New Avenues and Major Achievements in Phytocompounds Research for Glioblastoma Therapy.

Phytocompounds have been evaluated for their anti-glioblastoma actions for decades, with promising results from preclinical studies but only limited translation into clinics. Indeed, by targeting multiple signaling pathways deregulated in cancer, they often show high efficacy in the in vitro studies, but their poor bioavailability, low tumor accumulation, and rapid clearance compromise their efficacy in vivo. Here, we present the new avenues in phytocompound research for the improvement of glioblastoma therapy, including the ways to enhance the response to temozolomide using phytochemicals, the current focus on phytocompound-based immunotherapy, or the use of phytocompounds as photosensitizers in photodynamic therapy. Moreover, we present new, intensively evaluated approaches, such as chemical modifications of phytochemicals or encapsulation into numerous types of nanoformulations, to improve their bioavailability and delivery to the brain. Finally, we present the clinical trials evaluating the role of phytocompounds or phytocompound-derived drugs in glioblastoma therapy and the less studied phytocompounds or plant extracts that have only recently been found to possess promising anti-glioblastoma properties. Overall, recent advancements in phytocompound research are encouraging; however, only with more 3D glioblastoma models, in vivo studies, and clinical trials it is possible to upgrade the role of phytocompounds in glioblastoma treatment to a satisfactory level.

CRISPR/Cas9-induced knockout reveals the role of ABCB1 in the response to temozolomide, carmustine and lomustine in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor with limited therapeutic options. Besides surgery, chemotherapy using temozolomide, carmustine or lomustine is the main pillar of therapy. However, therapy success is limited and prognosis still is very poor. One restraining factor is drug resistance caused by drug transporters of the ATP-binding cassette family, e.g. ABCB1 and ABCG2, located at the blood-brain barrier and on tumor cells. The active efflux of xenobiotics including drugs, e.g. temozolomide, leads to low intracellular drug concentrations and subsequently insufficient anti-tumor effects. Nevertheless, the role of efflux transporters in GBM is controversially discussed. In the present study, we analyzed the role of ABCB1 and ABCG2 in GBM cells showing that ABCB1, but marginally ABCG2, is relevant. Applying a CRISPR/Cas9-derived ABCB1 knockout, the response to temozolomide was significantly augmented demonstrated by decreased cell number (p < 0.001) and proliferation rate (p = 0.04), while apoptosis was increased (p = 0.04). For carmustine, a decrease of cells in G1-phase was detected pointing to cell cycle arrest in the ABCB1 knockout (p = 0.006). For lomustine, however, loss of ABCB1 did not alter the response to the treatment. Overall, this study shows that ABCB1 is involved in the active transport of temozolomide out of the tumor cells diminishing the response to temozolomide. Interestingly, loss of ABCB1 also affected the response to the lipophilic drug carmustine. These findings show that ABCB1 is not only relevant at the blood-brain barrier, but also in the tumor cells diminishing success of chemotherapy.

Cannabidiol and Its Combinations with Nonsteroidal Anti-Inflammatory Drugs Induce Apoptosis and Inhibit Activation of NF-κB Signaling in Vulvar Squamous Cell Carcinoma.

Vulvar squamous cell carcinoma (VSCC) is a rare malignancy with a relatively good prognosis. However, the prognosis remains poor for elderly patients and those with a significant depth of tumor invasion; thus, novel treatment modalities are needed. The aim of this study was to analyze the impact of cannabidiol (CBD) and its combination with NSAIDs, diclofenac (DIC) and ibuprofen (IBU) on VSCC cells. In this regard, the MTT test was applied for cytotoxicity analysis. Moreover, the influence of CBD, DIC and IBU, as well as their combinations, on apoptosis and cell cycle distribution were analyzed by flow cytometry. The mechanisms of action of the analyzed compounds, including their impact on NF-κB signaling, p53 and COX-2 expression were evaluated using Western blot. This study shows that CBD and its combinations with NSAIDs are cytotoxic to A431 cells, but they also reduce, in a dose-dependent manner, the viability of immortalized keratinocyte HaCaT cells, and human umbilical vein cell line, EA.hy926. Moreover, the compounds and their combinations induced apoptosis, diminished the NF-κB signaling activation and reduced COX-2 expression. We conclude that CBD and its combination with DIC or IBU are promising candidates for the adjuvant treatment of high-risk VSCC patients. However, their impact on non-cancerous cells requires careful evaluation.

COXIBs and 2,5-dimethylcelecoxib counteract the hyperactivated Wnt/ß-catenin pathway and COX-2/PGE2/EP4 signaling in glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) is the deadliest and the most common primary brain tumor in adults. The invasiveness and proliferation of GBM cells can be decreased through the inhibition of Wnt/ß-catenin pathway. In this regard, celecoxib is a promising agent, but other COXIBs and 2,5-dimethylcelecoxib (2,5-DMC) await elucidation. Thus, the aim of this study was to analyze the impact of celecoxib, 2,5-DMC, etori-, rofe-, and valdecoxib on GBM cell viability and the activity of Wnt/ß-catenin pathway. In addition, the combination of the compounds with temozolomide (TMZ) was also evaluated. Cell cycle distribution and apoptosis, MGMT methylation level, COX-2 and PGE2 EP4 protein levels were also determined in order to better understand the molecular mechanisms exerted by these compounds and to find out which of them can serve best in GBM therapy. METHODS: Celecoxib, 2,5-DMC, etori-, rofe- and valdecoxib were evaluated using three commercially available and two patient-derived GBM cell lines. Cell viability was analyzed using MTT assay, whereas alterations in MGMT methylation level were determined using MS-HRM method. The impact of COXIBs, in the presence and absence of TMZ, on Wnt pathway was measured on the basis of the expression of ß-catenin target genes. Cell cycle distribution and apoptosis analysis were performed using flow cytometry. COX-2 and PGE2 EP4 receptor expression were evaluated using Western blot analysis. RESULTS: Wnt/ß-catenin pathway was attenuated by COXIBs and 2,5-DMC irrespective of the COX-2 expression profile of the treated cells, their MGMT methylation status, or radio/chemoresistance. Celecoxib and 2,5-DMC were the most cytotoxic. Cell cycle distribution was altered, and apoptosis was induced after the treatment with celecoxib, 2,5-DMC, etori- and valdecoxib in T98G cell line. COXIBs and 2,5-DMC did not influence MGMT methylation status, but inhibited COX-2/PGE2/EP4 pathway. CONCLUSIONS: Not only celecoxib, but also 2,5-DMC, etori-, rofe- and valdecoxib should be further investigated as potential good anti-GBM therapeutics.

Sodium Butyrate Enhances Curcuminoids Permeability through the Blood-Brain Barrier, Restores Wnt/ß-Catenin Pathway Antagonists Gene Expression and Reduces the Viability of Glioblastoma Cells.

Glioblastoma (GBM) is an extremely aggressive brain tumor awaiting novel, efficient, and minimally toxic treatment. Curcuminoids (CCM), polyphenols from Curcuma longa, and sodium butyrate (NaBu), a histone deacetylase inhibitor naturally occurring in the human body, await elucidation as potential anti-GBM agents. Thus, the aim of this study was to analyze CCM and NaBu both separately and as a combination treatment using three GBM cell lines. MTT was used for cytotoxicity evaluation, and the combination index was calculated for synergism prediction. Cell cycle, apoptosis, and reactive oxygen species (ROS) generation were analyzed using flow cytometry. DNA methylation was verified by MS-HRM and mRNA expression by qPCR. The permeability through the blood-brain barrier (BBB) and through the nasal cavity was evaluated using PAMPA model. The results of this study indicate that CCM and NaBu synergistically reduce the viability of GBM cells inducing apoptosis and cell cycle arrest. These effects are mediated via ROS generation and changes in gene expression, including upregulation of Wnt/ß-catenin pathway antagonists, SFRP1, and RUNX3, and downregulation of UHRF1, the key epigenetic regulator. Moreover, NaBu ameliorated CCM permeability through the BBB and the nasal cavity. We conclude that CCM and NaBu are promising agents with anti-GBM properties.

The methylation of a panel of genes differentiates low-grade from high-grade gliomas.

Epigenetic changes play an important role in the pathogenesis of gliomas and have the potential to become clinically useful biomarkers. The aim of this study was the evaluation of the profile of promoter methylation of 13 genes selected based on their anticipated diagnostic and/or prognostic value. Methylation-specific PCR (MSP) was used to assess the methylation status of MGMT, ERCC1, hMLH1, ATM, CDKN2B (p15INK4B), p14ARF, CDKN2A (p16INK4A), RASSF1A, RUNX3, GATA6, NDRG2, PTEN, and RARß in a subset of 95 gliomas of different grades. Additionally, the methylation status of MGMT and NDRG2 was analyzed using pyrosequencing (PSQ). The results revealed that the methylation index of individual glioma patients correlates with World Health Organization (WHO) tumor grade and patient's age. RASSF1A, RUNX3, GATA6, and MGMT were most frequently methylated, whereas the INK4B-ARF-INK4A locus, PTEN, RARß, and ATM were methylated to a lesser extent. ERCC1, hMLH1, and NDRG2 were unmethylated. RUNX3 methylation correlated with WHO tumor grade and patient's age. PSQ confirmed significantly higher methylation levels of MGMT and NDRG2 as compared with normal, non-cancerous brain tissue. To conclude, DNA methylation of a whole panel of selected genes can serve as a tool for glioma aggressiveness prediction.

Chromatinopathies: insight in clinical aspects and underlying epigenetic changes.

Chromatinopathies (CPs), a group of rare inborn defects characterized by chromatin state imbalance, have evolved from initially resembling Cornelia de Lange syndrome to encompass a wide array of genetic diseases with diverse clinical presentations. The CPs classification now includes human developmental disorders caused by germline mutations in epigenes, genes that regulate the epigenome. Recent advances in next-generation sequencing have enabled the association of 154 epigenes with CPs, revealing distinctive DNA methylation patterns known as episignatures.It has been shown that episignatures are unique for a particular CP or share similarities among specific CP subgroup. Consequently, these episignatures have emerged as promising biomarkers for diagnosing and treating CPs, differentiating subtypes, evaluating variants of unknown significance, and facilitating targeted therapies tailored to the underlying epigenetic dysregulation.The following review was conducted to collect, summarize, and analyze data regarding CPs in such aspects as clinical evaluation encompassing long-term patient care, underlying epigenetic changes, and innovative molecular and bioinformatic methodologies that have been devised for the assessment of CPs. We have also shed light on promising novel treatment options that have surfaced in recent research and presented a synthesis of ongoing clinical trials, contributing to the current understanding of the dynamic and evolving nature of CPs investigation.

[New cytochrome P450 isoforms as cancer biomarkers and targets for chemopreventive and chemotherapeutic agents]. / Nowe cytochromy P450 jako biomarkery i potencjalne cele oddzialywania w chemioprewencji i terapii nowotworów.

Cytochromes P450 (P450) are a multigene family of enzymes possessing a central role in the oxidative metabolism of a wide range of xenobiotics, including anticancer drugs, and endogenous compounds. The activity of different P450 isoforms varies within specific tissues and cell types and is selectively regulated together with their gene expression. Moreover, differential expression of certain P450 isoforms' genes in tumor cells compared to normal tissues can be observed. This creates the potential for the use of these isozymes as tumor markers or selective prodrug activators. This article discusses the characteristics and function of five isoforms of cytochrome P450 (P450 1B1, P450 2W1, P450 2S1, P450 2R1, P450 2U1) that could be potential targets for tumor therapeutic and preventive strategies. These isoforms have been chosen because their level of expression in tumor tissues is definitely higher than in normal tissues.

Tannins in cancer prevention and therapy.

Tannins are a heterogenous class of polyphenolic natural products with promising cancer chemopreventive and therapeutic potential. Studies undertaken over the last 30 years have demonstrated their capacity to target many cellular pathways and molecules important in the development of cancer. Recently, new mechanisms that might be important in anti-carcinogenic activity, such as inhibition of epithelial-to-mesenchymal transition, reduction of cancer stem cell creation, and modulation of cancer cells metabolism have been described. Along with the mechanisms underlying the anti-cancer activity of tannins, this review focuses on their possible application as chemosensitizers in adjuvant therapy and countering multidrug resistance. Furthermore, characteristic physicochemical properties of some tannins, particularly tannic acid, are useful in the formation of nanovehicles for anticancer drugs or the isolation of circulating cancer cells. These new potential applications of tannins deserve further studies. Well-designed clinical trials, which are scarce, are needed to assess the therapeutic effects of tannins themselves or as adjuvants in cancer treatment.

Targeting Nrf2 Signaling Pathway in Cancer Prevention and Treatment: The Role of Cannabis Compounds.

The development and progression of cancer are associated with the dysregulation of multiple pathways involved in cell proliferation and survival, as well as dysfunction in redox balance, immune response, and inflammation. The master antioxidant pathway, known as the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, regulates the cellular defense against oxidative stress and inflammation, making it a promising cancer prevention and treatment target. Cannabinoids have demonstrated anti-tumor and anti-inflammatory properties, affecting signaling pathways, including Nrf2. Increased oxidative stress following exposure to anti-cancer therapy prompts cancer cells to activate antioxidant mechanisms. This indicates the dual effect of Nrf2 in cancer cells-influencing proliferation and apoptotic processes and protecting against the toxicity of anti-cancer therapy. Therefore, understanding the complex role of cannabinoids in modulating Nrf2 might shed light on its potential implementation as an anti-cancer support. In this review, we aim to highlight the impact of cannabinoids on Nrf2-related factors, with a focus on cancer prevention and treatment. Additionally, we have presented the results of several research studies that combined cannabidiol (CBD) with other compounds targeting Nrf2. Further studies should be directed toward exploring the anti-inflammatory effects of cannabinoids in the context of cancer prevention and therapy.

Honokiol-Loaded Nanoemulsion for Glioblastoma Treatment: Statistical Optimization, Physicochemical Characterization, and an In Vitro Toxicity Assay.

BACKGROUND: Glioblastoma (GBM) is an extremely invasive and heterogenous malignant brain tumor. Despite advances in current anticancer therapy, treatment options for glioblastoma remain limited, and tumor recurrence is inevitable. Therefore, alternative therapies or new active compounds that can be used as adjuvant therapy are needed. This study aimed to develop, optimize, and characterize honokiol-loaded nanoemulsions intended for intravenous administration in glioblastoma therapy. METHODS: Honokiol-loaded nanoemulsion was developed by incorporating honokiol into Lipofundin MCT/LCT 20% using a horizontal shaker. The Box-Behnken design, coupled with response surface methodology, was used to optimize the incorporation process. The effect of the developed formulation on glioblastoma cell viability was determined using the MTT test. Long-term and short-term stress tests were performed to evaluate the effect of honokiol on the stability of the oil-in-water system and the effect of different stress factors on the stability of honokiol, respectively. Its physicochemical properties, such as MDD, PDI, ZP, OSM, pH, and loading efficiency (LE%), were determined. RESULTS: The optimized honokiol-loaded nanoemulsion was characterized by an MDD of 201.4 (0.7) nm with a PDI of 0.07 (0.02) and a ZP of -28.5 (0.9) mV. The LE% of honokiol was above 95%, and pH and OSM were sufficient for intravenous administration. The developed formulation was characterized by good stability and a satisfactory toxicity effect of the glioblastoma cell lines. CONCLUSIONS: The honokiol-loaded nanoemulsion is a promising pharmaceutical formulation for further development in the adjuvant therapy of glioblastoma.

Etoricoxib-Cannabidiol Combo: Potential Role in Glioblastoma Treatment and Development of PLGA-Based Nanoparticles.

BACKGROUND: Glioblastoma (GBM) is the most frequently occurring primary malignant central nervous system tumor, with a poor prognosis and median survival below two years. Administration of a combination of non-steroidal anti-inflammatory drugs and natural compounds that exhibit a curative or prophylactic effect in cancer is a new approach to GBM treatment. This study aimed to investigate the synergistic antitumor activity of etoricoxib (ETO) and cannabidiol (CBD) in a GBM cell line model, and to develop poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) for these two substances. METHODS: The activity of ETO+CBD was determined using the MTT test, cell-cycle distribution assay, and apoptosis analysis using two GBM cell lines, namely, T98G and U-138 MG. The PLGA-based NPs were developed using the emulsification and solvent evaporation method. Their physicochemical properties, such as shape, size, entrapment efficiency (EE%), in vitro drug release, and quality attributes, were determined using scanning electron microscopy, diffraction light scattering, high-performance liquid chromatography, infrared spectroscopy, and differential scanning calorimetry. RESULTS: The combination of ETO and CBD reduced the viability of cells in a dose-dependent manner and induced apoptosis in both tested GBM cell lines. The developed method allowed for the preparation of ETO+CBD-NPs with a spherical shape, mean particle size (MPS) below 400 nm, zeta potential (ZP) values from -11 to -17.4 mV, polydispersity index (PDI) values in the range from 0.029 to 0.256, and sufficient EE% of both drugs (78.43% for CBD, 10.94% for ETO). CONCLUSIONS: The combination of ETO and CBD is a promising adjuvant therapeutic in the treatment of GBM, and the prepared ETO+CBD-NPs exhibit a high potential for further pharmaceutical formulation development.

ABCB1 Is Frequently Methylated in Higher-Grade Gliomas and May Serve as a Diagnostic Biomarker of More Aggressive Tumors.

ABCB1 belongs to a superfamily of membrane transporters that use ATP hydrolysis to efflux various endogenous compounds and drugs outside the cell. Cancer cells upregulate ABCB1 expression as an adaptive response to evade chemotherapy-mediated cell death. On the other hand, several reports highlight the role of the epigenetic regulation of ABCB1 expression. In fact, the promoter methylation of ABCB1 was found to be methylated in several tumor types, including gliomas, but its role as a biomarker is not fully established yet. Thus, the aim of this study was to analyze the methylation of the ABCB1 promoter in tumor tissues from 50 glioma patients to verify its incidence and to semi-quantitively detect ABCB1 methylation levels in order to establish its utility as a potential biomarker. The results of this study show a high interindividual variability in the ABCB1 methylation level of the samples derived from gliomas of different grades. Additionally, a positive correlation between ABCB1 methylation, the WHO tumor grade, and an IDH1 wild-type status has been observed. Thus, ABCB1 methylation can be regarded as a potential diagnostic or prognostic biomarker for glioma patients, indicating more aggressive tumors.

Is Caperatic Acid the Only Compound Responsible for Activity of Lichen Platismatia glauca within the Nervous System?

Lichens are a source of various biologically active compounds. However, the knowledge about them is still scarce, and their use in medicine is limited. This study aimed to investigate the therapeutic potential of the lichen Platismatia glauca and its major metabolite caperatic acid in regard to their potential application in the treatment of central nervous system diseases, especially neurodegenerative diseases and brain tumours, such as glioblastoma. First, we performed the phytochemical analysis of the tested P. glauca extracts based on FT-IR derivative spectroscopic and gas chromatographic results. Next the antioxidant properties were determined, and moderate anti-radical activity, strong chelating properties of Cu2+ and Fe2+ ions, and a mild effect on the antioxidant enzymes of the tested extracts and caperatic acid were proved. Subsequently, the influence of the tested extracts and caperatic acid on cholinergic transmission was determined by in vitro and in silico studies confirming that inhibitory effect on butyrylcholinesterase is stronger than against acetylcholinesterase. We also confirmed the anti-inflammatory properties of P. glauca extracts and caperatic acid using a COX-2 and hyaluronidase inhibition models. Moreover, our studies show the cytotoxic and pro-apoptotic activity of the P. glauca extracts against T98G and U-138 MG glioblastoma multiforme cell lines. In conclusion, it is possible to assume that P. glauca extracts and especially caperatic acid can be regarded as the source of the valuable substances to finding new therapies of central nervous system diseases.

Lichen Secondary Metabolites Inhibit the Wnt/ß-Catenin Pathway in Glioblastoma Cells and Improve the Anticancer Effects of Temozolomide.

Lichens are a source of secondary metabolites with significant pharmacological potential. Data regarding their possible application in glioblastoma (GBM) treatment are, however, scarce. The study aimed at analyzing the mechanism of action of six lichen secondary metabolites: atranorin, caperatic acid, physodic acid, squamatic acid, salazinic acid, and lecanoric acid using two- and three-dimensional GBM cell line models. The parallel artificial membrane permeation assay was used to predict the blood-brain barrier penetration ability of the tested compounds. Their cytotoxicity was analyzed using the MTT test on A-172, T98G, and U-138 MG cells. Flow cytometry was applied to the analysis of oxidative stress, cell cycle distribution, and apoptosis, whereas qPCR and microarrays detected the induced transcriptomic changes. Our data confirm the ability of lichen secondary metabolites to cross the blood-brain barrier and exert cytotoxicity against GBM cells. Moreover, the compounds generated oxidative stress, interfered with the cell cycle, and induced apoptosis in T98G cells. They also inhibited the Wnt/ß-catenin pathway, and this effect was even stronger in case of a co-treatment with temozolomide. Transcriptomic changes in cancer related genes induced by caperatic acid and temozolomide were the most pronounced. Lichen secondary metabolites, caperatic acid in particular, should be further analyzed as potential anti-GBM agents.

Natural Compounds in Liposomal Nanoformulations of Potential Clinical Application in Glioblastoma.

Glioblastoma (GBM) is the most common malignant neoplasm in adults among all CNS gliomas, with the 5-year survival rate being as low as 5%. Among nanocarriers, liposomal nanoformulations are considered as a promising tool for precise drug delivery. The herein presented study demonstrates the possibility of encapsulating four selected natural compounds (curcumin, bisdemethoxycurcumin, acteoside, and orientin) and their mixtures in cationic liposomal nanoformulation composed of two lipid types (DOTAP:POPC). In order to determine the physicochemical properties of the new drug carriers, specific measurements, including particle size, Zeta Potential, and PDI index, were applied. In addition, NMR and EPR studies were carried out for a more in-depth characterization of nanoparticles. Within biological research, the prepared formulations were evaluated on T98G and U-138 MG glioblastoma cell lines in vitro, as well as on a non-cancerous human lung fibroblast cell line (MRC-5) using the MTT test to determine their potential as anticancer agents. The highest activity was exhibited by liposome-entrapped acteoside towards the T98G cell line with IC50 equal 2.9 ± 0.9 µM after 24 hours of incubation. Noteworthy, curcumin and orientin mixture in liposomal formulation exhibited a synergistic effect against GBM. Moreover, the impact on the expression of apoptosis-associated proteins (p53 and Caspase-3) of acteoside as well as curcumin and orientin mixture, as the most potent agents, was assessed, showing nearly 40% increase as compared to control U-138 MG and T98G cells. It should be emphasized that a new and alternative method of extrusion of the studied liposomes was developed.

Novel Approaches to Epigenetic Therapies: From Drug Combinations to Epigenetic Editing.

Cancer development involves both genetic and epigenetic alterations. Aberrant epigenetic modifications are reversible, allowing excellent opportunities for therapeutic intervention. Nowadays, several epigenetic drugs are used worldwide to treat, e.g., myelodysplastic syndromes and leukemias. However, overcoming resistance and widening the therapeutic profiles are the most important challenges faced by traditional epigenetic drugs. Recently, novel approaches to epigenetic therapies have been proposed. Next-generation epigenetic drugs, with longer half-life and better bioavailability, are being developed and tested. Since epigenetic phenomena are interdependent, treatment modalities include co-administration of two different epigenetic drugs. In order to sensitize cancer cells to chemotherapy, epigenetic drugs are administered prior to chemotherapy, or both epigenetic drug and chemotherapy are used together to achieve synergistic effects and maximize treatment efficacy. The combinations of epigenetic drug with immunotherapy are being tested, because they have proved to enhance antitumor immune responses. The next approach involves targeting the metabolic causes of epigenetic changes, i.e., enzymes which, when mutated, produce oncometabolites. Finally, epigenome editing makes it possible to modify individual chromatin marks at a defined region with unprecedented specificity and efficiency. This review summarizes the above attempts in fulfilling the promise of epigenetic drugs in the effective cancer treatment.

Lichen-Derived Compounds and Extracts as Biologically Active Substances with Anticancer and Neuroprotective Properties.

Lichens are a source of chemical compounds with valuable biological properties, structurally predisposed to penetration into the central nervous system (CNS). Hence, our research aimed to examine the biological potential of lipophilic extracts of Parmelia sulcata, Evernia prunastri, Cladonia uncialis, and their major secondary metabolites, in the context of searching for new therapies for CNS diseases, mainly glioblastoma multiforme (GBM). The extracts selected for the study were standardized for their content of salazinic acid, evernic acid, and (-)-usnic acid, respectively. The extracts and lichen metabolites were evaluated in terms of their anti-tumor activity, i.e., cytotoxicity against A-172 and T98G cell lines and anti-IDO1, IDO2, TDO activity, their anti-inflammatory properties exerted by anti-COX-2 and anti-hyaluronidase activity, antioxidant activity, and anti-acetylcholinesterase and anti-butyrylcholinesterase activity. The results of this study indicate that lichen-derived compounds and extracts exert significant cytotoxicity against GBM cells, inhibit the kynurenine pathway enzymes, and have anti-inflammatory properties and weak antioxidant and anti-cholinesterase properties. Moreover, evernic acid and (-)-usnic acid were shown to be able to cross the blood-brain barrier. These results demonstrate that lichen-derived extracts and compounds, especially (-)-usnic acid, can be regarded as prototypes of pharmacologically active compounds within the CNS, especially suitable for the treatment of GBM.

Permeability of Hypogymnia physodes Extract Component-Physodic Acid through the Blood-Brain Barrier as an Important Argument for Its Anticancer and Neuroprotective Activity within the Central Nervous System.

Lichen secondary metabolites are characterized by huge pharmacological potential. Our research focused on assessing the anticancer and neuroprotective activity of Hypogymnia physodes acetone extract (HP extract) and physodic acid, its major component. The antitumor properties were evaluated by cytotoxicity analysis using A-172, T98G, and U-138 MG glioblastoma cell lines and by hyaluronidase and cyclooxygenase-2 (COX-2) inhibition. The neuroprotective potential was examined using COX-2, tyrosinase, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) activity tests. Moreover, the antioxidant potential of the tested substances was examined, and the chemical composition of the extract was analyzed. For physodic acid, the permeability through the blood-brain barrier using Parallel Artificial Membrane Permeability Assay for the Blood-Brain Barrier assay (PAMPA-BBB) was assessed. Our study shows that the tested substances strongly inhibited glioblastoma cell proliferation and hyaluronidase activity. Besides, HP extract diminished COX-2 and tyrosinase activity. However, the AChE and BChE inhibitory activity of HP extract and physodic acid were mild. The examined substances exhibited strong antioxidant activity. Importantly, we proved that physodic acid crosses the blood-brain barrier. We conclude that physodic acid and H. physodes should be regarded as promising agents with anticancer, chemopreventive, and neuroprotective activities, especially regarding the central nervous system diseases.

Methoxy-stilbenes downregulate the transcription of Wnt/ß-catenin-dependent genes and lead to cell cycle arrest and apoptosis in human T98G glioblastoma cells.

PURPOSE: Glioblastoma is the most common and the deadliest brain cancer. The aim of this study was to analyze the impact of resveratrol and its five analogs: 3,4,4'-trimethoxy, 3,4,2'-trimethoxy, 3,4,2',4'-tetramethoxy, 3,4,2',6'-tetramethoxy and 3,4,2',4',6'-pentamethoxy-trans-stilbenes (MS) on human glioblastoma T98G cells. MATERIALS AND METHODS: The Parallel Artificial Membrane Permeation Assay (PAMPA) was used for the prediction of blood-brain barrier penetration ability of the tested stilbenes (PAMPA-BBB). MTT test was applied to analyze the cytotoxicity of the compounds, whereas their ability to inhibit Wnt/ß-catenin target genes expression was verified using qPCR. The potential DNA demethylating properties of the analyzed compounds were tested by Methylation-Sensitive High Resolution Melting (MS-HRM). Cell cycle distribution was tested using Fluorescence-Activated Cell Sorting (FACS), whereas apoptosis was analyzed using FITC Annexin V/propidium iodide double staining assay and Western blot. RESULTS: High blood-brain barrier permeability coefficient was obtained for both resveratrol as well as methoxy-stilbenes. Their ability to downregulate the expression of Wnt/ß-catenin pathway-related genes was confirmed. The 4'-methoxy substituted derivatives showed higher activity, whereas 3,4,4'-tri-MS was the most potent Wnt/ß-catenin pathway inhibitor. None of the compounds affected DNA methylation level of MGMT, SFRP1, or RUNX3, despite inducing moderate changes in the level of expression of epigenetic modifiers DNMT3B and TET1-3. Importantly, treatment with 3,4,4'-tri-MS and 3,4,2',4'-tetra-MS led to cycle arrest in the S phase and induced apoptosis. CONCLUSIONS: Both, resveratrol, as well as its synthetic methoxy-derivatives, should be further studied as promising adjuvants in glioblastoma treatment.