Michele Paulo

Photocytotoxic activity of a nitrosyl phthalocyanine ruthenium complex--a system capable of producing nitric oxide and singlet oxygen.

The synthesis, structural aspects, pharmacological assays, and in vitro photoinduced cytotoxic properties of [Ru(NO)(ONO)(pc)] (pc=phthalocyanine) are described. Its biological effect on the B16F10 cell line was studied in the presence and absence of visible light irradiation. At comparable irradiation levels, [Ru(NO)(ONO)(pc)] was more effective than [Ru(pc)] at inhibiting cell growth, suggesting that occurrence of nitric oxide release following singlet oxygen production upon light irradiation may be an important mechanism by which the nitrosyl ruthenium complex exhibits enhanced biological activity in cells. Following visible light activation, the [Ru(NO)(ONO)(pc)] complex displayed increased potency in B16F10 cells upon modifications to the photoinduced dose; indeed, enhanced potency was detected when the nitrosyl ruthenium complex was encapsulated in a drug delivery system. The liposome containing the [Ru(NO)(ONO)(pc)] complex was over 25% more active than the corresponding ruthenium complex in phosphate buffer solution. The activity of the complex was directly proportional to the ruthenium amount present inside the cell, as determined by inductively coupled plasma mass spectroscopy. Flow cytometry analysis revealed that the photocytotoxic activity was mainly due to apoptosis. Furthermore, the vasorelaxation induced by [Ru(NO)(ONO)(pc)], proposed as NO carrier, was studied in rat isolated aorta. The observed vasodilation was concentration-dependent. Taken together, the present findings demonstrate that the [Ru(NO)(ONO)(pc)] complex induces vascular relaxation and could be a potent anti-tumor agent. Nitric oxide release following singlet oxygen production upon visible light irradiation on a nitrosyl ruthenium complex produces two radicals and may elicit phototoxic responses that may find useful applications in photodynamic therapy.

Dose-dependent effect of Resveratrol on bladder cancer cells: Chemoprevention and oxidative stress

BACKGROUND Over 6 million people die annually in the world because of cancer. Several groups are focused on studying cancer chemoprevention approaches. Resveratrol, a polyphenol, at high dosages, has been reported as antitumor and chemopreventive. However, it has a dose-dependent effect on cell death, even on some cancer cells. OBJECTIVES Our aim was to investigate this dose-dependent effect on human bladder carcinoma ECV304 cells during oxidative stress condition. METHODS For this purpose, ECV304 cells incubated with different Resveratrol concentrations were analyzed as for their metabolic rate, membrane permeability, DNA fragmentation, anti/proapoptotic protein levels and phosphatidylserine exposure after oxidative stress. RESULTS Resveratrol induced cell death at high concentrations (>20 μM), but not at low ones (0.1-20 μM). Pretreatment with 2.5 μM protected the cells from oxidative damage, whereas 50 μM intensified the cell death and significantly increased Bad/Bcl-2 ratio (proapoptotic/antiapoptotic proteins). Resveratrol was able to modulate NO and PGE(2) secretion and performed an anti-adhesion activity of neutrophils on PMA-activated ECV304 cells. CONCLUSIONS Resveratrol at high doses induces cell death of ECV304 cells whereas low doses induce protection. Modulation of Bcl-2 protein induced by Resveratrol could be mediating this effect. This information about the role of Resveratrol on cancer alerts us about its dose-dependent effects and could lead the design of future chemoprevention strategies.

Nitric oxide synthesis and biological functions of nitric oxide released from ruthenium compounds

During three decades, an enormous number of studies have demonstrated the critical role of nitric oxide (NO) as a second messenger engaged in the activation of many systems including vascular smooth muscle relaxation. The underlying cellular mechanisms involved in vasodilatation are essentially due to soluble guanylyl-cyclase (sGC) modulation in the cytoplasm of vascular smooth cells. sGC activation culminates in cyclic GMP (cGMP) production, which in turn leads to protein kinase G (PKG) activation. NO binds to the sGC heme moiety, thereby activating this enzyme. Activation of the NO-sGC-cGMP-PKG pathway entails Ca(2+) signaling reduction and vasodilatation. Endothelium dysfunction leads to decreased production or bioavailability of endogenous NO that could contribute to vascular diseases. Nitrosyl ruthenium complexes have been studied as a new class of NO donors with potential therapeutic use in order to supply the NO deficiency. In this context, this article shall provide a brief review of the effects exerted by the NO that is enzymatically produced via endothelial NO-synthase (eNOS) activation and by the NO released from NO donor compounds in the vascular smooth muscle cells on both conduit and resistance arteries, as well as veins. In addition, the involvement of the nitrite molecule as an endogenous NO reservoir engaged in vasodilatation will be described.

A new NO donor failed to release NO and to induce relaxation in the rat basilar artery.

Nitric oxide (NO)-donors are pharmacologically active substances that in vivo or in vitro release NO. Their most common side effect is headache caused by cerebral vasodilatation. We previously demonstrated that the new NO-donor Ru(terpy)(bdq)NO](3+) (Terpy), synthesized in our laboratory, induces relaxation of rat aorta. This study aimed to verify the effect of Terpy and sodium nitroprusside (SNP) in basilar artery. We conducted vascular reactivity experiments on endothelium-denuded basilar rings. The concentrations of iron (Fe) and ruthenium (Ru) complex were analyzed in basilar artery lysates after incubation with NO donors by mass spectrometry. We also evaluated the NO released from SNP and Terpy by using confocal microscopy. Interestingly, Terpy did not induce relaxation of the basilar artery. SNP induced relaxation in a concentration-dependent way. NO donors cross the membrane of vascular smooth muscle and entered the cell. In spite of its permeability, Terpy did not release NO in the basilar artery. Otherwise, SNP released NO in the basilar artery cells cytoplasm. Taken together, our results demonstrate that the new NO donor (Terpy) failed to release NO and to induce relaxation in the basilar artery. The NO donor SNP induces vascular relaxation due to NO release in the vascular smooth muscle cells.

The environmental cofactors in carcinogenesis in high risk HPV/HIV-positive women

The objective of the present study was to assess the presence of human papilloma virus (HPV) in HIV-infected women, with comparison between the Papanicolaou cytologic technique and the molecular PCR technique, as well as to determine the type of HPV, to measure cellular immunocompetence and to identify the presence of risk factors for the acquisition of HPV infection. Thirty HIV-infected women were selected. Vaginal and endocervical samples were collected from 27 of them. The smears were examined by 3 experienced cytologists to diagnose the presence of HPV by the Papanicolaou technique and the results were compared to HPV detection and typing by PCR. HPV-infected patients were interviewed in order to identify the presence of risk factors for the acquisition of the virus. Eight of the 27 patients analyzed (29%) presented HPV in endocervical samples submitted to PCR, 6 of them (75%) presented HPV involving a high risk of development of cervical cancer. For 5 of these patients, the cytologic diagnosis was not confirmed by PCR. When cellular immunocompetence was related to HPV infection, PCR revealed a diagnosis of HPV in 37.50% of the patients at intermediate risk for HPV infection and in 83.33% of the patients at high risk for HPV infection. These immunologically compromised HPV-infected patients are at higher risk of developing cervical neoplasia. We showed here that PCR is adequate for HPV detection and that, if only the Papanicolaou method is used for the follow-up of these patients, we will not provide good prevention of cervical cancer.

Nitric oxide generated by the compound RuBPY promotes the vascular smooth cell membrane hyperpolarization.

The cis-[Ru(bpy)(2)(py)NO(2)](PF(6)) specie (RuBPY) has been used as nitric oxide (NO) delivery agent. It is an NO reservoir and it is thermodynamically stable in aqueous solution. This study aimed to evaluate the NO specie generated by RuBPY as compared to NO released from sodium nitroprusside (SNP) and to study the cellular mechanisms specially focusing the activation of soluble guanylyl-cyclase (sGC), K(+) channels and the cell membrane hyperpolarization, which are the main targets for NO-inducing vascular relaxation. NO generated by RuBPY and the vascular smooth muscle cell (VSMC) membrane potential were measured by confocal microscopy. The cellular mechanisms of aorta relaxation were investigated using K(+) channel blockers and sGC inhibitor. NO released from RuBPY was higher than NO released from SNP. RuBPY released only radicalar NO(0) and SNP released both NO(-) and NO(0). The concentration-effect curves for RuBPY-induced relaxation was shifted to the right by inhibition of sGC with ODQ and by the non-selective blockade of K(+) channels with TEA. The simultaneous combination of ODQ and TEA abolished the vasorelaxation induced by RuBPY. The membrane potential measured by the sensitive dye 4-Di-ANNEPS demonstrated that RuBPY induces cell membrane hyperpolarization. Taken together, our results indicate that the large amount of NO(0) specie generated by RuBPY induces vasorelaxation due to activation of sGC, K(+) channels sensitive to TEA, and cell membrane hyperpolarization. These results indicate that NO(0) generated from RuBPY can also directly activate the K(+) channels in an independent way of sGC.

Nitric Oxide Signaling and the Cross Talk with Prostanoids Pathways in Vascular System

BACKGROUND This review provides an overview of the cellular signaling of nitric oxide (NO) and prostanoids in vascular cells and the possible cross talk between their pathways, mainly in hypertension, since the imbalance of these two systems has been attributed to development of some cardiovascular diseases. It also deals with the modulation of vasodilation induced by NO donors. NO is a well-known second messenger involved in many cellular functions. CONCLUSION In the vascular system, the NO produced by endothelial NO-synthase (eNOS) or released by NO donors acts in vascular smooth muscle cells, the binding of NO to Fe2+-heme of soluble guanylyl-cyclase (sGC) activates sGC and the production of cyclic guanosine-3-5-monophosphate (cGMP). The second messenger (cGMP) activates protein kinase G and the signaling cascade, including K+ channels. Activation of K+ channels leads to cell membrane hyperpolarization and Ca2+ channels blockade, which induce vascular relaxation. Moreover, the enzyme cyclooxygenase (COX) is also an important regulator of the vascular function by prostanoids production such as thromboxane A2 (TXA2) and prostacyclin (PGI2), which classically induce contraction and relaxation, respectively. Additionaly, studies indicate that the activity of both enzymes can be modulated by their products and reactive oxygen species (ROS) in cardiovascular diseases such as hypertension. The interaction of NO with cellular molecules, particularly the reaction of NO with ROS, determines the biological mechanisms of action and short half-life of NO. We have been working on the vascular effects of ruthenium-derived complexes that release NO. Our research group has published works on the vasodilating effects of ruthenium-derived NO donors and the mechanisms of vascular cells involved in the relaxation of the vascular smooth muscle in health and hypertensive rats. In our previous studies, we have compared the new NO donors synthesized by our group to SNP. It shows the cellular signaling of NO in the endothelial and vascular smooth muscle cells. OBJECTIVE This work focuses on the cellular mechanisms involved in the vasodilation induced by NO and the role of prostanoids in contractile or relaxing vascular responses. Since the NO is produced by NO-synthase (NOS) or released from NO donors we also discussed the perspectives to cross talk between NO and COX pathways on the vascular tone control.

A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium.

Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases.

Sodium nitroprusside activates potassium channels in the vena cava in normotensive but not in hypertensive rats.

Despite the importance of the venous system in the regulation of blood pressure, there are few studies that evaluate venous function in health and disease, and the effects of drugs on venous function. Blood pressure depends directly on the peripheral resistance and cardiac output. Unlike the peripheral resistance, in which the contractile activity of the arteries is the key factor, cardiac output depends primarily on the venomotor tone. An increase in cardiac blood pressure can be caused by an increase in blood volume, structural changes in the walls of the veins, leading to a reduced compliance thereof, or an increase in the contractile activity of venous smooth muscle. This study examined the effect of sodium nitroprusside (SNP), a classical nitric oxide donor, on vascular relaxation in the vena cava from normotensive (2K) and renal hypertensive (2K-1C) rats. We studied the effect of this compound in vena cava rings from normotensive and renal hypertensive rats. We showed for the first time that the vascular relaxation induced by SNP is impaired in vena cavas from hypertensive rats because of an impaired functional activity of potassium channels. Another relevant finding of this study is that the sarcoplasmic reticulum Ca2+ ATPase is not involved in the venorelaxation induced by SNP.

Enhancing vascular relaxing effects of nitric oxide-donor ruthenium complexes

Ruthenium-derived complexes have emerged as new nitric oxide (NO) donors that may help circumvent the NO deficiency that impairs vasodilation. NO in vessels can be produced by the endothelial cells and/or released by NO donors. NO interacts with soluble guanylyl-cyclase to produce cGMP to activate the kinase-G pathway. As a result, conductance arteries, veins and resistance arteries dilate, whereas the cytosolic Ca(2+) levels in the smooth muscle cells decrease. NO also reacts with oxygen or the superoxide anion, to generate reactive oxygen species that modulate NO-induced vasodilation. In this article, we focus on NO production by NO synthase and discuss the vascular changes taking place during hypertension originating from endothelial dysfunction. We will describe how the NO released from ruthenium-derived complexes enhances the vascular effects arising from failed NO generation or lack of NO bioavailability. In addition, how ruthenium-derived NO donors induce the hypotensive effect by vasodilation is also discussed.