning rat cell sublines based upon protein concentration normalized profiles between 5,000 and 20,000 Da. The preparations from the three cells lines showed clear differences when the extracts from the metastatic sublines (AT-1 and MLL) were compared to the benign subline (G) for proteins with molecular weights of 9 kDa (decrease), 12 kDa (significant decrease), 14 kDa (decrease), and 17 kDa (significant gain). After pre-processing extracts with ammonium sulfate and molecular ultrafiltration, the molecular profile changes from one subline to the next became more apparent. Our results were reproducible using multiple runs including from Dunning cells cultured in a separate laboratory, and using different lots of SELDI ProteinChips. CONCLUSIONS: The application of SELDI-TOF to a series of Dunning rat prostate cancer cell lines illustrated apparent changes in protein profiles among the three cell lines with known differences in metastatic biologic activity. SELDI-TOF identified four reproducible changes in protein expression in the AT1 and MLL metastatic cell sublines. Three of the expression changes were manifested as decreases, but one protein (17 kDa) was over-expressed in the AT1 and MLL cell lines. Emphasis will be placed on the isolation, purification, and characterization of the 17 kDa over-expressed protein and its potential role in PCa metastasis.