Anticancer Activity of Essential Oils and Other Extracts from Aromatic Plants Grown in Greece

Aromatic plants have a long and significant history in the traditional medicine of many countries. Nowadays, there is an increasing interest in investigating the biological properties of aromatic plant extracts mainly due to their diversity, high availability, and low toxicity. Greece is abundant in aromatic plants, which can be attributed to the country's geographical position, the morphology of its landscape, and its numerous mountainous and insular areas. In the past 15 years, a number of aromatic plant extracts of Greek origin have been studied for their bioactivities, including their antiproliferative potential against different types of cancer. Although the pharmacological activities of specific species of Greek origin have been reviewed before, no gathered information on explicitly Greek species exist. In this review, we summarize existing data on the antiproliferative activity of extracts isolated from Greek aromatic plants and discuss their molecular mode(s) of action, where available, in order to identify promising extracts for future research and link chemical constituents responsible for their activity. We conclude that essentials oils are the most frequently studied plant extracts exhibiting high diversity in their composition and anticancer potential, but also other extracts appear to be worthy of further investigation for cancer chemoprevention

Molecular dynamics simulations of tight junction proteins

Tight junctions (TJs) are specialised cell-cell structures that serve primarily as a barrier to molecular transport through the intercellular space between the cells. The claudin family of proteins are the main structural and functional components of the TJ strands that circumscribe the cells. The detailed molecular organisation at the TJs is not entirely resolved, being relatively inaccessible by current experimental methods. Here, we have employed molecular dynamics simulations using both atomistic and coarse-grained models to investigate the TJ structure formed by claudin-1 using self-assembly coupled with free energy calculations and enhanced sampling techniques. A feature of the studies is that the self-assembly simulations have been carried out using atomistic detail (a first) by simulating only the extracellular domains of claudin-1 in an implied membrane. The results show that the cis-interaction can occur in the absence of trans-interacting partners and that a claudin dimer is the smallest stable unit. The dimers further form higher-order aggregates with a plethora of interacting dimeric interfaces. The transinteraction of claudins resulted in a compact structure with a minimal pore size confirming the barrier properties of claudin-1. The simulations also enabled the identification of the key regions of the claudin responsible for the trans-interaction, with the identified important amino acids being in agreement with experimental studies. The role of the lipid environment, with a focus on the skin lipids in the stratum granulosum, was also investigated, along with the effect of single-point mutations in claudin-1. The single-point mutation studies were consistent with experimental results. The simulation studies have enhanced our understanding of the assembly and structure of claudin-1 TJs, a notable finding being that kinetic locking is likely to be important in determining the TJ structure. The single-point mutation studies suggest that simulations could serve as screens towards defining potential gene-therapy strategies

Biological functions of therapy-induced senescence in cancer

Therapy-induced cellular senescence is a state of stable growth arrest induced by common cancer treatments such as chemotherapy and radiation. In an oncogenic context, therapy-induced senescence can have different consequences. By blocking cellular proliferation and by facilitating immune cell infiltration, it functions as tumor suppressive mechanism. By fueling the proliferation of bystander cells and facilitating metastasis, it acts as a tumor promoting factor. This dual role is mainly attributed to the differential expression and secretion of a set of pro-inflammatory cytokines and tissue remodeling factors, collectively known as the Senescence-Associated Secretory Phenotype (SASP). Here, we describe cell-autonomous and non-cell-autonomous mechanisms that senescent cells activate in response to chemotherapy and radiation leading to tumor suppression and tumor promotion. We present the current state of knowledge on the stimuli that affect the activation of these opposing mechanisms and the effect of senescent cells on their micro-environment eg. by regulating the functions of immune cells in tumor clearance as well as strategies to eliminate senescent tumor cells before exerting their deleterious side-effects

Antioxidant and Antiproliferative Properties of the Essential Oils of Satureja thymbra and Satureja parnassica and their Major Constituents

Aim: The biopotential of the essential oils of the Greek aromatic plants Satureja thymbra and Satureja parnassica were investigated, together with their major components carvacrol, thymol, γ-terpinene and p-cymene. Materials and Methods: Antioxidant and cancer cell cytotoxic properties were determined using 2,2-diphenyl-1-picrylhydrazyl and sulforhodamine B assays, respectively. The antiproliferative potential was studied against the MCF-7, A549, HepG2 and Hep3B cell lines. Results: S. thymbra oil possessed stronger antioxidant and antiproliferative capacity when tested on MCF-7 cells compared to S. parnassica oil. Thymol exhibited two-fold greater antioxidant potency than carvacrol, whereas γ-terpinene and p-cymene had no significant effect. Carvacrol was the most potent antiproliferative agent against A549 cells, while Hep3B cells were most sensitive to thymol. p-Cymene and γ-terpinene demonstrated negligible bioactivity. Conclusion: S. thymbra and S. parnassica essential oils exhibit significant but diverse antioxidant and antiproliferative activities, mainly attributed to their main components, carvacrol and thymol

Chemical Composition and Evaluation of the Biological Properties of the Essential Oil of the Dietary Phytochemical Lippia citriodora

The aim of the study was to characterize the chemical composition and biological properties of the essential oil from the plant Lippia citriodora grown in Greece. The essential oil volatiles were analyzed by gas chromatography–mass spectrometry GC-MS indicating citral as the major component. Τhe antimicrobial properties were assayed using the disk diffusion method and the minimum inhibitory and non-inhibitory concentration values were determined. Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus, Saccharomyces cerevisiae, and Aspergillus niger were sensitive to Lippia citriodora oil, but not Escherichia coli, Salmonella Enteritidis, Salmonella typhimurium, and Pseudomonas fragi. Adversely, all microbes tested were sensitive to citral. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays were used to assess direct antioxidant activity, which proved to be weak for both agents, while comet assay was utilized to study the cytoprotective effects against H2O2-induced oxidative damage in Jurkat cells. Interestingly, the oil showed a more profound cytoprotective effect compared to citral. The antiproliferative activity was evaluated in a panel of cancer cell lines using the sulforhodamine B (SRB) and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-S-(phenylamino) carbonyl-2-tetrazolium hydroxide (XTT) assays and both agents demonstrated potent antiproliferative activity with citral being more cytotoxic than the oil. Taken together, the essential oil of Lippia citriodora and its major component, citral, exert diverse biological properties worthy of further investigation

COVID-19: Is There Evidence for the Use of Herbal Medicines as Adjuvant Symptomatic Therapy?

Background: Current recommendations for the self-management of SARS-Cov-2 disease (COVID-19) include self-isolation, rest, hydration, and the use of NSAID in case of high fever only. It is expected that many patients will add other symptomatic/adjuvant treatments, such as herbal medicines. Aims: To provide a benefits/risks assessment of selected herbal medicines traditionally indicated for “respiratory diseases” within the current frame of the COVID-19 pandemic as an adjuvant treatment. Method: The plant selection was primarily based on species listed by the WHO and EMA, but some other herbal remedies were considered due to their widespread use in respiratory conditions. Preclinical and clinical data on their efficacy and safety were collected from authoritative sources. The target population were adults with early and mild flu symptoms without underlying conditions. These were evaluated according to a modified PrOACT-URL method with paracetamol, ibuprofen, and codeine as reference drugs. The benefits/risks balance of the treatments was classified as positive, promising, negative, and unknown. Results: A total of 39 herbal medicines were identified as very likely to appeal to the COVID-19 patient. According to our method, the benefits/risks assessment of the herbal medicines was found to be positive in 5 cases (Althaea officinalis, Commiphora molmol, Glycyrrhiza glabra, Hedera helix, and Sambucus nigra), promising in 12 cases (Allium sativum, Andrographis paniculata, Echinacea angustifolia, Echinacea purpurea, Eucalyptus globulus essential oil, Justicia pectoralis, Magnolia officinalis, Mikania glomerata, Pelargonium sidoides, Pimpinella anisum, Salix sp, Zingiber officinale), and unknown for the rest. On the same grounds, only ibuprofen resulted promising, but we could not find compelling evidence to endorse the use of paracetamol and/or codeine. Conclusions: Our work suggests that several herbal medicines have safety margins superior to those of reference drugs and enough levels of evidence to start a clinical discussion about their potential use as adjuvants in the treatment of early/mild common flu in otherwise healthy adults within the context of COVID-19. While these herbal medicines will not cure or prevent the flu, they may both improve general patient well-being and offer them an opportunity to personalize the therapeutic approaches

Atomistic simulations of tight junction proteins

The surface cells of the body are joined together by specific junctions, some which keep the cells together (adherens and desmosomes) and others that prevent or regulate flow of molecules between the cells. The latter are the so called tight junctions. Tight junctions (TJs) limit the passage of permeants through the inter-cellular space between adjacent epithelial and endothelial cells (Zihni, Mills, Matter, & Balda, 2016). They act as either barriers or selective gates, allowing the permeation of molecules in a size and charge selective way and help multicellular organisms maintain homeostasis. The primary proteins involved are the claudins. Claudin 1, the primary protein of interest here, is a ubiquitous claudin found in most tissues and located in the skin’s epidermis layer contributing in the skin barrier (Günzel & Alan, 2013). Claudins are transmembrane proteins and have external loops which come together to form the barrier or selective gate. The barrier function of TJs is regulated by the specific cisinteractions (in the same cell membrane) and trans- interaction (on adjacent cells membranes) of TJ proteins. The interaction can be either homo- or hetero- depending on whether it is between same or different proteins respectively. The fundamental question is how do these proteins interact at a molecular level resulting in the formation of the barrier? We need to get a better understanding of the assembly of the TJ proteins, their resulting architectural organisation, and the molecular level mechanism of the barrier function (Krause, Protze, & Piontek, 2015). In attempting to understand the assembly and resulting architecture of the TJ proteins, the atomistic resolution offered by molecular dynamics simulations can add considerable value. A molecular dynamics simulation of a system of molecules yields trajectories of the behaviour of molecules. The simulations yield structural information, dynamics and thermodynamic quantities characterising the system. The project investigates the behaviour of the extracellular loops (ECLs) of claudin 1 via their simulation in a box containing water and counterions. Subsequently, multiple loops are aligned in space as if they were attached to membranes as in cells. The loops are left to interact freely in the environment that imitates their natural one and analyse their selfassemble into higher order structures. Through these simulations, we should be able to investigate the packing of the ECLs and where appropriate investigate the nature of the selectivity with respect to permeability (Anderson & Van Itallie, 2009). Furthermore, we anticipate to model interactions of proteins with other molecules that can compromise the barrier

Characterization of the biological properties of the active components of essential oils

Aromatic plants have been used since antiquity for their diverse properties, which are mainly attributed to essential oils that are formed as secondary metabolites and have protective roles. Today, essential oils are studied for their biological properties, including their chemopreventive potential, mainly meaning a combination of activities like antioxidant, anti-inflammatory and pro-apoptotic for cancer prevention. The present thesis aimed at screening a large collection of plant extracts of Greek origin for their potential chemopreventive capacity and further explore the molecular modes of action of the most promising ones. For the screening process, fifty plant extracts including essential oils were initially tested for their antioxidant capacity in vitro (DPPH and ABTS assays). Essential oils were tested for their antiproliferative activity against a panel of human cancer cell lines (Caco2, HepG2, MCF-7) using the SRB assay and their chemical composition was also determined. The majority of the oils had terpenes and terpenoids as main components and the oils from Pistacia lentiscus var. Chia (mastic oil, MO), Lippia citriodora (lemon verbena oil, LVO), Origanum dittany and Satureja thymbra stood out for displaying promising biological properties, but only mastic and lemon verbena oils and their major components were chosen for further study. α-Pinene and myrcene, the major components of MO were weak radical scavengers in vitro, while LVO exhibited higher antioxidant activity than its major component, citral. With the exception of myrcene, all phytochemicals exhibited significant antiproliferative activity in vitro, likely attributed to their DNA damaging effect, while both oils presented tumor suppressive capacity in vivo, too. Next, using flow cytometry, it was shown that the oils and their major components induce a G2/M arrest in different human colon cancer cell lines, followed by a concomitant change in the expression levels of cell cycle-related genes in the case of LVO and citral (cyclinA, B1, D, E, p53, p21, survivin). Furthermore, the oils and citral (at the range of concentrations and time points used) caused caspase-independent cell death in Caco2 cells, as lack of increase in pro-apoptotic cell population and caspase-3 enzymatic activity was observed. In addition, no significant alterations were detected in the levels of apoptosis-related proteins apart from cleaved Caspase-3, cIAP-2, Livin, catalase, p21, p27 and TRAIL R1 and R2 for MO and survivin, claspin, TRAILR2, clusterin, HSP60 and Fas for LVO, which together with citral may induce autophagy based on experimental evidence. Finally, it was shown that LVO possesses higher capacity than citral at potentiating the activity of the conventional chemotherapeutic agents oxaliplatin and irinotecan in Caco2 and HT-29 cells. In conclusion, our findings suggest that plant extracts of Greek origin, and especially mastic and lemon verbena oils, possess promising bioactivities that are worthy of further investigation for their future use as chemopreventive and chemotherapeutic agents.Τα αρωματικά φυτά είναι γνωστά για τις ποικίλες ιδιότητές τους από την αρχαιότητα ένα μεγάλο μέρος των οποίων οφείλεται στα αιθέρια έλαια που αποτελούν δευτερογενείς μεταβολίτες. Σήμερα, τα αιθέρια ελαία μελετώνται για τις βιολογικές τους ιδιότητες, ανάμεσα στις οποίες συγκαταλέγεται η χημειοπροληπτική τους δράση, δηλαδή ένας συνδυασμός ενδοκυτταρικών μηχανισμών δράσης συμπεριλαμβανομένων της αντιοξειδωτικής, αντιφλεγμονώδους και προ-αποπτωτικής. Στόχος της έρευνάς μας ήταν να ελέγξουμε έναν αριθμό φυσικών εκχυλισμάτων από ελληνικά φυτά για την πιθανή χημειοπροληπτική τους δράση και να μελετήσουμε περαιτέρω τα πιο υποσχόμενα για τους μοριακούς μηχανισμούς δράσης τους. Πενήντα φυτικά εκχυλίσματα ελέγχθηκαν για την ικανότητα τους να απενεργοποιούν ελεύθερες ρίζες in vitro (τεχνικές DPPH και ABTS). Παράλληλα, 10 αιθέρια έλαια ελέγχθηκαν για την αντιπολλαπλασιαστική τους δράση εναντίον διαφορετικών καρκινικών κυτταρικών σειρών in vitro (Caco2, HepG2, MCF-7) (τεχνική SRB) και αναλύθηκε η χημική τους σύσταση. Τα περισσότερα αιθέρια έλαια είχαν ως κύρια συστατικά τερπένια και τερπενοειδή, ενώ συνολικά την σημαντικότερη δράση επέδειξαν τα έλαια των φυτών Satureja thymbra, Origanum dittany Lippia citriodora (λουίζα) και Pistacia lentiscus var. Chia (μαστίχα). Μετά το τέλος της σάρωσης επιλέξαμε τα δύο τελευταία, καθώς και τα κυριότερα συστατικά τους για περαιτέρω μελέτες. Τα δύο κυριότερα συστατικά του μαστιχέλαιου, α-πινένιο και μυρκένιο, δεν παρουσίασαν σημαντική αντιοξειδωτική δράση, ενώ το έλαιο της λουίζας, παρουσίασε ισχυρότερη δράση από το κύριο συστατικό του, citral. Με εξαίρεση το μυρκένιο, όλοι οι παράγοντες εμφάνισαν υψηλή αντιπολλαπλασιαστική ικανότητα in vitro, η οποία συνδέεται με την ικανότητά τους να προκαλούν βλάβες στο DNA, ενώ τα δύο έλαια επέδειξαν και σημαντική ογκοκατασταλτική δράση in vivo. Χρησιμοποιώντας κυτταρομετρία ροής παρατηρήθηκε αναστολή του κυτταρικού κύκλου στη φάση G2/M σε διαφορετικές κυτταρικές σειρές, με παράλληλη αλλαγή στην έκφραση γονιδίων που σχετίζονται με τον κυτταρικό κύκλο (cyclinA, B1, D, E, p53, p21, survivin) για τη λουίζα και το citral. Επίσης, παρατηρήσαμε πως κανένα φυτοχημικό δεν επάγει την κλασσική απόκριση της απόπτωσης σε καρκινικά κύτταρα παχέος εντέρου (για τις δεδομένες χρονικές επωάσεις και συγκεντρώσεις που χρησιμοποιήθηκαν), καθώς δεν ανιχνεύσαμε ούτε αύξηση του πληθυσμού των προ-αποπτωτικών κυττάρων, ούτε και αύξηση της ενεργότητας του ενζύμου κασπάση-3. Παράλληλα, δεν προκλήθηκε σημαντική αλλαγή στα επίπεδα πρωτεϊνών που σχετίζονται με την απόπτωση εκτός από τις ακόλουθες πρωτεΐνες: ενεργή κασπάση-3, cIAP-2, Livin, catalase, p21, p27 και TRAIL R1 και R2 για το έλαιο της μαστίχας και τις survivin, claspin, TRAILR2, clusterin, HSP60 και Fas για το έλαιο της λουίζας, που μαζί με το citral πιθανόν να ενεργοποιούν το μονοπάτι της αυτοφαγίας βάσει πειραματικών ενδείξεων. Τέλος, η λουίζα διαθέτει μεγαλύτερη ικανότητα ενίσχυσης της δράσης των δύο χημειοθεραπευτικών παραγόντων oxaliplatin και irinotecan στις κυτταρικές σειρές Caco2 και HT-29 σε σχέση με το citral. Συμπερασματικά, τα ευρήματα μας υποδεικνύουν πως τα φυσικά εκχυλίσματα που μελετήσαμε, και κυρίως τα έλαια μαστίχας και λουίζας, διαθέτουν πολλά υποσχόμενες δραστικότητες που αξίζουν περαιτέρω διερεύνησης για τη μελλοντική χρήση τους ως χημειοπροληπτικοί και χημειοθεραπευτικοί παράγοντες

Αξιολόγηση του Δεκτικού και Εκφραστικού Λεξιλογίου Παιδιών Τυπικής και Μη Τυπικής Ανάπτυξης 6 μηνών έως 3,5 ετών μέσω Ερωτηματολογίου Γονικής Αναφοράς

Η παρούσα μελέτη πραγματοποιήθηκε με στόχο τη διερεύνηση του δεκτικού και εκφραστικού λεξιλογίου παιδιών τυπικής και μη τυπικής ανάπτυξης έξι μηνών έως τρεισήμισι ετών, όπως αυτό αξιολογείται από γονικές αναφορές. Απώτερος σκοπός της έρευνας υπήρξε η στάθμιση του εργαλείου CYLEX (Cypriot-Greek Lexical Acquisition Checklist) στον Ελληνικό πληθυσμό, προκειμένου μελλοντικά αυτό να συμβάλλει στην πρώιμη ανίχνευση και αντιμετώπιση γλωσσικών ελλειμμάτων. Το δείγμα αποτέλεσαν, μέσω των γονικών αναφορών, 98 βρέφη και νήπια, εκ των οποίων τα 10 φοιτούσαν σε ειδικό πλαίσιο. Στους γονείς χορηγήθηκε το ερωτηματολόγιο CYLEX, το οποίο αποτελείται από 611 λέξεις, χωρισμένες σε 18 σημασιολογικές κατηγορίες. Μέσα από αυτό διερευνήθηκαν, επίσης, τυχόν διαφοροποιήσεις στο λεξιλόγιο σε σχέση με το φύλο, την ηλικία, τη φοίτηση σε προσχολικές μονάδες και τον ειδικό πληθυσμό. Τα ευρήματα που προέκυψαν έδειξαν σύνδεση της ηλικίας με την ανάπτυξη του εκφραστικού και δεκτικού λεξιλογίου, ενώ παρόμοια δεδομένα δεν αναδείχθηκαν για το φύλο, τη φοίτηση σε προσχολικές μονάδες ή τον ειδικό πληθυσμό. Συγκεκριμένα, για τη μεταβλητή της ηλικίας φάνηκε πως, όσο αυξανόταν η ηλικία, αυξανόταν και το μέγεθος του εκφραστικού και δεκτικού λεξιλογίου, ενώ λεξικολογική έκρηξη παρατηρήθηκε να πραγματοποιείται περίπου στους 18 με 23 μήνες. Τέλος, μέσα από την παρούσα μελέτη εμφανής έγινε και η χρονική και ποσοτική διαφορά ανάμεσα σε κατανόηση και έκφραση, επιβεβαιώνοντας την εμφάνιση της κατανόησης πρωτύτερα και περισσότερο στο λεξιλόγιο των βρεφών. Τα αποτελέσματα αξιολογήθηκαν και συζητούνται με βάση την υπάρχουσα βιβλιογραφία στον τομέα της γλωσσικής ανάπτυξης.The present study was conducted to investigate the receptive and expressive vocabulary of typically and non-typically developing children aged six months to three and a half years, as assessed by parental reports. The ultimate aim of the study was the validation of the CYLEX (Cypriot-Greek Lexical Acquisition Checklist) tool in the Greek population in order to contribute to the early detection and treatment of language deficits in the future. The sample, retrieved from parental reports, consisted of 98 infants and toddlers, 10 of which were attending a special educational setting. The parents were administered the CYLEX questionnaire, which consists of 611 words divided into 18 semantic categories. Through this, any differences in vocabulary in relation to gender, age, attendance in preschool and special population were also investigated. The findings revealed an association between age and development of expressive and receptive vocabulary, whereas similar data did not emerge for gender, pre-school attendance or special population. In particular, regarding the age variable, it was demonstrated that the more the age increased, the more the size of expressive and receptive vocabulary expanded; while «vocabulary spurt» was observed to occur at approximately 18 to 23 months. Finally, through the current study, the temporal and quantitative difference between perception and expression emerged, asserting the earlier appearance of comprehension, especially in the infants' vocabulary. The results were evaluated and discussed based on the existing literature in the field of language development

The mutual interactions of the extracellular domains of tight junction proteins

Tight junctions (TJs) are complex multiprotein structures found in epithelial and endothelial cells that serve as selective barriers and regulate the diffusion of small molecules and ions through the intercellular space (Alberts et al., 2015). TJs are composed of strands that encircle the cells like a belt-like network, and thus control the unlimited diffusion of permeants. The backbone of the TJ strands is composed of the family of proteins called claudins. The crystal structure of claudin 15 (Suzuki et al., 2014) and of fragments of other members of the claudin family complexed with toxins (Saitoh et al., 2015; Shinoda et al., 2016) have been resolved. However, the molecular organization of the TJ strands is unknown but is essential to understanding normal physiological function (Zihni, Mills, Matter, & Balda, 2016) as well as dysfunction in pathological states, including viral interactions. Furthermore, the development of new therapeutic strategies will rely on a molecular level understanding of the TJ barrier mechanism. Here we investigate the behaviour of the extracellular loops of claudin 1, which is essential for the epidermal barrier (Kirschner, Houdek, Fromm, Moll, & Brandner, 2010). We have employed atomistic and coarse-grained simulations of the extracellular loop domains restrained on a 2-d plane to mimic their natural occurrence in a lipid bilayer. The systems comprise large grids of the protein loop domains (8x8) randomly rotated and restrained on a plane, surrounded by water and counterions. Their cis (in the same lipid bilayer) and trans (between opposing bilayers) interactions were simulated and characterised. The domains reveal a tendency to form linear structures, though no specific cis-interaction appears to dominate. The individual loops do not show any particular regions with strong binding affinities. The residues with similar physical properties (i.e. either strongly positively or negatively charged, or non-polar) are dispersed throughout the loop structures and do not occur in local contiguous regions. The binding energy of interaction between the most frequent dimers observed in our simulations was also characterised by umbrella sampling. The lack of a specific, strong interaction underpinning any organisational motif for the cis- interaction suggests that the loops interactions are not the determinants of the molecular organisation of TJs. The next step is to investigate the self-assembly of the whole claudin monomers embedded in lipid bilayers with a view to identifying the key interactions and packing that gives rise to the formation of TJ strands