Identification of Fibulin-1 as a Human Bone Marrow Stromal (HS-5) Cell-Derived Factor That Induces Human Prostate Cancer Cell Death.

BACKGROUND: Previous studies showed that human bone marrow stromal HS-5 cells secreted unidentified factor(s) inducing PCa cell death. Herein, the HS-5-derived factor (HS-5 DF) was characterized and identified. METHODS: Conditioned media from confluent HS-5 cells were collected and modified for biochemical characteristic testing of HS-5 DF. Cell survival was measured by apoptosis assay and live/dead assay. Fibulin-1 was identified from gel electrophoresis and mass spectrometry. The validation of Fibulin-1 as a HS-5 DF was done by immunoprecipitation (IP) and genetic knockdown by CRISPR/Cas9 system. RESULTS: HS-5 DF was trypsin and heat sensitive, but pH stable. The tentative size of the factor fell between 30 kDa and 100 kDa. TGF-ß1 treatment led to a suppression of HS-5 DF activity, a property consistent with bone metastasis in prostate cancer. Examination of TGF-ß1 down regulated proteins led to identification of fibulin-1 as a candidate for the DF. IP of Fibulin-1 from HS-5 CM and CRISPR knockdown of Fibulin-1 showed a significant reduction of HS-5 CM-derived PCa cell death. These results strongly support a role for fibulin-1 in HS-5 bone marrow stromal cell induction of PCa cell death. CONCLUSION: Our data indicate that Fibulin-1 functions as a HS-5 bone marrow stromal cell-derived factor inducing prostate cancer cell death. Prostate 77:729-742, 2017. © 2016 Wiley Periodicals, Inc.

Transforming growth factor-ß signaling induced during prostate cancer cell death and neuroendocrine differentiation is mediated by bone marrow stromal cells.

INTRODUCTION: Prostate cancer that has metastasized to bone undergoes critical interactions with bone marrow stromal cells (BMSCs), ultimately promoting tumor survival. Previous studies have shown that BMSCs secrete factors that promote prostate cancer apoptosis or neuroendocrine differentiation. Because of the significance of transforming growth factor-ß (TGF-ß) family cytokines in cytostasis and bone metastasis, the role of TGF-ß signaling in the context of prostate cancer-BMSC interactions was investigated. METHODS: The role of TGF-ß family signaling in BMSC-induced apoptosis of lineage-related prostate cancer cells was investigated in live/dead assays. SMAD phosphorylation or activity during apoptosis and neuroendocrine differentiation was investigated using immunofluorescence, Western blotting, and luciferase reporter assays, along with the ALK-4, -5, -7 kinase inhibitor, SB-431542. RESULTS: Treatment of castration-resistant prostate cancer cells with SB-431542 resulted in significant reduction of apoptosis mediated by HS-5 BMSCs, supporting the involvement of TGF-ß/SMAD signaling during this event. Interestingly, however, pre-treatment of BMSCs with TGF-ß1 (5 ng/mL) yielded a conditioned medium that elicited a marked reduction in prostate cancer death. Phosphorylated-SMAD2 (P-SMAD2) was activated in BMSC-triggered transdifferentiated prostate cancer cells, as demonstrated through immunoblotting and luciferase reporter assays. However, SB-431542 did not restore androgen receptor and prostate specific antigen levels down-regulated by BMSC-secreted factors. Prostate cancer cells induced to undergo neuroendocrine differentiation in a BMSC-independent mechanism also showed elevated levels of P-SMAD2. DISCUSSION: Collectively, our findings indicate that: (1) TGF-ß family cytokines or regulated factors secreted from BMSCs are involved in prostate cancer apoptosis; (2) TGF-ß signaling in prostate cancer cells is induced during neuroendocrine differentiation; and (3) TGF-ß1 stimulation of BMSCs alters paracrine signaling to create a permissive environment for prostate cancer survival, suggesting a mechanism for prostate cancer-mediated colonization of bone. CONCLUSIONS: TGF-ß signaling resulting in activation of SMAD2 in prostate cancer may be an indicator of cellular stress in the presence of toxic paracrine factors released from the bone marrow stroma, ultimately fostering prostate cancer colonization of bone.

Dynamic bioinspired coculture model for probing ER+ breast cancer dormancy in the bone marrow niche.

Late recurrences of breast cancer are hypothesized to arise from disseminated tumor cells (DTCs) that reactivate after dormancy and occur most frequently with estrogen receptor-positive (ER+) breast cancer cells (BCCs) in bone marrow (BM). Interactions between the BM niche and BCCs are thought to play a pivotal role in recurrence, and relevant model systems are needed for mechanistic insights and improved treatments. We examined dormant DTCs in vivo and observed DTCs near bone lining cells and exhibiting autophagy. To study underlying cell-cell interactions, we established a well-defined, bioinspired dynamic indirect coculture model of ER+ BCCs with BM niche cells, human mesenchymal stem cells (hMSCs) and fetal osteoblasts (hFOBs). hMSCs promoted BCC growth, whereas hFOBs promoted dormancy and autophagy, regulated in part by tumor necrosis factor-α and monocyte chemoattractant protein 1 receptor signaling. This dormancy was reversible by dynamically changing the microenvironment or inhibiting autophagy, presenting further opportunities for mechanistic and targeting studies to prevent late recurrence.

Increased TGF-ß1-mediated suppression of growth and motility in castrate-resistant prostate cancer cells is consistent with Smad2/3 signaling.

BACKGROUND: Elevated TGF-ß levels are associated with prostate cancer progression. Although TGF-ß is a tumor suppressor for normal epithelial and early-stage cancer cells, it may act paradoxically as a tumor promoter in more advanced cancers, although its effects are largely cell and context dependent. This study analyzed prostate cancer responses to TGF-ß signaling in an isogenic model of androgen-sensitive and castration-resistant prostate cancer cells. METHODS: Phosphorylation and nuclear translocation of Smad2 and Smad3 were analyzed using immunoblotting. Proliferation and cell cycle responses to TGF-ß1 (5 ng/ml) were assessed using growth assays and flow cytometry for DNA content, as well as Western blot and immunoprecipitation of cell cycle proteins. RESULTS: Both androgen-sensitive (LNCaP) and castration-resistant (C4-2 and C4-2B) prostate cancer cell lines demonstrated TGF-ß1-induced phosphorylation and nuclear translocation of Smad2/3 that was robust in metastatic lines. Smad phosphorylation was completely abrogated with inhibition of ALK-5 kinase activity using the kinase inhibitor, SB-431542. Increased sensitivity to TGF-ß1-mediated growth inhibition was observed in C4-2 and C4-2B cells, as compared to LNCaP cells. This was paralleled with downregulation of Cyclin D and increased association of p15(Ink4b) or p27(Kip) with CDK's. Additionally, TGF-ß1 inhibited motility and invasion of metastatic cell lines. CONCLUSIONS: TGF-ß-mediated suppression of growth and motility is enhanced in metastatic, castration-resistant prostate cancer cells. Enhanced TGF-ß1-induced Smad2 and -3 signaling in prostate cancer cells may correlate with tumor suppressive activity. Therefore, the direct effects of TGF-ß1 on prostate cancer cells post-castration may be anti-tumorigenic and growth-suppressive.

Overexpression of the VSSC-associated CAM, ß-2, enhances LNCaP cell metastasis associated behavior.

BACKGROUND: Prostate cancer (PCa) is the second-leading cause of cancer death in American men. This is due largely to the "silent" nature of the disease until it has progressed to a highly metastatic and castrate resistant state. Voltage sensitive sodium channels (VSSCs) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two ß subunits. The ß-subunits modulate surface expression and gating kinetics of the channels but also have inherent cell adhesion molecule (CAM) functions. We hypothesize that PCa cells use VSSC ß-subunits as CAMs during PCa progression and metastasis. METHODS: We overexpressed the beta-2 isoform as a C-terminal fusion protein with enhanced cyan fluorescence protein (ECFP) in the weakly metastatic LNCaP cells. The effect of beta-2 overexpression on cell morphology was examined using confocal microscopy while metastasis-associated behavior was tested by performing several in vitro metastatic functional assays and in vivo subcutaneous tumor studies. RESULTS: We found that cells overexpressing beta-2 (2BECFP) converted to a bipolar fibroblastic morphology. 2BECFP cells were more adhesive than control (ECFP) to vitronectin (twofold) and Matrigel® (1.3-fold), more invasive through Matrigel® (3.6-fold in 72 hr), and had enhanced migration (2.1-fold in 96 hr) independent of proliferation in wound-healing assays. In contrast, 2BECFP cells have a reduced tumor-take and tumor volume in vivo even though the overexpression of beta-2 was maintained. CONCLUSIONS: Functional overexpression of VSSC ß-subunits in PCa may be one mechanism leading to increased metastatic behavior while decreasing the ability to form localized tumor masses.

Paracrine factors produced by bone marrow stromal cells induce apoptosis and neuroendocrine differentiation in prostate cancer cells.

BACKGROUND: Preferential bony metastasis of human prostate cancer (PCa) cells contributes to disease mortality and morbidity. Local factors in bone stromal extracellular matrix microenvironment affect tumor growth through paracrine interactions between tumor and stromal cells. METHODS: Using co-culture and medium transfer, we used several methods to assess interactions between PCa and bone stromal cells using three PCa cell lines: PC3, LNCaP, and the LNCaP derivative, C4-2B. RESULTS: Co-culture of LNCaP and C4-2B cells with bone marrow stromal cell lines, HS27a and HS5, decreased cell number, as did culture with conditioned medium (CM) harvested from these two cell lines suggesting a soluble paracrine factor was responsible. PC3 cell growth was unaffected. CM harvested from bone stromal cell lines triggered apoptosis in LNCaP and C4-2B cell lines, but not in PC3 cells. Surviving C4-2B cells grown in bone stromal cell CM over several days were growth arrested, suggesting presence of a growth inhibitor. Apoptosis induced by CM was dose-dependent. Flow cytometry demonstrated that over a 5-day culture period in stromal cell CM, LNCaP, and C4-2B cell lines, but not PC3 cells, underwent greater apoptosis than parallel cultures in SF medium. The LNCaP and C4-2B cells showed morphology and biomarker expression consistent with transdifferentiation towards a neuroendocrine phenotype after exposure to stromal cell CM. CONCLUSIONS: The reactive bone stromal microenvironment initially is hostile to PCa cells producing widespread apoptosis. Activation of transdifferentiation in a subset of apoptotic resistant cells may support phenotypic adaptation during disease progression in bone, eventually favoring lethal disease.

The developmental expression profile of PAX2 in the murine prostate.

BACKGROUND: Nine transcription factors comprise the PAX gene family that regulate organogenesis. The urogenital system of PAX2 null male mice fails to develop properly. PAX2 is overexpressed in PC3 cells. Therefore, PAX2 is implicated in both prostate organogenesis and cancer. However, the expression pattern/profile of PAX2 in the prostate is unknown. METHODS: PAX2/5/8 expression was surveyed in E16.5 male urogenital sinus (UGS) by RT-PCR. Prostate samples from 10 developmental stages in C3H male mice were used in quantitative reverse-transcript PCR (Q-PCR) and Western blotting (WB). RT-PCR and WB measured PAX2 expression in prostatic lobes or UGS layers, to identify local-regional expression patterns. Cytoplasmic versus nuclear expression was examined by WB. A castration series in adult C3H male mice and R1881 treatment in serum-free LNCaP cells examined androgen control of PAX2. RESULTS: PAX2 mRNA levels are higher in early developmental stages as compared to postpubertal prostates. RT-PCR and/or WB indicated a dorsal epithelial-nuclear localization of PAX2. PAX2 mRNA and protein increase postcastration. R1881 decreases expression of PAX2 mRNA in LNCaP cells as compared to controls. CONCLUSIONS: The expression profile of PAX2 indicates that it may regulate early, androgen-independent stages of murine prostate development, particularly for dorsally derived prostate glands. PAX2 expression appears to be associated with a dorsally localized epithelial cell population that is castration insensitive and retains proliferative and differentiative potential. Such a population of cells may represent a subset of stem-like cells having some characteristics in common with castrate-resistant prostate cancer cells.

Short-term intermittent PTH 1-34 administration enhances bone formation in SCID/Beige mice.

The anabolic effect of intermittent PTH on bone is variable depending on the species studied, duration/mode of administration, and location of skeletal response investigated. We tested the hypothesis low dose, short term, intermittent PTH 1-34 administration is sufficient to enhance bone formation without altering bone resorption. To test our hypothesis, mice were treated intermittently with one of three concentrations of PTH 1-34 (1 microg/kg; low, 10 microg/kg, or 20 microg/kg; high) for three weeks. The skeletal response was identified by quantifying: serum markers of bone turnover, cancellous bone parameters in distal femur, proximal tibia, and lumbar vertebrae by microCT, and number of osteoblasts and osteoclasts in distal femur. Mice receiving 20 microg/kg of PTH 1-34 demonstrated a 30% increase in serum osteocalcin, but no differences in serum calcium, type I collagen teleopeptides, or TRACP 5b. For all bones, microCT analysis suggested mice receiving 20 microg/kg of PTH 1-34 had increased cancellous bone mineral density, trabecular thickness and spacing, but decreased trabecular number. A 60% increase in the number of alkaline phosphatase positive osteoblasts in the distal femur was also observed in tissue sections; however, the number of TRAP positive osteoclasts was not different between test and control groups. While animals administered 10 microg/kg demonstrated similar trends for all bone turnover indices, such alterations were not observed in animals administered PTH 1-34 at 1 microg/kg per day. Thus, PTH 1-34, administered intermittently for three weeks at 20 microg/kg is sufficient to enhance bone formation without enhancing resorption.

Quantitative modeling and analysis of the transforming growth factor beta signaling pathway.

Transforming growth factor beta (TGF-beta) signaling, which regulates multiple cellular processes including proliferation, apoptosis, and differentiation, plays an important but incompletely understood role in normal and cancerous tissues. For instance, although TGF-beta functions as a tumor suppressor in the premalignant stages of tumorigenesis, paradoxically, it also seems to act as a tumor promoter in advanced cancer leading to metastasis. The mechanisms by which TGF-beta elicits such diverse responses during cancer progression are still not entirely clear. As a first step toward understanding TGF-beta signaling quantitatively, we have developed a comprehensive, dynamic model of the canonical TGF-beta pathway via Smad transcription factors. By describing how an extracellular signal of the TGF-beta ligand is sensed by receptors and transmitted into the nucleus through intracellular Smad proteins, the model provides quantitative insight into how TGF-beta-induced responses are modulated and regulated. Subsequent model analysis shows that mechanisms associated with Smad activation by ligand-activated receptor, nuclear complex formation among Smad proteins, and inactivation of ligand-activated Smad (e.g., degradation, dephosphorylation) may be critical for regulating TGF-beta-targeted functional responses. The model was also used to predict dynamic characteristics of the Smad-mediated pathway in abnormal cells, from which we generated four testable hypotheses regarding potential mechanisms by which TGF-beta's tumor-suppressive roles may appear to morph into tumor-promotion during cancer progression.

Androgen-independent growth and tumorigenesis of prostate cancer cells are enhanced by the presence of PKA-differentiated neuroendocrine cells.

The neuroendocrine status of prostatic adenocarcinomas is considered a prognostic indicator for development of aggressive, androgen-independent disease. Neuroendocrine-like cells are thought to function by providing growth and survival signals to surrounding tumor cells, particularly following androgen ablation therapy. To test this hypothesis directly, LNCaP cells were engineered to inducibly express a constitutively activated form of the cyclic AMP-dependent protein kinase A catalytic subunit (caPKA), which was previously found upon transient transfection to be sufficient for acquisition of neuroendocrine-like characteristics and loss of mitotic activity. Clonal cells that inducibly expressed caPKA enhanced the growth of prostate tumor cells in anchorage-dependent and anchorage-independent in vitro assays as well as the growth of prostate tumor xenografts in vivo, with the greatest effects seen under conditions of androgen deprivation. These results suggest that neuroendocrine-like cells of prostatic tumors have the potential to enhance androgen-independent tumor growth in a paracrine manner, thereby contributing to progression of the disease.

Novel surface expression of reticulocalbin 1 on bone endothelial cells and human prostate cancer cells is regulated by TNF-alpha.

An unbiased cDNA expression phage library derived from bone-marrow endothelial cells was used to identify novel surface adhesion molecules that might participate in metastasis. Herein we report that reticulocalbin 1 (RCN1) is a cell surface-associated protein on both endothelial (EC) and prostate cancer (PCa) cell lines. RCN1 is an H/KDEL protein with six EF-hand, calcium-binding motifs, found in the endoplasmic reticulum. Our data indicate that RCN1 also is expressed on the cell surface of several endothelial cell lines, including human dermal microvascular endothelial cells (HDMVECs), bone marrow endothelial cells (BMEC), and transformed human bone marrow endothelial cells (TrHBMEC). While RCN1 protein levels were highest in lysates from HDMVEC, this difference was not statistically significant compared BMEC and TrHBMEC. Given preferential adhesion of PCa to bone-marrow EC, these data suggest that RCN1 is unlikely to account for the preferential metastasis of PCa to bone. In addition, there was not a statistically significant difference in total RCN1 protein expression among the PCa cell lines. RCN1 also was expressed on the surface of several PCa cell lines, including those of the LNCaP human PCa progression model and the highly metastatic PC-3 cell line. Interestingly, RCN1 expression on the cell surface was upregulated by tumor necrosis factor alpha treatment of bone-marrow endothelial cells. Taken together, we show cell surface localization of RCN1 that has not been described previously for either PCa or BMEC and that the surface expression on BMEC is regulated by pro-inflammatory TNF-alpha.

Purinergic Receptor Expression and Cellular Responses to Purinergic Agonists in Human Prostate Cancer Cells.

BACKGROUND: Anticancer activity of extracellular nucleotides has been investigated in many types of cancer. Herein, the effects of extracellular nucleotides and the receptor profile for these nucleotides on prostate cancer (PCa) were elaborated. MATERIALS AND METHODS: PCa cell lines representing different stages of PCa were used. The effects of ATP and adenosine on PCa growth and migration on different extracellular matrix proteins were examined by MTT and wound-healing assays. Purinergic receptor profiling was carried out by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: A growth-inhibitory effect of ATP and adenosine was observed on all PCa cell lines tested. Several ATP-recognized P2 receptors and adenosine receptors were commonly expressed in PCa cell lines. Neither ATP nor adenosine had any significant effect on PCa migration. CONCLUSION: ATP and adenosine had an antiproliferative effect on PCa cells without affecting their motility, indicating their potential as a novel therapy for PCa.

Gene expression in the LNCaP human prostate cancer progression model: progression associated expression in vitro corresponds to expression changes associated with prostate cancer progression in vivo.

Identification of the genes involved in prostate cancer (PCa) progression to a virulent and androgen-independent (AI) form is a major focus in the field. cDNA microarray was used to compare the gene expression profile of the indolent, androgen sensitive (AS) LNCaP PCa cell line to the aggressively metastatic, AI C4-2. Thirty-eight unique sequences from a 6388 cDNA array were found differentially expressed (> or =2-fold, 95% CI). The expression of 14 genes was lower in C4-2 than in LNCaP cells, while the reverse was true for 24 genes. Twelve genes were validated using Q-PCR, Western blotting and immunohistochemistry (IHC) of LNCaP and C4-2 xenograft. Q-PCR showed that 10 of 12 (83.3%) genes had similar patterns of expression to the array (LNCaP>C4-2: TMEFF2, ATP1B1, IL-8, BTG1, BChE, NKX3.1; LNCaP

Met-Independent Hepatocyte Growth Factor-mediated regulation of cell adhesion in human prostate cancer cells.

BACKGROUND: Prostate cancer cells communicate reciprocally with the stromal cells surrounding them, inside the prostate, and after metastasis, within the bone. Each tissue secretes factors for interpretation by the other. One stromally-derived factor, Hepatocyte Growth Factor (HGF), was found twenty years ago to regulate invasion and growth of carcinoma cells. Working with the LNCaP prostate cancer progression model, we found that these cells could respond to HGF stimulation, even in the absence of Met, the only known HGF receptor. The new HGF binding partner we find on the cell surface may help to clarify conflicts in the past literature about Met expression and HGF response in cancer cells. METHODS: We searched for Met or any HGF binding partner on the cells of the PC3 and LNCaP prostate cancer cell models, using HGF immobilized on agarose beads. By using mass spectrometry analyses and sequencing we have identified nucleolin protein as a novel HGF binding partner. Antibodies against nucleolin (or HGF) were able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. Western blots, RT-PCR, and immunohistochemistry were used to assess nucleolin levels during prostate cancer progression in both LNCaP and PC3 models. RESULTS: We have identified HGF as a major signaling component of prostate stromal-conditioned media (SCM) and have implicated the protein nucleolin in HGF signal reception by the LNCaP model prostate cancer cells. Antibodies that silence either HGF (in SCM) or nucleolin (on the cell surfaces) eliminate the adhesion-stimulatory effects of the SCM. Likewise, addition of purified HGF to control media mimics the action of SCM. C4-2, an LNCaP lineage-derived, androgen-independent human prostate cancer cell line, responds to HGF in a concentration-dependent manner by increasing its adhesion and reducing its migration on laminin substratum. These HGF effects are not due to shifts in the expression levels of laminin-binding integrins, nor can they be linked to expression of the known HGF receptor Met, as neither LNCaP nor clonally-derived C4-2 sub-line contain any detectable Met protein. Even in the absence of Met, small GTPases are activated, linking HGF stimulation to membrane protrusion and integrin activation. Membrane-localized nucelolin levels increase during cancer progression, as modeled by both the PC3 and LNCaP prostate cancer progression cell lines. CONCLUSION: We propose that cell surface localized nucleolin protein may function in these cells as a novel HGF receptor. Membrane localized nucleolin binds heparin-bound growth factors (including HGF) and appears upregulated during prostate cancer progression. Antibodies against nucleolin are able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. HGF-nucleolin interactions could be partially responsible for the complexity of HGF responses and met expression reported in the literature.

Constitutive activation of the Ras/mitogen-activated protein kinase signaling pathway promotes androgen hypersensitivity in LNCaP prostate cancer cells.

Progression of prostate cancer ultimately results in a disease that is refractory to hormone ablation therapy but nevertheless continues to require the androgen receptor. Progression to hormone refractory disease is often correlated with overexpression of growth factors and receptors capable of establishing autocrine and/or paracrine growth-stimulatory loops. Many of these growth factor receptors engage the Ras/mitogen-activated protein (MAP) kinase pathway as part of their signaling activities. This raises the possibility that chronic activation of Ras/MAP kinase signaling could cause or contribute to the progression of prostate cancer. We have demonstrated previously that MAP kinase activation correlates with the progression to advanced hormone refractory disease in patient samples. Here we demonstrate that stable expression of Ras effector-loop mutants that activate the Ras/MAP kinase pathway is sufficient to reduce the androgen requirement of LNCaP prostate cancer cells for growth, prostate-specific antigen expression, and tumorigenicity. We propose that chronic activation of endogenous c-Ras by autocrine and paracrine growth factor stimulation sensitizes the androgen receptor transcriptional complex to subphysiological levels of androgen. This provides a common mechanism for prostate cancer progression driven by diverse agonists.

Increased insulin-like growth factor I receptor expression and signaling are components of androgen-independent progression in a lineage-derived prostate cancer progression model.

Apoptosis and inhibition of mitosis are primary mechanisms mediating androgen ablation therapy-induced regression of prostate cancer (PCa). However, PCa readily becomes androgen independent, leading to fatal disease. Up-regulated growth and survival signaling is implicated in development of resistance to androgen ablation therapy. We are testing the hypothesis that insulin-like growth factor (IGF) responsiveness is required for androgen-independent (AI) progression. Using the LNCaP human PCa progression model, we have determined that IGF-I-mediated protection from apoptotic stress and enhanced mitotic activity is androgen dependent in LNCaP cells but is androgen independent in lineage-derived C4-2 cells. Both cell lines exhibit androgen-responsive patterns of IGF-I receptor (IGF-IR) expression, activation, and signaling to insulin receptor substrate-2 and AKT. However, C4-2 cells express higher levels of IGF-IR mRNA and protein and exhibit enhanced IGF-I-mediated phosphorylation and downstream signaling under androgen-deprived conditions. In comparisons of naive and AI metastatic human PCa specimens, we have confirmed that IGF-IR levels are elevated in advanced disease. Together with our LNCaP/C4-2 AI progression model data, these results indicate that increased IGF-IR expression is associated with AI antiapoptotic and promitotic IGF signaling in PCa disease progression.

Voltage-gated sodium channel blockers as cytostatic inhibitors of the androgen-independent prostate cancer cell line PC-3.

The recent discovery of sodium (Na(+)) channel expression in human prostate cancer (PCa) cells led us to investigate the potential use of neuronal Na(+) channel blockers as inhibitors of PCa cells. Our initial studies discovered two classes of Na(+) channel blockers that were effective inhibitors of PCa cell proliferation. Both hydroxyamides (compounds 1 and 4) and a hydantoin (compound 5) were shown to inhibit the androgen-independent PCa cell line PC-3 in vitro. Electrophysiology showed that all compounds functionally block brain type II voltage-gated Na(+) channels (Nav1.2) expressed in Xenopus laevis oocytes. Long-term growth assays in androgen-independent PC-3 cells showed remarkable inhibition of cell growth, with cells growing to a maximum of 30% of controls with analogue 1. Further, our analogues demonstrated only marginal impact on cell viability over the same treatment interval.

Cell-based assays using calcein acetoxymethyl ester show variation in fluorescence with treatment conditions.

The use of fluorogenic compounds in cell and molecular biology has increased in both frequency and range of applications. However, such compounds may introduce artifacts in intracellular fluorescence and cell number estimations as a consequence of interaction with exogenous stimulants, necessitating the use of adequate controls for accurate measurements and valid conclusions. Using calcein acetoxymethyl ester (AM) in combination with various exogenous cellular treatments, we report that the standard practice of direct normalization of experimental values to controls is insufficient for fluorogenic measurements. Treatments applied to cells may influence intracellular conversion of the fluorogenic compound, thereby enhancing or decreasing fluorescence relative to controls. We hereby encourage caution and recommend normalization of cellular fluorescence within each treatment group before comparison to controls.

Changes in extracellular matrix (ECM) and ECM-associated proteins in the metastatic progression of prostate cancer.

Prostate cancer (PCa) is no exception to the multi-step process of metastasis. As PCa progresses, changes occur within the microenvironments of both the malignant cells and their targeted site of metastasis, enabling the necessary responses that result in successful translocation. The majority of patients with progressing prostate cancers develop skeletal metastases. Despite advancing efforts in early detection and management, there remains no effective, long-term cure for metastatic PCa. Therefore, the elucidation of the mechanism of PCa metastasis and preferential establishment of lesions in bone is an intensive area of investigation that promises to generate new targets for therapeutic intervention. This review will survey what is currently know concerning PCa interaction with the extracellular matrix (ECM) and the roles of factors within the tumor and ECM microenvironments that contribute to metastasis. These will be discussed within the context of changes in expression and functional heterodimerization patterns of integrins, changes in ECM expression and reorganization by proteases facilitating invasion. In this context we also provide a brief summary of how growth factors (GFs), cytokines and regulatory signaling pathways favor PCa metastasis to bone.

The role of insulin-like growth factor-II in cancer growth and progression evidenced by the use of ribozymes and prostate cancer progression models.

Towards understanding the IGF system during cancer growth and progression, progressive prostate cancer models, such as SV40 large T antigen immortalized human prostate epithelial cells (P69, M2182, M2205, and M12) and LNCaP sublines (C4, C4-2, and C4-2B4), were used. IGF-II mRNA levels progressively increase as prostate cancer cells become more tumorigenic and metastatic, suggesting that IGF-II contributes in part to prostate cancer progression. The role of IGF-II in cancer cell growth was evaluated in LNCaP, PC3, and M12 prostate cancer cell lines and MCF-7 breast cancer cell line by ribozyme/antisense strategies which were previously shown to suppress endogenous IGF-II expression and cell growth in PC-3 cells [Xu et al., Endocrinol 140 (1999) 2134]. Retroviral mediated transient expression of IGF-II-specific ribozyme (RZ) caused extensive cell death. In stably cloned cell lines, both RZ and mutant ribozyme (MRZ) inhibited cancer cell growth, suggesting that antisense effects of MRZ may be sufficient for cell growth inhibition. These results confirm an important role of IGF-II in cancer cell growth and progression, and support further development of gene therapy targeting IGF-II.