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Objective: Rhenium-188-HEDP is an effective radiopharmaceutical for the treatment of painful bone metastases from prostate cancer. The effectiveness of the β-radiation emitted by 188 Re might be enhanced by combination with chemotherapy, using the radiosensitization concept. Therefore, the authors investigated the combined treatment of the taxanes, docetaxel and cabazitaxel, with 188 Re in prostate carcinoma cell lines., Materials and Methods: The cytotoxic effects of single and combined treatment with taxanes and 188 Re were investigated in three human prostate carcinoma cell lines (PC-3, DU 145, and LNCaP), using the colony-forming assay. The half maximal effective concentration (EC50) of all individual agents was determined. The combined treatment was studied at 0.25, 0.5, 1, 2, and 4 times the EC50 of each agent. The interaction was investigated with a regression model., Results: The survival curves showed dose-dependent cell growth inhibition for both the taxanes and 188 Re. The regression model showed a good capability of explaining the data. It proved additivity in all combination experiments and confirmed a general trend to a slight subadditive effect., Conclusions: This proof-of-mechanism study exploring radiosensitization by combining 188 Re and taxanes showed no synergism, but significant additivity. This encourages the design of in vivo studies. Future research should explore the potential added value of concomitant treatment of bone metastases with chemotherapy and 188 Re-HEDP.

The centrosome plays a key role in cancer invasion and metastasis. However, it is unclear how abnormal centrosome numbers are regulated when prostate cancer (PCa) cells become metastatic. CP110 was previously described for its contribution of centrosome amplification (CA) and early development of aggressive cell behaviour. However its regulation in metastatic cells remains unclear. Here we identified miR-129-3p as a novel metastatic microRNA. CP110 was identified as its target protein. In PCa cells that have metastatic capacity, CP110 expression was repressed by miR-129-3p. High miR-129-3p expression levels increased cell invasion, while increasing CP110 levels decreased cell invasion. Overexpression of CP110 in metastatic PCa cells resulted in a decrease in the number of metastasis. In tissues of PCa patients, low CP110 and high miR-129-3p expression levels correlated with metastasis, but not with the expression of genes related to EMT. Furthermore, overexpression of CP110 in metastatic PCa cells resulted in excessive-CA (E-CA), and a change in F-actin distribution which is in agreement with their reduced metastatic capacity. Our data demonstrate that miR-129-3p functions as a CA gatekeeper in metastatic PCa cells by maintaining pro-metastatic centrosome amplification (CA) and preventing anti-metastatic E-CA.

Urine exosomes (extracellular vesicles; EVs) contain (micro)RNA (miRNA) and protein biomarkers that are useful for the non-invasive diagnosis of various urological diseases. However, the urinary Tamm-Horsfall protein (THP) complex, which forms at reduced temperatures, may affect EV isolation and may also lead to contamination by other molecules including microRNAs (miRNAs). Therefore, we compared the levels of three miRNAs within the purified EV fraction and THP- protein-network. Urine was collected from healthy donors and EVs were isolated by ultracentrifugation (UC), two commercial kits or sepharose size-exclusion chromatography (SEC). SEC enables the separation of EVs from protein-complexes in urine. After UC, the isolation of EV-miRNA was compared with two commercial kits. The EV isolation efficiency was evaluated by measuring the EV protein markers, Alix and TSG101, CD63 by Western blotting, or miR-375, miR-204 and miR-21 by RT-qPCR. By using commercial kits, EV isolation resulted in either low yields or dissimilar miRNA levels. Via SEC, the EVs were separated from the protein-complex fraction. Importantly, a different ratio was observed between the three miRNAs in the protein fraction compared to the EV fraction. Thus, protein-complexes within urine may influence EV-biomarker studies. Therefore, the characterization of the isolated EV fraction is important to obtain reproducible results., Competing Interests: The authors report no conflict of interest.

Background: Prognosis of patients with colorectal cancer liver metastasis (CRCLM) is estimated based on clinicopathological models. Stratifying patients based on tumor biology may have additional value., Methods: Tissue micro-arrays (TMAs), containing resected CRCLM and corresponding primary tumors from a multi-institutional cohort of 507 patients, were immunohistochemically stained for 18 candidate biomarkers. Cross-validated hazard rate ratios (HRRs) for overall survival (OS) and the proportion of HRRs with opposite effect (P(HRR < 1) or P(HRR > 1)) were calculated. A classifier was constructed by classification and regression tree (CART) analysis and its prognostic value determined by permutation analysis. Correlations between protein expression in primary tumor-CRCLM pairs were calculated., Results: Based on their putative prognostic value, EGFR (P(HRR < 1) = .02), AURKA (P(HRR < 1) = .02), VEGFA (P(HRR < 1) = .02), PTGS2 (P(HRR < 1) = .01), SLC2A1 (P(HRR > 1) < 01), HIF1α (P(HRR > 1) = .06), KCNQ1 (P(HRR > 1) = .09), CEA (P (HRR > 1) = .05) and MMP9 (P(HRR < 1) = .07) were included in the CART analysis (n = 201). The resulting classifier was based on AURKA, PTGS2 and MMP9 expression and was associated with OS (HRR 2.79, p < .001), also after multivariate analysis (HRR 3.57, p < .001). The prognostic value of the biomarker-based classifier was superior to the clinicopathological model (p = .001). Prognostic value was highest for colon cancer patients (HRR 5.71, p < .001) and patients not treated with systemic therapy (HRR 3.48, p < .01). Classification based on protein expression in primary tumors could be based on AURKA expression only (HRR 2.59, p = .04)., Conclusion: A classifier was generated for patients with CRCLM with improved prognostic value compared to the standard clinicopathological prognostic parameters, which may aid selection of patients who may benefit from adjuvant systemic therapy.

Introduction: Survival of patients after resection of colorectal cancer liver metastasis (CRCLM) is 36%-58%. Positron emission tomography (PET) tracers, imaging the expression of prognostic biomarkers, may contribute to assign appropriate management to individual patients. Aurora kinase A (AURKA) expression is associated with survival of patients after CRCLM resection., Methods: We synthesized [(3)H]alisertib and [(11)C]alisertib, starting from [(3)H]methyl nosylate and [(11)C]methyl iodide, respectively. We measured in vitro uptake of [(3)H]alisertib in cancer cells with high (Caco2), moderate (A431, HCT116, SW480) and low (MKN45) AURKA expression, before and after siRNA-mediated AURKA downmodulation, as well as after inhibition of P-glycoprotein (P-gp) activity. We measured in vivo uptake and biodistribution of [(11)C]alisertib in nude mice, xenografted with A431, HCT116 or MKN45 cells, or P-gp knockout mice., Results: [(3)H]Alisertib was synthesized with an overall yield of 42% and [(11)C]alisertib with an overall yield of 23%±9% (radiochemical purity ≥99%). Uptake of [(3)H]alisertib in Caco2 cells was higher than in A431 cells (P=.02) and higher than in SW480, HCT116 and MKN45 cells (P<.01). Uptake in A431 cells was higher than in SW480, HCT116 and MKN45 cells (P<.01). Downmodulation of AURKA expression reduced [(3)H]alisertib uptake in Caco2 cells (P<.01). P-gp inhibition increased [(3)H]alisertib uptake in Caco2 (P<.01) and MKN45 (P<.01) cells. In vivo stability of [(11)C]alisertib 90min post-injection was 94.7%±1.3% and tumor-to-background ratios were 2.3±0.8 (A431), 1.6±0.5 (HCT116) and 1.9±0.5 (MKN45). In brains of P-gp knockout mice [(11)C]alisertib uptake was increased compared to uptake in wild-type mice (P<.01) CONCLUSIONS: Radiolabeled alisertib can be synthesized and may have potential for the imaging of AURKA, particularly when AURKA expression is high. However, the exact mechanisms underlying alisertib accumulation need further investigation., Advances in Knowledge and Implications for Patient Care: Radiolabeled alisertib may be used for non-invasively measuring AURKA protein expression and to stratify patients for treatment accordingly., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: To investigate the individual and combined prognostic value of HIF1α, SLC2A1, and vascular endothelial growth factor A (VEGFA) in a multi-institutional cohort of patients with resected colorectal cancer liver metastasis (CRCLM)., Background: In the majority of patients with CRCLM, resection seems not to be curative, despite its curative intent. Overexpression of hypoxia-inducible factor 1α (HIF1α), glucose transporter 1 (SLC2A1; also known as GLUT1), and VEGFA has been associated with tumor progression and poor prognosis of patients with colorectal cancer (CRC)., Methods: Tissue microarrays were generated using CRCLM and patient-matched primary CRC from patients who underwent CRCLM resection between 1990 and 2010. Prognostic value of HIF1α, SLC2A1, and VEGFA was determined by immunohistochemistry. A 500-fold cross-validated hazard rate ratio (HRRav) for overall survival was calculated., Results: HIF1α, SLC2A1, and VEGFA expression could be evaluated in 328, 350, and 335 patients, respectively. High SLC2A1 expression was associated with good prognosis (HRRav, 0.67; P (HRR >1)  < 0.01) and high VEGFA expression to poor prognosis (HRRav, 1.84; P (HRR < 1)  = 0.02), also after multivariate analysis including established clinicopathological prognostic variables (HRRav, 0.67; P (HRR > 1)  < 0.01 and HRRav, 1.50; P (HRR < 1)  = 0.02, respectively). SLC2A1 showed prognostic value particularly in patients treated with systemic therapy (P < 0.01), whereas the prognostic value of VEGFA expression was mainly observed in patients not treated with systemic therapy (P < 0.01). Prognosis was especially poor in patients with both low SLC2A1 and high VEGFA expression (P < 0.01). HIF1α expression was not associated with survival., Conclusions: SLC2A1 and VEGFA expression are prognostic molecular biomarkers for patients with CRCLM with added value to established clinicopathological variables.

Purpose: The objective of the present study is to determine whether uptake of [(18)F]fluoromethylcholine ([(18)F]FCH) in comparison with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) accurately reflects chemotherapy efficacy at the tumor cell level in prostate cancer (PC)., Procedures: The effects of docetaxel and cabazitaxel on viable tumor cell number were explored in four PC cell lines. Cellular uptake of [(18)F]FDG and [(18)F]FCH was compared with the effects measured using sulforhodamine B (SRB) assay, cell counting and colony formation assay (CFA), as proximators of viable tumor cell number. Agreement between uptake and cell numbers was assessed by Bland-Altman plots., Results: [(18)F]FCH uptake in all PC cell lines significantly correlated to the cell numbers surviving the respective drug concentrations. Bland-Altman analysis showed that [(18)F]FDG uptake resulted in signal overestimation and higher variability after chemotherapy., Conclusions: [(18)F]FCH uptake correlates well with viable tumor cell numbers remaining after docetaxel and cabazitaxel exposure. Radiolabeled choline is a potential response monitoring biomarker after chemotherapy for PC.

Background: Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening., Methods: We developed a new high-throughput screening method for determining radiation susceptibility. Fast and uniform irradiation of batches up to 30 microplates was achieved using a Perspex container and a clinically employed linear accelerator. The readout was done by automated counting of fluorescently stained nuclei using the Acumen eX3 laser scanning cytometer. Assay performance was compared to that of the CFA and the CellTiter-Blue homogeneous uniform-well cell viability assay. The assay was validated in a whole-genome siRNA library screening setting using PC-3 prostate cancer cells., Results: On 4 different cancer cell lines, the automated cell counting assay produced radiation dose response curves that followed a linear-quadratic equation and that exhibited a better correlation to the results of the CFA than did the cell viability assay. Moreover, the cell counting assay could be used to detect radiosensitization by silencing DNA-PKcs or by adding caffeine. In a high-throughput screening setting, using 4 Gy irradiated and control PC-3 cells, the effects of DNA-PKcs siRNA and non-targeting control siRNA could be clearly discriminated., Conclusions: We developed a simple assay for radiation susceptibility that can be used for high-throughput screening. This will aid the identification of molecular targets for radiosensitization, thereby contributing to improving the efficacy of radiotherapy.

Background: Botulinum neurotoxin A (BoNT-A) is a highly neurotoxic drug and frequently used in patients. Knowledge on the optimal way of administration of BoNT-A and its subsequent distribution is still rather limited. An accurate method for monitoring these processes might be the use of radiolabelled BoNT-A. In this paper, we report our feasibility study on labelling BoNT-A with high-dose iodine-125 ((125)I) via IODOGEN-coated BoNT-A method., Methods: Using cetuximab as model substrate for BoNT-A, a miniaturization of the IODOGEN-coated mAb method was developed with special attention to the minimum required amount of the oxidant IODOGEN, while the amount of substrate, reaction volume and reaction time were downsized. Labelling efficiency and radiochemical purity were determined by TLC, integrity by SDS-PAGE and HPLC and immunoreactivity by cell-binding assay. BoNT-A (50 μg) was labelled with (125)I by coating with 2.5 μg IODOGEN, in a total reaction volume of 250 μL and a reaction time of 90 s. (125)I-BoNT-A was purified by size exclusion chromatography (PD10 column) using ascorbic acid solution (5 mg/ml, pH = 5) as eluent. Quality analysis of (125)I-BoNT-A was performed by an in vitro bladder strip model, an electrochemiluminescence assay and an Endopep assay., Results: Cetuximab (50 μg) labelling with (125)I (15 to 150 MBq) resulted in a labelling efficiency of 70% to 80%, a radiochemical purity of >99%, an immunoreactivity of >95% and a retained integrity on SDS; HPLC analysis revealed partly affected integrity when 110 to 150 MBq (125)I was used, i.e. when the averaged I/mAb molar ratio exceeded 3. Addition of HEPES (20 mM) and lactose (1.25%) (lyophilized BoNT-A contains HEPES and lactose) decreased the labelling efficiency to 44% to 54%. BoNT-A (50 μg) labelling with (125)I (97.2 to 98.3 MBq) resulted in labelling efficiency of 51% to 52% with a radiochemical purity >98.5%, a specific activity of 150.5 to 152.9 MBq/nmol and an I/BoNT-A molar ratio of 1.86 to 1.90. The in vitro bladder strip model showed no bioactivity of (125)I-BoNT-A when compared to unlabelled BoNT-A. The electrochemiluminescence and Endopep assay demonstrated around 10% and 15% bioactivity of (125)I-BoNT-A compared to unlabelled BoNT-A, respectively. The remaining bioactivity correlates within the Poisson distribution with the amount of BoNT-A molecules that does not bear an iodine atom., Conclusions: BoNT-A was successfully radio-iodinated with an activity high enough to enable in vivo measurement of nanograms of BoNT-A, which could be used in studying optimization of administration techniques of BoNT-A. The bioactivity of a BoNT-A molecule is, however, lost upon the introduction of an iodine atom into the tyrosine moiety of this sensitive molecule.

Background: Resection of colorectal cancer liver metastasis (CRCLM) with curative intent has long-term benefit in ~40% of cases. Prognostic biomarkers are needed to improve clinical management and reduce futile surgeries. Expression of epidermal growth factor receptor (EGFR) and prostaglandin-endoperoxide synthase 2 (PTGS2; also known as cyclooxygenase-2) has been associated with carcinogenesis and survival. We investigated the prognostic value of EGFR and PTGS2 expression in patients with resected CRCLM., Methods: Formalin-fixed paraffin-embedded CRCLM tissue and corresponding primary tumour specimens from a multi-institutional cohort of patients who underwent liver resection between 1990 and 2010 were incorporated into tissue microarrays (TMAs). TMAs were stained for EGFR and PTGS2 by immunohistochemistry. The hazard rate ratio (HRR) for the association between expression in CRCLM and overall survival was calculated using a 500-fold cross-validation procedure., Results: EGFR and PTGS2 expression could be evaluated in 323 and 351 patients, respectively. EGFR expression in CRCLM was associated with poor prognosis (HRR 1.54; P<0.01) with a cross-validated HRR of 1.47 (P=0.03). PTGS2 expression was also associated with poor prognosis (HRR 1.60; P<0.01) with a cross-validated HRR of 1.63 (P<0.01). Expression of EGFR and PTGS2 remained prognostic after multivariate analysis with standard clinicopathological variables (cross-validated HRR 1.51; P=0.02 and cross-validated HRR 1.59; P=0.01, respectively). Stratification for the commonly applied systemic therapy regimens demonstrated prognostic value for EGFR and PTGS2 only in the subgroup of patients who were not treated with systemic therapy (HRR 1.78; P<0.01 and HRR 1.64; P=0.04, respectively), with worst prognosis when both EGFR and PTGS2 were highly expressed (HRR 3.08; P<0.01). Expression of PTGS2 in CRCLM was correlated to expression in patient-matched primary tumours (P=0.02, 69.2% concordance)., Conclusions: EGFR and PTGS2 expressions are prognostic molecular biomarkers with added value to standard clinicopathological variables for patients with resectable CRCLM.

Prophylactic vaccination is arguably the most effective medical preventative method. After local inoculation, vaccines induce antigen-specific systemic immunity, protecting the whole body. Systemic antitumour immunity can cure advanced cancer, but will therapeutic vaccination suffice? A vaccine for castration-refractory prostate cancer (CRPC) was approved by regulatory authority, but its evidence is disputed. We critically reviewed the clinical efficacy of therapeutic cancer vaccines for prostate cancer, including the results of 31 clinical studies employing vaccines-only, and another 10 studies combining vaccines with immune co-stimulation. Vaccinations yielded immunological responses, but no study showed evidence for clinically relevant therapeutic improvement. Clinical failure of therapeutic vaccination is discussed in the light of immunological dogmas and mechanisms of antitumour therapies. We propose that cancer immunotherapy might be improved by immunological danger, i.e. disturbing tumour homeostasis by destroying the tumour tissue or inducing local inflammation. Such danger might override immunological tolerance, and thereby allow clinically relevant anticancer results., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Botulinum toxin A (BoNT-A) is a new treatment modality in various causes of bladder dysfunction; like neurogenic detrusor overactivity and overactive bladder. The best technique of administrating BoNT-A in patients is unknown. A validated in vitro model could be used to investigate newer intravesical administration techniques of BoNT-A. In this study, we describe the development and validation of in vitro model to measure inhibitory effects of BoNT-A on bladder strip contractions., Methods: Rat bladder strips were mounted in organ baths filled with Krebs' solution. The strips were stimulated chemically (80 mM potassium chloride, 1 μM carbachol) and electrically (Electrical Field Stimulation (EFS) 100 shocks, 50 V, 20 Hz, every 3 minutes). The viability of the strips was measured by carbachol stimulation at the beginning and at the end of the experiments. The strips were incubated in various concentrations of BoNT-A (0.03, 0.2, 0.3 nM). Controls were incubated in Krebs' solution only. The inhibition of strip contraction induced by EFS was measured. These measurements were statistically analyzed with a log-logistic model representing diffusion., Results: All strips remained viable during the experiments. Inhibition of strip contraction was observed after incubation with 0.3 nM BoNT-A. The measurements fitted to a log-logistic model describing diffusion of BoNT-A in the bladder strip. The parameters of the log-logistic model representing diffusion were significant for 0.3 nM BoNT-A. Incubation with 0.2 nM BoNT-A showed insignificant results for 2 out of 3 runs. Incubation with 0.03 nM BoNT-A did not result in significant inhibition of strip contractions., Conclusions: An in vitro model was developed and validated in which the inhibitory effect of low concentrations of BoNT-A on bladder strip contractions can be measured.

Aim: To characterize neurotoxicity induced by oxaliplatin, bortezomib, and epothilone-B as well as protection against their neurotoxicity using an in vitro model., Materials and Methods: Neurotoxicity was evaluated using the neurite outgrowth method in PC12 rat pheochromo-cytoma cells differentiated towards a mature neuronal phenotype, while neuroprotection was explored by simultaneous exposure to 0.5 mM amifostine. The potential markers of neuronal differentiation, cyclin-B2 (Ccnb2) and baculoviral inhibitor of apoptosis repeat-containing 5 (Birc5), were evaluated by quantitative reverse transcription polymerase chain reaction (RT-PCR)., Results: Bortezomib, epothilone-B, and oxaliplatin reduced neurite length to 68%, 78% and 66%, respectively (p<0.05). The percentage of neurite-forming-cells (discriminating neurotoxicity from general cytotoxicity) decreased from 70% (control) to 55% (bortezomib), 46% (epothilone-B), and 51% (oxaliplatin). Amifostine was neuroprotective against oxaliplatin-induced neurotoxicity, increasing both neurite length and neurite-forming-cells. Quantitative-RT-PCR showed a 2.7-fold decrease in Ccnb2 expression in differentiated PC12 vs. undifferentiated cells., Conclusion: Oxaliplatin, bortezomib, and epothilone-B are neurotoxic in the PC12 model. Amifostine has a neuroprotective effect only against oxaliplatin-induced neurotoxicity, suggesting that these compounds have different mechanisms of neurotoxicity.

Background: Cancer cells are able to change the protein expression and behavior of non-cancerous surrounding cells. Exosomes, secreted by prostate cancer (PCa) cells, may have a functional role in cancer metastasis and present a promising source for protein biomarkers. The aim of the present study was to identify which proteins in exosomes can influence non-cancerous cells, and to determine whether we can use urine exosomal proteins to identify high-risk PCa patients., Method: Exosomes were isolated by ultracentrifugation. Migration and invasion were studied by the transwell (invasion) assay. Proteomics was performed by LC-MS/MS and identified proteins were validated by Western blotting. Cellular uptake of fluorescent labeled PKH67-exosomes was measured by FACS., Results: Based on comparative protein profiling by mass spectrometry-based proteomics of LNCaP- and PC3-exosomes, we selected ITGA3 and ITGB1, involved in migration/invasion, for further analyses. Inhibition of exosomal ITGA3 reduced the migration and invasion of non-cancerous prostate epithelial cells (prEC) almost completely. Cellular uptake of exosomes by prEC was higher with PC3-exosomes compared to LNCaP exosomes. Finally, ITGA3 and ITGB1 were more abundant in urine exosomes of metastatic patients (p<0.05), compared to benign prostate hyperplasia or PCa., Conclusion: These data indicate exosomal ITGA3 and ITGB1 may play a role in manipulating non-cancerous surrounding cells and that measurement of ITGA3 and ITGB1 in urine exosomes has the potential to identify patients with metastatic PCa in a non-invasive manner.

Background: Five-year survival after resection of colorectal cancer liver metastasis (CRLCM) is <30%. We recently found that aurora kinase A (AURKA) drives 20q gain-associated tumour progression and is associated with disease recurrence. This study evaluates the prognostic value of AURKA expression in CRCLM of patients who underwent liver resection., Methods: Tissue microarrays (TMAs) were generated using formalin-fixed paraffin-embedded CRCLM and matched primary tumour from a multi-institutional cohort of patients with CRCLM who underwent liver resection between 1990 and 2010. Tissue microarrays were stained for AURKA using immunohistochemistry, and a hazard rate ratio (HRR) for the association between overall survival (OS) and nuclear AURKA expression in CRCLM was calculated. Results were validated by 500-fold cross-validation., Results: The expression of AURKA was evaluated in CRCLM of 343 patients. High AURKA expression was associated with poor OS (HRR 1.55, P<0.01), with a cross-validated average HRR of 1.57 (P=0.02). Average HRR was adjusted for the established prognostic clinicopathological variables in a multivariate analysis (average HRR 1.66; P=0.02). The expression of AURKA in CRCLM was correlated to its expression in corresponding primary tumour (P<0.01)., Conclusion: The expression of AURKA protein is a molecular biomarker with prognostic value for patients with CRCLM, independent of established clinicopathological variables.

Expression of the neuroendocrine peptide calcitonin (CT) and its receptor (CTR) is frequently elevated in prostate cancers (PCs), and activation of the CT-CTR axis in non-invasive PC cells induces an invasive phenotype. We aimed to link CT/CTR expression in prostate specimens to clinicopathological parameters of PC. We analyzed CT and CTR expression in cohorts of benign prostates and primary PCs with/without metastatic disease by immunohistochemistry. Furthermore, we correlated CT/CTR expression with several clinicopathological parameters. CT/CTR immunostaining in benign prostate acini was predominantly localized to basal epithelium. However, this spatial specificity was lost in malignant prostates. PC sections displayed a remarkable increase in cell populations expressing CT/CTR and their staining intensity. Tumors with higher CT/CTR expression consistently displayed metastatic disease and poor clinical outcome. High CT/CTR expression in primary prostate tumors may serve as a prognostic indicator of disease aggressiveness and poor clinical outcome.

To determine the relationship between ionizing radiation-induced levels of γ-H2AX foci and cell survival in cultured prostate cancer cell lines, three prostate cancer cell lines: LNCaP (wt TP53), DU145 (mut TP53) and PC3 (TP53 null), were studied. For γ-H2AX foci induction, cells were irradiated with a single dose of 2 Gy and foci levels were studied at 30 min and 24 h after irradiation. Cell survival was determined by clonogenic assay, directly and 24 h after irradiation with doses ranging from 0 to 8 Gy. Irradiation was performed with a Siemens Stabilipan 250 KeV X-ray machine at a dose rate of approximately 3 Gy/min. Survival curves were analyzed using the linear-quadratic model S(D)/S(0)=exp-(αD+βD2). LNCaP cells clearly demonstrated potentially lethal damage repair (PLDR) which was assessed as increased survival levels after delayed plating as compared to cells plated immediately after irradiation. DU145 cells demonstrated only a slight PLDR and PC3 cells did not show PLDR at all. Levels of γ-H2AX foci were significantly decreased in all cell lines at 24 h after irradiation, compared to levels after 30 min. The LNCaP cells which demonstrated a clear PLDR also showed the largest decay in the number of γ-H2AX foci. In addition, the PC cells which did not show PLDR had the lowest decay of γ-H2AX foci. A clear correlation was demonstrated between the degree of decay of γ-H2AX foci and PLDR.

Purpose: Prostate cells are dependent on androgens for growth and proliferation. Androgen deprivation therapy is the recommended treatment for advanced/metastatic prostate cancer. Under this therapy, prostate cancer will inevitably progress to castration resistant prostate cancer (CRPC). Despite putative castration resistance, testosterone might still play a crucial role in the progression of CRPC. The goal of this study was to determine the role of testosterone in the formation of metastases of CRPC in both in vitro and in vivo settings., Methods: In vitro, the effect of testosterone and the non-aromatizable androgen methyltrienolone on migration, invasion and proliferation of a castration-resistant prostate cancer rat cell line (Dunning R3327-MATLyLu) was assessed using a transwell assay and a sulforhodamine B assay and immunohistochemical detection of ki67. Androgen receptor status was determined using Western blot. In vivo, Copenhagen rats were divided in four groups (males, females, castrated males and females with testosterone suppletion) and inoculated with MATLyLu cells. Tumor size was assessed daily., Results: Testosterone increased cell migration and invasion in a concentration-dependent manner in vitro. Testosterone did not affect in vitro cell proliferation. No difference was shown between the effect of testosterone and methyltrienolone. In vivo, in groups with higher levels of circulating testosterone, more rats had (micro)metastases compared with groups with low levels of testosterone. No effect was observed on primary tumor size/growth., Conclusions: Despite assumed castration resistance, progression of prostate cancer is still influenced by androgens. Therefore, continuous suppression of serum testosterone in patients who show disease progression during castration therapy is still warranted.

Background: This study aimed to investigate cabazitaxel efficacy in a model for docetaxel-resistant prostate cancer cells and to evaluate the involvement of ATP-cassette binding protein 4 (ABCC4) with regard to multidrug resistance., Materials and Methods: Docetaxel and cabazitaxel sensitivity was measured in PC3 and R3327-MATLyLu (MLL) cell lines, using the sulforhodamine B (SRB) assay. ABCC4 expression was examined by western blotting and its functional involvement in drug sensitivity by blocking with MK571 inhibitor., Results: The docetaxel-resistant MLL cells (4.5-fold compared to cabazitaxel; p<0.001) were shown to express high levels of ABCC4, while non-resistant PC3 cells had no detectable ABCC4 expression. Functional inhibition of ABCC4 in MLL cells resulted in a two-fold decrease in effective concentration of docetaxel and had no effect on toxicity of cabazitaxel., Conclusion: Cabazitaxel showed an improved therapeutic efficacy over docetaxel in ABCC4-expressing prostate cancer cells. ABCC4 appears to be an important determinant of docetaxel resistance, since its inhibition almost completely reversed resistance.

Radiotherapy is one of the treatment options for locally or regionally advanced prostate cancer, but radioresistance of prostate cancer cells is a practical limitation of radiotherapy. The identification of molecular targets of radioresistance in prostate cancer is important to improve therapeutic intervention. The aim of this review is to give more biological insight into some well known processes involved in radioresistance of prostate cancer especially Apoptotic pathway; DNA damage response; and NF- κB(nuclear factor kappalight- chain-enhancer of activated B cells) signaling pathway. This review integrates salient, published, research findings with underlying molecular mechanisms, preclinical efficacy, and potential clinical applications of combining radiotherapy with these molecular targeted agents for the treatment of prostate cancer.

Background: It is important to identify markers that predict whether prostate cancer will metastasise. The adjacent noncancerous cells (influenced by the tumour cells) may also express potential markers. The objective of this study was to determine the influence of cancer cells on noncancerous cells and to assess the value of the cell-communication protein connexin-26 (Cx26) as a marker to predict the development of metastasis., Methods: The effect of conditioned medium (CM) from PrCa cells on in vitro noncancerous cell proliferation, migration and invasion and Cx26 expression was determined. Connexin-26 expression was investigated in prostatectomy tissues from 51 PrCa patients by immunohistochemistry and compared with various clinicopathological parameters., Results: Proliferation, migration and invasion of noncancerous cells were influenced by CM from the PrCa cell lines. Importantly, a clear relation was found between low Cx26 expression in the noncancerous tissue in prostatectomy sections and the risk of development of metastasis (P<0.0002). Kaplan-Meier analysis showed a relation between low Cx26 expression in noncancerous tissues and time to biochemical recurrence (P=0.0002)., Conclusion: Measuring Cx26 expression in the adjacent noncancerous tissues (rather than cancer tissues) of prostatectomy sections could help to identify high-risk patients who may benefit from adjuvant therapy to decrease the risk of metastasis.

Purpose: To perform a longitudinal analysis of changes in lymph node volume and apparent diffusion coefficient (ADC) in healthy, metastatic, and hyperplastic lymph nodes., Materials and Methods: Three groups of four female Copenhagen rats were studied. Metastasis was induced by injecting cells with a high metastatic potential in their left hind footpad. Reactive nodes were induced by injecting Complete Freund Adjuvant (CFA). Imaging was performed at baseline and at 2, 5, 8, 11, and 14 days after tumor cell injection. Finally, lymph nodes were examined histopathologically., Results: The model was highly efficient in inducing lymphadenopathy: subcutaneous cell or CFA inoculation resulted in ipsilateral metastatic or reactive popliteal lymph nodes in all rats. Metastatic nodal volumes increased exponentially from 5-7 mm(3) at baseline to 25 mm(3) at day 14, while the control node remained 5 mm(3). The hyperplastic nodes showed a rapid volume increase reaching a plateau at day 6. The ADC of metastatic nodes significantly decreased (range 13%-32%), but this decrease was also seen in reactive nodes., Conclusion: Metastatic and hyperplastic lymph nodes differed in terms of enlargement patterns and ADC changes. Enlarged reactive or malignant nodes could not be differentiated based on their ADC values., (Copyright © 2011 Wiley-Liss, Inc.)

Today's treatment and diagnosis of prostate cancer still exhibit major limitations. The search for new and additional prognostic markers is therefore still an actual field of interest. Potential markers involved in numerous biological processes in the tumor cell have been investigated intensively. For therapeutic interventions it is important to distinguish between harmless and aggressive disease in an early stage. Therefore the subject of this review is limited to markers associated with those functional processes, which discriminate early stage aggressive, metastatic cancer from harmless disease. Important processes in this respect are: altered cell adhesion and cellular migration. E-cadherin, N-cadherin, beta-catenin, integrins, focal adhesion kinase, connexins and matrix metalloproteinases all appear promising biological markers associated with the early stage metastatic process in prostate cancer. Here we discuss their potential to become valid biological markers based on literature data. Thus far, none of these markers proved to be a valid individual marker by itself due to prostate cancer heterogeneity and transient expression. Analyzing a combination of the potential markers discussed in this review is expected to be a better approach toward discriminating high- from low-risk tumors in an early stage of prostate cancer.

Multicellular tumor spheroids are used as a model to assess the efficacy of replicating oncolytic adenoviruses. As most assays used to assess cellular viability are unsuitable for oncolytic viruses because of ongoing viral replication, we have used positron emission tomography (PET) to sequentially determine the incorporation of 18F-labeled deoxyglucose (18F-DG) as a measure of viability and compared the results to more commonly used assays for measuring the effect of oncolytic therapy. Glioma monolayer cultures and spheroids were infected with wild-type replicating adenovirus and viability was measured by 18F-DG incorporation, WST-1 assay, crystal violet assay, and spheroid volume 2 to 10 days following infection. Results show that volume measurements in adenovirus-infected spheroids are confounded by the cytopathic effect occurring in infected cells. 18F-DG PET provides a useful method to assess small differences in cell number and viability following oncolytic viral therapy in glioma monolayer cultures and spheroids without the need for disintegration of these cultures. Moreover, using 18F-DG PET, repeated sequential measurements of spheroid viability can be made, decreasing the required number of spheroids per experiment. This is a valuable feature when using spheroids derived from limited amounts of patient material.

Introduction: P-glycoprotein (P-gp) is an efflux transporter responsible for the transport of various drugs across the blood-brain barrier (BBB). Loss of P-gp function with age may be one factor in the development and progression of neurodegenerative diseases. The aim of this study was to assess the effect of aging on BBB P-gp function. Furthermore, the relationship between BBB P-gp activity and peripheral P-gp activity in CD3-positive leukocytes was investigated. Finally, plasma pharmacokinetics of carbon 11-labeled (R)-verapamil was evaluated., Methods: (R)-[(11)C]verapamil and positron emission tomography were used to assess gray matter P-gp function. Because (R)-[(11)C]verapamil is a substrate for P-gp, the volume of distribution of (R)-[(11)C]verapamil in the brain inversely reflects P-gp function in the BBB., Results: Mean volume of distribution values for 5 young healthy volunteers (age range, 21-27 years) and 5 elderly healthy volunteers (age range, 59-68 years) were 0.62+/-0.10 and 0.73+/-0.07, respectively (P=.03). The activity index of P-gp activity in CD3-positive leukocytes was 2.88+/-0.77 in young volunteers and 1.76+/-0.58 in elderly volunteers (P=.02)., Conclusion: This study showed decreased P-gp activity during aging. Consequently, the brain may be exposed to higher drug and toxin levels in elderly subjects.

We completed a multicenter study of the effects of pomegranate cold-pressed (Oil) or supercritical CO(2)-extracted (S) seed oil, fermented juice polyphenols (W), and pericarp polyphenols (P) on human prostate cancer cell xenograft growth in vivo, and/or proliferation, cell cycle distribution, apoptosis, gene expression, and invasion across Matrigel, in vitro. Oil, W, and P each acutely inhibited in vitro proliferation of LNCaP, PC-3, and DU 145 human cancer cell lines. The dose of P required to inhibit cell proliferation of the prostate cancer cell line LNCaP by 50% (ED(50)) was 70 microg/mL, whereas normal prostate epithelial cells (hPrEC) were significantly less affected (ED(50) = 250 g/mL). These effects were mediated by changes in both cell cycle distribution and induction of apoptosis. For example, the androgen-independent cell line DU 145 showed a significant increase from 11% to 22% in G(2)/M cells (P <.05) by treatment with Oil (35 microg/mL) with a modest induction of apoptosis. In other cell lines/treatments, the apoptotic response predominated, for example, in PC-3 cells treated with P, at least partially through a caspase 3-mediated pathway. These cellular effects coincided with rapid changes in mRNA levels of gene targets. Thus, 4-hour treatment of DU 145 cells with Oil (35 microg/mL) resulted in significant 2.3 +/- 0.001-fold (mean +/- SEM) up-regulation of the cyclin-dependent kinase inhibitor p21((waf1/cip1)) (P <.01) and 0.6 +/- 0.14-fold down-regulation of c-myc (P <.05). In parallel, all agents potently suppressed PC-3 invasion through Matrigel, and furthermore P and S demonstrated potent inhibition of PC-3 xenograft growth in athymic mice. Overall, this study demonstrates significant antitumor activity of pomegranate-derived materials against human prostate cancer.

Background: Peripheral neurotoxicity is a dose-limiting side-effect of a number of effective chemotherapeutic agents. Neuroprotective agents may help to reduce neurotoxicity, thus allowing the intensification of cytostatic therapy in patients., Materials and Methods: In this in vitro study, using the rat pheochromocytoma cell line PC-12 neurite-outgrowth assay, the potential of amifostine to protect against cisplatin-, paclitaxel- and vincristine-induced neurotoxicity was investigated Amifostine is described as selectively protecting normal tissue and not tumour tissue. The effect of amifostine on tumour cell kill was investigated using the XTT and colony forming assay., Results: Paclitaxel and vincristine both caused a significant reduction in the percentage of cells expressing neurites. Co-incubation with amifostine significantly increased this percentage of neurites in paclitaxel-induced neurotoxicity, but not in vincristine-induced neurotoxicity. Post-incubation of amifostine also proved to partly reverse already existing cisplatin-induced neurotoxicity, but not paclitaxel-, or vincristine-induced neurotoxicity. Amifostine did not protect tumour cells against cisplatin- and paclitaxel-induced tumour cytotoxicity, using the XTT assay. However, a stimulation of clonogenic capacity was observed when amifostine was coincubated with cisplatin., Conclusion: Amifostine protects against paclitaxel-induced neurotoxicity, but not against vincristine-induced neurotoxicity in this in vitro model. Furthermore, amifostine has potential to reverse already existing cisplatin-induced neurotoxicity. The role of amifostine in the proliferative potential of tumour cells in vitro needs further investigation.

Purpose: To test the hypothesis that radiation-induced, transient G2/M arrest could potentially sensitize tumor cells to a subsequent, well-timed radiation dose., Methods: PC-3 human prostate cancer cells were treated using either radiotherapy or (186)Re-labeled hydroxyethylidene diphosphonate ((186)Re-HEDP) treatment in different combinations. The resulting cell cycle shift and clonogenic cell death were analyzed by DNA flow cytometry and colony forming cell assay, respectively., Results: Radiation doses of 4 Gy and 8 Gy induced a transient G2/M arrest, with a maximum after approximately 16 h. The presence of 2 mM pentoxifylline effectively abrogated this radiation-induced G2 M arrest, confirming a cell-cycle checkpoint-mediated effect. A second dose of 4 Gy, timed at the height of the G2/M arrest, significantly increased clonogenic cell-kill compared to delivery after a suboptimal interval (10 h, 20 h or 25 h after the first radiation fraction). Moreover, timed second doses of 2 Gy, 3 Gy or 4 Gy yielded improved normalized treatment effects compared to non-pretreated control. Radionuclide treatment of PC-3 cells, using (186)Re-HEDP (0.74 MBq/ml and 1.48 MBq/ml; total dose: 4.1 and 8.2 Gy, respectively) also induced a dose-dependent G2/M accumulation, which sensitized the cells to a subsequent external radiation dose of 2 Gy or 4 Gy. The observed pattern of cell-cycle shift towards a predominance of the G2/M phase is in line with the lack of functional p53 in this cell line., Conclusions: Radiation-induced cell-cycle shift was shown to effectively confer increased radiosensitivity to prostate tumor cells. Optimally timed combination of radiotherapy and radionuclide therapy could thus significantly increase treatment efficacy.

Starting from different regional samples taken from a heterogeneous follicular thyroid cancer recurrence in a male patient, a series of cell cultures was initiated. Three stable cancer cell lines were successfully established (TT2609-A02, TT2609-B02, and TT2609-C02) and kept in continuous culture for more than 3 years. The lines are each characterized by a unique set of biological parameters such as morphology, ploidy state, cell proliferation rate, ultrastructure, thyroid marker expression, p53 expression, karyogram, agar clonogenic capacity and tumorigenicity as xenografts in nude mice. These characterization studies point to a marked heterogeneity at the level of the clinical tumor recurrence. Karyotype analysis of the cell lines showed a pattern of aberrations indicating that the lines are clonally related and that the A02 and C02 lines are subsequently derived from the more "original" tumor cell type B02 after a tetraploidization event. It is concluded that the obtained cell lines represent an in vitro/in vivo model for human follicular thyroid cancer. The availability of a series of cell lines for human follicular thyroid cancer, mimicking the biological heterogeneity observed in patient tumors, enables both detailed fundamental investigation of thyroid cancer cell biology and the experimental exploration of new treatment approaches.

Background: Regulators of cell cycle phase transitions could be important targets for cancer treatment using cytostatic chemotherapy. Therefore, the extent of cell cycle arrest induced by different cytostatic agents has to be correlated with ultimate clonogenic tumor cell death. Especially the value of early cell cycle perturbations as indicators for the clinical efficacy of drugs should be a matter of investigation., Methods: In vitro PC-3 human prostate carcinoma cells were incubated for 24 hours with a panel of six different chemotherapeutic drugs in various concentrations (Aplidine, Cisplatin, Isohomohalichondrin B (IHB), Taxol, Vincristine and Vinorelbine). The short term effects on the cell cycle distribution were determined by DNA flowcytometry while the clonogenic capacity of these cells was quantitated to measure the cytotoxic treatment efficacy., Results: Significant decreases of clonogenic survival proved to be strongly correlated with cell cycle perturbations. IHB, Taxol, Vincristine and Vinorelbine resulted in accumulation (up to 87-92%) in the G2M phase, while Cisplatin and Aplidine led to increases in the S-phase fraction and in both G2M- as well as S-phase fractions, respectively., Conclusion: Cell cycle phase perturbations appear to be suitable, early markers for cytotoxic drug efficacy.

Cisplatin-induced neuropathy is a major dose-limiting toxicity. Counteraction by amifostine and its metabolite WR1065 may reduce peripheral neurotoxicity in a number of patients. Using the nerve growth factor (NGF)-dependent neurite outgrowth from the PC12 pheochromocytoma cell line as an in vitro assay for neurotoxicity, the protective effects of amifostine and WR1065 against cisplatin action on neurite formation by PC12 cells were studied. Cisplatin in a concentration of 10 microg/ml significantly decreased the percentage of neurite forming cells from 84% to 40%. Amifostine in doses of 0.4 and 0.8 mM proved to protect significantly against the cisplatin-induced decrease in neurite formation, when co-incubated with cisplatin. Also the metabolite WR1065 protected significantly against cisplatin neurotoxicity in a dose of 0.12 mM. Our results show a significant protection by amifostine and its main metabolite WR1065 against cisplatin-induced neurotoxicity using an in vitro model.

Unlabelled: Radionuclide therapy has proven to be an efficacious palliative treatment for metastatic prostate cancer. Its potential therapeutic possibilities may be substantially increased by combining it with effective radiosensitizing drugs., Methods: This study explores the radiosensitizing properties of cisplatin when combined with 186Re-labeled hydroxyethylidene diphosphonate (HEDP) in the treatment of R3327-MATLyLu prostate cancer cells in vitro. A concomitant incubation during 4 d, combining various concentrations of cisplatin (0, 0.42, 0.83 and 1.67 micromol/L) and 186Re-HEDP (0, 1.84 and 3.69 MBq/mL [0, 50 and 100 microCi/mL, respectively]) was followed by the determination of the cell numbers surviving and the replating of these cells in semisolid agar., Results: The surviving fraction of clonogenic tumor cells after combination treatment clearly showed synergism when analyzed by a panel of three different published analytical methods. In addition, analysis of variance demonstrated a significant interaction between radionuclide therapy and cisplatin-based chemotherapy (P < 0.001). Treatment with 186Re-HEDP and cisplatin by sequential incubation yielded similar, but never superior results., Conclusion: It is concluded that radionuclide therapy in combination with cisplatin is able, in principle, to improve therapeutic success rate in metastatic prostate cancer in a more than additive way.

Purpose: New classes of anticancer drugs, isolated from marine organisms, have been shown to possess cytotoxic activity against multiple tumor types. Aplidine, didemnin B, and isohomohalichondrin B (IHB), among the more promising antitumor candidates, have been evaluated in the present study on a comparative basis in terms of their antiproliferative activity and neurotoxic effects in vitro., Methods: Using a panel of different human, prostatic cancer cell lines (DU 145, PC-3 and LNCaP-FGC) the effects of Aplidine, didemnin B, and IHB on tumor cell proliferation were tested in a colorimetric (XTT) assay and compared with the effects of vincristine, vinorelbine, and Taxol. Under analogous in vitro conditions these drugs were also monitored for neurocytotoxic effects using a PC 12 cell line based model., Results: Didemnin B and - especially - Aplidine were more effective in the inhibition of prostate cancer cell proliferation than vincristine, vinorelbine or Taxol at concentration levels between 5 and 50 pmol/ml. At these same concentrations, however, Didemnin B and Aplidine were also most potent in the in vitro neurotoxicity assays. IHB was found to exert even more potent antiproliferative activity (at concentration levels between 0.05 and 0.1 pmol/ml). However, neurotoxic effects were also found to be present at these levels. After drug withdrawal, the neurotoxic damage, inflicted by aplidine or IHB appeared to be more long lasting than after vincristine or vinorelbine exposure., Conclusions: These results point to high antiproliferative activity of aplidine and IHB in prostate cancer. At the same time, the data urge some caution in the clinical use of these agents because of potential neurotoxic side-effects. The use of a newly formulated Aplidine may involve a more favorable therapeutic profile.

Background: A combination of local irradiation and systemic cytotoxic treatment could improve therapeutic efficacy in metastatic prostate cancer. Radiosensitization can augment the treatment response to standard doses of radiation or enable lower treatment doses to be given; thus decreasing possible side effects. Intracellular glutathione has been implicated in the mechanism of such radio- sensitizing effects., Materials and Methods: In the present study, R3327-MATLyLu prostate tumor cells were treated with cisplatin (0.0325 microM, 0.1625 microM, 0.325 microM and control "0 microM") in combination with irradiation (2, 4, 6, 8 Gy and control "0 Gy"). The survival of clonogenic tumor cells in agar was determined. In another experiment the irradiation was carried out after a 3 hours pretreatment with cisplatin concentrations (1.63 microM, 3.25 microM, 6.5 microM and control "0 microM") both in the presence and absence of Glutathione., Results: In both experimental conditions the combination of cisplatin with irradiation yielded significant supra-additive treatment effects., Conclusions: The analysis of combination treatment effects, using two different methods confirmed the existence of synergism. The presence of a high level of extracellular glutathione did not alter the radiosensitization effects observed without glutathione, suggesting that the presence of glutathione may not be a major limiting factor in the radiosensitization effects observed in these investigations.

Neurotoxicity forms a major limitation in many clinical applications of vincristine and other powerful vinca alkaloid anticancer drugs. Using the nerve growth factor (NGF) dependent neurite outgrowth from the PC12 pheochromocytoma cell line as an in vitro assay for neurotoxicity, the effect of different concentrations of vincristine (0.55; 1.1 and 11 nM) was compared with that of vindesine and vinblastine. Vincristine in comparatively low concentration (0.55 nM) could significantly decrease the percentage of neurite forming cells from 74% to 32% within a three day incubation period. Especially the longer neurites (> 2 x cell body) proved to be extremely sensitive for vincristine effects. Vinblastine and vindesine were also able to decrease, dose dependently and significantly, the percentage of neurite forming cells. However, the effects observed were less severe than that of vincristine. The sequentially increasing level of neurotoxicity due to vinblastine, vindesine and vincristine, as observed in the neurite outgrowth inhibition correlates well with previous findings from animal models and with data from the clinical practice. Withdrawal of vincristine resulted in a rapid restoration of neurite formation, suggesting the potential reversibility of neurotoxic effects in these cells. These results provide a validation of the PC12 neurite outgrowth assay as a suitable and reliable model for predicting neurotoxicity.

Objectives: The present study was initiated to explore the effects of hydroxyethylidene diphosphonate labeled with rhenium 186(186Re-HEDP) treatment on the progression of lumbar skeletal metastasis in an animal model and to correlate the eventual treatment efficacy with the radioisotope tissue distribution., Methods: The effect of 186Re-HEDP on the progression of lumbar metastasis from prostate cancer was investigated in the Copenhagen rat model. Metastatic prostate tumor deposits were induced in male rats by tail vein injection of R3327-MATLyLu prostate tumor cells under concomitant clamping of the inferior caval vein. The development of clinical symptoms such as onset of hind leg paralysis and urinary bladder swelling was monitored and related to the presence of tumor cells within histologic sections of L-5 and L-6 vertebrae., Results: The 186Re-HEDP administration, given either 1 day or 8 days after surgical induction of lumbar metastasis, could significantly increase the symptom-free survival of the animals. These results were confirmed by a significant decrease in the presence of histologically detectable tumor tissue. Biodistribution studies demonstrated the uptake of the major part of the radioisotope within bone tissue. Uptake of radioactivity within the lumbar vertebrae on a microscopic scale, as shown by phosphor screen autoradiography, was concentrated in areas of bone formation and turnover. Signs of radiotoxicity, such as bone marrow replacement by fat cells and the absence of megakaryocytes, were observed., Conclusions: The results show that radionuclide treatment using 186Re-HEDP is a potentially efficacious treatment option in prostate cancer disseminated to the skeleton. The optimal treatment dose should be determined carefully and aimed at acceptable levels of myelotoxicity.

While skeletal metastasis in prostate cancer is a major and frequent complication, literature data on the mechanisms involved are quite confusing. Recent efforts, however, to establish appropriate animal models for skeletal metastasis have finally yielded positive results which may provide clarity in this discussion. Models involving both human prostate cancer cell transplantation in nude mice as well as syngeneic rat models have contributed to the accumulated evidence in favor of the hypothesis of Batson. According to this hypothesis, prostate tumor cells reach the vertebral venous plexus of the spine especially under transient conditions of increased intraabdominal pressure and lead to metastatic tumor deposits in the vertebrae. Animal models displaying skeletal metastasis in conjunction with analysis of pathological findings have been instrumental in validating this concept. It is to be expected that such animal models will contribute to the development and optimization of new treatment approaches for prostate cancer bone metastasis.

The prevalence of nerve growth factor (NGF) production in different human prostatic tumor cell lines (DU145, PC-3, LNCaP-FGC) was investigated using a specific enzyme-linked immunosorbent assay (ELISA) and compared to that of different human and rat prostatic tissue samples. In addition, the biological effects of NGF beta addition to the human prostatic cancer cell cultures were investigated. The ELISA technique showed the DU145 cell line to secrete measurable levels of NGF in the culture medium. When neurite-outgrowth determination in a pheochromocytoma cell line was used as a bioassay, the NGF synthesized by DU145 cells was confirmed to exhibit functional biological activity. No effect of exogenously added NGF could be established on tumor cell proliferation, on the basis of either colorimetric tetrazolium-based staining assay or bromodeoxyuridine incorporation. Also the expression of prostate specific acid phosphatase was not influenced by NGF addition. However, the in vitro invasive capacity (Matrigel) of DU145 cells was significantly increased by inclusion of 50 ng or 100 ng NGF beta/ml culture medium. In view of the clinically well-known perineural invasion of prostate cancer cells, the possible involvement of NGF as a (paracrine) factor in prostatic cancer metastatic behavior should be investigated further.

The radiosensitizing effect of platinum compounds has been demonstrated in a number of tumors. In prostate cancer, clinical and preclinical data concerning an eventual efficacy of the concept of radiosensitization are lacking. In the present study cisplatin and carboplatin have been used as a model to explore radiosensitization in in vitro prostate cancer cell lines. Human (DU-145) and rat (R3327-MATLyLu) prostate tumor cells were irradiated with doses ranging from 0 to 8 Gy in the presence of various concentrations of either cisplatin or carboplatin. For the evaluation of the combined effect of the two treatment modalities, a simple model is presented. Supra-additive treatment effects of combinations of platinum drugs with radiotherapy, both at clinically achievable doses, were shown on the basis of surviving fractions of tumor cells and proved to be significant. These data strongly suggest that radiotherapy may be effectively combined with radiosensitizers such as platinum drugs in prostate cancer therapy, to yield synergism in treatment efficacy.

Over 80% of clinically manifested prostate cancers respond to androgen withdrawal. Several alternatives to castration have been explored. Since a growth promoting role for androstenedione has been suggested, we investigated the effect of inhibition of 3 beta-hydroxy-steroid-dehydrogenase (3 beta-HSD), a key enzyme involved in the biosynthesis of practically all steroids. In a previous study a reduced proliferation rate of androgen responsive R3327-H tumor was demonstrated after in vivo treatment with 17 beta-N,N-diethylcarbamoyl-4-aza-5 alpha-androstan-3-one (4MA) - a putative 5 alpha-reductase inhibitor. In the present investigation 3 beta-HSD enzyme activity derived from human placenta, testis and ovarian cancer cell line and from rat testis was determined using radiolabeled dehydroepiandrosterone (DHEA) or pregnenolone. Among different synthetic compounds known to interfere with steroidogenesis, only 4MA was shown to potently inhibit in vitro 3 beta-HSD activity from all tissue sources. 4MA was administered to male Copenhagen rats bearing R3327-H androgen dependent prostate tumors and levels of different androgens in serum and prostate tumor were measured using reversed phase HPLC and radioimmunoassay. The decreased content of androstenedione in serum and tumor tissue with DHEA accumulation in prostate tumor tissue showed an effective 3 beta-HSD inhibition by 4MA occurring in vivo as well. These observations unequivocally demonstrate a 3 beta-HSD inhibiting effect of 4MA in vitro as well as in vivo and point to a role for androstenedione in the promotion of cell proliferation in androgen sensitive tumors. 3 beta-HSD dependent androstenedione production could thus constitute a proper target -eventually combined with other endocrine treatment - for the treatment of hormone dependent prostate cancer.

The rat clonal pheochromocytoma cell line PC12 can be induced to display neurite outgrowth upon induction with nerve growth factor (NGF). This NGF-dependent neurite outgrowth assay looks a promising model for research on toxic neuropathies. Using this assay we demonstrated that cisplatin caused a dose-dependent reduction of NGF-dependent neurite formation. Increasing doses of NGF, however, proved to exert protective activity against this cisplatin effect at an intermediate and clinically relevant cisplatin concentration of 1 micrograms/ml. Even at a high cisplatin concentration (10 micrograms/ml), the protective action of NGF, although less adequate, was observed. The value and strength of this model for screening neuropathogenic effects of anticancer agents at the cellular level and the possibly therapeutic action of neurotrophins are discussed and demonstrated. Furthermore, in the light of the urgent need for adequate models for neuropathy research, the PC12 neurite outgrowth protection assay may contribute to our knowledge of the mechanisms underlying the development of neuropathy.

Prostate tumor cell metastasis to the axial skeleton was induced in male Copenhagen rats using the intravenous injection of syngeneic metastatic R3327-MATLyLu tumor cells and concomitant caval vein clamping. The proliferation of tumor cells in the lumbar region was monitored by the progression, within 19 days post tumor cell injection, of hindleg paralysis which appeared in these animals. Histology confirmed the presence of tumor cells within the lumbar spine in 100% of cases displaying hindleg paralysis. Treatment with either of the bisphosphonate drugs, Cl2MDP or APD, suppressed and delayed the development of hind leg paralysis. Bisphosphonate treatment may be expected to delay the onset of axial skeletal metastasis effects in prostate cancer patients.

Histopathological changes in rat pancreas were induced by cyclic periods of experimental malnutrition or by cassava (manioc) feeding for 11 weeks. Decline of body weight was correlated with decrease in testicular fat pad weight as a measure of body fat stores. A marked decrease in pancreatic weight in the cassava-fed group was correlated with shrinkage of acinar structures and degenerative features in exocrine pancreas. In the malnutrition group vacuolisation and loss of tissue architecture were observed in some parts of the organ. No signs of ductal obstruction as a tentative cause of pancreatic pathology after malnutrition could be detected. Loss of islets tissue was occasionally seen in degenerative areas. It is concluded that histopathological changes in exocrine pancreas result from malnutrition and cassava feeding differentially and precede ultimate degenerative processes of pancreas endocrine tissue. Tropical malnutrition type diabetes and low protein related diabetes may in their etiology be different entities, but may coincide in practice and aggravate each other to yield severe and irreversible morbidity.

Enzyme activities of N-acetylglucosaminyltransferase (GlcNAc-Tase) I-V involved in N-linked complex-type carbohydrate synthesis were determined in a non-metastatic hormone-dependent rat prostate tumor (R3327-H) and a related, hormone-independent variant metastasizing to lymph nodes and lungs (R3327-MatLyLu). In the metastasizing variant a significantly increased activity of both GlcNAc-Tase III and GlcNAc-Tase V was observed, whereas the activities of GlcNAc-Tase I and II were essentially unchanged. The increase in activity of GlcNAc-Tase III is particularly noteworthy since it indicates that elevated expression of this enzyme cannot be considered as an exclusive marker of hepatic malignancy.

A rat model for benign prostatic hyperplasia in man (BPH) was investigated. Citral treatment of male Copenhagen rats for 4 months via the transdermal route resulted in a marked hyperplasia of glandular epithelium and interglandular stroma in the ventral prostate. Despite the cellular hyperplasia there was not a significant increase in prostate weight. Investigations of the mechanism of action of citral showed that application of citral directly to the vagina in female, ovariectomized rats resulted in an increased proliferation of vaginal epithelium and a significant increase in the BrdUrd incorporation in vaginal epithelial cells, in short a similar effect to that of estrogen application. In an in vitro assay citral proved to inhibit estrogen binding to estrogen receptors, while no such inhibition was observed with testosterone for androgen receptors. These observations together with the estrogen implication in the BPH and the reported incidence of gynecomastia following exposure to geraniol, a precursor of citral, strongly suggest that the prostatic hyperplasia-inducing capacity of citral may be due to its estrogenic action.

We investigated the effect of 17 beta-N,N-diethylcarbamoyl-4-methyl-4aza- 5 alpha-androstan-3-one (4MA), a 5 alpha-reductase inhibitor, on growth inhibition of androgen-sensitive rat prostatic tumour (R3327-H) and correlated it with changes in weight of normal androgen target tissues and with levels of androgens. Groups of male Copenhagen rats were treated for 28 days with a daily injection of various, increasing doses of 4MA (0.01-4.0 mg/day) and the results were compared with control (vehicle-treated) and with castrated animals. 4MA decreased tumour growth rate in a dose-dependent manner, which was reflected in a decreased incorporation of BrdUrd in DNA of glandular epithelial cells in the tumour. Normal prostate wet weight was also decreased after high-dose 4MA treatment while serum testosterone levels were not affected by 4MA treatment. Contrary to expectations, however, tissue levels of dihydrotestosterone in tumour and ventral prostate were still considerable in 4MA-treated animals. The tumour-inhibiting action of 4MA, therefore, has to be interpreted as not being purely due to 5 alpha-reductase inhibition. On the other hand, it was not possible to demonstrate any direct tumoricidal effect of 4MA in vitro. The relevance of these findings in terms of the endocrine mechanism of action of 4MA on tumour growth is discussed.

The radiosensitivity of two variants of the Dunning Copenhagen rat prostatic tumor R3327 was investigated. The R3327-AT variant, which is a poorly differentiated anaplastic, fast-growing tumor, was irradiated both in vivo and in vitro. Following irradiation, monodispersed cells were plated in vitro and colonies were counted after 7 days. The survival curve of R3327-AT cells irradiated in vivo showed an initial shoulder (Dq-value 0.97 Gy), followed by two exponential parts. The D0-value for the first part of the curve (0-10 Gy) was 2.76 Gy and for the second part of the curve (greater than 10 gy) 9.05 Gy. Extrapolation of the second part of the curve to the Y-axis indicated that the proportion of more radioresistant cells was about 10%. The survival curve for R3327-AT cells irradiated in vitro also suggested the presence of a radioresistant subpopulation, although the proportion was lower (about 3%). This difference might be due to the presence of an hypoxic fraction in the tumors irradiated in vivo, but not in vitro. Tumor cells from the R3327 tumor variant metastatic to lymph nodes and lungs (R3327-MATLyLu), were irradiated in vitro. The radiation effect was evaluated by in vitro colony formation in agar and by in vivo lung colony assay. The colony formation in agar yielded a D0-value of 1.09 Gy. No radioresistant subpopulation was identified in this variant. A similar radiosensitivity was observed by the in vivo lung colony assay (D0 1.39 Gy). The mean inactivation dose calculated for R3327-AT cells (3.45 Gy) was significantly higher than for the metastatic variant (2.00 Gy).

Death related to prostate cancer is invariably due to the tumor metastasis (to lungs, skeleton and lymph nodes). Tumor cell metastatic behaviour is still poorly understood. The R3327 prostate tumor model in the male Copenhagen rat offers an opportunity to investigate the different aspects of metastatic processes in vivo and to evaluate effects of current and new treatment approaches. Lymphatic spread of cancer cells can be measured by determining volume of local load in successive draining lymph node stations. Surgical removal of primary tumor and inguinal lymph node shows that lymphatic metastasis in the R3327-MATLyLu tumor variant is a continuous progressive phenomenon, which starts already in early stages of tumor growth after subcutaneous transplantation. Spread to the lungs can be quantified by enumeration of macroscopically visible pleural lung colonies. Metastatic spread to the lungs can be mimicked by intravenous injection of monodispersed tumor cell suspension. Within 10 days pleural tumor colonies become visible. Effects of different agents and treatments like chemotherapeutic drugs, heparin, surgery and high energy shock wave (HESW) treatment have been described using these methods. Recently, metastasis to the vertebral column was induced by temporal occlusion of venous blood flow through the caval veins while injecting tumor cells in the lateral tail vein. The resulting osteoblastic metastatic lesions in the lumbar vertebrae and the concomitant spinal cord compression led in time to the loss of motoric and sensory function of the hind legs. These observations permit investigation of the mechanisms of bone metastasis based on biological functions, i.e. sensory and motoric function of the hind legs. Finally, it can be said that the various variants of the R3327 rat prostate tumor offer an appropriate model to study various aspects of prostate cancer and metastasis.