Procjena cito-/genotoksičnosti irinotekana u V79-stanicama primjenom komet-testa, mikronukleus-testa i testa kromosomskih aberacija

Irinotecan is a topoisomerase I interactive agent, widely used in the treatment of metastatic colorectal cancer. The genotoxic effects of the maximum single dose (18 μg mL-1), recommended monotherapy dose (9 μg mL-1), and recommended combined therapy dose (4.5 μg mL-1) of irinotecan were studied on V79 cells using the comet assay, chromosome aberration assay, and micronucleus test. The cells were treated with irinotecan for 2 h or 24 h. The statistical signifi cance of the results was determined using the one-way ANOVA test and a nonparametric Mann Whitney U test. The comet assay did not show dose-dependent or time-dependent effects. The chromosome aberration analysis showed large DNA rearrangements, i.e., chromosome exchanges. Although the exposed cultures showed a signifi cant increase in micronucleated cells in respect to control, no dose-dependent relation was established among the treated cultures. Timedependent effect was also not observed.Irinotekan je citotoksični lijek koji inhibira enzim DNA-topoizomerazu I. U širokoj je primjeni u terapiji metastatskog karcinoma kolona i rektuma. U uvjetima in vitro primjenom komet-testa, analize kromosomskih aberacija i mikronukleus-testa na V79-stanicama istražili smo genotoksični učinak maksimalne pojedinačne doze (18 μg mL-1), preporučene monoterapijske doze (9 μg mL-1) i preporučene doze irinotekana za kombiniranu terapiju (4,5 μg mL-1). Kulture stanica bile su tretirane irinotekanom 2 h i 24 h. Statistička značajnost određivana je jednosmjernim ANOVA-testom i neparametrijskim Mann Whitneyevim U-testom. Komet-testom nije utvrđen učinak koncentracije i/ili vremena izloženosti. Analiza kromosomskih aberacija pokazala je prisutnost izmjena kromatida, tj. porast broja triradijusa i tetraradijusa. Iako je u kulturama stanica izloženi irinotekanu opažen značajan porast broja mikronukleusa u odnosu na kontrolu, nije uočena ovisnost o dozi lijeka ni o vremenu izloženosti u opisanim eksperimentalnim uvjetima. Dobiveni rezultati upućuju na genotoksičnost irinotekana za V79-stanice. Nijednom od primijenjenih metoda nije utvrđena ovisnost učinka irinotekana o vremenu ili dozi

A new class of glycomimetic drugs to prevent free fatty acid-induced endothelial dysfunction

Background: Carbohydrates play a major role in cell signaling in many biological processes. We have developed a set of glycomimetic drugs that mimic the structure of carbohydrates and represent a novel source of therapeutics for endothelial dysfunction, a key initiating factor in cardiovascular complications. Purpose: Our objective was to determine the protective effects of small molecule glycomimetics against free fatty acid­induced endothelial dysfunction, focusing on nitric oxide (NO) and oxidative stress pathways. Methods: Four glycomimetics were synthesized by the stepwise transformation of 2,5­dihydroxybenzoic acid to a range of 2,5­substituted benzoic acid derivatives, incorporating the key sulfate groups to mimic the interactions of heparan sulfate. Endothelial function was assessed using acetylcholine­induced, endotheliumdependent relaxation in mouse thoracic aortic rings using wire myography. Human umbilical vein endothelial cell (HUVEC) behavior was evaluated in the presence or absence of the free fatty acid, palmitate, with or without glycomimetics (1µM). DAF­2 and H2DCF­DA assays were used to determine nitric oxide (NO) and reactive oxygen species (ROS) production, respectively. Lipid peroxidation colorimetric and antioxidant enzyme activity assays were also carried out. RT­PCR and western blotting were utilized to measure Akt, eNOS, Nrf­2, NQO­1 and HO­1 expression. Results: Ex vivo endothelium­dependent relaxation was significantly improved by the glycomimetics under palmitate­induced oxidative stress. In vitro studies showed that the glycomimetics protected HUVECs against the palmitate­induced oxidative stress and enhanced NO production. We demonstrate that the protective effects of pre­incubation with glycomimetics occurred via upregulation of Akt/eNOS signaling, activation of the Nrf2/ARE pathway, and suppression of ROS­induced lipid peroxidation. Conclusion: We have developed a novel set of small molecule glycomimetics that protect against free fatty acidinduced endothelial dysfunction and thus, represent a new category of therapeutic drugs to target endothelial damage, the first line of defense against cardiovascular disease

Inhibitor of apoptosis proteins are required for effective fusion of autophagosomes with lysosomes

Inhibitor of Apoptosis Proteins act as E3 ubiquitin ligases to regulate NF-κB signalling from multiple pattern recognition receptors including NOD2, as well as TNF Receptor Superfamily members. Loss of XIAP in humans causes X-linked Lymphoproliferative disease type 2 (XLP-2) and is often associated with Crohn’s disease. Crohn’s disease is also caused by mutations in the gene encoding NOD2 but the mechanisms behind Crohn’s disease development in XIAP and NOD2 deficient-patients are still unknown. Numerous other mutations causing Crohn’s Disease occur in genes controlling various aspects of autophagy, suggesting a strong involvement of autophagy in preventing Crohn’s disease. Here we show that the IAP proteins cIAP2 and XIAP are required for efficient fusion of lysosomes with autophagosomes. IAP inhibition or loss of both cIAP2 and XIAP resulted in a strong blockage in autophagic flux and mitophagy, suggesting that XIAP deficiency may also drive Crohn’s Disease due to defects in autophagy