"Cladonia uncialis" as a valuable raw material of biosynthetic compounds against clinical strains of bacteria and fungi

Cladonia uncialis is a lichen species with confirmed antibacterial activity and whose genome has been recently sequenced, enabling first attempts in its functional characterization. In this work, we investigated activity of the C. uncialis acetone extract (CUE) and usnic acid (UA) enantiomers against ten clinical microbial strains causing skin infections. The results showed that CUE, containing (–)-UA and squamatic acid, assayed at the same concentrations as UA, was noticeably more active than (–)-UA alone, in its pure form. The studied CUE displayed an activity that was comparable to that of (+)-UA observed for Staphylococcus epidermidis and Enterococcus faecium (18–24 mm zone of growth inhibition), but did not display any activity against fungal strains. The CUE demonstrated low cytotoxicity against HaCaT cells, in comparison to UA enantiomers, which is important for its therapeutic use. Results of the antioxidant assay (DPPH) indicated low antioxidant activity (IC50>200 µg/mL) of CUE, while the total phenolic content was 70.36 mg Gallic Acid Equivalent/g of the dry extrac

Prognostic significance of the methylation of Wnt pathway antagonists-CXXC4, DACT2, and the inhibitors of sonic hedgehog signaling-ZIC1, ZIC4, and HHIP in head and neck squamous cell carcinomas

Aberrations in Wnt and Shh signaling pathways are related to the pathogenesis of head and neck carcinomas, and their activation frequently results from epigenetic alterations. This study aimed to assess the frequency of methylation of negative regulators of Wnt signaling: CXXC4, DACT2, HDPR1, and FBXW11 and Shh signaling: HHIP, PTCH1, SUFU, ZIC1, and ZIC4 and correlate it with clinicopathological features in this group of patients.Methylation-specific PCR was used to detect gene promoter methylation, and real-time PCR was used to assess gene expression level.The analysis of the occurrence of gene promoter methylation in head and neck carcinoma cell lines indicated that CXXC4, DACT2, HHIP, ZIC1, and ZIC4 are methylated in these tumors. These genes were further analyzed in tumor sections from oral and laryngeal cancer patients. Gene methylation rate was higher in laryngeal tumors. The methylation index in tumor samples correlated with the overall survival in a subgroup of oral cancer patients who died of the disease. Moreover, ZIC4 methylation correlated with lymph node involvement in oral cancer patients.Our findings corroborate that the activation of Wnt signaling in head and neck squamous cell carcinoma (HNSCC) is related to epigenetic silencing of its negative regulators. Moreover, the results indicate that the same mechanism of activation may operate in the case of Shh signaling.The methylation of ZIC4 may be considered a new prognostic marker in oral cavity and oropharyngeal tumors. Further investigations should determine the diagnostic significance of methylation of ZIC4, HHIP, and DACT2 in head and neck carcinomas

Therapeutic targeting of alternative splicing

Gene transcription leads to the generation of pre-mRNA molecules which contain both coding sequences (exons) and intervening non-coding sequences (introns). The primary transcript needs further processing which involves the excision of introns and ligation of exons. This process is called RNA splicing. Nearly all primary transcripts undergo alternative forms of splicing, which may lead to exon skipping or intron inclusion in the final mRNA. Thus, the translation of alternatively spliced RNA molecules results in the formation of slightly different proteins, which may, in some cases, exert antagonistic activity. Splicing is a multistage process which is conducted by a complex machinery comprising small nuclear RNA molecules and many proteins called splicing factors. The process undergoes precise regulation by means of cis acting internal RNA sequences and trans acting protein factors which may either enhance or silence the splicing of an exon. Many diseases are associated with aberrations of alternative splicing and its modulation may be used therapeutically, e.g. for the treatment of spinal muscular atrophy (SMA) or Duchenne muscular dystrophy (DMD). This article presents current knowledge of the ways of pharmacological modulation of alternative splicing. The focus is on the use of those therapeutics which have been already approved for clinical application or have entered clinical trials. The chemistry and mechanism of action of specific splice switching oligonucleotides is presented. Nusinersen promotes exon inclusion during splicing of SMN2 and is used for SMA treatment. On the other hand, eteplirsen is an oligonucleotide promoting exon skipping during splicing of mutated DMD and has been conditionally approved for DMD treatment. Moreover, small molecule modulators of alternative splicing (e.g. branaplam) are also described. The dynamic developments in this field should result in the approval of new drugs acting by the modulation of alternative splicing in the nearest future.Powstająca w wyniku transkrypcji genu cząsteczka pre-mRNA zawiera odcinki kodujące (eksony) poprzedzielane odcinkami niekodującymi (intronami). Pierwotny transkrypt wymaga obróbki, która polega między innymi na wycinaniu intronów i łączeniu eksonów. Proces ten nazywany jest składaniem RNA. W przypadku niemal wszystkich transkryptów, składanie pre-mRNA przebiega w komórkach alternatywnymi drogami, na przykład prowadząc do wykluczenia jednego z eksonów z ostatecznego transkryptu, lub włączenia jednego z intronów. Proces składania RNA przeprowadzany jest przez skomplikowaną maszynerię złożoną z małych cząsteczek RNA i białek, i podlega precyzyjnej regulacji. Wiele chorób związanych jest z nieprawidłowościami alternatywnego składania RNA, a modulacja tego procesu może być wykorzystana terapeutycznie, między innymi w leczeniu rdzeniowego zaniku mięśni (SMA, ang. spinal muscular atrophy) czy dystrofii mięśniowej Duchenne’a (DMD). Celem artykułu jest przedstawienie wiedzy na temat farmakologicznych możliwości wpływania na proces składania RNA. Nacisk położono na scharakteryzowanie tych terapeutyków, które zostały już zarejestrowane do użytku klinicznego, lub które są w trakcie zaawansowanych badań klinicznych. Zaprezentowano budowę chemiczną i mechanizm działania oligonukleotydowych przełączników składania RNA. Nusinersen stymuluje włączanie eksonu 7 w trakcie składania SMN2 i jest wykorzystywany w leczeniu SMA. Eteplirsen, który stymuluje pomijanie eksonu 51 w trakcie składania zmutowanych wariantów DMD, został warunkowo dopuszczony do leczenia DMD w USA. Opisano także drobnocząsteczkowe modulatory alternatywnego składnia RNA, np. branaplam. Dynamiczny rozwój tego obszaru badań stwarza szansę na wprowadzone w najbliższej przyszłości do lecznictwa kolejnych leków, których mechanizm działania oparty będzie na modulacji alternatywnego składania RNA

The Significance of the Dysregulation of Canonical Wnt Signaling in Head and Neck Squamous Cell Carcinomas

The knowledge about the molecular alterations which are found in head and neck squamous cell carcinomas (HNSCC) has much increased in recent years. However, we are still awaiting the translation of this knowledge to new diagnostic and therapeutic options. Among the many molecular changes that are detected in head and neck cancer, the abnormalities in several signaling pathways, which regulate cell proliferation, cell death and stemness, seem to be especially promising with regard to the development of targeted therapies. Canonical Wnt signaling is a pathway engaged in the formation of head and neck tissues, however it is not active in adult somatic mucosal cells. The aim of this review paper is to bring together significant data related to the current knowledge on the mechanisms and functional significance of the dysregulation of the Wnt/β-catenin pathway in head and neck tumors. Research evidence related to the role of Wnt signaling activation in the stimulation of cell proliferation, migration and inhibition of apoptosis in HNSCC is presented. Moreover, its role in promoting stemness traits in head and neck cancer stem-like cells is described. Evidence corroborating the hypothesis that the Wnt signaling pathway is a very promising target of novel therapeutic interventions in HNSCC is also discussed

The Wnt Signaling Pathway Inhibitors Improve the Therapeutic Activity of Glycolysis Modulators against Tongue Cancer Cells

Excessive glucose metabolism and disruptions in Wnt signaling are important molecular changes present in oral cancer cells. The aim of this study was to evaluate the effects of the combinatorial use of glycolysis and Wnt signaling inhibitors on viability, cytotoxicity, apoptosis induction, cell cycle distribution and the glycolytic activity of tongue carcinoma cells. CAL 27, SCC-25 and BICR 22 tongue cancer cell lines were used. Cells were treated with inhibitors of glycolysis (2-deoxyglucose and lonidamine) and of Wnt signaling (PRI-724 and IWP-O1). The effects of the compounds on cell viability and cytotoxicity were evaluated with MTS and CellTox Green tests, respectively. Apoptosis was evaluated by MitoPotential Dye staining and cell cycle distribution by staining with propidium iodide, followed by flow cytometric cell analysis. Glucose and lactate concentrations in a culture medium were evaluated luminometrically. Combinations of 2-deoxyglucose and lonidamine with Wnt pathway inhibitors were similarly effective in the impairment of oral cancer cells’ survival. However, the inhibition of the canonical Wnt pathway by PRI-724 was more beneficial, based on the glycolytic activity of the cells. The results point to the therapeutic potential of the combination of low concentrations of glycolytic modulators with Wnt pathway inhibitors in oral cancer cells

The Emerging Significance of Histone Lysine Demethylases as Prognostic Markers and Therapeutic Targets in Head and Neck Cancers

Epigenetic aberrations, associated with altered DNA methylation profiles and global changes in the level of histone modifications, are commonly detected in head and neck squamous cell carcinomas (HNSCC). Recently, histone lysine demethylases have been implicated in the pathogenesis of HNSCC and emerged as potential molecular targets. Histone lysine demethylases (KDMs) catalyze the removal of methyl groups from lysine residues in histones. By affecting the methylation of H3K4, H3K9, H3K27, or H3K36, these enzymes take part in transcriptional regulation, which may result in changes in the level of expression of tumor suppressor genes and protooncogenes. KDMs are involved in many biological processes, including cell cycle control, senescence, DNA damage response, and heterochromatin formation. They are also important regulators of pluripotency. The overexpression of most KDMs has been observed in HNSCC, and their inhibition affects cell proliferation, apoptosis, cell motility, invasiveness, and stemness. Of all KDMs, KDM1, KDM4, KDM5, and KDM6 proteins are currently regarded as the most promising prognostic and therapeutic targets in head and neck cancers. The aim of this review is to present up-to-date knowledge on the significance of histone lysine demethylases in head and neck carcinogenesis and to discuss the possibility of using them as prognostic markers and pharmacological targets in patients’ treatment

THE EFFECT OF NICLOSAMIDE ON THE HEAD AND NECK CARCINOMA CELLS SURVIVAL AND THE EXPRESSION OF WNT/β-CATENIN SIGNALING AND GLYCOLYSIS PATHWAY COMPONENTS

The aim of this study was to evaluate the effect of niclosamide, an antihelminthic drug recently identified as potential anti-cancer agent, on head and neck squamous carcinoma cells (HNSCC) viability, cell cycle distribution and apoptosis. The expression of key components of Wnt (CTNNB1, GSK-3β, CCND1, c-MYC, MMP7, BIRC5, Axin2) and glycolysis (GLUT1, MCT1, HK2, PFKM, PKM2, PDHA1, PDK1, LDHA) pathways was also examined to assess possible involvement in niclosamide anti-carcinogenic activity. HNSCC cells (FaDu, BICR6, H314 lines) were used in the research. Niclosamide treatment affected hypopharyngeal FaDu cells to the most extent (IC50 = 0.40 µM), while H314 cells derived from the floor of mouth were the least sensitive (IC50 = 0.94 µM). In FaDu cells the increased percentage of the cells in the S phase was observed along with the induction of apoptosis. Treatment with niclosamide in FaDu cells reduced the expression of MMP7 and the majority of glycolytic genes except increased LDHA. These results indicate that niclosamide is efficient inhibitor of HNSCC cells viability, however this effect depends on the cell type. In FaDu cells, the most sensitive to its anti-proliferative effect and prone to cell cycle arrest and apoptosis, this effect might be related to slightly modulation of canonical Wnt signaling and increased expression of LDHA

THE COMPARISON OF THE EFFECTS OF PANOBINOSTAT AND PKF118-310 ON β-CATENIN-DEPENDENT TRANSCRIPTION IN HEAD AND NECK SQUAMOUS CELL CARCINOMA CELL LINES

Advanced head and neck squamous cell cancers (HNSCC) have unfavorable prognosis and new therapeutic options are necessary to improve treatment outcomes. The Wnt pathway plays an important role in the pathogenesis and progression of HNSCC. The aim of this study was to assess the effects of a histone deacetylase inhibitor – panobinostat on Wnt-dependent gene expression and on cell migration. Cell viability in HNSCC cell lines (BICR6, CAL27, FaDu, H314, SCC-25) was evaluated by MTT assay. The expression of β-catenin-target genes was assessed by qPCR and TCF/LEF-dependent reporter assay. Protein content was evaluated by Western blot. Cell migration was analyzed by the wound healing assay. Panobinostat showed differential modulation of gene expression. It reduced the level of Axin2 in CAL27 and SCC-25 cells but upregulated its expression in BICR6 and H314 cell lines. Moreover, it diminished the expression of MMP7 in BICR6, H314 and CAL27 cell lines. In contrast, the inhibitor of β-catenin transcriptional activity – PKF118-310 down-regulated the expression of β-catenin-target genes in HNSCC cell lines. Interestingly, panobinostat had opposite effects on cell migration in CAL27 and FaDu where it inhibited or stimulated migration, respectively. On the other hand, PKF118-310 reduced cell migration. The anti-cancer effects of panobinostat in HNSCC cells are rather not related to the inhibition of Wnt signaling. PKF118-310 attenuates Wnt signaling, but only in a limited number of HNSCC cell lines. Importantly, the inhibition of Wnt pathway reduces the capacity of cells for migration suggesting that it may potentially therapeutically reduce cell invasion

Analysies of implemetation nanocrystalic materials in power electronics

W artykule przedstawiono analizę możliwości zastosowania nowoczesnych, nanokrystalicznych materiałów magnetycznych w przekształtnikach energoelektronicznych. Wyodrębniono dwa obszary zastosowań. Pierwszy z nich to elementy filtrów LCL pozwalających na kształtowanie prądu w układach prostowników aktywnych, z jednoczesną poprawą niekorzystnego wpływu takich przekształtników na sieć zasilającą. Drugi obszar to transformatory podwyższonej częstotliwości w układach przetwarzania energii, zwłaszcza ładowarkach akumulatorów dużej mocy. W obu tych przypadkach zastosowanie materiałów nanokrystalicznych mogłoby poprawić właściwości układów przez redukcję wymiarów, zmniejszenie strat czy hałasu. Zaproponowano także dwa stanowiska testowe pozwalające na weryfikację wyżej przedstawionej tezy.The paper presents an analyses of possible application the modern nanocomposite materials in the power electronics. The applications area was divided into the two subsystems. The first application is input LCL filters in active front end converters for energy recuperation. The second one is HF transformers for DC/DC converters, mainly in high power battery chargers. In the both application area the nanocomposite materials could improve its properties by mass and noise reduction or higher efficiency of energy conversion. In the end of the paper the two setups was proposed which can proof the thesis

Prognostic factors in oral and oropharyngeal cancer based on ultrastructural analysis and DNA methylation of the tumor and surgical margin

Oral and oropharyngeal cancers are characterized by relatively low 5- year survival rates due to many factors, including local recurrence. The identification of new molecular markers may serve for the estimation of prognosis and thus augment treatment decisions and affect therapy outcome. The aim of this study was to describe the morphological characteristics and the DNA methylation status of the CDKN2A,CDH1, ATM, FHIT and RAR- genes in the central and peripheral part of the tumor and the surgical margin and evaluate their prognostic significance. 53 patients with oral and oropharyngeal cancer were enrolled to the prospective study, and had been primarily treated surgically. Correlations between morphological data, hypermethylation status and clinicopathological data, as well as prognosis, were assessed. Nuclei polymorphism highly correlated with T stage (p < 0.0001), N stage (p < 0.046), and metastases to the lymph nodes pN (p < 0.004 ). Also, the number of cells in irregular mitosis correlated with T stage (p < 0.004), and highly with pN (p < 0.009). The significance of CDKN2A hypermethylation as a good prognostic factor was also established in the Kaplan-Meir test. The ultrastructural analysis showed that none of the examined tumors had homogenous texture and that resection margin specimens clean in HE stained tissue samples frequently contained single tumor cells or few cells in groups surrounded by connective tissue. This indicates the superiority of electron microscopy over standard histopathological analysis. Thus, a combination of such morphological examination with epigenetic parameters described herein could result in the discovery of promising new prognostic markers of the disease