Proteomic analysis of cancer stem cells in human prostate cancer cells

Results from recent studies support the hypothesis that cancer stem cells (CSCs) are responsible for tumor initiation and formation. Here, we applied a proteome profiling approach to investigate the mechanisms of CSCs and to identify potential biomarkers in the prostate cancer cell line DU145. Using MACS, the DU145 prostate cancer cell line was isolated into CD44+ or CD44− cells. In sphere culture, CD44+ cells possessed stem cell characteristics and highly expressed genes known to be important in stem cell maintenance. In addition, they showed strong tumorigenic potential in the clonogenic assay and soft agar colony formation assay. We then analyzed and identified proteins that were differentially expressed between CD44+ and CD44− using two-dimensional gel electrophoresis and LC-MS/MS. Cofilin and Annexin A5, which are associated with proliferation or metastasis in cancer, were found to be positively correlated with CD44 expression. These results provide information that will be important to the development of new cancer diagnostic tools and understanding the mechanisms of CSCs although a more detailed study is necessary to investigate the roles of Cofilin and Annexin A5 in CSCs.     

v-rasH expression confers hormone-independent in vitro growth to LNCaP prostate carcinoma cells

The LNCaP human prostate cancer cell line is dependent on androgen for in vitro growth. To discover genes that may be responsible for progression of prostate cancer from hormone dependence to hormone independence, we transfected LNCaP cells with expression vectors that contained either the v-rasH or c-rasH gene under the control of the cadmium (Cd2+)-inducible human metallothionein-IIA promoter. Numerous derivative cell lines were isolated which manifested inducible expression of rasH p21 protein when the cells were treated with Cd2+. None of the cell lines transfected with c-rasH were found to have an altered growth phenotype. Several derivative cell lines expressing inducible v-rasH manifested hormone-independent growth in culture when treated with 10(-7) M Cd2+ . Cd2+ induction of v-rasH p21 was also shown to increase anchorage-independent colony formation of the v-rasH-expressing cell lines tested. Expression of a dominant mutated oncogene can change the hormone-dependent growth phenotype of prostate cancer cells.     

Regulation of Na,K-pump-mediated transport by prolactin in cultured human prostate epithelial cells.

The prostate gland is unique in its ability to secrete large amounts of zinc and citrate, suggesting that it employs unusual transport mechanisms. Intracellular ionic homeostasis in prostate is likely to be mediated by the Na,K-pump, yet there have been few studies of its regulation in this tissue. Accordingly, we explored the expression of the Na,K-pump in PC3 cells, an established cell line of human prostate epithelial cells. Total RNA from confluent monolayers of PC3 cells was isolated, reverse transcribed, and the resulting complementary DNA was amplified by polymerase chain reaction using primers specific for each of the pump's constituent subunits. The amplification revealed a complex pattern of Na,K-pump expression, with detection of mRNAs encoding the alpha1-, alpha3-, alpha4-, betal-, beta2- and beta3-isoforms. We next examined the effect on pump activity of prolactin, an important mediator of cell proliferation in prostate cancer. Monolayers exposed to 10 nM prolactin for 24 hr revealed an inhibition of 40% in ouabain-sensitive 86Rb+ uptake, a sensitive measure of pump-mediated transport. These experiments suggest that the unique transport properties of prostate may depend, at least in part, on a complicated pattern of Na,K-pump expression and regulation.     

Antiandrogen-induced cell death in LNCaP human prostate cancer cells

Antiandrogens such as Casodex (Bicalutamide) are designed to treat advance stage prostate cancer by interfering with androgen receptor-mediated cell survival and by initiating cell death. Treatment of androgen sensitive, non-metastatic LNCaP human prostate cancer cells with 0-100 microM Casodex or 0-10 ng/ml TNF-alpha induces cell death in 20-60% of the cells by 48 h in a dose-dependent manner. In cells treated with TNF-alpha, this is accompanied by the loss of mitochondrial membrane potential (DeltaPsim) and cell adhesion. In contrast, cells treated with Casodex display loss of cell adhesion, but sustained mitochondrial dehydrogenase activity. Overexpression of Bcl-2 in LNCaP cells attenuates the induction of cell death by TNF-alpha but not Casodex, suggesting that mitochondria depolarization is not required for the induction of cell death by Casodex. While both TNF-alpha and Casodex-induced release of cytochrome c in LNCaP cell is predominantely associated with the translocation and cleavage of Bax, our data also suggest that Casodex induces cell death by acting on components downstream of decline of DeltaPsim and upstream of cytochrome c release. Furthermore, while induction of both caspase-3 and caspase-8 activities are observed in TNF-alpha and Casodex-treated cells, a novel cleavage product of procaspase-8 is seen in Casodex-treated cells. Taken together, these data support the hypothesis that Casodex induces cell death by a pathway that is independent of changes in DeltaPsim and Bcl-2 actions and results in an extended lag phase of cell survival that may promote the induction of an invasive phenotype after treatment.     

Human osteocalcin: a strong promoter for nitric oxide synthase gene therapy, with specificity for hormone refractory prostate cancer

BACKGROUND: Gene therapy has been identified as a promising treatment strategy for hormone refractory prostate cancer (HRPC). We report, for the first time, the use of the human osteocalcin (hOC) promoter to control inducible nitric oxide synthase (iNOS) transgene expression in HRPC. METHODS: Human prostate carcinoma cells (PC3, DU145, LNCaP), colon cancer cells (HT29) and human microvascular endothelial cells (HMEC-1) were transfected in vitro with constitutively driven CMV/iNOS or hOC/iNOS plasmid DNA by cationic lipid vector. End points of these experiments were Western blotting, NO(.) generation using the Greiss test to measure accumulated nitrite, and clonogenic assay. RESULTS: Transfection of the hOC/iNOS plasmid increased iNOS protein and total nitrite levels in PC3 and DU145 cells, but not LNCaP or HT29. Transfection with CMV/iNOS or hOC/iNOS resulted in no additional cytotoxicity in androgen-dependent LNCaP cells or in the non-prostate cell lines. However, transfection with either construct resulted in a greatly reduced cell survival (to 10-20%) in the androgen-independent PC3 and DU145 cell lines. CONCLUSIONS: Utilising the tumour-type specific properties of the hOC promoter in tandem with the iNOS gene, we have demonstrated target cell specificity, and transgene activation, in the androgen-independent prostate cancer cell lines (PC3 and DU145), an effect absent in normal and androgen-dependent cells. Furthermore, the levels of NO(.) generated are comparable with those seen generated with constitutively (CMV)-driven iNOS. The data obtained from this study provide a basis for future development of hOC/iNOS gene therapy.     

Mitostatin is down-regulated in human prostate cancer and suppresses the invasive phenotype of prostate cancer cells

MITOSTATIN, a novel putative tumor suppressor gene induced by decorin overexpression, is expressed in most normal human tissues but is markedly down-regulated in advanced stages of mammary and bladder carcinomas. Mitostatin negatively affects cell growth, induces cell death and regulates the expression and activation levels of Hsp27. In this study, we demonstrated that ectopic expression of Mitostatin in PC3, DU145, and LNCaP prostate cancer cells not only induced a significant reduction in cell growth, but also inhibited migration and invasion. Moreover, Mitostatin inhibited colony formation in soft-agar of PC3 and LNCaP cells as well as tumorigenicity of LNCaP cells in nude mice. Conversely, targeting endogenous Mitostatin by siRNA and anti-sense strategies in PC3 and DU145 prostate cancer cells enhanced the malignant phenotype in both cell lines. In agreement of these anti-oncogenic roles, we discovered that Mitostatin was absent in ∼35% (n = 124) of prostate tumor samples and its overall reduction was associated with advanced cancer stages. Collectively, our findings indicate that MITOSTATIN may acts as a tumor suppressor gene in prostate cancer and provide a novel cellular and molecular mechanism to be further exploited and deciphered in our understanding of prostate cancer progression.     

Growth factors in human prostate cancer cells: implications for an improved treatment of prostate cancer

It has been previously shown that estrogens may exert their action on human breast cancer cells through coordinated control of secreted growth factors which act in an autocrine and paracrine fashion. Growth stimulation of the androgen receptor negative prostate carcinoma cell line DU-145 by dihydrotestosterone in the presence of the androgen-responsive human prostate carcinoma cell line LNCaP now indicates that androgens may regulate growth of prostate carcinoma cells through related mechanisms. A variety of androgen-regulated growth modulatory activities with autocrine and paracrine potential can be detected in conditioned media from LNCaP cells partially purified by ion exchange chromatography. Androgen-induced growth of LNCaP cells is partially inhibited by the polyanions suramin and dextran sulfates which antagonize growth factor action. These data suggest the existence of at least two different mechanisms of growth regulation by androgen which can be distinguished by their different sensitivity to growth factor inhibitory agents. We conclude that the combination of antipeptidergic substances and androgen withdrawal would represent a new and promising strategy for treatment of human prostate cancer.     

Dynamic chromatin remodeling on the HER2 promoter in human breast cancer cells.

Deregulation of the HER2 oncogene occurs in 30% of human breast cancers and correlates with poor prognosis and increased propensity for metastasis. Since the molecular basis of HER2 overexpression in human cancers is not known, we sought to determine whether chromatin remodeling pathways are involved in the regulation of HER2 expression. We report that compared with breast cancer cells expressing a low level of HER2, HER2-overexpressing breast cancer cells contained significantly higher levels of acetylated and phosphorylated histone H3, and acetylated histone H4 associated with the HER2 promoter. Decreased recruitment of histone deacetylases in the promoter is also noted in the HER2-overexpressing cell. The association of acetylated histone H4 with HER2 gene chromatin and HER2 expression in breast cancer cells was upregulated by an inhibitor of histone deacetylases. Treatment with histone deacetylase inhibitor also reduced the association of histone deacetylase-1 and -2 with the HER2 promoter. In addition, the tumor promoters 12-O-tetradecanoylphorbol-13-acetate and okadaic acid stimulated the association of phosphorylated histone H3 on serine 10 with the HER2 promoter and also stimulated HER2 expression. These findings identify histone acetylation and histone phosphorylation as novel regulatory modifications that target HER2 gene chromatin, and suggest that elevated levels of these chromatin-relaxing components in the vicinity of the HER2 gene promoter may constitute an important non-genomic mechanism of HER2 overexpression in human breast cancer.     

Insensitivity to transforming growth factor-beta results from promoter methylation of cognate receptors in human prostate cancer cells (LNCaP)

Prostate cancers often develop insensitivity to TGF-beta to gain a growth advantage. In this study, we explored the status of promoter methylation of TGF-beta receptors (TbetaRs) in a prostate cancer cell line, LNCaP, which is insensitive to TGF-beta. Sensitivity to TGF-beta was restored in cells treated with 5-Aza-2'-deoxycytidine (5-Aza), as indicated by an increase in the expression of phosphorylated Smad-2, type I (TbetaRI), and type II (TbetaRII) TGF-beta receptors, and a reduced rate of proliferation. The same treatment did not significantly affect a benign prostate cell line, RWPE-1, which is sensitive to TGF-beta. Mapping of methylation sites was performed by screening 82 potential CpG methylation sites in the promoter of TbetaRI and 33 sites in TbetaRII using methylation-specific PCR and sequence analysis. There were six methylation sites (-365, -356, -348, -251, -244, -231) in the promoter of TbetaRI. The -244 site was located in an activator protein (AP)-2 box. There were three methylated sites (-140, +27, +32) in the TbetaRII promoter and the -140 site was located in one of the Sp1 boxes. Chromatin immunoprecipitation analysis demonstrated DNA binding activity of AP-2 in the TbetaRI promoter and of Sp1 in the TbetaRII promoter after treatment with 5-Aza. To test whether promoter methylation is present in clinical specimens, we analyzed human prostate specimens that showed negative staining for either TbetaRI or TbetaRII in a tissue microarray system. DNA samples were isolated from the microarray after laser capture microdissection. Methylation-specific PCR was performed for TbetaRI (six sites) and TbetaRII (three sites) promoters as identified in LNCaP cells. A significant number of clinical prostate cancer specimens lacked expression of either TbetaRI and/or TbetaRII, especially those with high Gleason's scores. In those specimens showing a loss of TbetaR expression, a promoter methylation pattern similar to that of LNCaP cells was a frequent event. These results demonstrate that insensitivity to TGF-beta in some prostate cancer cells is due to promoter methylation in TbetaRs.     

Transport of methotrexate into LNCaP human prostate cancer cells

Uptake of methotrexate into the LNCaP human prostate cancer cells was linear for the first 60 min. The initial rate of methotrexate uptake was highest at extracellular pH 4.5 and decreased markedly until pH 7.0 to 8.0. Transport of methotrexate into LNCaP cells showed two components, one saturable -K(m) = 0.13 +/- 0.06 microM and V(max) = 1.20 +/- 0.16 pmol x 45 min(-1) x mg(-1) protein at low concentrations and the other apparently not saturable up to 10 microM. Uptake of methotrexate was inhibited by structural analogs with the K(i) values being 6.53, 12.4, and 85.6 microM for 5-formyltetrahydrofolate, 5-methyltetrahydrofolate, and folic acid, respectively. Uptake of methotrexate into LNCaP cells was not inhibited by the energy poisons in contrast to methotrexate uptake into PC-3 prostate cancer cells. Uptake was inhibited by increasing concentrations of sulfate and phosphate ions and by the organic anions probenecid and DIDS, suggesting that methotrexate may be transported by an anion-exchange mechanism. These results show that methotrexate is transported into LNCaP prostate cancer cells by a carrier-mediated process.     

Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin

Background  

Uterine serpins are members of the serine proteinase inhibitor superfamily. Like some other serpins, these proteins do not appear to be functional proteinase inhibitors. The most studied member of the group, ovine uterine serpin (OvUS), inhibits proliferation of several cell types including activated lymphocytes, bovine preimplantation embryos, and cell lines for lymphoma, canine primary osteosarcoma and human prostate cancer (PC-3) cells. The goal for the present study was to evaluate the mechanism by which OvUS inhibits cell proliferation. In particular, it was tested whether inhibition of DNA synthesis in PC-3 cells involves cytotoxic actions of OvUS or the induction of apoptosis. The effect of OvUS in the production of the autocrine and angiogenic cytokine interleukin (IL)-8 by PC-3 cells was also determined. Finally, it was tested whether OvUS blocks specific steps in the cell cycle using both PC-3 cells and lymphocytes.     

Blueberry flavonoids inhibit matrix metalloproteinase activity in DU145 human prostate cancer cells.

Regulation of the matrix metalloproteinases (MMPs), the major mediators of extracellular matrix (ECM) degradation, is crucial to regulate ECM proteolysis, which is important in metastasis. This study examined the effects of 3 flavonoid-enriched fractions (a crude fraction, an anthocyanin-enriched fraction, and a proanthocyanidin-enriched fraction), which were prepared from lowbush blueberries (Vaccinium angustifolium), on MMP activity in DU145 human prostate cancer cells in vitro. Using gelatin gel electrophoresis, MMP activity was evaluated from cells after 24-hr exposure to blueberry fractions. All fractions elicited an ability to decrease the activity of MMP-2 and MMP-9. Of the fractions tested, the proanthocyanidin-enriched fraction was found to be the most effective at inhibiting MMP activity in these cells. No induction of either necrotic or apoptotic cell death was noted in these cells in response to treatment with the blueberry fractions. These findings indicate that flavonoids from blueberry possess the ability to effectively decrease MMP activity, which may decrease overall ECM degradation. This ability may be important in controlling tumor metastasis formation.     

Effect of clomiphene on Ca movement in human prostate cancer cells

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca²⁺ indicator. Clomiphene at concentrations between 10-50 μM increased [Ca²⁺]_i in a concentration-dependent manner. The [Ca²⁺]_i signal was biphasic with an initial rise and a slow decay. Ca²⁺ removal inhibited the Ca²⁺ signal by 41%. Adding 3 mM Ca²⁺ increased [Ca²⁺]_i in cells pretreated with clomiphene in Ca²⁺-free medium, confirming that clomiphene induced Ca²⁺ entry. In Ca²⁺-free medium, pretreatment with 50 μM brefeldin A (to permeabilize the Golgi complex), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca²⁺ pump), and 2 μM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 μM clomiphene-induced store Ca²⁺ release. Conversely, pretreatment with 50 μM clomiphene in Ca²⁺-free medium abolished the [Ca²⁺]_i increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 μM clomiphene-induced Ca²⁺release was unaltered by inhibiting phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 μM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca²⁺]_i increases in PC3 cells by releasing store Ca²⁺ from multiple stores in an phospholipase C-independent manner, and by activating Ca²⁺ influx; and clomiphene was of mild cytotoxicity.     

Sp1 and Sp3 transcription factors upregulate the proximal promoter of the human prostate-specific antigen gene in prostate cancer cells

The serum level of prostate-specific antigen (PSA) is useful as a clinical marker for diagnosis and assessment of the progression of prostate cancer, and in evaluating the effectiveness of treatment. We characterized four Sp1/Sp3 binding sites in the proximal promoter of the PSA gene. In a luciferase assay, these sites contributed to the basal promoter activity in prostate cancer cells. In an electrophoretic mobility shift assay and chromatin immunoprecipitation assay, we confirmed that Sp1 and Sp3 bind to these sites. Overexpression of wild-type Sp1 and Sp3 further upregulated the promoter activity, whereas overexpression of the Sp1 dominant-negative form or addition of mithramycin A significantly reduced the promoter activity and the endogenous mRNA level of PSA. Among the four binding sites, a GC box located at nucleotides -53 to -48 was especially critical for basal promoter activity. These results indicate that Sp1 and Sp3 are involved in the basal expression of PSA in prostate cancer cells.