Avaliação do potencial antitumoral do bozepinib livre ou em nanocápsulas em ensaios pré-clínicos de glioblastoma

O Glioblastoma (GBM) é o mais comum e agressivo tumor cerebral que acomete adultos e, apesar dos avanços em seu diagnóstico e tratamento, não houve aumento significativo na sobrevida dos pacientes. O bozepinib (BZP) é um novo composto com atividade anticâncer que vem sendo estudado por suas promissoras atividades farmacológicas, principalmente sua potente e seletiva atividade antitumoral e antimetastática in vitro e in vivo em câncer de mama, cólon e melanoma. Nesse contexto, este estudo teve como objetivo avaliar o efeito citotóxico do BZP em linhagens de GBM, bem como elucidar seu papel frente a vias de proliferação, resistência e morte celular. Além disso desenvolvemos e caracterizamos nanocápsulas de núcleo lipídico carregadas com BZP e revestidas com quitosana (BZP-LNC+) a fim de melhorar as características físico-químicas desse composto com o objetivo de avaliar sua atividade in vivo. No primeiro set de experimentos, o BZP apresentou atividade citotóxicas em células GBM (C6 e U138) com baixos valores de IC50 quando comparado a células não tumorais (MRC-5). Identificamos que o tratamento com este composto induz morte celular por apoptose dependente de caspase, com formação de autofagossomos, ativação da via NF-kB, sem qualquer alteração significativa na progressão do ciclo celular e na via Akt. Além disso, o BZP foi capaz de modular o sistema purinérgico, aumentando a expressão e a atividade da enzima CD39, enquanto exibiu atividade inibitória da enzima CD73 in sílico e in vitro, reduzindo a hidrólise de AMP e a formação de adenosina Além disso, a exposição durante 24h com BZP foi capaz de selecionar células CD133+/NF-kB resistentes e em apenas dois ciclos de tratamento, todas as células foram eliminadas. A seguir, desenvolvemos e validamos com sucesso um método analítico sensível para quantificar BZP-LNC+ e, as formulações apresentaram características físico-químicas com tamanhos de partícula adequadas, estreita distribuição de tamanho, carga superficial positiva e, alta eficiência de encapsulação. BZPLNC+ apresentou citotoxicidade contra células GBM após 72h de tratamento, com valores de IC50 de aproximadamente 0,4 μM, induzindo morte celular por apoptose. Além disso, o tratamento combinado entre BZP-LNC+ e TMZ mostrou um efeito citotóxico sinérgico em células GBM, potencializando a indução de apoptose mesmo com concentrações reduzidas de ambas as drogas testadas. O co-tratamento com TMZ + BZP-LNC+ reduziu o crescimento do glioma in vivo em cerca de 81% quando comparado ao grupo controle. Em conjunto, concluímos que o BZP-LNC+ é um interessante candidato para o tratamento do GBM, encorajando mais estudos sobre o potencial terapêutico, a fim de melhor compreendermos o seu mecanismo de ação in vivo.Glioblastoma (GBM) is the most common and aggressive brain tumor affecting adults, and despite advances in its diagnosis and treatment, there has been no significant increase in patient survival. Bozepinib (BZP) is a new compound with anticancer activity that has been studied for its promising pharmacological activities, especially its potent and selective antitumor and antimetastatic activity in vitro and in vivo against breast, colon and melanoma cancer. In this context, this study aimed to evaluate the cytotoxic effect of BZP in GBM cells, as well as to elucidate its role in proliferation, resistance and cell death pathways. In addition, we developed and characterized lipid-core nanocapsules loaded with BZP and coated with chitosan (BZPLNC+) to improve the physical-chemical characteristics of this compound in order to evaluate its GBM activity in vivo. In the first set of experiments, BZP showed cytotoxic activity in GBM cells (C6 and U138) with low IC50 values when compared to non-tumor cells (MRC-5). We identified that BZP treatment induces cell death by apoptosis caspase-dependent, with autophagosomes formation, NF-kB activation, without any significant alteration in the cell cycle progression and in the Akt pathway. In addition, BZP was able to modulate the purinergic system, increasing the expression and activity of the CD39 enzyme, while exhibiting inhibitory activity of the CD73 enzyme in sílico and in vitro, reducing AMP hydrolysis and adenosine formation. Furthermore, exposure for 24h with BZP was able to select CD133+/ NF-kB resistant cells and, in only two treatment cycles, all cells were eliminated. Next, we successfully developed and validated a sensitive analytical method to quantify BZP-LNC+ and, the formulations showed physicochemical characteristics with adequate particle sizes, narrow size distribution, positive zeta potential, and high encapsulation efficiency. BZP-LNC+ exhibited cytotoxicity against GBM cells after 72 h of treatment, with IC50 values of approximately 0.4 μM, inducing cell death by apoptosis. In addition, the combined treatment between BZP-LNC+ and TMZ showed a synergistic cytotoxic effect in GBM cells, potentiating the induction of apoptosis even with reduced concentrations of both drugs tested. Co-treatment with TMZ + BZP-LNC+ reduced glioma growth in vivo by about 81% when compared to control group. Together, we conclude that BZP-LNC+ is an interesting candidate for GBM treatment, encouraging further studies on the therapeutic potential to better understand its mechanism of action in vivo

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

Lymphocytes from B-acute lymphoblastic leukemia patients present diferential regulation of the adenosinergic axis depending on risk stratifcation

Purpose Although risk-stratifed chemotherapy regimens improve B-cell acute lymphoblastic leukemia (B-ALL) clinical outcome, relapse occurs in a signifcant number of cases. The identifcation of new therapeutic targets as well as prog nostic and diagnostic biomarkers can improve B-ALL patients’ clinical outcomes. Purinergic signaling is an important pathway in cancer progression, however the expression of ectonucleotidases and their impact on immune cells in B-ALL lacks exploration. We aimed to analyze the expression of ectonucleotidases in B-ALL patients’ lymphocyte subpopulations. Methods Peripheral blood samples from 15 patients diagnosed with B-ALL were analyzed. Flow cytometry was used to analyze cellularity, expression level of CD38, CD39, and CD73, and frequency of CD38+∕CD73+, and CD39+∕CD73+ in lymphocyte subpopulations. Plasma was used for cytokines (by CBA kit) and adenine nucleosides/nucleotides detection (by HPLC). Results Comparing B-ALL patients to health donors, we observed an increase of CD4+and CD8+T-cells. In addition, a decrease in CD38 expression in B and Treg subpopulations and an increase in CD39+ CD73+ frequency in Breg and CD8+ T-cells. Analyzing cytokines and adenine nucleosides/nucleotides, we found a decrease in TNF, IL-1β, and ADO concentrations, together with an increase in AMP in B-ALL patients’ plasma. Conclusion: As immunomodulators, the expression of ectonucleotidases might be associated with activation states, as well as the abundance of diferent cellular subsets. We observed a pro-tumor immunity expression profle in B-ALL patients at diagnosis, being associated with cell exhaustion and immune evasion in B-ALL

Wellbore integrity in a saline aquifer : experimental steel-cement interface degradation under supercritical CO2 conditions representative of Brazil’s Parana basin

From our work, significant progress has been made in understanding the degradation of cement-casing systems. The CO2 degradation process was evaluated in specimens with a large interfacial defect, such as large annular spaces, voids and/or channels, which may be the result of a poor cementing job. From the experiments showing no interfacial defect, no signs of degradation were observed, while from experiments showing interfacial defect, both the cement and steel undergo significant degradation. In the well casing, the CO2-rich brine affects the steel phase, leaching Fe2+ ions into solution and promoting FeCO3 precipitation on the material surface, while on the cement sheath, two processes are occurring: (i) the portlandite dissolution and (ii) the cement carbonation process. Then, iron (Fe2+) starts to migrate into the cement structure, compromising the material’s self-healing and pore-blocking features, while calcium (Ca2+) starts to compose the corrosion film from the formation of mixed carbonates (FexCayCO3) so reducing the corrosion layer’s protection. Finally, both ions (Ca2+ and Fe2+) become so abundant in the material vicinity that they may form calcium carbonate (CaCO3) on the corrosion layer and iron carbonate (FeCO3) in the cement matrix. Thus, from our results, the degradation mechanisms of the cement-casing system in CO2-rich brine was revised

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Lymphocytes from B-acute lymphoblastic leukemia patients present differential regulation of the adenosinergic axis depending on risk stratification

Abstract Purpose Although risk-stratified chemotherapy regimens improve B-cell acute lymphoblastic leukemia (B-ALL) clinical outcome, relapse occurs in a significant number of cases. The identification of new therapeutic targets as well as prognostic and diagnostic biomarkers can improve B-ALL patients' clinical outcomes. Purinergic signaling is an important pathway in cancer progression, however the expression of ectonucleotidases and their impact on immune cells in B-ALL lacks exploration. We aimed to analyze the expression of ectonucleotidases in B-ALL patients’ lymphocyte subpopulations. Methods Peripheral blood samples from 15 patients diagnosed with B-ALL were analyzed. Flow cytometry was used to analyze cellularity, expression level of CD38, CD39, and CD73, and frequency of $${\mathrm{CD}38}^{+}{/\mathrm{CD}73}^{+}$$ CD 38 + / CD 73 + , and $${\mathrm{CD}39}^{+}{/\mathrm{CD}73}^{+}$$ CD 39 + / CD 73 + in lymphocyte subpopulations. Plasma was used for cytokines (by CBA kit) and adenine nucleosides/nucleotides detection (by HPLC). Results Comparing B-ALL patients to health donors, we observed an increase of CD4 + and CD8 + T-cells. In addition, a decrease in CD38 expression in B and Treg subpopulations and an increase in CD39+ CD73+  frequency in Breg and CD8+ T-cells. Analyzing cytokines and adenine nucleosides/nucleotides, we found a decrease in TNF, IL-1β, and ADO concentrations, together with an increase in AMP in B-ALL patients' plasma. Conclusion As immunomodulators, the expression of ectonucleotidases might be associated with activation states, as well as the abundance of different cellular subsets. We observed a pro-tumor immunity expression profile in B-ALL patients at diagnosis, being associated with cell exhaustion and immune evasion in B-ALL