Prostate Cancer 3 Research Articles

Dietary Melatonin and Omega-3 Fatty Acids Induce Human Cancer Xenograft Regression In Vivo in Rats by Suppressing Linoleic Acid Uptake and Metabolism.

Melatonin, the circadian nighttime neurohormone, and eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA), which are omega-3 fatty acids (FA) found in high concentrations in fish oil (FO) and plants, abrogate the oncogenic effects of linoleic acid (LA), an omega-6 FA, on the growth of rodent tumors and human breast, prostate, and head and neck squamous cell carcinoma (HNSCC) xenografts in vivo. Here we determined and compared the long-term effects of these inhibitory agents on tumor regression and LA uptake and metabolism to the mitogenic agent 13-[S]-hydroxyoctadecadienoic acid (13-[S]-HODE) in human prostate cancer 3 (PC3) and FaDu HNSCC xenografts in tumor-bearing male nude rats. Rats in this study were split into 3 groups and fed one of 2 diets: one diet containing 5% corn oil (CO, high LA), 5% CO oil and melatonin (2 μg/mL) or an alternative diet 5% FO (low LA). Rats whose diet contained melatonin had a faster rate of regression of PC3 prostate cancer xenografts than those receiving the FO diet, while both in the melatonin and FO groups induced the same rate of regression of HNSCC xenografts. The results also demonstrated that dietary intake of melatonin or FO significantly inhibited tumor LA uptake, cAMP content, 13-[S]-HODE formation, [³H]-thymidine incorporation into tumor DNA, and tumor DNA content. Therefore, long-term ingestion of either melatonin or FO can induce regression of PC3 prostate and HNSCC xenografts via a mechanism involving the suppression of LA uptake and metabolism by the tumor cells.

Isoleucine with secondary sulfonamide functionality as anticancer, antibacterial and antifungal agents

Isoleucine substituted analogues with secondary sulfonamide group (I1–I6) have been synthesized. Structures of synthesized analogues have been confirmed by Fourier Transform-Infrared Red, Nuclear Magnetic Resonance (1H and 13C) and ESI-MS spectroscopic tools. Cytotoxic screenings of synthesized analogues have been done on MCF-7 (breast), Prostate Cancer-3 (PC-3) and A549 (lung) cancer cell lines. N-(1-isobutyl-2-oxo-2-anilinoethyl) p-toluene sulfonamide (I5) screened to be better cytotoxic agent on MCF-7 and A549 cell lines whereas N-(1-isobutyl-2-oxo-2-p-chloroanilino ethyl) benzene sulfonamide (I3) against PC-3 cell line. Cell cycle analysis of N-(1-isobutyl-2-oxo-2-anilinoethyl) p-toluene sulfonamide (I5) analogue has been carried out on A549 cell line in comparison to control and Vinblastine (standard drug). Complete arrest in G0 and G1 phase along with mild disturbance in S-phase of cell cycle has been observed. The screened analogues (I1–I6) also showed good antifungal and antibacterial potential against gram positive as well as gram negative strains. Computer simulation indicated good bioactivity prediction by the ‘Lipinski rule’ and synthesized analogues did not violate this rule. Docking study of isoleucine sulfonamide analogues (I1–I6) were carried out to determine the possible interaction sites of the analogues with p53 tumor suppressor-DNA complex and demonstrate that the analogues confirmed binding and inhibition with the most mutated residues of p53. Density functional theory has been used to correlate the electronic and chemical properties of analogues and they were found to be stable and chemically reactive. Thus the results suggest that isoleucine substituted sulfonamide analogues can serve as a structural model for the design of anticancer agents, antibacterial agents as well as antifungal agents with better inhibitory potential. Communicated by Ramaswamy H. Sarma

EVALUATION OF [11C]MPC-6827 AS A MICROTUBULE TARGETING PET RADIOTRACER IN CANCER CELL LINES

Objective: The objective of this study was to evaluate the uptake and specificity of [11C]MPC-6827, a MT targeted PET ligand in prostate, glioblastoma and breast cancer cells.
 Methods: [11C]MPC-6827 was synthesized by reacting corresponding desmethyl precursors with [11C]CH3I in a GE-FX2MeI/FX2M radiochemistry module. In vitro binding of [11C]MPC-6827 was performed in breast cancer MDA-MB-231, glioblastoma (GBM) patient-derived tumor (GBM-PDX), GBM U251 and prostate cancer 3 (PC3) cell lines at 37 °C in quadruplicate at 5, 15, 30, 60, and 90 minute incubation time. The nonspecific bindings were determined by incubation with unlabeled microtubule targeting agents MPC-6827, HD-800, colchicine, paclitaxel and docetaxel (5.0 mM).
 Results: [11C]MPC-6827 provided the highest binding in the breast cancer cell, MDA-MB-231, among all the cells studied, with 90% specific binding. [11C]MPC-6827 binds to glioblastoma PDX and U251 cells with ~50% and 40% specific binding, whereas, prostate cancer cell line, PC3 cells showed 40% specific binding. [11C]MPC-6827 also exhibits binding to the taxane and colchicine binding sites of MTs, in MDA-MB-231 cells.
 Conclusion: These data indicate that [11C]MPC-6827 can be a promising PET radiotracer for preclinical imaging of the brain and peripheral cancers.

Design, Synthesis and Biological Evaluation of 4-Aryl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one Derivatives as a PI3Kα Inhibitor.

A novel series of 4-aryl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives were designed as a phosphoinositide 3-kinase α (PI3Kα) inhibitor by scaffold hopping. The target compounds, characterized by 1H-NMR, 13C-NMR and high resolution (HR)-MS, were synthesized from diethyl malonate and ethyl chloroacetate by nucleophilic substitution, ring-closure, chlorination and Suzuki reaction, etc. The biological activities were evaluated with cytotoxic activity in vitro on Uppsala 87 Malignant Glioma (U87MG) and prostate cancer-3 (PC-3) by Cell Counting Kit-8 (CCK-8). The results showed that compound 9c displayed the higher inhibition than the positive control PI-103, and high PI3Kα inhibitory activity with IC50 of 113 ± 9 nM in the same order of magnitude as BEZ235. In addition, the Log Kow values and molecular docking studies were performed to further investigate the drug-like properties of target compounds and interactions between 9c and PI3Kα.

Palladium nanoparticle-decorated 2-D graphene oxide for effective photodynamic and photothermal therapy of prostate solid tumors.

Intratumoral injection of nanoparticles is a viable alternative for treating solid tumors. In this study, we used intratumorally-injected palladium nanoparticle (Pd NP)-decorated graphene oxide (GO) (GO-Pd NPs) for the treatment of solid prostate tumors. GO was synthesized using the modified Hummer's method and GO-Pd NPs were prepared using the one pot synthesis method. Studies on physicochemical characterization and in vitro/in vivo anticancer properties were performed using GO-Pd NPs. Successful preparation of GO-Pd NPs was confirmed by transmission electron microscopy, Fourier transform infrared spectroscopy, energy dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy. Compared to GO or Pd NPs alone, GO-Pd NPs showed higher cytotoxic effects in prostate cancer 3 (PC3) cells. Irradiation of treated cells with near infrared (NIR) laser considerably enhanced apoptosis induced by synergistic photothermal effect and reactive oxygen species (ROS) generation. Intratumorally-injected GO-Pd NPs showed promising in vivo localized distribution, photothermal ablation, and anti-tumor effects in the PC3 xenograft mouse model. Furthermore, the minimal organ toxicity of GO-Pd NPs was an added advantage. Hence, GO-Pd NPs could be a potential formulation for localized treatment of prostate solid tumors.

Evaluation of a short RNA within Prostate Cancer Gene 3 in the predictive role for future cancer using non-malignant prostate biopsies.

BackgroundProstate Cancer 3 (PCA3) is a long non-coding RNA (ncRNA) upregulated in prostate cancer (PCa). We recently identified a short ncRNA expressed from intron 1 of PCA3. Here we test the ability of this ncRNA to predict the presence of cancer in men with a biopsy without PCa.MethodsWe selected men whose initial biopsy did not identify PCa and selected matched cohorts whose subsequent biopsies revealed PCa or benign tissue. We extracted RNA from the initial biopsy and measured PCA3-shRNA2, PCA3 and PSA (qRT-PCR).ResultsWe identified 116 men with and 94 men without an eventual diagnosis of PCa in 2–5 biopsies (mean 26 months), collected from 2002–2008. The cohorts were similar for age, PSA and surveillance period. We detected PSA and PCA3-shRNA2 RNA in all samples, and PCA3 RNA in 90% of biopsies. The expression of PCA3 and PCA3-shRNA2 were correlated (Pearson’s r = 0.37, p<0.01). There was upregulation of PCA3 (2.1-fold, t-test p = 0.02) and PCA3-shRNA2 (1.5-fold) in men with PCa on subsequent biopsy, although this was not significant for the latter RNA (p = 0.2). PCA3 was associated with the future detection of PCa (C-index 0.61, p = 0.01). This was not the case for PCA3-shRNA2 (C-index 0.55, p = 0.2).ConclusionsPCA3 and PCA3-shRNA2 expression are detectable in historic biopsies and their expression is correlated suggesting co-expression. PCA3 expression was upregulated in men with PCa diagnosed at a future date, the same did not hold for PCA3-shRNA2. Futures studies should explore expression in urine and look at a time course between biopsy and PCa detection.

Photobleaching of fluorescein as a probe for oxidative stress in single cells

BackgroundROS are involved in the regulation of many physiological and pathological processes. Apoptosis and necrosis are processes that are induced by changes in concentrations of Reactive Oxygen Species (ROS). This study aims to detect and quantify the cellular response to changing ROS concentrations in the scope of apoptosis and necrosis. MethodsPhotobleaching of the fluorescent substrate fluorescein is used as a probe to detect the response of individual Jurkat-T-lymphocytes and Prostate-Cancer-3(PC-3) cells to oxidative stress, induced by hydrogen peroxide (H2O2). A kinetic model is proposed to describe changes in intracellular dye quantities due to photobleaching, dye hydrolysis, influx and leakage, yielding a single time-dependent decaying exponent+constant. ResultsFluorescein photobleaching is controlled and used to detect intracellular ROS. An increase in the decay time of fluorescence of intracellular fluorescein (slow photobleaching) was measured from cells incubated with H2O2 at 50μM. At higher H2O2 concentrations a decrease in the decay time was measured (fast photobleaching), in contrast to in vitro results with fluorescein and H2O2 in phosphate buffer saline (PBS), where the addition of H2O2 decreases the decay time, regardless of the irradiation dose used. ConclusionsThe anomalous, ROS-concentration dependent reduction of the photobleaching rate in cells, as opposed to solutions, might indicate on the regulation of the activity of intracellular oxidative-stress protective mechanisms, as seen earlier with other methods. SignificanceAssessing photobleaching via the time decay of the fluorescence intensity of an ROS-sensitive fluorophore may be adapted to monitor oxidative stress or ROS-related processes in cells.

English

Introduction Src kinase activation has been rep orted in unrelated human cancers and considered a possible target of anti-invasive therapies. Our aim here is to identify the relationship of Src kinase with CD44-signalling. Although CD44 is implicated in the invasion of cancer cells, its role in invadopodia formation needs further elucidation. Materials and Methods To study the role of Src, PC3 cells were transfected with Src constructs by adenoviral-mediated delivery. Gelatin degradation assay was performed to determine the invasive property of PC3 cells. Immunoblotting and immunostaining analyses were used to determine complex interaction of proteins with CD44. PC3 cells knockdown of CD44 were used to characterize the function of CD44 in invadopodia formation and invasion. Results In this study, we show that expression of constitutively-active Src in prostate cancer 3 (PC3) cells significantly increased the invasiveness of PC3 cells via increasing the number of invadopodia as a result of CD44-associated complex (Src-Cortactin-WiskottAldrich Syndrome Protein WASP) forma tion. The inhibition of invadopodial structures in CD44 knockdown PC3 cells resulted in reduced invasion. Conclusion Based on these results, we conclude that CD44-Src-cortactin-WASP is an invasion promoting complex. Proteins in this complex are relevant targets for intervening invadopodia formation and migration/invasion processes in prostate cancer cells. Introduction Prostate cancer is a disease of extensive metastases, with secondary lesions in lymph nodes, bones and sometimes in visceral organs, including liver, lungs and even the brain. Actin dynamics is a critical process that modulates cellular activity via regulation of signalling pathways through receptors and cell adhesion to the extracellular matrix (ECM). CD44 is a cell surface receptor and regulator of cell migration and tumour metastasis1–5. We have shown pre viously in prostate cancer cells (PC3) that surface expression of CD44 and formation of CD44-Matrix metalloproteinase 9 (MMP9) complex generates motility enhancing signals through the degradation of ECM proteins6,7. These cells use invadopodia-like structures for invasion into ECM. Localisation of MMP9 in the invadopodia assists in the focal degradation of matrix during invasion7. Signalling and actin-binding proteins (e.g. Src, cortactin, WASP and Arp2/3) have been shown to contribute to the formation of invadopodia8–12. CD44 receptor has the potential to inte grate adhesive and signalling activities to modulate migration/invasion processes during cancer progression. CD44 shares some properties with the integrin receptor. Similar to integrin, the cytoplasmic domain of CD44 associates with several proteins involved in signalling and actin dynamics13,14. However, the role of CD44 in the form ation of invadopodia is not clear. PC3 cells knockdown of MMP9 (PC3/MMP9-/-) are highly adhesive which is due to the expression of highly glycosylated CD44 variant isoform v6. These cells are less invasive due to a deficiency in the expression of standard CD44 (CD44s) and failure in the formation invadopodia2. Pertinent to proteins that CD44 interacts with, it is highly possible that CD44 might regulate the formation of invadopodia. Therefore, we hypothesise that similar to integrins, CD44 associates with proteins involved in signalling, and these, in turn, are associated with actin and actin-binding proteins. We show here that Src has a direct role in the formation of invadopodia through its interaction with CD44. CD44-associated signalling complex (Src, WASP, cortactin and Arp2/3) regulates the process of actin polymerisation involved in invadopodia formation. Materials and Methods The protocol of this study has been approved by the relevant ethical committee related to our institution in which it was performed. Antibodies towards CD44, MMP9, Src, WASP, cortactin and cortactin (Y421) were bought from Santo Cruz Biotechnology, Inc. (Santa Cruz, CA). An antibody towards phosphoSrc Y418 was obtained from Cell Signalling (Beverly, MA). Rhodamine phalloidin and other chemicals were purchased from Sigma. CY2 and CY3 conjugated secondary antibodies were purchased from Jackson Immuno Research Laboratories, Inc. (West Grove, PA).

TU‐A‐144‐01: Clinical Delivery and Advanced Novel Technologies/Strategies

High Intensity Focused ultrasound, (HIFU), is a novel medical procedure using acoustic energy, delivered in a focused manner and under guidance of ultrasound or MRI, to a targeted volume of living tissue. The energy can induces mechanical, thermal and biological outcomes determined by the acoustic parameters used and their interaction with the tissue. The most common and already clinically used effect is heating in an accurate, localized and non‐invasive manner the tissue to a high enough temperature thereby inducing thermal necrosis. During this session we will cover current and future trends of MR guided HIFU in Radiation Oncology and would take an in‐depth look at HIFU usage for treatment of prostate cancer, bone metastasis and MRI guided hyperthermia. Current and future trends in HIFU for radiation oncology: HIFU already has a proven track record for the treatment of liver tumors, pancreatic and breast cancer as well as a significant potential to improve the care of brain cancer patients. We will cover the clinical and regulatory status of this method for the listed clinical indications as well as existing devices. Prostate cancer: Ultrasound guided High Intensity Focused Ultrasound for prostate treatment has been used clinically since 1992. To date over 42,000 men have received HIFU treatment for localized prostate cancer: Primarily two commercial devices provide US guided HIFU treatment for prostate cancer. The Sonablate 500 (manufactured by SonaCare Medical, Indianapolis, IN) and Ablatherm (manufactured by EDAP, Lyon, France). The prostate cancer presentation will cover imaging and localization of prostate, treatment planning using 2D and 3D ultrasound images, HIFU treatment and real time treatment monitoring with Tissue Change Monitoring derived from backscattered RF pulse/echo signals. Clinical analysis will include efficacy and safety data for whole gland and focal treatment. Bone Metastasis: The current status of palliation for painful bone metastases including the role of MRgFUS in this sphere will be reviewed. Results of clinical trials including a recently completed phase III trial demonstrating high rates of pain relief and improvement in quality of life with use of MR guided Focused Ultrasound, (MRgFUS), for patients who had recurrent or persistent pain following radiation of for whom radiation therapy was otherwise contraindicated will be presented. Future directions including novel new applicators and use as primary treatment with or without radiation will be discussed. MRI guided hyperthermia and Ultrasound Triggered Local Drug Delivery: we will review the delivery, monitoring and control techniques as well as promising usages of HIFU for localized drug delivery using hyperthermia.Learning Objectives:1. Know the current status and understand the potential of HIFU in the field of oncology2. Understand in details the usage of HIFU for the treatment of prostate cancer3. Understand in details the usage of HIFU for the palliation of painful bone metastasis4. Understand the principle and potential of HIFU for MR guided localized drug delivery.My trip will be supported by my current employer, the focused ultrasound foundation. I own InSightec stocks.

The Bioactive Compounds α-Chaconine and Gallic Acid in Potato Extracts Decrease Survival and Induce Apoptosis in LNCaP and PC3 Prostate Cancer Cells

We recently reported that colored potato extracts and an anthocyanin rich fraction suppressed lymph-node carcinoma of the prostate (LNCaP) and prostate cancer-3 (PC-3) prostate cancer cell proliferation and induced apoptosis via caspase-dependent and caspase-independent pathways. Chlorogenic acid, caffeic acid, gallic acid, catechin, malvidin, and glycoalkaloids (α-chaconine and solanine) have now been identified as the major bioactive components of potato, and their effects on LNCaP and PC-3 cell proliferation and apoptosis have been investigated. α-chaconine (5 μg/ml) and gallic acid (15 μg/ml) exhibited potent antiproliferative properties and increased cyclin-dependent kinase inhibitor p27 levels in both cell lines. Both α-chaconine and gallic acid induced poly [adenosine diphosphate (ADP)] ribose polymerase cleavage and caspase-dependent apoptosis in LNCaP cells; however, caspase-independent apoptosis through nuclear translocation of endonuclease G was observed in both LNCaP and PC-3 cells. α-chaconine and gallic acid activated c-Jun N-terminal protein kinase (JNK), and this response played a major role in induction of caspase-dependent apoptosis in LNCaP cells; whereas modulation of JNK and mitogen-activated protein kinase did not affect α-chaconine- and gallic acid-induced caspase-independent apoptosis. These results suggest that apoptosis induced by whole potato extracts in prostate cancer cell lines may be in part due to α-chaconine and gallic acid.

Granulocyte Macrophage Colony-Stimulating Factor May Modulate the Post-transcription Pathway of Interleukin-6 Expression in Prostate Carcinoma Cells

Interleukin-6 (IL-6) can stimulate a variety of tumors including prostatic carcinoma. Research has recently shown that IL-6 may act to stimulate the progression of prostatic cancer. To date, little research has been performed to better understand the nature of granulocyte macrophage colony-stimulating factor (GM-CSF) and the expression of IL-6. The aim of this study was to evaluate the effects of GM-CSF on the expression of IL-6 in prostate cancer-3 (PC-3) cells. The bone-derived PC-3 cell line was used in this study. Reverse transcription polymerase chain reaction (RT-PCR) and real- time PCR were performed to detect IL-6 mRNA expression. The IL-6 protein was measured by enzyme-linked immunosorbent assay (ELISA) after treatment with hGM-CSF. Our data indicated that IL-6 mRNA expression did not increase after treatment with hGM-CSF in comparison to the control group. However, the expression of IL-6 protein was increased compared to the control group. GM-CSF may modulate the post-transcription pathway of IL-6 expression in prostate carcinoma cells. Our data suggest that GM-CSF may have a role in IL-6-mediated development of prostate cancer.

Reduced tumor growth in vivo and increased c-Abl activity in PC3 prostate cancer cells overexpressing the Shb adapter protein

BackgroundInduction of apoptosis is one strategy for treatment of prostate cancer. The Shb adapter protein has been found to regulate apoptosis in various cell types and consequently human prostate cancer 3 (PC3) cells were transfected to obtain cells overexpressing Shb in order to increase our understanding of the mechanisms regulating PC3 cell apoptosis.MethodsHuman prostate cancer cells (PC3) were transfected with control vector or a vector containing the Shb cDNA. Clones overexpressing Shb were studied with respect to apoptosis (Dapi, M30) and c-Abl activation (Western blot for pY-245-Abl). The cells were exposed to the anti-tumor agent 2-methoxyestradiol (2-ME) and the p38 MAPK and c-Abl inhibitors SB203580 and STI-571, respectively, after which cell death was determined. In vivo tumor growth and tumor cell proliferation (Ki-67 staining) or apoptosis (active caspase 3 staining) were also determined in nude mice.ResultsPC3 cells overexpressing Shb exhibited increased rates of apoptosis in the presence of the anti-tumor agent 2-ME. The Shb cells displayed increased activity of the pro-apoptotic kinase c-Abl. Pre-treatment with p38 MAPK (SB203580) or c-Abl (STI-571) inhibitors completely blocked 2-ME-induced apoptosis, implicating these two pathways in the response. The PC3-Shb cells displayed reduced tumor growth in vivo, an effect occurring as a consequence of increased apoptosis and reduced DNA synthesis.ConclusionIt is concluded that Shb promotes 2-ME-induced PC3 cell apoptosis by increased pro-apoptotic signaling via the c-Abl pathway and that this causes reduced tumor growth in vivo.

Effects of Flutamide on [Methyl- 3H]-Choline Uptake in Human Prostate Cancer-3 Cells: A Pilot Study

Background: Positron emission tomography using [methyl- 11C]-choline is effective in imaging many types of cancer, especially prostate cancer (PC). The antiandrogen flutamide is often used as part of the initial treatment of PC. Data on the effect of flutamide on and methylcholine incorporation into PC-3 cells are lacking in the experimental and literature work. Objectives: The aims of this study were to assess whether human PC-3 cells are susceptible to flutamide and whether the drug modulates the uptake of [methyl- 3H]-choline into these cells. Methods: PC-3 cells were treated for 3 days with flutamide (≤100 nmol/L), inhibiting growth by 20% to 70% with control cells included. Two viability tests (cytotoxic analyses), the thiazole blue assay and the trypan blue exclusion method, were used to determine the median inhibitory concentration for flutamide (10 nmol/L). Control and flutamide-treated cells were incubated with [methyl- 3H]-choline for 10 minutes and then in nonradioactive medium for 10 minutes to simulate the rapid blood clearance of [methyl- 11C]-choline tracer that occurs within 5 to 20 minutes, and then extracted using organic and aqueous solvents to determine the intracellular distribution of the tracer. Protein assay and flow-cytometry analysis were used to determine protein content and DNA synthesis in both control and treated cells. The uptake of [methyl- 3H]-choline was normalized to protein content and expressed as mean (SD) dpm/1Jg protein (n = 6). Results: PC-3 cell proliferation was inhibited with flutamide treatment. After treatment of PC-3 cells with flutamide 10 nmol/L for 3 days, cells accumulated DNA during the S phase. Mean (SD) [methyl- 3H]-choline uptake was found to be significantly lower with flutamide 10-nmol/L-treated cells compared with control cells (65.95 [0.72] vs 114.21 [0.57] dpm/1Jg protein; P < 0.001); the difference between the 5-nmol/L-treated cells and controls was nonsignificant. Conclusions: In this pilot study, flutamide inhibited tumor cell growth and proliferation and decreased (modulated) the uptake of [methyl- 3H]-choline into androgen receptor-negative PC-3 cells. These results suggest that flutamide might inhibit proliferation by an androgen-independent mechanism.

Emmprin is a Biomarker of Prostate Cancer Progression in TRAMP Mice

The five-year survival rate for treated patients with localized prostate carcinoma (PCa) is almost 100%; however, this rate drops to 33% with metastatic disease. Treatment is considered curative for localized PCa, but no effective treatment for patients with metastatic disease is currently available. The successful identification of PCa patients with an increased risk of metastasis during the early stages of the disease will allow clinicians to enhance the survival rate and reduce the number of unnecessary biopsies. Knock-down of extracellular matrix metalloproteinase inducer (Emmprin), a member of the Ig-like superfamily of cell adhesion molecules, in prostate cancer-3 (PC-3) cells reduces their invasive properties by reducing secretions of matrix metalloproteinases-2 and -9 in vitro (1). To further characterize the role of Emmprin as a possible in vivo biomarker of progression, we performed immunohistochemical (IHC) analysis of transgenic adenocarcinoma of mouse prostate (TRAMP) model prostate sections at different stages of cancer progression. IHC analysis revealed increased Emmprin expression in prostatic intraepithelial neoplasia (PIN) and undifferentiated PCa when compared to adjacent normal tissue. In conclusion, Emmprin may serve as a possible PCa progression biomarker in the TRAMP model but further characterization of its clinical relevance is needed. Support: NIH RO1-CA10757501 and University of Kentucky Lyman T. Johnson Minority Fellowship1

Gene expression in cancer cells is influenced by contact with bone cells in a novel coculture system that models bone metastasis.

Contact between bone cells and cancer cells (heterotypic cell contact) is thought to play a central role in the initial growth and progression of metastatic cells. Attempts at studying heterotypic contact in vitro and in vivo have been confounded by difficulty in controlling how and when heterotypic contact occurs between unlike cells. A novel model, the micropatterned coculture system, is described that quantifies and controls heterotypic contact between cancer cells and bone cells in vitro. The micropatterned coculture system is biocompatible, and is modified easily to accommodate two or more different populations of cells. Immunofluorescence of cocultures of prostate cancer-3 cells and osteoblasts show the precise control of cell interactions. Ribonucleic acid of sufficient quantity and quality is isolated readily from cells cocultured on the micropatterned coculture system. The expression of the metastasis associated genes urokinase plasminogen activator, insulinlike growth factor binding protein-1 and insulinlike growth factor binding protein-3 are regulated in response to heterotypic contact and soluble factors respectively. A model of bone metastasis based on the micropatterned coculture system technology will streamline the process for testing therapeutic agents, so that more molecules can be identified for animal and clinical testing at less cost and in less time than using conventional methods.