Mast Cells as a Target-A Comprehensive Review of Recent Therapeutic Approaches.

Mast cells (MCs) are the immune cells distributed throughout nearly all tissues, mainly in the skin, near blood vessels and lymph vessels, nerves, lungs, and the intestines. Although MCs are essential to the healthy immune response, their overactivity and pathological states can lead to numerous health hazards. The side effect of mast cell activity is usually caused by degranulation. It can be triggered by immunological factors, such as immunoglobulins, lymphocytes, or antigen-antibody complexes, and non-immune factors, such as radiation and pathogens. An intensive reaction of mast cells can even lead to anaphylaxis, one of the most life-threatening allergic reactions. What is more, mast cells play a role in the tumor microenvironment by modulating various events of tumor biology, such as cell proliferation and survival, angiogenesis, invasiveness, and metastasis. The mechanisms of the mast cell actions are still poorly understood, making it difficult to develop therapies for their pathological condition. This review focuses on the possible therapies targeting mast cell degranulation, anaphylaxis, and MC-derived tumors.

Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing.

Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). The aim of this study was to show whether silencing of the CK2α gene affects TS and DHFR expression in A-549 cells. Additionally, we attempted to identify the endogenous kinases that phosphorylate TS and DHFR in CCRF-CEM and A-549 cells. We used immunodetection, immunofluorescence/confocal analyses, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), in-gel kinase assay, and mass spectrometry analysis. Our results demonstrate that silencing of the CK2α gene in lung adenocarcinoma cells significantly increases both TS and DHFR expression and affects their cellular distribution. Additionally, we show for the first time that both TS and DHFR are very likely phosphorylated by endogenous CK2 in two types of cancer cells, i.e., acute lymphoblastic leukaemia and lung adenocarcinoma. Moreover, our studies indicate that DHFR is phosphorylated intracellularly by CK2 to a greater extent in leukaemia cells than in lung adenocarcinoma cells. Interestingly, in-gel kinase assay results indicate that the CK2α' isoform was more active than the CK2α subunit. Our results confirm the previous studies concerning the physiological relevance of CK2-mediated phosphorylation of TS and DHFR.

Aptamer-based Advances in Skin Cancer Research.

Cancer diseases have been one of the biggest health threats for the last two decades. Approximately 9% of all diagnosed cancers are skin cancers, including melanoma and non-melanoma. In all cancer cases, early diagnosis is essential to achieve efficient treatment. New solutions and advanced techniques for rapid diagnosis are constantly being sought. Aptamers are single-stranded RNA or DNA synthetic sequences or peptides, which offer novel possibilities to this area of research by specifically binding selected molecules, the so-called cancer biomarkers. Nowadays, they are widely used as diagnostic probes in imaging and targeted therapy. In this review, we have summarized the recently made advances in diagnostics and treatment of skin cancers, which have been achieved by combining aptamers with basic or modern technologies.

Anandamide-Modulated Changes in Metabolism, Glycosylation Profile and Migration of Metastatic Melanoma Cells.

An effective therapy for advanced melanoma, a skin cancer with the highest mortality, has not yet been developed. The endocannabinoid system is considered to be an attractive target for cancer treatment. The use of endocannabinoids, such as anandamide (AEA), is considered to be much greater than as a palliative agent. Thus, we checked its influence on various signaling pathways in melanoma cells. Our investigation was performed on four commercial cell lines derived from different progression stages (radial WM35 and vertical WM115 growth phases, lymph node WM266-4 metastasis, solid tumor A375-P metastasis). Cell viability, glucose uptake, quantification of reactive oxygen species production, expression of selected genes encoding glycosyltransferases, quantification of glycoproteins production and changes in the glycosylation profile and migration, as well as in cell elastic properties were analyzed. The cell glycosylation profile was investigated using the biophysical profiling method-the quartz crystal microbalance with dissipation monitoring (QCM-D). Anandamide treatment of only metastatic cells resulted in: an increase in the cell metabolism, a decrease in GFAT-1 and DPM1 expression, followed by a decrease in L1-CAM glycoprotein production, which further influenced the reduction in the cell glycosylation profile and migration. Considering our results, AEA usage is highly recommended in the combined therapy of advanced melanoma.

Novel diagnostic and prognostic factors for the advanced melanoma based on the glycosylation-related changes studied by biophysical profiling methods.

Melanoma is a life-threatening disease due to the early onset of metastasis and frequent resistance to the applied treatment. For now, no single histological, immunohistochemical or serological biomarker was able to provide a precise predictive value for the aggressive behavior in melanoma patients. Thus, the search for quantifying methods allowing a simultaneous diagnosis and prognosis of melanoma patients is highly desirable. By investigating specific molecular interactions with some biosensor-based techniques, one can determine novel prognostic factors for this tumor. In our previous study, we have shown the possibility of a qualitative in vitro distinguishing the commercially available melanoma cells at different progression stages based on the measurements of the lectin Concanavalin A interacting with surface glycans present on cells. Here, we present the results of the quantitative diagnostic and prognostic study of both commercial and patient-derived melanoma cells based on the evaluation of two novel factors: lectin affinity and glycan viscoelastic index obtained from the quartz crystal microbalance with dissipation monitoring (QCM-D) measurements. Two approaches to the QCM-D measurements were applied, the first uses the ability of melanoma cells to grow as a monolayer of cells on the sensor (cell-based sensors), and the second shortens the time of the analysis (suspension cell based-sensors). The results were confirmed by the complementary label-free (atomic force microscopy, AFM; and surface plasmon resonance, SPR) and labeling (lectin-ELISA; and microscale thermophoresis, MST) techniques. This new approach provides additional quantitative diagnosis and a personalized prognosis which can be done simultaneously to the traditional histopathological analysis.

The Multifaceted Roles of Mast Cells in Immune Homeostasis, Infections and Cancers.

Mast cells (MCs) play important roles in normal immune responses and pathological states. The location of MCs on the boundaries between tissues and the external environment, including gut mucosal surfaces, lungs, skin, and around blood vessels, suggests a multitude of immunological functions. Thus, MCs are pivotal for host defense against different antigens, including allergens and microbial pathogens. MCs can produce and respond to physiological mediators and chemokines to modulate inflammation. As long-lived, tissue-resident cells, MCs indeed mediate acute inflammatory responses such as those evident in allergic reactions. Furthermore, MCs participate in innate and adaptive immune responses to bacteria, viruses, fungi, and parasites. The control of MC activation or stabilization is a powerful tool in regulating tissue homeostasis and pathogen clearance. Moreover, MCs contribute to maintaining the homeostatic equilibrium between host and resident microbiota, and they engage in crosstalk between the resident and recruited hematopoietic cells. In this review, we provide a comprehensive overview of the functions of MCs in health and disease. Further, we discuss how mouse models of MC deficiency have become useful tools for establishing MCs as a potential cellular target for treating inflammatory disorders.

Studying Viscoelastic Changes of Skin Cells Using QCM-D Measurements.

The viscoelastic properties of cells are responsible for the adhesion process to different surfaces and for cell motility. Therefore, it is very important to develop specific, label-free biosensors with the use of whole cells to study the effect of various factors on the survival and properties of selected type of normal and pathological cells. The quartz crystal microbalance with dissipation energy monitoring (QCM-D) is a technique which enables to track these changes in cells during real-time experiments. One of the applied procedures of the evaluation of the cells' viscoelastic changes is based on the investigations of interactions between specific, different glycans, present on the surface of the primary tumor and its metastases with specific lectins. Two procedures have been developed to detect the differences in the cellular glycosylation profile using cell-based sensors (adherent cells cultured on sensors) and suspension cell-based sensors (adherent cells mechanically detached and inserted into the QCM-D chamber with a sensor). Furthermore, in this work some cell-based sensor regeneration protocols have been described and a lectin-ELISA assay with a fluorescently labeled lectin, thus enabling a qualitative and quantitative tracking of each step of the lectin-glycan binding and unbinding process performed on whole cells.

A Novel and Effective Method for Human Primary Skin Melanocytes and Metastatic Melanoma Cell Isolation.

The development of an effective method of melanocyte isolation and culture is necessary for basic and clinical studies concerning skin diseases, including skin pigmentation disorders and melanoma. In this paper, we describe a novel, non-enzymatic and effective method of skin melanocyte and metastatic melanoma cell isolation and culture (along with the spontaneous spheroid creation) from skin or lymph node explants. The method is based on the selective harvesting of melanocytes and melanoma cells emigrating from the cultured explants. Thereby, isolated cells retain their natural phenotypical features, such as expression of tyrosinase and Melan-A as well as melanin production and are not contaminated by keratinocytes and fibroblasts. Such melanocyte and melanoma cell cultures may be very useful for medical and cosmetology studies, including studies of antitumor therapies.

Biophysical characterization of melanoma cell phenotype markers during metastatic progression.

Melanoma is the most fatal form of skin cancer, with increasing prevalence worldwide. The most common melanoma genetic driver is mutation of the proto-oncogene serine/threonine kinase BRAF; thus, the inhibition of its MAP kinase pathway by specific inhibitors is a commonly applied therapy. However, many patients are resistant, or develop resistance to this type of monotherapy, and therefore combined therapies which target other signaling pathways through various molecular mechanisms are required. A possible strategy may involve targeting cellular energy metabolism, which has been recognized as crucial for cancer development and progression and which connects through glycolysis to cell surface glycan biosynthetic pathways. Protein glycosylation is a hallmark of more than 50% of the human proteome and it has been recognized that altered glycosylation occurs during the metastatic progression of melanoma cells which, in turn facilitates their migration. This review provides a description of recent advances in the search for factors able to remodel cell metabolism between glycolysis and oxidative phosphorylation, and of changes in specific markers and in the biophysical properties of cells during melanoma development from a nevus to metastasis. This development is accompanied by changes in the expression of surface glycans, with corresponding changes in ligand-receptor affinity, giving rise to structural features and viscoelastic parameters particularly well suited to study by label-free biophysical methods.

Tracking of Glycans Structure and Metallomics Profiles in BRAF Mutated Melanoma Cells Treated with Vemurafenib.

Nearly half of patients with advanced and metastatic melanomas harbor a BRAF mutation. Vemurafenib (VEM), a BRAF inhibitor, is used to treat such patients, however, responses to VEM are very short-lived due to intrinsic, adaptive and/or acquired resistance. In this context, we present the action of the B-Raf serine-threonine protein kinase inhibitor (vemurafenib) on the glycans structure and metallomics profiles in melanoma cells without (MeWo) and with (G-361) BRAF mutations. The studies were performed using α1-acid glycoprotein (AGP), a well-known acute-phase protein, and concanavalin A (Con A), which served as the model receptor. The detection of changes in the structure of glycans can be successfully carried out based on the frequency shifts and the charge transfer resistance after interaction of AGP with Con A in different VEM treatments using QCM-D and EIS measurements. These changes were also proved based on the cell ultrastructure examined by TEM and SEM. The LA-ICP-MS studies provided details on the metallomics profile in melanoma cells treated with and without VEM. The studies evidence that vemurafenib modifies the glycans structures and metallomics profile in melanoma cells harboring BRAF mutation that can be further implied in the resistance phenomenon. Therefore, our data opens a new avenue for further studies in the short-term addressing novel targets that hopefully can be used to improve the therapeutic regiment in advanced melanoma patients. The innovating potential of this study is fully credible and has a real impact on the global patient society suffering from advanced and metastatic melanomas.

AFM and QCM-D as tools for the distinction of melanoma cells with a different metastatic potential.

Malignant melanoma is one of the most dangerous skin cancer originating from melanocytes. Thus, an early and proper melanoma diagnosis influences significantly the therapy efficiency. The melanoma recognition is still difficult, and generally, relies on subjective assessments. In particular, there is a lack of quantitative methods used in melanoma diagnosis and in the monitoring of tumour progression. One such method can be the atomic force microscopy (AFM) working in the force spectroscopy mode combined with quartz crystal microbalance (QCM), both applied to quantify the molecular interactions. In our study we have compared the recognition of mannose type glycans in melanocytes (HEMa-LP) and melanoma cells originating from the radial growth phase (WM35) and from lung metastasis (A375-P). The glycosylation level on their surfaces was probed using lectin concanavalin A (Con A) from Canavalia ensiformis. The interactions of Con A with surface glycans were quantified with both AFM and QCM techniques that revealed the presence of various glycan structural groups in a cell-dependent manner. The Con A - mannose (or glucose) type glycans present on WM35 cell surface are rather short and less ramified while in A375-P cells, Con A binds to long, branched mannose and glucose types of oligosaccharides.

The effect of delphinidin on the mechanical properties of keratinocytes exposed to UVB radiation.

The usage of active compounds of dietary phytochemicals in prevention of UV-induced skin diseases is increasingly gaining attention in the development of skin care products. The purpose of this study was to measure the influence of delphinidin (as a botanical agent) on the cell mechanical properties evaluated by the atomic force microscopy (AFM) technique in the immortalized human keratinocyte cell line (HaCaT) exposed to UVB radiation. The cells were treated with various doses of UVB radiation with and without pre and post-treatment with selected concentrations of delphinidin. The measurements of the elastic properties revealed that the exposure of HaCaT cells to high dose of the UVB radiation (100mJ/cm2) caused a decrease in the cell elastic modulus. It was accompanied by the decrease of metabolic activity, rearrangement of actin cytoskeleton and disappearance of the cell repair marker 53BP1. Both pre-treatment and post-treatment with delphinidin at non-cytotoxic concentrations (5 or 10µM), restored the elastic modulus of irradiated keratinocytes. A direct AFM analysis showed that the UVB-mediated decrease of the cell stiffness was restored more effectively when cells were treated with delphinidin after the UVB irradiation. The results demonstrate the regenerative effect of delphinidin on the mechanical properties of cells exposed to UVB radiation (100mJ/cm2), which may be due to antioxidant and inhibitory effect on matrix metalloproteinases activation.