Search

Haemophagocytic syndrome, diffuse alveolar haemorrhage, catastrophic antiphospholipid syndrome and various types of thrombotic microangiopathies are rare conditions with significant morbidity and mortality. A common feature is late diagnosis, which can affect the success of treatment. The aim of this review article is to summarize the basic diagnostic and therapeutic steps of the present subpopulation of critically ill patients.

Metabolic changes driven by the hostile tumor microenvironment surrounding cancer cells and the effect of these changes on tumorigenesis and metastatic potential have been known for a long time. The usual point of interest is glucose and changes in its utilization by cancer cells, mainly in the form of the Warburg effect. However, amino acids, both intra- and extracellular, also represent an important aspect of tumour microenvironment, which can have a significant effect on cancer cell metabolism and overall development of the tumor. Namely, alterations in the metabolism of amino acids glutamine, sarcosine, aspartate, methionine and cysteine have been previously connected to the tumor progression and aggressivity of cancer. : The aim of this review is to pinpoint current gaps in our knowledge of the role of amino acids as a part of the tumor microenvironment and to show the effect of various amino acids on cancer cell metabolism and metastatic potential. This review shows limitations and exceptions from the traditionally accepted model of Warburg effect in some cancer tissues, with the emphasis on prostate cancer, because the traditional definition of Warburg effect as a metabolic switch to aerobic glycolysis does not always apply. Prostatic tissue both in a healthy and transformed state significantly differs in many metabolic aspects, including the metabolisms of glucose and amino acids, from the metabolism of other tissues. Findings from different tissues are, therefore, not always interchangeable and have to be taken into account during experimentation modifying the environment of tumor tissue by amino acid supplementation or depletion, which could potentially serve as a new therapeutic approach.

Background: Squamous cell carcinoma of the oral cavity is generally caused by the long-term impact of known risk factors, e.g. tobacco and alcohol, along with chronic traumatisation. A number of studies now implicate HPV infection in head and neck tumour carcinogenesis but the exact role of HPV infection in the oral cavity remains unclear. Methods: In this study, we evaluated 78 patients with oral squamous cell carcinoma (OSCC) for the expression of protein p16 in the context of HPV positivity and its influence on the overall survival rate, disease location, staging and grading. Results: Regarding the tumour location, no significant difference was found between HPV-positive and HPV-negative patients, nor between p16-positive and p16-negative patients. There was also no trend in terms of HPV status and stage, and differentiation of carcinoma. There was no effect on HPV-positive patients relative to the time to progression (P=0.84) and overall survival rate (P=0.78). P16 positivity was not found to have an effect on the overall survival rate of patients (P=0.41) and there was no correlation between p16 positivity relative to the time to progression (P=0.66). Conclusions: In summary, the data suggest that there is no effect of HPV status on the prognosis of OSCC patients compared to other HNSCC locations.

We investigate the potential of Brillouin Light Scattering (BLS) Microspectroscopy for fast non-invasive all-optical assessment of the mechanical properties of viscose fibers and bleached softwood pulp. Using an optimized Brillouin spectrometer, we demonstrate fast spatial mapping of the complex longitudinal modulus over extended areas (> 100 µm). Our results reveal that while the softwood pulp has a relatively uniform moduli, the viscous fibers have significant spatial heterogeneous in the moduli. Specifically, the viscose fibers exhibited a regular pattern of increasing and decreasing modulus normal to the fiber axis. The potential influence of a locally changing refractive index is investigated by holographic phase microscopy and ruled out. We discuss our results in light of the anisotropic mechanical properties of the fibers and are able to estimate the relative difference between the modulus along the fiber axis and that perpendicular to it. Results are presented alongside reference measurements of the quasi-static mechanical properties transverse to the fiber axes obtained using AFM-nanoindentation which reveal a similar trend, hinting at the potential usefulness of BLS for mechanical characterization applications. However, more detailed investigations are called for to uncover all the factors influencing the measured high-frequency BLS modulus and its significance in relation to physical properties of the fiber that may be of practical interest.

Head and neck squamous cell carcinomas (HNSCC) belong among severe and highly complex malignant diseases showing a high level of heterogeneity and consequently also a variance in therapeutic response, regardless of clinical stage. Our study implies that the progression of HNSCC may be supported by cancer-associated fibroblasts (CAFs) in the tumour microenvironment (TME) and the heterogeneity of this disease may lie in the level of cooperation between CAFs and epithelial cancer cells, as communication between CAFs and epithelial cancer cells seems to be a key factor for the sustained growth of the tumour mass. In this study, we investigated how CAFs derived from tumours of different mRNA subtypes influence the proliferation of cancer cells and their metabolic and biomechanical reprogramming. We also investigated the clinicopathological significance of the expression of these metabolism-related genes in tissue samples of HNSCC patients to identify a possible gene signature typical for HNSCC progression. We found that the right kind of cooperation between cancer cells and CAFs is needed for tumour growth and progression, and only specific mRNA subtypes can support the growth of primary cancer cells or metastases. Specifically, during coculture, cancer cell colony supporting effect and effect of CAFs on cell stiffness of cancer cells are driven by the mRNA subtype of the tumour from which the CAFs are derived. The degree of colony-forming support is reflected in cancer cell glycolysis levels and lactate shuttle-related transporters.

Cell viscoelastic properties are affected by the cell cycle, differentiation, pathological processes such as malignant transformation. Therefore, evaluation of the mechanical properties of the cells proved to be an approach to obtaining information on the functional state of the cells. Most of the currently used methods for cell mechanophenotypisation are limited by low robustness or the need for highly expert operation. In this paper, the system and method for viscoelasticity measurement using shear stress induction by fluid flow is described and tested. Quantitative Phase Imaging (QPI) is used for image acquisition because this technique enables to quantify optical path length delays introduced by the sample, thus providing a label-free objective measure of morphology and dynamics. Viscosity and elasticity determination were refined using a new approach based on the linear system model and parametric deconvolution. The proposed method allows high-throughput measurements during live cell experiments and even through a time-lapse, where we demonstrated the possibility of simultaneous extraction of shear modulus, viscosity, cell morphology, and QPI-derived cell parameters like circularity or cell mass. Additionally, the proposed method provides a simple approach to measure cell refractive index with the same setup, which is required for reliable cell height measurement with QPI, an essential parameter for viscoelasticity calculation. Reliability of the proposed viscoelasticity measurement system was tested in several experiments including cell types of different Young/shear modulus and treatment with cytochalasin D or docetaxel, and an agreement with atomic force microscopy was observed. The applicability of the proposed approach was also confirmed by a time-lapse experiment with cytochalasin D washout, where an increase of stiffness corresponded to actin repolymerisation in time.SIGNIFICANCEWe present an approach for viscoelasticity measurement using QPI and shear stress induction by fluid flow. Our system builds and extends a recently published approach by parametric deconvolution, which allows us to eliminate the influence of the fluidic system and reliably measure both the shear modulus and viscosity of the cells in high throughput. Additionally, the proposed method enables to simultaneously determine cell refractive index map, cell dry mass map, and morphology, thereby enabling a multimodal cellular characterisation in a single measurement.

Background Within the tumour microenvironment, tumour cells are exposed to different mechanical stimuli such as compression stress, cell-cell and cell-extracellular matrix traction forces, interstitial fluid pressure, and shear stress. Cells actively sense and process this information by the mechanism of mechanotransduction to make decisions about their growth, motility, and differentiation. Indeed, the mechanical properties of the tumour microenvironment can deeply influence the behaviour of cancer cells and promote cancerogenesis. During tumour progression, desmoplasia arises and a positive feedback loop between the stiffening extracellular matrix and the properties enabling tumour expansion is established. Tumour cells can use mechanic stimuli to promote proliferation, increase their migratory and invasive potential, and induce therapeutic resistance. Mechanobio-logy is a progressive multidisciplinary field which studies how mechanical forces influence the behaviour of cells or tissues and may provide some interesting targets for cancer therapy. Purpose In this review, we discuss the mechanical properties of cancer cells and describe the tumour promoting effect of the transformed extracellular matrix. We propose that the differences in the mechanobio-logy of cells and extracellular matrix are significant enough to facilitate tumorigenesis and may provide interesting targets for cancer therapy.

In this paper, a novel U-Net-based method for robust adherent cell segmentation for quantitative phase microscopy image is designed and optimised. We designed and evaluated four specific post-processing pipelines. To increase the transferability to different cell types, non-deep learning transfer with adjustable parameters is used in the post-processing step. Additionally, we proposed a self-supervised pretraining technique using nonlabelled data, which is trained to reconstruct multiple image distortions and improved the segmentation performance from 0.67 to 0.70 of object-wise intersection over union. Moreover, we publish a new dataset of manually labelled images suitable for this task together with the unlabelled data for self-supervised pretraining.

In this paper, U-Net-based method for robust adherent cell segmentation for quantitative phase microscopy image is designed and optimised. We designed and evaluated four specific post-processing pipelines. To increase the transferability to different cell types, non-deep learning transfer with adjustable parameters is used in the post-processing step. Additionally, we proposed a self-supervised pretraining technique using nonlabelled data, which is trained to reconstruct multiple image distortions and improved the segmentation performance by from 0.67 to 0.70 of Object-wise Intersection over Union. Moreover, we publish a new dataset of manually labelled images suitable for this task together with the unlabelled data for self-supervised pretraining.Graphical AbstractHighlightsFour strategies for instance cell segmentation with U-Net were compared.Specialised post-processing pipelines with tunable/optimizable parameters were designed for each segmentation strategy.Transferability to different cell types by optimisation of post-processing parameters was tested.The proposed self-supervised pretraining method improved both segmentation performance and transferability to different cell types.A new manually labelled quantitative phase imaging dataset for cell segmentation with unlabelled data for self-supervised pretraining was created.

In this contribution, we focused on optimising a dynamic flow-based shear stress system to achieve a reliable platform for cell shear modulus (stiffness) and viscosity assessment using quantitative phase imaging. The estimation of cell viscoelastic properties is influenced by distortion of the shear stress waveform, which is caused by the properties of the flow system components (i.e., syringe, flow chamber and tubing). We observed that these components have a significant influence on the measured cell viscoelastic characteristics. To suppress this effect, we applied a correction method utilizing parametric deconvolution of the flow system’s optimized impulse response. Achieved results were compared with the direct fitting of the Kelvin-Voigt viscoelastic model and the basic steady-state model. The results showed that our novel parametric deconvolution approach is more robust and provides a more reliable estimation of viscosity with respect to changes in the syringe’s compliance compared to Kelvin-Voigt model.

In order to maximize post-therapeutic quality of life, radio(chemo)therapy becomes preferred over surgery in head-and-neck tumor (HNT) treatment. However, the therapy selection is only based on the clinical experience and patient#39;s preferences as the radiosensitivity markers remain unknown. New possibilities of deciding on the best primary therapy, moving us towards personalized medicine based on quantifiable biomarkers, have been opened by studies on DNA radiation damage and repair in individual patients tumors. Together with the importance of radiotherapy in HNT oncology, we discuss here our preliminary results revealing the existence of several HNT groups with respect to genome stability and repair ability of tumor cells after irradiation. Monitoring of the formation and disappearance of#947;H2AX/53BP1 foci in tumor cell primo-cultures derived from individual patients suggests that DNA repair capacity of the identified groups correlates with the tumor cell radiosensitivity. Our findings thus improve understanding of HNT biology; nevertheless, the relationship between the repair groups and in vivo response of tumors to radiotherapy must be further studied. Since most HNTs do not suffer from repair defects, although their viability varies after irradiation, pre-therapeutic tests covering the full spectrum of HNT radiosensitivity causes will require the use of a combination of multiple, still undiscovered biomarkers.

DNA double-strand breaks, marked by Ionizing Radiation-Induced (Repair) Foci (IRIF), are the most serious DNA lesions, dangerous to human health. IRIF quantification based on confocal microscopy represents the most sensitive and gold standard method in radiation biodosimetry and allows research of DSB induction and repair at the molecular and a single cell level. In this study, we introduce DeepFoci - a deep learning-based fully-automatic method for IRIF counting and its morphometric analysis. DeepFoci is designed to work with 3D multichannel data (trained for 53BP1 and γH2AX) and uses U-Net for the nucleus segmentation and IRIF detection, together with maximally stable extremal region-based IRIF segmentation.The proposed method was trained and tested on challenging datasets consisting of mixtures of non-irradiated and irradiated cells of different types and IRIF characteristics - permanent cell lines (NHDF, U-87) and cell primary cultures prepared from tumors and adjacent normal tissues of head and neck cancer patients. The cells were dosed with 1-4 Gy gamma-rays and fixed at multiple (0-24 h) post-irradiation times. Upon all circumstances, DeepFoci was able to quantify the number of IRIF foci with the highest accuracy among current advanced algorithms. Moreover, while the detection error of DeepFoci remained comparable to the variability between two experienced experts, the software kept its sensitivity and fidelity across dramatically different IRIF counts per nucleus. In addition, information was extracted on IRIF 3D morphometric features and repair protein colocalization within IRIFs. This allowed multiparameter IRIF categorization, thereby refining the analysis of DSB repair processes and classification of patient tumors with a potential to identify specific cell subclones.The developed software improves IRIF quantification for various practical applications (radiotherapy monitoring, biodosimetry, etc.) and opens the door to an advanced DSB focus analysis and, in turn, a better understanding of (radiation) DNA damaging and repair.HighlightsNew method for DSB repair focus (IRIF) detection and multi-parameter analysisTrainable deep learning-based methodFully automated analysis of multichannel 3D datasetsTrained and tested on extremely challenging datasets (tumor primary cultures)Comparable to an expert analysis and superb to available methodsGraphical Abstract

Expansins facilitate cell expansion via mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in the control of biomechanical CW properties in the tissue and organ context remains elusive. We determined hormonal responsiveness and specificity of expression and localization of expansins predicted to be direct targets of cytokinin signalling. We found EXPA1 homogenously distributed throughout the CW of columella/ lateral root cap, while EXPA10 and EXPA14 localized predominantly at the three-cell boundaries of epidermis/cortex in various root zones. Cell type-specific localization of EXPA15 overlaps with higher CW stiffness measured via Brillouin light scattering microscopy. As indicated by both Brillouin frequency shift and AFM-measured Young’s modulus,EXPA1overexpression upregulated CW stiffness, associated with shortening of the root apical meristem and root growth arrest. We propose that root growth inArabidopsisrequires delicate orchestration of biomechanical CW properties via tight regulation of various expansins’ localization to specific cell types and extracellular domains.

Background Despite progress in anticancer therapies, head and neck squamous cell carcinoma (HNSCC) has still a low survival rate. Recent studies have shown that tumour stroma may play an important role in the pathogenesis of this malignant disease. Fibroblasts are a major component of the tumour microenvironment and may significantly influence HNSCC progression as indicated by the contribution they make to important hallmarks of cancer, such as inflammation, non-restricted growth, angiogenesis, invasion, metastasis, and therapy resistance. It is well known that tumour cells can confer a cancer-associated fibroblast (CAF) phenotype that supports the growth and dissemination of cancer cells. CAFs can stimulate cancer progression through cell-cell contacts and communication, remodelling of extracellular matrix, and production of many signal molecules and matrix metalloproteinases. Consequently, genetic changes in epithelial cells are probably not the only factor that drives HNSCC carcinogenesis. Non-genetic changes in the tumour stroma can also be significantly involved. Stress-induced signals can induce a multicellular program, creating a field of tissue that is predisposed to malignant transformation. The “field cancerization” concept represents a process of active evolution of intercellular interactions and feedback loops between tumour and stromal cells. This model paves the way to study cancer from a new perspective and identify new therapeutic targets. Purpose In this review, we discuss current knowledge about CAFs, such as their cellular origin, phenotypical plasticity and functional heterogeneity, and stress their contribution to HNSCC progression. This article was supported by the project AZV 16-29835A. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 18. 6. 2019 Accepted: 9. 9. 2019.

To date, there has been no evidence regarding the association between urinary sarcosine content and prostate cancer survival. Our main objective was to investigate whether levels of post‐treatment urinary sarcosine are associated with relapse. The inclusion criteria were (in accordance with EAU 2017) as follows: histopathologically verified adenocarcinoma in prostate biopsy cores or specimens from transurethral resection of the prostate (TURP) or prostatectomy for benign prostatic enlargement (BPE) with retained ability to urinate. The median follow‐up was 53 months. In the study, we retrospectively evaluated a cohort of 511 patients with prostate cancer with various risk factors and treatment strategies. Post‐treatment sarcosine levels were elevated in 266 (52%) patients and highly elevated (≥200 nmol/L) in 71 (13%) patients. Urinary sarcosine content was significantly associated with number of relapses that patients experienced, P = 0.002 for sarcosine ≥200 vs ≤30 nmol/L. Multivariate analysis revealed that sarcosine was an independent predictor of recurrent relapses (≥2 relapses with an intermediate period of remission), HR = 3.89 (95% CI 1.29‐11.7) for sarcosine >200 vs

Background: Anthracycline antibiotic doxorubicin (DOX) is a very potent and extensively prescribed chemotherapeutic drug. It is widely utilized in the therapy of variety of haematological and solid tumours, although its administration is commonly accompanied with several severe side effects. The most serious one is a development of dose-dependent and cumulative cardiotoxicity. In the course of time, many strategies have been investigated in order to avoid or at least to diminish DOX-induced cardiac dysfunction; these include reduction of toxic effect by co-administration with iron chelators (dexrazoxane), trastuzumab, taxanes, statins, and ACE-inhibitors. However, the attenuation of cardiotoxic effect is still not satisfactory yet. Objective: This review represents an overall appraisal of studies concerning with the utilization of various doxorubicin-loaded nanoparticles in the cancer treatment with specific emphasis on those studies evaluating their influence on the reduction of heart tissue damage. Conclusion: Introduction of nanoscale drug delivery systems undoubtedly represents nowadays one of the most promising tools for lowering systemic toxicity. Nanoparticles enable to target the therapeutic payload directly towards the tumor tissue, thus leading to the increased accumulation of the drug in the desired tissue and simultaneously protecting surrounding healthy tissues.

BACKGROUND: Failure in intracellular zinc accumulation is a key process in prostate carcinogenesis. Nevertheless, epidemiological studies of zinc administration have provided contradicting results. In order to examine the impact of the artificial intracellular increase of zinc(II) ions on prostate cancer metabolism, PNT1A, 22Rv1, and PC-3 prostatic cell lines—depicting different stages of cancer progression—and their zinc-resistant counterparts were used. To determine “benign” and “malignant” metabolic profiles, amino acid patterns, gene expression, and antioxidant capacity of these cell lines were assessed. METHODS: Amino acid profiles were examined using an ion-exchange liquid chromatography. Intracellular zinc content was measured by atomic absorption spectrometry. Metallothionein was quantified using differential pulse voltammetry. The content of reduced glutathione was determined using high performance liquid chromatography coupled with an electrochemical detector. Cellular antioxidant capacity was determined by the ABTS test and gene expression analysis was performed by qRT-PCR. RESULTS AND CONCLUSIONS: Long-term zinc treatment was shown to reroute cell metabolism from benign to more malignant type. Long-term application of high concentration of zinc(II) significantly enhanced cisplatin resistance, invasiveness, cellular antioxidant capacity, synthesis of glutathione, and expression of treatment resistance- and stemness-associated genes (SOX2, POU5F1, BIRC5). Tumorous cell lines universally displayed high accumulation of aspartate and sarcosine and depletion of essential amino acids. Increased aspartate/threonine, aspartate/methionine, and sarcosine/serine ratios were associated with cancer phenotype with high levels of sensitivity and specificity.

Metabolic phenotypes of cancer cells are heterogeneous and flexible as a tumor mass is a hurriedly evolving system capable of constant adaptation to oxygen and nutrient availability. The exact type of cancer metabolism arises from the combined effects of factors intrinsic to the cancer cells and factors proposed by the tumor microenvironment. As a result, a condition termed oncogenic metabolic symbiosis in which components of the tumor microenvironment (TME) promote tumor growth often occurs. Understanding how oncogenic metabolic symbiosis emerges and evolves is crucial for perceiving tumorigenesis. The process by which tumor cells reprogram their TME involves many mechanisms, including changes in intercellular communication, alterations in metabolic phenotypes of TME cells, and rearrangement of the extracellular matrix. It is possible that one molecule with a pleiotropic effect such as Caveolin-1 may affect many of these pathways. Here, we discuss the significance of Caveolin-1 in establishing metabolic symbiosis in TME.

Cisplatin is a widely used chemotherapeutic agent that is clinically approved to fight both carcinomas and sarcomas. It has relatively high efficiency in treating ovarian cancers and metastatic testicular cancers. It is generally accepted that the major mechanism of cisplatin anti-cancer action is DNA damage. However, cisplatin is also effective in metastatic cancers and should, therefore, affect slow-cycling cancer stem cells in some way. In this review, we focused on the alternative effects of cisplatin that can support a good therapeutic response. First, attention was paid to the effects of cisplatin at the cellular level such as changes in intracellular pH and cellular mechanical properties. Alternative cellular targets of cisplatin, and the effects of cisplatin on cancer cell metabolism and ER stress were also discussed. Furthermore, the impacts of cisplatin on the tumor microenvironment and in the whole organism context were reviewed. In this review, we try to reveal possible causes of the unexpected effectiveness of this anti-cancer drug.

Cell viability and cytotoxicity assays are highly important for drug screening and cytotoxicity tests of antineoplastic or other therapeutic drugs. Even though biochemical-based tests are very helpful to obtain preliminary preview, their results should be confirmed by methods based on direct cell death assessment. In this study, time-dependent changes in quantitative phase-based parameters during cell death were determined and methodology useable for rapid and label-free assessment of direct cell death was introduced. Our method utilizes Quantitative Phase Imaging (QPI) which enables the time-lapse observation of subtle changes in cell mass distribution. According to our results, morphological and dynamical features extracted from QPI micrographs are suitable for cell death detection (76% accuracy in comparison with manual annotation). Furthermore, based on QPI data alone and machine learning, we were able to classify typical dynamical changes of cell morphology during both caspase 3,7-dependent and independent cell death subroutines. The main parameters used for label-free detection of these cell death modalities were cell density (pg/pixel) and average intensity change of cell pixels further designated as Cell Dynamic Score (CDS). To the best of our knowledge, this is the first study introducing CDS and cell density as a parameter typical for individual cell death subroutines with prediction accuracy 75.4 % for caspase 3,7-dependent and -independent cell death.

We focused on the biomechanical and morphological characteristics of prostate cancer cells and their changes resulting from the effect of docetaxel, cisplatin, and long-term zinc supplementation. Cell population surviving the treatment was characterized as follows: cell stiffness was assessed by atomic force microscopy, cell motility and invasion capacity were determined by colony forming assay, wound healing assay, coherence-controlled holographic microscopy, and real-time cell analysis. Cells of metastatic origin exhibited lower height than cells derived from the primary tumour. Cell dry mass and CAV1 gene expression followed similar trends as cell stiffness. Docetaxel- and cisplatin-surviving cells had higher stiffness, and decreased motility and invasive potential as compared to non-treated cells. This effect was not observed in zinc(II)-treated cells. We presume that cell stiffness changes may represent an important overlooked effect of cisplatin-based anti-cancer drugs. Atomic force microscopy and confocal microscopy data images used in our study are available for download in the Zenodo repository (https://zenodo.org/, Digital Object Identifiers:10.5281/zenodo.1494935).

Metals are known for playing essential roles in human physiology. Copper and zinc are trace elements closely dependent on one another and are involved in cell proliferation, growth, gene expression, apoptosis and other processes. Their homeostasis is crucial and tightly controlled by a resourceful system of transporters and transport proteins which deliver copper and zinc ions to their target sites. Abnormal zinc and copper homeostasis can be seen in a number of malignancies and also in head and neck cancer. Imbalance in this homeostasis is observed as an elevation or decrease of copper and zinc ions in serum or tissue levels in patients with cancer. In head and neck cancer these altered levels stand out from those of other malignancies which makes them an object of interest and therefore zinc and copper ions might be a good target for further research of head and neck cancer development and progression. This review aims to summarize the physiological roles of copper and zinc, its binding and transport mechanisms, and based on those, its role in head and neck cancer. To provide stronger evidence, dysregulation of levels is analysed by a meta-analytical approach.

BACKGROUND Sarcosine (N-methylglycine) was previously delineated as a substantial oncometabolite of prostate cancer (PCa) and its metabolism seems to be significantly involved in PCa development and behavior. METHODS We focused on investigation whether the exposure of prostate cells (PNT1A, 22Rv1, and PC-3) to sarcosine-related amino acids (glycine, dimethylglycine, and sarcosine) affects their aggressiveness (cell mobility and division rates, using real-time cell based assay). The effect of supplementation on expression of glycine-N-methyltransferase (GNMT) mRNA was examined using qRT-PCR. Finally, post-treatment amino acids patterns were determined with consequent statistical processing using the Ward's method, factorial ANOVA and principal component analysis (P

Aim: New ischemic lesions in the brain can be detected in approximately 50% of patients undergoing carotid artery stenting (CAS). We wished to discover the laboratory-based predictors of new infarctions in the brain after CAS. Methods: All consecutive patients with internal carotid artery stenosis of ≥ 70% with indication for CAS were enrolled in a prospective study for 16 months. All patients used dual antiplatelet therapy for ≥ 7 days before CAS. Neurologic examination and magnetic resonance imaging (MRI) of the brain were undertaken before and at 24 h after CAS. Samples of venous blood were collected at < 24 h before CAS for the evaluation of hematology, reticulocytes, coagulation markers (PT, APTT, Fbg, Clauss), vWF antigen, PAI-1 activity, PAI-1 polymorphism 4G/5G, and the multiplate (aspirin and clopidogrel) resistance test. Blood samples for the assessment of anti-Xa activity were collected during CAS. Differences in the values of laboratory markers between patients with and without new ischemic lesions of the brain on control MRI were evaluated. Results: The cohort comprised 81 patients (53 males; mean age, 67.3 ± 7.2 years). New ischemic infarctions in the brain on control MRI were found in 46 (56.8%) patients. Three of seven patients with resistance to aspirin or clopidogrel had a new ischemic infarction in the brain. No significant differences for particular markers were found between patients with and without an ischemic lesion in the brain. Conclusion: A high risk of a new ischemic infarction in the brain was detected in patients undergoing CAS, but a laboratory-based predictor of such an infarction could not be identified.

OBJECTIVES: We aimed on effect of supernatant derived from prostate cancer cell line PC-3 on M1/M2 functional polarization in macrophages. BACKGROUND: Cytokines play an important role in carcinogenesis. Most of them are produced by macrophages. Macrophages are divided into groups M1 or M2. Classical phenotype macrophages M1 support pro-inflammatory effects and produce pro-inflammatory cytokines, such as interleukin 6 (IL-6), interleukin 12 (IL-12), tumor necrosis factor alpha (TNF-alpha). Macrophages exhibiting a phenotype M2 secrete anti-inflammatory cytokines, e.g. interleukin 10 (IL-10), transforming growth factor beta (TGF-beta). METHODS: Peripheral blood monocytes were cultivated for 7 days and during this time went through a differentiation into macrophages. Macrophages were stimulated for 24 hours by lipopolysaccharide (LPS) as a positive control and cultivated with supernatant for another 24 hours. RESULTS: Macrophages cultivated without LPS and without supernatant were used as negative control. Relative expression of IL-6, IL-10, IL-12 and TNF-alpha was measured by Quantitative real-time PCR. Expression of pro-inflammatory cytokines was lower in macrophages with supernatant compared to positive control. CONCLUSION: Expression of pro-inflammatory cytokines was lower in macrophages with supernatant (M phi+sup) compared to positive control (M phi+LPS). Effect of the supernatant on expression of IL-6, IL-10, IL-12 and TNF-alpha was not confirmed (Tab. 1, Fig. 5, Ref. 15). Text in PDF www.elis.sk.

This meta-analysis aims to evaluate the effects of high c-Met levels in head and neck squamous cell carcinomas (HNSCC) on survival and clinicopathological features. Publications concerned with the clinical significance of c-Met protein expression in HNSCC were identified from the Scopus and Web of Science database searches. To elucidate the relationship between c-Met expression and clinical outcomes, a meta-analysis of the selected articles was conducted. Seventeen publications involving a total of 1724 patients met the inclusion criteria. c-Met overexpression was significantly correlated with poor overall survival (hazard ratio (HR) = 2.19, 95% confidence interval (CI) = 1.55–3.10). c-Met immunohistochemical staining positivity was also associated with worse relapse-free survival (HR = 1.64, 95% CI = 1.24–2.17) and presence of regional lymph node metastases (odds ratio (OR) = 1.76, 95% CI = 1.26–2.45). High levels of c-Met expression in HNSCC predict unfavorable prognosis associated with common clinicopathological features.

Fluorescence microscopy is a golden standard for contemporary biological studies. However, since fluorescent dyes cross-react with biological processes, a label-free approach is more desirable. The aim of this study is to create artificial, fluorescence-like nuclei labeling from label-free images using Convolution Neural Network (CNN), where training data are easy to obtain if simultaneous label-free and fluorescence acquisition is available. This approach was tested on holographic microscopic image set of prostate non-tumor tissue (PNT1A) and metastatic tumor tissue (DU145) cells. SegNet and U-Net were tested and provide “synthetic” fluorescence staining, which are qualitatively sufficient for further analysis. Improvement was achieved with addition of bright-field image (by-product of holographic quantitative phase imaging) into analysis and two step learning approach, without and with augmentation, were introduced. Reconstructed staining was used for nucleus segmentation where 0.784 and 0.781 dice coefficient (for DU145 and PNT1A) were achieved.

Approximately 90 % of head and neck cancers are squamous cell carcinomas (HNSCC), and the overall 5-year survival rate is not higher than 50 %. There is much evidence that human papillomavirus (HPV) infection may influence the expression of commonly studied HNSCC markers. Our study was focused on the possible HPV-specificity of molecular markers that could be key players in important steps of cancerogenesis (MKI67, EGF, EGFR, BCL-2, BAX, FOS, JUN, TP53, MT1A, MT2A, VEGFA, FLT1, MMP2, MMP9, and POU5F). qRT-PCR analysis of these selected genes was performed on 74 biopsy samples of tumors from patients with histologically verified HNSCC (22 HPV-, 52 HPV+). Kaplan-Meier analysis was done to determine the relevance of these selected markers for HNSCC prognosis. In conclusion, our study confirms the impact of HPV infection on commonly studied HNSCC markers MT2A, MMP9, FLT1, VEGFA, and POU5F that were more highly expressed in HPV-negative HNSCC patients and also shows the relevance of studied markers in HPV-positive and HPV-negative HNSCC patients.

Even with significant advances in operative skills and adjuvant therapies, the overall survival of patients suffering with head and neck squamous cancers (HNSCC) is unsatisfactory. Accordingly, no clinically useful prognostic biomarkers have been found yet for HNSCC. Many studies analysed the expression of potential markers in tumour tissues compared to adjacent tissues. Nevertheless, due to the sharing of the same microenvironment, adjacent tissues show molecular similarity to tumour tissues. Thus, gene expression patterns of 94 HNSCC tumorous tissues were compared with 31 adjacent tissues and with 10 tonsillectomy specimens of non-cancer individuals. The genes analysed at RNA level using quantitative RT-PCR and correlated with clinico-pathological conditions were as follows: EGF, EGFR, MKI67, BCL2, BAX, FOS, JUN, TP53, VEGF, FLT1, MMP2, MMP9, MT1A and MT2A. The elevated MT2A, BAX, EGF and JUN expression was associated with the influence of tumour cells on the rearrangement of healthy tissues, as well as a significant shift in the BAX/BCL2 ratio. Our investigation also indicated that adjacent tissues play an important role in cancerogenesis by releasing several tumour-supporting factors such as EGF. A gradual increase in the metallothionein expression, from the lowest one in tonsillectomy samples to the highest ones in tumour samples, suggests that MT expression might be tissue reaction to the presence of tumour cells. The results of this study confirmed the significance of metallothionein in tumori-genesis and gave evidences for its use as a potential HNSCC biomarker. Furthermore, this study highlighted the importance of histologically normal tumour-adjacent tissue in prediction of HNSCC progress.

Breast cancer patients negative for the nuclear oestrogen receptor α have a particularly poor prognosis. Therefore, the 4T1 cell line (considered as a triple-negative model) was chosen to induce malignancy in mice. The aim of the present study was to assess if zinc ions, provided in excess, may significantly modify the process of mammary oncogenesis. Zn(II) ions were chosen because of their docu- mented antitumour effects. Zn(II) is also known to induce the expression of metallothioneins (MT) and glutathion (GSH). A total dose of zinc sulphate per one gram of mouse weight used in the experiment was 0.15 mg. We studied the expression of MT1, MT2, TP53 and MTF-1 genes and also examined the effect of the tumour on antioxidant capacity. Tumour-free mice had significantly higher expression levels of the studied genes ( p

In order to improve patients’ post-treatment quality of life, a shift from surgery to non-surgical (chemo)radio-treatment is recognized in head and neck oncology. However, about half of HNSCC tumors are resistant to irradiation and an efficient marker of individual tumor radiosensitivity is still missing. We analyzed whether various parameters of DNA double strand break (DSB) repair determined in vitro can predict, prior to clinical treatment initiation, the radiosensitivity of tumors. We compared formation and decrease of γH2AX/53BP1 foci in 48 h after irradiating tumor cell primocultures with 2 Gy of γ-rays. To better understand complex tumor behavior, three different cell type primocultures – CD90−, CD90+, and a mixed culture of these cells – were isolated from 1 clinically radioresistant, 2 radiosensitive, and 4 undetermined HPV–HNSCC tumors and followed separately. While DSB repair was delayed and the number of persisting DSBs increased in the radiosensitive tumors, the results for the radioresistant tumor were similar to cultured normal human skin fibroblasts. Hence, DSB repair kinetics/efficiency may correlate with clinical response to radiotherapy for a subset of HNSCC tumors but the size (and therefore practical relevance) of this subset remains to be determined. The same is true for contribution of different cell type primocultures to tumor radioresistance.

Introduction Metallothioneins (MTs) are a class of ubiquitously occurring low-molecular-weight cysteine- and metal-rich proteins containing sulfur-based metal clusters. MT-3 exhibits neuro-inhibitory activity. The possibility to enhance the expression of MT-3 or protect it from degradation is an attractive therapeutic target, because low levels of MT-3 were found in brains of Alzheimer's disease (AD) patients. Objectives The primary objective of this study was to test an enhancement of MT-3 cellular concentration after MT-3 binding treatment, which could prevent MT-3 degradation. Methods MTT assay, flow-cytometry, fluorescence microscopy, quantitative real-time polymerase chain reaction, and immunodetection of MT3 were used for analysis of effect of STOCK1N-26544, STOCK1N-26929, and STOCK1N-72593 on immortalized human microglia-SV40 cell line. Results All three tested compounds enhanced concentration of MT-3 protein in cells and surprisingly also mRNA concentration. IC50 values of tested molecules exceeded about ten times the concentration that was needed for induction of MT-3 expression. The tested compound Benzothiazolone-2 enhanced apoptosis and necrosis, but it was not of severe effect. About 80% of cells were still viable. There was no serious ROS-generation and no severe decrease in mitochondria numbers or stress induced endoplasmic reticulum changes after test treatments. The selected compound showed stable hydrophobic and electrostatic interaction during MT-3 ligand interaction. Conclusion Benzothiazolone-2 compounds significantly enhanced MT-3 protein and mRNA levels. The compounds can be looked upon as one of the probable lead compounds for future drug designing experiments in the treatment of Alzheimer's disease.

Doxorubicin is an effective chemotherapeutic drug, however, its toxicity is a significant limitation in therapy. Encapsulation of doxorubicin inside liposomes or ferritin cages decreases cardiotoxicity while maintaining anticancer potency. We synthesized novel apoferritin- and liposome-encapsulated forms of doxorubicin (“Apodox” and “lip-8-dox”) and compared its toxicity with doxorubicin and Myocet on prostate cell lines. Three different prostatic cell lines PNT1A, 22Rv1, and LNCaP were chosen. The toxicity of the modified doxorubicin forms was compared to conventional doxorubicin using the MTT assay, real-time cell impedance-based cell growth method (RTCA), and flow cytometry. The efficiency of doxorubicin entrapment was 56% in apoferritin cages and 42% in the liposome carrier. The accuracy of the RTCA system was verified by flow-cytometric analysis of cell viability. The doxorubicin half maximal inhibition concentrations (IC50) were determined as 170.5, 234.0, and 169.0 nM for PNT1A, 22Rv1, and LNCaP, respectively by RTCA. Lip8-dox is less toxic on the non-tumor cell line PNT1A compared to doxorubicin, while still maintaining the toxicity to tumorous cell lines similar to doxorubicin or epirubicin (IC50 = 2076.7 nM for PNT1A vs. 935.3 and 729.0 nM for 22Rv1 and LNCaP). Apodox IC50 was determined as follows: 603.1, 1344.2, and 931.2 nM for PNT1A, 22Rv1, and LNCaP.

The present study suggests and describes the application of a delivery system for antisense oligonucleotides against mRNA encoding estrogen receptor proteins α and β. The delivery system is composed of a cationic liposome enve- lope containing 17β-estradiol (E2) in its structure. Cationic liposomes protect cargo against the extracellular matrix, and E2 can increase its shuttling efficiency into cells. Using MCF-7 cells derived from estrogen receptor-positive ductal carci- noma, treatment with liposomes against ERα was found to decrease MCF-7 proliferation, and importantly the application of both the antisense against ERα and β exhibited an antipro- liferative effect expressed as cell viability. Using qRT-PCR, it was shown that MT1A, NF-κB1 and K-ras genes, but not TFF1, were downregulated using E2-based liposomes (evaluated at P=0.05). Further indicators of oxidative stress were employed to assess the effect on treatment efficiency. Glutathione (GSH/ GSSG redox ratio), metallothionein (MT) and malondialde - hyde (MDA) confirmed a positive effect of antisense therapy resulting in their decreased levels in the MCF-7 cells. Based on these data, we suggest that E2-based liposomes offer sufficient transfer efficiency and moreover, due to the effect on NF-κB1, MT and GSH, tumor cells can be chemosensitized to increase treatment effectiveness.

Metallothioneins (MTs) are a class of metal-binding proteins characterized by a high cysteine content and low molecular weight. MTs play an important role in metal metabolism and protect cells against the toxic effects of radiation, alkylating agents and oxygen free radicals. The evidence that individual genetic characteristics of MTs play an important role in physiological and pathological processes associated with antioxidant defense and detoxification inspired targeted studies of genetic polymorphisms in a clinical context. In recent years, common MT polymorphisms were identified and associated with, particularly, western lifestyle diseases such as cancer, complications of atherosclerosis, and type 2 diabetes mellitus along with related complications. This review summarizes all evidence regarding MT polymorphisms of major human MTs (MT1, MT2, MT3 and MT4), their relation to pathological processes, and outlines specific applications of MTs as a set of genetic markers for certain pathologies.

Interactions of silver phosphate nanoparticles (SPNPs) and selenium nanoparticles (SeNPs) with Staphylococcus aureus cultures have been studied at the cellular, molecular and protein level. Significant antibacterial effects of both SPNPs and SeNPs on S. aureus were observed. At a concentration of 300 μM, SPNPs caused 37.5% inhibition of bacterial growth and SeNPs totally inhibited bacterial growth. As these effects might have been performed due to the interactions of nanoparticles with DNA and proteins, the interaction of SPNPs or SeNPs with the amplified zntR gene was studied. The presence of nanoparticles decreased the melting temperatures of the nanoparticle complexes with the zntR gene by 23% for SeNPs and by 12% for SPNPs in comparison with the control value. The concentration of bacterial metallothionein was 87% lower in bacteria after application of SPNPs (6.3 μg mg(-1) protein) but was increased by 29% after addition of SeNPs (63 μg mg(-1) protein) compared with the S. aureus control (49 μg mg(-1) protein). Significant antimicrobial effects of the nanoparticles on bacterial growth and DNA integrity provide a promising approach to reducing the risk of bacterial infections that cannot be controlled by the usual antibiotic treatments.

Differences in the antioxidant system, apoptotic mechanism and in cell cycle between prostatic cell lines could partially elucidate the development of cisplatin resistance. The aim of this study was to identify the most characteristic parameter for a particular cell line and/or a particular cisplatin treatment using a general regression model and to assess whether it is possible to use measured parameters as markers of cisplatin resistance. This study integrates the results of viability, antioxidant, flow cytometric and quantitative PCR assays in order to characterize the resistance of prostate cancer to cisplatin. Cell growth using metabolic- (MTT) and impedance-based assays, the expression of key cell death signaling proteins (p53, Bax and Bcl-2), cell cycle, activity of antioxidant system-related proteins (superoxide dismutase, glutathione peroxidase, glutathione reductase and metallothionein) and free radical scavenging capacity assays [free radicals (FR), ferric reducing antioxidant power (FRAP), ABTS] were analyzed in the cell lines 22Rv1, PC-3 and PNT1A with respect to rising concentrations (0-150 µM) and different length of cisplatin treatment (12-72 h). The non-functional-p53 PC-3 cell line showed decreased BAX (p0.05) and, in contrast to PNT1A and 22Rv1, no cisplatin-induced effects on cell cycle. All cell lines showed increasing levels of free radical scavenging activity by ABTS, FRAP and FR assays in a time- and dose-dependent manner (r0.76 at p0.001 for ABTS, FRAP and FR at p0.001). PC-3 showed increased (p0.05) levels of free radical scavenging activity by ABTS and FR methods. These findings, together with significantly elevated MT, decreased p53 and Bax indicate PC-3 to be cisplatin-resistant. The differences in the antioxidant system and apoptotic mechanisms in PC-3 cells may elucidate the development of cisplatin resistance and indicate that this cell line may be further studied as a model of cytostatic resistance.

Determination of serum mRNA gained a lot of attention in recent years, particularly from the perspective of disease markers. Streptavidin-modified paramagnetic particles (SMPs) seem an interesting technique, mainly due to possible automated isolation and high efficiency. The aim of this study was to optimize serum isolation protocol to reduce the consumption of chemicals and sample volume. The following factors were optimized: amounts of (i) paramagnetic particles, (ii) oligo(dT)20 probe, (iii) serum, and (iv) the binding sequence (SMPs, oligo(dT)20 , serum vs. oligo(dT)20 , serum and SMPs). RNA content was measured, and the expression of metallothionein-2A as possible prostate cancer marker was analyzed to demonstrate measurable RNA content with ability for RT-PCR detection. Isolation is possible on serum volume range (10-200 μL) without altering of efficiency or purity. Amount of SMPs can be reduced up to 5 μL, with optimal results within 10-30 μL SMPs. Volume of oligo(dT)20 does not affect efficiency, when used within 0.1-0.4 μL. This optimized protocol was also modified to fit needs of automated one-step single-tube analysis with identical efficiency compared to conventional setup. One-step analysis protocol is considered a promising simplification, making RNA isolation suitable for automatable process.

We analyzed 1156 multiple myeloma (MM) patients treated with thalidomide. The overall response rate was 63.6%, with complete remission in 13.4%. Combined regimens had better outcomes than thalidomide plus dexamethasone or single agent thalidomide. Thalidomide was not able to overcome adverse cytogenetics. Superior results were seen in patients undergoing subsequent autologous stem cell transplantation. The rate of adverse events was low. Thalidomide has a strong potential to improve response and survival measures in patients with standard risk MM. Combined regimens should be used, with lower doses of thalidomide. High risk myelomas should be treated individually.

Altered expression of microRNAs (miRNAs) has been shown in many types of malignancies including the head and neck squamous cell carcinoma (HNSCC). Although there are many new and innovative approaches in the treatment of HNSCC, a clear marker of this disease is still missing. Three candidate miRNAs (miR-29c-3p, miR-200b-5p and miR-375-3p) were studied in connection with HNSCC using quantitative real-time PCR expression levels in 42 tissue samples of HNSCC patients and histologically normal tumour-adjacent tissue samples of these patients. Primary HNSCC carcinoma tissues can be distinguished from histologically normal-matched noncancerous tumour-adjacent tissues based on hsa-miR-375-3p expression (sensitivity 87.5 %, specificity 65 %). Additionally, a significant decrease of hsa-miR-200b-5p expression was revealed in tumour-adjacent tissue samples of patients with node positivity. Lower expression of hsa-miR-200b-5p and hsa-miR-29c-3p in HNSCC tumour tissue was associated with higher tumour grade. Consequently, survival analysis was performed. Lower expression of hsa-miR-29c-3p in tumour-adjacent tissue was associated with worse overall and disease-specific survivals. Lower expression of miR-29c-3p in tumourous tissue was associated with worse relapse-free survival. hsa-miR-375-3p seems to be a relatively promising diagnostic marker in HNSCC but is not suitable for prognosis of patients. Furthermore, this study highlighted the importance of histologically normal tumour-adjacent tissue in HNSCC progress (significant decrease of hsa-miR-200b-5p expression in tumour-adjacent tissue of patients with node positivity and low expression of hsa-miR-29c-3p in HNSCC tumour-adjacent tissue associated with worse prognosis).

Metallothionein is significantly elevated in various tumors, notably in prostate cancer on both mRNA and protein level. We demonstrated a strong predictive potential of free circulating metallothionein 2A isoform mRNA for patients with this cancer. Circulating mRNA detection relies on expensive equipment and requires high level of expertise. In this work we developed compact "all-in-one" laboratory system which replace microvolume spectrophotometer, thermocycler and realtime PCR machines. We managed to design and construct a microprocessor controlled heating/cooling chamber that ensures required temperature gradient. The chamber includes implemented optical system to enable fluorescence excitation and fluorescence analysis using a smart-phone.

Herein, we present a study focused on the determination of the influence of long-distance (53 km) bicycle riding on levels of chosen biochemical urinary and serum prostate cancer (PCa) biomarkers total prostate-specific antigen (tPSA), free PSA (fPSA) and sarcosine. Fourteen healthy participants with no evidence of prostate diseases, in the age range from 49–57 years with a median of 52 years, underwent physical exercise (mean race time of 150 20 min, elevation increase of 472 m) and pre- and post-ride blood/urine sampling. It was found that bicycle riding resulted in elevated serum uric acid (p = 0.001, median 271.76 vs. 308.44 mol/L pre- and post-ride, respectively), lactate (p = 0.01, median 2.98 vs. 4.8 mmol/L) and C-reactive protein (p = 0.01, 0.0–0.01 mg/L). It is noteworthy that our work supports the studies demonstrating an increased PSA after mechanical manipulation of the prostate. The subjects exhibited either significantly higher post-ride tPSA (p = 0.002, median 0.69 vs. 1.1 ng/mL pre- and post-ride, respectively) and fPSA (p = 0.028, median 0.25 vs. 0.35 ng/mL). Contrary to that, sarcosine levels were not significantly affected by physical exercise (p = 0.20, median 1.64 vs. 1.92 mol/mL for serum sarcosine, and p = 0.15, median 0.02 mol/mmol of creatinine vs. 0.01 mol/mmol of creatinine for urinary sarcosine). Taken together, our pilot study provides the first evidence that the potential biomarker of PCa—sarcosine does not have a drawback by means of a bicycle riding-induced false positivity, as was shown in the case of PSA. Uvádíme zde studii zaměřenou na stanovení vlivu jízd na kole do delších vzádelností (53 km) na úrovni vybrané biochemické moči a séra rakoviny prostaty (DPS) biomarker prostatický specifický antigen (tPSA), volné PSA (fPSA) a sarcosine. Čtrnáct zdravých účastníků bez známek onemocnění prostaty, ve věkovém rozmezí od 49-57 let s mediánem 52 let, podstoupili fyzickou aktivituí (střední doba závodu 150 20 min, převýšení 472 m) a odběr vzorků krve/moči před a po jízdě. Bylo zjištěno, že jízdy na kole ústí ve zvýšené serum kyseliny močové (p = 0,001, medián 271.76 vs. 308.44 mol/L před a po jízdě, respektive), laktát (p = 0,01, medián 2,98 versus 4,8 mmol/L) a C - reaktivního proteinu (p = 0,01, 0,0 – 0,01 mg/L). Stojí za zmínku,že naši práci podporuje studie, které prokazují zvýšené PSA po mechanické manipulaci s prostatou. Subjekty byly vystaveny buď podstatně vyšší post- jízdě tPSA (p = 0,002, medián 0,69 vs. 1,1 ng/mL, před a po jízdě, respektive) a fPSA (p = 0.028, medián 0,25-0,35 ng/mL). Na rozdíl od toho, úroveň sarkosinu nebyla výrazně ovlivněna fyzickou aktivitou. (p = 0,20, medián 1,64 vs. 1.92 mol/mL sérum sarcosine, a p = 0,15, medián0,02 mol/mmol kreatininu vs. 0,01 mol/mmol kreatininu za močového sarcosine). Čistě vazto našepilotní studie poukazuje na první důkazy, že potenciální biomarker DPS – sarcosine nemá záporné účinky při jízdě nakole tak jak bylo ukázáno v případě PSA.

Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent aggressive cancers in humans. Well-known risk factors include HPV infection, tobacco smoking, and alcohol consumption. HNSCC overall survival rate is one of the lowest among human malignancies. The poor prognosis of HNSCC often results from late-stage diagnosis, therapeutic resistance, high rates of recurrence, and frequent metastases to lymph nodes. To date, the TNM classification is still the best evaluation of disease progress; however, this method of staging does not pay attention to the molecular basis of tumorigenesis. An improvement in treatment efficacy and diagnostic capabilities will be realized through a better understanding of the pathogenesis and characteristics of HNSCC, a disease that has come to be characterized by confounding heterogeneity. This chapter is focused on molecular markers derived from key processes of cancerogenesis that are involved in metastasis, treatment resistance, avoidance of immune detection, inflammation, induction of angiogenesis, genome instability, dysregulation of cellular energetics, cell death, cancer stem cell biology, and rearrangement of tissues adjacent to the tumor. We will discuss biomarkers identified at different levels of cellular regulation (DNA, RNA, miRNA, and protein markers).

Guinea pigs (Cavia porcellus) were treated with haloperidol (HP), and free radical (FR) and ferric reducing antioxidant power (FRAP) assays were used to determine oxidative stress levels. Furthermore, the superoxide dismutase (SOD), glutathione reductase (GR) and glutathione-S-trans- ferase (GST) activity levels were detected and glucose levels and the reduced and oxidized glutathione (GSH/GSSG) ratio were measured in HP-treated and untreated guinea pigs. The present study demonstrated that the administration of HP causes significant oxidative stress in guinea pigs (P=0.022). In animals treated with HP, the activity of GST was significantly increased compared with a placebo (P=0.007). The elevation of SOD and GR activity levels and increase in the levels of glutathione (GSH) in HP-treated animals were not statistically significant. In the HP‑untreated animals, a significant positive correlation was observed between oxidative stress detected by the FR method and GST (r=0.88, P=0.008) and SOD (r=0.86, P=0.01) activity levels, respectively. A significant negative correlation between the levels of plasma glucose and oxidative stress detected by the FRAP method was observed (r=‑0.78, P=0.04). Notably, no significant correlations were observed in the treated animals. In the HP-treated group, two subgroups of animals were identified according to their responses to oxida- tive stress. The group with higher levels of plasma HP had higher enzyme activity and reactive oxygen species production compared with the group with lower plasma levels of HP. The greatest difference in activity (U/µl) between the two groups of animals was for GR.

The present paper is focused on zinc(ii) treatment effects on prostatic cell lines PC-3 (tumour) and PNT1A (non-tumour). Oxidative status of cells was monitored by evaluation of expression of metallothionein (MT) isoforms 1A and 2A at the mRNA and protein level, glutathione (oxidised and reduced), and intracellular zinc(ii) after exposition to zinc(ii) treatment at concentrations of 0-150 μM using electrochemical methods, western blotting and fluorescent microscopy. A novel real-time impedance-based growth monitoring system was compared with widely used end-point MTT assay. Impedance-based IC(50) for zinc(ii) is 55.5 and 150.8 μM for PC-3 and PNT1A, respectively. MTT-determined IC(50) are1.3-fold higher. Impedance-based viability correlates with viable count (r0.92; p0.03), not with MTT. Two-fold lower intracellular zinc(ii) in the tumour PC-3 cell line was found. After zinc(ii) treatment2.6-fold increase of intracellular zinc(ii) was observed in non-tumour PNT1A and in tumour PC-3 cells. In PC-3 cells, free and bound zinc(ii) levels were enhanced more markedly as compared to PNT1A. PNT1A produced 4.2-fold less MT compared to PC3. PNT1A cells showed a 4.8-fold increase trend (r = 0.94; p = 0.005); PC-3 did show a significant trend at MT1 and MT2 protein levels (r = 0.93; p = 0.02) with nearly ten-fold increase after 100 μM zinc(ii) treatment. In terms of redox state, PNT1A had a predominance of reduced GSH forms (GSH : GSSG ratio1), when exposed to zinc(ii) compared to PC3, where predominance of oxidised forms remains at all concentrations. IC(50) differs significantly when determined by MTT and real-time impedance-based assays due to dependence of impedance on cell morphology and adhesion. When real-time growth monitoring, precise electrochemical methods and fluorescent microscopy are performed together, accurate information for metal fluxes, their buffering by thiol compounds and monitoring of the redox state become a powerful tool for understanding the role of oxidative stress in carcinogenesis.

MicroRNAs (miRNAs) are a large class of single-stranded RNA molecules involved in post-transcriptional gene silencing. miRNAs not only regulate various developmental and physiological processes but also are involved in cancer development. Additionally, they can be considered as biomarkers of some pathological processes. The aim of this study was to determine the expression levels of selected miRNA and zinc(II)-related genes (ZIP-1, BAX, MT2A and MT1A) in the non-tumor PNT1A prostate cell line in comparison with the prostate cancer cell lines 22Rv1, PC-3 and LNCaP after zinc(II) treatment. Using bioinformatic approaches we selected miRNAs with putative binding sites in the 3'UTR regions in Metallothionein 1A and 2A as miRNA 23a, 141, 224, 296-3p, 320, 375 and 376. We observed significantly higher expression of miRNA 23a in all tumor lines compared to non-tumor PNT1A (13.6-fold in 22Rv1, 7.3-fold in PC-3, 8.3-fold in LNCaP, p0.01). We also observed that the 22Rv1 cell line has significantly higher expression of miRNA 224 in comparison to other cell lines. In addition, all tumor cell lines expressed significantly higher levels of miRNA 375 in comparison to non-tumor PNT1A (87.1‑fold in 22Rv1, nearly 2,000-fold in PC-3, 56.3‑fold in LNCaP, p0.01). Nevertheless, miRNA 375 and 23a expression levels strongly suggest their potential to contribute to the diagnosis of prostate cancer and miRNA 224 eventually may be suitable for classification of primary tumors. The expression of miRNA 224 in 22Rv1 cell line was negatively correlated with increasing zinc(II) concentration only. Our experiments revealed significant negative correlation of miRNA 376 and MT2A in 22Rv1 and a negative correlation between miRNA 224 and MT1A in PC-3 cells which may denote possible direct regulation of MT genes by specific miRNAs in prostate cancer.

Current diagnostic techniques are inefficient in distinguishing latent and low-risk forms of prostate cancer from high-risk forms. The present study is focused on determination of putative tumor markers of aggressive high-grade forms of prostate cancer. Potential markers were determined in blood sera of 133 patients (82 cases and 51 controls) and in cell lines (Gleason score 9-derived 22Rv1 and normal tissue derived PNT1A) on mRNA and protein levels. Alpha-methylacyl-CoA racemase (AMACR), metallothionein classes 1A and 2A (MT1A and MT2A) were determined and compared to prostate specific antigen (PSA) levels. On mRNA level, significantly increased expression of MT2A (2.4-fold), PSA (2.6-fold) and AMACR (8.4-fold) and insignificantly (1.9-fold) elevated MT1A in 22Rv1 compared to non-tumor PNT1A were determined. On protein level, significant enhancement of free PSA and total PSA in tumor cell line was evident. AMACR protein was 1.5-fold elevated in tumor line (below the level of significance). Contrary to mRNA, significantly (p = 0.01) reduced level of MT protein in tumor lines was determined. In the case of serum level, significantly enhanced MT level (4.5-fold) in patients' sera was found. No significant changes were observed in the case of AMACR. These findings indicate possible alternative role of MT to PSA prostate cancer marker. In addition, level of AMACR is distinctly higher in the Gleason score 9 in serum of patients and MT shows a descending trend in relation to Gleason score.