ABSTRACT
Small molecules are considered a source of novel medicines targeting carcinogenic intracellular pathways including epidermal growth factor receptor (EGFR) signaling. The main goal of the study is to assess whether LHT-17-19 could be considered an effective target molecule against EGFR-expressing tumor cells in silico, in vitro, and in vivo. This was an in vivo, ex vivo, and in vivo experimental study. LHT-17-19 affinity to EGFR's kinase domain was assessed by the ligand's molecular docking. EGFR-expressing Hs746T human gastric cancer cell culture and patient-derived organoid (PDO) model of EGFR-positive breast cancer (BC) were used for in vitro assessment of the molecule anticancer property. IC50 and GI50 indexes were estimated using MTT- and MTS-based tests, respectively. Anticancer activity of LHT-17-19 against EGFR-expressing mutant lung carcinoma was studied on patient-derived xenograft (PDX) model established in 10 humanized BALB/c male mice. Continuous variables were presented as a mean ± standard deviation. Intergroup differences were assessed by two-way t-test. Kaplan-Meier's curves were used for survival analysis. High affinity of LHT-17-19 for the EGFR kinase domain with dG score -7.9 kcal/mol, EDoc-5.45 kcal/mol, and Ki 101.24 uM was due to intermolecular π-σ bonds formation and the ligand intramolecular transformation. LHT-17-19 induced anti-EGFR-expressing gastric cancer cells cytotoxicity with IC50 0.32 µM (95% confidence interval [CI] 0.11-0.54 µM). The derivative inhibited growth of EGFR-expressing BC PDO with GI50 16.25 µM (95% CI 4.44-28.04 µM). 2 mg/kg LHT-17-19 intravenously daily during 7 days inhibited PDX tumor growth and metastatic activity, prolonged animals' survival, and eliminated EGFR-mutant lung cancer cells from residual tumor's node. LHT-17-19 may be considered a molecular platform for further search of promising molecules, EGFR-expressing cancer cell inhibitors.
ABSTRACT
A non-surgical pharmacological approach to control cellular vitality and functionality during ischemic and/or reperfusion-induced phases of strokes remains extremely important. The synthesis of 2-ethyl-6-methyl-3-hydroxypyridinium gammalactone-2,3-dehydro-L-gulonate (3-EA) was performed using a topochemical reaction. The cell-protective effects of 3-EA were studied on a model of glutamate excitotoxicity (GluTox) and glucose-oxygen deprivation (OGD) in a culture of NMRI mice cortical cells. Ca2+ dynamics was studied using fluorescent bioimaging and a Fura-2 probe, cell viability was assessed using cytochemical staining with propidium iodide, and gene expression was assessed by a real-time polymerase chain reaction. The compound anti-ischemic efficacy in vivo was evaluated on a model of irreversible middle cerebral artery (MCA) occlusion in Sprague-Dawley male rats. Brain morphological changes and antioxidant capacity were assessed one week after the pathology onset. The severity of neurological disorder was evaluated dynamically. 3-EA suppressed cortical cell death in a dose-dependent manner under the excitotoxic effect of glutamate and ischemia/reoxygenation. Pre-incubation of cerebral cortex cells with 10-100 µM 3-EA led to significant stagnation in Ca2+ concentration in a cytosol ([Ca2+]i) of neurons and astrocytes suffering GluTox and OGD. Decreasing intracellular Ca2+ and establishing a lower [Ca2+]i baseline inhibited necrotic cell death in an acute experiment. The mechanism of 3-EA cytoprotective action involved changes in the baseline and ischemia/reoxygenation-induced expression of genes encoding anti-apoptotic proteins and proteins of the oxidative status; this led to inhibition of the late irreversible stages of apoptosis. Incubation of brain cortex cells with 3-EA induced an overexpression of the anti-apoptotic genes BCL-2, STAT3, and SOCS3, whereas the expression of genes regulating necrosis and inflammation (TRAIL, MLKL, Cas-1, Cas-3, IL-1ß and TNFa) were suppressed. 3-EA 18.0 mg/kg intravenous daily administration for 7 days following MCA occlusion preserved rats' cortex neuron population, decreased the severity of neurological deficit, and spared antioxidant capacity of damaged tissues. 3-EA demonstrated proven short-term anti-ischemic activity in vivo and in vitro, which can be associated with antioxidant activity and the ability to target necrotic and apoptotic death. The compound may be considered a potential neuroprotective molecule for further pre-clinical investigation.
