Daniel Pens Gelain

Comparison between proliferative and neuron-like SH-SY5Y cells as an in vitro model for Parkinson disease studies

The molecular mechanisms underlying the cellular lost found in the nigrostriatal pathway during the progression of Parkinsons disease (PD) are not completely understood. Human neuroblastoma cell line SH-SY5Y challenged with 6-hydroxydopamine (6-OHDA) has been widely used as an in vitro model for PD. Although this cell line differentiates to dopaminergic neuron-like cells in response to low serum and retinoic acid (RA) treatment, there are few studies investigating the differences between proliferative and RA-differentiated SH-SY5Y cells. Here we evaluate morphological and biochemical changes which occurs during the differentiation of SH-SY5Y cells, and their responsiveness to 6-OHDA toxicity. Exponentially growing SH-SY5Y cells were maintained with DMEM/F12 medium plus 10% of fetal bovine serum (FBS). Differentiation was triggered by the combination of 10 microM RA plus 1% of FBS during 4, 7 and 10 days in culture. We found that SH-SY5Y cells differentiated for 7 days show an increase immunocontent of several relevant neuronal markers with the concomitant decrease in non-differentiated cell marker. Moreover, cells became two-fold more sensitive to 6-OHDA toxicity during the differentiation process. Time course experiments showed loss of mitochondrial membrane potential triggered by 6-OHDA (mitochondrial dysfunction parameter), which firstly occurs in proliferative than neuron-like differentiated cells. This finding could be related to the increase in the immunocontent of the neuroprotective protein DJ-1 during differentiation. Our data suggest that SH-SY5Y cells differentiated by 7 days with the protocol described here represent a more suitable experimental model for studying the molecular and cellular mechanisms underlying the pathophysiology of PD.

NFκB inhibitors induce cell death in glioblastomas

Identification of novel target pathways in glioblastoma (GBM) remains critical due to poor prognosis, inefficient therapies and recurrence associated with these tumors. In this work, we evaluated the role of nuclear-factor-kappa-B (NFκB) in the growth of GBM cells, and the potential of NFκB inhibitors as antiglioma agents. NFκB pathway was found overstimulated in GBM cell lines and in tumor specimens compared to normal astrocytes and healthy brain tissues, respectively. Treatment of a panel of established GBM cell lines (U138MG, U87, U373 and C6) with pharmacological NFκB inhibitors (BAY117082, parthenolide, MG132, curcumin and arsenic trioxide) and NFκB-p65 siRNA markedly decreased the viability of GBMs as compared to inhibitors of other signaling pathways such as MAPKs (ERK, JNK and p38), PKC, EGFR and PI3K/Akt. In addition, NFκB inhibitors presented a low toxicity to normal astrocytes, indicating selectivity to cancerous cells. In GBMs, mitochondrial dysfunction (membrane depolarization, bcl-xL downregulation and cytochrome c release) and arrest in the G2/M phase were observed at the early steps of NFκB inhibitors treatment. These events preceded sub-G1 detection, apoptotic body formation and caspase-3 activation. Also, NFκB was found overstimulated in cisplatin-resistant C6 cells, and treatment of GBMs with NFκB inhibitors overcame cisplatin resistance besides potentiating the effects of the chemotherapeutics, cisplatin and doxorubicin. These findings support NFκB as a potential target to cell death induction in GBMs, and that the NFκB inhibitors may be considered for in vivo testing on animal models and possibly on GBM therapy.

Characterization of blood oxidative stress in type 2 diabetes mellitus patients: increase in lipid peroxidation and SOD activity.

This study evaluated the oxidative stress through enzymatic and nonenzymatic biomarkers in diabetic patients with and without hypertension and prediabetics. The SOD and CAT (in erythrocytes) and GPx (in plasma) enzymatic activities, plasma levels of lipid peroxidation, and total thiols were measured in the blood of 55 subjects with type 2 diabetes and 38 subjects without diabetes (9 pre-diabetics and 29 controls) aged 40–86 years. The total SOD activity and the lipid peroxidation were higher in diabetics compared to nondiabetics. In stratified groups, the total SOD activity was different for the hypertensive diabetics compared to the prediabetics and normotensive controls. Lipid peroxidation was significantly higher in both groups of diabetics (hypertensive and normotensive) compared to prediabetic groups and hypertensive and normotensive controls. There was no significant difference in the CAT and GPx activities, as well as in the concentration of total thiols in the groups studied. Present data strongly suggest the involvement of oxidative stress in the pathophysiology of diabetes, revealing that the increased lipid peroxidation has a close relationship with high glucose levels, as observed by the fasting glucose and HbA1c levels. The results evidence the correlation between lipid peroxidation and DM, irrespective of the presence of hypertension.

Improvement of p-cymene antinociceptive and anti-inflammatory effects by inclusion in β-cyclodextrin.

Previously, we have demonstrated the analgesic-like property of p-cymene in rodents. Short half-life is a limitation for p-cymene application and several approaches have been used to improve pharmaceutical properties of monoterpenes, including the employment of drug-delivery systems. Here, we used p-cymene/β-cyclodextrin (β-CD) complex and p-cymene (PC) isolated to evaluated whether the complex formulation is able to improve the antinociceptive activity of this monoterpene. Male mice (26-30g) were pretreated with PC/β-CD (20 or 40mg/kg, p.o.), PC (20 or 40mg/kg, p.o.) or vehicle (distilled water), 0.5h before painful tests and antinociceptive effect was evaluated at times: 0.5, 1, 2, 4, 8, and 16h after treatment. We evaluated the analgesic-like effect of PC/β-CD and PC in acetic acid-induced abdominal writhes, hot-plate, carrageenan-induced paw edema and in rota-rod apparatus. Our results demonstrated that acute treatment with complex PC/β-CD produced an antinocicepitve effect (p<0.01 or p<0.001) for 8h followed whereas isolated PC produced the same effect for 2h. Similar results were obtained in hot-plate test, PC/β-CD, in all doses, significantly reduces (p<0.01 or p<0.001) nociceptive behavior for 8h while isolated PC for 1h, did so only in higher dose. Such results were unlikely to be caused by motor abnormality. Systemic pretreatment with PC/β-CD and PC inhibited the development paw edema by carrageenan 1%, but PC/β-CD did so during a longer period when compared with isolated monoterpene alone. Our results provide evidence to propose that the complex with β-CD improved analgesic and anti-inflammatory effects of p-cymene.

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.

Matrix Metalloproteinase-2 and Metalloproteinase-9 Activities are Associated with Blood–Brain Barrier Dysfunction in an Animal Model of Severe Sepsis

There is no description on the mechanisms associated with blood–brain barrier (BBB) disruption during sepsis development. Thus, we here determined changes in permeability of the BBB in an animal model of severe sepsis and the role of matrix metalloproteinase (MMP)-2 and MMP-9 in the dysfunction of the BBB. Sepsis was induced in Wistar rats by cecal ligation and perforation. BBB permeability was assessed using the Evans blue dye method. The content of MMP-2 and MMP-9 in the cerebral microvessels was determined by western blot. The activity of MMP-2 and MMP-9 was determined using zymography. An inhibitor of MMP-2 and MMP-9 or specific inhibitors of MMP-2 or MMP-9 were administered to define the role of MMPs on BBB permeability, brain inflammatory response, and sepsis-induced cognitive alterations. The increase of BBB permeability is time-related to the increase of MMP-9 and MMP-2 in the microvessels, both in cortex and hippocampus. Using an MMP-2 and MMP-9 inhibitor, or specific MMP-2 or MMP-9 inhibitors, the increase in the permeability of the BBB was reversed. This was associated with lower brain levels of interleukin (IL)-6 and lower oxidative damage. In contrast, only the inhibition of both MMP-9 and MMP-2 was able to improve acute cognitive alterations associated with sepsis. In conclusion, MMP-2 and MMP-9 activation seems to be a major step in BBB dysfunction, but BBB dysfunction seems not to be associated with acute cognitive dysfunction during sepsis development.

Serum heat shock protein 70 levels, oxidant status, and mortality in sepsis

Animal studies as well as prospective randomized clinical trials associated sepsis with redox imbalance and oxidative stress, but other studies failed to establish a correlation between antioxidant-based therapies and improvement of sepsis condition. This is also true for studies on the role of the chaperone heat shock protein 70 (HSP70), which is increased in serum during sepsis. Heat shock protein 70 is affected at several levels by oxidative stress, but this relationship has never been studied in sepsis. Here, we evaluated the relationship between serum HSP70 immunocontent and oxidant status in sepsis. Patients with severe sepsis were followed up for 28 days after diagnosis, or until death. Up to a maximum of 12 h after sepsis diagnosis, serum was collected for determination of HSP70 immunocontent by Western blot and evaluation of oxidative parameters (TRAP [total radical-trapping antioxidant parameter], TBARSs [thiobarbituric acid-reactive substances], and carbonyl levels). Serum of sepsis patients presented enhanced HSP70 levels. Analysis of oxidative parameters revealed that septic patients with pronounced oxidative damage in serum had also increased HSP70 serum levels. Sepsis patients in whom serum oxidative stress markers were not different from control presented normal serum HSP70. Analysis of septic patients according to survival outcome also indicated that patients with increased HSP70 serum levels presented increased mortality. We concluded that serum HSP70 levels are modulated according to the patient oxidant status, and increased serum HSP70 is associated to mortality in sepsis.

β-Cyclodextrin-complexed (-)-linalool produces antinociceptive effect superior to that of (-)-linalool in experimental pain protocols.

Many plants produce (−)‐linalool, a plant‐derived monoterpene alcohol, including members of the Lamiaceae (mints) and Lauraceae family (laurels, cinnamon, rosewood). The anti‐inflammatory and analgesic effects of (−)‐linalool have been widely suggested for various studies. Poor chemical stability and short half‐life restrain the clinical applications of some essential oil and monoterpenes, including (−)‐linalool. However, β‐cyclodextrin (β‐CD) has been used to increase solubility and stability of lipophilic compounds and also to improve the pharmacological effects. In this study, the antinociceptive effect of (−)‐linalool and (−)‐linalool/β‐CD was examined using the acetic acid writhing reflex, formalin and hotplate tests in rodents. (−)‐Linalool and (−)‐linalool/β‐CD demonstrated strong antinociceptive activity in all the chemical‐ and heat‐induced mice models (p < 0.01 or p < 0.001). These findings imply the involvement of both peripheral and central antinociceptive mechanisms. In peritonitis induced by carrageenan, isolated monoterpene or β‐CD complex also reduced total leucocyte migration and TNF‐α levels in peritoneal fluid. The inclusion complexes, (−)‐linalool/β‐CD, revealed that the antinociceptive effect was significantly (p < 0.01) improved when compared with (−)‐linalool alone. Such results were unlikely to be provoked by any motor abnormality. Together, our results suggest that β‐CD might represent an important tool for improvement of analgesic and anti‐inflammatory profiles of (−)‐linalool and other water‐insoluble compounds, such as lipophilic monoterpenes or essential oils.

Redox properties and cytoprotective actions of atranorin, a lichen secondary metabolite.

Atranorin (ATR) is a lichenic secondary metabolite with potential uses in pharmacology. Antinociceptive and antiinflammatory actions have been reported, and the use of atranorin-enriched lichen extracts in folk medicine is widespread. Nonetheless, very few data on ATR biological actions are available. Here, we evaluated free radical scavenging activities and antioxidant potential of ATR using various in vitro assays for scavenging activity against hydroxyl radicals, hydrogen peroxide, superoxide radicals, and nitric oxide. The total reactive antioxidant potential (TRAP) and total antioxidant reactivity (TAR) indexes and in vitro lipoperoxidation were also evaluated. Besides, we determined the cytoprotective effect of ATR on H(2)O(2)-challenged SH-SY5Y cells by the MTT assay. ATR exerts differential effects towards reactive species production, enhancing hydrogen peroxide and nitric oxide production and acting as a superoxide scavenger; no activity toward hydroxyl radical production/scavenging was observed. Besides, TRAP/TAR analysis indicated that atranorin acts as a general antioxidant, although it demonstrated to enhance peroxyl radical-induced lipoperoxidation in vitro. ATR was not cytotoxic, and also protected SH-SY5Y cells against H(2)O(2)-induced cell viability impairment. Our results suggest that ATR has a relevant redox-active action, acting as a pro-oxidant or antioxidant agent depending on the radical. Also, it will exert cytoprotective effects on cells under oxidative stress induced by H(2)O(2).

Antioxidant Activity and Mechanisms of Action of Natural Compounds Isolated from Lichens: A Systematic Review

Chronic diseases such as cancer, diabetes, neurodegenerative and cardiovascular diseases are characterized by an enhanced state of oxidative stress, which may result from the overproduction of reactive species and/or a decrease in antioxidant defenses. The search for new chemical entities with antioxidant profile is still thus an emerging field on ongoing interest. Due to the lack of reviews concerning the antioxidant activity of lichen-derived natural compounds, we performed a review of the antioxidant potential and mechanisms of action of natural compounds isolated from lichens. The search terms “lichens”, “antioxidants” and “antioxidant response elements” were used to retrieve articles in LILACS, PubMed and Web of Science published until February 2014. From a total of 319 articles surveyed, 32 met the established inclusion and exclusion criteria. It was observed that the most common isolated compound studied was usnic acid, cited in 14 out of the 32 articles. The most often described antioxidant assays for the study of in vitro antioxidant activity were mainly DPPH, LPO and SOD. The most suggested mechanisms of action were scavenging of reactive species, enzymatic activation and inhibition of iNOS. Thus, compounds isolated from lichens are possible candidates for the management of oxidative stress, and may be useful in the treatment of chronic diseases.