[Show abstract][Hide abstract] ABSTRACT: We evaluated the effects of curcumin treatment on protein oxidation (PO), lipid peroxidation (LP) and brain-derived neurotrophic factor (BDNF) levels in the hippocampus and frontal cortex (FC) of diabetic db/db mice (DM) and in sera of obese humans. Thus, DM were treated daily with 50 mg/kg of curcumin during an 8-week period. Obese human were treated daily with 500 and 750 mg of curcumin that was administered orally for 12 weeks; BDNF, PO and LP levels in sera were determined at in weeks 0, 2, 6 and 12 of treatment. BDNF levels decreased in hippocampus and FC of DM as compared with untreated wild-type mice. Curcumin improved or restored BDNF levels to normal levels in DM, but curcumin did not have any effect on BDNF levels in sera of obese humans. In hippocampus and FC of DM, hyperglycaemia and curcumin did not have effect on LP levels. Hyperglycaemia increased PO levels in hippocampus and FC, whereas curcumin decreased these levels in hippocampus but not in FC. In sera of obese humans, the 500-mg dose decreased LP levels in weeks 6 and 12 when compared with basal levels, but the 750-mg dose did not have any effect; both doses of curcumin decreased PO levels in weeks 2, 6 and 12 of treatment when compared with basal levels. Present results suggest a therapeutic potential of curcumin to decrease oxidation caused by obesity in humans and also show that curcumin restores BDNF levels in DM.
[Show abstract][Hide abstract] ABSTRACT: Background aims
Glioblastoma multiforme (GBM) is the most common and lethal primary brain tumor and current treatments have not improved its prognosis. Therefore, new strategies and therapeutic agents should be investigated. Growth arrest specific-1 (Gas1) is a protein that induces cell arrest and apoptosis of gliomas and a soluble form, tGas1, increases these effects acting in both autocrine and paracrine manners. Moreover, neural stem cells (NSCs) can be used as a vehicle to transport therapeutic molecules because they have innate tropism towards tumors.
Lentiviral vectors were used to obtain NSCs capable of expressing tGas1 in a regulated manner. The ability of engineered NSCs to track and reach GBM in vivo, produce tGas1, and their efficacy decreasing tumor growth and increasing the overall health and survival time of nude mice implanted with GBM were assessed.
The overexpression of tGas1 from NSCs decreased viability and induced cell arrest and apoptosis of GBM cells and also, albeit in a reduced manner, of NSCs themselves. NSCs migrate from one cerebral hemisphere to the contralateral, reach GBM, express the tGas1 transgene when induced by tetracycline and produce the protein. Tumor volume decreased by 77% compared with controls, and tGas1 improved the overall health and increased the survival time of mice implanted with GBM by 75%.
We demonstrated that tGas1 has an antineoplastic effect, and the results support the potential of tGas1 as an adjuvant for the treatment of gliomas.
[Show abstract][Hide abstract] ABSTRACT: Growth arrest specific 1 (GAS1) is a pleiotropic protein that induces apoptosis and cell arrest in different tumors, but it is also involved in the development of the nervous system and other tissues and organs. This dual ability is likely caused by its capacity to interact both by inhibiting the intracellular signaling cascade induced by glial cell-line derived neurotrophic factor and by facilitating the activity of the sonic hedgehog pathway. The presence of GAS1 mRNA has been described in adult mouse brain, and here we corroborated this observation. We then proceeded to determine the distribution of the protein in the adult central nervous system (CNS). We detected, by western blot analysis, expression of GAS1 in olfactory bulb, caudate-putamen, cerebral cortex, hippocampus, mesencephalon, medulla oblongata, cerebellum, and cervical spinal cord. To more carefully map the expression of GAS1, we performed double-label immunohistochemistry and noticed expression of GAS1 in neurons in all brain areas examined. We also observed expression of GAS1 in astroglial cells, albeit the pattern of expression was more restricted than that seen in neurons. Briefly, in the present article, we report the widespread distribution and cellular localization of the GAS1 native protein in adult mammalian CNS.
Journal of Histochemistry and Cytochemistry 06/2013; · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Parkinson's disease is a neurodegenerative disease, resulting from deterioration of the substantia nigra which in turn leads to a decrease of dopamine levels in the striatum. Clinically the syndrome is characterized by motor alterations that are treated by the oral administration of levodopa. However, this treatment typically loses efficacy over time and therefore new treatments that procure a steady long term supplement of dopamine are needed. Here we tested the expression of a tyrosine hydroxilase (TH) transgene in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated primates own astrocytes. The transgene, whose expression of TH cDNA was controlled by a glial fibrillary acidic protein (GFAP) promoter, was injected into MPTP treated primate's brains using liposomes as a delivery system. Monkeys were tested before and after MPTP administration, and after gene therapy treatment on the HALLWAY behavioral task. Results showed both transgene expression and significant behavioral improvements in the hallway task after the TH cDNA transfer. The behavioral recovery observed in the primates whose astrocytes expressed rat TH, is a first step that warrant further studies using primate's astrocytes as a good cell lineage to express therapeutic molecules.
[Show abstract][Hide abstract] ABSTRACT: Huntington's disease (HD) is a neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms. The most characteristic structural feature of this disease is neurodegeneration accompanied by gliosis in the striatum. BDNF has been proposed to protect striatal neurons from degeneration, because it is an important survival factor for these neurons from development to adulthood. Considering the extensive gliosis and the survival effects of BDNF, we constructed an adenovirus to express a BDNF cDNA in astrocyte cells using a promoter of the glial fibrillary acidic protein gene. Cells stably transfected in vitro with a BDNF cDNA driven by this promoter expressed BDNF and responded to external stimuli increasing BDNF production. When the vector was applied into the striata of mice transgenic for HD, long-term expression of the transgene was observed, associated with a delay of onset of the motor phenotype of the R6/2 HD transgenic mice. The present data indicate that the striatal expression of BDNF is a potential adjuvant for the treatment of HD.
Cellular and Molecular Neurobiology 06/2011; 31(8):1229-43. · 2.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous clinical observations suggested that coffee may have beneficial effects on the liver. In fact, an inverse relationship between coffee consumption and liver cirrhosis has been reported in humans. However, the causative role of coffee has not been established; therefore, the aim of this work was to study the effect of coffee in an experimental model of liver damage.
In this work, cirrhosis was induced by chronic CCl(4) administration and soluble or grain coffee (SC, GC, respectively) were co-administered for 8 weeks.
CCl(4) administration elevated serum alkaline phosphatase and alanine aminotranspherase, liver lipid peroxidation, collagen content (fourfold) and TGF-β mRNA, and protein levels; depleted liver glycogen and reduced glutathione (GSH) content. Coffee prevented most of the changes produced by CCl(4). Histopathological analysis was in agreement with biochemical and molecular data. The best effect was produced by GC. It is worth noting that GC preserved the normal collagen content as well as the normal TGF-β mRNA and protein levels.
Our results suggest (1) that coffee plays a causative role in preventing cirrhosis (at least experimental cirrhosis); (2) that action mechanisms are probably associated with down regulation of the profibrogenic cytokine TGF-β and to its antioxidant properties and, (3) that GC is more potent than SC. These findings suggest a beneficial effect of coffee on the liver. However, more clinical and basic studies must be performed before reaching a final recommendation.
Hepatology International 01/2011; 5(3):857-63. · 2.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gliomas are the most frequent primary tumors of the central nervous system, and their clinical prognosis remains very poor. Because of the characteristics of gliomas, gene therapy appears as a potentially relevant strategy for their treatment. However, the inability of viral vectors to transfer the therapeutic genes to a significantly high number of tumor cells, due to their limited diffusion and distribution, remains a critical obstacle for their application treating gliomas. We have demonstrated that the overexpression of growth arrest specific1 (Gas1) induces cell arrest and apoptosis and eliminates glioma cells in vitro and when implanted in mice. To improve the therapeutic range of Gas1, we generated lentiviral vectors coding for a soluble form of Gas1. Here, we show that cells infected with this virus produce the mutant protein, that acting both in autocrine and paracrine manners, causes death of infected and neighbor cells, thus importantly enhancing the effect of Gas1. Furthermore, the administration of this vector, or cells expressing it, inhibit the growth of tumors inoculated in mice. We present a gene therapy strategy that increases the effect of the therapeutic molecule by eliminating not just the infected cells that express Gas1, but neighbor non-infected cells.
Cancer gene therapy 10/2010; 18(2):87-99. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transforming growth factor-beta (TGF-beta) plays a pivotal role in liver fibrosis, because it activates hepatic stellate cells, stimulating extracellular matrix deposition. Cyclooxygenase-2 (COX-2) has been associated with TGF-beta because its inhibition decreases TGF-beta expression and collagen production in some cultured cell types.
The aim of this work was to evaluate the ability of celecoxib (a selective COX-2 inhibitor) to prevent and to reverse the liver fibrosis induced by CCl(4).
We established experimental groups of rats including vehicle and drug controls, damage induced by chronic CCl(4) administration and CCl(4) plus pharmacological treatment in both prevention and reversion models. We determined: alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transpeptidase, COX and metalloproteinase-2 and -9 activities, lipid peroxidation, glutathione levels, glycogen and collagen content and TGF-beta expression.
Celecoxib prevented and aided to the recovery of livers with necrotic and cholestatic damage. Celecoxib exhibited anti-oxidant properties by restoring the redox equilibrium (lipid peroxidation and glutathione levels). Glycogen was decreased by CCl(4), while celecoxib partially prevented and reversed this effect. Celecoxib inhibited COX-2 activity, decreased TGF-beta expression, induced metalloproteinase-2 activity and, consequently, prevented and reversed collagen accumulation.
Our findings indicate that celecoxib exerts strong antifibrogenic and fibrolytic effects in the CCl(4) model of cirrhosis.
Liver international: official journal of the International Association for the Study of the Liver 08/2010; 30(7):969-78. · 3.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interference with nitric oxide production is a possible mechanism for lead neurotoxicity. In this work, we studied the effects of sub-acute lead administration on the distribution of NOS isoforms in the hippocampus with respect to blood and hippocampal lead levels. Lead acetate (125, 250 and 500ppm) was given via drinking water to adult male Wistar rats for 14 days. We determined blood and hippocampal lead levels by atomic absorption spectrophotometry. Antibodies against three isoforms of NOS were used to analyze expression and immunolocalization using western blotting and immunohistochemistry, respectively. Blood and hippocampal lead levels were increased in a dose-dependent manner in groups treated with lead acetate. We found diminished expression and immunoreactivity of nNOS and eNOS at 500ppm as compared to the control group. No expression and immunoreactivity was observed in hippocampus for iNOS. The observed high levels of lead in the blood reflect free physiological access to this metal to the organism and were related to diminished expression and immunoreactivity for nNOS and eNOS.
Experimental and toxicologic pathology: official journal of the Gesellschaft fur Toxikologische Pathologie 07/2009; 62(3):311-6. · 1.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Growth arrest specific1 (Gas1) is a protein expressed during development and when cells arrest their growth. The potential of Gas1 as an adjuvant in the treatment of cancer, and its role as a tumor suppressor have also been proposed. In this work we are addressing the molecular mechanisms by which Gas1 induces cell arrest and apoptosis of cancer cells, using primary cultures of human gliomas as a model. We had previously demonstrated the structural relationship between Gas1 and the alpha receptors for the Glial-cell line-Derived Neurotrophic Factor (GDNF) family of ligands, and showed that Gas1 acts by inhibiting the intracellular signaling induced by GDNF. There are also reports indicating that Gas1 positively cooperates with Sonic Hedgehog (Shh) during embryonic development and in this paper we analyzed the potential interactions between Gas1 and Shh. We show that human gliomas do not express Shh, whereas GDNF and the molecular components necessary to transduce its signaling are present in human gliomas. Furthermore, the over-expression of Gas1 induces cell arrest, apoptosis and prevents the activation of Akt, a crucial mediator of survival and cellular proliferation pathways. In the present work, we present evidence demonstrating that Gas1 exerts its effects inhibiting cell growth and inducing apoptosis of glioma cells in the absence of Shh.
International journal of developmental neuroscience: the official journal of the International Society for Developmental Neuroscience 07/2009; 27(4):305-13. · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: N-acetylcysteine (NAC) is an antioxidant, a precursor of reduced glutathione, and an inhibitor of the profibrotic cytokine liver transforming growth factor-beta (TGF-beta). Carbon tetrachloride (CCl4) cirrhosis is characterized by oxidative stress and fibrosis. Therefore, the aim of this work was to study the effect of NAC on experimental cirrhosis.
CCl4 was chronically administered for 8 weeks along with 300 mg/kg of NAC orally once a day. Alkaline phosphatase, alanine aminotransferase, and gamma-glutamyltranspeptidase were measured in plasma. Hydroxyproline, glycogen, lipid peroxidation, glutathione were determined in liver samples by colorimetric methods. TGF-beta was evaluated by western blotting, and a histopathological analysis was performed.
Serum markers of liver damage increased by CCl4 intoxication (P<0.05), whereas cotreatment with NAC prevented these increases (P<0.05); glycogen was depleted in the cirrhotic group (P<0.05), but preserved by NAC (P<0.05). Lipid peroxidation increased and glutathione decreased by the administration of CCl4 (P<0.05), again NAC prevented both effects (P<0.05). Importantly, collagen increased by about seven-fold in the CCl4 group (P<0.05); administration of NAC preserved the normal levels of collagen (P<0.05). Biochemical determinations were corroborated by hematoxylin and eosin, and trichromic stains. Western blots revealed a four-fold increase in TGF-beta in the group receiving CCl4, NAC cotreatment abolished TGF-beta signal (P<0.05).
Our results strongly suggest that NAC prevents experimental cirrhosis by two mechanisms: by preventing oxidative stress and by downregulating the profibrogenic cytokine TGF-beta. As NAC is currently used in humans intoxicated with paracetamol, it can be tested in fibrotic or cirrhotic patients under controlled trials.
European journal of gastroenterology & hepatology 04/2009; 21(8):908-14. · 1.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular studies have revealed the presence of R-type voltage-gated Ca(2+) channels at pre- and postsynaptic regions; however, no evidence for the participation of these channels in transmitter release has been presented for the spinal cord. Here we characterize the effects of SNX-482, a selective R channel blocker, on the monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in motoneurons by stimulation of dorsolateral funiculus (DLF) terminals in a slice preparation from the adult turtle spinal cord. SNX-482 inhibited neurotransmission in a dose-dependent manner, with an IC(50) of approximately 9 +/- 1 nM. The EPSP time course and membrane time constant of the motoneurons were not altered, suggesting a presynaptic mechanism. The toxin inhibited the residual component of the EPSPs recorded in the presence of N- and P/Q-type Ca(2+) channel blockers, strongly suggesting a role for the R channels in neurotransmission at the spinal cord DLF terminals. Consistently with this, RT-PCR analysis of turtle spinal cord segments revealed the expression of the Ca(V)2.3 pore-forming (alpha(1E)) subunit of R channels, whereas the use of anti-alpha(1E)-specific antibodies resulted in its localization in the DLF fibers as demonstrated by immunohistochemistry coupled with laser confocal microscopy.
The Journal of Comparative Neurology 02/2009; 513(2):188-96. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cirrhosis is a very common disease and its treatment is limited due to lack of effective drugs. Some studies indicate that this disease is associated with oxidative stress. Therefore, we decided to study the effect of trolox, an effective antioxidant, on experimental cirrhosis. Cirrhosis was induced by CCl4 administration (0.4 g/kg, intraperitoneally, three times per week, for 8 weeks) to Wistar male rats. Trolox was administered daily (50 mg/kg, orally). Fibrosis was assessed histologically and by measuring liver hydroxyproline content. Glutathione, lipid peroxidation and glycogen were measured in liver; serum markers of liver damage were also quantified. Transforming growth factor-beta (TGF-beta) was determined by Western blot and quantified densitometrically. Alkaline phosphatase, gamma-glutamyl transpeptidase and alanine aminotransferase increased in the group receiving CCl4; trolox completely or partially prevented these alterations. Glycogen was almost depleted by CCl4 but was partially preserved by trolox. Lipid peroxidation increased while glutathione decreased by CCl4 administration; trolox corrected both effects. Histology showed thick bands of collagen, necrosis and distortion of the hepatic parenchyma in the CCl4 group, such effects were prevented by trolox. Hydroxyproline content increased 5-fold by CCl4, while the group receiving both CCl4 and trolox showed no significant difference compared to the control group. CCl4 increased 3-fold TGF-beta, while trolox completely prevented this increase. We found that trolox effectively prevented cirrhosis induced with CCl4 in the rat. Our results suggest that the beneficial effects of trolox may be associated to its antioxidant properties and to its ability to reduce the profibrogenic cytokine TGF-beta expression.
[Show abstract][Hide abstract] ABSTRACT: The effects of hypothyroidism on lipid peroxidation (LP), reactive oxygen species (ROS), and nitric oxide synthase (NOS), levels and expression, in rat brain were examined. Hypothyroidism was induced by administering methimazole in drinking water (60 mg/kg/day). In striatum, motor cortex and cerebellum of hypothyroid rats LP was not modified, whereas LP and ROS increased in amygdala and hippocampus of hypothyroid rats at the third week of treatment with methimazole as compared to euthyroid group values. Regarding NOS participation, only hippocampal constitutive-NOS activity was increased, accompanied by an augmentation in nNOS expression. Results show that hypothyroidism induces selective oxidative stress in both the hippocampus and amygdala, where the nitrergic system is involved.
[Show abstract][Hide abstract] ABSTRACT: The present results show that the expression of Growth Arrest Specific1 (Gas1) in SH-SY5Y neuroblastoma cells significantly inhibits the increased phosphorylation of tyrosine 1062 of the Ret receptor tyrosine kinase induced by glial-cell-line-derived neurotrophic factor (GDNF). We also observed that Gas1 significantly reduces the activation of Akt. GDNF and members of its family of ligands (GFLs), signal through a molecular complex consisting of one of its receptors (GFRalphas) and the Ret receptor tyrosine kinase. GDNF is a key component to preserve several cell populations in the nervous system, including dopaminergic and motor neurons, and also participates in the survival and differentiation of peripheral neurons such as enteric, sympathetic and parasympathetic. On the other hand, Gas1 is a molecule involved in cell arrest that can induce apoptosis when over-expressed in different cell lines, including cells of neuronal and glial origin. Although, Gas1 is widely expressed during development, its role in vivo has not yet been clearly defined. We recently showed the structural homology between Gas1 and GFRalphas, thus suggesting that the physiological role of Gas1 is that of modulating the biological responses induced by GDNF and/or other members of this family of signaling molecules. The results of this work are consistent with the hypothesis of Gas1 acting as a negative modulator of GDNF signaling.
International Journal of Developmental Neuroscience 09/2008; 26(5):497-503. · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Curcumin is a phytophenolic compound, which is highly efficacious for treating several inflammatory diseases. The aim of this study was to evaluate the efficacy of curcumin in preventing or reversing liver cirrhosis. A 4-week bile duct ligation (BDL) rat model was used to test the ability of curcumin (100 mg/kg, p.o., daily) to prevent cirrhosis. To reverse cirrhosis, CCl(4) was administered chronically for 3 months, and then it was withdrawn and curcumin administered for 2 months. Alanine aminotransferase, gamma-glutamyl transpeptidase, liver histopathology, bilirubin, glycogen, reduced and oxidized glutathione, and TGF-beta (mRNA and protein) levels were assessed. Curcumin preserved normal values of markers of liver damage in BDL rats. Fibrosis, assessed by measuring hydroxyproline levels and histopathology, increased nearly fivefold after BDL and this effect was partially but significantly prevented by curcumin. BDL increased transforming growth factor-beta (TGF-beta) levels (mRNA and proteins), while curcumin partially suppressed this mediator of fibrosis. Curcumin also partially reversed the fibrosis induced by CCl(4). Curcumin was effective in preventing and reversing cirrhosis, probably by its ability of reducing TGF-beta expression. These data suggest that curcumin might be an effective antifibrotic and fibrolitic drug in the treatment of chronic hepatic diseases.
Fundamental and Clinical Pharmacology 09/2008; 22(4):417-27. · 1.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Liver fibrosis is characterized by an excess of collagen fiber deposition, and it is known that Kupffer cells play an important role by immunomodulation of the toxic response. Methyl palmitate (MP) is an effective Kupffer cell inhibitor. The aim of this work was to evaluate the effect of MP on experimental liver fibrosis. Four groups were formed: the control group, which received the vehicles only; CCl(4) group (0.4 g kg(-1), i.p., three times a week, for eight weeks); CCl(4) plus MP (300 mg kg(-1), i.p., daily); and MP alone. Alanine aminotransferase was increased by CCl(4), and MP did not prevent this increase. Lipid peroxidation was increased markedly by CCl(4); again, MP was not able to prevent this effect. Fibrosis increased nearly 6-fold (measured as liver hydroxyproline content) in the CCl(4) group; MP preserved the normal content of collagen. These results were corroborated by histopathology. To elucidate the antifibrogenic mechanism of MP, we measured the production of TGF-beta; CCl(4) increased this cytokine several-fold, and MP abolished this increase. Collectively the present results indicate that MP possesses a strong antifibrogenic effect at least in the CCl(4) model of fibrosis. The antifibrotic effect of MP is probably associated with its ability to reduce TGF-beta content, maybe by immunomodulation of Kupffer cells functioning.
Journal of Applied Toxicology 07/2008; 28(8):1021-6. · 2.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Resveratrol is a nonflavonoid polyphenol with antioxidant, anticancer and antiinflammatory properties. Moreover, it has been reported that this compound inhibits NF-kappaB, which regulates the transcription of several genes including cytokines such as the profibrogenic TGF-beta. The aim of this work was to evaluate the pharmacological effects of resveratrol on CCl(4)-induced cirrhosis in the rat. Four groups were formed: the control group that received the vehicles only; the CCl(4) group that received the toxin (0.4 g kg(-1), i.p., three times a week, for 8 weeks); the CCl(4) plus resveratrol (10 mg kg(-1), daily) group; and the resveratrol alone group. Alanine aminotransferase, alkaline phosphatase and bilirubins were increased by CCl(4), but resveratrol afforded some degree of protection. Glycogen was decreased markedly by CCl(4) and resveratrol prevented almost completely this effect. No antioxidant effect of resveratrol was observed. One of the most prominent effects was on fibrosis which increased near 5-fold (hydroxyproline) in the CCl(4) group; resveratrol preserved the content of collagen. These results were corroborated by histopathology. To elucidate the antifibrogenic mechanism of resveratrol, the activation of NF-kappaB and the production of TGF-beta were measured; in both cases CCl(4) increased them and resveratrol abolished them; however, changes in NF-kappaB were modest and did not reach statistical significance, while the increase in TGF-beta was about three fold and resveratrol decreased it under control values. Together, the present results indicate that resveratrol possesses a strong antifibrogenic effect at least in the CCl(4) model of cirrhosis. Moreover, the action mechanism is probably associated with its ability to reduce NF-kappaB activation and TGF-beta content.
Journal of Applied Toxicology 02/2008; 28(1):35-43. · 2.60 Impact Factor