Plant-Derived Natural Products for the Treatment of Bacterial Infections.

Bacterial infections are a significant public health concern, and the emergence of antibiotic-resistant bacteria (ARB) has become a major challenge for modern medicine. The overuse and misuse of antibiotics have contributed to the development of ARB, which has led to the need for alternative therapies. Plant-derived natural products (PNPs) have been extensively studied for their potential as alternative therapies for the treatment of bacterial infections. The diverse chemical compounds found in plants have shown significant antibacterial properties, making them a promising source of novel antibacterial agents. The use of PNPs as antibacterial agents is particularly appealing because they offer a relatively safe and cost-effective approach to the treatment of bacterial infections. This chapter aims to provide an overview of the current state of research on PNPs as antibacterial agents. It will cover the mechanisms of action of the main PNPs against bacterial pathogens and discuss their potential to be used as complementary therapies to combat ARB. This chapter will also highlight the most common screening methodologies to discover new PNPs and the challenges and future prospects in the development of these compounds as antibacterial agents.

Influence of Drying Temperature and Harvesting Season on Phenolic Content and Antioxidant and Antiproliferative Activities of Olive (Olea europaea) Leaf Extracts.

Interest in plant compounds has increased, given recent evidence regarding their role in human health due to their pleiotropic effects. For example, plant bioactive compounds present in food products, including polyphenols, are associated with preventive effects in various diseases, such as cancer or inflammation. Breast and colorectal cancers are among the most commonly diagnosed cancers globally. Although appreciable advances have been made in treatments, new therapeutic approaches are still needed. Thus, in this study, up to 28 olive leaf extracts were obtained during different seasons and using different drying temperatures. The influence of these conditions on total polyphenolic content (measured using Folin-Ciocalteu assays), antioxidant activity (using Trolox Equivalent Antioxidant Capacity and Ferric Reducing Ability of Plasma assays) and antiproliferative capacity (using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT assays) was tested in breast and colorectal cancer cells. Increased phenolic composition and antioxidant and antiproliferative capacity are noted in the extracts obtained from leaves harvested in autumn, followed by summer, spring and winter. Regarding drying conditions, although there is not a general trend, conditions using the highest temperatures lead to the optimal phenolic content and antioxidant and antiproliferative activities in most cases. These results confirm previously published studies and provide evidence in support of the influence of both harvesting and drying conditions on the biological activity of olive leaf extracts.

The metabolic and vascular protective effects of olive (Olea europaea L.) leaf extract in diet-induced obesity in mice are related to the amelioration of gut microbiota dysbiosis and to its immunomodulatory properties.

INTRODUCTION: Many studies have showed the beneficial effects of the olive (Olea europaea) leaf extract (OLE) in experimental models of metabolic syndrome, which have been ascribed to the presence of phenolic compounds, like oleuropeoside. This study evaluated the effects of a chemically characterized OLE in high fat diet (HFD)-induced obesity in mice, describing the underlying mechanisms involved in the beneficial effects, with special attention to vascular dysfunction and gut microbiota composition. METHODS: C57BL/6J mice were distributed in different groups: control, control-treated, obese and obese-treated with OLE (1, 10 and 25 mg/kg/day). Control mice received a standard diet, whereas obese mice were fed HFD. The treatment was followed for 5 weeks, and animal body weight periodically assessed. At the end of the treatment, metabolic plasma analysis (including lipid profile) as well as glucose and insulin levels were performed. The HFD-induced inflammatory status was studied in liver and fat, by determining the RNA expression of different inflammatory mediators by qPCR; also, different markers of intestinal epithelial barrier function were determined in colonic tissue by qPCR. Additionally, flow cytometry of immune cells from adipose tissue, endothelial dysfunction in aortic rings as well as gut microbiota composition were evaluated. Faecal microbiota transplantation (FMT) to antibiotic-treated mice fed with HFD was performed. RESULTS: OLE administration reduced body weight gain, basal glycaemia and insulin resistance, and showed improvement in plasma lipid profile when compared with HFD-fed mice. The extract significantly ameliorated the HFD-induced altered expression of key adipogenic genes, like PPARs, adiponectin and leptin receptor, in adipose tissue. Furthermore, the extract reduced the RNA expression of Tnf-α, Il-1ß, Il-6 in liver and adipose tissue, thus improving the tissue inflammatory status associated to obesity. The flow cytometry analysis in adipose tissue corroborated these observations. Additionally, the characterization of the colonic microbiota by sequencing showed that OLE administration was able to counteract the dysbiosis associated to obesity. The extract reversed the endothelial dysfunction observed in the aortic rings of obese mice. FMT from donors HFD-OLE to recipient mice fed an HFD prevented the development of obesity, glucose intolerance, insulin resistance and endothelial dysfunction. CONCLUSION: OLE exerts beneficial effects in HFD-induced obesity in mice, which was associated to an improvement in plasma and tissue metabolic profile, inflammatory status, gut microbiota composition and vascular dysfunction.

Intestinal Permeability Study of Clinically Relevant Formulations of Silibinin in Caco-2 Cell Monolayers.

An ever-growing number of preclinical studies have investigated the tumoricidal activity of the milk thistle flavonolignan silibinin. The clinical value of silibinin as a bona fide anti-cancer therapy, however, remains uncertain with respect to its bioavailability and blood⁻brain barrier (BBB) permeability. To shed some light on the absorption and bioavailability of silibinin, we utilized the Caco-2 cell monolayer model of human intestinal absorption to evaluate the permeation properties of three different formulations of silibinin: silibinin-meglumine, a water-soluble form of silibinin complexed with the amino-sugar meglumine; silibinin-phosphatidylcholine, the phytolipid delivery system Siliphos; and Eurosil85/Euromed, a milk thistle extract that is the active component of the nutraceutical Legasil with enhanced bioavailability. Our approach predicted differential mechanisms of transport and blood⁻brain barrier permeabilities between the silibinin formulations tested. Our assessment might provide valuable information about an idoneous silibinin formulation capable of reaching target cancer tissues and accounting for the observed clinical effects of silibinin, including a recently reported meaningful central nervous system activity against brain metastases.

Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells.

Targeting tumor-initiating, drug-resistant populations of cancer stem cells (CSC) with phytochemicals is a novel paradigm for cancer prevention and treatment. We herein employed a phenotypic drug discovery approach coupled to mechanism-of-action profiling and target deconvolution to identify phenolic components of extra virgin olive oil (EVOO) capable of suppressing the functional traits of CSC in breast cancer (BC). In vitro screening revealed that the secoiridoid decarboxymethyl oleuropein aglycone (DOA) could selectively target subpopulations of epithelial-like, aldehyde dehydrogenase (ALDH)-positive and mesenchymal-like, CD44+CD24-/low CSC. DOA could potently block the formation of multicellular tumorspheres generated from single-founder stem-like cells in a panel of genetically diverse BC models. Pretreatment of BC populations with noncytotoxic doses of DOA dramatically reduced subsequent tumor-forming capacity in vivo. Mice orthotopically injected with CSC-enriched BC-cell populations pretreated with DOA remained tumor-free for several months. Phenotype microarray-based screening pointed to a synergistic interaction of DOA with the mTOR inhibitor rapamycin and the DNA methyltransferase (DNMT) inhibitor 5-azacytidine. In silico computational studies indicated that DOA binds and inhibits the ATP-binding kinase domain site of mTOR and the S-adenosyl-l-methionine (SAM) cofactor-binding pocket of DNMTs. FRET-based Z-LYTE™ and AlphaScreen-based in vitro assays confirmed the ability of DOA to function as an ATP-competitive mTOR inhibitor and to block the SAM-dependent methylation activity of DNMTs. Our systematic in vitro, in vivo and in silico approaches establish the phenol-conjugated oleoside DOA as a dual mTOR/DNMT inhibitor naturally occurring in EVOO that functionally suppresses CSC-like states responsible for maintaining tumor-initiating cell properties within BC populations.

New Mammalian Target of Rapamycin (mTOR) Modulators Derived from Natural Product Databases and Marine Extracts by Using Molecular Docking Techniques.

Mammalian target of rapamycin (mTOR) is a PI3K-related serine/threonine protein kinase that functions as a master regulator of cellular growth and metabolism, in response to nutrient and hormonal stimuli. mTOR functions in two distinct complexes-mTORC1 is sensitive to rapamycin, while, mTORC2 is insensitive to this drug. Deregulation of mTOR's enzymatic activity has roles in cancer, obesity, and aging. Rapamycin and its chemical derivatives are the only drugs that inhibit the hyperactivity of mTOR, but numerous side effects have been described due to its therapeutic use. The purpose of this study was to identify new compounds of natural origin that can lead to drugs with fewer side effects. We have used computational techniques (molecular docking and calculated ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) parameters) that have enabled the selection of candidate compounds, derived from marine natural products, SuperNatural II, and ZINC natural products, for inhibitors targeting, both, the ATP and the rapamycin binding sites of mTOR. We have shown experimental evidence of the inhibitory activity of eleven selected compounds against mTOR. We have also discovered the inhibitory activity of a new marine extract against this enzyme. The results have been discussed concerning the necessity to identify new molecules for therapeutic use, especially against aging, and with fewer side effects.

An Updated Review on Marine Anticancer Compounds: The Use of Virtual Screening for the Discovery of Small-Molecule Cancer Drugs.

Marine secondary metabolites are a promising source of unexploited drugs that have a wide structural diversity and have shown a variety of biological activities. These compounds are produced in response to the harsh and competitive conditions that occur in the marine environment. Invertebrates are considered to be among the groups with the richest biodiversity. To date, a significant number of marine natural products (MNPs) have been established as antineoplastic drugs. This review gives an overview of MNPs, both in research or clinical stages, from diverse organisms that were reported as being active or potentially active in cancer treatment in the past seventeen years (from January 2000 until April 2017) and describes their putative mechanisms of action. The structural diversity of MNPs is also highlighted and compared with the small-molecule anticancer drugs in clinical use. In addition, this review examines the use of virtual screening for MNP-based drug discovery and reveals that classical approaches for the selection of drug candidates based on ADMET (absorption, distribution, metabolism, excretion, and toxicity) filtering may miss potential anticancer lead compounds. Finally, we introduce a novel and publically accessible chemical library of MNPs for virtual screening purposes.

Further exploring the absorption and enterocyte metabolism of quercetin forms in the Caco-2 model using nano-LC-TOF-MS.

When using the Caco-2 intestinal model, the low uptake, intracellular presence at low levels as well as generation of trace metabolites may limit the analysis of flavonoids. To overcome these limitations, we performed a simple but sensitive methodology based on nano-LC-TOF-MS, using an on-line trapping step. The analytical method was validated for quercetin, quercetin 3-O-glucoside, and quercetin 3-O-glucuronide and the reliability for characterization using lock-mass calibration was also assessed along the linear range. Afterwards, the in vitro absorption, metabolism, and cellular occurrence were explored with the proposed methodology. The apparent permeability coefficient in the absorptive direction and the cellular accumulation were higher for quercetin aglycone, with a value of 2.61 × 10(-6) cm/s and relative amounts of 0.73 and 1.17% in the cytosolic and solid particle fraction at the end of the assay, respectively. Alternatively, the net efflux ratio was lower for quercetin than for their derivatives. Moreover, depending on the structure of the parent compound, metabolites were generated by glucuronidation, sulfation, and methylation.

Effect of polyphenol supplements on redox status of blood cells: a randomized controlled exercise training trial.

PURPOSE: The effect of endogenous antioxidants can be either an immediate response (relying on enzymatic activities) or a long-term adaptation (relying on gene modulation events), both susceptible to be modified by antioxidants from diet and supplementation. The aim of this work was to delve in these aspects in circulating white blood cells in a group of volunteers (n = 33, 20-22 years) performing eccentric exercises and consuming or not (n = 8) different polyphenolic antioxidants (Lippia citriodora extract-PLX(®) n = 8, almond beverage n = 9 or a mixture of both n = 8) during 21 days. METHODS: We have designed a single-blind, parallel-group, randomized controlled trial. Antioxidant enzyme activities, oxidative stress markers, and antioxidant gene expression were determined. RESULTS: Neutrophils and lymphocytes expressed high amounts of oxidative markers compared to plasma. Concerning enzymatic activities, increased superoxide dismutase levels were detected when certain supplements were consumed. However, catalase levels did not change. As for glutathione peroxidase levels, no differences were detected in lymphocytes, while neutrophils expressed increased levels in both placebo and PLX(®) groups. Glutathione reductase activity was decreased in all groups, except in neutrophils of PLX(®) group. At the level of gene expression, neither PLX(®) nor the almond beverage interfered with the expression of genes coding for the corresponding enzymes. However, the combined intake of both supplements affected the expression of glutathione reductase and Cu-Zn and Mn-superoxide dismutases in neutrophils. CONCLUSIONS: Altogether, these results suggest that blood cell types respond and adapt differently to exercise-induced oxidative damage.

The promiscuous and synergic molecular interaction of polyphenols in bactericidal activity: an opportunity to improve the performance of antibiotics?

Plant polyphenols are a potential source of new antimicrobial molecules against bacteria because most newly developed antimicrobial agents do not improve the clinical management of infectious diseases. The potential synergism between the major polyphenolic compounds present in a Cistus salviifolius extract, which was characterized by HPLC-ESI-MS/MS, was investigated by the isobole method and the fractional inhibitory concentration index determination. Pairwise combinations of selected flavonoids and ellagitannins present in C. salviifolius extract were assayed against the in vitro growth of Staphylococcus aureus. Some combinations revealed synergic effects, resulting in a reduction of the minimum inhibitory concentration required to inhibit 50% growth (MIC50 ) up to 20 times lower as compared with the individual compounds. Some of the combinations exhibited MIC50 values close to drug potency level (0.5-1 µg/mL). Punicalagin and myricetin were the major contributors in the combinations. The proportion between the compounds in the synergic mixtures is crucial and may explain the superior antimicrobial activity displayed by this extract when compared with other botanical extracts. The rational optimization of these combinations could lead to the design of potent antimicrobial phytopharmaceuticals, which may improve the performance of current antibiotics, taking advantage of the multi-targeted and synergic molecular interactions of selected polyphenols.

Permeability Study of Polyphenols Derived from a Phenolic-Enriched Hibiscus sabdariffa Extract by UHPLC-ESI-UHR-Qq-TOF-MS.

Previous findings on the capacity of Hibiscus sabdariffa (HS) polyphenols to ameliorate metabolic disturbances justify the necessity of studies oriented to find the potential metabolites responsible for such an effect. The present study examined the intestinal epithelial membrane permeability of polyphenols present in a phenolic-enriched Hibiscus sabdariffa extract (PEHS), free and encapsulated, using the Caco-2 cell line. Additionally, selected polyphenols (quercetin, quercetin-3-glucoside, quercetin-3-glucuronide, and N-feruloyltyramine) were also studied in the same absorption model. The powerful analytical platform used ultra-high-performance liquid chromatography coupled with ultra-high-resolution quadrupole time-of-flight mass spectrometry (UHPLC-ESI-UHR-Qq-TOF-MS), and enabled the characterization of seven new compounds in PEHS. In the permeation study, only a few compounds were able to cross the cell monolayer and the permeability was lower when the extract was in an encapsulated form. Pure compounds showed a moderate absorption in all cases. Nevertheless, these preliminary results may need further research to understand the complete absorption mechanism of Hibiscus polyphenols.

Comprehensive foodomics study on the mechanisms operating at various molecular levels in cancer cells in response to individual rosemary polyphenols.

In this work, the contribution of carnosic acid (CA) and carnosol (CS), two major compounds present in rosemary, against colon cancer HT-29 cells proliferation is investigated using a comprehensive Foodomics approach. The Foodomics study reveals that CA induces transcriptional activation of genes that encode detoxifying enzymes and altered the expression of genes linked to transport and biosynthesis of terpenoids in the colon cancer cell line. Functional analysis highlighted the activation of the ROS metabolism and alteration of several genes involved in pathways describing oxidative degradation of relevant endogenous metabolites, providing new evidence about the transcriptional change induced by CA in HT-29 cells. Metabolomics analysis showed that the treatment with CA affected the intracellular levels of glutathione. Elevated levels of GSH provided additional evidence to transcriptomic results regarding chemopreventive response of cells to CA treatment. Moreover, the Foodomics approach was useful to establish the links between decreased levels of N-acetylputrescine and its degradation pathway at the gene level. The findings from this work and the predictions based on microarray data will help explore novel metabolic processes and potential signaling pathways to further elucidate the effect of CA in colon cancer cells.

Polyphenols and the modulation of gene expression pathways: can we eat our way out of the danger of chronic disease?

Plant-derived dietary polyphenols may improve some disease states and promote health. Experimental evidence suggests that this is partially attributable to changes in gene expression. The rational use of bioactive food components may therefore present an opportunity to activate or repress selected gene expression pathways and, consequently, to manage or prevent disease. It remains to be determined whether this use of bioactive food components can be done safely. This article reviews the associated controversies and limitations of polyphenol therapy. There is a paucity of clinical data on the rational use of polyphenols, including a lack of knowledge on effective dosage, actual chemical formulations, bioavailability, distribution in tissues, the effect of genetic variations, differences in gut microflora, the synergistic (or antagonistic) effects observed in extracts, and the possible interaction between polyphenols and lipid domains of cell membranes that may alter the function of relevant receptors. The seminal question of why plants make substances that benefit humans remains unanswered, and there is still much to learn in terms of correlative versus causal effects of human exposure to various nutrients. The available data strongly suggest significant effects at the molecular level that represent interactions with the epigenome. The advent of relatively simple technologies is helping the field of epigenetics progress and facilitating the acquisition of multiple types of data that were previously difficult to obtain. In this review, we summarize the molecular basis of the epigenetic regulation of gene expression and the epigenetic changes associated with the consumption of polyphenols that illustrate how modifications in human nutrition may become relevant to health and disease.

Molecular promiscuity of plant polyphenols in the management of age-related diseases: far beyond their antioxidant properties.

The use of plant-derived polyphenols for the management of diseases has been under debate in the last decades. Most studies have focused on the specific effects of polyphenols on particular targets, while ignoring their pleiotropic character. The multitargeted character of polyphenols, a plausible consequence of their molecular promiscuity, may suppose an opportunity to fight multifactorial diseases. Therefore, a wider perspective is urgently needed to elucidate whether their rational use as bioactive food components may be valid for the management of diseases. In this chapter, we discuss the most likely targets of polyphenols that may account for their salutary effects from a global perspective. Among these targets, the modulation of signalling and energy-sensitive pathways, oxidative stress and inflammation-related processes, mitochondrial functionality, epigenetic machinery, histone acetylation and membrane-dependent processes play central roles in polyphenols' mechanisms of action.Sufficient evidence on polyphenols has accumulated for them to be considered a serious option for the management of non-communicable diseases, such as cancer and obesity, as well as infectious diseases. The remaining unresolved issues that must be seriously addressed are their bioavailability, metabolite detection, specific molecular targets, interactions and toxicity. The Xenohormesis hypothesis, which postulates that polyphenols are the product of plant evolutive adaptation to stress and conferee their resistance to mammals, offers a reasonable explanation to justify the beneficial and non-toxic effects of plant mixtures, but do not fully meet expectations. Hence, future research must be supported by the use of complex polypharmacology approaches and synergic studies focused on the understanding of the pleiotropic effects of polyphenols. Revisiting polyphenol mechanisms of action with the help of these techniques may allow for the improvement of human health and wellness by using intelligent nutritional intervention.

Analysis of Lemon Verbena Polyphenol Metabolome and Its Correlation with Oxidative Stress under Glucotoxic Conditions in Adipocyte.

Lemon verbena has been shown to ameliorate obesity-related oxidative stress, but the intracellular final effectors underlying its antioxidant activity are still unknown. The purpose of this study was to correlate the antioxidant capacity of plasma metabolites of lemon verbena (verbascoside, isoverbascoside, hydroxytyrosol, caffeic acid, ferulic acid, homoprotocatechuic acid, and luteolin-7-diglucuronide) with their uptake and intracellular metabolism in hypertrophic adipocytes under glucotoxic conditions. To this end, intracellular ROS levels were measured, and the intracellular metabolites were identified and quantified by high-performance liquid chromatography with a diode array detector coupled to mass spectrometry (HPLC-DAD-MS). The results showed that the plasma metabolites of lemon verbena are absorbed by adipocytes and metabolized through phase II reactions and that the intracellular appearance of these metabolites correlates with the decrease in the level of glucotoxicity-induced oxidative stress. It is postulated that the biotransformation and accumulation of these metabolites in adipocytes contribute to the long-term antioxidant activity of the extract.

Acute Antioxidant Response to Two Types of Exercises: 2000 M Run vs. Burpee Test.

Physical activity results in oxidative stress, as evidenced by the increased production of reactive oxygen, nitrogen species, and inflammatory mediators. The management of these components is instrumental for antioxidant adaptation to exercise and post-exercise recovery. Therefore, the present report aims to study the antioxidant response to two types of exercise (a 2000 m run and a burpee test) in healthy volunteers after a long period of inactivity (1-2 months). Antioxidant enzyme activities and oxidative stress markers (protein carbonyls and malondialdehyde content) were measured in neutrophils, peripheral blood mononuclear cells, and plasma. These parameters were determined under basal conditions and immediately post-exercise. Compared to those in basal state, neutrophil superoxide dismutase (28.3 vs. 22.9 pkat/109 cells), glutathione peroxidase (147.5 vs. 120.1 nkat/109 cells), and catalase (106.3 vs. 57.9 k/109 cells) were activated significantly (p < 0.05) after the burpee test. Peripheral blood mononuclear cells exhibited only significant (p < 0.05) catalase activation (113.6 vs. 89.4 k/109 cells) after the burpee test. Other enzymes, such as glutathione reductase and myeloperoxidase, tended to increase post-exercise, although the differences from baseline were not significant. Finally, compared to basal conditions, the protein carbonyl (24.5 vs. 14.5 mmol/L) and malondialdehyde (39.6 vs. 18.3 mmol/L) contents increased significantly (p < 0.05) in neutrophils and in plasma (115.1 vs. 97.8 and 130.2 vs. 123.4 µmol/L, respectively) after the burpee test. In conclusion, high-intensity exercise seems to induce immediate oxidative stress in inactive individuals, and the acute antioxidant response was slightly greater after the burpee test than after the 2000 m run. Glutathione-dependent antioxidant systems are activated immediately as protective mechanisms.

Evaluation of Bioactive Effects of Five Plant Extracts with Different Phenolic Compositions against Different Therapeutic Targets.

Plant extracts rich in phenolic compounds have been reported to exert different bioactive properties. Despite the fact that there are plant extracts with completely different phenolic compositions, many of them have been reported to have similar beneficial properties. Thus, the structure-bioactivity relationship mechanisms are not yet known in detail for specific classes of phenolic compounds. In this context, this work aims to demonstrate the relationship of extracts with different phenolic compositions versus different bioactive targets. For this purpose, five plant matrices (Theobroma cacao, Hibiscus sabdariffa, Silybum marianum, Lippia citriodora, and Olea europaea) were selected to cover different phenolic compositions, which were confirmed by the phytochemical characterization analysis performed by HPLC-ESI-qTOF-MS. The bioactive targets evaluated were the antioxidant potential, the free radical scavenging potential, and the inhibitory capacity of different enzymes involved in inflammatory processes, skin aging, and neuroprotection. The results showed that despite the different phenolic compositions of the five matrices, they all showed a bioactive positive effect in most of the evaluated assays. In particular, matrices with very different phenolic contents, such as T. cacao and S. marianum, exerted a similar inhibitory power in enzymes involved in inflammatory processes and skin aging. It should also be noted that H. sabdariffa and T. cacao extracts had a low phenolic content but nevertheless stood out for their bioactive antioxidant and anti-radical capacity. Hence, this research highlights the shared bioactive properties among phenolic compounds found in diverse matrices. The abundance of different phenolic compound families highlights their elevated bioactivity against diverse biological targets.

Silibinin is a suppressor of the metastasis-promoting transcription factor ID3.

BACKGROUND: ID3 (inhibitor of DNA binding/differentiation-3) is a transcription factor that enables metastasis by promoting stem cell-like properties in endothelial and tumor cells. The milk thistle flavonolignan silibinin is a phytochemical with anti-metastatic potential through largely unknown mechanisms. HYPOTHESIS/PURPOSE: We have mechanistically investigated the ability of silibinin to inhibit the aberrant activation of ID3 in brain endothelium and non-small cell lung cancer (NSCLC) models. METHODS: Bioinformatic analyses were performed to investigate the co-expression correlation between ID3 and bone morphogenic protein (BMP) ligands/BMP receptors (BMPRs) genes in NSCLC patient datasets. ID3 expression was assessed by immunoblotting and qRT-PCR. Luciferase reporter assays were used to evaluate the gene sequences targeted by silibinin to regulate ID3 transcription. In silico computational modeling and LanthaScreen TR-FRET kinase assays were used to characterize and validate the BMPR inhibitory activity of silibinin. Tumor tissues from NSCLC xenograft models treated with oral silibinin were used to evaluate the in vivo anti-ID3 effects of silibinin. RESULTS: Analysis of lung cancer patient datasets revealed a top-ranked positive association of ID3 with the BMP9 endothelial receptor ACVRL1/ALK1 and the BMP ligand BMP6. Silibinin treatment blocked the BMP9-induced activation of the ALK1-phospho-SMAD1/5-ID3 axis in brain endothelial cells. Constitutive, acquired, and adaptive expression of ID3 in NSCLC cells were all significantly downregulated in response to silibinin. Silibinin blocked ID3 transcription via BMP-responsive elements in ID3 gene enhancers. Silibinin inhibited the kinase activities of BMPRs in the micromolar range, with the lower IC50 values occurring against ACVRL1/ALK1 and BMPR2. In an in vivo NSCLC xenograft model, tumoral overexpression of ID3 was completely suppressed by systematically achievable oral doses of silibinin. CONCLUSIONS: ID3 is a largely undruggable metastasis-promoting transcription factor. Silibinin is a novel suppressor of ID3 that may be explored as a novel therapeutic approach to interfere with the metastatic dissemination capacity of NSCLC.

Plant-derived polyphenols regulate expression of miRNA paralogs miR-103/107 and miR-122 and prevent diet-induced fatty liver disease in hyperlipidemic mice.

BACKGROUND: MicroRNAs have the potential for clinical application. Probable modulation by plant-derived polyphenols might open preventive measures using simple dietary recommendations. METHODS: We assessed the ability of continuous administration of high-dose polyphenols to modulate hepatic metabolism and microRNA expression in diet-induced fatty liver disease in commercially available hyperlipidemic mice using well-established and accepted procedures that included the development of new antibodies against modified quercetin. RESULTS: Weight gain, liver steatosis, changes in the composition of liver tissue, and insulin resistance were all attenuated by the continuous administration of polyphenols. We also demonstrated that metabolites of polyphenols accumulate in immune cells and at the surface of hepatic lipid droplets indicating not only bioavailability but a direct likely action on liver cells. The addition of polyphenols also resulted in changes in the expression of miR-103, miR-107 and miR-122. CONCLUSIONS: Polyphenols prevent fatty liver disease under these conditions. The differential expression of mRNAs and miRNAs was also associated with changes in lipid and glucose metabolism and with the activation of 5'-adenosine monophosphate-activated protein kinase, effects that are not necessarily connected. miRNAs function via different mechanisms and miRNA-mRNA interactions are difficult to ascertain with current knowledge. Further, cell models usually elicit contradictory results with those obtained in animal models. GENERAL SIGNIFICANCE: Our data indicate that plant-derived polyphenols should be tested in humans as preventive rather than therapeutic agents in the regulation of hepatic fatty acid utilization. A multi-faceted mechanism of action is likely and the regulation of liver miRNA expression blaze new trails in further research.

Immunoliposome encapsulation increases cytotoxic activity and selectivity of curcumin and resveratrol against HER2 overexpressing human breast cancer cells.

Natural compounds have been studied as a source of countless bioactive compounds with diverse activities. Among them, many dietary phytochemicals have been thoroughly studied for their cytotoxic or apoptotic effects in several cellular models in order to explain their anticancer capacity. Curcumin and resveratrol are two natural compounds with a large body of evidence showing their cytotoxic activity against a wide variety of cancer cells; however, their poor absorption, bioavailability, and low selectivity have limited their clinical use. With the aim of improving bioavailability and selectivity, the antiproliferative effects of free-, liposomed-, and immunoliposomed-curcumin and/or resveratrol formulations have been compared in two human breast cancer cell lines with different HER2 expression levels. The results demonstrate that when HER2-targeted immunoliposomes are coupled to trastuzumab there is a dramatic increase in the antiproliferative effects of curcumin and resveratrol in HER2 positive human breast cancer cells in comparison to regular liposomed or free forms, indicating an increase of its therapeutic effect. The enhancement of the cytotoxic effects was also correlated to the uptake of curcumin at intracellular level, as shown by using ImageStream technique. The striking efficacy of the immunoliposomed formulation containing both resveratrol and curcumin suggests a multitargeted mechanism of action that deserves further study. These findings show the potential of HER2-targeted nanovesicles to develop new drug delivery systems for cancer therapy based on these compounds and justify further preclinical trials.