RESUMEN
Membrane active peptides are known to porate lipid bilayers, but their exact permeabilization mechanism and the structure of the nanoaggregates they form in membranes have often been difficult to determine experimentally. For many sequences at lower peptide concentrations, transient leakage is observed in experiments, suggesting the existence of transient pores. For two well-know peptides, alamethicin and melittin, we show here that molecular mechanics simulations i) can directly distinguish equilibrium poration and non-equilibrium transient leakage processes, and ii) can be used to observe the detailed pore structures and mechanism of permeabilization in both cases. Our results are in very high agreement with numerous experimental evidence for these two peptides. This suggests that molecular simulations can capture key membrane poration phenomena directly and in the future may develop to be a useful tool that can assist experimental peptide design.
Asunto(s)
Membrana Dobles de Lípidos , Meliteno , Simulación de Dinámica Molecular , Meliteno/química , Meliteno/metabolismo , Membrana Dobles de Lípidos/metabolismo , Membrana Dobles de Lípidos/química , Alameticina/química , Alameticina/metabolismo , Permeabilidad de la Membrana Celular , PermeabilidadRESUMEN
In previous studies, we developed a novel fusion protein named "melittin-MIL-2" which exhibited more anti-tumor activity. However, it remains unclear whether melittin-MIL-2 possesses antitumor immune effect on lung adenocarcinoma. In this study, the immune effect and mechanism of melittin-MIL-2 inhibiting the growth and invasion of lung adenocarcinoma will be investigated, in order to provide novel perspectives for the immunotherapy of lung cancer. The results indicated that melittin-MIL-2 promoted T cell proliferation, enhanced NK cell cytotoxicity, and boosted IFN-γ secretion in PBMCs. After melittin-MIL-2 stimulation, perforin expression and LAK/NK-like killing activities of human PBMCs and NK cells were significantly enhanced. Melittin-MIL-2 is capable of hampering the development and proliferation of lung adenocarcinoma cell A549. ICAM-1 and Fas expression in A549 cells exposed to melittin-MIL-2 rose significantly. The expression levels of TLR8 and VEGF in A549 cells decreased significantly after melittin-MIL-2 stimulation. In vivo, melittin-MIL-2 substantially impeded the growth of lung adenocarcinoma and formed an immune-stimulating microenvironment locally in tumor tissues. In conclusion, the novel fusion protein melittin-MIL-2 exhibits strong anti-tumor immune effect in lung adenocarcinoma cell A549 via activating the LFA-1/ICAM-1 and Fas/FasL pathways to enhance cytolytic activity, upregulating the secretion of IFN-γ and perforin, and boosting LAK/NK-like killing activities. Immuno-effector cells and their secreted cytokines can form immune stimulation microenvironment locally in lung adenocarcinoma Lewis mice tissue.
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Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Meliteno , Meliteno/farmacología , Humanos , Animales , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Ratones , Células A549 , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/inmunología , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/genética , Proliferación Celular/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/efectos de los fármacos , Interleucina-2/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/genética , Inmunoterapia/métodosRESUMEN
CONTEXT: Molecularly imprinted polymers (MIPs) have promising applications as synthetic antibodies for protein and peptide recognition. A critical aspect of MIP design is the selection of functional monomers and their adequate proportions to achieve materials with high recognition capacity toward their targets. To contribute to this goal, we calibrated a molecular dynamics protocol to reproduce the experimental trends in peptide recognition of 13 pre-polymerization mixtures reported in the literature for the peptide toxin melittin. METHODS: Three simulation conditions were tested for each mixture by changing the box size and the number of monomers and cross-linkers surrounding the template in a solvent-explicit environment. Fully atomistic MD simulations of 350 ns were conducted with the AMBER20 software, with ff19SB parameters for the peptide, gaff2 parameters for the monomers and cross-linkers, and the OPC water model. Template-monomer interaction energies under the LIE approach showed significant differences between high-affinity and low-affinity mixtures. Simulation systems containing 100 monomers plus cross-linkers in a cubic box of 90 Å3 successfully ranked the mixtures according to their experimental performance. Systems with higher monomer densities resulted in non-specific intermolecular contacts that could not account for the experimental trends in melittin recognition. The mixture with the best recognition capacity showed preferential binding to the 13-26-α-helix, suggesting a relevant role for this segment in melittin imprinting and recognition. Our findings provide insightful information to assist the computational design of molecularly imprinted materials with a validated protocol that can be easily extended to other templates.
Asunto(s)
Simulación de Dinámica Molecular , Péptidos , Péptidos/química , Meliteno/química , Polimerizacion , Polímeros Impresos Molecularmente/química , Impresión Molecular/métodosRESUMEN
Rheumatoid arthritis (RA) involves chronic joint inflammation. Combining acupuncture and medication for RA treatment faces challenges like spatiotemporal variability, limited drug loading in acupuncture needles, and premature or untargeted drug release. Here, we designed a new type of tubular acupuncture needles, with an etched hollow honeycomb-like structure to enable the high loading of therapeutics, integrating the traditional acupuncture and drug repository into an all-in-one therapeutic platform. In these proof-of-concept experiments, we fabricated injectable hollow honeycomb electroacupuncture needles (HC-EA) loaded with melittin hydrogel (MLT-Gel), enabling the combination treatment of acupuncture stimulation and melittin therapy in a spatiotemporally synchronous manner. Since the RA microenvironment is mildly acidic, the acid-responsive chitosan (CS)/sodium beta-glycerophosphate (ß-GP)/ hyaluronic acid (HA) composited hydrogel (CS/GP/HA) was utilized to perform acupuncture stimulation and achieve the targeted release of injected therapeutics into the specific lesion site. Testing our therapeutic platform involved a mouse model of RA and bioinformatics analysis. MLT-Gel@HC-EA treatment restored Th17/Treg-mediated immunity balance, reduced inflammatory factor release (TNF-α, IL-6, IL-1ß), and alleviated inflammation at the lesion site. This novel combination of modified acupuncture needle and medication, specifically melittin hydrogel, holds promise as a therapeutic strategy for RA treatment.
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Terapia por Acupuntura , Artritis Reumatoide , Hidrogeles , Meliteno , Agujas , Animales , Meliteno/farmacología , Meliteno/química , Ratones , Artritis Reumatoide/terapia , Artritis Reumatoide/tratamiento farmacológico , Hidrogeles/química , Terapia por Acupuntura/métodos , Quitosano/química , Ácido Hialurónico/química , Masculino , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
HCC is the most common fatal malignancy. Although surgical resection is the primary treatment strategy, most patients are not eligible for resection due to tumor heterogeneity, underlying liver disease, or comorbidities. Therefore, this study explores the possibility of multi-molecular targeted drug delivery in treating HCC. In this study, we constructed the recombinant adenovirus co-expressing apoptin and melittin (MEL) genes. The inhibitory effect of the recombinant adenovirus on hepatocellular carcinoma cells was detected through experiments on cell apoptosis, migration, invasion, and other factors. The tumor inhibitory effect in vivo was assessed using subcutaneous HCC mice. Results showed that recombinant adenovirus co-expressing anti-tumor genes TAT and apoptin, RGD and MEL can significantly inhibit the proliferation, migration, and invasion of HCC cells by inducing an increase in reactive oxygen species (ROS) levels, upregulation of apoptotic proteins such as Bax, cleaved caspase-3, and cleaved caspase-9, and downregulation of the anti-apoptotic protein Bcl-2. In subcutaneous HCC mice, recombinant adenovirus induced significant apoptosis in tumor, and inhibited tumor growth. In conclusion, recombinant adenovirus co-expressing apoptin and MEL can inhibit the growth and proliferation of tumor cells both in vivo and in vitro.
Asunto(s)
Adenoviridae , Apoptosis , Proteínas de la Cápside , Carcinoma Hepatocelular , Proliferación Celular , Neoplasias Hepáticas , Meliteno , Meliteno/farmacología , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Adenoviridae/genética , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/genética , Humanos , Proteínas de la Cápside/genética , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ratones , Movimiento Celular/efectos de los fármacos , Ratones Desnudos , Ratones Endogámicos BALB C , Línea Celular Tumoral , Especies Reactivas de Oxígeno/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Terapia Genética/métodosRESUMEN
Leucine residues are commonly found in the hydrophobic face of antimicrobial peptides (AMPs) and are crucial for membrane permeabilization, leading to the cell death of invading pathogens. Melittin, which contains four leucine residues, demonstrates broad-spectrum antimicrobial properties but also significant cytotoxicity against mammalian cells. To enhance the cell selectivity of melittin, this study synthesized five analogs by replacing leucine with its structural isomer, 6-aminohexanoic acid. Among these analogs, Mel-LX3 exhibited potent antibacterial activity against both Gram-positive and Gram-negative bacteria. Importantly, Mel-LX3 displayed significantly reduced hemolytic and cytotoxic effects compared to melittin. Mechanistic studies, including membrane depolarization, SYTOX green uptake, FACScan analysis, and inner/outer membrane permeation assays, demonstrated that Mel-LX3 effectively permeabilized bacterial membranes similar to melittin. Notably, Mel-LX3 showed robust antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Furthermore, Mel-LX3 effectively inhibited biofilm formation and eradicated existing biofilms of MDRPA. With its improved selective antimicrobial and antibiofilm activities, Mel-LX3 emerges as a promising candidate for the development of novel antimicrobial agents. We propose that the substitution of leucine with 6-aminohexanoic acid in AMPs represents a significant strategy for combating resistant bacteria.
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Antibacterianos , Biopelículas , Meliteno , Staphylococcus aureus Resistente a Meticilina , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa , Meliteno/farmacología , Meliteno/química , Biopelículas/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Humanos , Hemólisis/efectos de los fármacos , Ácido Aminocaproico/química , Ácido Aminocaproico/farmacología , Bacterias Gramnegativas/efectos de los fármacos , AnimalesRESUMEN
We have evolved the nanopore-forming macrolittin peptides from the bee venom peptide melittin using successive generations of synthetic molecular evolution. Despite their sequence similarity to the broadly membrane permeabilizing cytolytic melittin, the macrolittins have potent membrane selectivity. They form nanopores in synthetic bilayers made from 1-palmitoyl, 2-oleoyl-phosphatidylcholine (POPC) at extremely low peptide concentrations and yet have essentially no cytolytic activity against any cell membrane, even at high concentration. Here, we explore the structural determinants of macrolittin nanopore stability in POPC bilayers using atomistic molecular dynamics simulations and experiments on macrolittins and single-site variants. Simulations of macrolittin nanopores in POPC bilayers show that they are stabilized by an extensive, cooperative hydrogen bond network comprised of the many charged and polar side chains interacting with each other via bridges of water molecules and lipid headgroups. Lipid molecules with unusual conformations participate in the H-bond network and are an integral part of the nanopore structure. To explore the role of this H-bond network on membrane selectivity, we swapped three critical polar residues with the nonpolar residues found in melittin. All variants have potency, membrane selectivity, and cytotoxicity that were intermediate between a cytotoxic melittin variant called MelP5 and the macrolittins. Simulations showed that the variants had less organized H-bond networks of waters and lipids with unusual structures. The membrane-spanning, cooperative H-bond network is a critical determinant of macrolittin nanopore stability and membrane selectivity. The results described here will help guide the future design and optimization of peptide nanopore-based applications.
Asunto(s)
Meliteno , Simulación de Dinámica Molecular , Nanoporos , Fosfatidilcolinas , Meliteno/química , Fosfatidilcolinas/química , Membrana Dobles de Lípidos/química , Enlace de Hidrógeno , Péptidos/química , HumanosRESUMEN
BACKGROUND: NSCLC is one of the most common causes of death. The hypoxia microenvironment contributes to cancer progression. The purpose was to explore the effects and mechanism of melittin on NSCLC cells in the hypoxic microenvironment. METHODS: NSCLC cell lines (A549 and H1299) were cultured in normoxia or hypoxia conditions with or without melittin treatment. The viability of the cells was detected via MTT assay and the proliferation ability was evaluated by EdU assay. QRT-PCR was performed to evaluate GLUT1, LDHA, HK2, VEGF and LATS2 mRNA levels. Glucose transport was assessed by the 2-NBDG uptake assay. The angiogenesis was determined by the tubule formation assay. The protein expressions of GLUT1, LDHA, HK2, VEGF, LATS2, YAP, p-YAP and HIF-1α were detected via western blotting assay. The tumor formation assay was conducted to examine the roles of melittin and LATS2 in vivo. RESULTS: Melittin inhibited hypoxia-induced cell viability, proliferation, glycolysis and angiogenesis as well as suppressed YAP binding to HIF-1α in NSCLC. Melittin inactivated the YAP/HIF-1α pathway via up-regulation of LATS2, ultimately inhibiting cancer progression of NSCLC. Moreover, melittin suppressed tumor growth via up-regulation of LATS2 in vivo. CONCLUSION: Melittin inactivated the YAP/HIF-1α pathway via up-regulation of LATS2 to contribute to the development of NSCLC. Therefore, melittin is expected to become a potential prognostic drug for the therapy of NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Proliferación Celular , Glucólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Neoplasias Pulmonares , Meliteno , Neovascularización Patológica , Proteínas Serina-Treonina Quinasas , Proteínas Supresoras de Tumor , Regulación hacia Arriba , Proteínas Señalizadoras YAP , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/irrigación sanguínea , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Glucólisis/efectos de los fármacos , Proteínas Supresoras de Tumor/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas Señalizadoras YAP/metabolismo , Meliteno/farmacología , Meliteno/uso terapéutico , Línea Celular Tumoral , Factores de Transcripción/metabolismo , Animales , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transducción de Señal/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fosfoproteínas/metabolismo , AngiogénesisRESUMEN
This research aimed to explore the healing impacts of Melittin treatment on gastrocnemius muscle wasting caused by immobilization with a cast in rabbits. Twenty-four rabbits were randomly allocated to four groups. The procedures included different injections: 0.2 mL of normal saline to Group 1 (G1-NS); 4 µg/kg of Melittin to Group 2 (G2-4 µg/kg Melittin); 20 µg/kg of Melittin to Group 3 (G3-20 µg/kg Melittin); and 100 µg/kg of Melittin to Group 4 (G4-100 µg/kg Melittin). Ultrasound was used to guide the injections into the rabbits' atrophied calf muscles following two weeks of immobilization via casting. Clinical measurements, including the length of the calf, the compound muscle action potential (CMAP) of the tibial nerve, and the gastrocnemius muscle thickness, were assessed. Additionally, cross-sectional slices of gastrocnemius muscle fibers were examined, and immunohistochemistry and Western blot analyses were performed following two weeks of therapy. The mean regenerative changes, as indicated by clinical parameters, in Group 4 were significantly more pronounced than in the other groups (p < 0.05). Furthermore, the cross-sectional area of the gastrocnemius muscle fibers and immunohistochemical indicators in Group 4 exceeded those in the remaining groups (p < 0.05). Western blot analysis also showed a more significant presence of anti-inflammatory and angiogenic cytokines in Group 4 compared to the others (p < 0.05). Melittin therapy at a higher dosage can more efficiently activate regeneration in atrophied gastrocnemius muscle compared to lower doses of Melittin or normal saline.
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Meliteno , Músculo Esquelético , Atrofia Muscular , Regeneración , Animales , Conejos , Meliteno/farmacología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Regeneración/efectos de los fármacos , Atrofia Muscular/tratamiento farmacológico , Atrofia Muscular/metabolismo , Atrofia Muscular/etiología , Atrofia Muscular/patología , MasculinoAsunto(s)
Carcinoma de Células Escamosas , Meliteno , Neoplasias de la Boca , Meliteno/uso terapéutico , Meliteno/farmacología , Humanos , Neoplasias de la Boca/terapia , Neoplasias de la Boca/tratamiento farmacológico , Carcinoma de Células Escamosas/terapia , Carcinoma de Células Escamosas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Bacteriófagos , RatonesRESUMEN
Despite exhibiting potent anticancer activity, the strong hemolytic properties of melittin (MEL) significantly restrict its delivery efficiency and clinical applications. To address this issue, we have devised a strategy wherein homologous dopamine (DA), an essential component of bee venom, is harnessed as a vehicle for the synthesis of MEL-polydopamine (PDA) nanoparticles (MP NPs). The ingenious approach lies in the fact that MEL is a basic polypeptide, and the polymerization of DA is also conducted under alkaline conditions, indicating the distinctive advantages of PDA in MEL encapsulation. Furthermore, MP NPs are modified with folic acid to fabricate tumor-targeted nanomedicine (MPF NPs). MPF NPs can ameliorate the hemolysis of MEL in drug delivery and undergo degradation triggered by high levels of reactive oxygen species (ROS) within solid tumors, thereby facilitating MEL release and subsequent restoration of anticancer activity. After cellular uptake, MPF NPs induce cell apoptosis through the PI3K/Akt-mediated p53 signaling pathway. The tumor growth inhibitory rate of MPF NPs in FA receptor-positive 4T1 and CT26 xenograft mice reached 78.04% and 81.66%, which was significantly higher compared to that in FA receptor-negative HepG2 xenograft mice (45.79%). Homologous vehicles provide a new perspective for nanomedicine design.
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Antineoplásicos , Hemólisis , Indoles , Meliteno , Polímeros , Meliteno/química , Meliteno/farmacología , Animales , Humanos , Indoles/química , Indoles/farmacología , Polímeros/química , Polímeros/farmacología , Antineoplásicos/farmacología , Antineoplásicos/química , Ratones , Hemólisis/efectos de los fármacos , Nanopartículas/química , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ratones Endogámicos BALB C , Ensayos de Selección de Medicamentos Antitumorales , Ratones Desnudos , Tamaño de la PartículaRESUMEN
Melittin (Mel) is considered a promising candidate drug for the treatment of triple negative breast cancer (TNBC) due to its various antitumor effects. However, its clinical application is hampered by notable limitations, including hemolytic activity, rapid clearance, and a lack of tumor selectivity. Here, we designed novel biomimetic nanoparticles based on homologous tumor cell membranes and poly(lactic-co-glycolic acid) (PLGA)/poly(beta-aminoester) (PBAE), denoted MDM@TPP, which efficiently coloaded the cytolytic peptide Mel and the photosensitizer mTHPC. Both in vitro and in vivo, the MDM@TPP nanoparticles effectively mitigated the acute toxicity of melittin and exhibited strong TNBC targeting ability due to the homologous targeting effect of the tumor cell membrane. Under laser irradiation, the MDM@TPP nanoparticles showed excellent photodynamic performance and thus accelerated the release of Mel by disrupting cell membrane integrity. Moreover, Mel combined with photodynamic therapy (PDT) can synergistically kill tumor cells and induce significant immunogenic cell death, thereby stimulating the maturation of dendritic cells (DCs). In 4T1 tumor-bearing mice, MDM@TPP nanoparticles effectively inhibited the growth and metastasis of primary tumors and finally prevented tumor recurrence by improving the immune response.
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Meliteno , Nanopartículas , Fotoquimioterapia , Fármacos Fotosensibilizantes , Neoplasias de la Mama Triple Negativas , Meliteno/química , Meliteno/farmacología , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Nanopartículas/química , Animales , Ratones , Femenino , Humanos , Antineoplásicos/química , Antineoplásicos/farmacología , Ratones Endogámicos BALB C , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayos de Selección de Medicamentos AntitumoralesRESUMEN
BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease lacking a definitive cure. Although conventional treatments such as dexamethasone and methotrexate are prevalent, their usage is constrained by potential adverse effects. Melittin (MLT) has emerged as a promising natural anti-rheumatic drug; however, studies focusing on the role of MLT in modulating the expression and metabolism of RA-related genes are scarce. METHOD: Arthritis was induced in rats using Complete Freund's Adjuvant (CFA), followed by MLT injections for treatment. Post-treatment, the inflammatory status of each group was assessed, and the mechanistic underpinnings of MLT's ameliorative effects on RA were elucidated through transcriptomic and metabolomic analyses. Additionally, this study conducted qRT-PCR validation of key therapeutic genes and characterized the molecular docking interactions of MLT with key receptor proteins (TNF-α and IL-1ß) using the AutoDock Vina software. RESULT: MLT significantly diminished redness and swelling in affected joints, ameliorated inflammatory cell infiltration, and mitigated joint damage. Integration of transcriptomic and metabolomic data revealed that MLT predominantly regulated the transcription levels of pathways and genes related to cytokines and immune responses, and the metabolic biomarkers of Sphingomyelin, fatty acid, and flavonoid. qRT-PCR confirmed MLT's downregulation of inflammation-related genes such as Il6, Jak2, Stat3, and Ptx3. Molecular docking simulations demonstrated the stable binding of MLT to TNF-α and IL-1ß. CONCLUSION: MLT demonstrated significant efficacy in alleviating RA. This study provides a comprehensive summary of MLT's impact on gene expression and metabolic processes associated with RA.
Asunto(s)
Artritis Experimental , Artritis Reumatoide , Meliteno , Metaboloma , Simulación del Acoplamiento Molecular , Transcriptoma , Animales , Ratas , Transcriptoma/efectos de los fármacos , Meliteno/farmacología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Artritis Reumatoide/inducido químicamente , Metaboloma/efectos de los fármacos , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/metabolismo , Artritis Experimental/inducido químicamente , Artritis Experimental/genética , Adyuvante de Freund , Masculino , Regulación de la Expresión Génica/efectos de los fármacos , Perfilación de la Expresión GénicaRESUMEN
Melittin is a powerful toxin present in honeybee venom that is active in a wide range of animals, from insects to humans. Melittin exerts numerous biological, toxicological, and pharmacological effects, the most important of which is destruction of the cell membrane. The phospholipase activity of melittin and its ability to activate phospholipases in the venom contribute to these actions. Using analytical methods, we discovered that the honeybee Apis mellifera produces melittin not only in the venom gland but also in its fat body cells, which remain resistant to this toxin's effects. We suggest that melittin acts as an anti-bacterial agent, since its gene expression is significantly upregulated when honeybees are infected with Escherichia coli and Listeria monocytogenes bacteria; additionally, melittin effectively kills these bacteria in the disc diffusion test. We hypothesize that the chemical and physicochemical properties of the melittin molecule (hydrophilicity, lipophilicity, and capacity to form tetramers) in combination with reactive conditions (melittin concentration, salt concentration, pH, and temperature) are responsible for the targeted destruction of bacterial cells and apparent tolerance towards own tissue cells. Considering that melittin is an important current and, importantly, potential broad-spectrum medication, a thorough understanding of the observed phenomena may significantly increase its use in clinical practice.
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Antibacterianos , Venenos de Abeja , Escherichia coli , Cuerpo Adiposo , Meliteno , Animales , Antibacterianos/farmacología , Antibacterianos/toxicidad , Venenos de Abeja/farmacología , Venenos de Abeja/toxicidad , Abejas , Escherichia coli/efectos de los fármacos , Cuerpo Adiposo/metabolismo , Proteínas de Insectos/metabolismo , Listeria monocytogenes/efectos de los fármacos , Meliteno/farmacología , Meliteno/toxicidadRESUMEN
BACKGROUND: Development of promising medicines from natural sources, specially venom, is of highly necessitated to combat against life-threatening cancers. Non-small cell lung cancer (NSCLC) has a significant percentage of mortalities. Melittin, from bee venom, is a potent anticancer peptide but its toxicity has limited its therapeutic applications. Accordingly, this study aims to synthesize niosomes with suitable stability and capacity for carrying melittin as a drug. Additionally, it seeks to evaluate the anti-cancer activity of melittin-loaded niosomes on non-small cell lung cancer. METHODS: The niosome was prepared by thin film hydration method. Cytotoxicity and apoptosis were assessed on A549, Calu-3, and MRC5 cells. Real-time PCR was used to determine expression of apoptotic and pro-apoptotic Bax, Bcl2, and Casp3 genes. Immunocytochemistry (ICC) was also used to confirm expression of the abovementioned genes. Furthermore, wound healing assay was performed to compare inhibition effects of melittin-loaded niosomes with free melittin on migration of cancer cells. RESULTS: IC50 values of melittin-loaded niosomes for A549, Calu-3, and MRC5 cells were respectively 0.69 µg/mL, 1.02 µg/mL, and 2.56 µg/mL after 72 h. Expression level of Bax and Casp3 increased '10 and 8' and '9 and 10.5' fold in A549 and Calu-3, whereas Bcl2 gene expression decreased 0.19 and 0.18 fold in the mentioned cell lines. The cell migration inhibited by melittin-loaded niosomes. CONCLUSIONS: Melittin-loaded niosomes had more anti-cancer effects and less toxicity on normal cells than free melittin. Furthermore, it induced apoptosis and inhibited cancer cells migration. Our results showed that melittin-loaded niosomes may be a drug lead and it has the potential to be future developed for lung cancer treatment.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Meliteno/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Liposomas , Caspasa 3 , Proteína X Asociada a bcl-2/genética , Neoplasias Pulmonares/tratamiento farmacológicoRESUMEN
Enterotoxins are a type of toxins that primarily affect the intestines. Understanding their harmful effects is essential for food safety and medical research. Current methods lack high-throughput, robust, and translatable models capable of characterizing toxin-specific epithelial damage. Pressing concerns regarding enterotoxin contamination of foods and emerging interest in clinical applications of enterotoxins emphasize the need for new platforms. Here, we demonstrate how Caco-2 tubules can be used to study the effect of enterotoxins on the human intestinal epithelium, reflecting toxins' distinct pathogenic mechanisms. After exposure of the model to toxins nigericin, ochratoxin A, patulin and melittin, we observed dose-dependent reductions in barrier permeability as measured by TEER, which were detected with higher sensitivity than previous studies using conventional models. Combination of LDH release assays and DRAQ7 staining allowed comprehensive evaluation of toxin cytotoxicity, which was only observed after exposure to melittin and ochratoxin A. Furthermore, the study of actin cytoskeleton allowed to assess toxin-induced changes in cell morphology, which were only caused by nigericin. Altogether, our study highlights the potential of our Caco-2 tubular model in becoming a multi-parametric and high-throughput tool to bridge the gap between current enterotoxin research and translatable in vivo models of the human intestinal epithelium.
Asunto(s)
Toxinas Bacterianas , Enterotoxinas , Humanos , Enterotoxinas/toxicidad , Toxinas Bacterianas/toxicidad , Células CACO-2 , Meliteno/farmacología , Nigericina/farmacología , Mucosa Intestinal/patologíaRESUMEN
Among the various natural compounds used in alternative and Oriental medicine, toxins isolated from different organisms have had their application for many years, and Apis mellifera venom has been studied the most extensively. Numerous studies dealing with the positive assets of bee venom (BV) indicated its beneficial properties. The usage of bee products to prevent the occurrence of diseases and for their treatment is often referred to as apitherapy and is based mainly on the experience of the traditional system of medical practice in diverse ethnic communities. Today, a large number of studies are focused on the antitumor effects of BV, which are mainly attributed to its basic polypeptide melittin (MEL). Previous studies have indicated that BV and its major constituent MEL cause a strong toxic effect on different cancer cells, such as liver, lung, bladder, kidney, prostate, breast, and leukemia cells, while a less pronounced effect was observed in normal non-target cells. Their proposed mechanisms of action, such as the effect on proliferation and growth inhibition, cell cycle alterations, and induction of cell death through several cancer cell death mechanisms, are associated with the activation of phospholipase A2 (PLA2), caspases, and matrix metalloproteinases that destroy cancer cells. Numerous cellular effects of BV and MEL need to be elucidated on the molecular level, while the key issue has to do with the trigger of the apoptotic cascade. Apoptosis could be either a consequence of the plasmatic membrane fenestration or the result of the direct interaction of the BV components with pro-apoptotic and anti-apoptotic factors. The interaction of BV peptides and enzymes with the plasma membrane is a crucial step in the whole process. However, before its possible application as a remedy, it is crucial to identify the correct route of exposure and dosage of BV and MEL for potential therapeutic use as well as potential side effects on normal cells and tissues to avoid any possible adverse event.
Asunto(s)
Venenos de Abeja , Masculino , Animales , Abejas , Meliteno , Membrana Celular , Apoptosis , Muerte CelularRESUMEN
Background: The potential anticancer effect of melittin has motivated scientists to find its exact molecular mechanism of action. There are few data on the effect of melittin on the UPR and autophagy as two critical pathways involved in tumorigenesis of colorectal and drug resistance. This study aimed to investigate the effect of melittin on these pathways in the colorectal cancer (CRC) HCT116 cells. Methods: MTT method was carried out to assess the cytotoxicity of melittin on the HCT116 cell line for 24, 48, and 72 h. After selecting the optimal concentrations and treatment times, the gene expression of autophagy flux markers (LC3-ßII and P62) and UPR markers (CHOP and XBP-1s) were determined using qRT-PCR. The protein level of autophagy initiation marker (Beclin1) was also determined by Western blotting. Results: MTT assay showed a cytotoxic effect of melittin on the HCT116 cells. The increase in LC3-ßII and decrease in P62 mRNA expression levels, along with the elevation in the Beclin1 protein level, indicated the stimulatory role of melittin on the autophagy. Melittin also significantly enhanced the CHOP and XBP-1s expressions at mRNA level, suggesting the positive role of the melittin on the UPR activation. Conclusion: This study shows that UPR and autophagy can potentially be considered as two key signaling pathways in tumorigenesis, which can be targeted by the BV melittin in the HCT116 cells. Further in vivo evaluations are recommended to verify the obtained results.
Asunto(s)
Neoplasias Colorrectales , Meliteno , Humanos , Células HCT116 , Meliteno/farmacología , Meliteno/genética , Meliteno/metabolismo , Beclina-1/genética , Beclina-1/metabolismo , Respuesta de Proteína Desplegada , Autofagia , ARN Mensajero/metabolismo , Carcinogénesis , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genéticaRESUMEN
Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer for which effective therapies are lacking. Targeted remodeling of the immunosuppressive tumor microenvironment (TME) and activation of the body's immune system to fight tumors with well-designed nanoparticles have emerged as pivotal breakthroughs in tumor treatment. To simultaneously remodel the immunosuppressive TME and trigger immune responses, we designed two potential therapeutic nanodelivery systems to inhibit TNBC. First, the bromodomain-containing protein 4 (BRD4) inhibitor JQ1 and the cyclooxygenase-2 (COX-2) inhibitor celecoxib (CXB) were coloaded into chondroitin sulfate (CS) to obtain CS@JQ1/CXB nanoparticles (NPs). Then, the biomimetic nanosystem MM@P3 was prepared by coating branched polymer poly(ß-amino ester) self-assembled NPs with melittin embedded macrophage membranes (MM). Both in vitro and in vivo, the CS@JQ1/CXB and MM@P3 NPs showed excellent immune activation efficiencies. Combination treatment exhibited synergistic cytotoxicity, antimigration ability, and apoptosis-inducing and immune activation effects on TNBC cells and effectively suppressed tumor growth and metastasis in TNBC tumor-bearing mice by activating the tumor immune response and inhibiting angiogenesis. In summary, this study offers a novel combinatorial immunotherapeutic strategy for the clinical TNBC treatment.
Asunto(s)
Azepinas , Celecoxib , Triazoles , Neoplasias de la Mama Triple Negativas , Microambiente Tumoral , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patología , Microambiente Tumoral/efectos de los fármacos , Animales , Femenino , Ratones , Humanos , Celecoxib/administración & dosificación , Línea Celular Tumoral , Sulfatos de Condroitina/química , Sulfatos de Condroitina/administración & dosificación , Nanopartículas/química , Nanopartículas/administración & dosificación , Meliteno/administración & dosificación , Meliteno/química , Apoptosis/efectos de los fármacos , Sistema de Administración de Fármacos con Nanopartículas/química , Ensayos Antitumor por Modelo de Xenoinjerto , Ratones Endogámicos BALB C , Inhibidores de la Ciclooxigenasa 2/administración & dosificación , Inhibidores de la Ciclooxigenasa 2/farmacología , Inhibidores de la Ciclooxigenasa 2/uso terapéutico , Polímeros/química , Ratones Desnudos , Sistemas de Liberación de Medicamentos/métodosRESUMEN
The primary constituents of honeybee venom, melittin and phospholipase A2 (PLA2), display toxin synergism in which the PLA2 activity is significantly enhanced by the presence of melittin. It has been shown previously that this is accomplished by the disruption in lipid packing, which allows PLA2 to become processive on the membrane surface. In this work, we show that melittin is capable of driving miscibility phase transition in giant unilamellar vesicles (GUVs) and that it raises the miscibility transition temperature (Tmisc) in a concentration-dependent manner. The induced phase separation enhances the processivity of PLA2, particularly at its boundaries, where a substantial difference in domain thickness creates a membrane discontinuity. The catalytic action of PLA2, in response, induces changes in the membrane, rendering it more conducive to melittin binding. This, in turn, facilitates further lipid phase separation and eventual vesicle lysis. Overall, our results show that melittin has powerful membrane-altering capabilities that activate PLA2 in various membrane contexts. More broadly, they exemplify how this biochemical system actively modulates and capitalizes on the spatial distribution of membrane lipids to efficiently achieve its objectives.