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Projeto de investigação
Inibição da Tiorredoxina para Reduzir o Glioblastoma: Etilmercúrio e Timerosal na Terapia do Cancro
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Thioredoxin Reductase Inhibitors as Potential Antitumors: Mercury Compounds Efficacy in Glioma Cells
Publication . Pires, Vanessa; Bramatti, Isabella; Aschner, Michael; Branco, Vasco; Carvalho, Cristina
Glioblastoma multiforme (GBM) is the most aggressive and common form of glioma. GBM, like many other tumors, expresses high levels of redox proteins, such as thioredoxin (Trx) and thioredoxin reductase (TrxR), allowing tumor cells to cope with high levels of reactive oxygen species (ROS) and resist chemotherapy and radiotherapy. Thus, tackling the activity of these enzymes is a strategy to reduce cell viability and proliferation and most importantly achieve tumor cell death. Mercury (Hg) compounds are among the most effective inhibitors of TrxR and Trx due to their high affinity for binding thiols and selenols. Moreover, organomercurials such as thimerosal, have a history of clinical use in humans. Thimerosal effectively crosses the blood–brain barrier (BBB), thus reaching effective concentrations for the treatment of GBM. Therefore, this study evaluated the effects of thimerosal (TmHg) and its metabolite ethylmercury (EtHg) over the mouse glioma cell line (GL261), namely, the inhibition of the thioredoxin system and the occurrence of oxidative cellular stress. The results showed that both TmHg and EtHg increased oxidative events and triggered cell death primarily by apoptosis, leading to a significant reduction in GL261 cell viability. Moreover, the cytotoxicity of TmHg and ETHg in GL261 was significantly higher when compared to temozolomide (TMZ). These results indicate that EtHg and TmHg have the potential to be used in GBM therapy since they strongly reduce the redox capability of tumor cells at exceedingly low exposure levels.
N-Acetylcysteine or Sodium Selenite Prevent the p38-Mediated Production of Proinflammatory Cytokines by Microglia during Exposure to Mercury (II)
Publication . Branco, Vasco; Coppo, Lucia; Aschner, Michael; Carvalho, Cristina
Mercury (Hg) is known for its neurotoxicity and is reported to activate microglia cells at low exposure levels. Since mercury decreases the activity of the glutathione and thioredoxin systems, we hypothesize that Hg would, in turn, disrupt microglia homeostasis by interfering with redox regulation of signaling pathways. Thus, in this work, we analyzed the effect of exposure to Hg2+ on nuclear translocation and activation of NF-kB (p50) and p38 and pro-inflammatory gene transcription (IL-1ß; iNOS, TNF-alpha) considering the interaction of Hg with the glutathione system and thioredoxin systems in microglial cells. N9 (mouse) microglia cells were exposed to different concentrations of Hg2+ and the 24 h EC50 for a reduction in viability was 42.1 ± 3.7 μM. Subsequent experiments showed that at sub-cytotoxic levels of Hg2+, there was a general increase in ROS (≈40%) accompanied by a significant depletion (60–90%) of glutathione (GSH) and thioredoxin reductase (TrxR) activity. Upon 6 h of exposure to Hg2+, p38 (but not p50) accumulated in the nucleus (50% higher than in control), which was accompanied by an increase in its phosphorylation. Transcript levels of both IL1-ß and iNOS were increased over two-fold relative to the control. Furthermore, pre-exposure of cells to the p38 inhibitor SB 239063 hindered the activation of cytokine transcription by Hg2+. These results show that disruption of redox systems by Hg2+ prompts the activation of p38 leading to transcription of pro-inflammatory genes in microglia cells. Treatment of N9 cells with NAC or sodium selenite—which caused an increase in basal GSH and TrxR levels, respectively, prevented the activation of p38 and the transcription of pro-inflammatory cytokines. This result demonstrates the importance of an adequate nutritional status to minimize the toxicity resulting from Hg exposure in human populations at risk.
Thioredoxin reductase inhibitors in anticancer therapy : thimerosal efficacy in Glioblastoma
Publication . Bramatti, Isabella; Carvalho, Cristina; Branco, Vasco; Gonçalves, João Manuel Braz
Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor with a low life expectancy. The standard treatment includes resection surgery, radiotherapy and chemotherapy with temozolomide (TMZ). TMZ is considered the first choice and standard treatment, but therapeutic success is limited due to neoangiogenesis, intratumor heterogeneity and therapy resistance, with overall patient survival increasing by just 14 months. This poor outcome of current therapies increases the demand for new, more effective therapeutic options, including drug repurposing.
Tumor hypoxia involves the development of non-functional blood vessels within neoplastic tissue, leading to tumor survival, invasion and metastasis. Cancer growth and resistance to therapy are frequently related to overexpression of antioxidant systems such as the thioredoxin system. This system includes the disulfide reductase, thioredoxin (Trx), the selenoenzyme thioredoxin reductase (TrxR) and NADPH. This system is fundamental for cell survival and proliferation with a key role in maintaining redox homeostasis, being able to provide electrons to a large range of enzymes critical for DNA synthesis and defense against oxidative stress, regulating hypoxia inducible factor-1alpha (HIF-1α) activity, which in turn controls vascular endothelial growth factor (VEGF), an indispensable factor for tumor invasiveness, and microenvironment tumor maintenance. HIF-1α can also be regulated by the signal transducer and activator of transcription 3 (STAT3), an oncogene stimulated by pro-inflammatory cytokines and growth factors.
Indeed, targeting of antioxidant systems arises as a potential strategy to tackle tumors since they are more sensitive to loss of redox homeostasis than normal cells and those systems are involved in more than one of the considered hallmarks of cancer. The thioredoxin system has several known inhibitors including mercury compounds such as thimerosal (TmHg), an ethylmercury-containing compound, that readily crosses the blood-brain barrier (BBB) and accumulates in the brain. Despite having a history of human use, epidemiological evidence of TmHg neurotoxicity was never obtained.
Therefore, since elevation of oxidative stress triggers different pathways that will lead to apoptosis of glioma cells and the overall survival for this cancer is still low even with emerging therapies, the goal of this thesis is to study the use of thioredoxin’ inhibitors, in this case thimerosal, as a possible candidate for hard repurposing towards GBM as well as to overcome therapy resistance to TMZ.
Results showed that TmHg significantly decreased the cell viability (> 50%) of two different glioblastoma cell lines (GL261 and U87 cell line) at much lower concentrations than TMZ, and strongly inhibited the Trx system. In fact, in GL261 cells besides an inhibition of cytosolic TrxR1/Trx1 there was a strong inhibition of mitochondrial TrxR2/Trx2 expression, predominantly with TmHg. In U87 cells, exposure to TmHg decreased cell migration, HIF-1α expression (35%) and in co-exposure with TMZ reduced HIF-1α, VEGF, and phosphorylation of STAT3, showing that tumour invasiveness related factors are significantly hampered. Furthermore, TmHg elicit N9 microglial inflammation markers - increased levels of IL1β, iNOS and TNFα and the autophagic process. Moreover, in co-cultures of microglia and GBM cells, the cytotoxicity of TmHg towards GL261 was higher.
The effectiveness of EtHg and TmHg in reducing GBM viability and affecting the Trx system could also be related with its interaction with TrxR. We demonstrated that EtHg, unlike other Hg compounds such as methylmercury, is a partial inhibitor of this selenoenzyme, forming a selenium-compromised thioredoxin reductase-derived apoptotic proteins (SecTRAPs), which enhances ROS-production and cytotoxicity.
Also, a mouse (C57BL/6) model was used in a distribution study following TmHg administration (i.v.; 20, 40 and 100 µM) for 14 days. We observed a concentration-dependent Hg accumulation in the brain, kidney and liver. Nevertheless, no neurobehavioral alterations were noticed as well as no histopathological damage in the organs, which led to the optimization and testing in a GBM model encompassing stereotactic injection of GBM cells.
Overall, thimerosal arose as a good candidate for hard repurposing towards GBM and the results obtained indicate its potential to be used alone as well as to overcome therapy resistance to TMZ.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/MED-FAR/31136/2017