Extracellular HSP70 Activates ERK1/2, NF-kB and Pro-Inflammatory Gene Transcription Through Binding with RAGE in A549 Human Lung Cancer Cells.

BACKGROUND/AIMS: Heat shock protein 70 (HSP70) has been recently described with extracellular actions, where it is actively released in inflammatory conditions. Acting as DAMPs (damage associated molecular pattern), extracellular HSP70 (eHSP70) interacts with membrane receptors and activates inflammatory pathways. At this context, the receptor for advanced glycation endproducts (RAGE) emerges as a possible candidate for interaction with eHSP70. RAGE is a pattern-recognition receptor and its expression is increased in several diseases related to a chronic pro-inflammatory state. One of the main consequences of RAGE ligand-binding is the ERK1/2 (extracellular signal-regulated kinases)-dependent activation of NF-kB (nuclear factor kappa B), which leads to expression of TNF-α (tumor necrosis factor alpha) and other cytokines. The purpose of this work is to elucidate if eHSP70 is able to evoke RAGE-dependent signaling using A549 human lung cancer cells, which constitutively express RAGE. METHODS: Immunoprecipitation and protein proximity assay were utilized to demonstrate the linkage between RAGE and eHSP70. To investigate RAGE relevance on cell response to eHSP70, siRNA was used to knockdown the receptor expression. Signaling pathways activation were evaluated by western blotting, gene reporter luciferase and real time quantitative PCR. RESULTS: Protein eHSP70 shown to be interacting physically with the receptor RAGE in our cell model. Treatment with eHSP70 caused ERK1/2 activation and NF-κB transactivation impaired by RAGE knockdown. Moreover, the stimulation of pro-inflammatory cytokines expression by eHSP70 was inhibited in RAGE-silenced cells. Finally, conditioned medium of eHSP70-treated A549 cells caused differential effects in monocytes cytokine expression when A549 RAGE expression is inhibited. CONCLUSIONS: Our results evidence eHSP70 as a novel RAGE agonist capable of influence the cross-talk between cancer and immune system cells.

Retinoic acid downregulates thiol antioxidant defences and homologous recombination while promotes A549 cells sensitization to cisplatin.

Recent studies have investigated the use of retinoic acid (RA) molecule in combined chemotherapies to cancer cells as an attempt to increase treatment efficiency and circumvent cell resistance. Positive results were obtained in clinical trials from lung cancer patients treated with RA and cisplatin. Meanwhile, the signalling process that results from the interaction of both molecules remains unclear. One of the pathways that RA is able to modulate is the activity of NRF2 transcription factor, which is highly associated with tumour progression and resistance. Therefore, the aim of this work was to investigate molecular mechanism of RA and cisplatin co-treatment in A549 cells, focusing in NRF2 pathway. To this end, we investigated NRF2 and NRF2-target genes expression, cellular redox status, cisplatin-induced apoptosis, autophagy and DNA repair through homologous recombination. RA demonstrated to have an inhibitory effect over NRF2 activation, which regulates the expression of thiol antioxidants enzymes. Moreover, RA increased reactive species production associated with increased oxidation of thiol groups within the cells. The expression of proteins associated with DNA repair through homologous recombination was also suppressed by RA pre-treatment. All combined, these effects appear to create a more sensitive cellular environment to cisplatin treatment, increasing apoptosis frequency. Interestingly, autophagy was also increased by combination therapy, suggesting a resistance mechanism by A549 cells. In conclusion, these results provided new information about molecular mechanisms of RA and cisplatin treatment contributing to chemotherapy optimization.

Nuclear RXRα and RXRß receptors exert distinct and opposite effects on RA-mediated neuroblastoma differentiation.

Retinoic acid (RA) promotes differentiation in multiple neurogenic cell types by promoting gene reprogramming through retinoid receptors and also by inducing cytosolic signaling events. The nuclear RXR receptors are one of the main mediators of RA cellular effects, classically by joining the direct receptors of RA, the nuclear RAR receptors, in RAR/RXR dimers which act as transcription factors. Distinct RXR genes lead to RXRα, RXRß and RXRγ subtypes, but their specific roles in neuronal differentiation remain unclear. We firstly investigated both RXRs and RARs expression profiles during RA-mediated neuronal differentiation of human neuroblastoma cell line SH-SY5Y, and found varying levels of retinoid receptors transcript and protein contents along the process. In order to understand the roles of the expression of distinct RXR subtypes to RA signal transduction, we performed siRNA-mediated silencing of RXRα and RXRß during the first stages of SH-SY5Y differentiation. Our results showed that RXRα is required for RA-induced neuronal differentiation of SH-SY5Y cells, since its silencing compromised cell cycle arrest and prevented the upregulation of neuronal markers and the adoption of neuronal morphology. Besides, silencing of RXRα affected the phosphorylation of ERK1/2. By contrast, silencing of RXRß improved neurite extension and led to increased expression of tau and synaptophysin, suggesting that RXRß may negatively regulate neuronal parameters related to neurite outgrowth and function. Our results indicate distinct functions for RXR subtypes during RA-dependent neuronal differentiation and reveal new perspectives for studying such receptors as clinical targets in therapies aiming at restoring neuronal function.

Changes in Cell Cycle and Up-Regulation of Neuronal Markers During SH-SY5Y Neurodifferentiation by Retinoic Acid are Mediated by Reactive Species Production and Oxidative Stress.

Human neuroblastoma SH-SY5Y cells have been used as an in vitro model for neurodegenerative disorders such as Parkinson's disease and can be induced to a mature neuronal phenotype through retinoic acid (RA) differentiation. However, mechanisms of RA-induced differentiation remain unclear. Here, we investigate the role of reactive species (RS) on SH-SY5Y neuroblastoma cells under RA differentiation, using the antioxidant Trolox® as co-treatment. We found that RA treatment for 7 days reduced the cell number and proliferative capacity and induced the expression of adult catecholaminergic/neuronal markers such as tyrosine hydroxylase (TH), ß-III tubulin, and enolase-2. Evaluation of intracellular RS production by DCFH oxidation assay and quantification of cell non-enzymatic antioxidant activity by TRAP demonstrated that RA increases RS production. Furthermore, mitochondrial NADH oxidation showed to be inhibited under differentiation with RA. Cells subjected to co-treatment with antioxidant Trolox® demonstrated a remaining proliferative capacity and a decrease in the pro-oxidant state and RS production. Besides, antioxidant treatment restores the mitochondrial NADH oxidation. Importantly, Trolox® co-treatment inhibited the appearance of morphological characteristics such as neurite extension and branching, and decreased the expression of TH, ß-III tubulin, and enolase-2 after a seven-day differentiation with RA, indicating that RS production is a necessary step in this process. Trolox® also inhibited the phosphorylation of Akt and ERK1/2, which are involved in differentiation and survival, respectively, of these cells. Altogether, these data indicate the presence of a redox-dependent mechanism in SH-SY5Y RA-differentiation process and can be a useful insight to improve understanding of neuronal differentiation signaling.

The kinetic mechanism of Human Thymidine Phosphorylase - a molecular target for cancer drug development.

Human Thymidine Phosphorylase (HTP), also known as the platelet-derived endothelial cell growth factor (PD-ECGF) or gliostatin, catalyzes the reversible phosphorolysis of thymidine (dThd) to thymine and 2-deoxy-α-d-ribose-1-phosphate (2dR1P). HTP is a key enzyme in the pyrimidine salvage pathway involved in dThd homeostasis in cells. HTP is a target for anticancer drug development as its enzymatic activity promotes angiogenesis. Here, we describe cloning, expression, and purification to homogeneity of recombinant TYMP-encoded HTP. Peptide fingerprinting and the molecular mass value of the homogenous protein confirmed its identity as HTP assessed by mass spectrometry. Size exclusion chromatography showed that HTP is a dimer in solution. Kinetic studies revealed that HTP displayed substrate inhibition for dThd. Initial velocity and isothermal titration calorimetry (ITC) studies suggest that HTP catalysis follows a rapid-equilibrium random bi-bi kinetic mechanism. ITC measurements also showed that dThd and Pi binding are favorable processes. The pH-rate profiles indicated that maximal enzyme activity was achieved at low pH values. Functional groups with apparent pK values of 5.2 and 9.0 are involved in dThd binding and groups with pK values of 6.1 and 7.8 are involved in phosphate binding.