Antiproliferative effects of mistletoe (Viscum album L.) extract in urinary bladder carcinoma cell lines

BACKGROUND: The aim of the study was to evaluate the antiproliferative potency of Viscum album extract (VA-E) in human bladder carcinoma cell lines with regard to its possible use for intravesical therapy of superficial bladder cancer. MATERIALS AND METHODS: Proliferation (MTT-test or 3H-thymidine incorporation), necrotic disintegration (3H-thymidine release of prelabelled cells) and portions of apoptotic and/or necrotic cells (Annexin-V binding, propidium iodide (PI) labelling and DNA-fluorescence profiles by flow cytometry) were measured in four different human bladder carcinoma cell lines (T24, TCCSUP, J82 and UM-UC3) cultured in vitro. RESULTS: Antiproliferative effects of VA-E were observed in the four bladder carcinoma cell lines tested. Metabolic activity could also be completely abrogated by short-time contact of the cells with VA-E. Apoptosis and necrosis, as underlying mechanisms of action, were differentially expressed by the different cell lines. CONCLUSION: VA-E and cytotoxic proteins, i.e., mistletoe lectins (ML) and viscotoxins (VT), were able to block the growth of bladder carcinoma cells. Together with the immunomodulating properties of VA-E, the observed antiproliferative potency might give a rationale for the topical intravesical application of VA-E for the treatment of superficial bladder cancer.

The stem cell gene "inhibitor of differentiation 1" (ID1) is frequently expressed in non-small cell lung cancer

Inhibitor of differentiation 1 (ID1) plays a role in cellular differentiation, proliferation, angiogenesis and tumor invasion. As shown recently, ID1 is positively regulated by the tyrosine kinase SRC in lung carcinoma cell lines and with that appears as a potential new therapeutic target in non-small cell carcinoma (NSCLC). To substantiate this hypothesis we examined ID1, SRC and matrix metalloproteinase-9 (MMP-9) immunohistochemically in human NSCLC specimens.

Nuclear cysteine cathepsin variants in thyroid carcinoma cells

The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in thyroid prohormone processing initiated in the follicular lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through, e.g., ECM degradation. In this study, immunofluorescence and biochemical data from subcellular fractionation revealed that cathepsin B, in its single- and two-chain forms, is localized to endo-lysosomes in the papillary thyroid carcinoma cell line KTC-1 and in the anaplastic thyroid carcinoma cell lines HTh7 and HTh74. This distribution is not affected by thyroid stimulating hormone (TSH) incubation of HTh74, the only cell line that expresses a functional TSH-receptor. Immunofluorescence data disclosed an additional nuclear localization of cathepsin B immunoreactivity. This was supported by biochemical data showing a proteolytically active variant slightly smaller than the cathepsin B proform in nuclear fractions. We also demonstrate that immunoreactions specific for cathepsin V, but not cathepsin L, are localized to the nucleus in HTh74 in peri-nucleolar patterns. As deduced from co-localization studies and in vitro degradation assays, we suggest that nuclear variants of cathepsins are involved in the development of thyroid malignancies through modification of DNA-associated proteins.

High interstitial fluid pressure is associated with low tumour penetration of diagnostic monoclonal antibodies applied for molecular imaging purposes

The human epithelial cell adhesion molecule (EpCAM) is highly expressed in a variety of clinical tumour entities. Although an antibody against EpCAM has successfully been used as an adjuvant therapy in colon cancer, this therapy has never gained wide-spread use. We have therefore investigated the possibilities and limitations for EpCAM as possible molecular imaging target using a panel of preclinical cancer models. Twelve human cancer cell lines representing six tumour entities were tested for their EpCAM expression by qPCR, flow cytometry analysis and immunocytochemistry. In addition, EpCAM expression was analyzed in vivo in xenograft models for tumours derived from these cells. Except for melanoma, all cell lines expressed EpCAM mRNA and protein when grown in vitro. Although they exhibited different mRNA levels, all cell lines showed similar EpCAM protein levels upon detection with monoclonal antibodies. When grown in vivo, the EpCAM expression was unaffected compared to in vitro except for the pancreatic carcinoma cell line 5072 which lost its EpCAM expression in vivo. Intravenously applied radio-labelled anti EpCAM MOC31 antibody was enriched in HT29 primary tumour xenografts indicating that EpCAM binding sites are accessible in vivo. However, bound antibody could only be immunohistochemically detected in the vicinity of perfused blood vessels. Investigation of the fine structure of the HT29 tumour blood vessels showed that they were immature and prone for higher fluid flux into the interstitial space. Consistent with this hypothesis, a higher interstitial fluid pressure of about 12 mbar was measured in the HT29 primary tumour via "wick-in-needle" technique which could explain the limited diffusion of the antibody into the tumour observed by immunohistochemistry.

The tumor suppressor gene hypermethylated in cancer 1 is transcriptionally regulated by E2F1

The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that cooperates with p53 to suppress cancer development. We and others recently showed that HIC1 is a transcriptional target of p53. To identify additional transcriptional regulators of HIC1, we screened a set of transcription factors for regulation of a human HIC1 promoter reporter. We found that E2F1 strongly activates the full-length HIC1 promoter reporter. Promoter deletions and mutations identified two E2F responsive elements in the HIC1 core promoter region. Moreover, in vivo binding of E2F1 to the HIC1 promoter was shown by chromatin immunoprecipitation assays in human TIG3 fibroblasts expressing tamoxifen-activated E2F1. In agreement, activation of E2F1 in TIG3-E2F1 cells markedly increased HIC1 expression. Interestingly, expression of E2F1 in the p53(-/-) hepatocellular carcinoma cell line Hep3B led to an increase of endogenous HIC1 mRNA, although bisulfite genomic sequencing of the HIC1 promoter revealed that the region bearing the two E2F1 binding sites is hypermethylated. In addition, endogenous E2F1 induced by etoposide treatment bound to the HIC1 promoter. Moreover, inhibition of E2F1 strongly reduced the expression of etoposide-induced HIC1. In conclusion, we identified HIC1 as novel E2F1 transcriptional target in DNA damage responses.

Cathepsin D specifically cleaves the chemokines macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta, and SLC that are expressed in human breast cancer

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1 alpha (CCL3), MIP-1 beta (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1 alpha and MIP-1 beta degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu(58) to Trp(59) bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1 beta hybrids indicated that processing of MIP-1 beta might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1 alpha, MIP-1 beta, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes.

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

Fusion toxins used for cancer-related therapy have demonstrated short circulation half-lives, which impairs tumor localization and, hence, efficacy. Here, we demonstrate that the pharmacokinetics of a fusion toxin composed of a designed ankyrin repeat protein (DARPin) and domain I–truncated Pseudomonas Exotoxin A (PE40/ETA″) can be significantly improved by facile bioorthogonal conjugation with a polyethylene glycol (PEG) polymer at a unique position. Fusion of the anti-EpCAM DARPin Ec1 to ETA″ and expression in methionine-auxotrophic E. coli enabled introduction of the nonnatural amino acid azidohomoalanine (Aha) at position 1 for strain-promoted click PEGylation. PEGylated Ec1-ETA″ was characterized by detailed biochemical analysis, and its potential for tumor targeting was assessed using carcinoma cell lines of various histotypes in vitro, and subcutaneous and orthotopic tumor xenografts in vivo. The mild click reaction resulted in a well-defined mono-PEGylated product, which could be readily purified to homogeneity. Despite an increased hydrodynamic radius resulting from the polymer, the fusion toxin demonstrated high EpCAM-binding activity and retained cytotoxicity in the femtomolar range. Pharmacologic analysis in mice unveiled an almost 6-fold increase in the elimination half-life (14 vs. 82 minutes) and a more than 7-fold increase in the area under the curve (AUC) compared with non-PEGylated Ec1-ETA″, which directly translated in increased and longer-lasting effects on established tumor xenografts. Our data underline the great potential of combining the inherent advantages of the DARPin format with bioorthogonal click chemistry to overcome the limitations of engineering fusion toxins with enhanced efficacy for cancer-related therapy.

Chondroitin sulfate proteoglycan CSPG4 as a novel hypoxia-sensitive marker in pancreatic tumors.

CSPG4 marks pericytes, undifferentiated precursors and tumor cells. We assessed whether the shed ectodomain of CSPG4 (sCSPG4) might circulate and reflect potential changes in CSPG4 tissue expression (pCSPG4) due to desmoplastic and malignant aberrations occurring in pancreatic tumors. Serum sCSPG4 was measured using ELISA in test (n = 83) and validation (n = 221) cohorts comprising donors (n = 11+26) and patients with chronic pancreatitis (n = 11+20) or neoplasms: benign (serous cystadenoma SCA, n = 13+20), premalignant (intraductal dysplastic IPMNs, n = 9+55), and malignant (IPMN-associated invasive carcinomas, n = 4+14; ductal adenocarcinomas, n = 35+86). Pancreatic pCSPG4 expression was evaluated using qRT-PCR (n = 139), western blot analysis and immunohistochemistry. sCSPG4 was found in circulation, but its level was significantly lower in pancreatic patients than in donors. Selective maintenance was observed in advanced IPMNs and PDACs and showed a nodal association while lacking prognostic relevance. Pancreatic pCSPG4 expression was preserved or elevated, whereby neoplastic cells lacked pCSPG4 or tended to overexpress without shedding. Extreme pancreatic overexpression, membranous exposure and tissue(high)/sera(low)-discordance highlighted stroma-poor benign cystic neoplasm. SCA is known to display hypoxic markers and coincide with von-Hippel-Lindau and Peutz-Jeghers syndromes, in which pVHL and LBK1 mutations affect hypoxic signaling pathways. In vitro testing confined pCSPG4 overexpression to normal mesenchymal but not epithelial cells, and a third of tested carcinoma cell lines; however, only the latter showed pCSPG4-responsiveness to chronic hypoxia. siRNA-based knockdowns failed to reduce the malignant potential of either normoxic or hypoxic cells. Thus, overexpression of the newly established conditional hypoxic indicator, CSPG4, is apparently non-pathogenic in pancreatic malignancies but might mark distinct epithelial lineage and contribute to cell polarity disorders. Surficial retention on tumor cells renders CSPG4 an attractive therapeutic target. Systemic 'drop and restoration' alterations accompanying IPMN and PDAC progression indicate that the interference of pancreatic diseases with local and remote shedding/release of sCSPG4 into circulation deserves broad diagnostic exploration.

Interplay between the prostaglandin transporter OATP2A1 and prostaglandin E2-mediated cellular effects.

Prostaglandins such as prostaglandin E2 (PGE2) play a pivotal role in physiological and pathophysiological pathways in gastric mucosa. Little is known about the interrelation of the prostaglandin E (EP) receptors with the prostaglandin transporter OATP2A1 in the gastric mucosa and gastric carcinoma. Therefore, we first investigated the expression of OATP2A1 and EP4 in normal and carcinoma gastric mucosa. Different PGE2-mediated cellular pathways and mechanisms were investigated using human embryonic kidney cells (HEK293) and the human gastric carcinoma cell line AGS stably transfected with OATP2A1. Colocalization and expression of OATP2A1 and EP4 were detected in mucosa of normal gastric tissue and of gastric carcinomas. OATP2A1 reduced the PGE2-mediated cAMP production in HEK293 and AGS cells overexpressing EP4 and OATP2A1. The expression of OATP2A1 in AGS cells resulted in a reduction of [(3)H]-thymidine incorporation which was in line with a higher accumulation of AGS-OATP2A1 cells in S-phase of the cell cycle compared to control cells. In contrast, the expression of OATP2A1 in HEK293 cells had no influence on the distribution in the S-phase compared to control cells. OATP2A1 also diminished the PGE2-mediated expression of interleukin-8 mRNA (IL-8) and hypoxia-inducible-factor 1α (HIF1α) protein in AGS-OATP2A1 cells. The expression of OATP2A1 increased the sensitivity of AGS cells against irinotecan which led to reduced cell viability. Taken together, these data show that OATP2A1 influences PGE2-mediated cellular pathways. Therefore, OATP2A1 needs to be considered as a key determinant for the understanding of the physiology and pathophysiology of prostaglandins in healthy and tumorous gastric mucosa.

Quantitative analysis of extracapsular extension of metastatic lymph nodes and its significance in radiotherapy planning in head and neck squamous cell carcinoma

We performed a histopathologic analysis to assess the extent of the extracapsular extension (ECE) beyond the capsule of metastatic lymph nodes (LN) in head and neck cancer to determine appropriate clinical target volume (CTV) expansions.

Lysyl oxidase expression is an independent marker of prognosis and a predictor of lymph node metastasis in oral and oropharyngeal squamous cell carcinoma (OSCC)

Proteins of the lysyl oxidase (LOX) family are important modulators of the extracellular matrix. However, they have an important role in the tumour development as well as in tumour progression. To evaluate the diagnostic and prognostic value of the LOX protein in oral and oropharyngeal squamous cell carcinoma (OSCC) we performed QRT-PCR and immunohistochemical analysis on two tissue microarrays (622 tissue samples in total). Significantly higher LOX expression was detected in high grade dysplastic oral mucosa as well as in OSCC when compared to normal oral mucosa (P < 0.001). High LOX expression was correlated with clinical TNM stage (P = 0.020), lymph node metastases for the entire cohort (P < 0.001), as well as in the subgroup of small primary tumours (T1/T2, P < 0.001). Moreover, high LOX expression was correlated with poor overall survival (P = 0.004) and disease specific survival (P = 0.037). In a multivariate analysis, high LOX expression was an independent prognostic factor, predicting unfavourable overall survival. In summary, LOX expression is an independent prognostic biomarker and a predictor of lymph node metastasis in OSCC. Moreover, LOX overexpression may be an early phenomenon in the pathogenesis of OSCC and thus an attractive novel target for chemopreventive and therapeutic strategies.

Incidence of small lymph node metastases with evidence of extracapsular extension: clinical implications in patients with head and neck squamous cell carcinoma

Small lymph nodes (LN) show evidence of extracapsular extension (ECE) in a significant number of patients. This study was performed to determine the impact of ECE in LN 7 mm as compared with ECE in larger LN.

VHL-gene deletion in single renal tubular epithelial cells and renal tubular cysts: further evidence for a cyst-dependent progression pathway of clear cell renal carcinoma in von Hippel-Lindau disease

Inheritance of a mutant allele of the von Hippel-Lindau tumor suppressor gene predisposes affected individuals to develop renal cysts and clear cell renal cell carcinoma. Von Hippel-Lindau gene inactivation in single renal tubular cells has indirectly been showed by immunohistochemical staining for the hypoxia-inducible factor alpha target gene product carbonic anhydrase IX. In this study we were able to show von Hippel-Lindau gene deletion in carbonic anhydrase IX positive nonneoplastic renal tubular cells, in epithelial cells lining renal cysts and in a clear cell renal cell carcinoma of a von Hippel-Lindau patient. This was carried out by means of laser confocal microscopy and immunohistochemistry in combination with fluorescence in situ hybridization. Carbonic anhydrase IX negative normal renal tubular cells carried no von Hippel-Lindau gene deletion. Furthermore, recent studies have indicated that the von Hippel-Lindau gene product is necessary for the maintenance of primary cilia stability in renal epithelial cells and that disruption of the cilia structure by von Hippel-Lindau gene inactivation induces renal cyst formation. In our study, we show a significant shortening of primary cilia in epithelial cells lining renal cysts, whereas, single tubular cells with a von Hippel-Lindau gene deletion display to a far lesser extent signs of cilia shortening. Our in vivo results support a model in which renal cysts represent precursor lesions for clear cell renal cell carcinoma and arise from single renal tubular epithelial cells owing to von Hippel-Lindau gene deletion.

PTCH promoter methylation at low level in sporadic basal cell carcinoma analysed by three different approaches

Please cite this paper as: PTCH promoter methylation at low level in sporadic basal cell carcinoma analysed by three different approaches. Experimental Dermatology 2010. Abstract: Basal cell carcinoma (BCC) is the most common form of skin cancer. Mutations of the PTCH hallmark gene are detected in about 50-60% of BCCs, which raises the question whether other mechanisms such as promoter methylation can inactivate PTCH. Therefore, we performed methylation analysis of the PTCH promoter in a total of 56 BCCs. The sensitivity of three different methods, including direct bisulphite sequencing PCR, MethyLight and high-resolution melting (HRM), was applied and compared. We found that HRM analysis of DNA from fresh tissue [rather than formalin-fixed and paraffin-embedded tissue (FFPE)] was the most sensitive method to detect methylation. Low-level methylation of the PTCH promoter was detected in five out of 16 analysed BCCs (31%) on DNA from fresh tissue but only in two (13%) samples on short-time stored FFPE DNA from the very same tumors. In contrast, we were unable to detect methylation by HRM on long-time stored DNA in any of the remaining 40 BCC samples. Our data suggest that (i) HRM on DNA extracted from fresh tissue is the most sensitive method to detect methylation and (ii) methylation of the PTCH promoter may only play a minor role in BCC carcinogenesis.