Carmen Diniz

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.

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.

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.

The Adenosinergic System as a Therapeutic Target in the Vasculature: New Ligands and Challenges

Adenosine is an adenine base purine with actions as a modulator of neurotransmission, smooth muscle contraction, and immune response in several systems of the human body, including the cardiovascular system. In the vasculature, four P1-receptors or adenosine receptors—A1, A2A, A2B and A3—have been identified. Adenosine receptors are membrane G-protein receptors that trigger their actions through several signaling pathways and present differential affinity requirements. Adenosine is an endogenous ligand whose extracellular levels can reach concentrations high enough to activate the adenosine receptors. This nucleoside is a product of enzymatic breakdown of extra and intracellular adenine nucleotides and also of S-adenosylhomocysteine. Adenosine availability is also dependent on the activity of nucleoside transporters (NTs). The interplay between NTs and adenosine receptors’ activities are debated and a particular attention is given to the paramount importance of the disruption of this interplay in vascular pathophysiology, namely in hypertension., The integration of important functional aspects of individual adenosine receptor pharmacology (such as in vasoconstriction/vasodilation) and morphological features (within the three vascular layers) in vessels will be discussed, hopefully clarifying the importance of adenosine receptors/NTs for modulating peripheral mesenteric vascular resistance. In recent years, an increase interest in purine physiology/pharmacology has led to the development of new ligands for adenosine receptors. Some of them have been patented as having promising therapeutic activities and some have been chosen to undergo on clinical trials. Increased levels of endogenous adenosine near a specific subtype can lead to its activation, constituting an indirect receptor targeting approach either by inhibition of NT or, alternatively, by increasing the activity of enzymes responsible for ATP breakdown. These findings highlight the putative role of adenosinergic players as attractive therapeutic targets for cardiovascular pathologies, namely hypertension, heart failure or stroke. Nevertheless, several aspects are still to be explored, creating new challenges to be addressed in future studies, particularly the development of strategies able to circumvent the predicted side effects of these therapies.

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.

Cytotoxic and COX-2 Inhibition Properties of Hydroxycinnamic Derivatives

The anticancer (cytotoxicity against human mammary gland adenocarcinoma cells) and antiinflammatory properties (COX-2 inhibition) of the hydroxycinnamic derivatives trans-3-(3,4,5trihydroxyphenyl)-2-propenoic acid, trans-ethyl(3,4,5-trihydroxyphenyl)-2-propenoate and diethyl 2-(3,4,5trihydroxy-phenylmethylene)malonate were screened. Data point out a putative correlation between antiinflammatory and anticancer properties and suggest hydroxycinnamic derivatives as promising lead compounds for the development of anti-inflammatory/chemopreventive agents.

Differential inhibition of noradrenaline release mediated by inhibitory A1-adenosine receptors in the mesenteric vein and artery from normotensive and hypertensive rats

Mesenteric arteries and veins are densely innervated by sympathetic nerves and are crucial in the regulation of peripheral resistance and capacitance, respectively, thus, in the control of blood pressure. Presynaptic adenosine receptors are involved in vascular tonus regulation, by modulating noradrenaline release from vascular postganglionic sympathetic nerve endings. Some studies also suggest that adenosine receptors (AR) may have a role in hypertension. We aim at investigating the role of presynaptic adenosine receptors in mesenteric vessels and establish a relationship between their effects (in mesenteric vessels) and hypertension, using the spontaneously hypertensive rats (SHR) as a model of hypertension. Adenosine receptor-mediated modulation of noradrenaline release was investigated through the effects of selective agonists and antagonists on electrically-evoked [(3)H]-noradrenaline overflow. CPA (A1AR selective agonist: 1-100 nM) inhibited tritium overflow, but the inhibition was lower in SHR mesenteric vessels. IB-MECA (A3AR selective agonist: 1-100 nM) also inhibited tritium overflow but only in WKY mesenteric veins. CGS 21680 (A2AAR selective agonist: up to 100 nM) failed to facilitate noradrenaline release in mesenteric veins, from both strains, but induced a similar facilitation in the mesenteric arteries. NECA (non-selective AR agonist: 1, 3 and 10μM), in the presence of A1 (DPCPX, 20 nM) and A3 (MRS 1523, 1 μM) AR selective antagonists, failed to change tritium overflow. In summary, the modulatory effects mediated by presynaptic adenosine receptors were characterized, for the first time, in mesenteric vessels: a major inhibition exerted by the A1 subtype in both vessels; a slight inhibition mediated by A3 receptors in mesenteric vein; a facilitation mediated by A2A receptors only in mesenteric artery (from both strains). The less efficient prejunctional adenosine receptor mediated inhibitory effects can contribute to an increase of noradrenaline in the synaptic cleft (both in arteries and veins), which might conduce to increased vascular reactivity.

REGIONAL DIFFERENCES IN EXTRACELLULAR PURINE DEGRADATION IN THE PROSTATIC AND EPIDIDYMAL PORTIONS OF THE RAT VAS DEFERENS

1. The aim of the present study was to compare ecto‐nucleotidase activities in rat bisected vas deferens using 1,N6‐etheno(ε)‐nucleotides (ε‐ATP and ε‐AMP) as substrates. Degradation was estimated by measuring the disappearance of the substrate and the appearance of its metabolites using HPLC with fluorescence detection. Incubation of tissue preparations (prostatic or epididymal portions) with 300 nmol/L ε‐ATP at 37°C caused a partial disappearance of ε‐ATP and appearance of its metabolites (ε‐ADP, ε‐AMP and ε‐adenosine). Incubation at 25°C reduced ε‐ATP degradation more in the prostatic than in the epididymal portion.