Enhancing PSMA-uptake with androgen deprivation therapy - a new way to detect prostate cancer metastases?

PURPOSE: 68Ga-PSMA PET/CT imaging is a promising modality for the staging of recurrent prostate cancer (PCa). Current evidence suggests limited diagnostic value of the 68Ga-PSMA PET/CT in PSA-levels ≤0.3ng/mL. Experimental data have demonstrated na increase in PSMA-expression in PCa metastases by androgen deprivation in vitro. The aim of the current study was to investigate a possible enhancing effect of PSMA with low-dose androgen deprivation in patients with BCR and low PSA-levels. MATERIALS AND METHODS: Five patients with PCa and BCR, following radical prostatectomy, underwent 68Ga-PSMA PET/CT. A consecutive 68Ga-PSMA PET/CT was performed 6 to 11 days after injection of 80mg of Degarelix (Firmagon®). We recorded PSA and testosterone serum-levels and changes of PSMA-uptake in 68Ga-PSMA PET/CT images. RESULTS: Median PSA prior 68Ga-PSMA PET/CT was 0.27ng/mL. All patients had a decrease in testosterone serum levels from median 2.95µg/l to 0.16µg/l following Degarelix injection. We observed an increase in the standardized uptake value (SUV) in PSMA-positive lymphogenous and osseous lesions in two patients following androgen deprivation. In another two patients, no PSMA positive signals were detected in either the fi rst or the second scan. CONCLUSION: Our preliminary results of this feasibility assessment indicate a possible enhancing effect of PSMA-imaging induced by low-dose ADT. Despite several limitations and the small number of patients, this could be a new approach to improve staging by 68Ga-PSMA PET/CT in PCa patients with BCR after primary therapy. Further prospective studies with larger number of patients are needed to validate our findings.

Valproic acid inhibits the proliferation of cancer cells by re-expressing cyclin D2.

In this study, primary murine prostate cancer (PCa) cells were derived using the well-established TRAMP model. These PCa cells were treated with the histone deacetylase inhibitor, valproic acid (VPA), and we demonstrated that VPA treatment has an antimigrative, antiinvasive and antiproliferative effect on PCa cells. Using microarray analyses, we discovered several candidate genes that could contribute to the cellular effects we observed. In this study, we could demonstrate that VPA treatment of PCa cells causes the re-expression of cyclin D2, a known regulator that is frequently lost in PCa as we could show using immunohistochemical analyses on PCa specimens. We demonstrate that VPA specifically induces the re-expression of cyclin D2, one of the highly conserved D-type cyclin family members, in several cancer cell lines with weak or no cyclin D2 expression. Interestingly, VPA treatment had no effect in fibroblasts, which typically have high basal levels of cyclin D2 expression. The re-expression of cyclin D2 observed in PCa cells is activated by increased histone acetylation in the promoter region of the Ccnd2 gene and represents one underlying molecular mechanism of VPA treatment that inhibits the proliferation of cancer cells. Altogether, our results confirm that VPA is an anticancer therapeutic drug for the treatment of tumors with epigenetically repressed cyclin D2 expression.

Testosterone boosts for treatment of castration resistant prostate cancer: an experimental implementation of intermittent androgen deprivation.

BACKGROUND: The primary therapeutic target for non-organ-confined prostate cancer is the androgen receptor (AR). Main strategies to ablate AR function are androgen depletion and direct receptor blockade by AR antagonists. However, incurable castration resistant prostate cancer (CRPC) develops resistance mechanisms to cope with trace amounts of androgen including AR overexpression and mutation in the AR ligand binding domain. METHODS: The CRPC cell model VCaP derivative of a prostate cancer bone metastasis was used in vitro and in nude mice in vivo to examine the effects of immediate testosterone boost on CRPC cells. In addition, a testosterone tolerant cell model was established by incremental acclimatization of VCaP cells to 1 nM testosterone. The effects of androgen withdrawal and testosterone boosts on gene expression were assessed by quantitative real-time polymerase chain reaction, ELISA, and Western blots. Tumor cell proliferation was evaluated with a BrdU test. RESULTS: Testosterone boosts on CRPC VCaP cells eliminate tumor cells to a higher extent than androgen withdrawal in androgen tolerant cells. The pronounced decrease of tumor cell proliferation was accompanied by a marked downregulation of AR expression regarding full-length AR and splice variant AR V7. CONCLUSIONS: Acquiring castration resistance of prostate cancer cells by AR overexpression and amplification obviously sensitizes such cells to testosterone concentrations as low as physiological values. This introduces novel therapeutic means to treat CRPC with non-toxic measures and may find clinical implementation in intermittent androgen deprivation regimens.

Amyloid precursor protein is a biomarker for transformed human pluripotent stem cells.

Increasing evidence suggests an important function of the ß-amyloid precursor protein (APP) in malignant disease in humans; however, the biological basis for this evidence is not well understood at present. To understand the role of APP in transformed pluripotent stem cells, we studied its expression levels in human testicular germ cell tumors using patient tissues, model cell lines, and an established xenograft mouse model. In the present study, we demonstrate the cooperative expression of APP with prominent pluripotency-related genes such as Sox2, NANOG, and POU5F1 (Oct3/4). The closest homologue family member, APLP2, showed no correlation to these stem cell factors. In addition, treatment with histone deacetylase (HDAC) inhibitors suppressed the levels of APP and stem cell markers. Loss of pluripotency, either spontaneously or as a consequence of treatment with an HDAC inhibitor, was accompanied by decreased APP protein levels both in vitro and in vivo. These observations suggest that APP represents a novel and specific biomarker in human transformed pluripotent stem cells that can be selectively modulated by HDAC inhibitors.

A common MLP (muscle LIM protein) variant is associated with cardiomyopathy.

RATIONALE: We previously discovered the human 10T-->C (Trp4Arg) missense mutation in exon 2 of the muscle LIM protein (MLP, CSRP3) gene. OBJECTIVE: We sought to study the effects of this single-nucleotide polymorphism in the in vivo situation. METHODS AND RESULTS: We now report the generation and detailed analysis of the corresponding Mlp(W4R/+) and Mlp(W4R/W4R) knock-in animals, which develop an age- and gene dosage-dependent hypertrophic cardiomyopathy and heart failure phenotype, characterized by almost complete loss of contractile reserve under catecholamine induced stress. In addition, evidence for skeletal muscle pathology, which might have implications for human mutation carriers, was observed. Importantly, we found significantly reduced MLP mRNA and MLP protein expression levels in hearts of heterozygous and homozygous W4R-MLP knock-in animals. We also detected a weaker in vitro interaction of telethonin with W4R-MLP than with wild-type MLP. These alterations may contribute to an increased nuclear localization of W4R-MLP, which was observed by immunohistochemistry. CONCLUSIONS: Given the well-known high frequency of this mutation in Caucasians of up to 1%, our data suggest that (W4R-MLP) might contribute significantly to human cardiovascular disease.

N-cadherin expression in malignant germ cell tumours of the testis.

BACKGROUND: Testicular germ cell tumours (TGCTs) are the most common malignancy in young men aged 18-35 years. They are clinically and histologically subdivided into seminomas and non-seminomas. Cadherins are calcium-dependent transmembrane proteins of the group of adhesion proteins. They play a role in the stabilization of cell-cell contacts, the embryonic morphogenesis, in the maintenance of cell polarity and signal transduction. N-cadherin (CDH2), the neuronal cadherin, stimulates cell-cell contacts during migration and invasion of cells and is able to suppress tumour cell growth. METHODS: Tumour tissues were acquired from 113 male patients and investigated by immunohistochemistry, as were the three TGCT cell lines NCCIT, NTERA-2 and Tcam2. A monoclonal antibody against N-cadherin was used. RESULTS: Tumour-free testis and intratubular germ cell neoplasias (unclassified) (IGCNU) strongly expressed N-cadherin within the cytoplasm. In all seminomas investigated, N-cadherin expression displayed a membrane-bound location. In addition, the teratomas and yolk sac tumours investigated also differentially expressed N-cadherin. In contrast, no N-cadherin could be detected in any of the embryonal carcinomas and chorionic carcinomas examined. This expression pattern was also seen in the investigated mixed tumours consisting of seminomas, teratomas, and embryonal carcinoma. CONCLUSIONS: N-cadherin expression can be used to differentiate embryonal carcinomas and chorionic carcinomas from other histological subtypes of TGCT.

The plasminogen activator inhibitor system in colon cancer cell lines is influenced by the CO2 pneumoperitoneum.

PURPOSE: Laparoscopic surgery in the treatment of colon carcinoma causes pH value alterations as well as changes in fibrinolytic activity. This results in enhanced proliferation of colon carcinoma cells in vitro and also in enhanced growth of liver metastasis when compared to isobaric (gasless) laparoscopy in vivo. So far, the direct influence of CO(2) pneumoperitoneum on the invasiveness and metastatic capabilities of colon cancer cells remains unclear. We therefore evaluated transcripts of the uPA system. METHODS: The influence of CO(2) pneumoperitoneum on the gene expression of plasminogen activator inhibitor-1 (PAI-1), urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA) was investigated in colon carcinoma cell lines (HT116, SW48, and WiDr) and mesothelial cells employing a pneumoperitoneum chamber in vitro. Quantitative gene expression data were collected using real-time RT-PCR and statistical analysis was performed by means of analysis of variance and Bonferroni correction. RESULTS: The expression of uPA and PAI-1 was increased in colon carcinoma cell lines when cultivated at pH 6.1, a value corresponding to intraabdominal pH values during CO(2) insufflation. Elevated PAI-1 mRNA levels were also observed when CO(2) was simultaneously applied with a pressure of 10 mmHg. In contrast, there were no significant changes in mesothelial cells in the investigated parameter. CONCLUSION: The conditions of CO(2) pneumoperitoneum cause changes in the expression of genes controlling the fibrinolytic activity. The increase of PAI-1 and uPA can contribute to the enhancement of metastasis and invasive potential of tumour cells. Therefore, changes in the conditions of laparoscopy may well optimise laparoscopic therapy in colon cancer.

Gamma radiation sterilization of N95 respirators leads to decreased respirator performance.

In response to personal protective equipment (PPE) shortages in the United States due to the Coronavirus Disease 2019, two models of N95 respirators were evaluated for reuse after gamma radiation sterilization. Gamma sterilization is attractive for PPE reuse because it can sterilize large quantities of material through hermetically sealed packaging, providing safety and logistic benefits. The Gamma Irradiation Facility at Sandia National Laboratories was used to irradiate N95 filtering facepiece respirators to a sterilization dose of 25 kGy(tissue). Aerosol particle filtration performance testing and electrostatic field measurements were used to determine the efficacy of the respirators after irradiation. Both respirator models exhibited statistically significant decreases in particle filtering efficiencies and electrostatic potential after irradiation. The largest decrease in capture efficiency was 40-50% and peaked near the 200 nm particle size. The key contribution of this effort is correlating the electrostatic potential change of individual filtration layer of the respirator with the decrease filtration efficiency after irradiation. This observation occurred in both variations of N95 respirator that we tested. Electrostatic potential measurement of the filtration layer is a key indicator for predicting filtration efficiency loss.

Modulating the Heat Sensitivity of Prostate Cancer Cell Lines In Vitro: A New Impact for Focal Therapies.

Focal therapies such as high-intensity focused ultrasound (HiFU) are an emerging therapeutic option for prostate cancer (PCA). Thermal or mechanical effects mediate most therapies. Moreover, locally administered drugs such as bicalutamide or docetaxel are new focal therapeutic options. We assessed the impact of such focal medical treatments on cell viability and heat sensitivity by pre-treating PCA cell lines and then gradually exposing them to heat. The individual heat response of the cell lines tested differed largely. Vertebral-Cancer of the Prostate (VCaP) cells showed an increase in metabolic activity at 40-50 °C. Androgen receptor (AR)-negative PC3 cells showed an increase at 51.3 °C and were overall more resistant to higher temperatures. Pre-treatment of VCaP cells with testosterone (VCaPrev) leads to a more PC3-like kinetic of the heat response. Pre-treatment with finasteride and bicalutamide did not cause changes in heat sensitivity in any cell line. Mitoxantrone treatment, however, shifted heat-induced proliferation loss to lower temperature in VCaP cells. Further analysis via RNAseq identified a possible correlation of heat resistance with H3K27me3-dependent gene regulation, which could be related to an increase in the histone methyltransferase EZH2 and a possible neuroendocrine differentiation. Pre-treatment with mitoxantrone might be a perspective for HiFU treatment. Further studies are needed to evaluate possible combinations with Hsp90 or EZH2 inhibitors.

DNA methylation biomarkers of prostate cancer: confirmation of candidates and evidence urine is the most sensitive body fluid for non-invasive detection.

BACKGROUND: A prostate cancer (PCa) biomarker with improved specificity relative to PSA is a public health priority. Hypermethylated DNA can be detected in body fluids from PCa patients and may be a useful biomarker, although clinical performance varies between studies. We investigated the performance of candidate PCa DNA methylation biomarkers identified through a genome-wide search. METHODS: Real-time PCR was used to measure four DNA methylation biomarkers: GSTP1 and three previously unreported candidates associated with the genes RASSF2, HIST1H4K, and TFAP2E in sodium bisulfite-modified DNA. Matched plasma and urine collected prospectively from 142 patients referred for prostate biopsy and 50 young asymptomatic males were analyzed. RESULTS: Analysis of all biomarkers in urine DNA significantly discriminated PCa from biopsy negative patients. The biomarkers discriminated PCa from biopsy negative patients with AUCs ranging from 0.64 for HIST1H4K (95% CI 0.55-0.72, P < 0.00001) to 0.69 for GSTP1 (95% CI 0.60-0.77, P < 0.00001). All biomarkers showed minimal correlation with PSA. Multivariate analysis did not yield a panel that significantly improved performance over that of single biomarkers. All biomarkers showed greater sensitivity for PCa in urine than in plasma DNA. CONCLUSIONS: Analysis of the biomarkers in urine DNA significantly discriminated PCa from biopsy negative patients. The biomarkers provided information independent of PSA and may warrant inclusion in nomograms for predicting prostate biopsy outcome. The biomarkers' PCa sensitivity was greater for urine than plasma DNA. The biomarker performances in urine DNA should next be validated in formal training and test studies.

Leupaxin, a novel coactivator of the androgen receptor, is expressed in prostate cancer and plays a role in adhesion and invasion of prostate carcinoma cells.

In the present study, we demonstrate that leupaxin mRNA is overexpressed in prostate cancer (PCa) as compared with normal prostate tissue by using cDNA arrays and quantitative RT-PCR analyses. Moderate to strong expression of leupaxin protein was detected in approximately 22% of the PCa tissue sections analyzed, and leupaxin expression intensities were found to be significantly correlated with Gleason patterns/scores. In addition, different leupaxin expression levels were observed in PCa cell lines, and at the subcellular level, leupaxin was usually localized in focal adhesion sites. Furthermore, mutational analysis and transfection experiments of LNCaP cells using different green fluorescent protein-leupaxin constructs demonstrated that leupaxin contains functional nuclear export signals in its LD3 and LD4 motifs, thus shuttling between the cytoplasm and the nucleus. We could also demonstrate for the first time that leupaxin interacts with the androgen receptor in a ligand-dependent manner and serves as a transcriptional activator of this hormone receptor in PCa cells. Down-regulation of leupaxin expression using RNA interference in LNCaP cells resulted in a high rate of morphological changes, detachment, spontaneous apoptosis, and a reduction of prostate-specific antigen secretion. In contrast, knockdown of leupaxin expression in androgen-independent PC-3 and DU 145 cells induced a significant decrease of both the invasive capacity and motility. Our results therefore indicate that leupaxin could serve as a potential progression marker for a subset of PCa and may represent a novel coactivator of the androgen receptor. Leupaxin could function as a putative target for therapeutic interventions of a subset of advanced PCa.

Functional analysis of NKX3.1 in LNCaP prostate cancer cells by RNA interference.

The function of the androgen-regulated homeobox protein NKX3.1 in prostate cancer is controversial. NKX3.1 is necessary for correct prostate development and undergoes frequent allelic loss in prostate cancer. However, no mutations occur in the coding region and some particularly aggressive cancers over-express the protein. Nevertheless NKX3.1 is often referred to as candidate tumor suppressor gene. Recent findings suggest a function in protection against oxidative damage involved in prostate carcinogenesis. Thus NKX3.1 may act differently at various stages of prostate cancer. Unlike a classical tumor suppressor NKX3.1 is up-regulated by androgens and down-regulated by phytoestrogens. In this study we performed RNAi based functional analysis by knocking down NKX3.1 expression in LNCaP prostate cancer cells and analyzing the impact of NKX3.1 on gene expression and cell proliferation. Knock-down of NKX3.1 evoked a massive down-regulation of NKX3.1 expression, followed by reduction in mRNA expression of the androdrogen receptor (AR) and the insulin-like growth factor receptor (IGF-1R). Western blot analysis showed strong decreases of NKX3.1, AR, and IGF-1R protein expression. Concomitantly, cell proliferation decreased and expression of prostate-specific antigen (PSA) mRNA and its secretion were diminished, whereas expression of IGF binding protein 3 (IGFBP-3) and MMP tissue inhibitor 3 (TIMP-3) was up-regulated. In tumor cells not deprived of NKX3.1 expression this gene still has a function which might differ from its role in prostate development and carcinogenesis. NKX3.1 knock-down altered the expression of genes highly relevant in prostate cancer cell proliferation and apoptosis. In LNCaP NKX3.1 most probably plays the role of an androgen-regulated transcription factor whose down-regulation is paralleled by anti-proliferative and pro-apoptotic effects. Since NKX3.1 can regulate AR expression it may become a target for interference in hormone refractory prostate carcinoma.

The relevance of estrogen receptor-beta expression to the antiproliferative effects observed with histone deacetylase inhibitors and phytoestrogens in prostate cancer treatment.

In the prostate, estrogen receptor beta (ERbeta), the preferred receptor for phytoestrogens, has features of a tumor suppressor. To investigate the mechanisms underlying the beneficial effects on prostate cancer of histone deacetylase inhibitor valproic acid (VPA) and phytoestrogen tectorigenin, we analyzed the expression of ERbeta after tectorigenin or VPA treatment. For further functional analysis, we knocked down ERbeta expression by RNA interference. LNCaP prostate cancer cells were treated with 5 mmol/L VPA or 100 micromol/L tectorigenin and transfected with small interfering RNA (siRNA) against ERbeta. Control transfections were done with luciferase (LUC) siRNA. Expression of ERbeta was assessed by Western blot. mRNA expression was quantitated by real-time reverse transcription-PCR. Expression of ERbeta mRNA and protein markedly increased after VPA or tectorigenin treatment. When ERbeta was knocked down by siRNA, the expression of prostate-derived Ets factor, prostate-specific antigen, prostate cancer-specific indicator gene DD3(PCA3), insulin-like growth factor-1 receptor, the catalytic subunit of the telomerase, and ERalpha was up-regulated and the tectorigenin effects were abrogated. ERbeta levels were diminished in prostate cancer and loss of ERbeta was associated with proliferation. Here, we show that siRNA-mediated knockdown of ERbeta increases the expression of genes highly relevant to tumor cell proliferation. In addition, we show that one prominent result of treatment with VPA or tectorigenin is the up-regulation of ERbeta resulting in antiproliferative effects. Thus, these drugs, by restoring the regulatory function of ERbeta in tumor cells, could become useful in the intervention of prostate cancer.

Testosterone metabolites inhibit proliferation of castration- and therapy-resistant prostate cancer.

Novel treatments for castration-resistant prostate cancer (CRPC) such as abiraterone acetate (AA) or enzalutamide effectively target the androgen pathway to arrest aberrant signalling and cell proliferation. Testosterone is able to inhibit tumour cell growth in CRPC. Estrogen receptor-beta (ERß) binds the testosterone-metabolites 3ß-androstanediol and 3α-androstanediol in parallel to the canonical estradiol. In the prostate it is widely accepted that ERß regulates estrogen signalling, mediating anti-proliferative effects. We used the prostate cancer cell lines LNCaP, PC-3, VCaP, and the non-neoplastic BPH-1. VCaP cells were treated with 1 nmol/L testosterone over 20 passages, yielding the cell line VCaPrev, sensitive to hormone therapies. In contrast, LNCaP cells were grown for more than 100 passages yielding a high passage therapy resistant cell line (hiPLNCaP). VCaP and hiPLNCaP cell lines were treated with 5 µmol/L AA for more than 20 passages, respectively, generating the AA-tolerant-subtypes VCaPAA and hiPLNCaPAA. Cell lines were treated with testosterone, dihydrotestosterone (DHT), R1881, and the androgen-metabolites 3ß-androstanediol and 3α-androstanediol. 3ß-androstanediol or 3α-androstanediol significantly reduced proliferation in all cell lines except the BPH-1 and androgen receptor-negative PC-3 and markedly downregulated AR and estrogen receptor alpha (ERα). Whereas ERß expression was increased in all cell lines except BPH-1 or PC-3. In summary, 3ß-adiol or 3α-adiol, as well as DHT and R1881, significantly reduced tumour cell growth in CRPC cells. Thus, these compounds represent novel potential therapeutic approaches to overcome drug-resistance in CRPC, especially with regard to AR-V7 function in therapy resistance. Furthermore, these data confirm the tumour suppressor properties of ERß in CRPC.

Establishment and characterization of two distinct malignant mesothelioma cell lines with common clonal origin.

We describe two newly established malignant mesothelioma (MM) cell lines derived from a pleural effusion of a male. One cell line, designated as MM-Z03E, reveals an epithelioid cobblestone morphology, while the second one, designated as MM-Z03S and subcloned after in vivo selection, exhibits a sarcomatoid storiform growth pattern. Both cell lines showed the immunologic profile characteristic for MM (i.e., expression of cytokeratin, CK18, calretinin, and vimentin in both phenotypes). Cytogenetics, multicolor fluorescence in situ hybridization, comparative genomic hybridization, and oligonucleotide array CGH were performed on both cell lines. Aberrations shared by both cell lines included chromosomal losses of 1q34 approximately qter, 4, 9p, 10p, 13, 14, 16q, 18, and 22, as well as a complex structural aberration involving chromosome 17. Aberrations exclusive to MM-Z03E included gains of 3q11q27 and 5p, while gain of 9q and losses of 3q27qter, 11q, and 18 in MM-Z03S were exclusive to MM-Z03E. Both cell lines were able to develop solid transplant tumors in nude mice within 16 weeks, and immunophenotyping of tumor xenografts revealed an overall retained expression profile of the markers used. Remarkably, one xenograft from MM-Z03E revealed overexpression of p53 and widely invasive growth. In conclusion, both cell lines are useful in vivo and in vitro model systems to study the underlying genetic mechanisms of biphasic differentiation in MM, which can be of certain value considering the increasing relevance of assessing MM tumor biology for the clinical management of this disease.

Prospects of estrogen receptor ß activation in the treatment of castration-resistant prostate cancer.

Advanced prostate cancer can develop into castration-resistant prostate cancer (CRPC). This process is mediated either by intratumoral ligand synthesis or by mutations or aberrations of the androgen receptor (AR) or its cofactors. To date, no curative therapy for CRPC is available, as AR-targeted therapies eventually result in the development of resistance. The human prostate cancer cell line VCaP (vertebral cancer of the prostate) overexpresses AR and its splice variants (ARVs) as a mechanism of resistance to androgen-deprivation therapy (ADT) of external and intratumoral origin. In the present study, we demonstrate that stimulating estrogen receptor ß activity with the specific agonist 8ß-VE2 in VCaP cells in successive stages of ADT induced a time- and dose-dependent decrease in cell survival and an increase in apoptosis. Furthermore, 8ß-VE2 treatment reduced the overexpression of the AR as well as ARVs in VCaP cells under maximum ADT. Our results indicate that decreased survival of the androgen-dependent CRPC cells employing apoptosis together with the regulative effect on AR expression could have beneficial effects over current AR-targeting therapies.

Role of N-cadherin in proliferation, migration, and invasion of germ cell tumours.

Germ cell tumors (GCTs) are the most common malignancies in young men. Most patients with GCT can be cured with cisplatin-based combination chemotherapy, even in metastatic disease. In case of therapy resistance, prognosis is usually poor. We investigated the potential of N-cadherin inhibition as a therapeutic strategy. We analyzed the GCT cell lines NCCIT, NTERA-2, TCam-2, and the cisplatin-resistant sublines NCCIT-R and NTERA-2R. Effects of a blocking antibody or siRNA against N-cadherin on proliferation, migration, and invasion were investigated. Mouse xenografts of GCT cell lines were analyzed by immunohistochemistry for N-cadherin expression. All investigated GCT cell lines were found to express N-cadherin protein in vitro and in vivo. Downregulation of N-cadherin in vitro leads to a significant inhibition of proliferation, migration, and invasion. N-cadherin-downregulation leads to a significantly higher level of pERK. N-cadherin-inhibition resulted in significantly higher rates of apoptotic cells in caspase-3 staining. Expression of N-cadherin is preserved in cisplatin-resistant GCT cells, pointing to an important physiological role in cell survival. N-cadherin-downregulation results in a significant decrease of proliferation, migration, and invasion and stimulates apoptosis in cisplatin-naive and resistant GCT cell lines. Therefore, targeting N-cadherin may be a promising therapeutic approach, particularly in cisplatin-resistant, therapy refractory and metastatic GCT.

The balance between MMP-2/-9 and TIMP-1/-2 is shifted towards MMP in renal cell carcinomas and can be further disturbed by hydrogen peroxide.

In malignant tumors the balance of matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinases (TIMP) is disturbed. Radical oxygen species (ROS) and hydrogen peroxide potentially influence this balance. Therefore, we analyzed the balance of MMP and TIMP in renal cell carcinoma (RCC) specimens and cell lines. In RCC specimens MMP-2, MMP-9, TIMP-1, and TIMP-2 were immunohistochemically detected. Tumor-associated macrophages (TAM) as a potential source of ROS were characterized with an anti-CD68 antibody. Three RCC cell lines were treated with sublethal concentrations of hydrogen peroxide to simulate the effects of radical oxygen species. MMP-2 and MMP-9 protein expression was measured by zymography. mRNA expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 was assessed by quantitative reverse transcriptase polymerase chain reaction. Tumor cell-derived reactive oxygen species were measured by FACS analysis and dihydrorhodamine 123 oxidation. In RCCs the MMP and TIMP expression profile was variable. The balance between MMP and TIMP was shifted towards MMP in comparison to matched normal controls. TAM were localized in a close vicinity to MMP expresssing tumor cells. As in RCC specimens, the expression of MMP and TIMP in the analyzed RCC cell lines varied. Hydrogen peroxide induced MMP-2 and -9 mRNA and protein expression, whereas TIMP-1 and TIMP-2 mRNA levels remained unaffected in cell lines. Thus, the ratio between MMP and TIMP was shifted towards MMP. Tumor cells did not increase the production of reactive oxygen species stimulation with phorbol ester or hydrogen peroxide. In RCC the balance between MMP and TIMP is disturbed. Oxidative stress potentially increases this imbalance. TAM might be one source of hydrogen peroxide thus supporting the invasive properties of RCCs.

cDNA microarray analysis with amplified RNA after isolation of intact cellular RNA from neoplastic and non-neoplastic prostate tissue separated by laser microdissections.

Laser microdissection is a valuable tool to prepare pure cell populations from complex tissues for further analyses. Gene expression studies by real-time RT-PCR and cDNA arrays of microdissected tissues are becoming widely used methods. The integrity and quantity of prepared RNA must be proven to ensure reliable results in subsequent applications such as quantitative RT-PCR and cDNA-arrays. In the present study we used RNAlater trade mark protected prostate tissue for laser microdissection of tumor and tumor-free tissues. RNA quality and quantity was assessed using automated capillary gel electrophoresis. By using quantitative real time-RT-PCR before and after mRNA amplification the insulin-like growth factor binding protein-3 (IGFBP-3) gene expression was shown to be down-regulated in three out of five cases and DD3 was up-regulated in cancer tissues in all cases. The up-regulation of DD3 and the down-regulation of IGFBP-3 gene expression in cancer tissue were conserved after RNA amplification. A cDNA microarray also revealed an IGFBP-3 down-regulation in cancer tissue as well as several genes known to be differerentially expressed in prostate cancer. Taken together, we present a novel method for the isolation of intact RNA from laser microdissection-derived prostate cancer tissue useful for downstream applications of real-time RT-PCR and cDNA microarrays.

Expressional changes after histone deacetylase inhibition by valproic acid in LNCaP human prostate cancer cells.

Pathogenesis of prostate cancer is paralleled by aberrant transcriptional regulation which involves gene silencing by histone deacetylases. In cancer cells, inhibitors of histone deacetylases such as valproic acid can act as differentiation agents which relieve pro-apoptotic factors from transcriptional repression. We investigated the potential of the well-tolerated anticonvulsant valproic acid in prostate cancer cell line LNCaP and analyzed the activation of pro-apoptotic factors and resulting apoptosis. We used real time RT-PCR to quantify the mRNA expression of prostate-specific antigen, prostate-derived Ets transcription factor, tissue inhibitor of matrix metalloproteinase-3 and insulin-like growth factor binding protein-3. An automated sandwich-ELISA was used to measure secretion of prostate-specific antigen in conditioned cell culture media of LNCaP prostate cancer cells. Apoptotic cells were detected cytochemically and by applying immunocytochemistry. Activity of histone deacetylases in nuclear extracts was measured with a colorimetric assay kit. Valproic acid treatment caused a marked inhibition of histone deacetylases activity. Expression of prostate-derived Ets transcription factor and consequently prostate-specific antigen were down-regulated to basal levels in LNCaP cells. Pro-apoptotic factor caspase-3, tissue inhibitor of matrix metalloproteinase-3 and insulin-like growth factor binding protein-3 were up-regulated resulting in apoptosis of tumor cells. Valproic acid mediates marked effects on the expression of genes relevant in proliferation and apoptosis. Our study provides strong evidence that prostate cancer may benefit particularly from anti-proliferative stimuli from this well established drug.