ABSTRACT
While surgery is the definitive treatment for early-stage melanoma, the current therapies against advanced melanoma do not yet provide an effective, long-lasting control of the lesions and a satisfactory impact on patient survival. Thus, research is also focused on novel treatments that could potentiate the current therapies. In the present study, we evaluated the effect of potassium ascorbate with ribose (PAR) treatment on the human melanoma cell line, A375, in 2D and 3D models. In the 2D model, in line with the current literature, the pharmacological treatment with PAR decreased cell proliferation and viability. In addition, an increase in Connexin 43 mRNA and protein was observed. This novel finding was confirmed in PAR-treated melanoma cells cultured in 3D, where an increase in functional gap junctions and a higher spheroid compactness were observed. Moreover, in the 3D model, a remarkable decrease in the size and volume of spheroids was observed, further supporting the treatment efficacy observed in the 2D model. In conclusion, our results suggest that PAR could be used as a safe adjuvant approach in support to conventional therapies for the treatment of melanoma.
Subject(s)
Antineoplastic Agents/pharmacology , Ascorbic Acid/chemistry , Cell Proliferation/drug effects , Potassium/chemistry , Ribose/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Survival/drug effects , Connexin 43/genetics , Connexin 43/metabolism , Humans , Melanoma/drug therapy , Melanoma/pathology , Microscopy, Electron, Scanning , Spheroids, Cellular/drug effects , Spheroids, Cellular/ultrastructureABSTRACT
The structural features of the complexes that alpha-bungarotoxin forms with three different synthetic peptides, mimotopes of the nicotinic acetylcholine receptor binding site, have been compared to the corresponding nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) data. For the considered peptides, the observed different affinities towards the toxin could not be accounted simply by static structural considerations. A combined analysis of the SPR- and NMR-derived dynamic parameters shows new correlations between complex formation and dissociation and the overall pattern of intramolecular and intermolecular nuclear Overhauser effects. These features could be crucial for a rational design of protein ligands.
Subject(s)
Bungarotoxins/chemistry , Bungarotoxins/metabolism , Nuclear Magnetic Resonance, Biomolecular/methods , Peptides/chemistry , Peptides/metabolism , Surface Plasmon Resonance/methods , Amino Acid Sequence , Animals , Binding Sites , Models, Molecular , Molecular Mimicry , Protein Binding , Protein Conformation , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/metabolismABSTRACT
A combinatorial library approach was used to produce synthetic peptides mimicking the snake neurotoxin binding site of nicotinic receptors. Among the sequences, which inhibited binding of alpha-bungarotoxin to muscle and neuronal nicotinic receptors, HRYYESSLPWYPD, a 14-amino acid peptide with considerably higher toxin-binding affinity than the other synthesized peptides, was selected, and the structure of its complex with the toxin was analyzed by NMR. Comparison of the solution structure of the free toxin and its complex with this peptide indicated that complex formation induced extensive conformational rearrangements mainly at finger II and the carboxy terminus of the protein. The peptidyl residues P10 and Y4 seemed to be critical for peptide folding and complex stability, respectively. The latter residue of the peptide strongly interacted with the protein by entering a small pocket delimited by D30, C33, S34, R36, and V39 toxin side chains.
