guanosine-triphosphate and Prostatic-Neoplasms

A large number of aggressive cancer cell lines display elevated levels of activated Rac1, a small GTPase widely implicated in cytoskeleton reorganization, cell motility, and metastatic dissemination. A commonly accepted methodological approach for detecting Rac1 activation in cancer cells involves the use of a conformation-sensitive antibody that detects the active (GTP-bound) Rac1 without interacting with the GDP-bound inactive form. This antibody has been extensively used in fixed cell immunofluorescence and immunohistochemistry. Taking advantage of prostate and pancreatic cancer cell models known to have high basal Rac1-GTP levels, here we have established that this antibody does not recognize Rac1 but rather detects the intermediate filament protein vimentin. Indeed, Rac1-null PC3 prostate cancer cells or cancer models with low levels of Rac1 activation still show a high signal with the anti-Rac1-GTP antibody, which is lost upon silencing of vimentin expression. Moreover, this antibody was unable to detect activated Rac1 in membrane ruffles induced by epidermal growth factor stimulation. These results have profound implications for the study of this key GTPase in cancer, particularly because a large number of cancer cell lines with characteristic mesenchymal features show simultaneous up-regulation of vimentin and high basal Rac1-GTP levels when measured biochemically. This misleading correlation can lead to assumptions about the validity of this antibody and inaccurate conclusions that may affect the development of appropriate therapeutic approaches for targeting the Rac1 pathway.

Topics: Cell Membrane; Guanosine Triphosphate; Humans; Male; Microscopy, Fluorescence; Neoplasm Proteins; PC-3 Cells; Prostatic Neoplasms; rac1 GTP-Binding Protein

We previously reported that pheophorbide a (PhA), excited by 630 nm light, significantly inhibited the growth of prostate cancer cells. In this study, we employed whole-cell proteomics to investigate photodynamic treatment (PDT)-related proteins.. Two-dimensional gel electrophoresis (2-DE) coupled with tandem mass spectrometry was employed to reveal the proteins involved in PhA-mediated PDT in LNCaP and PC-3 prostate cancer cells.. After PhA-PDT treatment, decreased expression of translationally-controlled tumor protein (TCTP) was found in both PC-3 and LNCaP whole-cell proteomes. In contrast, human rab GDP dissociation inhibitor (GDI) in LNCaP cells and ras-related homologs GDI in PC-3 cells were up-regulated.. GDP-GTP exchange is an underlying target of photodynamic treatment in prostate cancer cells.

Topics: Cell Line, Tumor; Chlorophyll; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Male; PC-3 Cells; Photochemotherapy; Photosensitizing Agents; Prostatic Neoplasms; Proteomics; ras Proteins; Tandem Mass Spectrometry; Tumor Protein, Translationally-Controlled 1; Two-Dimensional Difference Gel Electrophoresis

The pi-class glutathione S-transferase (GSTP1) actively protect cells from carcinogens and electrophilic compounds. Loss of GSTP1 expression via promoter hypermethylation is the most common epigenetic alteration observed in human prostate cancer. Silencing of GSTP1 can increase generation of reactive oxygen species (ROS) and DNA damage in cells. In this study we investigated whether loss of GSTP1 contributes to increased DNA damage that may predispose men to a higher risk of prostate cancer. We found significantly elevated (103%; P < 0.0001) levels of 8-oxo-2'-deoxogunosine (8-OHdG), an oxidative DNA damage marker, in adenocarcinomas, compared to benign counterparts, which positively correlated (r = 0.2) with loss of GSTP1 activity (34%; P < 0.0001). Silencing of GSTP1 using siRNA approach in normal human prostate epithelial RWPE1 cells caused increased intracellular production of ROS and higher susceptibility of cells to H2 O2 -mediated oxidative stress. Additionally, human prostate carcinoma LNCaP cells, which contain a silenced GSTP1 gene, were genetically modified to constitutively express high levels of GSTP1. Induction of GSTP1 activity lowered endogenous ROS levels in LNCaP-pLPCX-GSTP1 cells, and when exposed to H2 O2 , these cells exhibited significantly reduced production of ROS and 8-OHdG levels, compared to vector control LNCaP-pLPCX cells. Furthermore, exposure of LNCaP cells to green tea polyphenols caused reexpression of GSTP1, which protected the cells from H2 O2 -mediated DNA damage through decreased ROS production compared to nonexposed cells. These results suggest that loss of GSTP1 expression in human prostate cells, a process that increases their susceptibility to oxidative stress-induced DNA damage, may be an important target for primary prevention of prostate cancer.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Catechin; Cell Line; Deoxyguanosine; DNA Damage; Enzyme Activation; Epithelial Cells; Gene Expression Regulation, Neoplastic; Glutathione S-Transferase pi; Guanosine Triphosphate; Humans; Hydrogen Peroxide; Male; Oxidative Stress; Prostate; Prostatic Neoplasms; Reactive Oxygen Species

The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Shape; Female; Focal Adhesions; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Humans; Integrin beta1; Male; Microtubules; Neoplasm Invasiveness; Neoplasm Proteins; Prostatic Neoplasms; Pseudopodia; rac1 GTP-Binding Protein; Recombinant Fusion Proteins; Rho Guanine Nucleotide Exchange Factors; rhoB GTP-Binding Protein; RNA, Small Interfering

CD97, an adhesion-linked G-protein-coupled receptor (GPCR), is induced in multiple epithelial cancer lineages. We address here the signaling properties and the functional significance of CD97 expression in prostate cancer. Our findings show that CD97 signals through Gα12/13 to increase RHO-GTP levels. CD97 functioned to mediate invasion in prostate cancer cells, at least in part, by associating with lysophosphatidic acid receptor 1 (LPAR1), leading to enhanced LPA-dependent RHO and extracellular signal-regulated kinase activation. Consistent with its role in invasion, depletion of CD97 in PC3 cells resulted in decreased bone metastasis without affecting subcutaneous tumor growth. Furthermore, CD97 heterodimerized and functionally synergized with LPAR1, a GPCR implicated in cancer progression. We also found that CD97 and LPAR expression were significantly correlated in clinical prostate cancer specimens. Taken together, these findings support the investigation of CD97 as a potential therapeutic cancer target.

Topics: Antigens, CD; Cell Line, Tumor; Disease Progression; Extracellular Signal-Regulated MAP Kinases; GTP-Binding Protein alpha Subunits, G12-G13; Guanosine Triphosphate; Humans; Lysophospholipids; Male; Neoplasm Invasiveness; Neoplasm Metastasis; Prostatic Neoplasms; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; rhoA GTP-Binding Protein; Signal Transduction

Epigenetic silencing of gluthathione-S-transferase pi (GSTP1) is recognized as being a molecular hallmark of human prostate cancer. We investigated the effects of green tea polyphenols (GTPs) on GSTP1 re-expression and further elucidated its mechanism of action and long-term safety, compared with nucleoside-analog inhibitor of DNA methyltransferase (DNMT), 5-aza-2'-deoxycitidine. Exposure of human prostate cancer LNCaP cells to 1-10 microg/ml of GTP for 1-7 days caused a concentration- and time-dependent re-expression of GSTP1, which correlated with DNMT1 inhibition. Methyl-specific-PCR and sequencing revealed extensive demethylation in the proximal GSTP1 promoter and regions distal to the transcription factor binding sites. GTP exposure in a time-dependent fashion diminished the mRNA and protein levels of MBD1, MBD4 and MeCP2; HDAC 1-3 and increased the levels of acetylated histone H3 (LysH9/18) and H4. Chromatin immunoprecipitation assays demonstrated that cells treated with GTP have reduced MBD2 association with accessible Sp1 binding sites leading to increased binding and transcriptional activation of the GSTP1 gene. Exposure of cells to GTP did not result in global hypomethylation, as demonstrated by methyl-specific PCR for LINE-1 promoter; rather GTP promotes maintenance of genomic integrity. Furthermore, exposure of cells to GTP did not cause activation of the prometaststic gene S100P, a reverse response noted after exposure of cells to 5-aza-2'deoxycitidine. Our results, for the first time, demonstrate that GTP has dual potential to alter DNA methylation and chromatin modeling, the 2 global epigenetic mechanisms of gene regulation and their lack of toxicity makes them excellent candidates for the chemoprevention of prostate cancer.

Topics: Cell Line, Tumor; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Epithelial Cells; Flavonoids; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Glutathione S-Transferase pi; Guanosine Triphosphate; Histones; Humans; Immunoglobulins; Immunohistochemistry; Male; Phenols; Polyphenols; Promoter Regions, Genetic; Prostate; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Tea

Green tea polyphenols (GTP) have been reported to inhibit DNA methylation in cultured cells. Here, we tested whether oral consumption of GTPs affects normal or cancer-specific DNA methylation in vivo, using mice. Wild-type (WT) and transgenic adenocarcinoma of mouse prostate (TRAMP) mice were given 0.3% GTPs in drinking water beginning at 4 weeks of age. To monitor DNA methylation, we measured 5-methyl-deoxycytidine (5mdC) levels, methylation of the B1 repetitive element, and methylation of the Mage-a8 gene. Each of these parameters were unchanged in prostate, gut, and liver from WT mice at both 12 and 24 weeks of age, with the single exception of a decrease of 5mdC in the liver at 12 weeks. In GTP-treated TRAMP mice, 5mdC levels and the methylation status of four loci hypermethylated during tumor progression were unaltered in TRAMP prostates at 12 or 24 weeks. Quite surprisingly, GTP treatment did not inhibit tumor progression in TRAMP mice, although known pharmacodynamic markers of GTPs were altered in both WT and TRAMP prostates. We also administered 0.1%, 0.3%, or 0.6% GTPs to TRAMP mice for 12 weeks and measured 5mdC levels and methylation of B1 and Mage-a8 in prostate, gut, and liver tissues. No dose-dependent alterations in DNA methylation status were observed. Genome-wide DNA methylation profiling using the HpaII tiny fragment enrichment by ligation-mediated PCR assay also revealed no significant hypomethylating effect of GTP. These data indicate that oral administration of GTPs does not affect normal or cancer-specific DNA methylation in the murine prostate.

Topics: Adenocarcinoma; Animals; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Flavonoids; Guanosine Triphosphate; Immunoenzyme Techniques; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phenols; Polyphenols; Prostate; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tea; Tissue Distribution

We recently showed that metastasis-promoting Mts1 gene (S100A4) and protein is overexpressed during progression of prostate cancer in humans. The purpose of this study was to assess the expression of S100A4 during autochthonous prostate cancer progression in transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Because oral consumption of green tea polyphenols (GTP) has been shown to inhibit metastasis and prostate cancer in TRAMP, we further assessed the significance of S100A4 during chemoprevention regimen.. Male TRAMP mice 8 weeks of age were equally divided into two groups. A freshly prepared 0.1% GTP solution in tap water was supplied thrice a week to experimental animals as the sole source of drinking fluid for 24 weeks, whereas the control group of animals received the same tap water throughout the experiment. The animals were sacrificed at 0, 8, 16, and 24 weeks of GTP feeding and were analyzed for S100A4 and E-cadherin. Additional untreated and treated nontransgenic controls were also included in the study.. With the progression of age and prostate cancer growth in TRAMP mice, an increase in the expression of S100A4 at mRNA and protein level in dorsolateral prostate, but not in nontransgenic mice, occurred. GTP feeding to TRAMP mice resulted in marked inhibition of prostate cancer progression, which was associated with reduction of S100A4 and restoration of E-cadherin.. S100A4 represents a promising marker for prostate cancer progression and could be employed as a biomarker in chemoprevention regimens.

Topics: Administration, Oral; Age Factors; Animals; Biomarkers; Blotting, Western; Cadherins; Cell Line, Tumor; Densitometry; Disease Progression; DNA, Complementary; Flavonoids; Guanosine Triphosphate; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Phenols; Polymerase Chain Reaction; Polyphenols; Prostatic Neoplasms; RNA; RNA, Messenger; S100 Calcium-Binding Protein A4; S100 Proteins; Tea; Time Factors; Transgenes

Animal, epidemiological and clinical studies have demonstrated the anti-tumor activity of pharmacological proteasome inhibitors and the cancer-preventive effects of green tea consumption. Previously, one of our laboratories reported that natural ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG] and (-)-gallocatechin-3-gallate [(-)-GCG], are potent and specific proteasome inhibitors. Another of our groups, for the first time, was able to enantioselectively synthesize (-)-EGCG as well as other analogs of this natural GTP. Our interest in designing and developing novel synthetic GTPs as proteasome inhibitors and potential cancer-preventive agents prompted our current study.. GTP analogs, (+)-EGCG, (+)-GCG, and a fully benzyl-protected (+)-EGCG [Bn-(+)-EGCG], were prepared by enantioselective synthesis. Inhibition of the proteasome or calpain (as a control) activities under cell-free conditions were measured by fluorogenic substrate assay. Inhibition of intact tumor cell proteasome activity was measured by accumulation of some proteasome target proteins (p27, I kappa B-alpha and Bax) using Western blot analysis. Inhibition of tumor cell proliferation and induction of apoptosis by synthetic GTPs were determined by G(1) arrest and caspase activation, respectively. Finally, inhibition of the transforming activity of human prostate cancer cells by synthetic GTPs was measured by a colony formation assay.. (+)-EGCG and (+)-GCG potently and specifically inhibit the chymotrypsin-like activity of purified 20S proteasome and the 26S proteasome in tumor cell lysates, while Bn-(+)-EGCG does not. Treatment of leukemic Jurkat T or prostate cancer LNCaP cells with either (+)-EGCG or (+)-GCG accumulated p27 and IkappaB-alpha proteins, associated with an increased G(1) population. (+)-EGCG treatment also accumulated the pro-apoptotic Bax protein and induced apoptosis in LNCaP cells expressing high basal levels of Bax, but not prostate cancer DU-145 cells with low Bax expression. Finally, synthetic GTPs significantly inhibited colony formation by LNCaP cancer cells.. Enantiomeric analogs of natural GTPs, (+)-EGCG and (+)-GCG, are able to potently and specifically inhibit the proteasome both, in vitro and in vivo, while protection of the hydroxyl groups on (+)-EGCG renders the compound completely inactive.

Topics: Adenocarcinoma; Apoptosis; bcl-2-Associated X Protein; Calpain; Carcinoma; Carrier Proteins; Caspases; Cell Line; Cysteine Endopeptidases; G1 Phase; Guanosine Triphosphate; Humans; Intracellular Signaling Peptides and Proteins; Jurkat Cells; Male; Microfilament Proteins; Multienzyme Complexes; Muscle Proteins; Phenols; Polymers; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Stereoisomerism; Tea; Transcriptional Elongation Factors

Prostate cancer cells derived from transgenic mice with adenocarcinoma of the prostate (TRAMP cells) were treated with the HMG-CoA reductase inhibitor, lovastatin. This caused inactivation of the small GTPase RhoA, actin stress fiber disassembly, cell rounding, growth arrest in the G1 phase of the cell cycle, cell detachment and apoptosis. Addition of geranylgeraniol (GGOL) in the presence of lovastatin, to stimulate protein geranylgeranylation, prevented lovastatin's effects. That is, RhoA was activated, actin stress fibers were assembled, the cells assumed a flat morphology and cell growth resumed. The following observations support an essential role for RhoA in TRAMP cell growth: (1) TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein displayed few actin stress fibers and grew at a slower rate than controls (35 h doubling time for cells expressing RhoA (T19N) vs 20 h for untransfected cells); (2) TRAMP cells expressing constitutively active RhoA (Q63L) mutant protein displayed a contractile phenotype and grew faster than controls (13 h doubling time). Interestingly, addition of farnesol (FOL) with lovastatin, to stimulate protein farnesylation, prevented lovastatin-induced cell rounding, cell detachment and apoptosis, and stimulated cell spreading to a spindle shaped morphology. However, RhoA remained inactive and growth arrest persisted. The morphological effects of FOL addition were prevented in TRAMP cells expressing dominant-negative H-Ras (T17N) mutant protein. Thus, it appears that H-Ras is capable of inducing cell spreading, but incapable of supporting cell proliferation, in the absence of geranylgeranylated proteins like RhoA.

Topics: Actin Cytoskeleton; Adenocarcinoma; Alkyl and Aryl Transferases; Animals; Antineoplastic Agents; Apoptosis; Cell Adhesion; Cell Division; Cell Size; Diterpenes; Drug Interactions; Enzyme Activation; Farnesol; G1 Phase; Genes, ras; GTP-Binding Proteins; Guanosine Triphosphate; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Male; Mevalonic Acid; Mice; Mice, Transgenic; Polyisoprenyl Phosphates; Prostatic Neoplasms; Protein Prenylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins p21(ras); rac GTP-Binding Proteins; rhoA GTP-Binding Protein; Sesquiterpenes; Tumor Cells, Cultured

The interaction of the antimitotic agent estramustine with bovine microtubule proteins and purified tubulin was investigated. Direct photoaffinity labeling of microtubule protein with [14C]estramustine resulted in the labeling of both alpha- and beta-tubulin, and this was inhibited with unlabeled estramustine in a dose-dependent manner. [14C]Estramustine was incorporated into both the soluble and polymerized forms of tubulin. The affinity constant for estramustine binding to tubulin was determined by equilibrium dialysis to be 23 +/- 5 mM. Estramustine did not affect [3H]vinblastine binding, and vinblastine had no effect on direct labeling with [14C]estramustine. Both rhizoxin and paclitaxel decreased the covalent labeling of tubulin with [14C]estramustine in a dose-dependent fashion and were noncompetitive inhibitors of the binding of estramustine to tubulin. The binding of colchicine to tubulin was not inhibited by estramustine as detected by fluorescence and DEAE filter assays. The estramustine binding site on tubulin is therefore distinct from that of colchicine and vinblastine and may at least partially overlap with the binding site for paclitaxel. In both bovine brain microtubules and cytoskeletal proteins from human prostatic carcinoma cells, the incorporation of [14C]estramustine into the beta III isotype of tubulin was found to occur with a reduced efficiency compared to that of the other beta-tubulin isotypes and alpha-tubulin. Since this isotype is overexpressed in estramustine resistant human prostate carcinoma cells, these results indicate that beta III-tubulin may play a role in the response to the effects of estramustine.

Topics: Animals; Antineoplastic Agents, Alkylating; Antineoplastic Agents, Hormonal; Binding Sites; Brain; Cattle; Colchicine; Estramustine; Guanosine Diphosphate; Guanosine Triphosphate; Humans; In Vitro Techniques; Male; Microtubule Proteins; Mitosis; Paclitaxel; Prostatic Neoplasms; Tubulin; Vinca Alkaloids

Transglutaminases with different functions and tissue distribution patterns can be distinguished by specific antibodies and by inhibition of enzyme activity in the presence of guanosine triphosphate (GTP). The most common form is the so-called tissue-type transglutaminase that is apparently involved in membrane stabilization processes, e.g. during apoptosis, and can be inhibited by incubation with GTP at low calcium concentrations. A secretory transglutaminase that cannot be inhibited by GTP is synthesized in an androgen-dependent manner in the dorsal prostate of the rat, the site suggested to represent the origin of the Dunning tumor used as an experimental model in prostate cancer research. Here we studied the expression of transglutaminases in different Dunning tumor lines--mainly in the highly differentiated H subline--and characterized the enzyme both biochemically and immunocytochemically. A very high enzyme activity was found only in the less well differentiated HI-F tumor line. Immunohistochemical reactions and Western blot analysis showed that there is no secretory transglutaminase present in any of the Dunning tumor lines studied. Transglutaminase activity of the Dunning tumor results from the so-called tissue-type enzyme that is non-organ specific. The absence of a secretory form of transglutaminase does not support the contention of a prostatic origin of the Dunning tumor.

Topics: Animals; Blotting, Western; Disease Models, Animal; Female; Genitalia, Male; Guanosine Triphosphate; Histocytochemistry; Immunohistochemistry; Male; Mammary Glands, Animal; Prostatic Neoplasms; Rats; Rats, Wistar; Tissue Distribution; Transglutaminases; Tumor Cells, Cultured