In Vivo Treatment with a Standardized Green Tea Extract Restores Cardiomyocyte Contractility in Diabetic Rats by Improving Mitochondrial Function through SIRT1 Activation.

Background. Green tea catechins are known to promote mitochondrial function, and to modulate gene expression and signalling pathways that are altered in the diabetic heart. We thus evaluated the effectiveness of the in vivo administration of a standardized green tea extract (GTE) in restoring cardiac performance, in a rat model of early streptozotocin-induced diabetes, with a focus on the underlying mechanisms. Methods. Twenty-five male adult Wistar rats were studied: the control (n = 9), untreated diabetic animals (n = 7) and diabetic rats subjected to daily GTE administration for 28 days (n = 9). Isolated ventricular cardiomyocytes were used for ex vivo measurements of cell mechanics and calcium transients, and molecular assays, including the analysis of functional protein and specific miRNA expression. Results. GTE treatment induced an almost complete recovery of cardiomyocyte contractility that was markedly impaired in the diabetic cells, by preserving mitochondrial function and energy availability, and modulating the expression of the sarcoplasmic reticulum calcium ATPase and phospholamban. Increased Sirtuin 1 (SIRT1) expression and activity substantially contributed to the observed cardioprotective effects. Conclusions. The data supported the hypothesis that green tea dietary polyphenols, by targeting SIRT1, can constitute an adjuvant strategy for counteracting the initial damage of the diabetic heart, before the occurrence of diabetic cardiomyopathy.

The Potential of Epigallocatechin Gallate (EGCG) in Targeting Autophagy for Cancer Treatment: A Narrative Review.

Autophagy is an evolutionarily conserved process for the degradation of redundant or damaged cellular material by means of a lysosome-dependent mechanism, contributing to cell homeostasis and survival. Autophagy plays a multifaceted and context-dependent role in cancer initiation, maintenance, and progression; it has a tumor suppressive role in the absence of disease and is upregulated in cancer cells to meet their elevated metabolic demands. Autophagy represents a promising but challenging target in cancer treatment. Green tea is a widely used beverage with healthy effects on several diseases, including cancer. The bioactive compounds of green tea are mainly catechins, and epigallocatechin-gallate (EGCG) is the most abundant and biologically active among them. In this review, evidence of autophagy modulation and anti-cancer effects induced by EGCG treatment in experimental cancer models is presented. Reviewed articles reveal that EGCG promotes cytotoxic autophagy often through the inactivation of PI3K/Akt/mTOR pathway, resulting in apoptosis induction. EGCG pro-oxidant activity has been postulated to be responsible for its anti-cancer effects. In combination therapy with a chemotherapy drug, EGCG inhibits cell growth and the drug-induced pro-survival autophagy. The selected studies rightly claim EGCG as a valuable agent in cancer chemoprevention.

Effects of Standardized Green Tea Extract and Its Main Component, EGCG, on Mitochondrial Function and Contractile Performance of Healthy Rat Cardiomyocytes.

We recently showed that the long-term in vivo administration of green tea catechin extract (GTE) resulted in hyperdynamic cardiomyocyte contractility. The present study investigates the mechanisms underlying GTE action in comparison to its major component, epigallocatechin-3-gallate (EGCG), given at the equivalent amount that would be in the entirety of GTE. Twenty-six male Wistar rats were given 40 mL/day of a tap water solution with either standardized GTE or pure EGCG for 4 weeks. Cardiomyocytes were then isolated for the study. Cellular bioenergetics was found to be significantly improved in both GTE- and EGCG-fed rats compared to that in controls as shown by measuring the maximal mitochondrial respiration rate and the cellular ATP level. Notably, the improvement of mitochondrial function was associated with increased levels of oxidative phosphorylation complexes, whereas the cellular mitochondrial mass was unchanged. However, only the GTE supplement improved cardiomyocyte mechanics and intracellular calcium dynamics, by lowering the expression of total phospholamban (PLB), which led to an increase of both the phosphorylated-PLB/PLB and the sarco-endoplasmic reticulum calcium ATPase/PLB ratios. Our findings suggest that GTE might be a valuable adjuvant tool for counteracting the occurrence and/or the progression of cardiomyopathies in which mitochondrial dysfunction and alteration of intracellular calcium dynamics constitute early pathogenic factors.

Flavonoids as Epigenetic Modulators for Prostate Cancer Prevention.

Prostate cancer (PCa) is a multifactorial disease with an unclear etiology. Due to its high prevalence, long latency, and slow progression, PCa is an ideal target for chemoprevention strategies. Many research studies have highlighted the positive effects of natural flavonoids on chronic diseases, including PCa. Different classes of dietary flavonoids exhibit anti-oxidative, anti-inflammatory, anti-mutagenic, anti-aging, cardioprotective, anti-viral/bacterial and anti-carcinogenic properties. We overviewed the most recent evidence of the antitumoral effects exerted by dietary flavonoids, with a special focus on their epigenetic action in PCa. Epigenetic alterations have been identified as key initiating events in several kinds of cancer. Many dietary flavonoids have been found to reverse DNA aberrations that promote neoplastic transformation, particularly for PCa. The epigenetic targets of the actions of flavonoids include oncogenes and tumor suppressor genes, indirectly controlled through the regulation of epigenetic enzymes such as DNA methyltransferase (DNMT), histone acetyltransferase (HAT), and histone deacetylase (HDAC). In addition, flavonoids were found capable of restoring miRNA and lncRNA expression that is altered during diseases. The optimization of the use of flavonoids as natural epigenetic modulators for chemoprevention and as a possible treatment of PCa and other kinds of cancers could represent a promising and valid strategy to inhibit carcinogenesis and fight cancer.

Molecular Targets of Epigallocatechin-Gallate (EGCG): A Special Focus on Signal Transduction and Cancer.

Green tea is a beverage that is widely consumed worldwide and is believed to exert effects on different diseases, including cancer. The major components of green tea are catechins, a family of polyphenols. Among them, epigallocatechin-gallate (EGCG) is the most abundant and biologically active. EGCG is widely studied for its anti-cancer properties. However, the cellular and molecular mechanisms explaining its action have not been completely understood, yet. EGCG is effective in vivo at micromolar concentrations, suggesting that its action is mediated by interaction with specific targets that are involved in the regulation of crucial steps of cell proliferation, survival, and metastatic spread. Recently, several proteins have been identified as EGCG direct interactors. Among them, the trans-membrane receptor 67LR has been identified as a high affinity EGCG receptor. 67LR is a master regulator of many pathways affecting cell proliferation or apoptosis, also regulating cancer stem cells (CSCs) activity. EGCG was also found to be interacting directly with Pin1, TGFR-II, and metalloproteinases (MMPs) (mainly MMP2 and MMP9), which respectively regulate EGCG-dependent inhibition of NF-kB, epithelial-mesenchimal transaction (EMT) and cellular invasion. EGCG interacts with DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), which modulates epigenetic changes. The bulk of this novel knowledge provides information about the mechanisms of action of EGCG and may explain its onco-suppressive function. The identification of crucial signalling pathways that are related to cancer onset and progression whose master regulators interacts with EGCG may disclose intriguing pharmacological targets, and eventually lead to novel combined treatments in which EGCG acts synergistically with known drugs.

Long-Term Oral Administration of Theaphenon-E Improves Cardiomyocyte Mechanics and Calcium Dynamics by Affecting Phospholamban Phosphorylation and ATP Production.

BACKGROUND/AIMS: Dietary polyphenols from green tea have been shown to possess cardio-protective activities in different experimental models of heart diseases and age-related ventricular dysfunction. The present study was aimed at evaluating whether long term in vivo administration of green tea extracts (GTE), can exert positive effects on the normal heart, with focus on the underlying mechanisms. METHODS: The study population consisted of 20 male adult Wistar rats. Ten animals were given 40 mL/day tap water solution of GTE (concentration 0.3%) for 4 weeks (GTE group). The same volume of water was administered to the 10 remaining control rats (CTRL). Then, in vivo and ex vivo measurements of cardiac function were performed in the same animal, at the organ (hemodynamics) and cellular (cardiomyocyte mechanical properties and intracellular calcium dynamics) levels. On cardiomyocytes and myocardial tissue samples collected from the same in vivo studied animals, we evaluated: (1) the intracellular content of ATP, (2) the endogenous mitochondrial respiration, (3) the expression levels of the Sarcoplasmic Reticulum Ca2+-dependent ATPase 2a (SERCA2), the Phospholamban (PLB) and the phosphorylated form of PLB, the L-type Ca2+ channel, the Na+-Ca2+ exchanger, and the ryanodine receptor 2. RESULTS: GTE cardiomyocytes exhibited a hyperdynamic contractility compared with CTRL (the rate of shortening and re-lengthening, the fraction of shortening, the amplitude of calcium transient, and the rate of cytosolic calcium removal were significantly increased). A faster isovolumic relaxation was also observed at the organ level. Consistent with functional data, we measured a significant increase in the intracellular ATP content supported by enhanced endogenous mitochondrial respiration in GTE cardiomyocytes, as well as higher values of the ratios phosphorylated-PLB/PLB and SERCA2/PLB. CONCLUSIONS: Long-term in vivo administration of GTE improves cell mechanical properties and intracellular calcium dynamics in normal cardiomyocytes, by increasing energy availability and removing the inhibitory effect of PLB on SERCA2.

A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.

Iron-sulfur (Fe/S) cluster enzymes are crucial to life. Their assembly requires a suite of proteins, some of which are specific for particular subsets of Fe/S enzymes. One such protein is yeast Iba57p, which aconitase and certain radical S-adenosylmethionine enzymes require for activity. Iba57p homologs occur in all domains of life; they belong to the COG0354 protein family and are structurally similar to various folate-dependent enzymes. We therefore investigated the possible relationship between folates and Fe/S cluster enzymes using the Escherichia coli Iba57p homolog, YgfZ. NMR analysis confirmed that purified YgfZ showed stereoselective folate binding. Inactivating ygfZ reduced the activities of the Fe/S tRNA modification enzyme MiaB and certain other Fe/S enzymes, although not aconitase. When successive steps in folate biosynthesis were ablated, folE (lacking pterins and folates) and folP (lacking folates) mutants mimicked the ygfZ mutant in having low MiaB activities, whereas folE thyA mutants supplemented with 5-formyltetrahydrofolate (lacking pterins and depleted in dihydrofolate) and gcvP glyA mutants (lacking one-carbon tetrahydrofolates) had intermediate MiaB activities. These data indicate that YgfZ requires a folate, most probably tetrahydrofolate. Importantly, the ygfZ mutant was hypersensitive to oxidative stress and grew poorly on minimal media. COG0354 genes of bacterial, archaeal, fungal, protistan, animal, or plant origin complemented one or both of these growth phenotypes as well as the MiaB activity phenotype. Comparative genomic analysis indicated widespread functional associations between COG0354 proteins and Fe/S cluster metabolism. Thus COG0354 proteins have an ancient, conserved, folate-dependent function in the activity of certain Fe/S cluster enzymes.

Plant Vacuolar ATP-binding Cassette Transporters That Translocate Folates and Antifolates in Vitro and Contribute to Antifolate Tolerance in Vivo.

The vacuoles of pea (Pisum sativum) leaves and red beet (Beta vulgaris) storage root are major sites for the intracellular compartmentation of folates. In the light of these findings and preliminary experiments indicating that some plant multidrug resistance-associated protein (MRP) subfamily ATP-binding cassette transporters are able to transport compounds of this type, the Arabidopsis thaliana vacuolar MRP, AtMRP1 (AtABCC1), and its functional equivalent(s) in vacuolar membrane vesicles purified from red beet storage root were studied. In so doing, it has been determined that heterologously expressed AtMRP1 and its equivalents in red beet vacuolar membranes are not only competent in the transport of glutathione conjugates but also folate monoglutamates and antifolates as exemplified by pteroyl-l-glutamic acid and methotrexate (MTX), respectively. In agreement with the results of these in vitro transport measurements, analyses of atmrp1 T-DNA insertion mutants of Arabidopsis ecotypes Wassilewskia and Columbia disclose an MTX-hypersensitive phenotype. atmrp1 knock-out mutants are more sensitive than wild-type plants to growth retardation by nanomolar concentrations of MTX, and this is associated with impaired vacuolar antifolate sequestration. The vacuoles of protoplasts isolated from the leaves of Wassilewskia atmrp1 mutants accumulate 50% less [(3)H]MTX than the vacuoles of protoplasts from wild-type plants when incubated in media containing nanomolar concentrations of this antifolate, and vacuolar membrane-enriched vesicles purified from the mutant catalyze MgATP-dependent [(3)H]MTX uptake at only 40% of the capacity of the equivalent membrane fraction from wild-type plants. AtMRP1 and its counterparts in other plant species therefore have the potential for participating in the vacuolar accumulation of folates and related compounds.

Pterin and folate salvage. Plants and Escherichia coli lack capacity to reduce oxidized pterins.

Dihydropterins are intermediates of folate synthesis and products of folate breakdown that are readily oxidized to their aromatic forms. In trypanosomatid parasites, reduction of such oxidized pterins is crucial for pterin and folate salvage. We therefore sought evidence for this reaction in plants. Three lines of evidence indicated its absence. First, when pterin-6-aldehyde or 6-hydroxymethylpterin was supplied to Arabidopsis (Arabidopsis thaliana), pea (Pisum sativum), or tomato (Lycopersicon esculentum) tissues, no reduction of the pterin ring was seen after 15 h, although reduction and oxidation of the side chain of pterin-6-aldehyde were readily detected. Second, no label was incorporated into folates when 6-[(3)H]hydroxymethylpterin was fed to cultured Arabidopsis plantlets for 7 d, whereas [(3)H]folate synthesis from p-[(3)H]aminobenzoate was extensive. Third, no NAD(P)H-dependent pterin ring reduction was found in tissue extracts. Genetic evidence showed a similar situation in Escherichia coli: a GTP cyclohydrolase I (folE) mutant, deficient in pterin synthesis, was rescued by dihydropterins but not by the corresponding oxidized forms. Expression of a trypanosomatid pterin reductase (PTR1) enabled rescue of the mutant by oxidized pterins, establishing that E. coli can take up oxidized pterins but cannot reduce them. Similarly, a GTP cyclohydrolase I (fol2) mutant of yeast (Saccharomyces cerevisiae) was rescued by dihydropterins but not by most oxidized pterins, 6-hydroxymethylpterin being an exception. These results show that the capacity to reduce oxidized pterins is not ubiquitous in folate-synthesizing organisms. If it is lacking, folate precursors or breakdown products that become oxidized will permanently exit the metabolically active pterin pool.