Análisis farmacogenético y funcional del gen Timidilato sintasa en cáncer gastrointestinal

Tesis (Maestría en Ciencias con Orientación Terminal en Biología Molecular e Ingeniería Genética) U.A.N.L., 2007.UANLhttp://www.uanl.mx

Proteoma y vías de señalización inducidas por AAS en células que expresan el VHC

RESUMEN El AAS disminuye la expresión del VHC por mecanismos aún desconocidos. Se compararon perfiles de expresión proteómica en células de hepatocarcinoma (Huh7) y células que expresan proteínas no estructuradas del VHC (Huh7-VHC) tratadas con AAS 4mM. Los perfiles obtenidos mediante electroforesis bidimensional fueron analizados por pI y PM. Se identificaron diferentes patrones entre células Huh7- VHC tratadas y no tratadas con AAS. Entre las proteínas diferencialmente expresadas encontramos proteínas relacionadas con proliferación celular (MTMR6, FAM22, HDGF y HCF-1) y sobreexpresión de angiotensina, PI4KA y STAT-1. A las 72h, identificamos sobreexpresión de adenil-succinato sintasa, 2’-3’-di-deoxiadenosina, proteína ligasa de ubiquitina E6A, adenilsuccinato-liasa y nibrina. El AAS induce diferentes patrones proteicos en células Huh7- VHC, promoviendo la activación de proteínas relacionadas con progresión celular, reparación de DNA, inhibición de apoptosis y estimulación del crecimiento. ABSTRACT ASA has been shown to downregulate HCV expression; however, the involved mechanisms are unknown. We used proteomic analysis to compare protein expression profiles between human hepatocarcinome cells (Huh7) and Huh7-HCV cells harboring expression of non-structural HCV proteins to elucidate the mechanisms involved in ASAmediated downregulation of HCV replication. Cell lines were treated with 4 mM ASA and harvested to isolate total proteins, which were resolved by 2-DE. Gels were analyzed and proteins elucidated by pI and MW patterns. Different protein patterns among hepatocytes expressing HCV-proteins in ASA treated and untreated cells were found. Among proteins differentially expressed we found proteins related to cellular proliferation (MTMR6, FAM22, HDGF y HCF-1) and overexpression of angiotensin (PI4KA y STAT-1). We found that ASA induces different protein patterns in Huh7-HCV cells promoting activation of proteins involved in cell progression, repair of double strand breaks, proliferation, inhibition of apoptosis and growth stimulation at the same time that it decreased HCV expression

Quantification of nitric oxide by high‑performance liquid chromatography‑fluorometric method in subgenomic hepatitis C virus‑replicon expressing Huh7 cells upon treatment with acetylsalicylic acid

As nitric oxide (NO) expression levels are lower in hepatocytes compared with other cell types, it is difficult to quantify this compound via Griess assay. The aim of the present study was to quantify NO concentration in the cell culture medium from a subgenomic hepatitis C virus (HCV)‑replicon expressing Huh‑7 cell system using a high‑performance liquid chromatography (HPLC)‑fluorescence detector in the presence or absence of acetylsalicylic acid (ASA) treatment. HCV‑replicon cells were incubated with ASA (4 mM) for 24, 48 and 72 h. Thereafter, the medium was collected to measure nitrites (NO2-) as an indirect indicator of NO levels using diaminonaphtalene as a derivate agent. NO levels were significantly higher (1.7‑fold) in Huh‑7 replicon cells treated with ASA (72 h post‑treatment) than untreated cells (P<0.05); NO inhibitor reduced ~30% the level of NO in Huh‑7 replicon cells treated with ASA (48 h post‑treatment; P<0.05). The findings suggested that the HPLC‑fluorescence method provided an accurate and efficient measurement of NO production in Huh‑7‑HCV‑replicon cells culture medium

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Use of proteomic analysis tools to identify HCV-proteins down-regulated by acetylsalicylic acid

Background and aim. Acetylsalicylic acid (ASA) has been shown to downregulate HCV expression; however, the involved mechanisms are unknown. We used proteomic analysis to compare protein expression profiles between human hepatocarcinoma cells (Huh7) and Huh7-HCV cells harboring expression of non-structural HCV proteins, to elucidate the mechanism(s) involved in ASA-mediated downregulation of HCV replication.Material and methods. Both cell lines were treated or untreated with 4 mM ASA and harvested at 0, 24, 48 and 72 h to isolate total proteins, which were resolved by two-dimensional gel electrophoresis (2DE) to separate them by isoelectric point (pI), followed by fractionation by molecular weight (MW). Gels were scanned and analyzed with PD-Quest software V8.0.1, and proteins were elucidated by the specific pI and MW using TAGIDENT software. Statistics analysis included the t-test.Results and Discussion. Different protein patterns among hepatocytes expressing HCV-proteins in ASA treated and untreated cells were found. Among proteins differentially expressed in Huh7-HCV cells, we found proteins related to cell proliferation (MTMR6, FAM22, HDGF and HCF–1) after 24 h of ASA treatment; and upregulation of angiostatin, PI4KA and STAT–1 after 48 h of treatment. Finally, at 72 h of ASA exposure, we identified overexpression of adenyl-succinate synthase, 2’–3’-di-deoxyadenosine, ubiquitin-protein-ligase E6A, adenylosuccinate-lyase and ni-brin (NBN).Conclusion. We found that ASA induces different protein patterns in Huh7-HCV cells promoting activation of proteins involved in cell progression, repair of double strand breaks, proliferation, inhibition of apoptosis and growth stimulation at the same time that it decreased HCV expression