Paula Fresco

The Anticancer Properties of Dietary Polyphenols and its Relation with Apoptosis

Aberrantly regulated apoptosis is involved in the pathogenesis of several diseases and defective apoptosis leads to uncontrolled cell proliferation and tumorigenesis. Cancer is an example of a pathologic condition where the normal mechanisms of cell cycle regulation are dysfunctional either by excessive cell proliferation, inhibited/suppressed apoptosis or both. Dietary habits are estimated to contribute to, at least, one third of all human cancers, showing that dietary components can exacerbate or interfere with carcinogenesis. However, several epidemiological studies have revealed that some dietary factors can decrease the risk of different types of cancer. Apoptosis is suggested to be a crucial mechanism for the chemopreventive properties associated with several dietary factors by eliminating potentially deleterious (damaged/mutated) cells. Food, a readily available item, contains several promising chemopreventive agents. Polyphenols are serious candidates since they are responsible for the cancer protective properties of a diet rich in vegetables and fruits: numerous phenolic compounds showed antiproliferative and cytotoxic effects, and more specifically pro-apoptotic activities, in several cancer cells lines and animal tumor models. The aim of the present review is to analyze and summarize several aspects related to the molecular mechanisms of apoptosis induced by dietary factors with particular emphasis on polyphenols. Dietary factors that can activate cell death signals and induce apoptosis, preferentially in precancerous or malignant cells, and the study of their apoptotic inducing targets can represent a mean to devise new strategies for cancer prevention in the future.

Synthesis and in vivo modulatory activity of protein kinase C of xanthone derivatives.

The modulatory activity of a series of 20 simple xanthones on isoforms alpha, betaI, delta, eta and zeta of protein kinase C (PKC) was evaluated using an in vivo yeast phenotypic assay. Hydroxy and/or methoxyxanthones were synthesised. The majority of these compounds caused an effect compatible with activation of PKC and some showed to be more effective than the standard PKC activator (PMA or arachidonic acid). The xanthones tested differ in their efficacy and potency towards individual PKC isoforms and some showed higher selectivities for PKC-delta, -eta or -zeta, suggesting that xanthone derivatives can become valuable research tools to elucidate the physiological roles of these isoforms.

Inhibition of protein kinase C by synthetic xanthone derivatives

The modulatory activity of two xanthones (3,4-dihydroxyxanthone and 1-formyl-4-hydroxy-3-methoxyxanthone) on isoforms alpha, betaI, delta, eta and zeta of protein kinase C (PKC) was evaluated using an in vivo yeast phenotypic assay. Both xanthones caused an effect compatible with PKC inhibition, similar to that elicited by known PKC inhibitors (chelerythrine and NPC 15437). PKC inhibition caused by xanthones was confirmed using an in vitro kinase assay. The yeast phenotypic assay revealed that xanthones present differences on their potency towards the distinct PKC isoforms tested. It is concluded that 3,4-dihydroxyxanthone and 1-formyl-4-hydroxy-3-methoxyxanthone may become useful PKC inhibitors and xanthone derivatives can be explored to develop new isoform-selective PKC inhibitors.

Release inhibitory receptors activation favours the A2A‐adenosine receptor‐mediated facilitation of noradrenaline release in isolated rat tail artery

Interactions between A2A‐adenosine receptors and α2‐, A1‐ and P2‐ release‐inhibitory receptors, on the modulation of noradrenaline release were studied in isolated rat tail artery. Preparations were labelled with [3H]‐noradrenaline, superfused with desipramine‐containing medium, and stimulated electrically (100 pulses at 5 Hz or 20 pulses at 50 Hz). Blockade of α2‐autoreceptors with yohimbine (1 μM) increased tritium overflow elicited by 100 pulses at 5 Hz but not by 20 pulses at 50 Hz. The selective A2A‐receptor agonist 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680; 1 – 100 nM) enhanced tritium overflow elicited by 100 pulses at 5 Hz. Yohimbine prevented the effect of CGS 21680, which was restored by the A1‐receptor agonist N6‐cyclopentyladenosine (CPA; 100 nM) or by the P2‐receptor agonist 2‐methylthioadenosine triphosphate (2‐MeSATP; 80 μM). CGS 21680 (100 nM) failed to increase tritium overflow elicited by 20 pulses at 50 Hz. The α2‐adrenoceptor agonist 5‐bromo‐6‐(2‐imidazolin‐2‐ylamino)‐quinoxaline (UK 14304; 30 nM), the A1‐receptor agonist CPA (100 nM) or the P2‐receptor agonist 2‐MeSATP (80 μM) reduced tritium overflow. In the presence of these agonists CGS 21680 elicited a facilitation of tritium overflow. Blockade of potassium channels with tetraethylammonium (TEA; 5 mM) increased tritium overflow elicited by 100 pulses at 5 Hz to values similar to those obtained in the presence of yohimbine but did not prevent the effect of CGS 21680 (100 nM) on tritium overflow. It is concluded that, in isolated rat tail artery, the facilitation of noradrenaline release mediated by A2A‐adenosine receptors is favoured by activation of release inhibitory receptors.

Isoform-selectivity of PKC Inhibitors Acting at the Regulatory and Catalytic Domain of Mammalian PKC-α, -βI, -δ, -η and -ζ

The aim of the present study was to compare the potency of a series of widely used PKC inhibitors acting either at the regulatory (NPC 15437, tamoxifen and d-sphingosine) or at the catalytic domain (Ro 32-0432, chelerythrine and rottlerin) on individual mammalian PKC isoforms of the classical (α and βI), novel (δ and η) and atypical (ζ) PKC families, using the yeast phenotypic assay, in order to determine their isoform-selectivity. The PKC inhibitors studied presented differences in their ability to reduce the effect of the appropriate PKC activator (estimated as EC50 ratios) which was interpreted as an index of PKC inhibitory potency. In general, the more marked inhibition was observed on novel PKC isoforms, particularly on PKC-η. This study indicates promising isoform-selectivity of some PKC inhibitors, namely NPC 15437 for PKC-η or rottlerin for both novel PKC isoforms. It also suggests that the PKC domain involved in the inhibition does not seem to be relevant for the potency and isoform-selectivity of PKC inhibitors.

Inhibition of α, βI, δ, η and ζ Protein Kinase C Isoforms by Xanthonolignoids

The effect of the xanthonolignoids trans-(±)-kielcorin C, cis-(±)-kielcorin C, trans-(±)-kielcorin D, trans-(±)-isokielcorin D and trans-(±)-kielcorin E on isoforms α, βI, δ, η and ζ of protein kinase C (PKC) was studied using the yeast phenotypic assay. All the compounds tested revealed an effect compatible with PKC inhibition, similar to that exhibited by the well established PKC inhibitor chelerythrine, and with differences in their potency towards the distinct isoforms tested, being, in general, potent inhibitors of the atypical PKC isoform (PKC-ζ). PKC inhibition caused by these kielcorins was confirmed using an in vitro kinase assay. The present study constitutes the first attempt to unravel the molecular mechanism of kielcorins activity, and shows that xanthonolignoids are a promising group of compounds to investigate for isoform selective PKC inhibitors.

Immunohistochemical characterization of adenosine receptors in rat aorta and tail arteries.

Adenosine plays an important role in the cardiovascular system, activating adenosine A1, A2A, A2B, and A3 receptors, and regulating blood flow either by acting directly on vascular cells or indirectly because of its effects on the central or peripheral nervous systems. The aim of the present study was to investigate whether the pattern of distribution of adenosine receptor subtypes is different on elastic and muscular, using abdominal aorta and tail arteries as models. Immunohistochemistry using anti‐A1, anti‐A2A, anti‐A2B, and anti‐A3 receptor antibodies was performed on perfused‐fixed/paraffin‐embedded arteries from Wistar rats. 3,3′‐Diaminobenzidine tetrahydrochloride (DAB; activated by hydrogen peroxide) staining revealed significant differences in the abundance of A1, A2A, and A3 receptors between abdominal aorta and tail artery and allowed the identification of distinct distribution patterns for A1, A2A, A2B, and A3 receptors in the tunica adventitia, media, and intima of muscular and elastic arteries. Data are compatible with several previous functional reports supporting that different adenosine receptor subtype expression and/or their distribution in the vessel wall may influence their respective contribution to the control of blood flow. Microsc. Res. Tech., 2008.

Lack of Endogenous Adenosine Tonus on Sympathetic Neurotransmission in Spontaneously Hypertensive Rat Mesenteric Artery

Background Increased sympathetic activity has been implicated in hypertension. Adenosine has been shown to play a role in blood flow regulation. In the present study, the endogenous adenosine neuromodulatory role, in mesenteric arteries from normotensive and spontaneously hypertensive rats, was investigated. Methods and Results The role of endogenous adenosine in sympathetic neurotransmission was studied using electrically-evoked [3H]-noradrenaline release experiments. Purine content was determined by HPLC with fluorescence detection. Localization of adenosine A1 or A2A receptors in adventitia of mesenteric arteries was investigated by Laser Scanning Confocal Microscopy. Results indicate a higher electrically-evoked noradrenaline release from hypertensive mesenteric arteries. The tonic inhibitory modulation of noradrenaline release is mediated by adenosine A1 receptors and is lacking in arteries from hypertensive animals, despite their purine levels being higher comparatively to those determined in normotensive ones. Tonic facilitatory adenosine A2A receptor-mediated effects were absent in arteries from both strains. Immunohistochemistry revealed an adenosine A1 receptors redistribution from sympathetic fibers to Schwann cells, in adventitia of hypertensive mesenteric arteries which can explain, at least in part, the absence of effects observed for these receptors. Conclusion Data highlight the role of purines in hypertension revealing that an increase in sympathetic activity in hypertensive arteries is occurring due to a higher noradrenaline/ATP release from sympathetic nerves and the loss of endogenous adenosine inhibitory tonus. The observed nerve-to-glial redistribution of inhibitory adenosine A1 receptors in hypertensive arteries may explain the latter effect.

Ligands and Therapeutic Perspectives of Adenosine A2A Receptors

Adenosine A(2A) receptors are members of the G protein-coupled receptor family and mediate multiple physiological effects of adenosine, both at the central nervous system (CNS) and at peripheral tissues, by activating several pathways or interacting with other receptors or proteins. Increasing evidence relate A(2A) receptors with pharmacological stress testing, neurodegenerative disorders (such as Parkinsons disease) and inflammation, renewing the interest in these receptors, increasingly viewed as promising therapeutic targets. Series of agonists and antagonists have been developed by medicinal chemistry artwork either by structure activity relationship (SAR) or quantitative structure activity relationship (QSAR) studies. These studies have allowed identification of the structural and electrostatic requirements for high affinity A(2A) receptor binding and, therefore, contributing to the rational design of A(2A) receptor ligands. Additional rational chemical modifications of the existing A(2A) receptor ligands may further improve their affinity/selectivity. The purpose of this review is to analize and summarize aspects related to the medicinal chemistry of A(2A) receptor ligands, their present and potencial therapeutic applications by exploring the molecular structure and physiological and pathophysiological roles of A(2A) receptors.

Potentiation of cytotoxicity of paclitaxel in combination with Cl-IB-MECA in human C32 metastatic melanoma cells: A new possible therapeutic strategy for melanoma.

Metastatic melanoma monotherapies with drugs such as dacarbazine, cisplatin or paclitaxel (PXT) are associated with significant toxicity and low efficacy rates. These facts reinforce the need for development of novel agents or combinatory strategies. Cl-IB-MECA is a small molecule, orally bioavailable, well tolerated and currently under clinical trials as an anticancer agent. Our aim was to investigate a possible combinatory therapeutic strategy using PXT and Cl-IB-MECA on human C32 melanoma cells and its underlying mechanisms. Cytotoxicity was evaluated using MTT reduction, lactate dehydrogenase leakage and neutral red uptake assays, for different concentrations and combinations of both agents, at 24 and 48 h. Apoptosis was also assessed using fluorescence microscopy and through the evaluation of caspases 8, 9, and 3 activities. We demonstrated, for the first time, that combination of PXT and Cl-IB-MECA significantly increases cytotoxicity for clinically relevant concentrations. This combination seems to act synergistically in disrupting membrane integrity, but also causing lysosomal and mitochondrial dysfunction. When using the lowest PTX concentration (10 ng/mL), co-incubation with CI-IB-MECA (micromolar concentrations) potentiated overall cytotoxic effects and morphological signs of apoptosis. All combinations studied enhanced caspase 8, 9, and 3 activities, suggesting the involvement of both intrinsic and extrinsic apoptotic pathways. The possibility that cytotoxicity elicited by Cl-IB-MECA, alone or in combination with PXT, involves adenosine receptor activation was discarded and results confirmed that oxidative stress is only involved in cytotoxicity after treatment with PXT, alone. Being melanoma a very apoptosis-resistance cancer, this combination seems to hold promise as a new therapeutic strategy for melanoma.