The role of HOXC6 in prostate cancer development.

BACKGROUND: Homeobox (HOX) genes, which are involved in organ development and homeostasis, have been shown to be involved in normal prostate- and PCa development. In this study, we investigate the expression levels of the HOX A-D genes in PCa. The functional relevance and potential of HOX gene as biomarkers are explored. METHODS: We evaluated HOX gene expression in prostate tissues of different grade and stage and related the outcome to clinical parameters. We analyzed AR regulation and function of HOXC6 in PCa cell lines. We developed a urine-based HOXC6 mRNA assay for diagnostic purposes. RESULTS: HOXC6 was one of the most upregulated HOX genes in all primary, metastasized, and castration-resistant PCa. HOXC6 upregulation was specific to the epithelial component of PCa, and HOXC6 was shown to be involved in epithelial cell proliferation. HOXC6 expression was not influenced by androgens nor by treatments targeting the AR signaling pathway. HOXC6 expression was not related to a prognosis after radical prostatectomy, that is, biochemical or local recurrence. We successfully developed an assay for HOXC6 mRNA detection in urine and confirmed that HOXC6 levels are higher in PCa patients. CONCLUSIONS: HOXC6 has a role in all PCa stages, particularly in PCa cell proliferation. Due to its stable expression, HOXC6 is a novel candidate biomarker for PCa not only in early detection but also for monitoring of progression or response to therapy.

Antitumor effects of cis-urocanic acid on experimental urothelial cell carcinoma of the bladder.

PURPOSE: We determined the effect of protodynamic therapy against bladder cancer cells in vitro and in vivo. We investigated cis-urocanic acid in rat bladder cancer cell cultures and in an orthotopic rat urothelial carcinoma model to assess its safety and antiproliferative activity. MATERIALS AND METHODS: The rat bladder cancer cell line AY-27 was exposed to cis-urocanic acid (BioCis Pharma, Turku, Finland) at pH 6.5 or 7.4 for 2 hours. Cell viability was measured by colorimetric assay at 24 and 48 hours. For in vivo experiments AY-27 cells were instilled into the acid treated bladder of 17 rats. After 4, 7 and 10 days 14 rats were treated intravesically with cis-urocanic acid 6% (weight per volume) or vehicle. Rats were sacrificed on day 12 and the bladders were dissected. Immunohistochemical staining was done to assess apoptosis (caspase-3) and cell proliferation (Ki-67) in vivo. RESULTS: Cis-urocanic acid caused dose dependent, pH dependent inhibition of AY-27 cell proliferation, showing the protodynamic action at concentrations of 0.5% and 1%. At higher cis-urocanic acid doses complete cell death was observed. All tumors detected in animals treated with vehicle were muscle invasive (stage T2 or greater) but only 43% of tumors were muscle invasive in the cis-urocanic acid treated group (p=0.049). There was no difference in the percent of apoptotic or proliferating tumor cells between treatment groups. No signs of toxicity were observed. CONCLUSIONS: Cis-urocanic acid showed direct antiproliferative activity against rat bladder cancer cells in vitro and antitumor effects in vivo. It may have therapeutic potential as an intravesical agent for nonmuscle invasive bladder cancer.

Pharmacokinetics and toxicity of intravesical TMX-101: a preclinical study in pigs.

OBJECTIVE: • To study the pharmacokinetic and toxicity profile of intravesically administered TMX-101, with its active ingredient R-837, a synthetic Toll-like receptor (TLR)-7 agonist, in a pig model. MATERIALS AND METHODS: • TLR-7 expression was determined by immunohistochemistry in human and pig bladder tissue. • Four groups of six pigs received a 1-h intravesical instillation with R-837 of different formulations. • Pharmacokinetic analysis was performed on plasma. Toxicity evaluation included monitoring the well-being of the animals, peripheral blood cell counts, and interleukin-6 and creatinine measurements. Urine was collected for R-837 measurement and dipstick analysis. • In total, three pigs per group were sacrificed 24 h post-treatment, and the remaining animals were sacrificed after 1 week. Histopathological examination of the bladder wall was performed. RESULTS: • TLR-7 was homogeneously expressed in human and pig urothelium. • R-837 and vehicle were well tolerated without deterioration in animal well-being. • Systemic R-837 absorption was low. • Mean maximum plasma concentration of R-837 differed depending on the formulation. Post-treatment, plasma levels were negligible at 24 h. • Histopathological examination of the bladders did not show significant abnormalities, apart from the intended inflammatory reaction in the R-837 treated groups. CONCLUSION: • Intravesically administered R-837 in pigs, which showed a similar TLR-7 distribution in bladder tissue as humans, is well tolerated and causes no bladder wall toxicity, and formulations with poloxamer and hydroxypropyl-ß-cyclodextrin showed less systemic absorption.

Molecular Phenotyping of AR Signaling for Predicting Targeted Therapy in Castration Resistant Prostate Cancer.

Castration-resistant prostate cancer (CRPC) is defined by resistance of the tumor to androgen deprivation therapy (ADT). Several molecular changes, particularly in the AR signaling cascade, have been described that may explain ADT resistance. The variety of changes may also explain why the response to novel therapies varies between patients. Testing the specific molecular changes may be a major step towards personalized treatment of CRPC patients. The aim of our study was to evaluate the molecular changes in the AR signaling cascade in CRPC patients. We have developed and validated several methods which are easy to use, and require little tissue material, for exploring AR signaling pathway changes simultaneously. We found that the AR signaling pathway is still active in the majority of our CRPC patients, due to molecular changes in AR signaling components. There was heterogeneity in the molecular changes observed, but we could classify the patients into 4 major subgroups which are: AR mutation, AR amplification, active intratumoral steroidogenesis, and combination of AR amplification and active intratumoral steroidogenesis. We suggest characterizing the AR signaling pathway in CRPC patients before beginning any new treatment, and a recent fresh tissue sample from the prostate or a metastatic site should be obtained for the purpose of this characterization.

Detailed analysis of histopathological parameters in radical prostatectomy specimens and PCA3 urine test results.

BACKGROUND: Due to the drawbacks of serum prostate-specific antigen, there is an ongoing search for new diagnostic and prognostic prostate cancer (PCa) markers. PCA3 has proven to be of value in the diagnosis of PCa. However, so far few attempts have been made to investigate the prognostic value of PCA3. Our objective was to further investigate the prognostic value of PCA3. METHODS: In this study we correlated the PCA3 score in urinary sediments after digital rectal examination in 62 men with the classical prognostic parameters assessed in radical prostatectomy specimens (i.e. Gleason score, pathological tumor stage and total tumor volume) and moreover, with the expression of three immunohistochemical markers for PCa biological aggressiveness (i.e. E-cadherin, alpha-catenin and EZH2). The results from this study serve as a reflection on and a valuable adjunct to recently published results. RESULTS: We did not find a significant correlation of the PCA3 score with the classical prognostic parameters assessed in radical prostatectomy specimens or the expression of the immunohistochemical markers for PCa biological aggressiveness. However, we did observe a trend for a higher PCA3 score in significant PCa versus insignificant PCa, aberrant E-cadherin staining versus normal E-cadherin staining and increased EZH2 staining versus normal EZH2 staining. CONCLUSIONS: In this study we could not prove the prognostic value of PCA3. Further research with large numbers of men and adequate follow-up is needed to provide a definitive answer to the question if PCA3 is not only a diagnostic but also a prognostic PCa marker.

Cadherin-11 is expressed in detrusor smooth muscle cells and myofibroblasts of normal human bladder.

OBJECTIVES: It has recently been found that detrusor smooth muscle cells and myofibroblasts are coupled via gap junctions. However, gap junctions cannot account for strong physical interaction between cells, which has prompted the search for intercellular adhesion molecules. Cadherin-11 is a candidate for such a molecule, since it mediates the interaction of dermal myofibroblasts in contractile wound granulation tissue. We therefore hypothesised that the physical adhesion between detrusor smooth muscle cells and myofibroblasts is mediated by cadherin-11. The aim of this study was to test this hypothesis. METHODS: Bladder biopsies from eight radical cystectomy specimens were snap-frozen, sectioned, and stained for E-cadherin; cadherin-11; alpha-catenin; beta-catenin; gamma-catenin; and smooth muscle cell/myofibroblast markers connexin-43, vimentin, desmin, smooth muscle actin, and smoothelin. Specimens were analysed by using binocular epifluorescent and confocal laser-scanning microscopy. RESULTS: Specific positive membranous expression of all adhesion complex molecules except E-cadherin was detected in detrusor suburothelial tissue. All biopsies showed a similar punctate pattern of expression for cadherin-11 within bundles of smooth muscle cells and a suburothelial layer of cells. Cadherin-11 was specifically located at the cell membrane, in distinct linear domains. CONCLUSIONS: To our knowledge this is the first time evidence has been provided for cadherin-mediated smooth muscle and suburothelial myofibroblast cell-cell interaction in the human bladder. Cadherin-11 most probably plays an important role in the intercellular physical coupling of detrusor smooth muscle cells and also of myofibroblasts.

Effect of hyperthermia on the cytotoxicity of 4 chemotherapeutic agents currently used for the treatment of transitional cell carcinoma of the bladder: an in vitro study.

PURPOSE: Hyperthermia combined with chemotherapy is not a novel cancer treatment. However, the working mechanism of this combination therapy is not fully understood. In the current in vitro study we investigated the differences in cytotoxicity of 4 chemotherapeutic agents at 37C or 43C. MATERIALS AND METHODS: The human transitional cell carcinoma cell lines used were RT4, RT112, 253J and T24. Cells were seeded in 96-well microtiter plates. After 24 hours cells were treated for 60 minutes with increasing concentrations of mitomycin C, epirubicin, gemcitabine and EO9 at a temperature of 37C or 43C. After treatment cells were rinsed 3 times and left for 24 hours in the incubator at 37C. The influence of chemotherapy and temperature on cell survival was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay. RESULTS: Decreased cell proliferation with increasing concentrations of chemotherapeutic agents was demonstrated. EO9 proved to be the most potent agent at each temperature. Hyperthermia alone did not demonstrate decreased cell proliferation. However, a synergistic effect on decreased cell proliferation was demonstrated in all cell lines and chemotherapeutic agents used, although each had a maximum at a different chemotherapy concentration and to a different extent. Synergism was most obvious in cell lines treated with low dose epirubicin. CONCLUSIONS: Synergism with hyperthermia and chemotherapy was clearly demonstrated for epirubicin, EO9, mitomycin C and to a lesser extent gemcitabine. Hyperthermia alone did not cause decreased cell proliferation. Synergism was most prominent with low drug doses and the most potent drug used in this in vitro study was EO9.

The effect of hyperthermia on mitomycin-C induced cytotoxicity in four human bladder cancer cell lines.

INTRODUCTION: Hyperthermia and mitomycin-C (MMC) have given very encouraging results in several clinical studies for the treatment of superficial transitional cell carcinoma of the bladder. However, a synergistic effect of hyperthermia and MMC on the decrease of cell proliferation has never been demonstrated accurately in vitro. We investigated the effect of MMC versus MMC combined with hyperthermia on the cytotoxicity in four human bladder cancer cell lines. MATERIAL AND METHODS: The RT112, RT4, 253J and T24 human bladder cancer cell lines were seeded in 96-well microtiter plates at 2.0 x 10(4) cells per well and were left to attach for 24 hours. The cells were then treated for 60 minutes with MMC concentrations ranging from 0 to 400 microg/ml at a temperature of 37 degrees C or 43 degrees C. After treatment cells were rinsed three times with culture medium and left for 24 hours in the incubator. Dimethyl thiazolyl tetrazolium (MTT) solution was added and after 4 hours of incubation the MTT containing media was aspired from all wells and 100 microl of dimethyl sulfoxide was added to each well. A spectrum analyses was performed at 595 nm light wavelength. RESULTS: A decrease of cell proliferation after treatment with increasing concentrations MMC was demonstrated. Hyperthermia has a synergistic effect on the decrease of cell proliferation by different concentrations MMC. In the cells treated without MMC no significant difference in the extent of cell killing at 37 degrees C and 43 degrees C was observed. Furthermore, no difference was observed between cells with a p53 protein mutation (RT112 and T24) or without a p53 protein mutation (253J and RT4). CONCLUSION: A clear synergistic effect of MMC and hyperthermia has been demonstrated in four human bladder cancer cell lines.