Invasion Of Human Prostate Cancer Cells Research Articles

Hypoxia and the Presence of Human Vascular Endothelial Cells Affect Prostate Cancer Cell Invasion and Metabolism

Tumor progression and metastasis are influenced by hypoxia, as well as by interactions between cancer cells and components of the stroma, such as endothelial cells. Here, we have used a magnetic resonance (MR)compatible invasion assay to further understand the effects of hypoxia on human prostate cancer cell invasion and metabolism in the presence and absence of human umbilical vein endothelial cells (HUVECs). Additionally, we compared endogenous activities of selected proteases related to invasion in PC-3 cells and HUVECs, profiled gene expression of PC-3 cells by microarray, evaluated cell proliferation of PC-3 cells and HUVECs by flow cytometry, under hypoxic and oxygenated conditions. The invasion of less-invasive DU-145 cells was not affected by either hypoxia or the presence of HUVECs. However, hypoxia significantly decreased the invasion of PC-3 cells. This hypoxia-induced decrease was attenuated by the presence of HUVECs, whereas under oxygenated conditions, HUVECs did not alter the invasion of PC-3 cells. Cell metabolism changed distinctly with hypoxia and invasion. The endogenous activity of selected extracellular proteases, although altered by hypoxia, did not fully explain the hypoxia-induced changes in invasion. Gene expression profiling indicated that hypoxia affects multiple cellular functions and pathways.

Alteration of the Methylation Status of Tumor-Promoting Genes Decreases Prostate Cancer Cell Invasiveness and Tumorigenesis In vitro and In vivo

We tested the hypothesis that cell invasiveness and tumorigenesis are driven by hypomethylation of genes involved in tumor progression. Highly invasive human prostate cancer cells PC-3 were treated with either the methyl donor S-adenosylmethionine (SAM) or methyl DNA-binding domain protein 2 antisense oligonucleotide (MBD2-AS). Both treatments resulted in a dose- and time-dependent inhibition of key genes, such as urokinase-type plasminogen activator (uPA), matrix metalloproteinase-2 (MMP-2), and vascular endothelial growth factor expression to decrease tumor cell invasion in vitro. No change in the levels of expression of genes already known to be methylated in late-stage prostate cancer cells, such as glutathione S-transferase P1 and androgen receptor, was seen. Inoculation of PC-3 cells pretreated with SAM and MBD2-AS into the flank of male BALB/c nu/nu mice resulted in the development of tumors of significantly smaller volume compared with animals inoculated with PC-3 cells treated with vehicle alone or MBD2 scrambled oligonucleotide. Immunohistochemical analysis of tumors showed the ability of SAM and MBD2-AS to significantly decrease tumoral uPA and MMP-2 expression along with levels of angiogenesis and survival pathway signaling molecules. Bisulfite sequencing analysis of tumoral genomic DNA showed that inhibition of both uPA and MMP-2 expression was due to methylation of their 5' regulatory region. These studies support the hypothesis that DNA hypomethylation controls the activation of multiple tumor-promoting genes and provide valuable insight into developing novel therapeutic strategies against this common disease, which target the demethylation machinery.

Protein kinase Cδ signaling downstream of the EGF receptor mediates migration and invasiveness of prostate cancer cells

Tumor progression to the invasive phenotype occurs secondary to upregulated signaling from growth factor receptors that drive key cellular responses like proliferation, migration, and invasion. We hypothesized that Protein kinase Cδ (PKCδ)-mediated transcellular contractility is required for migration and invasion of prostate tumor cells. Two invasive human prostate cancer cell lines, DU145 cells overexpressing wildtype human EGFR (DU145WT) and PC3 cells, were studied. PKCδ is overexpressed in these cells relative to normal prostate epithelial cells, and is activated downstream of EGFR leading to cell motility via modulation of myosin light chain activity. Abrogation of PKCδ using Rottlerin and specific siRNA significantly decreased migration and invasion of both cell lines in vitro. Both PKCδ and phosphorylated PKCδ protein levels were higher in human prostate cancer tissue relative to normal donor prostate as assessed by Western blotting and immunohistochemistry. Thus, we conclude that PKCδ inhibition can limit migration and invasion of prostate cancer cells.

Ganoderma lucidum suppresses angiogenesis through the inhibition of secretion of VEGF and TGF-β1 from prostate cancer cells

Ganoderma lucidum ( G. lucidum) is a popular medicinal mushroom that has been used as a home remedy for the general promotion of health and longevity in East Asia. The dried powder of G. lucidum, which was recommended as a cancer chemotherapy agent in traditional Chinese medicine, is currently popularly used worldwide in the form of dietary supplements. We have previously demonstrated that G. lucidum induces apoptosis, inhibits cell proliferation, and suppresses cell migration of highly invasive human prostate cancer cells PC-3. However, the molecular mechanism(s) responsible for the inhibitory effects of G. lucidum on the prostate cancer cells has not been fully elucidated. In the present study, we examined the effect of G. lucidum on angiogenesis related to prostate cancer. We found that G. lucidum inhibits the early event in angiogenesis, capillary morphogenesis of the human aortic endothelial cells. These effects are caused by the inhibition of constitutively active AP-1 in prostate cancer cells, resulting in the down-regulation of secretion of VEGF and TGF-β1 from PC-3 cells. Thus, G. lucidum modulates the phosphorylation of Erk1/2 and Akt kinases in PC-3 cells, which in turn inhibits the activity of AP-1. In summary, our results suggest that G. lucidum inhibits prostate cancer-dependent angiogenesis by modulating MAPK and Akt signaling and could have potential therapeutic use for the treatment of prostate cancer.

Evidence that the prostate-specific antigen (PSA)/Zn 2+ axis may play a role in human prostate cancer cell invasion

Prostate-specific antigen (PSA), which is used as a marker for the diagnosis and monitoring of prostate cancer, is a kallikrein protease which could potentially play a role in human prostate cancer cell invasion. Zinc ions are effective inhibitors of a number of proteases. The enzymatic activity of purified PSA was strongly inhibited by Zn 2+. The ability of LNCaP cells which express and secrete PSA to invade Matrigel was strongly suppressed by Zn 2+ at a concentration similar to that inhibiting the activity of purified PSA. Zn 2+ effectively inhibited the degradation of Matrigel by purified PSA. These results suggest that Zn 2+ in human prostate may suppress the invasion and metastasis of prostate cancer cells through the regulation of the proteolytic activity of PSA. Loss of inhibition of the proteolytic activity of PSA by Zn 2+ in prostate tumors could contribute to invasion.

Aspirin inhibits highly invasive prostate cancer cells

Cell adhesion, proteolytic degradation and cell migration are interrelated processes responsible for the invasion and metastasis of cancer. One of the crucial molecules involved in cancer metastasis is urokinase-type plasminogen activator (uPA). An elevated concentration of uPA is a strong indicator of poor prognosis. In addition to the proteolytic activity of uPA, which degrades the extracellular matrix, uPA also binds to its receptor (uPAR) and controls cell adhesion and migration through the reorganization of actin cytoskeleton. We have recently demonstrated that constitutively active nuclear factor-kappa B (NF-kappaB) is responsible for the increased secretion of uPA and that inhibition of NF-kappaB suppresses secretion of uPA and cell migration of highly invasive cancer cells. Aspirin and other nonsteroidal anti-inflammatory drugs have been recently shown to have a chemopreventive effect in colon and pancreatic cancers. Here we show that aspirin inhibits NF-kappaB, resulting in the suppression of uPA secretion from the highly invasive human prostate cancer cells PC-3. Furthermore, aspirin inhibited migration of PC-3 cells, suggesting an effect on the uPA-uPAR signaling complex. Finally, aspirin suppressed adhesion of PC-3 cells to fibronectin (FN), which binds to an alpha3beta1 integrin receptor, and to vitronectin (VN), which binds to alphavbeta3 integrin receptor. Altogether, our data suggests that aspirin inhibits the formation of uPA-uPAR-FN-alpha3beta1 and uPA-uPAR-VN-alphavbeta3 complexes, resulting in the suppression of cell adhesion and cell motility of the highly invasive prostate cancer cells PC-3. These results indicate that aspirin may contribute directly to reducing invasion and metastasis of prostate cancers by inhibiting cell migration and invasion.

A novel antisense inhibitor of MMP-9 attenuates angiogenesis, human prostate cancer cell invasion and tumorigenicity.

Androgen deprivation therapy causes a paradoxical elevation of matrix metalloproteinases (MMPs) including MMP-9 resulting in aggressive tumor phenotype in many patients with prostate cancer. In this study, we have evaluated a novel antisense phosphorodiamidate Morpholino oligomer (PMO) targeted against MMP-9 in models of angiogenesis and in human prostate xenograft in athymic mice. The treatment of androgen-independent DU145 human prostate cells with a 21-mer MMP-9 antisense PMO caused a dose-dependent inhibition of cell proliferation compared to scrambled or MMP-2 antisense PMO at similar concentrations. This was associated with decreases in MMP-9 expression, gelatinolytic activity and increased stability of the insulin-like growth factor-binding protein (IGFBP-3), a proapoptotic factor and MMP-9 substrate. In vitro invasion assays revealed a 40-60% inhibition of DU145 cell invasion in the presence of 25 microM MMP-9 antisense PMO. A significant decrease in endothelial cell migration and vascularization was observed in the Matrigel plug assay in mice when treated intraperitoneally with 300 microg/day MMP-9 antisense for 21 days. In the highly vascular DU145 tumor xenografts, MMP-9 inhibition caused decreased tumor growth with regression in 50% of the animals. Histological analysis revealed increased apoptosis and fibrous tissue deposits in the MMP-9 antisense-treated tumors compared to the scrambled and saline controls. No apparent toxicity or mortality was associated with the MMP-9 PMO treatment. In summary, the MMP-9 antisense PMO inhibited in vitro prostate cancer cell proliferation, invasion and in vivo angiogenesis. These data establish the feasibility of developing a site-directed, nontoxic antisense therapeutic agent for inhibiting local invasion and metastasis.

Hepatocyte growth factor (HGF) alters the expression of the HGF activators HGFA and matriptase-1 and the HGF activation inhibitors HAIs in invasive human prostate cancer cells

Hepatocyte growth factor (HGF) alters the expression of the HGF activators HGFA and matriptase-1 and the HGF activation inhibitors HAIs in invasive human prostate cancer cells

Activation of Pro-gelatinase B by Endometase/Matrilysin-2 Promotes Invasion of Human Prostate Cancer Cells

This work has explored a putative biochemical mechanism by which endometase/matrilysin-2/matrix metalloproteinase-26 (MMP-26) may promote human prostate cancer cell invasion. Here, we showed that the levels of MMP-26 protein in human prostate carcinomas from multiple patients were significantly higher than those in prostatitis, benign prostate hyperplasia, and normal prostate glandular tissues. The role of MMP-26 in prostate cancer progression is unknown. MMP-26 was capable of activating pro-MMP-9 by cleavage at the Ala(93)-Met(94) site of the prepro-enzyme. This activation proceeded in a time- and dose-dependent manner, facilitating the efficient cleavage of fibronectin by MMP-9. The activated MMP-9 products generated by MMP-26 appeared more stable than those cleaved by MMP-7 under the conditions tested. To investigate the contribution of MMP-26 to cancer cell invasion via the activation of MMP-9, highly invasive and metastatic human prostate carcinoma cells, androgen-repressed prostate cancer (ARCaP) cells were selected as a working model. ARCaP cells express both MMP-26 and MMP-9. Specific anti-MMP-26 and anti-MMP-9 functional blocking antibodies both reduced the invasiveness of ARCaP cells across fibronectin or type IV collagen. Furthermore, the introduction of MMP-26 antisense cDNA into ARCaP cells significantly reduced the MMP-26 protein level in these cells and strongly suppressed the invasiveness of ARCaP cells. Double immunofluorescence staining and confocal laser scanning microscopic images revealed that MMP-26 and MMP-9 were co-localized in parental and MMP-26 sense-transfected ARCaP cells. Moreover, MMP-26 and MMP-9 proteins were both expressed in the same human prostate carcinoma tissue samples examined. These results indicate that MMP-26 may be a physiological and pathological activator of pro-MMP-9.

Determination of cyclooxygenase and arachidonic acid metabolites in invasive human prostate cancer cells.

Cyclooxygenase (COX) metabolizes arachidonic acid (AA) to many biologically active eicosanoids such as prostaglandins (PGs), prostacyclins and thromboxane. Two forms of COX have been identified, a constitutive COX-1 and an inducible COX-2. COX-1 and COX-2 are expressed from different gene (1) and utilize different pools of AA. COX-1 is involved in cell-cell signaling and maintaining tissue homeostasis. COX-2 is expressed in only certain cell types (2-3) and regulated by specific stimuli such as growth factors, tumor promoters and cytokines. It is hypothesized that COX-2 is responsible for the synthesis of prostanoids involved in pain, inflammation and mitogenesis (4-5). There is strong evidence that COX-2 and AA metabolites have important roles in the development, growth and invasiveness of many types of cancer. COX-2 expression increases PGE2production and metastatic potential of human breast cancer cell lines (6). PGF2awas found to enhance the invasiveness of mouse melanoma and human fibrosarcoma (7).

Requirement of cyclooxygenase-2 expression and prostaglandins for human prostate cancer cell invasion.

The PC-3 Low Invasive cells and the PC-3 High Invasive cells were used to investigate the correlation of the COX-2 expression and its arachidonic acid metabolites, prostaglandins, with their invasiveness through Matrigel using a Boyden chamber assay. The COX-2 expression in PC-3 High Invasive cells was approximately 3-fold higher than in PC-3 Low Invasive cells while the COX-1 expression was similar in both cell sublines. When incubated with arachidonic acid, PGE2 was the major prostaglandin produced by these cells. PC-3 High Invasive cells produced PGE2 approximately 2.5-fold higher than PC-3 Low Invasive cells. PGD2 was the second most abundant prostaglandin produced by these cells. Both indomethacin (a nonspecific COX inhibitor) and NS-398 (a specific COX-2 inhibitor) inhibited the production of prostaglandins and the cell invasion. PGE2 alone did not induce the cell invasion of PC-3 Low Invasive cells. However, PGE2 reversed the inhibition of cell invasion by NS-398 and enhanced the cell invasion of the PC-3 High Invasive cells. In contrast, PGD2 slightly inhibited the cell invasion. These results suggest that in the PC-3 Low Invasive cells, COX-2-derived PGE2 may not be sufficient to induce cell invasion while in the PC-3 High Invasive cells, PGE2 may be sufficient to act as an enhancer for the cell invasion. Further, PGD2 may represent a weak inhibitor and counteracts the effect of PGE2 in the cell invasion.

Down-regulation of prostasin serine protease: a potential invasion suppressor in prostate cancer.

Prostasin is a serine protease predominantly expressed in normal prostate epithelial cells. The biological function of prostasin has not been determined. Western blot and RT-PCR analyses were used to examine the expression of prostasin in prostate cancer cell lines. Immunohistochemistry was used to evaluate prostasin protein expression in human prostate cancer. An in vitro Matrigel invasion assay was used to test the invasiveness of prostate cancer cell lines forced to express recombinant prostasin. Both prostasin protein and mRNA were found to be expressed in normal human prostate epithelial cells and a non-invasive human prostate cancer cell line, the LNCaP, but neither was found in invasive human prostate cancer cell lines DU-145 and PC-3. Prostasin mRNA expression was absent in invasive prostate cancer cell lines of a transgenic mouse model. Immunohistochemistry analysis showed that prostasin protein expression is down-regulated in high-grade prostate cancer. Transfection of DU-145 and PC-3 cells with a full-length human prostasin cDNA restored prostasin expression and reduced the in vitro invasiveness by 68 and 42%, respectively. Our data indicate that prostasin may be implicated in normal prostate biology and is able to suppress prostate cancer invasion in vitro.

Determination of cyclooxygenases and arachidonic acid metabolites in invasive human prostate cancer cells

Determination of cyclooxygenases and arachidonic acid metabolites in invasive human prostate cancer cells

Androgen regulation of the human pseudoautosomal geneMIC2, a potential marker for prostate cancer

Using the differential display-polymerase chain reaction technique to identify androgen-responsive genes in the human prostatic tumor cell line LNCaP, we cloned an expression tag homologous to the human pseudoautosomal gene MIC2. The role of MIC2 in the prostate had not previously been studied. We used a series of cell lines derived from LNCaP that varied in their degree of differentiation and metastatic potential to assess the relationship between MIC2 expression and androgen responsiveness in prostate cancer. The expression of MIC2 mRNA and its product E2 was upregulated by androgen in a dose- and time-dependent manner in the parental LNCaP line and correlated with the expression of prostate-specific antigen. In the LNCaP sublines and an androgen-repressed invasive human prostate cancer cell line (ARCaP), MIC2 gene expression was not regulated by androgen and was associated with poorer differentiation, decreased androgen sensitivity, and higher metastatic potential. Immunohistochemical analyses indicated that E2 was expressed in tissues from patients with primary prostate cancer (16 of 20), in fetal prostatic tissues (low levels in all 10 fetal tissues assessed), and sporadically in benign prostatic hyperplasia tissues (one of four). The normal prostate tissues did not show positive E2 staining, with the exception of one central-zone section from one of the eight normal prostate samples assessed. These findings suggest that deregulation of expression of the human pseudoautosomal gene MIC2 occurred in the prostate.