Simvastatin impairs murine melanoma growth

Background: Statins induces cell cycle arrest, apoptosis, reduction of angiogenic factors, inhibition of the endothelial growth factor, impairing tissue adhesion and attenuation of the resistance mechanisms. The aim of this study was evaluate the anti tumoral activity of simvastatin in a B16F10 melanoma-mouse model. Methods: Melanoma cells were treated with different concentrations of simvastatin and assessed by viability methods. Melanoma cells (5 x 10(4)) were implanted in two month old C57Bl6/J mice. Around 7 days after cells injection, the oral treatments were started with simvastatin (5 mg/kg/day, p.o.). Tumor size, hematological and biochemical analyses were evaluated. Results: Simvastatin at a concentration of 0.8 mu M, 1.2 mu M and 1.6 mu M had toxic effect. Concentration of 1.6 mu M induced a massive death in the first 24 h of incubation. Simvastatin at 0.8 mu M induces early cell cycle arrest in G0/G1, followed by increase of hypodiploidy. Tumor size were evaluated and the difference of treated group and control, after ten days, demonstrates that simvastatin inhibited the tumor expansion in 68%. Conclusion: Simvastatin at 1.6 mu M, presented cytototoxicity after 72 h of treatment, with an intense death. In vivo, simvastatin being potentially useful as an antiproliferative drug, with an impairment of growth after ten days.

A lipocalin sequence signature modulates cell survival

Lipocalins are beta-barrel proteins, which share three conserved motifs in their amino acid sequence. In this study, we identified by a peptide mapping approach, a seven-amino acid sequence related to one of these motifs (motif 2) that modulates cell survival. A synthetic peptide based on an insect lipocalin displayed cytoprotective activity in serum-deprived endothelial cells and leucocytes. This activity was dependent on nitric oxide synthase. This sequence was found within several lipocalins, including apolipoprotein D, retinol binding protein, lipocalin-type prostaglandin D synthase, and many unknown proteins, suggesting that it is a sequence signature and a lipocalin conserved property. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Lopap: A non-inflammatory and cytoprotective molecule in neutrophils and endothelial cells

Lopap (Lonomia obliqua prothrombin activator protease) is a member of the lipocalin family isolated from the extract of L obliqua bristles. Lopap displays serine protease-like activities, including coagulation disturbance, cytokine secretion and antiapoptotic activity in human cultured endothelial cells. Here, we have investigated the effects of the recombinant protein (rLopap) on the inflammatory and apoptotic processes of neutrophils and endothelial cells from male Wistar rats. We found that rLopap did not induce in vivo leukocyte-endothelial interactions in the microvasculature, initial steps of leukocyte recruitment during inflammation. Incubation of rLopap with neutrophils or endothelial cells prevented apoptosis evoked by serum deprivation and induced nitric oxide (NO) production in both cell types, and increased the expression of ICAM-1 by endothelial cells. Simultaneous incubation of endothelial cells or neutrophils with rLopap and N(omega)-nitro-L-arginine methyl ester (L-NAME), a non-specific inhibitor of NO synthases, inhibited NO production and impaired the protection on apoptosis. Differently, incubation of endothelial cells with monoclonal antibody anti ICAM-1 did not change the protection on apoptosis evoked by rLopap. Together, these results indicate that rLopap does not display inflammatory properties in vivo but inhibits apoptosis of neutrophils and endothelial cells depending, at least in part, on NO production. (C) 2009 Elsevier Ltd. All rights reserved.

Chemoprevention of rat hepatocarcinogenesis with histone deacetylase inhibitors: Efficacy of tributyrin, a butyric acid prodrug

Hepatocellular carcinoma (HCC) ranks in prevalence and mortality among top 10 cancers worldwide. Butyric acid (BA), a member of histone deacetylase inhibitors (HDACi) has been proposed as an anticareinogenic agent. However, its short half-life is a therapeutical limitation. This problem could be circumvented with tributyrin (TB), a proposed BA prodrug. To investigate TB effectiveness for chemoprevention, rats were treated with the compound during initial phases of ""resistant hepatocyte"" model of hepatocarcinogenesis, and cellular and molecular parameters were evaluated. TB inhibited (p < 0.05) development of hepatic preneoplastic lesions (PNL) including persistent ones considered HCC progression sites. TB increased (p < 0.05) PNL remodeling, a process whereby they tend to disappear. TB did not inhibit cell proliferation in PNL, but induced (p < 0.05) apoptosis in remodeling ones. Compared to controls, rats treated with TB presented increased (P < 0.05) hepatic levels of BA indicating its effectiveness as a prodrug. Molecular mechanisms of TB-induced hepatocarcinogenesis chemoprevention were investigated. TB increased (p < 0.05) hepatic nuclear histone H3K9 hyperacetylation specifically in PNL and p21 protein expression, which could be associated with inhibitory HDAC effects. Moreover, it reduced (p < 0.05) the frequency of persistent PNL with aberrant cytoplasmic p53 accumulation, an alteration associated with increased malignancy. Original data observed in our study support the effectiveness of TB as a prodrug of BA and as an HDACi in hepatocarcinogenesis chemoprevention. Besides histone acetylation and p21 restored expression, molecular mechanisms involved with TB anticarcinogenic actions could also be related to modulation of p53 pathways. (C) 2008 Wiley-Liss, Inc.

Antileukemic effects of Didemnum psammatodes (Tunicata: Ascidiacea) constituents

Chemical investigation of the methanolic extract of the ascidian Didemnum psammatodes has led to the identification of fourteen known compounds: three methyl esters (methyl myristate, methyl palmitate and methyl stearate), four steroids (cholesterol, campesterol, stigmasterol and beta-sitosterol), two fatty acids (palmitic acid and stearic acid), three glyceryl ethers {(1,2-propanediol, 3-(heptadecyloxy), batyl alcohol and 1,2-propanediol, 3-[(methyloctadecyl)oxy]) and two nucleosides (thymidine and 2`-deoxyguanosine). Their structures were proposed by NMR and comparison with literature data and GC analysis in comparison with authentic sample. The cytotoxic activity of these compounds was evaluated against human leukemia cell line panel using the MTT assay. The mixture of the three methyl esters was the most active group of compounds, showing antiproliferative and cytotoxic effects. Further studies on their mode of action suggest that these activities are connected with inhibition of DNA synthesis and induction of both necrosis and apoptosis. (C) 2007 Elsevier Inc. All rights reserved.

Cytotoxic activity of a dichloromethane extract and fractions obtained from Eudistoma vannamei (Tunicata: Ascidiacea)

This study consists of the bioassay-guided fractionation of the dichloromethane extract from Eudistoma vannamei and the pharmacological characterization of the active fractions. The dried hydromethanolic extract dissolved in aqueous methanol was partitioned with dichloromethane and chromatographed on a silica gel flash column. The anti-proliferative effect was monitored by the MTT assay. Four of the latest fractions, numbered 14 to 17, which held many chemical similarities amongst each other, were found to be the most active. The selected fractions were tested for viability, proliferation and death induction on cultures of HL-60 promycloblastic leukemia cells. The results suggested that the observed cytotoxicity is related to apoptosis induction. (C) 2007 Elsevier Inc. All rights reserved.

The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: Ensuing energetic and oxidative stress implications

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 mu M) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca(2+) efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP(+) transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. (C) 2011 Elsevier Inc. All rights reserved.

The anti-cancer agent nemorosone is a new potent protonophoric mitochondrial uncoupler

Nemorosone, a natural-occurring polycyclic polyprenylated acylphloroglucinol, has received increasing attention due to its strong in vitro anti-cancer action. Here, we have demonstrated the toxic effect of nemorosone (1-25 mu M) on HepG2 cells by means of the MTT assay, as well as early mitochondrial membrane potential dissipation and ATP depletion in this cancer cell line. In mitochondria isolated from rat liver, nemorosone (50-500 nM) displayed a protonophoric uncoupling activity, showing potency comparable to the classic protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Nemorosone enhanced the succinate-supported state 4 respiration rate, dissipated mitochondrial membrane potential, released Ca(2+) from Ca(2+)-loaded mitochondria, decreased Ca(2+) uptake and depleted ATP. The protonophoric property of nemorosone was attested by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium in the presence of valinomycin. In addition, uncoupling concentrations of nemorosone in the presence of Ca(2+) plus ruthenium red induced the mitochondrial permeability transition process. Therefore, nemorosone is a new potent protonophoric mitochondrial uncoupler and this property is potentially involved in its toxicity on cancer cells. (C) 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Visualizing inhibition of fatty acid synthase through mass spectrometric analysis of mitochondria from melanoma cells

Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid palmitate. In contrast to most normal cells, FASN is overexpressed in a variety of human cancers including cutaneous melanoma, in which its levels of expression are associated with a poor prognosis and depth of invasion. Recently, we have demonstrated the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells. Herein we compare, via electrospray ionization mass spectrometry (ESI-MS), free fatty acids (FFA) composition of mitochondria isolated from control (EtOH-treated cells) and Orlistat-treated B16-F10 mouse melanoma cells. Principal component analysis (PCA) was applied to the ESI-MS data and found to separate the two groups of samples. Mitochondria from control cells showed predominance of six ions, that is, those of m/z 157 (Pelargonic, 9:0), 255 (Palmitic, 16:0), 281 (Oleic, 18:1), 311 (Arachidic, 20:0), 327 (Docosahexaenoic, 22:6) and 339 (Behenic, 22:0). In contrast, FASN inhibition with Orlistat changes significantly mitochondrial FFA composition by reducing synthesis of palmitic acid, and its elongation and unsaturation products, such as arachidic and behenic acids, and oleic acid, respectively. ESI-MS of mitochondria isolated from Orlistat-treated cells presented therefore three major ions of m/z 157 (Pelargonic, 9:0), 193 (unknown) and 199 (Lauric, 12:0). These findings demonstrate therefore that FASN inhibition by Orlistat induces significant changes in the FFA composition of mitochondria. Copyright (C) 2011 John Wiley & Sons, Ltd.

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. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

Fluoxetine induces preventive and complex effects against colon cancer development in epithelial and stromal areas in rats

Fluoxetine (FIX) is a drug commonly used as antidepressant. However, its effects on tumorigenesis remain controversial. Aiming to evaluate the effects of FIX treatment on early malignant changes, we analyzed serotonin (5-HT) metabolism and recognition, aberrant crypt foci (ACF), proliferative process, microvessels, vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2) expression in colon tissue. Male Wistar rats received a daily FLX-gavage (30 mg kg(-1)) and, a single dose of 1.2 dimethylhydrazine (DMH; i.p., 125 mg kg(-1)). After 6 weeks of FIX-treatment, our results revealed that FIX and nor-fluoxetine (N-FIX) are present in colon tissue, which was related to significant increase in serotonin (5-HT) levels (P < 0.05) possibly through a blockade in SERT mRNA (serotonin reuptake transporter; P < 0.05) resulting in lower 5-hydroxyindoleacetic acid (5-HIAA) levels (P < 0.01) and, 5-HT2C receptor mRNA expressions. FIX-treatment decreased dysplastic ACF development (P < 0.01) and proliferative process (P < 0.001) in epithelia. We observed a significant decrease in the development of malignant microvessels (P < 0.05), VEGF (P < 0.001), and COX-2 expression (P < 0.01). These findings suggest that FIX may have oncostatic effects on carcinogenic colon tissue, probably due to its modulatory activity on 5-HT metabolism and/or its ability to reduce colonic malignant events. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Dehydromonocrotaline induces cyclosporine A-insensitive mitochondrial permeability transition/cytochrome c release

Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the genus Crotalaria that causes cytotoxicity and genotoxicity in animals and humans. It is well established that the toxicity of MCT results from its hepatic bioactivation to dehydromonocrotaline (DHM), an alkylating agent, but the exact mechanism of action remains unknown. In a previous study, we demonstrated DHM`s inhibition of mitochondrial NADH-dehydrogenase activity at micromolar concentrations, which is an effect associated with a significant reduction in ATP synthesis. As a follow-up study, we have evaluated the ability of DHM to induce mitochondrial permeability transition (MPT) and its associated processes in isolated rat liver mitochondria. In the presence of 10 mu M Ca(2+), DHM (50-250 mu M) elicited MPT in a concentration-dependent, but cyclosporine A-independent manner, as assessed by mitochondrial swelling, which is associated with mitochondrial Ca(2+) efflux and cytochrome c release. DHM (50-250 mu M) did not cause hydrogen peroxide accumulation but did deplete endogenous glutathione and NAD(P)H, while oxidizing protein thiol groups. These results potentially indicate the involvement of mitochondria, via apoptosis, in the well-documented cytotoxicity of monocrotaline. (C) 2009 Elsevier Ltd. All rights reserved.

Effects on mitochondria of mitochondria-induced nitric oxide release from a ruthenium nitrosyl complex

The ruthenium nitrosyl complex trans-[Ru(NO)(NH(3))(4)(py)](PF(6))(3) (pyNO), a nitric oxide (NO) donor, was studied in regard to the release of NO and its impact both on isolated mitochondria and HepG2 cells. In isolated mitochondria, NO release from pyNO was concomitant with NAD(P)H oxidation and, in the 25-100 mu M range, it resulted in dissipation of mitochondrial membrane potential, inhibition of state 3 respiration, ATP depletion and reactive oxygen species (ROS) generation. In the presence of Ca(2+), mitochondrial permeability transition (MPT), an unspecific membrane permeabilization involved in cell necrosis and some types of apoptosis, was elicited. As demonstrated by externalization of phosphatidylserine and activation of caspase-9 and caspase-3, pyNO (50-100 mu M) induced HepG2 cell death, mainly by apoptosis. The combined action of the NO itself, the peroxynitrite yielded by NO in the presence of reactive oxygen species (ROS) and the oxidative stress generated by the NAD(P)H oxidation is proposed to be involved in cell death by pyNO, both via respiratory chain inhibition and ROS levels increase, or even via MPT, if Ca(2+) is present. (c) 2008 Elsevier Inc. All rights reserved.

The Aspergillus nidulans nucA(EndoG) Homologue Is Not Involved in Cell Death

Upon apoptosis induction, translocation of mammalian mitochondrial endonuclease G (EndoG) to the nucleus coincides with large-scale DNA fragmentation. Here, we describe for the first time a homologue of EndoG in filamentous fungi by investigating if the Aspergillus nidulans homologue of the EndoG gene, named nucA(EndoG), is being activated during farnesol-induced cell death. Our results suggest that NucA is not involved in cell death, but it plays a role in the DNA-damaging response in A. nidulans.

Ligand Reduces Galectin-1 Sensitivity to Oxidative Inactivation by Enhancing Dimer Formation

Galectin-1 (Gal-1) regulates leukocyte turnover by inducing the cell surface exposure of phosphatidylserine (PS), a ligand that targets cells for phagocytic removal, in the absence of apoptosis. Gal-1 monomer- dimer equilibrium appears to modulate Gal-1-induced PS exposure, although the mechanism underlying this regulation remains unclear. Here we show that monomer- dimer equilibrium regulates Gal-1 sensitivity to oxidation. A mutant form of Gal-1, containing C2S and V5D mutations (mGal-1), exhibits impaired dimerization and fails to induce cell surface PS exposure while retaining the ability to recognize carbohydrates and signal Ca(2+) flux in leukocytes. mGal-1 also displayed enhanced sensitivity to oxidation, whereas ligand, which partially protected Gal-1 from oxidation, enhanced Gal-1 dimerization. Continual incubation of leukocytes with Gal-1 resulted in gradual oxidative inactivation with concomitant loss of cell surface PS, whereas rapid oxidation prevented mGal-1 from inducing PS exposure. Stabilization of Gal-1 or mGal-1 with iodoacetamide fully protected Gal-1 and mGal-1 from oxidation. Alkylation-induced stabilization allowed Gal-1 to signal sustained PS exposure in leukocytes and mGal-1 to signal both Ca(2+) flux and PS exposure. Taken together, these results demonstrate that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidative inactivation and provides a mechanism whereby ligand partially protects Gal-1 from oxidation.