Usnic Acid Isolated from Usnea antarctica (Du Rietz) Reduced In Vitro Angiogenesis in VEGF- and bFGF-Stimulated HUVECs and Ex Ovo in Quail Chorioallantoic Membrane (CAM) Assay.

Natural products include a diverse set of compounds of drug discovery that are currently being actively used to target tumor angiogenesis. In the present study, we evaluated the anti-angiogenic activities of secondary metabolite usnic acid isolated from Usena antarctica. We investigated the in vitro effects on proliferation, migration, and tube formation of VEGF- and bFGF-stimulated HUVECs. Ex ovo anti-angiogenic activity was evaluated using the CAM assay. Our findings demonstrated that usnic acid in the concentration of 33.57 µM inhibited VEGF (25 ng/mL) and bFGF (30 ng/mL)-induced HUVECs proliferation, migration, and tube formation. The ex ovo CAM model was used to confirm the results obtained from in vitro studies. VEGF- and bFGF-induced vessel formation was inhibited by usnic acid after 72 h in over 2-fold higher concentrations compared to in vitro. Subsequently, histological sections of affected chorioallantoic membranes were stained with hematoxylin-eosin and alcian blue to determine the number and diameter of vessels as well as the thickness of the individual CAM layers (ectoderm, mesoderm, endoderm). Usnic acid was able to suppress the formation of VEGF- and bFGF-induced vessels with a diameter of less than 100 µm, which was demonstrated by the reduction of mesoderm thickness as well.

Potential Effect of Pseudevernia furfuracea (L.) Zopf Extract and Metabolite Physodic Acid on Tumour Microenvironment Modulation in MCF-10A Cells.

Lichens comprise a number of unique secondary metabolites with remarkable biological activities and have become an interesting research topic for cancer therapy. However, only a few of these metabolites have been assessed for their effectiveness against various in vitro models. Therefore, the aim of the present study was to assess the effect of extract Pseudevernia furfuracea (L.) Zopf (PSE) and its metabolite physodic acid (Phy) on tumour microenvironment (TME) modulation, focusing on epithelial-mesenchymal transition (EMT), cancer-associated fibroblasts (CAFs) transformation and angiogenesis. Here, we demonstrate, by using flow cytometry, Western blot and immunofluorescence microscopy, that tested compounds inhibited the EMT process in MCF-10A breast cells through decreasing the level of different mesenchymal markers in a time- and dose-dependent manner. By the same mechanisms, PSE and Phy suppressed the function of Transforming growth factor beta (TGF-ß)-stimulated fibroblasts. Moreover, PSE and Phy resulted in a decreasing level of the TGF-ß canonical pathway Smad2/3, which is essential for tumour growth. Furthermore, PSE and Phy inhibited angiogenesis ex ovo in a quail embryo chorioallantoic model, which indicates their potential anti-angiogenic activity. These results also provided the first evidence of the modulation of TME by these substances.

Zinc oxide nanoparticles phytotoxicity on halophyte from genus Salicornia.

This study deals with the effect of zinc oxide nanoparticles (ZnO NPs) on halophyte from the genus Salicornia. The presence of ZnO nanoparticles (100 and 1000 mg/L) in the solid culture medium resulted in the negative effects on plant growth in the concentration-dependent manner. The shoot length of plant cultivated with 1000 mg/L ZnO NPs decreased by more than 50% compared to non-treated plants. The phytotoxicity was associated with the release of free zinc(II) ions, which was determined by atomic absorption spectroscopy and fluorescence microscopy. Another mechanism involved in ZnO NPs phytotoxicity was closely connected with generation of reactive oxygen species (ROS), which was accompanied by changes in activities and amounts of antioxidant enzymes. Histochemical evaluation showed that ROS were present also in the shoot of plant, which was not in direct contact with NPs. The reduction of activity and amount of antioxidant enzymes such as gamma-ESC, GR, SOD, PER, APX and higher concentration of ROS lead to lipid peroxidation, the latter being almost 3 times higher for the plant treated with 1000 mg/L NPs compared to control. The misbalance in zinc homeostasis and creation of ROS with subsequent oxidative stress led to the initiation of processes of programmed cell death, which was demonstrated by the loss of mitochondrial potential and increase of intracellular calcium (II) ions. Despite halophytes exhibit higher stress resistance than glycophytes, they are prone to negative changes if incubated in the environment containing ZnO nanoparticles.

Toxicity of Diclofenac in the Fern Azolla filiculoides and the Lichen Xanthoria parietina.

This study investigated the occurrence of toxicity, expressed as damage to the photosynthetic apparatus, in the aquatic fern Azolla filiculoides and the lichen Xanthoria parietina following treatments with diclofenac at different concentrations (0.1, 1, 10 and 100 mg/L) and different exposure times (24, 48, 72 and 240 h). Measurements of photosynthetic efficiency, chlorophyll content and chlorophyll degradation indicated dose- and time-dependent toxicity, since significant differences with control samples as well as among treatments, emerged mainly for the highest concentration (100 mg/L) and the longest time (240 h). In addition, also the mycobiont of the lichen X. parietina showed similar toxic effects, expressed as ergosterol content. The absence of relevant alterations at the lowest concentration (0.1 mg/L) suggested a very limited susceptibility of these species to environmentally relevant levels of this pharmaceutical.

Host use of a specialist lichen-feeder: dealing with lichen secondary metabolites.

Host use by herbivores is largely determined by host properties such as nutrient content and chemical defence against foragers. The impacts of these attributes on a herbivore may largely depend on its life cycle stage. Lichen species are known to differ in nutritional quality and level of chemical defence and, consequently, vary as fodder for herbivores. The aim of this study was to explore the impact of several lichen species and the presence of their secondary metabolites on their use as hosts by a specialist lichen-feeder, Cleorodes lichenaria. This study also addressed, for the first time, how a specialist lichen-feeder deals with different lichen secondary metabolites. In the beginning of their development, larvae grew better on Xanthoria parietina than on the other host lichens, whereas older larvae grew best on Ramalina fraxinea. Lichen secondary chemicals in R. fraxinea and Parmelia sulcata hindered larval growth in the beginning but after 75 days lichen secondary chemicals had no impact on the mass of larvae. Physodic acids in Hypogymnia physodes were lethal to larvae. In general, larvae metabolized 70-95% of ingested lichen secondary chemicals and the rest of these were excreted in frass. Lichen secondary metabolites in P. sulcata restrict and in H. physodes prevent their use as a host for C. lichenaria larvae. Our main finding, the ability of larvae to metabolize several lichen secondary metabolites, indicates digestive adaptation to these chemicals. No signs of sequestration of these chemicals were found.

Comparison of the phytotoxic effects of usnic acid on cultures of free-living alga Scenedesmus quadricauda and aposymbiotically grown lichen photobiont Trebouxia erici.

The phytotoxic effects of the lichen secondary metabolite-usnic acid on cultures of free living alga-Scenedesmus quadricauda (UTEX 76) and aposymbiotically grown lichen photobiont Trebouxia erici (UTEX 911) were assessed. We found a relatively strong inhibition effect of usnic acid on the growth of alga Scenedesmus, accompanied by an increase of cell size, an alteration of assimilation pigment composition, followed by strong degradation of chlorophyll a, a decrease of chlorophyll a fluorescence, and an increase of reactive oxygen species in the cells. The content of soluble proteins remained a stable parameter. Phytotoxicity of usnic acid on cultures of Trebouxia photobiont was significantly lower. Usnic acid in lichens may act as an allochemical that controls the division of photobiont cells, thereby regulating the balance between the photobiont and mycobiont forming thallus. Higher tolerance to usnic acid in Trebouxia cultures may be an adaptation resulting from the long term co-evolution of these algae with fungi that produce secondary metabolites.

Photobiont diversity in lichens from metal-rich substrata based on ITS rDNA sequences.

The photobiont is considered as the more sensitive partner of lichen symbiosis in metal pollution. For this reason the presence of a metal tolerant photobiont in lichens may be a key factor of ecological success of lichens growing on metal polluted substrata. The photobiont inventory was examined for terricolous lichen community growing in Cu mine-spoil heaps derived by historical mining. Sequences of internal transcribed spacer (ITS) were phylogenetically analyzed using maximum likelihood analyses. A total of 50 ITS algal sequences were obtained from 22 selected lichen taxa collected at three Cu mine-spoil heaps and two control localities. Algae associated with Cladonia and Stereocaulon were identified as members of several Asterochloris lineages, photobionts of cetrarioid lichens clustered with Trebouxia hypogymniae ined. We did not find close relationship between heavy metal content (in localities as well as lichen thalli) and photobiont diversity. Presence of multiple algal genotypes in single lichen thallus has been confirmed.