Effective Synergy of Sorafenib and Nutrient Shortage in Inducing Melanoma Cell Death through Energy Stress

Skin melanoma is one of the most aggressive and difficult-to-treat human malignancies, characterized by poor survival rates, thus requiring urgent novel therapeutic approaches. Although metabolic reprogramming has represented so far, a cancer hallmark, accumulating data indicate a high plasticity of cancer cells in modulating cellular metabolism to adapt to a heterogeneous and continuously changing microenvironment, suggesting a novel therapeutic approach for dietary manipulation in cancer therapy. To this aim, we exposed melanoma cells to combined nutrient-restriction/sorafenib. Results indicate that cell death was efficiently induced, with apoptosis representing the prominent feature. In contrast, autophagy was blocked in the final stage by this treatment, similarly to chloroquine, which also enhanced melanoma cell sensitization to combined treatment. Energy stress was evidenced by associated treatment with mitochondrial dysfunction and glycolysis impairment, suggesting metabolic stress determining melanoma cell death. A reduction of tumor growth after cycles of intermittent fasting together with sorafenib treatment was also observed in vivo, reinforcing that the nutrient shortage can potentiate anti-melanoma therapy. Our findings showed that the restriction of nutrients by intermittent fasting potentiates the effects of sorafenib due to the modulation of cellular metabolism, suggesting that it is possible to harness the energy of cancer cells for the treatment of melanoma

Structure-activity relationship of Trp-containing analogs of the antimicrobial peptide gomesin

Gomesin (Gm) has a broad antimicrobial activity making it of great interest for development of drugs. in this study, we analyzed three Gm analogs, [Trp(1)]-Gm, [Trp(7)]-Gm, and [Trp(9)]-Gm, in an attempt to gain insight into the contributions of different regions of the peptide sequence to its activity. the incorporation of the tryptophan residue in different positions has no effect on the antimicrobial and hemolytic activities of the Gm analogs in relation to Gm. Spectroscopic studies (circular dichroism, fluorescence and absorbance) of Gm and its analogs were performed in the presence of SDS, below and above its critical micelle concentration (CMC) (similar to 8mM), in order to monitor structural changes induced by the interaction with this anionic surfactant (0-15mM). Interestingly, we found that the analogs interact more strongly with SDS at low concentrations (0.3-6.0mM) than close to or above its CMC. This suggests that SDS monomers are able to cover the whole peptide, forming large detergent-peptide aggregates. On the other hand, the peptides interact differently with SDS micelles, inserting partially into the micelle core. Among the peptides, Trp in position 1 becomes more motionally-restricted in the presence of SDS, probably because this residue is located at the N-terminal region, which presents higher conformational freedom to interact stronger with SDS molecules. Trp residues in positions 7 and 9, close to and in the region of the turn of the molecule, respectively, induced a more constrained structure and the compounds cannot insert deeper into the micelle core or be completely buried by SDS monomers. Copyright (c) 2014 European Peptide Society and John Wiley & Sons, Ltd.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)INCT-FCxUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilUniv São Paulo, Inst Biomed Sci, Dept Parasitol, BR-05508900 São Paulo, BrazilUniv São Paulo, Sch Arts Sci & Humanities, BR-03828000 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

Substitution of some amino acid residues in the Gm structure reduces the cytotoxic ability, but did not modify the cell death mechanism.

<p>B16 cells were stimulated by Gm and analogues for 24 h. (<b>A</b>) Cytotoxic activities of Gm and its analogues were quantified by the MTT reduction test. (<b>B</b>) Cell death type identification caused by Gm was evaluated using annexin-V and 7-AAD assay by flow cytometry using the IC₅₀ values. (<b>C-H</b>) Apoptosis (Z-VAD) and necroptosis (necrostatin) inhibitors were unable to reduce cell death induced by Gm and analogues. Cells were incubated with inhibitors for 1 h before to stimulation with the peptides (IC₅₀ values for each peptide was used) and the viability was assessed by the MTT. Results are the means ± SEM of three independent experiments preformed in duplicate.</p

Primary structure of gomesin and its analogues.

a<p>Z  =  pyroglutamic acid, lowercase letters denote D-amino acids, X  =  Rh-Lys, Rh =  rhodamin; B  =  B₁₂-Lys, B₁₂ =  biotin. ^bThe observed m/z of the unresolved peak was compared with the calculated [M + H]⁺ average mass in Da.^cPercent purity as determined by HPLC analysis performed on a Waters Nova-Pak C₁₈ (2,1×150 mm, 60 Å, 3,5 µm); UV detection at 214 nm; 0.4 mL/min flow rate; [A]  =  0.1% TFA in H₂O and [B]  =  0.1% TFA in 60% MeCN/H2O; gradient  =  5–95%B in 30 min.</p

External or internal localization of Gm and analogues.

<p>B16 cells were incubated with 2 µM of the biotin labeled peptides for different times. Images correspond to a single focal plane performed by confocal microscopy. External membrane peptides were labeled with streptavidin-Alexa Fluor 488 conjugated while internal membrane peptides were labeled with streptavidin-Alexa Fluor 647 conjugated. Unviable cells were excluded using PI stain. Nuclei were stain with DAPI. (<b>A</b>) Typical image of labeled Gm is showed. The images XY, XZ and YZ correspond to a single plane. Typical images obtained with (<b>B</b>) Gm, (<b>C</b>) [D-Thr^2,6,11,15]-D-Gm, (<b>D</b>) [D-Thr^2,6,11,15, Pro⁹]-D-Gm, (<b>E</b>) [Thr^2,6,11,15, D-Pro⁹]-Gm, (<b>F</b>) [Ser^2,6,11,15]-Gm and (<b>G</b>) [Thr^2,6,11,15]-Gm are shown.</p

Potencies and efficacies of gomesin and its analogues.

*<p>To quantify internal or external amount fluorescence biotinylated peptides were used.</p><p># Starting peptide concentration (10⁻⁴ M) was considered as 100%.</p

Lysosomal enzymes participate in the cytotoxic reduction activities of the Gm analogues.

<p>(<b>A, C and D</b>) Cell viability was assessed by the MTT reduction test. (<b>A</b>) Cells were incubated with cytoskeletal inhibitor (cytochalasin D), and lysosomal inhibitor (chloroquine) for 1 h before to stimulation by Gm, and its analogues for 24 h. Chloroquine potentiated the cytotoxicity of peptides. (<b>B</b>) Since chloroquine was able to potentiate the cytotoxicity of the peptides, resistance to degradation of lysosomal enzymes was evaluated by LC/ESI-MS. Peptides were incubated at 37°C for different times. (<b>C</b>) Cells were incubated with the IC₅₀ concentration for each peptide. Cytotoxic activities after 24 and 72 h of the peptide incubation were compared.</p