Development of Bi- and Tri-Layer Nanofibrous Membranes Based on the Sulfated Polysaccharide Carrageenan for Periodontal Tissue Regeneration.

Periodontitis is a microbially-induced inflammation of the periodontium that is characterized by the destruction of the periodontal ligament (PDL) and alveolar bone and constitutes the principal cause of teeth loss in adults. Periodontal tissue regeneration can be achieved through guided tissue/bone regeneration (GTR/GBR) membranes that act as a physical barrier preventing epithelial infiltration and providing adequate time and space for PDL cells and osteoblasts to proliferate into the affected area. Electrospun nanofibrous scaffolds, simulating the natural architecture of the extracellular matrix (ECM), have attracted increasing attention in periodontal tissue engineering. Carrageenans are ideal candidates for the development of novel nanofibrous GTR/GBR membranes, since previous studies have highlighted the potential of carrageenans for bone regeneration by promoting the attachment and proliferation of osteoblasts. Herein, we report the development of bi- and tri-layer nanofibrous GTR/GBR membranes based on carrageenans and other biocompatible polymers for the regeneration of periodontal tissue. The fabricated membranes were morphologically characterized, and their thermal and mechanical properties were determined. Their periodontal tissue regeneration potential was investigated through the evaluation of cell attachment, biocompatibility, and osteogenic differentiation of human PDL cells seeded on the prepared membranes.

Regulation of Energy Metabolism and Anti-Inflammatory Activities of Mastiha Fractions from Pistacia lentiscus L. var. chia.

Pistacia lentiscus L. var. chia resin (Chios Mastiha), the first natural chewing gum, is widely used in Mediterranean cuisine and has been used in traditional medicine from ancient times. Regarding its chemical composition, Chios Mastiha is known to be rich in triterpenes. Triterpenes have a similar structure to glucocorticoids (GCs), the steroid hormones that exert strong anti-inflammatory activities and play crucial roles in the regulation of cellular metabolism. To simplify the characterization of the bioactive compounds of Mastiha resin, three different polarity fractions were isolated and were further analyzed regarding their main chemical composition and an assessment of their biological activities. The biological assessment focused on the evaluation of the potential anti-proliferative, anti-inflammatory, and apoptotic activities as well as the possible interference of the three different polarity Mastiha fractions with the glucocorticoid receptor signaling, with the aim of characterizing the biochemical mechanisms of the actions of the Mastiha fraction. Applying MTT cell viability assay, luciferase/ß-galactosidase assay, and Western blot analysis showed that Chios Mastiha apolar, medium-polar, and polar fractions reduced the HEK293 cell viability in a dose-dependent manner, possibly by mitochondrial-mediated induction of apoptosis. Medium-polar and polar Mastiha fractions also suppressed the GR and NF-κΒ transcriptional activation and the p65 protein levels. These activities were accompanied by the modulation of protein levels of regulatory molecules playing a crucial role in cellular energy homeostasis, such as GR, phosphoenolpyruvate carboxykinase (PEPCK), and/or peroxisome proliferator-activated receptor alpha (PPARα), and by the induction of phosphorylation and the activation of the AMP-activated protein kinase (AMPK). The medium-polar fraction was found to be enriched in triterpenes, such as lupeol, 24Z-masticadienonic acid methyl ester, and 24Z-isomasticadienonic acid methyl ester, and it was the most active one, so we propose that triterpenes in medium-polar fraction are possibly the bioactive compounds responsible for Mastiha's regulatory actions on energy metabolism and anti-inflammatory activities via interference with GR, NF-κΒ, and AMPK signaling. This highlights its potential applications in many fields of pharmaceutical, cosmetic, and nutraceutical interest.

Increased Expression of the Mitochondrial Glucocorticoid Receptor Enhances Tumor Aggressiveness in a Mouse Xenograft Model.

Mitochondria are important organelles for cellular physiology as they generate most of the energy requirements of the cell and orchestrate many biological functions. Dysregulation of mitochondrial function is associated with many pathological conditions, including cancer development. Mitochondrial glucocorticoid receptor (mtGR) is proposed as a crucial regulator of mitochondrial functions via its direct involvement in the regulation of mitochondrial transcription, oxidative phosphorylation (OXPHOS), enzymes biosynthesis, energy production, mitochondrial-dependent apoptosis, and regulation of oxidative stress. Moreover, recent observations revealed the interaction of mtGR with the pyruvate dehydrogenase (PDH), a key player in the metabolic switch observed in cancer, indicating direct involvement of mtGR in cancer development. In this study, by using a xenograft mouse model of mtGR-overexpressing hepatocarcinoma cells, we showed increased mtGR-associated tumor growth, which is accompanied by reduced OXPHOS biosynthesis, reduction in PDH activity, and alterations in the Krebs cycle and glucose metabolism, metabolic alterations similar to those observed in the Warburg effect. Moreover, autophagy activation is observed in mtGR-associated tumors, which further support tumor progression via increased precursors availability. Thus, we propose that increased mitochondrial localization of mtGR is associated with tumor progression possible via mtGR/PDH interaction, which could lead to suppression of PDH activity and modulation of mtGR-induced mitochondrial transcription that ends up in reduced OXPHOS biosynthesis and reduced oxidative phosphorylation versus glycolytic pathway energy production, in favor of cancer cells.

Apoptotic, Anti-Inflammatory Activities and Interference with the Glucocorticoid Receptor Signaling of Fractions from Pistacia lentiscus L. var. chia Leaves.

In this study acetonic extracts of leaves of Pistacia lentiscus L. var. chia (mastiha tree) grown in the south as well as in the north Chios Greek island were isolated and further fractionated to give three different polarity fractions: apolar, medium-polar, and polar. The isolated fractions were assessed as regards their main composition, cytotoxic, anti-inflammatory activities, and interference with the glucocorticoid receptor (GR) signaling, applying cytotoxic assay, luciferase assays, and Western blot analysis of apoptosis-, energy-, and inflammation-associated molecules. Differences in cell viability have been detected among different polarity leaf fractions as well as among fractions of different plant origin with polar fractions showing the highest cytotoxicity. Fractions-induced anti-inflammatory activities and suppressive effects on the dexamethasone (DEX)-induced GR transcriptional activation were unveiled. The partition protocol of leaves fractions applied uncovers the enhanced glucocorticoid-associated biological activities of the medium-polar fractions, which may be associated with their enrichment in the triterpenoids that showed structural similarity with the glucocorticoids. A reduction in GR protein levels is observed by the fraction which is shown to be associated with the medium polar-induced proteolytic degradation of the receptor. In addition, the enhanced cytotoxic, anti-inflammatory, and potential anti-glycemic activities of the fractions from the Southern P. lentiscus L. that exclusively produce the mastiha resin, is revealed, indicating that leaves fractions from mastiha tree, similarly to mastiha tree resin, may have the potential to be further analyzed for their potent applications in the pharmaceutical cosmetic and nutraceutical fields.

Proteomic analysis of the mitochondrial glucocorticoid receptor interacting proteins reveals pyruvate dehydrogenase and mitochondrial 60 kDa heat shock protein as potent binding partners.

Glucocorticoids are steroid hormones that regulate plethora biological actions such as growth and metabolism, immune response, and apoptosis. Glucocorticoids actions are mediated via glucocorticoid receptors which act mainly as transcription factors, but it is also found to be localized in mitochondria. Mitochondrial localization of the receptor indicates novel functions of the receptor. Characterization of the mitochondrial glucocorticoid receptor (mtGR) interacting proteins will shed light on these actions and the biochemical mechanisms that underlie mitochondrial glucocorticoid receptor import and functions. In this study, applying immunoprecipitation, mass spectrometry and Western blot analysis of the GR interacting proteins in total or mitochondrial extracts of HepG2 cells and of HepG2 cells overexpressing a mitochondrial targeted GR we found pyruvate dehydrogenase (PDH), chaperones such as and heat shock protein (HSP) -60, -70, -75 and -90, and 78 kDa glucose-regulated protein, mitochondrial transcription factors and enzymes involved in the regulation of the mitochondrial protein biosynthesis, lipid metabolism, ATP production and apoptosis as glucocorticoid receptor interacting proteins. Our results uncover potential novel mitochondrial partners of the receptor, suggesting possible new regulatory roles of mtGR in the control of mitochondrial-associated functions of the cell. SIGNIFICANCE: In this study the mitochondrial GR interacting proteins were characterized highlighting novel regulatory roles of the receptor in mitochondria. Detection of the mtGR/PDH and mtGR/HSP60 interaction in almost all the analyses performed uncovered PDH and HSP60 proteins as potent mtGR binding partners. The interesting finding of the PDH/mtGR interaction possibly indicates involvement of mtGR in the regulation of the balance between glycolytic and oxidative phosphorylation energy production. Characterization of the mitochondrial heat shock -60, -70, -75 and 78 proteins as mtGR binding partners contribute to the characterization of the biochemical mechanisms of the mitochondrial import of the receptor. Moreover, identification of mitochondrial heat shock proteins, metabolic enzymes, transcription factors, OXPHOS, and regulatory molecules in mitochondrial protein biosynthesis as mtGR binding partners indicates possible new regulatory roles of mtGR in the glucocorticoids-induced regulation and orchestration of nuclear and mitochondrial functions, the exact biochemical mechanism of which remain to be established. The study discloses potential new regulatory roles of the receptor in mitochondria, pointing out its importance as a promising target molecule for the control of the mitochondria-associated pathophysiology of the cell.

Anti-Apoptotic and Antioxidant Activities of the Mitochondrial Estrogen Receptor Beta in N2A Neuroblastoma Cells.

Estrogens are steroid hormones that play a crucial role in the regulation of the reproductive and non-reproductive system physiology. Among non-reproductive systems, the nervous system is mainly affected by estrogens due to their antioxidant, anti-apoptotic, and anti-inflammatory activities, which are mediated by membranous and nuclear estrogen receptors, and also by non-estrogen receptor-associated estrogen actions. Neuronal viability and functionality are also associated with the maintenance of mitochondrial functions. Recently, the localization of estrogen receptors, especially estrogen receptor beta, in the mitochondria of many types of neuronal cells is documented, indicating the direct involvement of the mitochondrial estrogen receptor beta (mtERß) in the maintenance of neuronal physiology. In this study, cell lines of N2A cells stably overexpressing a mitochondrial-targeted estrogen receptor beta were generated and further analyzed to study the direct involvement of mtERß in estrogen neuroprotective antioxidant and anti-apoptotic actions. Results from this study revealed that the presence of estrogen receptor beta in mitochondria render N2A cells more resistant to staurosporine- and H2O2-induced apoptotic stimuli, as indicated by the reduced activation of caspase-9 and -3, the increased cell viability, the increased ATP production, and the increased resistance to mitochondrial impairment in the presence or absence of 17-ß estradiol (E2). Thus, the direct involvement of mtERß in antioxidant and anti-apoptotic activities is documented, rendering mtERß a promising therapeutic target for mitochondrial dysfunction-associated degenerative diseases.

Boswellic acids and their derivatives as potent regulators of glucocorticoid receptor actions.

Glucocorticoid (GCs) hormones exert their actions via their cognate steroid receptors the Glucocorticoid Receptors (GR), by genomic or non-genomic mechanisms of actions. GCs regulate many cellular functions among them growth, metabolism, immune response and apoptosis. Due to their cell type specific induction of apoptosis GCs are used for the treatment of certain type of cancer. In addition, due to their anti-inflammatory actions, GCs are among the most highly prescribed drug to treat chronic inflammatory disorders, albeit to the many adverse side effects arising by their long term and high doses use. Thus, there is a high need for selective glucocorticoid receptor agonist - modulators (SEGRA- SGRMs) as effective as classic GCs, but with a reduced side effect profile. Boswellic acids (BAs) are triterpenes that show structural similarities with GCs and exhibit anti-inflammatory and anti-cancer activities. In this study we examined whether BA alpha and beta and certain BAs derivatives exert their actions, at least in part, through the regulation of GR activities. Applying docking analysis we found that BAs can bind stably into the deacylcortivazol (DAC) accommodation pocket of GR. Moreover we showed that certain boswellic acids derivatives induce glucocorticoid receptor nuclear translocation, no activation of GRE dependent luciferase gene expression, and suppression of the TNF-α induced NF-κB transcriptional activation in GR positive HeLa and HEK293 cells, but not in low GR level COS-7 cells. Furthermore, certain boswellic acids compounds exert antagonistic effect on the DEX-induced GR transcriptional activation and induce cell type specific mitochondrial dependent apoptosis. Our results indicate that certain BAs are potent selective glucocorticoid receptor regulators and could have great potential for therapeutic use.

Synthetic flavonoid derivatives targeting the glycogen phosphorylase inhibitor site: QM/MM-PBSA motivated synthesis of substituted 5,7-dihydroxyflavones, crystallography, in vitro kinetics and ex-vivo cellular experiments reveal novel potent inhibitors.

Glycogen phosphorylase (GP) is an important target for the development of new anti-hyperglycaemic agents. Flavonoids are novel inhibitors of GP, but their mode of action is unspecific in terms of the GP binding sites involved. Towards design of synthetic flavonoid analogues acting specifically at the inhibitor site and to exploit the site's hydrophobic pocket, chrysin has been employed as a lead compound for the in silico screening of 1169 new analogues with different B ring substitutions. QM/MM-PBSA binding free energy calculations guided the final selection of eight compounds, subsequently synthesised using a Baker-Venkataraman rearrangement-cyclisation approach. Kinetics experiments against rabbit muscle GPa and GPb together with human liver GPa, revealed three of these compounds (11, 20 and 43) among the most potent that bind at the site (Ki s < 4 µM for all three isoforms), and more potent than previously reported natural flavonoid inhibitors. Multiple inhibition studies revealed binding exclusively at the inhibitor site. The binding is synergistic with glucose suggesting that inhibition could be regulated by blood glucose levels and would decrease as normoglycaemia is achieved. Compound 43 was an effective inhibitor of glycogenolysis in hepatocytes (IC50 = 70 µM), further promoting these compounds for optimization of their drug-like potential. X-ray crystallography studies revealed the B-ring interactions responsible for the observed potencies.

Neurotoxic effects of aluminum are associated with its interference with estrogen receptors signaling.

Aluminum compounds have been observed in various brain regions, and their accumulation has been associated with many neurodegenerative disorders. Neurotoxic effects of aluminum are attributed to reactive oxygen species generation, induction of apoptosis and inflammatory reactions activation. Metalloestrogen activity of aluminum has also been linked to breast cancer progression and metastasis. In this study, taking into account the anti-apoptotic and anti-oxidant activities of estrogens in neuronal cells, which are mediated by estrogen receptors, the possible estrogenic activity of aluminum in SH-SY5Y neuroblastoma cells was studied. Our results showed that aluminum in the form of aluminum chlorohydrate (ACH) exhibited no effect on estrogen receptors transcriptional activation, and differential effect on estrogen receptor alpha (ERα) and estrogen receptor beta (ERß) protein levels. ACH caused reduction in ERß protein levels, and increase in its mitochondrial localization. ACH-induced reduction in ERß protein level may be linked, at least in part, to the ACH-induced increase in ERα protein level. This statement is based on our observations showing aluminum-induced reduction in the E2-induced increase in ERα S118 phosphorylation, in MCF-7 and SH-SH5Y cells. Phosphorylation at S118 residue is known to be associated with inhibition of the ubiquitin-induced proteolytic degradation of ERα, leading to its accumulation. Since it is known that ERα negatively regulate ERß expression, increase in ERα, may contribute to reduction in ERß levels and subsequent weakening of its anti-apoptotic and anti-oxidant activity, justified by the observed reduction in procaspase 9, mitochondrial cytochrome c, Bcl-2, Bcl-xL and mitochondrial thioredoxin protein level, as well as by the increase in proapoptotic BAX level, in ACH treated SH-SY5Y cells. In addition, increase in mitochondrial ERß localization may also trigger mitochondrial metabolism, suppress biosynthetic process of gluconeogenesis, as indicated by the observed reduction in the phosphoenolpyruvate carboxykinase protein level, and eventually lead to increase in reactive oxygen species (ROS) generation, known to be implicated in aluminum induced neurodegeneration. This statement was verified by the observed ACH-induced increase in ERß mitochondrial localization, induction of the mitochondrial membrane depolarization and increase in ROS production, in neuronal-like differentiated SH-SY5Y cells.

The architecture of hydrogen and sulfur σ-hole interactions explain differences in the inhibitory potency of C-ß-d-glucopyranosyl thiazoles, imidazoles and an N-ß-d glucopyranosyl tetrazole for human liver glycogen phosphorylase and offer new insights to structure-based design.

C-Glucopyranosyl imidazoles, thiazoles, and an N-glucopyranosyl tetrazole were assessed in vitro and ex vivo for their inhibitory efficiency against isoforms of glycogen phosphorylase (GP; a validated pharmacological target for the development of anti-hyperglycaemic agents). Imidazoles proved to be more potent inhibitors than the corresponding thiazoles or the tetrazole. The most potent derivative has a 2-naphthyl substituent, a Ki value of 3.2 µM for hepatic glycogen phosphorylase, displaying also 60% inhibition of GP activity in HepG2 cells, compared to control vehicle treated cells, at 100 µM. X-Ray crystallography studies of the protein - inhibitor complexes revealed the importance of the architecture of inhibitor associated hydrogen bonds or sulfur σ-hole bond interactions to Asn284 OD1, offering new insights to structure-based design efforts. Moreover, while the 2-glucopyranosyl-tetrazole seems to bind differently from the corresponding 1,2,3-triazole compound, the two inhibitors are equipotent.

Glucopyranosylidene-spiro-imidazolinones, a New Ring System: Synthesis and Evaluation as Glycogen Phosphorylase Inhibitors by Enzyme Kinetics and X-ray Crystallography.

Epimeric series of aryl-substituted glucopyranosylidene-spiro-imidazolinones, an unprecedented new ring system, were synthesized from the corresponding Schiff bases of O-perbenzoylated (gluculopyranosylamine)onamides by intramolecular ring closure of the aldimine moieties with the carboxamide group elicited by N-bromosuccinimide in pyridine. Test compounds were obtained by Zemplén O-debenzoylation. Stereochemistry and ring tautomers of the new compounds were investigated by NMR, time-dependent density functional theory (TDDFT)-electronic circular dichroism, and DFT-NMR methods. Kinetic studies with rabbit muscle and human liver glycogen phosphorylases showed that the (R)-imidazolinones were 14-216 times more potent than the (S) epimers. The 2-naphthyl-substituted (R)-imidazolinone was the best inhibitor of the human enzyme (Ki 1.7 µM) and also acted on HepG2 cells (IC50 177 µM). X-ray crystallography revealed that only the (R) epimers bound in the crystal. Their inhibitory efficacy is based on the hydrogen-bonding interactions of the carbonyl oxygen and the NH of the imidazolinone ring.

Potential Dissociative Glucocorticoid Receptor Activity for Protopanaxadiol and Protopanaxatriol.

Glucocorticoids are steroid hormones that regulate inflammation, growth, metabolism, and apoptosis via their cognate receptor, the glucocorticoid receptor (GR). GR, acting mainly as a transcription factor, activates or represses the expression of a large number of target genes, among them, many genes of anti-inflammatory and pro-inflammatory molecules, respectively. Transrepression activity of glucocorticoids also accounts for their anti-inflammatory activity, rendering them the most widely prescribed drug in medicine. However, chronic and high-dose use of glucocorticoids is accompanied with many undesirable side effects, attributed predominantly to GR transactivation activity. Thus, there is a high need for selective GR agonist, capable of dissociating transrepression from transactivation activity. Protopanaxadiol and protopanaxatriol are triterpenoids that share structural and functional similarities with glucocorticoids. The molecular mechanism of their actions is unclear. In this study applying induced-fit docking analysis, luciferase assay, immunofluorescence, and Western blot analysis, we showed that protopanaxadiol and more effectively protopanaxatriol are capable of binding to GR to activate its nuclear translocation, and to suppress the nuclear factor-kappa beta activity in GR-positive HeLa and HEK293 cells, but not in GR-low level COS-7 cells. Interestingly, no transactivation activity was observed, whereas suppression of the dexamethasone-induced transactivation of GR and induction of apoptosis in HeLa and HepG2 cells were observed. Thus, our results indicate that protopanaxadiol and protopanaxatriol could be considered as potent and selective GR agonist.

Potential interference of aluminum chlorohydrate with estrogen receptor signaling in breast cancer cells.

Aluminum salts are widely used as the active antiperspirant in underarm cosmetic. Experimental observations indicate that its long term application may correlate with breast cancer development and progression. This action is proposed to be attributed, among others, to aluminum possible estrogen-like activities. In this study we showed that aluminum, in the form of aluminum chlorohydrate (ACH), caused increase in estrogen receptor alpha (ERα) protein levels, in ERα-positive MCF-7 cells. This effect was accompanied by moderate activation of Estrogen Response Elements (ERE)-driven reporter gene expression and 20%-50% increase in certain estrogen responsive, ERE-independent genes expression. Genes affected were ERα, p53, cyclin D1, and c-fos, crucial regulators of breast cancer development and progression. ACH-induced genes expression was eliminated in the presence of the estrogen antagonist: ICI 182780, in MCF-7 cells, whereas it was not observed in ERα-negative MDA-MB-231 breast cancer cells, indicating aluminum interference with estrogen signaling. Moreover, ACH caused increase in the perinuclear localization of estrogen receptor alpha in MCF-7 breast cancer cells and increase in the mitochondrial Bcl-2 protein, possibly affecting receptors-mediated mitochondrial actions and mitochondrial-dependent apoptosis. ACH-induced perinuclear localization of estrogen receptor beta was also observed in MDA-MB-231. Our findings indicate that aluminum actions on estrogen receptors protein level and subcellular localization possibly affect receptors-mediated actions and thus, aluminum interference with estrogen signaling.