Susanne Fuessel

Gene signatures of pulmonary metastases of renal cell carcinoma reflect the disease‐free interval and the number of metastases per patient

Our understanding of metastatic spread is limited and molecular mechanisms causing particular characteristics of metastasis are largely unknown. Herein, transcriptome‐wide expression profiles of a unique cohort of 20 laser‐resected pulmonary metastases (Mets) of 18 patients with clear‐cell renal cell carcinoma (RCC) were analyzed to identify expression patterns associated with two important prognostic factors in RCC: the disease‐free interval (DFI) after nephrectomy and the number of Mets per patient. Differentially expressed genes were identified by comparing early (DFI ≤ 9 months) and late (DFI ≥ 5 years) Mets, and Mets derived from patients with few (≤8) and multiple (≥16) Mets. Early and late Mets could be separated by the expression of genes involved in metastasis‐associated processes, such as angiogenesis, cell migration and adhesion (e.g., PECAM1, KDR). Samples from patients with multiple Mets showed an elevated expression of genes associated with cell division and cell cycle (e.g., PBK, BIRC5, PTTG1) which indicates that a high number of Mets might result from an increased growth potential. Minimal sets of genes for the prediction of the DFI and the number of Mets per patient were identified. Microarray results were confirmed by quantitative PCR by including nine further pulmonary Mets of RCC. In summary, we showed that subgroups of Mets are distinguishable based on their expression profiles, which reflect the DFI and the number of Mets of a patient. To what extent the identified molecular factors contribute to the development of these characteristics of metastatic spread needs to be analyzed in further studies.

Aldehyde Dehydrogenase Is Regulated by β-Catenin/TCF and Promotes Radioresistance in Prostate Cancer Progenitor Cells

Radiotherapy is a curative treatment option in prostate cancer. Nevertheless, patients with high-risk prostate cancer are prone to relapse. Identification of the predictive biomarkers and molecular mechanisms of radioresistance bears promise to improve cancer therapies. In this study, we show that aldehyde dehydrogenase (ALDH) activity is indicative of radioresistant prostate progenitor cells with an enhanced DNA repair capacity and activation of epithelial-mesenchymal transition (EMT). Gene expression profiling of prostate cancer cells, their radioresistant derivatives, ALDH(+) and ALDH(-) cell populations revealed the mechanisms, which link tumor progenitors to radioresistance, including activation of the WNT/β-catenin signaling pathway. We found that expression of the ALDH1A1 gene is regulated by the WNT signaling pathway and co-occurs with expression of β-catenin in prostate tumor specimens. Inhibition of the WNT pathway led to a decrease in ALDH(+) tumor progenitor population and to radiosensitization of cancer cells. Taken together, our results indicate that ALDH(+) cells contribute to tumor radioresistance and their molecular targeting may enhance the effectiveness of radiotherapy.

Biological Activity of a Gallic Acid−Gelatin Conjugate

Goal of the present study was the characterization of the biological properties of a gelatin-gallic acid conjugate (Gel-GA) to evaluate its applicability in biomedicine and pharmacy. The macromolecular conjugate was synthesized by free radical grafting reaction between gelatin and gallic acid (GA) to form a covalent conjugate that was found to retain the antioxidant and enzymatic activities of free GA. In particular, the peroxynitrite scavenging power was found to be consistent with a IC(50) value of 2.17 ± 0.4 mg mL(-1). The enzymatic capacities of GA, which are regarded beneficial for cell functions, are partly retained in the Gel-GA conjugate. In particular, acetylcholinesterase inhibition (IC(50) of 7.1 ± 1.3 mg mL(-1)) implies the conjugates usefulness in the chemoprevention of Alzheimers disease, while the inhibition of α-amylase (IC(50) of 9.8 ± 1.1 mg mL(-1)) suggests that the conjugate can be a preferred alternative for inhibition of carbohydrate breakdown and control of glycemic index of food products. Finally, the anticancer activity of Gel-GA was proven in prostate carcinoma and renal cell carcinoma cell lines, confirming the potential of the proposed protein-polyphenol conjugate in medicine.

Detection of Circulating Tumor Cells in Peripheral Blood of Patients with Renal Cell Carcinoma Correlates with Prognosis

Purpose: The aim of this study was to evaluate the clinical relevance of the presence of disseminated tumor cells in peripheral blood (so-called circulating tumor cells) for renal cell carcinoma patients. Methods: Two hundred thirty-three peripheral blood samples from 154 renal cell carcinoma patients were investigated for the presence of disseminated tumor cells by autoMACS technique and immunocytochemical staining of cytokeratin. The frequency of circulating tumor cells was analyzed statistically for correlation with relevant clinical data. Results: Two kinds of tumor cells were detected: those with expression of cytokeratin 8/18 (CK+) and cells without a detectable cytokeratin expression, which we called large blue-stained cells with a tumorlike morphology. After following the CD45 autoMACS depletion protocol, we identified circulating tumor cells in 96 (41%) of 233 peripheral blood samples, which originated from 81 (53%) of 154 renal cell carcinoma patients. A significant correlation between the detection of circulating tumor cells and positive lymph node status (P < 0.001; χ2 test) and the presence of synchronous metastases at the time of primary tumor resection (P = 0.014; χ2 test) was found. In a multivariate Coxs regression hazard model, presence of CK+ circulating tumor cells was significantly correlated with poor overall survival for renal cell carcinoma patients (relative risk, 2.3; P = 0.048). Conclusions: The presence of circulating tumor cells correlated to lymph node status and presence of synchronous metastases in renal cell carcinoma. It is important to evaluate CK+ and blue-stained tumor cells together to determine the role of circulating tumor cells in tumor behavior and disease progression. Detection of CK+ circulating tumor cells in peripheral blood is a significant and independent prognostic factor for renal cell carcinoma.(Cancer Epidemiol Biomarkers Prev 2009;18(8):2190–4)

Delivery of carboplatin by carbon-based nanocontainers mediates increased cancer cell death

Since the activity of several conventional anticancer drugs is restricted by resistance mechanisms and dose-limiting side-effects, the design of nanocarriers seems to be an efficient and promising approach for drug delivery. Their chemical and mechanical stability and their possible multifunctionality render tubular nanomaterials, such as carbon nanotubes (CNTs) and carbon nanofibres (CNFs), promising delivery agents for anticancer drugs. The goal of the present study was to investigate CNTs and CNFs in order to deliver carboplatin in vitro. No significant intrinsic toxicity of unloaded materials was found, confirming their biocompatibility. Carboplatin was loaded onto CNTs and CNFs, revealing a loading yield of 0.20 mg (CNT-CP) and 0.13 mg (CNF-CP) platinum per milligram of material. The platinum release depended on the carrier material. Whereas CNF-CP marginally released the drug, CNT-CP functioned as a drug depot, constantly releasing up to 68% within 14 days. The cytotoxicity of CNT-CP and CNF-CP in urological tumour cell lines was dependent on the drug release. CNT-CP was identified to be more effective than CNF-CP concerning the impairment of proliferation and clonogenic survival of tumour cells. Moreover, carboplatin, which was delivered by CNT-CP, exhibited a higher anticancer activity than free carboplatin.

Expression profile of WNT molecules in prostate cancer and its regulation by aminobisphosphonates.

Skeletal metastases represent a frequent complication in patients with advanced prostate cancer (PCa) and often require bisphosphonate treatment to limit skeletal‐related events. Metastasized PCa cells disturb bone remodeling. Since the WNT signaling pathway regulates bone remodeling and has been implicated in tumor progression and osteomimicry, we analyzed the WNT profile of primary PCa tissues and PCa cell lines and assessed its regulation by bisphosphonates. Prostate tissue (n = 18) was obtained from patients with benign prostate hyperplasia (BPH) and PCa patients with different disease stages. Serum samples were collected from 62 patients. Skeletal metastases were present in 17 patients of whom 6 had been treated with zoledronic acid. The WNT profile and its regulation by bisphoshonates were analyzed in tissue RNA extracts and serum samples as well as in osteotropic (PC3) and non‐osteotropic (DU145, LNCaP) PCa cell lines. Several members of the WNT pathway, including WNT5A, FZD5, and DKK1 were highly up‐regulated in PCa tissue from patients with advanced PCa. Interestingly, osteotropic cells showed a distinct WNT profile compared to non‐osteotropic cells. While WNT5A, FZD5, and DKK1 were highly expressed in PC3 cells, WNT1 and SFRP1 mRNA levels were higher in DU145 cells. Moreover, zoledronic acid down‐regulated mRNA levels of WNT5A (−34%), FZD5 (−60%), and DKK1 (−46%) in PC3 cells. Interestingly, patients with skeletal metastases who received zoledronic acid had twofold higher DKK1 serum levels compared to bisphosphonate‐naive patients. The WNT signaling pathway is up‐regulated in advanced PCa, differentially expressed in osteotropic versus non‐osteotropic cells, and is regulated by zoledronic acid. J. Cell. Biochem. 112: 1593–1600, 2011.

Analyses of potential predictive markers and survival data for a response to sunitinib in patients with metastatic renal cell carcinoma.

Background Patients with metastatic clear cell renal cell carcinoma (ccRCC) are frequently treated with tyrosine kinase inhibitors (TKI) such as sunitinib. It inhibits angiogenic pathways by mainly targeting the receptors of VEGF and PDGF. In ccRCC, angiogenesis is characterized by the inactivation of the von Hippel-Lindau gene (VHL) which in turn leads to the induction of HIF1α target genes such as CA9 and VEGF. Furthermore, the angiogenic phenotype of ccRCC is also reflected by endothelial markers (CD31, CD34) or other tumor-promoting factors like Ki67 or survivin. Methods Tissue microarrays from primary tumor specimens of 42 patients with metastatic ccRCC under sunitinib therapy were immunohistochemically stained for selected markers related to angiogenesis. The prognostic and predictive potential of theses markers was assessed on the basis of the objective response rate which was evaluated according to the RECIST criteria after 3, 6, 9 months and after last report (12–54 months) of sunitinib treatment. Additionally, VHL copy number and mutation analyses were performed on DNA from cryo-preserved tumor tissues of 20 ccRCC patients. Results Immunostaining of HIF-1α, CA9, Ki67, CD31, pVEGFR1, VEGFR1 and -2, pPDGFRα and -β was significantly associated with the sunitinib response after 6 and 9 months as well as last report under therapy. Furthermore, HIF-1α, CA9, CD34, VEGFR1 and -3 and PDGRFα showed significant associations with progression-free survival (PFS) and overall survival (OS). In multivariate Cox proportional hazards regression analyses high CA9 membrane staining and a response after 9 months were independent prognostic factors for longer OS. Frequently observed copy number loss and mutation of VHL gene lead to altered expression of VHL, HIF-1α, CA9, and VEGF. Conclusions Immunoexpression of HIF-1α, CA9, Ki67, CD31, pVEGFR1, VEGFR1 and -2, pPDGFRα and -β in the primary tumors of metastatic ccRCC patients might support the prediction of a good response to sunitinib treatment.

Characterization of different carbon nanotubes for the development of a mucoadhesive drug delivery system for intravesical treatment of bladder cancer

In order to increase the effectiveness of therapeutics for bladder carcinoma (BCa) treatment, alternative strategies for intravesical applications are needed. The use of carbon nanotubes (CNTs) as basis for a multifunctional drug transporter is a promising possibility to combine traditional chemotherapeutics with innovative therapeutic agents such as antisense oligodeoxynucleotides or small interfering RNA. In the current study four CNT types varying in length and diameter (CNT-1, CNT-2, CNT-3, CNT-4) were synthesized and then characterized with different spectroscopic techniques. Compared to the pristine CNT-1 and CNT-3, the shortened CNT-2 and CNT-4 exhibited more defects and lower aspect ratios. To analyze their mucoadhesive properties, CNTs were exposed to mouse bladders ex vivo by using Franz diffusion cells. All four tested CNT types were able to adhere to the urothelium with a mean covering area of 5-10%. In vitro studies on UM-UC-3 and EJ28 BCa cells were conducted to evaluate the toxic potential of these CNTs. Viability and cytotoxicity assays revealed that the shortened CNT-2 and CNT-4 induced stronger inhibitory effects on BCa cells than CNT-1 and CNT-3. In conclusion, CNT-1 and CNT-3 showed the most promising properties for further optimization of a multifunctional drug transporter.

Elevated expression of prostate cancer-associated genes is linked to down-regulation of microRNAs

BackgroundRecent evidence suggests that the prostate cancer (PCa)-specific up-regulation of certain genes such as AMACR, EZH2, PSGR, PSMA and TRPM8 could be associated with an aberrant expression of non-coding microRNAs (miRNA).MethodsIn silico analyses were used to search for miRNAs being putative regulators of PCa-associated genes. The expression of nine selected miRNAs (hsa-miR-101, -138, -186, -224, -26a, -26b, -374a, -410, -660) as well as of the aforementioned PCa-associated genes was analyzed by quantitative PCR using 50 malignant (Tu) and matched non-malignant (Tf) tissue samples from prostatectomy specimens as well as 30 samples from patients with benign prostatic hyperplasia (BPH). Then, correlations between paired miRNA and target gene expression levels were analyzed. Furthermore, the effect of exogenously administered miR-26a on selected target genes was determined by quantitative PCR and Western Blot in various PCa cell lines. A luciferase reporter assay was used for target validation.ResultsThe expression of all selected miRNAs was decreased in PCa tissue samples compared to either control group (Tu vs Tf: -1.35 to -5.61-fold; Tu vs BPH: -1.17 to -5.49-fold). The down-regulation of most miRNAs inversely correlated with an up-regulation of their putative target genes with Spearman correlation coefficients ranging from -0.107 to -0.551. MiR-186 showed a significantly diminished expression in patients with non-organ confined PCa and initial metastases. Furthermore, over-expression of miR-26a reduced the mRNA and protein expression of its potential target gene AMACR in vitro. Using the luciferase reporter assay AMACR was validated as new target for miR-26a.ConclusionsThe findings of this study indicate that the expression of specific miRNAs is decreased in PCa and inversely correlates with the up-regulation of their putative target genes. Consequently, miRNAs could contribute to oncogenesis and progression of PCa via an altered miRNA-target gene-interaction.

Polyclonal antibodies against kallikrein-related peptidase 4 (KLK4): immunohistochemical assessment of KLK4 expression in healthy tissues and prostate cancer.

Abstract KLK4 is a member of the human kallikrein-related peptidase family of (chymo)trypsin-like serine proteases. The aim of the present study was to generate polyclonal antibodies (pAb) directed against KLK4 for the analysis of KLK4 by immunohistochemistry in human tissues. Recombinantly expressed human mature KLK4 was used for immunization of chickens. pAb 617A is an affinity-purified monospecific pAb fraction reacting with a linear epitope within a flexible surface-exposed loop of KLK4. pAb 617C is the KLK-directed pAb fraction completely depleted from pAb 617A. In healthy adult tissues, KLK4 was immunodetected by both antibody fractions in kidney, liver, and prostate, but not in other organs such as colon and lung. To evaluate protein expression of KLK4 in prostate cancer, samples of tumor tissue plus corresponding tumor-free areas of 44 prostate cancer patients, represented on a tissue microarray, were investigated. Distinct KLK4 immunostaining was observed with both antibodies in cancerous glandular epithelial cells, but not in surrounding stromal cells. KLK4 expression was lower in stage pT3+4 than in pT1+2 tumors, which was highly significant when employing pAb 617A. Thus, our results indicate that KLK4, which is expressed in the healthy prostate, is upregulated in early-stage but not late-stage prostate cancer.