RESUMO
Countless efforts have been made to prevent and suppress the formation and spread of melanoma. Natural astaxanthin (AST; extracted from the alga Haematococcus pluvialis) showed an antitumor effect on various cancer cell lines due to its interaction with the cell membrane. This study aimed to characterize the antitumor effect of AST against B16F10-Nex2 murine melanoma cells using cell viability assay and evaluate its mechanism of action using electron microscopy, western blotting analysis, terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, and mitochondrial membrane potential determination. Astaxanthin exhibited a significant cytotoxic effect in murine melanoma cells with features of apoptosis and autophagy. Astaxanthin also decreased cell migration and invasion in vitro assays at subtoxic concentrations. In addition, assays were conducted in metastatic cancer models in mice where AST significantly decreased the development of pulmonary nodules. In conclusion, AST has cytotoxic effect in melanoma cells and inhibits cell migration and invasion, indicating a promising use in cancer treatment.
Assuntos
Antineoplásicos , Melanoma Experimental , Camundongos , Animais , Linhagem Celular Tumoral , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Apoptose , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Autofagia , Proliferação de Células , Camundongos Endogâmicos C57BL , XantofilasRESUMO
BACKGROUND/AIM: The Brain-Specific Homeobox/POU Domain Protein 2 (BRN2) transcription factor supports melanoma progression by regulating the expression of several genes involved in cell migration and invasion. We hypothesized that a peptide designed based on the POU domain of BRN2 could block the BRN2 transcription activity and, consequently, reduce metastasis. MATERIALS AND METHODS: Cell viability was accessed by Trypan Blue exclusion dye assay and xCelligence platform. Wound-healing scratch assay and transwell invasion with matrigel membrane assay were performed to analyze cell migration and invasion. The internalization mechanism of the L13S peptide was investigated using confocal microscopy and wound-healing scratch assay. The impact of L13S on cell protein expression was analyzed through western blotting. In vivo assays were conducted to evaluate the protective effect and toxicity of L13S in a metastatic model using murine melanoma cells. RESULTS: Here, we show that the peptide named L13S can inhibit the migration and invasion of murine melanoma cells (B16F10-Nex2) as well as the migration of human melanoma cells (SK-MEL-25 and A375) by regulating the expression of proteins involved in motility. Mechanistically, we found that L13S is internalized by murine melanoma cells via macropinocytosis and binds actin filaments and nuclei. More importantly, in vivo studies indicated that the peptide was able to significantly inhibit lung metastasis in syngeneic models without off-target effects and with virtually no cytotoxicity toward normal organs. CONCLUSION: L13S peptide is a strong candidate for further development as an anticancer agent for the treatment of melanoma metastasis.
Assuntos
Antineoplásicos , Melanoma , Humanos , Camundongos , Animais , Melanoma/patologia , Antineoplásicos/farmacologia , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Movimento Celular , Linhagem Celular Tumoral , Proliferação de Células , Invasividade NeoplásicaRESUMO
Nanoparticles (NPs) are a promising strategy for delivering drugs to specific sites because of their tunable size and surface chemistry variety. Among the availablematerials, NPs prepared with biopolymers are of particular interest because of their biocompatibility and controlled release of encapsulated drugs. Poly lactic-co-glycolic acid (PLGA) is one of the most widely used biopolymers in biomedical applications. In addition to material choice modulation of the interaction between NPs and biological systems is essential for the safety and effective use of NPs. Therefore, this work focused on evaluating different surface functionalization strategies to promote cancer cell uptake and intracellular targeting of PLGA NPs. Herein, cell-penetrating peptides (CPPs) were shown to successfully drive PLGA NPs to the mitochondria and nuclei. Furthermore, the functionalization of PLGA NPs with peptide AC-1001 H3 (GQYGNLWFAY) was proven to be useful for targeting actin filaments. The PLGA NPs cell internalization mechanism by B16F10-Nex2 cells was identified as caveolae-mediated endocytosis, which could be inhibited by the presence of methyl-ß-cyclodextrin. Notably, when peptide C (CVNHPAFAC) was used to functionalize PLGA NPs, none of the tested inhibitors could avoid cell internalization of PLGA NPs. Therefore, we suggest this peptide as a promising surface modification agent for enhancing drug delivery to cancer cells. Finally, PLGA NPs showed slow release kinetics and low cytotoxic profile, which, combined with the surface functionalization strategies addressed in this study, highlight the potential of PLGA NPs as a drug delivery platform for improving cancer therapy.
Assuntos
Peptídeos Penetradores de Células , Nanopartículas , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Glicolatos , Glicóis , Ácido Láctico , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
Melanoma is a malignant proliferative disease originated in melanocytes, characterized by high metastatic activity and by the activation of oncogenes, such as B-RAF (40-60% of cases). Recent studies have shown that vemurafenib (a MAPK inhibitor) promoted disturbance of mitochondrial bioenergetics, although underlying mechanisms are not fully comprehended. Here we showed that MAPK inhibition by vemurafenib in B-RAFV600E-mutated human melanoma culminated in the inhibition of DRP1 phosphorylation, associated to a large mitochondrial network remodeling to the hyperfused phenotype, and increased oxidative phosphorylation capacity. Such alterations may be associated to melanoma resistance to vemurafenib, since the impairment of oxidative phosphorylation increased the vemurafenib cytotoxicity. These results point to the potential of mitochondrial dynamics as a targetable pathway in melanoma.
Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Vemurafenib/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Dinaminas/antagonistas & inibidores , Dinaminas/genética , Dinaminas/metabolismo , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanócitos/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Terapia de Alvo Molecular , Mutação , Fosforilação Oxidativa/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de SinaisRESUMO
Despite the positive results observed in vitro and in vivo, clinical trials with bioactive peptides are generally hampered by their fast degradation in the biological system. Two bioactive peptides, P20 (CSSRTMHHC) and the combined peptide C (CVNHPAFACGYGHTMYYHHYQHHL) have been identified as anticancer therapeutics. Combined peptide C consists of peptide C (CVNHPAFAC), a tumor-homing peptide, conjugated to the antiangiogenic peptide HTMYYHHYQHHL with a GYG. In this work, PLGA NPs with peptide C were applied as a dual-peptide carrier for application in cancer therapy. Peptide P20 was loaded into the NPs and combined peptide C was conjugated to the NPs surface. These NPs were evaluated as a therapeutic system to treat metastatic melanoma. In vivo assays showed that P20 encapsulation in PLGA NPs enhanced its antitumor activity. The inhibitory activity of P20-PLGANPs was similar to the activity of non-encapsulated P20 in a dose fivefold higher. The inhibitory activity was even higher when P20PLGA NPs were functionalized with combined peptide C. P20PLGAPepC NPs reduced in 28% the number of lung nodules in a syngeneic model of metastatic melanoma as compared to untreated animals. Additionally to the better tumor targeting and the in situ release of P20, it is expected that the therapeutic efficiency of the dual-peptide PLGA NPs was further enhanced by a synergistic effect between P20 and combined peptide C. Our encouraging results showed that by enabling the co-delivery of two peptides and promoting tumor targeting, PLGA NPs coupled with peptide C is a promising platform for peptide-based cancer therapy.
Assuntos
Antineoplásicos/química , Nanopartículas/química , Peptídeos/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Sinergismo Farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Transplante HomólogoRESUMO
The incidence of skin cancer has increased in recent decades, and melanoma is the most aggressive form with the lowest chance of successful treatment. Currently, drug design projects are in progress, but available treatments against metastatic melanoma have not significantly increased survival, and few patients are cured. Thus, new therapeutic agents should be developed as more effective therapeutic options for melanoma. High levels of the BRN2 transcription factor have been related to melanoma development. However, neither the three-dimensional (3D) structure of BRN2 protein nor its POU domain has been determined experimentally. Construction of the BRN2 3D structure, and the study of its interaction with its DNA target, are important strategies for increasing the structural and functional knowledge of this protein. Thus, the aim of this work was to study the interaction between BRN2 and MORE DNA through in silico methods. The full-length BRN2 3D structure was built using the PHYRE2 and Swiss-Model programs, and molecular dynamics of this protein in complex with MORE DNA was simulated for 20 ns by the NAMD program. The BRN2 model obtained includes helix and loop regions, and the BRN2 POU domain shares structural similarity with other members of the transcription factor family. No significant conformational change of this protein occurred during dynamics simulation. These analyses revealed BRN2 residues important for the specific interaction with nucleotide bases and with more than one DNA nucleotide. This study may contribute to the design of inhibitors against BRN2 or MORE DNA as molecular targets of melanoma skin cancer. Graphical Abstract Model of complete Brn2 protein in complex with MORE DNA after building through comparative modeling and refinement by molecular dynamics simulation.
Assuntos
DNA/química , Proteínas de Homeodomínio/química , Melanoma/fisiopatologia , Fatores do Domínio POU/química , Simulação por Computador , HumanosRESUMO
The isoprenoid metabolic pathway in protozoa of the Leishmania genus exhibits distinctive characteristics. These parasites, as well as other members of the Trypanosomatidae family, synthesize ergosterol, instead of cholesterol, as the main membrane sterol lipid. Leishmania has been shown to utilize leucine, instead of acetate as the main precursor for sterol biosynthesis. While mammalian dolichols are molecules containing 15-23 isoprene units, Leishmania amazonensis promastigotes synthesize dolichol of 11 and 12 units. In this paper, we show that the intracellular stages of L. amazonensis, amastigotes, synthesize mainly polyprenols of 9 isoprene units, instead of dolichol.