Evaluation of tissue and circulating mir-21 as potential biomarker of response to chemoradiotherapy in rectal cancer

This research received funding from European Structural & Investment Funds through the COMPETE Programme—Programa Operacional Regional de Lisboa—Programme Grant LISBOA-01-0145-FEDER-016405,and from National Funds through FCT—Fundação para a Ciência e a Tecnologia—Programme Grant SAICTPAC/0019/2015.Response to chemoradiotherapy (CRT) in patients with locally advanced rectal cancer (RC) is quite variable and it is urgent to find predictive biomarkers of response. We investigated miR-21 as tissue and plasma biomarker of response to CRT in a prospective cohort of RC patients; The expression of miR-21 was analyzed in pre-and post-CRT rectal tissue and plasma in 37 patients with RC. Two groups were defined: Pathological responders (TRG 0, 1 and 2) and non-responders (TRG 3). The association between miR-21, clinical and oncological outcomes was assessed; miR-21 was upregulated in tumor tissue and we found increased odds of overexpression in pre-CRT tumor tissue (OR: 1.63; 95% CI: 0.40–6.63, p = 0.498) and pre-CRT plasma (OR: 1.79; 95% CI: 0.45–7.19, p = 0.414) of non-responders. The overall recurrence risk increased with miR-21 overexpression in pre-CRT tumor tissue (HR: 2.175, p = 0.37); Significantly higher miR-21 expression is observed in tumor tissue comparing with non-neoplastic. Increased odds of non-response is reported in patients expressing higher miR-21, although without statistical significance. This is one of the first studies on circulating miR-21 as a potential biomarker of response to CRT in RC patients.publishersversionpublishe

Potential of miR-21 to Predict Incomplete Response to Chemoradiotherapy in Rectal Adenocarcinoma

Funding: This work has received funding from European Structural and Investment Funds through the COMPETE Programme Grant LISBOA-01-0145-FEDER-016405, from National Funds through Fundação para a Ciência e Tecnologia Programme grant SAICTPAC/0019/2015 and by a cholar from the Portuguese Society of Coloproctology as Investigation in Coloproctology Research Prize 2016–2018.Background: Patients with locally advanced rectal adenocarcinoma (LARC) are treated with neoadjuvant chemoradiotherapy (CRT). However, biomarkers for patient selection are lacking, and the association between miRNA expression and treatment response and oncological outcomes is unclear. Objectives: To investigate miRNAs as predictors of response to neoadjuvant CRT and its association with oncological outcomes. Methods: This retrospective study analyzed miRNA expression (miR-16, miR-21, miR-135b, miR-145, and miR-335) in pre- and post-chemoradiation rectal adenocarcinoma tissue and non-neoplastic mucosa in 91 patients treated with neoadjuvant CRT (50.4 Gy) and proctectomy. Two groups were defined: a pathological complete responders group (tumor regression grade—TRG 0) and a pathological incomplete responders group (TRG 1, 2, and 3). Results: miR-21 and miR-135b were upregulated in tumor tissue of incomplete responders comparing with non-neoplastic tissue (p = 0.008 and p < 0.0001, respectively). Multivariate analysis showed significant association between miR-21 in pre-CRT tumor tissue and response, with a 3.67 odds ratio (OR) of incomplete response in patients with higher miR-21 levels (p = 0.04). Although with no significance, patients treated with 5-fluorouracil (5-FU) presented reduced odds of incomplete response compared with those treated with capecitabine (OR = 0.19; 95% confidence interval (CI) 0.03–1.12, p = 0.05). Moreover, significant differences were seen in overall survival (OS) in relation to clinical TNM stage (p = 0.0004), cT (p = 0.0001), presence of distant disease (p = 0.002), mesorectal tumor deposits (p = 0.003), and tumor regression grade (p = 0.04). Conclusion: miR-21 may predict response to CRT in rectal cancer (RC).publishersversionpublishe

Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53

AbstractDrug resistance to 5-fluorouracil (5-FU) is still a major limitation to its clinical use. In addition, the clinical value of p53 as a predictive marker for 5-FU-based chemotherapy remains a matter of debate. Here, we used HCT116 human colorectal cancer cells expressing wild-type p53 and investigated whether inhibition of Fas expression by interference RNA modulates 5-FU-induced apoptosis. Cells were treated with 5-FU (1, 4 or 8 μM) for 8–48 h. Cell viability was evaluated by trypan blue dye exclusion. Apoptosis was assessed by changes in nuclear morphology and caspase activity. The interference RNA technology was used to silence Fas expression. Caspase activation, p53, Fas, cytochrome c, and Bcl-2 family protein expression was evaluated by immunoblotting. 5-FU was cytotoxic in HCT116 cells (p<0.001). Nuclear fragmentation and caspase-3, -8 and -9 activities were also markedly increased in HCT116 cells after 5-FU (p<0.001). In addition, wild-type p53 and Fas expression were 25- and 4-fold increased (p<0.05). Notably, when interference RNA was used to inhibit Fas, 5-FU-mediated nuclear fragmentation and caspase activity were markedly reduced in HCT116 cells. Finally, western blot analysis of mitochondrial extracts from HCT116 cells exposed to 5-FU showed a 6-fold increase in Bax, together with a 3-fold decrease in cytochrome c (p<0.001). In conclusion, 5-FU exerts its cytotoxic effects, in part, through a p53/Fas-dependent apoptotic pathway that involves Bax translocation and mitochondrial permeabilization

Electrochemical sensing of ecstasy with electropolymerized molecularly imprinted poly(o-phenylenediamine) polymer on the surface of disposable screen-printed carbon electrodes

This study demonstrates the ability of an electrochemical sensor based on molecularly imprinted polymers (MIPs) to selectively quantify 3,4-methylenedioxymethamphetamine (MDMA), also known as ecstasy, in biological samples. The device was constructed using ortho-phenylenediamine (o-PD) as the MIP’s building monomer at the surface of a screen-printed carbon electrode (SPCE). The step-by-step construction of the SPCE-MIP sensor was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Density functional theory (DFT) calculations and modelling were performed not only to understand template-monomer interaction but also to comprehend which possible polymer structure - linear or ramified poly(o-PD) – indeed interacts with the analyte. The prepared sensor worked by directly measuring the MDMA oxidation signal through square-wave voltammetry (SWV) after an incubation period of 10 min. Several parameters were optimized, such as the monomer/template ratio, the number of electropolymerization scanning cycles, and the incubation period, to obtain the best sensing efficiency. Optimized sensors exhibited suitable selectivity, repeatability (2.6%), reproducibility (7.7%) and up to one month of stable response. A linear range up to 0.2 mmol L−1 was found with an r2 of 0.9990 and a limit of detection (LOD) and quantification (LOQ) of 0.79 and 2.6 μmol L−1 (0.15 and 0.51 μg mL−1), respectively. The proposed sensor was successfully applied to human blood serum and urine samples, showing its potential for application in medicine and in forensic sciences.This work received financial support from FCT/MCTES through national funds and was co-financed by FEDER, under Partnership Agreement PT2020-UID/QUI/50006/2013-POCI/01/0145/FEDER/007265. RASC wishes to acknowledge FCT for her PhD fellowship (PD/BD/127797/2016) from the PhD Programme in Medicines and Pharmaceutical Innovation (i3DU). AACB (grants 2014/25770-6 and 2015/01491-3) and LMG (grant 2018/14425-7) thank the São Paulo Research Foundation (FAPESP) for financial support. AACB (grant 309715/2017-2) also thanks the Brazilian National Research Council (CNPq) for financial support and fellowships. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.info:eu-repo/semantics/publishedVersio

Impaired cross-talk between the thioredoxin and glutathione systems is related to ASK-1 mediated apoptosis in neuronal cells exposed to mercury

Mercury (Hg) compounds target both cysteine (Cys) and selenocysteine (Sec) residues in peptides and proteins. Thus, the components of the two major cellular antioxidant systems – glutathione (GSH) and thioredoxin (Trx) systems – are likely targets for mercurials. Hg exposure results in GSH depletion and Trx and thioredoxin reductase (TrxR) are prime targets for mercury. These systems have a wide-range of common functions and interaction between their components has been reported. However, toxic effects over both systems are normally treated as isolated events. To study how the interaction between the glutathione and thioredoxin systems is affected by Hg, human neuroblastoma (SH-SY5Y) cells were exposed to 1 and 5 μM of inorganic mercury (Hg2+), methylmercury (MeHg) or ethylmercury (EtHg) and examined for TrxR, GSH and Grx levels and activities, as well as for Trx redox state. Phosphorylation of apoptosis signalling kinase 1 (ASK1), caspase-3 activity and the number of apoptotic cells were evaluated to investigate the induction of Trx-mediated apoptotic cell death. Additionally, primary cerebellar neurons from mice depleted of mitochondrial Grx2 (mGrx2D) were used to examine the link between Grx activity and Trx function. Results showed that Trx was affected at higher exposure levels than TrxR, especially for EtHg. GSH levels were only significantly affected by exposure to a high concentration of EtHg. Depletion of GSH with buthionine sulfoximine (BSO) severely increased Trx oxidation by Hg. Notably, EtHg-induced oxidation of Trx was significantly enhanced in primary neurons of mGrx2D mice. Our results suggest that GSH/Grx acts as backups for TrxR in neuronal cells to maintain Trx turnover during Hg exposure, thus linking different mechanisms of molecular and cellular toxicity. Finally, Trx oxidation by Hg compounds was associated to apoptotic hallmarks, including increased ASK-1 phosphorylation, caspase-3 activation and increased number of apoptotic cells