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1.
Biomolecules ; 14(6)2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38927102

RESUMO

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.


Assuntos
Antibacterianos , Biofilmes , Meliteno , Staphylococcus aureus Resistente à Meticilina , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa , Meliteno/farmacologia , Meliteno/química , Biofilmes/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Humanos , Hemólise/efeitos dos fármacos , Ácido Aminocaproico/química , Ácido Aminocaproico/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Animais
2.
Clinics (Sao Paulo) ; 79: 100407, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38889502

RESUMO

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.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Proliferação de Células , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia , Neoplasias Pulmonares , Meliteno , Neovascularização Patológica , Proteínas Serina-Treonina Quinases , Proteínas Supressoras de Tumor , Regulação para Cima , Proteínas de Sinalização YAP , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Sinalização YAP/metabolismo , Meliteno/farmacologia , Meliteno/uso terapêutico , Linhagem Celular Tumoral , Fatores de Transcrição/metabolismo , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fosfoproteínas/metabolismo , Angiogênese
3.
ACS Nano ; 18(24): 15831-15844, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38844421

RESUMO

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.


Assuntos
Meliteno , Simulação de Dinâmica Molecular , Nanoporos , Fosfatidilcolinas , Meliteno/química , Fosfatidilcolinas/química , Bicamadas Lipídicas/química , Ligação de Hidrogênio , Peptídeos/química , Humanos
4.
J Mater Chem B ; 12(22): 5431-5438, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38726737

RESUMO

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.


Assuntos
Antineoplásicos , Hemólise , Indóis , Meliteno , Polímeros , Meliteno/química , Meliteno/farmacologia , Animais , Humanos , Indóis/química , Indóis/farmacologia , Polímeros/química , Polímeros/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Camundongos , Hemólise/efeitos dos fármacos , Nanopartículas/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Ensaios de Seleção de Medicamentos Antitumorais , Camundongos Nus , Tamanho da Partícula
5.
J Mater Chem B ; 12(22): 5465-5478, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38742364

RESUMO

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.


Assuntos
Meliteno , Nanopartículas , Fotoquimioterapia , Fármacos Fotossensibilizantes , Neoplasias de Mama Triplo Negativas , Meliteno/química , Meliteno/farmacologia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Nanopartículas/química , Animais , Camundongos , Feminino , Humanos , Antineoplásicos/química , Antineoplásicos/farmacologia , Camundongos Endogâmicos BALB C , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais
6.
Int J Biol Macromol ; 270(Pt 1): 132293, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38735618

RESUMO

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.


Assuntos
Artrite Experimental , Artrite Reumatoide , Meliteno , Metaboloma , Simulação de Acoplamento Molecular , Transcriptoma , Animais , Ratos , Transcriptoma/efeitos dos fármacos , Meliteno/farmacologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Artrite Reumatoide/induzido quimicamente , Metaboloma/efeitos dos fármacos , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Artrite Experimental/induzido quimicamente , Artrite Experimental/genética , Adjuvante de Freund , Masculino , Regulação da Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica
7.
Int J Mol Sci ; 25(9)2024 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-38732255

RESUMO

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.


Assuntos
Meliteno , Músculo Esquelético , Atrofia Muscular , Regeneração , Animais , Coelhos , Meliteno/farmacologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Regeneração/efeitos dos fármacos , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/metabolismo , Atrofia Muscular/etiologia , Atrofia Muscular/patologia , Masculino
9.
Artigo em Inglês | MEDLINE | ID: mdl-38649084

RESUMO

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.


Assuntos
Antibacterianos , Venenos de Abelha , Escherichia coli , Corpo Adiposo , Meliteno , Animais , Antibacterianos/farmacologia , Antibacterianos/toxicidade , Venenos de Abelha/farmacologia , Venenos de Abelha/toxicidade , Abelhas , Escherichia coli/efeitos dos fármacos , Corpo Adiposo/metabolismo , Proteínas de Insetos/metabolismo , Listeria monocytogenes/efeitos dos fármacos , Meliteno/farmacologia , Meliteno/toxicidade
10.
Langmuir ; 40(14): 7456-7462, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38546877

RESUMO

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.


Assuntos
Venenos de Abelha , Meliteno , Meliteno/farmacologia , Lipossomas Unilamelares , Fosfolipases A2 , Lipídeos de Membrana
11.
Iran Biomed J ; 28(1): 46-52, 2024 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-38445441

RESUMO

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.


Assuntos
Neoplasias Colorretais , Meliteno , Humanos , Células HCT116 , Meliteno/farmacologia , Meliteno/genética , Meliteno/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Resposta a Proteínas não Dobradas , Autofagia , RNA Mensageiro/metabolismo , Carcinogênese , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética
12.
Nucl Med Biol ; 132-133: 108905, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38555651

RESUMO

DOTATATE is a somatostatin peptide analog used in the clinic to detect somatostatin receptors which are highly expressed on neuroendocrine tumors. Somatostatin receptors are found naturally in the intestines, pancreas, lungs, and brain (mainly cortex). In vivo measurement of the somatostatin receptors in the cortex has been challenging because available tracers cannot cross the blood-brain barrier (BBB) due to their intrinsic polarity. A peptide called melittin, a main component of honeybee venom, has been shown to disrupt plasma membranes and increase the permeability of biological membranes. In this study, we assessed the feasibility of using melittin to facilitate the passage of [64Cu]Cu-DOTATATE through the BBB and its binding to somatostatin receptors in the cortex. Evaluation included in vitro autoradiography on Long Evans rat brains to estimate the binding affinity of [64Cu]Cu-DOTATATE to the somatostatin receptors in the cortex and an in vivo evaluation of [64Cu]Cu-DOTATATE binding in NMRI mice after injection of melittin. This study found an in vitro Bmax = 89 ± 4 nM and KD = 4.5 ± 0.6 nM in the cortex, resulting in a theoretical binding potential (BP) calculated as Bmax/KD ≈ 20, which is believed suitable for in vivo brain PET imaging. However, the in vivo results showed no significant difference between the control and melittin injected mice, indicating that the honeybee venom failed to open the BBB. Additional experiments, potentially involving faster injection rates are required to verify that melittin can increase brain uptake of non-BBB permeable PET tracers. Furthermore, an evaluation of whether a venom with a narrow therapeutic range can be used for clinical purposes needs to be considered.


Assuntos
Barreira Hematoencefálica , Estudos de Viabilidade , Meliteno , Compostos Organometálicos , Tomografia por Emissão de Pósitrons , Receptores de Somatostatina , Animais , Receptores de Somatostatina/metabolismo , Meliteno/química , Meliteno/metabolismo , Ratos , Tomografia por Emissão de Pósitrons/métodos , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Compostos Organometálicos/farmacocinética , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/diagnóstico por imagem , Masculino , Camundongos , Radioisótopos de Cobre , Octreotida/análogos & derivados
13.
Toxins (Basel) ; 16(3)2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38535786

RESUMO

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.


Assuntos
Venenos de Abelha , Masculino , Animais , Abelhas , Meliteno , Membrana Celular , Apoptose , Morte Celular
14.
Toxicon ; 241: 107673, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38432612

RESUMO

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.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Meliteno/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Lipossomos , Caspase 3 , Proteína X Associada a bcl-2/genética , Neoplasias Pulmonares/tratamento farmacológico
15.
Sci Rep ; 14(1): 5797, 2024 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-38461178

RESUMO

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.


Assuntos
Toxinas Bacterianas , Enterotoxinas , Humanos , Enterotoxinas/toxicidade , Toxinas Bacterianas/toxicidade , Células CACO-2 , Meliteno/farmacologia , Nigericina/farmacologia , Mucosa Intestinal/patologia
16.
Mol Pharm ; 21(5): 2148-2162, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38536949

RESUMO

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.


Assuntos
Azepinas , Celecoxib , Triazóis , Neoplasias de Mama Triplo Negativas , Microambiente Tumoral , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/efeitos dos fármacos , Animais , Feminino , Camundongos , Humanos , Celecoxib/administração & dosagem , Linhagem Celular Tumoral , Sulfatos de Condroitina/química , Sulfatos de Condroitina/administração & dosagem , Nanopartículas/química , Nanopartículas/administração & dosagem , Meliteno/administração & dosagem , Meliteno/química , Apoptose/efeitos dos fármacos , Sistemas de Liberação de Fármacos por Nanopartículas/química , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Endogâmicos BALB C , Inibidores de Ciclo-Oxigenase 2/administração & dosagem , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Polímeros/química , Camundongos Nus , Sistemas de Liberação de Medicamentos/métodos
17.
J Phys Chem Lett ; 15(7): 1930-1935, 2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38346015

RESUMO

Non-equilibrium kinetics techniques like pressure-jump nuclear magnetic resonance (NMR) are powerful in tracking changes in oligomeric populations and are not limited by relaxation rates for the time scales of exchange that can be probed. However, these techniques are less sensitive to minor, transient populations than are Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments. We integrated non-equilibrium pressure-jump and equilibrium CPMG relaxation dispersion data to fully map the kinetic landscape of melittin tetramerization. While monomeric peptides weakly form dimers (Kd,D/M ≈ 26 mM) whose population never exceeds 1.6% at 288 K, dimers associate tightly to form stable tetrameric species (Kd,T/D ≈ 740 nM). Exchange between the monomer and dimer, along with exchange between the dimer and tetramer, occurs on the millisecond time scale. The NMR approach developed herein can be readily applied to studying the folding and misfolding of a wide range of oligomeric assemblies.


Assuntos
Imageamento por Ressonância Magnética , Meliteno , Ressonância Magnética Nuclear Biomolecular/métodos , Modelos Moleculares , Espectroscopia de Ressonância Magnética
18.
Molecules ; 29(3)2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38338303

RESUMO

The development of antibiotic-resistant microorganisms is a major global health concern. Recently, there has been an increasing interest in antimicrobial peptides as a therapeutic option. This study aimed to evaluate the triple-action (broad-spectrum antibacterial, anti-biofilm, and anti-quorum sensing activities) of melittin, a membrane-active peptide present in bee venom. The minimum inhibitory concentration and minimum bactericidal concentration of the melittin were determined using the microdilution method and agar plate counting. Growth curve analysis revealed that melittin showed a concentration-dependent antibacterial activity. Scanning electron microscope analysis revealed that melittin treatment altered the morphology. Confocal laser scanning microscope revealed that melittin increased the membrane permeability and intracellular ROS generation in bacteria, all of which contribute to bacterial cell death. In addition, the crystal violet (CV) assay was used to test the anti-biofilm activity. The CV assay demonstrated that melittin inhibited biofilm formation and eradicated mature biofilms. Biofilm formation mediated by quorum sensing (QS) plays a major role in this regard, so molecular docking and molecular dynamics analysis confirmed that melittin interacts with LasR receptors through hydrogen bonds, and further evaluates the anti-QS activity of melittin through the production of virulence factors (pyocyanin, elastase, and rhamnolipid), exopolysaccharides secretion, and bacterial motility, that may be the key to inhibiting the biofilm formation mechanism. The present findings highlight the promising role of melittin as a broad-spectrum antibacterial, anti-biofilm agent, and potential QS inhibitor, providing a new perspective and theoretical basis for the development of alternative antibiotics.


Assuntos
Meliteno , Percepção de Quorum , Meliteno/farmacologia , Simulação de Acoplamento Molecular , Biofilmes , Antibacterianos/química , Fatores de Virulência/metabolismo , Pseudomonas aeruginosa/fisiologia
19.
J Arthroplasty ; 39(7): 1845-1855, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38336308

RESUMO

BACKGROUND: Aseptic loosening around the prosthesis is a common cause of failure in total joint arthroplasty. Polyethylene wear particles trigger the release of inflammatory factors by macrophages. Key mediators involved in osteoclastogenesis include interleukin-6, tumor necrosis factor-α, receptor activator of nuclear factor kappa B (RANK), RANK ligand (RANKL), and bone protection hormone (Osteoprotegerin [OPG]). The purpose of our experiment was to see whether melittin can slow down the release of inflammatory mediators through the NF-kB pathway, regulate the RANKL/OPG ratio, reduce osteoclast formation, and delay the onset of arthritis in rats. METHODS: A total of 20 male Sprague-Dawley rats (10 months, Specific Pathogen Free, 350 g ± 20 g) were randomly divided into 5 groups: sham group, model group, melittin concentration 1 group (0.2 mg/kg), concentration 2 group (0.4 mg/kg), and concentration 3 group (0.6 mg/kg). All rats were implanted with TA2 high-purity titanium rods. A drill was used to create a bone canal along the long axis of the femur in the intercondylar notch. The model group and experimental groups were exposed to polyethylene particles, while the sham group did not receive any particles. RESULTS: The melittin group exhibited significantly increased serum levels of serum P, calcium-phosphorus product, OPG, PINP, PINP/CTX-I, and OPG/RANKKL (P < .05). In the experimental group, micro computed tomography scanning results revealed a decrease in the amount of bone defect around the prosthesis. Immunofluorescence analysis demonstrated a decrease in the expression of IKKα and P65, while the expression of OPG showed an upward trend. Both Hematoxylin-Eosin and Tartrate-Resistant Acid Phosphatase staining revealed less osteoclast and inflammatory cell infiltration in bone resorption pits. CONCLUSIONS: Our study demonstrates that melittin has the ability to inhibit the NF-kB pathway in a rat model, and reduce the impact of RANKL/OPG, thereby delaying osteoclast activity and alleviating periprosthetic osteolysis.


Assuntos
Modelos Animais de Doenças , Meliteno , NF-kappa B , Osteólise , Osteoprotegerina , Ligante RANK , Ratos Sprague-Dawley , Animais , Masculino , Osteólise/etiologia , Osteólise/prevenção & controle , Ligante RANK/metabolismo , Osteoprotegerina/metabolismo , Ratos , Meliteno/farmacologia , NF-kappa B/metabolismo , Titânio , Osteoclastos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Polietileno , Falha de Prótese
20.
J Integr Med ; 22(1): 72-82, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38307819

RESUMO

OBJECTIVE: Melittin and its derivative have been developed to support effective gene delivery systems. Their ability to facilitate endosomal release enhances the delivery of nanoparticle-based gene therapy. Nevertheless, its potential application in the context of viral vectors has not received much attention. Therefore, we would like to optimize the rAAV vector by Melittin analog to improve the transduction efficiency of rAAV in liver cancer cells and explore the mechanism of Melittin analog on rAAV. METHODS: Various melittin-derived peptides were inserted into loop VIII of the capsid protein in recombinant adeno-associated virus vectors. These vectors carrying either gfp or fluc genes were subjected to quantitative polymerase chain reaction assays and transduction assays in human embryonic kidney 293 (HEK293T) cells to investigate the efficiency of vector production and gene delivery. In addition, the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice. Finally, the intricate details of the vector-mediated transduction mechanisms were explored by using pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle. RESULTS: A total of 76 melittin-related peptides were identified from existing literature. Among them, CMA-3, p5RHH and aAR3 were found to significantly inhibit transduction of rAAV2 vector crude lysate. The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV. Mechanistically, bafilomycin A1, a vacuolar endosome acidification inhibitor, completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors. Most importantly, p5RHH-rAAV8 vectors also increased hepatic transduction in vivo in C57BL/6 mice. CONCLUSION: The incorporation of melittin analogs into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression. While further modifications remain an area of interest, our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery. Please cite this article as: Meng J, He Y, Yang H, Zhou L, Wang S, Feng X, Al-shargi OY, Yu X, Zhu L, Ling, C. Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency. J Integr Med. 2024; 22(1): 72-82.


Assuntos
Dependovirus , Meliteno , Camundongos , Masculino , Animais , Humanos , Dependovirus/genética , Meliteno/farmacologia , Meliteno/genética , Transdução Genética , Células HEK293 , Camundongos Endogâmicos C57BL , Vetores Genéticos
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