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.

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.

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.

Affinity Crystallography Reveals the Bioactive Compounds of Industrial Juicing Byproducts of Punica granatum for Glycogen Phosphorylase.

BACKGROUND: Glycogen phosphorylase (GP) is a pharmaceutical target for the discovery of new antihyperglycaemic agents. Punica granatum is a well-known plant for its potent antioxidant and antimicrobial activities but so far has not been examined for antihyperglycaemic activity. OBJECTIVE: The aim was to examine the inhibitory potency of eighteen polyphenolic extracts obtained from Punica granatum fruits and industrial juicing byproducts against GP and discover their most bioactive ingredients. METHOD: Kinetic experiments were conducted to measure the IC50 values of the extracts while affinity crystallography was used to identify the most bioactive ingredient. The inhibitory effect of one of the polyphenolic extracts was also verified ex vivo, in HepG2 cells. RESULTS: All extracts exhibited significant in vitro inhibitory potency (IC50 values in the range of low µg/mL). Affinity crystallography revealed that the most bioactive ingredients of the extracts were chlorogenic and ellagic acids, found bound in the active and the inhibitor site of GP, respectively.While ellagic acid is an established GP inhibitor, the inhibition of chlorogenic acid is reported for the first time. Kinetic analysis indicated that chlorogenic acid is an inhibitor with Ki=2.5 x 10-3Mthat acts synergistically with ellagic acid. CONCLUSION: Our study provides the first evidence for a potential antidiabetic usage of Punica granatum extracts as antidiabetic food supplements. Although, more in vivo studies have to be performed before these extracts reach the stage of antidiabetic food supplements, our study provides a first positive step towards this process.

The triterpene echinocystic acid and its 3-O-glucoside derivative are revealed as potent and selective glucocorticoid receptor agonists.

Glucocorticoids are steroid hormones widely used to control many inflammatory conditions. These effects are primarily attributed to glucocorticoid receptor transrepressional activities but with concomitant receptor transactivation associated with considerable side effects. Accordingly, there is an immediate need for selective glucocorticoid receptor agonists able to dissociate transactivation from transrepression. Triterpenoids have structural similarities with glucocorticoids and exhibit anti-inflammatory and apoptotic activities via mechanisms that are not well-defined. In this study, we examined whether echinocystic acid and its 3-O-glucoside derivative act, at least in part, through the regulation of glucocorticoid receptor and whether they can constitute selective receptor activators. We showed that echinocystic acid and its glucoside induced glucocorticoid receptor nuclear translocation by 75% and 55%. They suppressed the nuclear factor-kappa beta transcriptional activity by 20% and 70%, respectively, whereas they have no glucocorticoid receptor transactivation capability and stimulatory effect on the expression of the phosphoenolopyruvate carboxykinase target gene in HeLa cells. Interestingly, their suppressive effect is diminished in glucocorticoid receptor low level COS-7 cells, verifying the receptor involvement in this process. Induced fit docking calculations predicted favorable binding in the ligand binding domain and structural characteristics which can be considered consistent with the experimental observations. Further, glucocorticoids exert apoptotic activities; we have demonstrated here that the echinocystic acids in combination with the synthetic glucocorticoid, dexamethasone, induce apoptosis. Taken together, our results indicate that echinocystic acids are potent glucocorticoid receptor regulators with selective transrepressional activities (dissociated from transactivation), highlighting the potential of echinocystic acid derivatives as more promising treatments for inflammatory conditions.

Biochemical and biological assessment of the inhibitory potency of extracts from vinification byproducts of Vitis vinifera extracts against glycogen phosphorylase.

The inhibitory potency of thirteen polyphenolic extracts obtained from vinification byproducts of Greek varieties of Vitis vinifera against glycogen phosphorylase (GP) has been studied by kinetic experiments. GP is an enzyme involved in glucose homeostasis and a molecular target for the discovery of new hypoglycemic agents. Studies have shown that all extracts display significant inhibitory potency for GP in vitro with IC50 values in the range of low µg/mL. X-ray crystallographic analysis of GP crystals soaked with two of these extracts revealed that the most active ingredient is quercetin which binds at novel binding site, distinct from the other known sites of the enzyme. One of the most potent of the studied extracts had also a moderate effect on glycogenolysis in the cellular lever with an IC50 value of 17.35 µg/mL. These results highlight the importance of natural resources in the quest for the discovery of new hypoglycemic agents, while at the same time they can serve as the starting point for their exploitation for antidiabetic usage and the development of novel biofunctional foods.