Rilzabrutinib, an Oral BTK Inhibitor, in Immune Thrombocytopenia.

BACKGROUND: Rilzabrutinib, an oral, reversible covalent inhibitor of Bruton's tyrosine kinase, may increase platelet counts in patients with immune thrombocytopenia by means of dual mechanisms of action: decreased macrophage (Fcγ receptor)-mediated platelet destruction and reduced production of pathogenic autoantibodies. METHODS: In an international, adaptive, open-label, dose-finding, phase 1-2 clinical trial, we evaluated rilzabrutinib therapy in previously treated patients with immune thrombocytopenia. We used intrapatient dose escalation of oral rilzabrutinib over a period of 24 weeks; the lowest starting dose was 200 mg once daily, with higher starting doses of 400 mg once daily, 300 mg twice daily, and 400 mg twice daily. The primary end points were safety and platelet response (defined as at least two consecutive platelet counts of ≥50×103 per cubic millimeter and an increase from baseline of ≥20×103 per cubic millimeter without the use of rescue medication). RESULTS: Sixty patients were enrolled. At baseline, the median platelet count was 15×103 per cubic millimeter, the median duration of disease was 6.3 years, and patients had received a median of four different immune thrombocytopenia therapies previously. All the treatment-related adverse events were of grade 1 or 2 and transient. There were no treatment-related bleeding or thrombotic events of grade 2 or higher. At a median of 167.5 days (range, 4 to 293) of treatment, 24 of 60 patients (40%) overall and 18 of the 45 patients (40%) who had started rilzabrutinib treatment at the highest dose met the primary end point of platelet response. The median time to the first platelet count of at least 50×103 per cubic millimeter was 11.5 days. Among patients with a primary platelet response, the mean percentage of weeks with a platelet count of at least 50×103 per cubic millimeter was 65%. CONCLUSIONS: Rilzabrutinib was active and associated with only low-level toxic effects at all dose levels. The dose of 400 mg twice daily was identified as the dose for further testing. Overall, rilzabrutinib showed a rapid and durable clinical activity that improved with length of treatment. (Funded by Sanofi; ClinicalTrials.gov number, NCT03395210; EudraCT number, 2017-004012-19.).

Hormone-regulated expansins: Expression, localization, and cell wall biomechanics in Arabidopsis root growth.

Expansins facilitate cell expansion by mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in controlling CW biomechanical properties in specific tissues and organs remains elusive. We monitored hormonal responsiveness and spatial specificity of expression and localization of expansins predicted to be the direct targets of cytokinin signaling in Arabidopsis (Arabidopsis thaliana). We found EXPANSIN1 (EXPA1) homogenously distributed throughout the CW of columella/lateral root cap, while EXPA10 and EXPA14 localized predominantly at 3-cell boundaries in the epidermis/cortex in various root zones. EXPA15 revealed cell-type-specific combination of homogenous vs. 3-cell boundaries localization. By comparing Brillouin frequency shift and AFM-measured Young's modulus, we demonstrated Brillouin light scattering (BLS) as a tool suitable for non-invasive in vivo quantitative assessment of CW viscoelasticity. Using both BLS and AFM, we showed that EXPA1 overexpression upregulated CW stiffness in the root transition zone (TZ). The dexamethasone-controlled EXPA1 overexpression induced fast changes in the transcription of numerous CW-associated genes, including several EXPAs and XYLOGLUCAN:XYLOGLUCOSYL TRANSFERASEs (XTHs), and associated with rapid pectin methylesterification determined by in situ Fourier-transform infrared spectroscopy in the root TZ. The EXPA1-induced CW remodeling is associated with the shortening of the root apical meristem, leading to root growth arrest. Based on our results, we propose that expansins control root growth by a delicate orchestration of CW biomechanical properties, possibly regulating both CW loosening and CW remodeling.

Autophagy modulators influence the content of important signalling molecules in PS-positive extracellular vesicles.

Extracellular vesicles (EVs) are important mediators of intercellular communication in the tumour microenvironment. Many studies suggest that cancer cells release higher amounts of EVs exposing phosphatidylserine (PS) at the surface. There are lots of interconnections between EVs biogenesis and autophagy machinery. Modulation of autophagy can probably affect not only the quantity of EVs but also their content, which can deeply influence the resulting pro-tumourigenic or anticancer effect of autophagy modulators. In this study, we found that autophagy modulators autophinib, CPD18, EACC, bafilomycin A1 (BAFA1), 3-hydroxychloroquine (HCQ), rapamycin, NVP-BEZ235, Torin1, and starvation significantly alter the composition of the protein content of phosphatidylserine-positive EVs (PS-EVs) produced by cancer cells. The greatest impact had HCQ, BAFA1, CPD18, and starvation. The most abundant proteins in PS-EVs were proteins typical for extracellular exosomes, cytosol, cytoplasm, and cell surface involved in cell adhesion and angiogenesis. PS-EVs protein content involved mitochondrial proteins and signalling molecules such as SQSTM1 and TGFß1 pro-protein. Interestingly, PS-EVs contained no commonly determined cytokines, such as IL-6, IL-8, GRO-α, MCP-1, RANTES, and GM-CSF, which indicates that secretion of these cytokines is not predominantly mediated through PS-EVs. Nevertheless, the altered protein content of PS-EVs can still participate in the modulation of the fibroblast metabolism and phenotype as p21 was accumulated in fibroblasts influenced by EVs derived from CPD18-treated FaDu cells. The altered protein content of PS-EVs (data are available via ProteomeXchange with identifier PXD037164) also provides information about the cellular compartments and processes that are affected by the applied autophagy modulators. Video Abstract.

Cancer cell viscoelasticity measurement by quantitative phase and flow stress induction.

Cell viscoelastic properties are affected by the cell cycle, differentiation, and 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 mechanophenotyping 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 one 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, whereby we demonstrated the possibility of simultaneous extraction of shear modulus, viscosity, cell morphology, and QPI-derived cell parameters such as 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, whereby an increase of stiffness corresponded to actin repolymerization in time.

Selected severe „haematological“ syndromes in adult intensive care patients.

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.

Sensitivity to Cisplatin in Head and Neck Cancer Cells Is Significantly Affected by Patient-Derived Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) are one of the most abundant and critical components of the tumor stroma. CAFs can impact many important steps of cancerogenesis and may also influence treatment resistance. Some of these effects need the direct contact of CAFs and cancer cells, while some involve paracrine signals. In this study, we investigated the ability of head and neck squamous cell carcinomas (HNSCC) patient-derived CAFs to promote or inhibit the colony-forming ability of HNSCC cells. The effect of cisplatin on this promoting or inhibiting influence was also studied. The subsequent analysis focused on changes in the expression of genes associated with cancer progression. We found that cisplatin response in model HNSCC cancer cells was modified by coculture with CAFs, was CAF-specific, and different patient-derived CAFs had a different "sensitizing ratio". Increased expression of VEGFA, PGE2S, COX2, EGFR, and NANOG in cancer cells was characteristic for the increase of resistance. On the other hand, CCL2 expression was associated with sensitizing effect. Significantly higher amounts of cisplatin were found in CAFs derived from patients who subsequently experienced a recurrence. In conclusion, our results showed that CAFs could promote and/or inhibit colony-forming capability and cisplatin resistance in HNSCC cells via paracrine effects and subsequent changes in gene expression of cancer-associated genes in cancer cells.

Quantitative Morphology of Cerebral Thrombi Related to Intravital Contraction and Clinical Features of Ischemic Stroke.

BACKGROUND AND PURPOSE: The purpose was to assess quantitatively and qualitatively the composition and structure of cerebral thrombi and correlate them with the signs of intravital clot contraction (retraction), as well as with etiology, severity, duration, and outcomes of acute ischemic stroke. METHODS: We quantified high-resolution scanning electron micrographs of 41 cerebral thrombi for their detailed cellular and noncellular composition and analyzed histological images for the overall structure with the emphasis on red blood cell compression, fibrin age, and the signs of inflammation. RESULTS: Cerebral thrombi were quite compact and had extremely low porosity. The prevailing cell type was polyhedral compressed erythrocytes (polyhedrocytes) in the core, and fibrin-platelet aggregates were concentrated at the periphery; both findings are indicative of intravital contraction of the thrombi. The content of polyhedrocytes directly correlated with the stroke severity. The prevalence of fibrin bundles was typical for more severe cases, while the content of fibrin sponge prevailed in cases with a more favorable course. The overall platelet content in cerebral thrombi was surprisingly small, while the higher content of platelet aggregates was a marker of stroke severity. Fibrillar types of fibrin prevailed in atherothrombogenic thrombi. Older fibrin prevailed in thrombi from the patients who received thrombolytics, and younger fibrin dominated in cardioembolic thrombi. Alternating layers of erythrocytes and fibrin mixed with platelets were common for thrombi from the patients with more favorable outcomes. Thrombi with a higher number of leukocytes were associated with fatal cases. CONCLUSIONS: Most cerebral thrombi undergo intravital clot contraction (retraction) that may be of underestimated clinical importance. Despite the high variability of the composition and structure of cerebral thrombi, the content of certain types of blood cells and fibrin structures combined with the morphological signs of intravital contraction correlate with the clinical course and outcomes of acute ischemic stroke.

Caveolin-1 in oncogenic metabolic symbiosis.

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.

DNA repair in head and neck tumor cells and possibilities of its monitoring to estimate individual tumor radioresistance and selection of optimal primary treatment.

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'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 γH2AX/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.

Cell segmentation methods for label-free contrast microscopy: review and comprehensive comparison.

BACKGROUND: Because of its non-destructive nature, label-free imaging is an important strategy for studying biological processes. However, routine microscopic techniques like phase contrast or DIC suffer from shadow-cast artifacts making automatic segmentation challenging. The aim of this study was to compare the segmentation efficacy of published steps of segmentation work-flow (image reconstruction, foreground segmentation, cell detection (seed-point extraction) and cell (instance) segmentation) on a dataset of the same cells from multiple contrast microscopic modalities. RESULTS: We built a collection of routines aimed at image segmentation of viable adherent cells grown on the culture dish acquired by phase contrast, differential interference contrast, Hoffman modulation contrast and quantitative phase imaging, and we performed a comprehensive comparison of available segmentation methods applicable for label-free data. We demonstrated that it is crucial to perform the image reconstruction step, enabling the use of segmentation methods originally not applicable on label-free images. Further we compared foreground segmentation methods (thresholding, feature-extraction, level-set, graph-cut, learning-based), seed-point extraction methods (Laplacian of Gaussians, radial symmetry and distance transform, iterative radial voting, maximally stable extremal region and learning-based) and single cell segmentation methods. We validated suitable set of methods for each microscopy modality and published them online. CONCLUSIONS: We demonstrate that image reconstruction step allows the use of segmentation methods not originally intended for label-free imaging. In addition to the comprehensive comparison of methods, raw and reconstructed annotated data and Matlab codes are provided.

The Role of von Willebrand Factor, ADAMTS13, and Cerebral Artery Thrombus Composition in Patient Outcome Following Mechanical Thrombectomy for Acute Ischemic Stroke.

BACKGROUND The aim of the study was to investigate the role of von Willebrand factor (vWF), the vWF-cleaving protease, ADAMTS13, the composition of thrombus, and patient outcome following mechanical cerebral artery thrombectomy in patients with acute ischemic stroke. MATERIAL AND METHODS A prospective cohort study included 131 patients with ischemic stroke (<6 hours) with or without intravenous thrombolysis. Interventional procedure parameters, hemocoagulation markers, vWF, ADAMTS13, and histological examination of the extracted thrombi were performed. The National Institutes of Health Stroke Scale (NIHSS) score was used on hospital admission, after 24 hours, at day 7; the three-month modified Rankin Scale score was used. RESULTS Mechanical thrombectomy resulted in a Treatment in Cerebral Ischemia (TICI) score of 2-3, with recanalization in 89% of patients. Intravenous thrombolysis was used in 101 (78%). Patients with and without intravenous thrombolysis therapy had a good clinical outcome (score 0-2) in 47% of cases (P=0.459) using the three-month modified Rankin Scale. Patients with a National Institutes of Health Stroke Scale (NIHSS) score ≥15 had significantly increased vWF levels (P=0.003), and a significantly increased vWF: ADAMTS13 ratio (P=0.038) on hospital admission. Significant correlation coefficients were found for plasma vWF and thrombo-embolus vWF (r=0.32), platelet (r=0.24), and fibrin (r=0.26) levels. In the removed thrombus, vWF levels were significantly correlated with platelet count (r=0.53), CD31-positive cells (r=0.38), and fibrin (r=0.48). CONCLUSIONS In patients with acute ischemic stroke, mechanical cerebral artery thrombectomy resulted in a good clinical outcome in 47% of cases, with and without intravenous thrombolysis therapy.

Amino Acid Profiling of Zinc Resistant Prostate Cancer Cell Lines: Associations With Cancer Progression.

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. Prostate 77: 604-616, 2017. © 2017 Wiley Periodicals, Inc.

VPA does not enhance platinum binding to DNA in cisplatin-resistant neuroblastoma cancer cells.

Neuroblastoma represents a malignancy of the sympathetic nervous system characteristic by biological heterogeneity. Thus, chemotherapy exhibits only low effectivity in curing high-risk forms. Previous studies revealed the cytotoxic potential of valproate on neuroblastoma cells. Nevertheless, these studies omitted effects of hypoxia, despite its undeniable tumorigenic role. In this study, we addressed the question whether valproate promotes binding of platinum-based anti-cancer drugs (cisplatin, carboplatin and oxaliplatin) to DNA and role of hypoxia, cellular antioxidant capacity and cisplatin resistance in this process. Following parameters differed significantly when cells were exposed to treatment with platinum-based drugs: elevation of platinum content bound to DNA, elevation of total thiol content, GSH/GSSG ratio, glutathione reductase and peroxidase, superoxide dismutase and elevation of antioxidant capacity. Hypoxia caused a decrease in cytosine/adenine peak, and no changes in platinum-DNA binding properties were observed. After valproate co-treatment, oxidative stress-related parameters and cytosine/adenine peak were only elevated. The amount of platinum bound to DNA was not changed significantly. Valproate is not able to enhance platinum binding to DNA in neuroblastoma cells, neither in case of intrinsic resistance (UKF-NB-4) nor in case of acquired resistance (UKF-NB-4CDDP). Therefore, another mechanism different from increase in platinum binding to DNA should be considered as a synergistic effect of valproate by cisplatin treatment.

Levels of heavy metals and their binding protein metallothionein in type 2 diabetics with kidney disease.

Hyperglycemia, a major metabolic disturbance present in diabetes, promotes oxidative stress. Activation of antioxidant defense is an important mechanism to prevent cell damage. Levels of heavy metals and their binding proteins can contribute to oxidative stress. Antiradical capacity and levels of metallothionein (MT), metals (zinc and copper), and selected antioxidants (bilirubin, cysteine, and glutathione) were determined in 70 type 2 diabetes mellitus (T2DM) subjects and 80 healthy subjects of Caucasian origin. Single nucleotide polymorphism (rs28366003) in MT gene was detected. Antiradical capacity, conjugated bilirubin, and copper were significantly increased in diabetics, whereas MT and glutathione were decreased. Genotype AA of rs28366003 was associated with higher zinc levels in the diabetic group. The studied parameters were not influenced by renal function. This is the first study comprehensively investigating differences in MT and metals relevant to oxidative stress in T2DM. Ascertained differences indicate increased oxidative stress in T2DM accompanied by abnormalities in non-enzymatic antioxidant defense systems.

Relation of exposure to amino acids involved in sarcosine metabolic pathway on behavior of non-tumor and malignant prostatic cell lines.

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 < 0.05). RESULTS: The highest migration induced sarcosine and glycine in metastatic PC-3 cells (a decrease in relative free area about 53% and 73%). The highest cell division was achieved after treatment of 22Rv1 and PC-3 cells with sarcosine (time required for division decreased by 65% or 45%, when compared to untreated cells). qRT-PCR revealed also significant effects on expression of GNMT. Finally, amino acid profiling shown specific amino acid patterns for each cell line. In both, treated and untreated PC-3 cells significantly higher levels of serine, glutamic acid, and aspartate, linked with prostate cancer progression were found. CONCLUSIONS: Sarcosine-related amino acids can exceptionally affect the behavior of benign and malignant prostate cells.

Expression profiles of miR-29c, miR-200b and miR-375 in tumour and tumour-adjacent tissues of head and neck cancers.

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).

Effect of HPV on tumor expression levels of the most commonly used markers in HNSCC.

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.

Influence of Long-Distance Bicycle Riding on Serum/Urinary Biomarkers of Prostate Cancer.

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 µmol/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 µmol/mL for serum sarcosine, and p = 0.15, median 0.02 µmol/mmol of creatinine vs. 0.01 µmol/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.

Prognostic significance of the tumour-adjacent tissue in head and neck cancers.

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.

Novel biophysical determination of miRNAs related to prostate and head and neck cancers.

In this study we have chosen a new approach and characterized three miRNAs (miR-23a, miR-34a and miR-320a) related to prostate cancer and head and neck cancer by spectral (circular dichroic and UV-absorption spectra) and electrochemical (voltammetry at graphite and mercury electrodes) methods. The spectral and voltammetric results, reflecting different nucleotide sequences of miRNAs, were complemented by the results of DNAs(U) having the same oligonucleotide sequences as miRNAs. The effect of the substitution of ribose for deoxyribose was shown and structural diversity was confirmed. The stability of RNA and DNA(U) was studied using CD and UV-absorption spectroscopy and melting points were calculated. MiRNA-320a with the highest content of guanine provided the highest melting point. With respect to the rapid progress of miRNA electrochemical sensors, our results will be useful for the research and development of sensitive, portable and time-efficient miRNA sensors, which will be able to diagnose cancer and other diseases.