The epidermal growth factor receptor (EGFR) is one of the most well-studied molecular targets in non-small cell lung cancer (NSCLC) and tyrosine kinase inhibitors have been shown to be effective in the treatment of advanced NSCLC. Nevertheless, the efficacy of tyrosine kinase inhibitors could be compromised by additional mutations in EGFR and compensatory activations of other pathways. Epigallocatechin-3-gallate (EGCG), the main bioactive molecule in green tea, acts as a tyrosine kinase inhibitor toward cancer cells overexpressing EGFR (wild-type). However, little information has been reported on the effect of EGCG on EGFR with activating mutations. In this study, we evaluated the ability of EGCG to inhibit EGFR signaling activation in three different NSCLC cell lines containing wild-type EGFR or EGFR with additional mutations. The effect on proliferation, apoptosis, migration, and vinculin expression was then studied. Overall, our results demonstrate that EGCG polyphenol inhibits cell proliferation and migration in NSCLC cell lines, although with different efficacy and mechanisms. These data may be of interest for an evaluation of the use of EGCG as an adjunct to NSCLC therapies.

Epigallocatechin-3-gallate (EGCG) has the highest antioxidant activity compared to the others catechins of green tea. However, the beneficial effects are mainly limited by its poor membrane permeability. A derivatization strategy to increase the EGCG interaction with lipid membranes is considered as one feasible approach to expand its application in lipophilic media, in particular the cellular absorption. At this purpose the hydrophilic EGCG was modified by inserting an aliphatic C18 chain linked to the gallate ring by an ethereal bond, the structure determined by NMR (Nuclear Magnetic Resonance) and confirmed by Density Functional Theory (DFT) calculations. The in vitro antioxidant activity of the mono-alkylated EGCG (C18-EGCG) was studied by the DPPH and Thiobarbituric Acid Reactive Substances (TBARS) assays, and its ability to protect cells towards oxidative stress was evaluated in Adult Retinal Pigmented Epithelium (ARPE-19) cells. Molecular Dynamics (MD) simulation and liposomal/buffer partition were used to study the interaction of the modified and unmodified antioxidants with a cell membrane model: the combined experimental-in silico approach shed light on the higher affinity of C18-EGCG toward lipid bilayer. Although the DPPH assay stated that the functionalization decreases the EGCG activity against free radicals, from cellular experiments it resulted that the lipid moiety increases the antioxidant protection of the new lipophilic derivative.

Non-small cell lung cancer (NSCLC) represents a difficult condition to treat, due to epidermal growth factor receptor (EGFR) kinase domain mutations, which lead to ligand-independent phosphorylation. Deletion of five amino acids (ELREA) in exon 19 and mutational change from leucine to arginine at position 858 (L858R) are responsible for tyrosine kinase domain aberrant activation. These two common types of EGFR-mutated forms are clinically associated with high response with Tyrosine Kinase Inhibitors (TKI); however, the secondary T790M mutation within the Tyrosine Kinase Domain (TKD) determines a resistance to these EGFR-TKIs. Using molecular dynamic simulation (MD), the present study investigated the architectural changes of wild-type and mutants EGFR’s kinase domains in order to detect any conformational differences that could be associated with a constitutively activated state and thus to evaluate the differences between the wild-type and its mutated forms. In addition, in order to evaluate to which extent the EGFR mutations affect its inhibition, Epigallocatechin 3-Gallate (EGCG) and Erlotinib (Erl), known EGFR-TKI, were included in our study. Their binding modes with the EGFR-TK domain were elucidated and the binding differences between EGFR wild-type and the mutated forms were evidenced. The aminoacids mutations directly influence the binding affinity of these two inhibitors, resulting in a different efficacy of Erl and EGCG inhibition. In particular, for the T790M/L858R EGFR, the binding modes of studied inhibitors were compromised by aminoacidic substitution confirming the experimental findings. These results may be useful for novel drug design strategies targeting the dimerization domain of the EGFR mutated forms, thus preventing receptor activation.

As part of a program aimed to obtain antioxidants able to interact with cell membrane, edaravone (EdV, 3-methyl-1-phenyl-2-pyrazolin-5-one), a well-known free radical scavenger, has been modified by alkylation at its allylic position (4) with a C-18 hydrocarbon chain, and the increased lipophilicity has been determined towards the interaction with liposomes. The obtained derivative has been studied by means of density functional theory (DFT) methods in order to characterize its lowest energy conformers and predict its antioxidant properties with respect to the parent compound EdV. The in vitro antioxidant activity of C18-edaravone was studied by means of the α,α-diphenyl-β-picrylhydrazyl (DPPH) assay and in lipid peroxidation experiments performed on artificial lipid membranes using water-soluble as well as lipid-soluble radical initiators. Moreover, since oxidative stress is involved in numerous retinal degenerative diseases, the ability of C18-edaravone to contrast 2,2-azobis (2-amidinopropane hydrochloride) (AAPH)-induced cell death was assessed in adult retinal pigmented epithelium (ARPE-19) cells. Overall, the results demonstrated that the newly synthesized molecule has a high affinity for lipid membrane, increasing the efficacy of the unmodified edaravone under stress conditions.

Glyoxalase II (GlxII) is an antioxidant glutathione-dependent enzyme, which catalyzes the hydrolysis of S-D-lactoylglutathione to form D-lactic acid and glutathione (GSH). The last product is the most important thiol reducing agent present in all eukaryotic cells that have mitochondria and chloroplasts. It is generally known that GSH plays a crucial role not only in the cellular redox state but also in various cellular processes. One of them is protein S-glutathionylation, a process that can occur through an oxidation reaction of proteins’ thiol groups by GSH. Changes in protein S-glutathionylation have been associated with a range of human diseases such as diabetes, cardiovascular and pulmonary diseases, neurodegenerative diseases and cancer. Within a major project aimed at elucidating the role of GlxII in the mechanism of S-glutathionylation, a reliable computational protocol consisting of a protein–protein docking approach followed by atomistic Molecular Dynamics (MD) simulations was developed and it was applied to the prediction of molecular associations between human GlxII (in the presence and absence of GSH) and some proteins that are known to be S-glutathionylated in vitro, such as actin, malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The computational results show a high propensity of GlxII to interact with actin and MDH through its active site and a high stability of the GlxII–protein systems when GSH is present. Moreover, close proximities of GSH with actin and MDH cysteine residues have been found, suggesting that GlxII could be able to perform protein S-glutathionylation by using the GSH molecule present in its catalytic site.