Rat Prostate Cancer Cell Lines Research Articles

Methylation Silencing of Transforming Growth Factor-β Receptor Type II in Rat Prostate Cancers

To identify methylation-silenced genes in prostate cancers, a microarray analysis for genes up-regulated by treatment with a demethylating agent, 5-aza-2'-deoxycytidine, was performed using three rat prostate cancer cell lines. Eight genes (Aebp1, Dysf, Gas6, LOC361288, Nnat, Ocm, RGD1308119, and Tgfbr2) were re-expressed at 16-fold or more, and their promoter CpG islands were shown to be densely methylated in the cancer cell lines. From the eight genes, Tgfbr2, a key mediator of transforming growth factor-beta (TGF-beta) signaling that has been strongly implicated in human and rat prostate carcinogenesis, was selected, and its silencing in primary samples was analyzed further. Tgfbr2 was methylated and markedly down-regulated in three of seven 3,2'-dimethyl-4-aminobiphenyl-induced invasive adenocarcinomas in the dorsolateral lobe of the rat prostate. In humans, marked down-regulation of TGFBR2 protein was observed in 12 of 20 high-grade prostatic intraepithelial neoplasia and 36 of 60 prostate cancers. DNA methylation of the human TGFBR2 promoter CpG islands repressed transcription, if present, but neither methylation nor mutation were detected in 27 human prostate cancers analyzed. Methylation silencing of rat Tgfbr2 was associated with histone H3 lysine 9 trimethylation, whereas decreased expression of human TGFBR2 was mainly due to decreased transcription activity, sometimes in concert with histone deacetylation and H3 lysine 27 trimethylation. The identification of methylation silencing of Tgfbr2 in rat prostate cancers, in accordance with TGFBR2 down-regulation in human prostate cancers, will enable us to analyze how aberrant methylation is induced in vivo and identify factors that promote and suppress the induction of aberrant methylation.

Pulsed diode laser-based monitor for singlet molecular oxygen

Photodynamic therapy (PDT) is a promising cancer treatment. PDT uses the affinity of photosensitizers to be selectively retained in malignant tumors. When tumors, pretreated with the photosensitizer, are irradiated with visible light, a photochemical reaction occurs and tumor cells are destroyed. Oxygen molecules in the metastable singlet delta state O2(1Delta) are believed to be the species that destroys cancerous cells during PDT. Monitoring singlet oxygen produced by PDT may lead to more precise and effective PDT treatments. Our approach uses a pulsed diode laser-based monitor with optical fibers and a fast data acquisition system to monitor singlet oxygen during PDT. We present results of in vitro singlet oxygen detection in solutions and in a rat prostate cancer cell line as well as PDT mechanism modeling.

Single cell adhesion measuring apparatus (SCAMA): application to cancer cell lines of different metastatic potential and voltage-gated Na+ channel expression

We have developed a simple yet effective apparatus, based upon negative pressure directed to the tip of a micro-pipette, to measure the adhesiveness of single cells. The "single cell adhesion measuring apparatus" (SCAMA) could differentiate between the adhesion of strongly versus weakly metastatic cancer cells as well as normal cells. Adhesion was quantified as "detachment negative pressure" (DNP) or "DNP relative to cell size" (DNPR) where a noticeable difference in cell size was apparent. Thus, for rat and human prostate and human breast cancer cell lines, adhesiveness (DNPR values) decreased in line with increased metastatic potential. Using the SCAMA, we investigated the effect of tetrodotoxin (TTX), a specific blocker of voltage-gated Na(+) channels (VGSCs), on the adhesion of rat and human prostate cancer cell lines of markedly different metastatic potential. Following pretreatment with TTX (48 h with 1 microM), the adhesion values for the Mat-LyLu cells increased significantly 4.3-fold; there was no effect on the AT-2 cells. For the strongly metastatic PC-3M cells, TTX treatment caused a significant (approximately 30%) increase in adhesion. The adhesion of PNT2-C2 ("normal") cells was not affected by the TTX pretreatment. The TTX-induced increase in the adhesiveness of the strongly metastatic cells was consistent with the functional VGSC expression in these cells and the proposed role of VGSC activity in metastatic cell behaviour. In conclusion, the SCAMA, which can be constructed easily and cheaply, offers a simple and effective method to characterise single-cell adhesion and its modulation.

DNA microarray analysis reveals metastasis-associated genes in rat prostate cancer cell lines.

The molecular and cellular mechanisms involved in prostate cancer progression towards a hormone-independent and highly invasive, metastatic phenotype, are not well understood. Cell lines with different metastatic potential, when analyzed by microarray techniques, offer valuable tools for identifying genes associated with the metastatic phenotype. Gene expression profiles were compared for two rat prostate cancer cell lines with differing metastatic abilities in order to better characterized molecular underpinnings of the prostate cancer metastatic process. Affymetrix arrays were used to analyze gene expression of two rat prostate cancer cell lines, MAT-LyLu and G. Microarray data were analyzed using pathway and functional group analysis. A selected set of genes was subjected to real-time polymerase chain reaction for validating the microarray data. Microarray data analysis revealed differential expression of genes from a number of signaling and metabolic pathways. Overexpression was detected in 48 genes and underexpression in 59 genes of the MAT-LyLu line compared to the standard G line. Genes were grouped into functional categories, including epithelial-extracellular matrix interaction, cell motility, cell proliferation, and transporters, among others. Many of these genes were not previously associated to prostate cancer metastasis. Many genes with altered expression associated with a metastatic prostate cancer phenotype were identified. Further validation of these genes in human prostate samples will determine their usefulness as biomarkers for early diagnosis of recurrence or metastasis of prostate cancer, as well as potential therapeutic targets for this disease.

Análisis de micromatrices de ADN revela genes asociados a metástasis en líneas celulares de cáncer de próstata de rata

The molecular and cellular mechanisms involved in prostate cancer progression towards a hormone-independent and highly invasive, metastatic phenotype, are not well understood. Cell lines with different metastatic potential, when analyzed by microarray techniques, offer valuable tools for identifying genes associated with the metastatic phenotype.Gene expression profiles were compared for two rat prostate cancer cell lines with differing metastatic abilities in order to better characterized molecular underpinnings of the prostate cancer metastatic process.Affymetrix arrays were used to analyze gene expression of two rat prostate cancer cell lines, MAT-LyLu and G. Microarray data were analyzed using pathway and functional group analysis. A selected set of genes was subjected to real-time polymerase chain reaction for validating the microarray data.Microarray data analysis revealed differential expression of genes from a number of signaling and metabolic pathways. Overexpression was detected in 48 genes and underexpression in 59 genes of the MAT-LyLu line compared to the standard G line. Genes were grouped into functional categories, including epithelial-extracellular matrix interaction, cell motility, cell proliferation, and transporters, among others. Many of these genes were not previously associated to prostate cancer metastasis.Many genes with altered expression associated with a metastatic prostate cancer phenotype were identified. Further validation of these genes in human prostate samples will determine their usefulness as biomarkers for early diagnosis of recurrence or metastasis of prostate cancer, as well as potential therapeutic targets for this disease.

Nerve growth factor enhances voltage‐gated Na+ channel activity and Transwell migration in Mat‐LyLu rat prostate cancer cell line

The highly dynamic nature of voltage-gated Na+ channel (VGSC) expression and its controlling mechanism(s) are not well understood. In this study, we investigated the possible involvement of nerve growth factor (NGF) in regulating VGSC activity in the strongly metastatic Mat-LyLu cell model of rat prostate cancer (PCa). NGF increased peak VGSC current density in a time- and dose-dependent manner. NGF also shifted voltage to peak and the half-activation voltage to more positive potentials, and produced currents with faster kinetics of activation; sensitivity to the VGSC blocker tetrodotoxin (TTX) was not affected. The NGF-induced increase in peak VGSC current density was suppressed by both the pan-trk antagonist K252a, and the protein kinase A (PKA) inhibitor KT5720. NGF did not affect the Nav1.7 mRNA level, but the total VGSC alpha-subunit protein level was upregulated. NGF potentiated the cells' migration in Transwell assays, and this was not affected by TTX. We concluded that NGF upregulated functional VGSC expression in Mat-LyLu cells, with PKA as a signaling intermediate, but enhancement of migration by NGF was independent of VGSC activity.

In vitro behavior of Porfimer sodium and Talaporfin sodium with high intensity pulsed irradiation

We studied pulse energy density dependence of two distinctive clinical photosensitizers, Porfimer sodium and Talaporfin sodium, in terms of oxygen consumption, photodegradation in these photosensitizer solutions, and rat prostate cancer cell line photocytotoxicity. The transient transmittances during the pulsed irradiation to these photosensitizer solutions were measured with the pulse energy densities ranging from 0.31 to 31 mJ/cm2. We revealed that Talaporfin sodium was easier to produce absorption saturation than Porfimer sodium. The significant suppression of Talaporfin sodium mediated oxygen consumption, photodegradation, and photocytotoxicity which were observed with pulse energy densities increasing from 0.5 to 10 mJ/cm2. This result could be mainly attributed to absorption saturation. On the other hand, Porfimer sodium did not display significant absorption saturation with the pulse energy densities ranging from 0.31 to 31 mJ/cm2. The photodegradation mechanism for Porfimer sodium changed at high pulse energy density. This phenomenon might accelerate the photodegradation and cause the photocytotoxicity suppression.

Cyclin-Dependent Kinase 5 Activity Controls Cell Motility and Metastatic Potential of Prostate Cancer Cells

We show here that cyclin-dependent kinase 5 (CDK5), a known regulator of migration in neuronal development, plays an important role in prostate cancer motility and metastasis. P35, an activator of CDK5 that is indicative of its activity, is expressed in a panel of human and rat prostate cancer cell lines, and is also expressed in 87.5% of the human metastatic prostate cancers we examined. Blocking of CDK5 activity with a dominant-negative CDK5 construct, small interfering RNA, or roscovitine resulted in changes in the microtubule cytoskeleton, loss of cellular polarity, and loss of motility. Expression of a dominant-negative CDK5 in the highly metastatic Dunning AT6.3 prostate cancer cell line also greatly impaired invasive capacity. CDK5 activity was important for spontaneous metastasis in vivo; xenografts of AT6.3 cells expressing dominant-negative CDK5 had less than one-fourth the number of lung metastases exhibited by AT6.3 cells expressing the empty vector. These results show that CDK5 activity controls cell motility and metastatic potential in prostate cancer.

Activity‐dependent regulation of voltage‐gated Na+ channel expression in Mat‐LyLu rat prostate cancer cell line

We have shown previously that voltage-gated Na(+) channels (VGSCs) are up-regulated in human metastatic disease (prostate, breast and small-cell lung cancers), and that VGSC activity potentiates metastatic cell behaviours. However, the mechanism(s) regulating functional VGSC expression in cancer cells remains unknown. We investigated the possibility of activity-dependent (auto)regulation of VGSC functional expression in the strongly metastatic Mat-LyLu model of rat prostate cancer. Pretreatment with tetrodotoxin (TTX) for 24-72 h subsequently suppressed peak VGSC current density without affecting voltage dependence. The hypothesis was tested that the VGSC auto-regulation occurred via VGSC-mediated Na(+) influx and subsequent activation of protein kinase A (PKA). Indeed, TTX pretreatment reduced the level of phosphorylated PKA, and the PKA inhibitor KT5720 decreased, whilst the adenylate cyclase activator forskolin and the Na(+) ionophore monensin both increased the peak VGSC current density. TTX reduced the mRNA level of Nav1.7, predominant in these cells, and VGSC protein expression at the plasma membrane, although the total VGSC protein level remained unchanged. TTX pretreatment eliminated the VGSC-dependent component of the cells' migration in Transwell assays. We concluded that the VGSC activity in Mat-LyLu rat prostate cancer cells was up-regulated in steady-state via a positive feedback mechanism involving PKA, and this enhanced the cells' migratory potential.

Constitutive activation of the 41‐ and 43‐kDa mitogen‐activated protein (MAP) kinases in the progression of prostate cancer to an androgen‐independent state

The 41- and 43-kDa mitogen-activated protein kinases (MAPK; ERK2 and ERK1, respectively) play pivotal roles in the mitogenic signal transduction pathway. We previously demonstrated that constitutive activation of the MAPK cascade was related to the carcinogenesis of human tumors. In this study, we examined whether constitutive activation of MAPK was related to the progression to androgen independence of prostate cancer. MAPK activation was examined by the appearance of phosphorylated forms and an in vitro kinase assay in four human (androgen-dependent and independent) prostate cancer cell lines and rat prostate cancer cell line Dunning (androgen-sensitive G line, and androgen-independent AT-3, AT-6 sublines). In addition, when androgen-dependent mouse Shionogi Carcinoma 115 (SC115) cells were serially cultured without androgen to obtain androgen-independent cells, the time and degree of MAPK activation were examined. One of three human androgen-independent cell lines (DU145) showed constitutive activation of MAPK, while an androgen-dependent cell line (LNCaP) did not show MAPK activation. While MAPK were not activated in an androgen-sensitive Dunning G cell line, MAPK were activated in androgen-independent sublines (AT-3 and AT-6) derived from a G cell line. In addition, when SC115 cells were serially cultured without androgen, the cells 16-24 weeks after androgen removal showed MAPK activation. Furthermore, in subcloned cells, MAPK activation was observed even in the cells maintained for 9 weeks in medium without testosterone. The present fi ndings suggest that constitutive activation of MAPK may be associated with the acquisition of hormone independence in prostate cancer and that clonal selection after androgen removal and hormone-independent growth through the MAPK signal transduction pathway could begin at a relatively early period in the individual cells.

Comparative studies of intracellular Ca 2+ in strongly and weakly metastatic rat prostate cancer cell lines

The metastatic ability of prostate cancer cells involves differential expression of ionic mechanisms. In the present study, using electrophysiological recordings and intracellular Ca 2+ measurements, we investigated Ca 2+ related signalling in two rat prostate cancer (MAT-LyLu and AT-2) cell lines of markedly different metastatic potential. Whole-cell voltage clamp experiments indicated the absence of an inward current carried through voltage-dependent Ca 2+ channels in either cell line. A Ca 2+-dependent component was also absent in the voltage-activated outward K + currents. Indo-1 microfluorimetry confirmed these results and also revealed marked differences in the resting level of intracellular Ca 2+ and the ability of the two cell lines to regulate intracellular Ca 2+. The weakly metastatic AT-2 cells displayed a significantly higher resting intracellular Ca 2+ than the related but strongly metastatic MAT-LyLu cell line. Increasing extracellular K + decreased intracellular Ca 2+ in the AT-2 but had no effect on intracellular Ca 2+ levels in the MAT-LyLu cells. Furthermore, increasing extracellular Ca 2+ increased intracellular Ca 2+ in AT-2 but, again, had no effect on MAT-LyLu cells. These results suggested the presence of a tonic, voltage-independent Ca 2+ permeation mechanism operating specifically in the AT-2 cells. The influx of Ca 2+ into the AT-2 cells was suppressed by both CdCl 2 (100–300 μM) and SKF-96365 (10–30 μM). It is concluded that the strongly metastatic MAT-LyLu cell line lacks a voltage-independent basal Ca 2+ influx mechanism that is present in the weakly metastatic AT-2 cells.

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

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. 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. 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. 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.

Serum concentration modifies amplitude and kinetics of voltage-gated Na + current in the Mat-LyLu cell line of rat prostate cancer

Voltage-gated Na + channel (VGSC) expression has previously been shown to be upregulated in strongly metastatic prostate cancer cells (rat and human) and its activity shown to potentiate a variety of cellular behaviours integral to the metastatic cascade. However, the mechanism(s) responsible for the Na + channel upregulation is not known. As a step towards evaluating the role of the extracellular biochemical environment in this regard, we have determined the effects of serum concentration on characteristics of Na + channel expressed in the strongly metastatic Mat-LyLu rat prostate cancer cell line. Whole-cell patch-clamp recording techniques were used to study the effects of serum concentrations, above and below the normal 1%. Both the amplitude and the kinetics of the currents were analysed. The following results were obtained: (1) Adding 1% foetal calf serum to cells starved of serum for 24 h increased Na + current density; however, increasing serum concentration further (to 5%) caused a reduction. (2) Serum-free medium produced Na + currents with slower kinetics of activation (time to peak) and inactivation (exponential decay). (3) Increased serum concentration (a) shifted steady-state inactivation to more positive potentials without affecting conductance and (b) increased tetrodotoxin sensitivity. It is concluded that serum concentration is an important determinant of the Na + channel characteristics leading to possible transcriptional and post-translational modifications of channel expression and/or activity. Experiments are now needed to determine which constituents (protein hormones, growth factors, etc.) are responsible for these effects.

Inhibition of prostate cancer metastatic colonization by ∼4.2 Mb of human chromosome 12

Our previous studies demonstrate that introduction of a approximately 70 cM region (now estimated at 63.75 Mb by the Human Genome Project) of human chromosome 12 into the highly metastatic Dunning rat prostate cancer cell line AT6.1 results in >30-fold (>/=90%) reduction in the number of overt metastases in spontaneous metastasis assays. We report the further localization and biological characterization of the metastasis-suppressor activity encoded by a reduced region of chromosome 12. To localize this metastasis-suppressor activity, a panel of AT6.1 microcell hybrids that retain varying portions of human chromosome 12 was constructed and subjected to sequence-tagged site (STS)-based PCR analysis and assessment of in vivo metastatic ability. Data from these complementary approaches localized the metastasis-suppressor activity to a approximately 4.2 Mb portion of human chromosome 12q24.3 comprised of 3 separate regions. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting were used for differential expression analyses to identify which characterized genes, predicted gene sequences and expressed sequence tags (ESTs) within this region could be responsible for the observed metastasis suppression. Comprehensive in vivo studies showed that suppressed AT6.1-12 hybrids that retain the metastasis-suppressor region on 12q24.3 are capable of arriving at the secondary site, but are not able to persist there. Thus, unlike other metastasis-suppressor genes characterized to date, the metastasis-suppressor gene encoded by this region appears to utilize a different biologic mechanism to suppress the growth of overt metastases at the secondary site.

Predominant expression of Kv1.3 voltage-gated K+ channel subunit in rat prostate cancer cell lines: electrophysiological, pharmacological and molecular characterisation.

Voltage-gated K+ currents expressed in two rat prostate cancer ("Dunning") cell lines of markedly different metastatic ability were characterised using electrophysiological, pharmacological and molecular approaches. Whole-cell patch-clamp recordings showed that both strongly metastatic MAT-LyLu and weakly metastatic AT-2 cell lines possessed outward (delayed-rectifier type) K+ currents, which activated at around -40 mV. From the parameters measured, several characteristics of the two cell lines were similar. However, a number of statistically significant differences were noted for MAT-LyLu versus the AT-2 cells as follows: (1) current densities were smaller; (2) the slope factor for channel activation was smaller; (3) the voltage at which current was half-inactivated, and the slope factor for channel inactivation were greater; (4) the time constants for current decay at -20 and 0 mV were smaller; and (5) the residual peak current was larger following 60 s of repetitive voltage pulses for stimulation frequencies in the range 0.05-0.2 Hz. On the other hand, the K+ currents in both cell lines showed similar pharmacological profiles. Thus, the currents were blocked by 4-aminopyridine, tetraethylammonium, verapamil, margatoxin, and charybdotoxin, with highly similar IC(50)s for given blockers. The electrophysiological and pharmacological data taken together suggested expression of voltage-gated K+ channels of the Kv1 family, expression of the Kv1.3 subunit being predominant. Western blot and RT-PCR tests both confirmed that the cells indeed expressed Kv1.3 and to a lesser extent Kv1.4 and Kv1.6 channel alpha-subunits. In view of the similarity of channel expression in the two cell lines, voltage-gated K+ channel activity may not be a primary determinant of metastatic potential in the rat model of prostate cancer, but the possible contribution of K+ channel activity to the metastatic process is discussed.

Tomato and Soy Polyphenols Reduce Insulin-Like Growth Factor-I–Stimulated Rat Prostate Cancer Cell Proliferation and Apoptotic Resistance In Vitro via Inhibition of Intracellular Signaling Pathways Involving Tyrosine Kinase

We examined the ability of polyphenols from tomatoes and soy (genistein, quercetin, kaempferol, biochanin A, daidzein and rutin) to modulate insulin-like growth factor-I (IGF-I)-induced in vitro proliferation and apoptotic resistance in the AT6.3 rat prostate cancer cell line. IGF-I at 50 micro g/L in serum-free medium produced maximum proliferation and minimized apoptosis. Polyphenols exhibited different abilities to modulate IGF-I-induced proliferation, cell cycle progression (flow cytometry) and apoptosis (Annexin V/propidium iodide and terminal deoxynucleotidyltransferase-mediated deoxyuridine 5'-triphosphate nick end labeling). Genistein, quercetin, kaempferol and biochanin A exhibited dose-dependent inhibition of growth with a 50% inhibitory concentration (IC(50)) between 25 and 40 micro mol/L, whereas rutin and daidzein were less potent with an IC(50) of >60 micro mol/L. Genistein and kaempferol potently induced G(2)/M cell cycle arrest. Genistein, quercetin, kaempferol and biochanin A, but not daidzein and rutin, counteracted the antiapoptotic effects of IGF-I. Human prostate epithelial cells grown in growth factor-supplemented medium were also sensitive to growth inhibition by polyphenols. Genistein, biochanin A, quercetin and kaempferol reduced the insulin receptor substrate-1 (IRS-1) content of AT6.3 cells and prevented the down-regulation of IGF-I receptor beta in response to IGF-I binding. IGF-I-stimulated proliferation was dependent on activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and phosphatidylinositide 3-kinase pathways. Western blotting demonstrated that ERK1/2 was constitutively phosphorylated in AT6.3 cells with no change in response to IGF-I, whereas IRS-1 and AKT were rapidly and sensitively phosphorylated after IGF-I stimulation. Several polyphenols suppressed phosphorylation of AKT and ERK1/2, and more potently inhibited IRS-1 tyrosyl phosphorylation after IGF-I exposure. In summary, polyphenols from soy and tomato products may counteract the ability of IGF-I to stimulate proliferation and prevent apoptosis via inhibition of multiple intracellular signaling pathways involving tyrosine kinase activity.

Prostate-specific antigen (PSA) protein does not affect growth of prostate cancer cells in vitro or prostate cancer xenografts in vivo.

Prostate-specific antigen (PSA) is produced in high amounts by normal and malignant prostate cancer cells. PSA is a serine protease with substrates that include semenogelin I and II, insulin-like growth factor binding protein 3, fibronectin, and laminin. PSA, via its enzymatic activity, may play a role in growth, invasion, and metastasis of prostate cancer cells. Recent data also suggest that the PSA protein itself, independent of enzymatic activity, may also function as an endothelial cell-specific inhibitor of angiogenesis. Human (PC3, DU145) and rat (AT2, AT6) prostate cancer cell lines were transfected with the full PSA gene encoding preproPSA protein. PSA-producing clones of each cell line were selected and the amount of enzymatically active PSA produced by each cell line determined using a PSA-specific fluorescent peptide substrate. In vitro and in vivo growth characteristics of PSA-producing transfectants were compared to neomycin controls and wild type cells. All selected clones produced and secreted PSA (5-120 ng/ml/10(5) cells). None of the PSA-transfected cell lines produced detectable amounts of enzymatically active PSA. Production of enzymatically inactive PSA by prostate cancer cell lines did not alter growth kinetics in vitro. PSA-producing xenograft doubling times in vivo were similar to neomycin controls and wild type. Although recent reports suggest the PSA protein itself may be antiangiogenic, our results demonstrate that production of PSA protein by prostate cancer cells does not significantly alter growth in vitro or in vivo.

A novel steroid receptor co-activator protein (SRAP) as an alternative form of steroid receptor RNA-activator gene: expression in prostate cancer cells and enhancement of androgen receptor activity.

We have cloned a cDNA coding for a novel steroid receptor co-activator protein termed SRAP from a rat prostate library. Although the nucleotide sequence of the SRAP has 78.2% identity to that of the human steroid receptor RNA activator (SRA), a novel RNA molecule which was reported to act as an RNA transcript without being translated into protein [Lanz, McKenna, Onate, Albrecht, Wong, Tsai, Tsai and O'Malley (1999) Cell 97, 17-27], the cDNA of SRAP is capable of generating a functional protein. Glutathione S-transferase pull-down assays showed that SRAP associates with the partial androgen receptor (AR) protein composed of a DNA-binding domain and an activation function 2. Luciferase assays demonstrated that SRAP enhances the transactivation activity of the AR, the glucocorticoid receptor and the peroxisome proliferator-activated receptor gamma(1) in a ligand-dependent manner. Using a green fluorescent protein (GFP) fusion-protein construct, we demonstrated in vivo translation of the GFP-SRAP fusion protein in HeLa cells co-transfected with pSG5AR and reporter gene in the presence of 5 alpha-dihydrotestosterone (DHT). Co-transfection of the GFP-SRAP fusion protein expression plasmid enhanced the transactivation activity of AR whereas incorporation of mutations in SRAP of the fusion protein resulted in loss of enhancement of the transactivation activity. Northern blot analysis and reverse transcriptase PCR assays showed that SRAP and SRA are expressed in rat and human prostate cancer cell lines respectively. In HeLa cells and the human prostate cancer cells line DU-145, co-transfected with SRAP, the DHT-dependent transactivation activities of AR were not completely inhibited by the anti-androgen flutamide, but the transactivation activities still remained high even in the presence of 5 microM flutamide, suggesting that SRAP may play an important role in enhancing AR activity in prostate cancer.

Antiproliferative and apoptotic effects of silibinin in rat prostate cancer cells.

The tremendous impact of prostate cancer (PCA) on the US male population has led to an increased attention on its prevention and on therapeutic intervention. Short-term models are needed to quickly screen the efficacy of promising agents against PCA. We have established recently several rat PCA cell lines from primary PCA in rats induced by a MNU-testosterone protocol, but their usefulness as a model for screening PCA preventive and therapeutic agents remains to be established. With the rationale that agents found effective in these cells could be promising for efficacy testing in long-term in vivo experiments, e.g., with MNU-testosterone-induced PCA in rats, the major goal of our study was to assess the antiproliferative and apoptotic efficacy in rat PCA cell lines of silibinin, a major active flavonoid component of silymarin, which consists of a group of flavonoid antioxidants occurring in milk thistle (Silybum marianum). Three rat PCA cell lines, namely H-7, I-8, and I-26, were treated with silibinin or silymarin, a crude silibinin-containing preparation, at various doses for varying lengths of time. Cell growth and viability studies were carried out by using hemocytometer and Trypan blue dye exclusion methods. Cell cycle distribution studies were conducted by using PI staining and flow cytometry analysis, and DNA synthesis was assessed by bromodeoxyuridine incorporation. Apoptotic cell death was assessed as DNA damage by using an enzyme-linked immunosorbent assay method and by annexin V and PI staining followed by flow cytometry analysis. Silibinin resulted in a significant growth inhibition and reduction in cell viability in each cell line studied in both a dose- and a time-dependent manner. Silibinin treatment of H-7 and I-8 cells at 100 microM dose for 12 and 24 hr resulted in a G1 arrest but caused S phase arrest after a 48-hr treatment period in each cell line studied. Similar silibinin treatment of I-26 cells resulted in a slight S phase arrest at all time points studied. Consistent with these findings, silibinin showed a strong inhibition of DNA synthesis. Silibinin also induced a substantial apoptotic death in each cell line studied. Similar to silibinin, silymarin induced growth inhibition and reduced viability in a dose- and time-dependent manner. This study demonstrates that silibinin as well as silymarin induce growth inhibition and apoptosis in rat PCA cells. These results form a strong rationale for PCA prevention and therapeutic intervention studies with silibinin and silymarin in animal models, such as the MNU-testosterone rat PCA model, to establish their efficacy and to further define their mechanisms of action under in vivo conditions.

Stat3 activation in prostatic carcinomas.

Activated Stat3 is found in various types of immortal cell lines and cancers. We and others have previously demonstrated that Stat3 is constitutively activated in rat and human prostate cancer cell lines, and that Stat3 activation is involved in IL-6-mediated signaling transduction in prostate cancer cells. The aim of this study is to examine quantitative Stat3 activity in benign and malignant human prostate tissues and analyze the association between Stat3 activity levels and the clinical and pathologic parameters. Stat3 activity levels were analyzed in a total of 104 human primary prostate tissues using electromobility shift assay and immunohistochemical staining for phosphorylated Stat3. The tissue samples used were 42 prostate carcinomas, 42 matched normal prostate tissues from patients with prostatic adenocarcinoma (normal adjacent to tumor), and 20 normal prostate tissues from organ donors. Significantly higher levels of constitutive Stat3 activity were detected in both prostate carcinomas and the matched normal prostate tissues adjacent to tumors compared to the normal prostates from donors without prostate cancer. There was no significant difference of Stat3 activity in foci of tumor and normal prostate tissue adjacent to tumor. No correlation was seen between Stat3 activity and Gleason grade or serum PSA levels in samples from prostate carcinomas. These results indicate that Stat3 is constitutively activated in prostate cancer. The high level of Stat3 activity in both the prostate carcinomas and the normal prostate tissues adjacent to tumors suggests that Stat3 activation may occur before detectable histological alterations of the prostate.