Guenther K. Bonn

Intracellular signaling pathways regulating radioresistance of human prostate carcinoma cells

Radiation therapy plays an important role in the management of prostate carcinoma. However, the problem of radioresistance and molecular mechanisms by which prostate carcinoma cells overcome cytotoxic effects of radiation therapy remains to be elucidated. In order to investigate possible intracellular mechanisms underlying the prostate carcinoma recurrences after radiotherapy, we have established three radiation‐resistant prostate cancer cell lines, LNCaP‐IRR, PC3‐IRR, and Du145‐IRR derived from the parental LNCaP, PC3, and Du145 prostate cancer cells by repetitive exposure to ionizing radiation. LNCaP‐IRR, PC3‐IRR, and Du145‐IRR cells (prostate carcinoma cells recurred after radiation exposure (IRR cells)) showed higher radioresistance and cell motility than parental cell lines. IRR cells exhibited higher levels of androgen and epidermal growth factor (EGF) receptors and activation of their downstream pathways, such as Ras‐mitogen‐activated protein kinase (MAPK) and phosphatidyl inositol 3‐kinase (PI3K)‐Akt and Jak‐STAT. In order to define additional mechanisms involved in the radioresistance development, we determined differences in the proteome profile of parental and IRR cells using 2‐D DIGE followed by computational image analysis and MS. Twenty‐seven proteins were found to be modulated in all three radioresistant cell lines compared to parental cells. Identified proteins revealed capacity to interact with EGF and androgen receptors related signal transduction pathways and were involved in the regulation of intracellular routs providing cell survival, increased motility, mutagenesis, and DNA repair. Our data suggest that radioresistance development is accompanied by multiple mechanisms, including activation of cell receptors and related downstream signal transduction pathways. Identified proteins regulated in the radioresistant prostate carcinoma cells can significantly intensify activation of intracellular signaling that govern cell survival, growth, proliferation, invasion, motility, and DNA repair. In addition, such analyses may be utilized in predicting cellular response to radiotherapy.

Proanthocyanidins: target compounds as antibacterial agents.

Grape seeds accumulate in huge quantities as byproduct during wine production and are therefore a cheap source for pharmacologically active agents. However, studies prove poor antibacterial activity, and results of analyses are sometimes contradictory. The aim of this study was, thus, to determine the antibacterial activity of grape seed extracts with special focus on the chromatographic characterization of active fractions. In the course of these investigations, extraction protocols were optimized so that microwave-assisted extraction (MAE) guaranteed highest preconcentration efficiency. Proanthocyanidins, monomeric flavonoid aglycones, as well as some of their glycosides could be identified within yielded extracts via high-performance liquid chromatography-mass spectrometry (HPLC-MS). By that means the coherence number of possible isomers of procyanidins was approximated by a newly developed equation. As far as antibacterial activity determined via screening tests is concerned, the extracts generally have been found to be positively responsive toward 10 different gram-positive and gram-negative bacteria strains. After fractionation of the raw extracts, proanthocyanidins P2, P3, P4 and gallate esters P2G and P3G (P = proanthocyanidin consisting of catechin and epicatechin units, n = oligomerization degree, G = gallate ester) were determined as active antibacterial agents toward 10 different pathogens. Only moderate activity was found for monomeric flavonoid fractions.

High capacity organic monoliths for the simultaneous application to biopolymer chromatography and the separation of small molecules.

A method for controlling the mesoporous structure of monolithic organic copolymers is presented by systematic variation in polymerisation time, employing poly(p-methylstyrene-co-1,2-(p-vinylphenyl)ethane) (MS/BVPE) as a representative styrene system. Decreasing the time of polymerisation introduces a considerable fraction of mesopores (up to 20% of the total pore volume), while keeping the support permeability reasonable high ( approximately 1.3x10(-14)m(2)). Monolith structures, prepared in such a manner, enable efficient (typically around 70,000plates/m) and fast separation of low-molecular-weight compounds, whereas their performance towards biopolymers is comparable to column supports, fabricated according to typically used protocols (polymerisation time >12h and thus monomer conversion >98%). The polymerisation time is hence a valuable tool to tailor the fraction of support flow-channels, macropores as well as mesopores, which is shown dramatically to influence the chromatographic separation characteristics of the respective column. This way, the preferred applicability of organic (styrene) monolithic copolymers can be extended to the separation of small molecules beyond biopolymer chromatography.

Proteomics profiling of microdissected low- and high-grade prostate tumors identifies Lamin A as a discriminatory biomarker.

Proteomics screening methods for the identification of diagnostic and prognostic biomarkers in cancer are still lagging behind DNA- or RNA-based analysis. We used two-dimensional differential gel electrophoresis (2D-DIGE) in combination with laser capture microdissection (LCM) and MALDI-TOF/TOF mass spectrometry to determine differentially abundant proteins and candidate biomarkers in prostate cancer. Paired (benign and tumor) samples were isolated from 23 Gleason Score 6 (GS 6) and 23 Gleason Score 8 and higher (GS 8+) radical prostatectomy specimens and subjected to 2D-DIGE analysis. Minimal fluorescent dye labeling was applied and electrophoresis performed with triple samples (paired benign and tumor; internal control) for each case of tumor. Nineteen differently abundant proteins were identified by mass spectrometry and further validated. One half of them were associated with glycolysis and the Warburg effect; these were upregulated in tumors. The upregulation correlated with tumor dedifferentiation and might be relevant for selection of therapeutic strategies. Among the other proteins, heat shock protein 60 (HSP60) was significantly upregulated in tumor tissue compared to its benign counterpart. Furthermore, lamin A was statistically highly discriminatory between low and high Gleason score tumors and might serve as a new biomarker of tumor differentiation and prognosis.

Epithelial-to-mesenchymal transition and c-myc expression are the determinants of cetuximab-induced enhancement of squamous cell carcinoma radioresponse

PURPOSE Radiation therapy cures malignant tumors of the head and neck region more effectively when it is combined with application of the anti-EGFR monoclonal antibody cetuximab. Despite the successes achieved, we still do not know how to select patients who will respond to this combination of anti-EGFR monoclonal antibody and radiation. This study was conducted to elucidate possible mechanisms which cause the combined treatment with cetuximab and irradiation to fail in some cases of squamous cell carcinomas. METHODS AND MATERIALS Mice bearing FaDu and A431 squamous cell carcinoma xenograft tumors were treated with cetuximab (total dose 3 mg, intraperitoneally), irradiation (10 Gy) or their combination at the same doses. Treatment was applied when tumors reached 8mm in size. To collect samples for further protein analysis (two-dimensional differential gel electrophoresis (2-D DIGE), mass spectrometry MALDI-TOF/TOF, Western blot analysis, and ELISA), mice from each group were sacrificed on the 8th day after the first injection of cetuximab. Other mice were subjected to tumor growth delay assay. RESULTS In FaDu xenografts, treatment with cetuximab alone was nearly as effective as cetuximab combined with ionizing radiation, whereas A431 tumors responded to the combined treatment with significantly enhanced delay in tumor growth. Tumors extracted from the untreated FaDu and A431 xenografts were analysed for protein expression, and 34 proteins that were differently expressed in the two tumor types were identified. The majority of these proteins are closely related to intratumoral angiogenesis, cell adhesion, motility, differentiation, epithelial-to-mesenchymal transition (EMT), c-myc signaling and DNA repair. CONCLUSIONS The failure of cetuximab to enhance radiation response in FaDu xenografts was associated with the initiation of the program of EMT and with c-myc up-regulation in the carcinoma cells. For this reason, c-myc and EMT-related proteins (E-cadherin, vimentin) may be considered as potential biomarkers to predict squamous cell carcinoma response after treatment with cetuximab in combination with radiation.

Identification of Endosomal Epidermal Growth Factor Receptor Signaling Targets by Functional Organelle Proteomics

Epidermal growth factor (EGF) receptor (EGFR) signal transduction is organized by scaffold and adaptor proteins, which have specific subcellular distribution. On a way from the plasma membrane to the lysosome EGFRs are still in their active state and can signal from distinct subcellular locations. To identify organelle-specific targets of EGF receptor signaling on endosomes a combination of subcellular fractionation, two-dimensional DIGE, fluorescence labeling of phosphoproteins, and MALDI-TOF/TOF mass spectrometry was applied. All together 23 EGF-regulated (phospho)proteins were identified as being differentially associated with endosomal fractions by functional organelle proteomics; among them were proteins known to be involved in endosomal trafficking and cytoskeleton rearrangement (Alix, myosin-9, myosin regulatory light chain, Trap1, moesin, cytokeratin 8, septins 2 and 11, and CapZβ). Interestingly R-Ras, a small GTPase of the Ras family that regulates cell survival and integrin activity, was associated with endosomes in a ligand-dependent manner. EGF-dependent association of R-Ras with late endosomes was confirmed by confocal laser scanning immunofluorescence microscopy and Western blotting of endosomal fractions. EGFR tyrosine kinase inhibitor gefitinib was used to confirm EGF-dependent regulation of all identified proteins. EGF-dependent association of signaling molecules, such as R-Ras, with late endosomes suggests signaling specification through intracellular organelles.

Quality assessment and quantitative analysis of flavonoids from tea samples of different origins by HPLC-DAD-ESI-MS.

Components of green tea ( Camellia sinensis) have been of considerable interest in recent years because of their potential utility as pharmaceutical agents, particularly for their antioxidant and anticarcinogenic activity. Responding to the increasing scientific validation of numerous health benefits of tea, a comprehensive approach was adopted to carry out analysis for the quality assessment of flavonoids in tea samples of different origins. For this purpose, extraction, separation, and mass spectrometric parameters were optimized. Extraction methods evaluated include reflux extraction, a modified accelerated solvent extraction (ASE), namely, Aquasolv extraction, and microwave-assisted extraction (MAE) using different percentages of solvents. Separation was performed by a specifically developed reversed phase high-performance liquid chromatography (RP-HPLC) method using different C18 and C8 stationary phases. Optimization of extraction techniques clearly proved the performance of MAE, which delivered highest yields in a very short time. Additionally, the comparison with Aquasolv extraction provided new insights, as variations in quantified amounts of target compounds between the extracts could be explained on the basis of thermal degradation and epimerization phenomena. Especially the epimerization phenomenon for catechin/epicatechin oligomers, that is, of procyanidins P 2 and P 3, was observed for the first time. Finally, an optimized extraction and separation system was used for qualitative and quantitative investigations of compounds from different green tea samples from Ceylon (cultivated under biologically controlled conditions), Japan, India, and China as well as from one black tea sample from India.

Au-Nanomaterials as a Superior Choice for Near-Infrared Photothermal Therapy

Photothermal therapy (PPT) is a platform to fight cancer by using multiplexed interactive plasmonic nanomaterials as probes in combination with the excellent therapeutic performance of near-infrared (NIR) light. With recent rapid developments in optics and nanotechnology, plasmonic materials have potential in cancer diagnosis and treatment, but there are some concerns regarding their clinical use. The primary concerns include the design of plasmonic nanomaterials which are taken up by the tissues, perform their function and then clear out from the body. Gold nanoparticles (Au NPs) can be developed in different morphologies and functionalized to assist the photothermal therapy in a way that they have clinical value. This review outlines the diverse Au morphologies, their distinctive characteristics, concerns and limitations to provide an idea of the requirements in the field of NIR-based therapeutics.

Characterization of normal and malignant prostate tissue by Fourier transform infrared microspectroscopy

Prostate cancer has become one of the most common malignancies worldwide. Morphological and histomorphological evaluation of this disease is a well established technique for the cancer classification and has remained relatively unchanged since several decades, although it remains a time consuming and subjective technique, with unsatisfactory levels of inter- and intra-observer discrepancy. Novel approaches for histological recognition are necessary to identify and to investigate cancer in detail. Fourier transform infrared (FTIR) spectroscopic imaging has become an essential tool for the detection, identification and characterization of the molecular components of biological processes, such as those responsible for the dynamic properties of cancer progression. Major advantage of this new technique is the acquisition of local molecular expression profiles while maintaining the topographic integrity of the tissue and avoiding time-consuming extraction, purification and separation steps. By using this method it is possible to investigate the spatial distribution of proteins, lipids, carbohydrates, cholesterols, nucleic acids, phospholipids and small molecules within biological systems by in situ analysis of tissue sections. We applied this technique on prostate cancer patients radical prostatectomy specimens in order to develop new tools for histomorphological analysis and the characterization of snap frozen prostate cancer tissues. As a first step, an optimization of sample preparation, tissue section thickness and IR slide material was performed. Special preparation methods for FTIR imaging are the essential requirements to maintain the spatial arrangement of compounds and avoid delocalization and degradation of the analytes. Subsequently, selected cancer samples were characterized with the prior optimized parameters and analyzed by univariate and cluster analysis. For the interpretation and calibration of the system we correlated the FTIR-images with the histopathological information. With this method it is possible to distinguish between cancer and noncancer areas within a prostate cancer tissue with a resolution of 6.25 μm × 6.25 μm on frozen sections.

Functionalized diamond nanopowder for phosphopeptides enrichment from complex biological fluids.

Diamond is known for its high affinity and biocompatibility towards biomolecules and is used exclusively in separation sciences and life science research. In present study, diamond nanopowder is derivatized as Immobilized Metal Ion Affinity Chromatographic (IMAC) material for the phosphopeptides enrichment and as Reversed Phase (C-18) media for the desalting of complex mixtures and human serum profiling through MALDI-TOF-MS. Functionalized diamond nanopowder is characterized by Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) spectroscopy. Diamond-IMAC is applied to the standard protein (β-casein), spiked human serum, egg yolk and non-fat milk for the phosphopeptides enrichment. Results show the selectivity of synthesized IMAC-diamond immobilized with Fe(3+) and La(3+) ions. To comprehend the elaborated use, diamond-IMAC is also applied to the serum samples from gall bladder carcinoma for the potential biomarkers. Database search is carried out by the Mascot program (www.matrixscience.com) for the assignment of phosphorylation sites. Diamond nanopowder is thus a separation media with multifunctional use and can be applied to cancer protein profiling for the diagnosis and biomarker identification.