Advancing the Design of Artificial Nano-organelles for Targeted Cellular Detoxification of Reactive Oxygen Species.

Artificial organelles (AnOs) are in the spotlight as systems to supplement biochemical pathways in cells. While polymersome-based artificial organelles containing enzymes to reduce reactive oxygen species (ROS) are known, applications requiring control of their enzymatic activity and cell-targeting to promote intracellular ROS detoxification are underexplored. Here, we introduce advanced AnOs where the chemical composition of the membrane supports the insertion of pore-forming melittin, enabling molecular exchange between the AnO cavity and the environment, while the encapsulated lactoperoxidase (LPO) maintains its catalytic function. We show that H2O2 outside AnOs penetrates through the melittin pores and is rapidly degraded by the encapsulated enzyme. As surface attachment of cell-penetrating peptides facilitates AnOs uptake by cells, electron spin resonance revealed a remarkable enhancement in intracellular ROS detoxification by these cell-targeted AnOs compared to nontargeted AnOs, thereby opening new avenues for a significant reduction of oxidative stress in cells.

Structures of calmodulin-melittin complexes show multiple binding modes lacking classical anchoring interactions.

Calmodulin (CaM) is a Ca2+ sensor protein found in all eukaryotic cells that regulates a large number of target proteins in a Ca2+ concentration-dependent manner. As a transient-type hub protein, it recognizes linear motifs of its targets, though for the Ca2+-dependent binding, no consensus sequence was identified. Its complex with melittin, a major component of bee venom, is often used as a model system of protein-protein complexes. Yet, the structural aspects of the binding are not well understood, as only diverse, low-resolution data are available concerning the association. We present the crystal structure of melittin in complex with Ca2+-saturated CaMs from two, evolutionarily distant species, Homo sapiens and Plasmodium falciparum, representing three binding modes of the peptide. Results-augmented by molecular dynamics simulations-indicate that multiple binding modes can exist for CaM-melittin complexes, as an intrinsic characteristic of the binding. While the helical structure of melittin remains, swapping of its salt bridges and partial unfolding of its C-terminal segment can occur. In contrast to the classical way of target recognition by CaM, we found that different sets of residues can anchor at the hydrophobic pockets of CaM, which were considered as main recognition sites. Finally, the nanomolar binding affinity of the CaM-melittin complex is created by an ensemble of arrangements of similar stability-tight binding is achieved not by optimized specific interactions but by simultaneously satisfying less optimal interaction patterns in co-existing different conformers.

The capsule of Bacillus anthracis protects it from the bactericidal activity of human defensins and other cationic antimicrobial peptides.

During infection, Bacillus anthracis bacilli encounter potent antimicrobial peptides (AMPs) such as defensins. We examined the role that B. anthracis capsule plays in protecting bacilli from defensins and other cationic AMPs by comparing their effects on a fully virulent encapsulated wild type (WT) strain and an isogenic capsule-deficient capA mutant strain. We identified several human defensins and non-human AMPs that were capable of killing B. anthracis. The human alpha defensins 1-6 (HNP-1-4, HD-5-6), the human beta defensins 1-4 (HBD-1-4), and the non-human AMPs, protegrin, gramicidin D, polymyxin B, nisin, and melittin were all capable of killing both encapsulated WT and non-encapsulated capA mutant B. anthracis. However, non-encapsulated capA mutant bacilli were significantly more susceptible than encapsulated WT bacilli to killing by nearly all of the AMPs tested. We demonstrated that purified capsule bound HBD-2, HBD-3, and HNP-1 in an electrophoretic mobility shift assay. Furthermore, we determined that the capsule layer enveloping WT bacilli bound and trapped HBD-3, substantially reducing the amount reaching the cell wall. To assess whether released capsule might also play a protective role, we pre-incubated HBD-2, HBD-3, or HNP-1 with purified capsule before their addition to non-encapsulated capA mutant bacilli. We found that free capsule completely rescued the capA mutant bacilli from killing by HBD-2 and -3 while killing by HNP-1 was reduced to the level observed with WT bacilli. Together, these results suggest an immune evasion mechanism by which the capsule, both that enveloping the bacilli and released fragments, contributes to virulence by binding to and inhibiting the antimicrobial activity of cationic AMPs.

A novel recombinant adenovirus expressing apoptin and melittin genes kills hepatocellular carcinoma cells and inhibits the growth of ectopic tumor.

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.

Neuroprotective Effects of Melittin Against Cerebral Ischemia and Inflammatory Injury via Upregulation of MCPIP1 to Suppress NF-κB Activation In Vivo and In Vitro.

Melittin, a principal constituent of honeybee venom, exhibits diverse biological effects, encompassing anti-inflammatory capabilities and neuroprotective actions against an array of neurological diseases. In this study, we probed the prospective protective influence of melittin on cerebral ischemia, focusing on its anti-inflammatory activity. Mechanistically, we explored whether monocyte chemotactic protein-induced protein 1 (MCPIP1, also known as ZC3H12A), a recently identified zinc-finger protein, played a role in melittin-mediated anti-inflammation and neuroprotection. Male C57/BL6 mice were subjected to distal middle cerebral artery occlusion to create a focal cerebral cortical ischemia model, with melittin administered intraperitoneally. We evaluated motor functions, brain infarct volume, cerebral blood flow, and inflammatory marker levels within brain tissue, employing quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assays, and western blotting. In vitro, an immortalized BV-2 microglia culture was stimulated with lipopolysaccharide (LPS) to establish an inflammatory cell model. Post-melittin exposure, cell viability, and cytokine expression were examined. MCPIP1 was silenced using siRNA in LPS-induced BV-2 cells, with the ensuing nuclear translocation of nuclear factor-κB assessed through cellular immunofluorescence. In vivo, melittin enhanced motor functions, diminished infarction, fostered blood flow restoration in ischemic brain regions, and markedly inhibited the expression of inflammatory cytokines (interleukin-1ß, interleukin-6, tumor necrosis factor-α, and nuclear factor-κB). In vitro, melittin augmented MCPIP1 expression in LPS-induced BV-2 cells and ameliorated inflammation-induced cell death. The neuroprotective effect conferred by melittin was attenuated upon MCPIP1 knockdown. Our findings establish that melittin-induced tolerance to ischemic injury is intrinsically linked with its anti-inflammatory capacity. Moreover, MCPIP1 is, at the very least, partially implicated in this process.

Combination Nano-Delivery Systems Remodel the Immunosuppressive Tumor Microenvironment for Metastatic Triple-Negative Breast Cancer Therapy.

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.

A comprehensive investigation of the medicinal efficacy of antimicrobial fusion peptides expressed in probiotic bacteria for the treatment of pan drug-resistant (PDR) infections.

The present work aimed to examine the intracellular antibacterial efficacy of Recombinant Lactobacillus acidophilus/antimicrobial peptides (AMPs) Melittin and Alyteserin-1a, specifically targeting Gram-negative bacteria. The first assessment was to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Recombinant L. acidophilus/AMPs versus Gram-negative and Gram-positive bacteria. In addition, the researchers examined the in vitro viability and safety of AMPs generated by L. acidophilus. The experiments included exposing the AMPs to elevated temperatures, proteases, cationic salts at physiological levels, and specific pH settings. The safety aspect was evaluated using hemolytic analysis utilizing sheep erythrocytes; cytotoxicity assays employing cell lines, and experiments on beneficial gut lactobacilli. An experiment was done using a time-kill method to assess the intracellular antibacterial efficacy of Recombinant L. acidophilus/AMPs compared to pathogenic varieties in HEp-2 cells. Previous investigations have shown that the MBC levels of recombinant L. acidophilus/AMPs were consistently two to four times higher than the equivalent MIC values when evaluated versus Gram-negative bacteria. Furthermore, the stability of the Recombinant L. acidophilus/AMPs showed variability when exposed to elevated temperatures (70 and 90 â„ƒ), treated with protease enzymes (proteinase K, lysozyme), exposed to higher concentrations of physiological salts (150 mM NaCl and 2 mM MgCl2), and varying pH levels (ranging from 4.0 to 9.0). The recombinant L. acidophilus/AMPs are non-hemolytic towards sheep erythrocytes, exhibit little cytotoxicity in RAW 264.7 and HEp-2 cells, and are considered safe when compared to beneficial gut lactobacilli. The research examined the intracellular bacteriostatic effects of recombinant L. acidophilus/AMPs on Gram-negative bacteria inside HEp-2 cells. Nevertheless, no notable bactericidal impact was seen on Gram-positive bacteria (P > 0.05). The research shows that recombinant L. acidophilus/AMPs, namely (L. acidophilus/melittin/Alyteserin-1a) as the focus of the investigation, effectively eliminate Gram-negative bacteria. Therefore, more investigation is necessary to elaborate on these discoveries.

Melittin-Phospholipase A2 Synergism Is Mediated by Liquid-Liquid Miscibility Phase Transition in Giant Unilamellar Vesicles.

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.

Standardized guidelines for Africanized honeybee venom production needed for development of new apilic antivenom.

Africanized bees have spread across the Americas since 1956 and consequently resulted in human and animal deaths attributed to massive attacks related to exposure from Argentina to the USA. In Brazil, more than 100,000 accidents were registered in the last 5 years with a total of 303 deaths. To treat such massive attacks, Brazilian researchers developed the first specific antivenom against Africanized honey bee sting exposure. This unique product, the first of its kind in the world, has been safely tested in 20 patients during a Phase 2 clinical trial. To develop the antivenom, a standardized process was undertaken to extract primary venom antigens from the Africanized bees for immunization of serum-producing horses. This process involved extracting, purifying, fractionating, characterizing, and identifying the venom (apitoxin) employing mass spectrometry to generate standardized antigen for hyperimmunization of horses using the major toxins (melittin and its isoforms and phospholipase A2). The current guide describes standardization of the entire production chain of venom antigens in compliance with good manufacturing practices (GMP) required by regulatory agencies. Emphasis is placed upon the welfare of bees and horses during this process, as well as the development of a new biopharmaceutical to ultimately save lives.

Upgrading of the general AMBER force field 2 for fluorinated alcohol biosolvents: A validation for water solutions and melittin solvation.

The standard GAFF2 force field parameterization has been refined for the fluorinated alcohols 2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and 1,1,1,3,3,3-hexafluoropropan-2-one (HFA), which are commonly used to study proteins and peptides in biomimetic media. The structural and dynamic properties of both proteins and peptides are significantly influenced by the biomimetic environment created by the presence of these cosolvents in aqueous solutions. Quantum mechanical calculations on stable conformers were used to parameterize the atomic charges. Different systems, such as pure liquids, aqueous solutions, and systems formed by melittin protein and cosolvent/water solutions, have been used to validate the new models. The calculated macroscopic and structural properties are in agreement with experimental findings, supporting the validity of the newly proposed models.

The combination of modified acupuncture needle and melittin hydrogel as a novel therapeutic approach for rheumatoid arthritis treatment.

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.

Neochloris oleoabundans cell wall rupture through melittin peptide: a new approach to increase lipid recovery.

OBJECTIVES: Microalgae cell wall affects the recovery of lipids, representing one of the main difficulties in the development of biofuel production. This work aimed to test a new method based on melittin peptide to induce a cellular disruption in N. oleoabundans. RESULTS: Neochloris oleoabundans cells were grown at 32 °C in the presence of a high concentration of nitrate-phosphate, causing a cell disruption extent of 83.6%. Further, a two-fold increase in lipid recovery following melittin treatment and solvent extraction was observed. Additionally, it was possible to verify the effects of melittin, both before and after treatment on the morphology of the cells. Scanning electron microscopy (SEM) and confocal images of the melittin-treated microalgae revealed extensive cell damage with degradation of the cell wall and release of intracellular material. CONCLUSIONS: Melittin produced a selective cell wall rupture effect in N. oleoabundans under some culture conditions. These results represent the first report on the effect of melittin on lipid recovery from microalgae.

Immunomodulatory activities and biomedical applications of melittin and its recent advances.

Melittin (MLT), a peptide containing 26 amino acids, is a key constituent of bee venom. It comprises ∼40%-60% of the venom's dry weight and is the main pricing index for bee venom, being the causative factor of pain. The unique properties of MLT extracted from bee venom have made it a very valuable active ingredient in the pharmaceutical industry as this cationic and amphipathic peptide has propitious effects on human health in diverse biological processes. It has the ability to strongly impact the membranes of cells and display hemolytic activity with anticancer characteristics. However, the clinical application of MLT has been limited by its severe hemolytic activity, which poses a challenge for therapeutic use. By employing more efficient mechanisms, such as modifying the MLT sequence, genetic engineering, and nano-delivery systems, it is anticipated that the limitations posed by MLT can be overcome, thereby enabling its wider application in therapeutic contexts. This review has outlined recent advancements in MLT's nano-delivery systems and genetically engineered cells expressing MLT and provided an overview of where the MLTMLT's platforms are and where they will go in the future with the challenges ahead. The focus is on exploring how these approaches can overcome the limitations associated with MLT's hemolytic activity and improve its selectivity and efficacy in targeting cancer cells. These advancements hold promise for the creation of innovative and enhanced therapeutic approaches based on MLT for the treatment of cancer.

Melittin Treats Periprosthetic Osteolysis in a Rat Model by Inhibiting the NF-kB Pathway and Regulating the Ratio of Receptor Activator of Nuclear Factor Kappa B Ligand/Osteoprotegerin.

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.

New N-Terminal Fatty-Acid-Modified Melittin Analogs with Potent Biological Activity.

Melittin, a natural antimicrobial peptide, has broad-spectrum antimicrobial activity. This has resulted in it gaining increasing attention as a potential antibiotic alternative; however, its practical use has been limited by its weak antimicrobial activity, high hemolytic activity, and low proteolytic stability. In this study, N-terminal fatty acid conjugation was used to develop new melittin-derived lipopeptides (MDLs) to improve the characteristics of melittin. Our results showed that compared with native melittin, the antimicrobial activity of MDLs was increased by 2 to 16 times, and the stability of these MDLs against trypsin and pepsin degradation was increased by 50 to 80%. However, the hemolytic activity of the MDLs decreased when the length of the carbon chain of fatty acids exceeded 10. Among the MDLs, the newly designed analog Mel-C8 showed optimal antimicrobial activity and protease stability. The antimicrobial mechanism studied revealed that the MDLs showed a rapid bactericidal effect by interacting with lipopolysaccharide (LPS) or lipoteichoic acid (LTA) and penetrating the bacterial cell membrane. In conclusion, we designed and synthesized a new class of MDLs with potent antimicrobial activity, high proteolytic stability, and low hemolytic activity through N-terminal fatty acid conjugation.

Comparison of Muscle Regeneration Effects at Different Melittin Concentrations in Rabbit Atrophied Muscle.

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.

Discovery of Melittin as Triple-Action Agent: Broad-Spectrum Antibacterial, Anti-Biofilm, and Potential Anti-Quorum Sensing Activities.

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.

Melittin inhibits tumor cell migration and enhances cisplatin sensitivity by suppressing IL-17 signaling pathway gene LCN2 in castration-resistant prostate cancer.

BACKGROUND: Melittin is a small molecule polypeptide extracted from the abdominal cavity of bees, which is used to treat inflammatory diseases and relieve pain. However, the antitumor effect of melittin and its mechanisms remain unclear, especially in castration-resistant prostate cancer (CRPC). METHODS: Through CCK-8 assay, colony formation assay, wound healing assay and Transwell migration assay, we explored the effect of melittin on CRPC cell lines. In addition, with microarray analysis, gene ontology analysis and kyoto encyclopedia of genes and genomes analysis, this study identified key genes and signaling pathways that influence the growth of PC-3 cells. Meanwhile, the effect of melittin on CRPC was also verified through subcutaneous tumor formation experiments. Finally, we also tested the relevant indicators of human prostate cancer (PCa) specimens through immunohistochemistry and H＆E stating. RESULTS: Here, melittin was verified to inhibit the cell proliferation and migration of CPRC. Moreover, RNA-sequence analysis demonstrated that Interleukin-17 (IL-17) signaling pathway gene Lipocalin-2 (LCN2) was downregulated by melittin treatment in CRPC. Further investigation revealed that overexpression of LCN2 was able to rescue tumor suppression and cisplatin sensitivity which melittin mediated. Interestingly, the expression of LCN2 is highly related to metastasis in PCa. CONCLUSIONS: In brief, our study indicates that LCN2 plays an oncogenic role in CRPC and melittin may be selected as an attractive candidate for CRPC therapy.

A combination of genetically engineered oncolytic virus and melittin-CpG for cancer viro-chemo-immunotherapy.

BACKGROUND: Immunotherapy has emerged as an efficient therapeutic approach for cancer management. However, stimulation of host immune system against cancer cells often fails to achieve promising clinical outcomes mainly owing to the immunosuppressive characteristics of the tumor microenvironment (TME). Combination therapeutics that can trigger sustained immunogenic cell death (ICD) have provided new opportunities for cancer treatment. METHODS: In this study, we designed and applied an ICD inducer regimen, including a genetically engineered oncolytic virus (miRNA-modified coxsackieviruses B3, miR-CVB3), a pore-forming lytic peptide (melittin, found in bee venom), and a synthetic toll-like receptor 9 ligand (CpG oligodeoxynucleotides), for breast cancer and melanoma treatment. We compared the anti-tumor efficacy of miR-CVB3 and CpG-melittin (CpGMel) alone and in combination (miR-CVB3 + CpGMel) and investigated possible mechanisms involved. RESULTS: We demonstrated that miR-CVB3 + CpGMel had no major impact on viral growth, while enhancing the cellular uptake of CpGMel in vitro. We further showed that combination therapy led to significant increases in tumor cell death and release of damage-associated molecular patterns compared with individual treatment. In vivo studies in 4T1 tumor-bearing Balb/c mice revealed that both primary and distant tumors were significantly suppressed, and the survival rate was significantly prolonged after administration of miR-CVB3 + CpGMel compared with single treatment. This anti-tumor effect was accompanied by increased ICD and immune cell infiltration into the TME. Safety analysis showed no significant pathological abnormalities in Balb/c mice. Furthermore, the developed therapeutic regimen also demonstrated a great anti-tumor activity in B16F10 melanoma tumor-bearing C57BL/6 J mice. CONCLUSIONS: Overall, our findings indicate that although single treatment using miR-CVB3 or CpGMel can efficiently delay tumor growth, combining oncolytic virus-based therapy can generate even stronger anti-tumor immunity, leading to a greater reduction in tumor size.

Bacteria primed by antimicrobial peptides develop tolerance and persist.

Antimicrobial peptides (AMPs) are key components of innate immune defenses. Because of the antibiotic crisis, AMPs have also come into focus as new drugs. Here, we explore whether prior exposure to sub-lethal doses of AMPs increases bacterial survival and abets the evolution of resistance. We show that Escherichia coli primed by sub-lethal doses of AMPs develop tolerance and increase persistence by producing curli or colanic acid, responses linked to biofilm formation. We develop a population dynamic model that predicts that priming delays the clearance of infections and fuels the evolution of resistance. The effects we describe should apply to many AMPs and other drugs that target the cell surface. The optimal strategy to tackle tolerant or persistent cells requires high concentrations of AMPs and fast and long-lasting expression. Our findings also offer a new understanding of non-inherited drug resistance as an adaptive response and could lead to measures that slow the evolution of resistance.