Ivi Juliana Bristot

Autophagy inhibition improves the efficacy of curcumin/temozolomide combination therapy in glioblastomas

Glioblastoma is a devastating primary brain tumor resistant to conventional therapies. In this study, we tested the efficacy of combining temozolomide with curcumin, a phytochemical known to inhibit glioblastoma growth, and investigated the mechanisms involved. The data showed that synergy between curcumin and temozolomide was not achieved due to redundant mechanisms that lead to activating protective autophagy both in vitro and in vivo. Autophagy preceded apoptosis, and blocking this response with autophagy inhibitors (3-methyl-adenine, ATG7 siRNA and chloroquine) rendered cells susceptible to temozolomide and curcumin alone or combinations by increasing apoptosis. While curcumin inhibited STAT3, NFκB and PI3K/Akt to affect survival, temozolomide-induced autophagy relied on the DNA damage response and repair components ATM and MSH6, as well as p38 and JNK1/2. However, the most interesting observation was that both temozolomide and curcumin required ERK1/2 to induce autophagy. Blocking this ERK1/2-mediated temozolomide and curcumin induced autophagy with resveratrol, a blood-brain barrier permeable drug, improved temozolomide/curcumin efficacy in brain-implanted tumors. Overall, the data presented demonstrate that autophagy impairs the efficacy of temozolomide/curcumin, and inhibiting this phenomenon could provide novel opportunities to improve brain tumor treatment.

Cofilin/Actin Rod Formation by Dysregulation of Cofilin-1 Activity as a Central Initial Step in Neurodegeneration

Cofilin-1 protein, which main function is to regulate actin cytoskeleton dynamics, appears to be involved with many steps in the neurotoxicity processes found in neurodegenerative diseases such as Alzheimers disease (AD), Parkinsons disease (PD) and Huntingtons disease (HD). As the dynamics of actin filaments play a major role in several cellular processes, the primary involvement of cofilin-1 dysfunctions in the pathophysiology of these disorders may be related to a cytoskeleton stress. However, recently cofilin-1 has also been related to other biological processes such as cell death by apoptosis. In both cases, ATP depletion associated with the presence of reactive species and other stressors regulate cofilin-1 by inducing the formation of aggregates composed primarily by actin and cofilin-1, known as cofilin/actin rods. These structures seem to be formed initially as a neuroprotective response to mitochondrial damage; but once the stressor persists they are thought to act as inducers of further impairments and loss of neuronal functions. Therefore, here we provide a brief overview of the current knowledge about the central role of cofilin/actin rods formation, where its dysregulation and malfunction might be the trigger to neurodegeneration.

Effects of 47C allele (rs4880) of the SOD2 gene in the production of intracellular reactive species in peripheral blood mononuclear cells with and without lipopolysaccharides induction

Abstract Challenging of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharides (LPS) has been shown to activate monocytes and macrophages, leading to the production of pro-inflammatory cytokines and reactive oxygen species (ROS). Manganese superoxide dismutase (MnSOD) is an important enzyme that may play a central role in the response to oxidative stress. 47C> T SNP of the SOD2 gene, the -9Val MnSOD is less efficient than the -9Ala version. We have previously characterized the cellular redox status of human PBMCs expressing either -9Ala (CC) or -9Val (TT) SOD2 and analyzed the responses of these cells to oxidative stress induced by LPS. Due to the observed alterations in the activities of these antioxidant enzymes, we decided to investigate their immunocontent and analyze the production of intracellular oxidants, as well as any resulting DNA damage. PBMCs were isolated from the blood of 30 healthy human volunteers (15 volunteers per allele). We then analyzed levels of nitrite, DNA damage by comet assay, TNF-α, carboxymethyl lysine and nitrotyrosine and assessed production of intracellular reactive species by the DCFH-DA-based assay and western blots were used to analyze protein levels. Our results show that there occurs an increase in nitric oxide production in both allele groups after challenge with LPS. A significant increase in DNA damage was observed in PBMCs after an 8-h LPS challenge. Cells expressing the SOD2 47C allele quickly adapt to a more intense metabolism by upregulating cellular detoxification mechanisms. However, when these cells are stressed over a long period, they accumulate a large quantity of toxic metabolic byproducts.

Effects of chronic caloric restriction on kidney and heart redox status and antioxidant enzyme activities in Wistar rats.

Caloric restriction (CR) has been associated with health benefits and these effects have been attributed, in part, to modulation of oxidative status by CR; however, data are still controversial. Here, we investigate the effects of seventeen weeks of chronic CR on parameters of oxidative damage/modification of proteins and on antioxidant enzyme activities in cardiac and kidney tissues. Our results demonstrate that CR induced an increase in protein carbonylation in the heart without changing the content of sulfhydryl groups or the activities of superoxide dismutase and catalase (CAT). Moreover, CR caused an increase in CAT activity in kidney, without changing other parameters. Protein carbonylation has been associated with oxidative damage and functional impairment; however, we cannot exclude the possibility that, under our conditions, this alteration indicates a different functional meaning in the heart tissue. In addition, we reinforce the idea that CR can increase CAT activity in the kidney. [BMB Reports 2012; 45(11): 671-676]

Sertraline reduces glutamate uptake in human platelets.

Mitochondrial damage and declines in ATP levels have been recently attributed to sertraline. The effects of sertraline on different parameters were investigated in washed platelets from 18 healthy male volunteers, after 24h of drug exposure. Sertraline toxicity was observed only at the highest concentrations, 30 and 100 μM, which significantly reduced platelet viability to 76 ± 3% and 20 ± 2%, respectively. The same concentrations significantly decreased total ATP to 73 ± 3% and 13 ± 2%, respectively. Basal values of glycogen were not significantly affected by sertraline treatment. Glutamate uptake was significantly reduced after treatment with 3, 30 and 100 μM, by 28 ± 6%, 32 ± 5% and 54 ± 4%, respectively. Our data showed that sertraline at therapeutic concentrations does not compromise platelet viability and ATP levels, but they suggest that in a situation where extracellular glutamate levels are potentially increased, sertraline might aggravate an excitotoxic condition.

Mimicking Parkinson’s Disease in a Dish: Merits and Pitfalls of the Most Commonly used Dopaminergic In Vitro Models

Parkinson’s disease (PD) is the second most common neurodegenerative disorder and has both unknown etiology and non-curative therapeutic options. Patients begin to present the classic motor symptoms of PD—tremor at rest, bradykinesia and rigidity—once 50–70% of the dopaminergic neurons of the nigrostriatal pathway have degenerated. As a consequence of this, it is difficult to investigate the early-stage events of disease pathogenesis. In vitro experimental models are used extensively in PD research because they present a controlled environment that enables the direct investigation of the early molecular mechanisms that are potentially involved with dopaminergic degeneration, as well as for the screening of potential therapeutic drugs. However, the establishment of PD in vitro models is a controversial issue for neuroscience research not only because it is challenging to mimic, in isolated cell systems, the physiological neuronal environment, but also the pathophysiological conditions experienced by human dopaminergic cells in vivo during the progression of the disease. Since no previous work has attempted to systematically review the literature regarding the establishment of an optimal in vitro model, and/or the features presented by available models used in the PD field, this review aims to summarize the merits and limitations of the most widely used dopaminergic in vitro models in PD research, which may help the PD researcher to choose the most appropriate model for studies directed at the elucidation of the early-stage molecular events underlying PD onset and progression.

Cannabinoid-Based Therapies and Brain Development: Potential Harmful Effect of Early Modulation of the Endocannabinoid System

The endocannabinoid retrograde signaling pathway is widely expressed in the central nervous system, where it plays major roles in regulating synaptic plasticity (excitatory and inhibitory) through long-term potentiation and long-term depression. The endocannabinoid system (ECS) components—cannabinoid receptors, endocannabinoids and synthesis/degradation enzymes—are expressed and are functional from early developmental stages and throughout adolescent cortical development, regulating progenitor cell fate, neural differentiation, migration and survival. This may potentially confer increased vulnerability to adverse outcomes from early cannabinoid exposure. Cannabidiol (CBD) is one of the most studied exogenous cannabinoids, and CBD-enriched Cannabis extracts have been widely (and successfully) used as adjuvants to treat children with refractory epilepsy, and there is even a Food and Drug Administration (FDA)-approved drug with purified CBD derived from Cannabis. However, there is insufficient information on possible long-term changes in the central nervous system caused by cannabinoid treatments during early childhood. Like the majority of cannabinoids, CBD is able to exert its effects directly and indirectly through the ECS, which can perturb the regulatory processes mediated by this system. In addition, CBD has a large number of non-endocannabinoid targets, which can explain CBD’s effects. Here, we review the current knowledge about CBD-based therapies—pure and CBD-enriched Cannabis extracts—in studies with pediatric patients, their side effects, and their mechanisms of action regarding the central nervous system and neurodevelopment aspects. Since Cannabis extracts contain Δ9-tetrahydrocannabinol (Δ9-THC), we consider that pure CBD is possibly safer for young patients. Nevertheless, CBD, as well as other natural and/or synthetic cannabinoids, should be studied in more detail as a therapeutic alternative to CBD-enriched Cannabis extracts for young patients.

Immunosenescence induced by plasma from individuals with obesity caused cell signaling dysfunction and inflammation

To evaluate the consequences of plasma from individuals with obesity on parameters associated with immunosenescence in unrelated healthy peripheral blood mononuclear cells (PBMC).

Abstract 804: Energetic metabolism and DNA damage response in fibroblasts from Li-Fraumeni syndrome patients: new insights into the molecular mechanisms of the disease

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Li-Fraumeni Syndrome (LFS) is a cancer predisposition syndrome associated with TP53 germline mutations and characterized by increased risk to multiple early-onset cancers. Studies in families from Southern and Southeastern Brazil have identified a germline founder mutation in the TP53 gene, the p.R337H, in high population prevalence (∼0.3%). Unlike the majority of the mutations in TP53, which are missense mutations located in the DNA-binding domain (DBD) of the protein, the TP53 p.R337H is located in the oligomerization domain (OD). Recently, we identified a new rare germline variant, the rs78378222 (A>C), in the 3′ UTR of the gene in 7/130 of the patients who tested negative for coding germline TP53 mutations. Based on the broad spectrum of p53 functions, which include metabolism and DNA damage response regulation, in the present study we aimed to characterize the functional impact of germline TP53 mutations identified in Brazilian families. Methods: Primary fibroblasts from p.R337H/WT (n = 2), p.R337H/p.R337H (n = 1), DBD mutations (n = 2), rs78378222[C] patients and from WT p53 controls (n = 2) were included. The DNA damage induction was performed using UVB (0.2W/m2) and ionizing irradiation (IR) (1 Gy). Results: First, we observed that all p53-mutated fibroblasts appeared to have lower p53 levels but higher levels of reactive oxygen species (ROS) and mitochondrial biomass compared to controls. Fibroblasts homozygous to p.R337H mutation presented more pronounced ROS production and, notably, higher expression of two antioxidant proteins, SOD2 and GPX1. On the other hand, the results of high-resolution respirometry showed higher oxygen consume in DBD-mutated fibroblasts. Second, using a specific multiplexed enzymatic DNA repair assay on biochip, we simultaneously investigated several DNA repair pathways. Overall, we observed a high DNA repair activation profile in p.R337H/p.R337H and poor DNA repair capacity in DBD-mutated fibroblasts. Interestingly, all p53-mutated fibroblasts showed higher repair activity of apurinic/pyrimidinic (AP) sites, a DNA lesion caused mainly by ROS attacks. Conclusions: Our data indicate important alterations in both metabolism and DNA damage response in normal cells (fibroblasts) from LFS patients, suggesting haploinsufficiency mechanism associated to these mutations. We were also able to demonstrate distinct phenotypes according to mutational status, which may explain, at least in part, the clinical variability in LFS. Citation Format: Gabriel Macedo, Sylvie Sauvaigo, Michele Silva Alves, Sylvain Caillat, Igor Araujo Vieira, Fernanda Timm, Cristina Brinckmann Oliveira Netto, Mauro Antonio Alves Castro, Ivi Bristot, Angela Fachel, Fabio Klamt, Patricia Ashton-Prolla. Energetic metabolism and DNA damage response in fibroblasts from Li-Fraumeni syndrome patients: new insights into the molecular mechanisms of the disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 804. doi:10.1158/1538-7445.AM2015-804