The Novel Fusion Protein Melittin-MIL-2 Exhibits Strong Antitumor Immune Effect in Lung Adenocarcinoma Cell A549.

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.

Molecular dynamics simulations in pre-polymerization mixtures for peptide recognition.

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.

Melittin inactivates YAP/HIF-1α pathway via up-regulation of LATS2 to inhibit hypoxia-induced proliferation, glycolysis and angiogenesis in NSCLC.

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.

Structural Determinants of Peptide Nanopore Formation.

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.

Homologous polydopamine ameliorates haemolysis of melittin for enhancing its anticancer efficacy.

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.

Homologous-targeting biomimetic nanoparticles co-loaded with melittin and a photosensitizer for the combination therapy of triple negative breast cancer.

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.

Melittin as an Activator of the Autophagy and Unfolded Protein Response Pathways in Colorectal HCT116 Cell Line.

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.

Bee Venom: Composition and Anticancer Properties.

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.

Highly in vitro anti-cancer activity of melittin-loaded niosomes on non-small cell lung cancer cells.

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.

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.

Investigation of imaging the somatostatin receptor by opening the blood-brain barrier with melittin - A feasibility study using positron emission tomography and [64Cu]Cu-DOTATATE.

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.

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.

The current landscape of the antimicrobial peptide melittin and its therapeutic potential.

Melittin, a main component of bee venom, is a cationic amphiphilic peptide with a linear α-helix structure. It has been reported that melittin can exert pharmacological effects, such as antitumor, antiviral and anti-inflammatory effects in vitro and in vivo. In particular, melittin may be beneficial for the treatment of diseases for which no specific clinical therapeutic agents exist. Melittin can effectively enhance the therapeutic properties of some first-line drugs. Elucidating the mechanism underlying melittin-mediated biological function can provide valuable insights for the application of melittin in disease intervention. However, in melittin, the positively charged amino acids enables it to directly punching holes in cell membranes. The hemolysis in red cells and the cytotoxicity triggered by melittin limit its applications. Melittin-based nanomodification, immuno-conjugation, structural regulation and gene technology strategies have been demonstrated to enhance the specificity, reduce the cytotoxicity and limit the off-target cytolysis of melittin, which suggests the potential of melittin to be used clinically. This article summarizes research progress on antiviral, antitumor and anti-inflammatory properties of melittin, and discusses the strategies of melittin-modification for its future potential clinical applications in preventing drug resistance, enhancing the selectivity to target cells and alleviating cytotoxic effects to normal cells.

Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency.

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.

How do Antimicrobial Peptides Interact with the Outer Membrane of Gram-Negative Bacteria? Role of Lipopolysaccharides in Peptide Binding, Anchoring, and Penetration.

Gram-negative bacteria possess a complex structural cell envelope that constitutes a barrier for antimicrobial peptides that neutralize the microbes by disrupting their cell membranes. Computational and experimental approaches were used to study a model outer membrane interaction with an antimicrobial peptide, melittin. The investigated membrane included di[3-deoxy-d-manno-octulosonyl]-lipid A (KLA) in the outer leaflet and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in the inner leaflet. Molecular dynamics simulations revealed that the positively charged helical C-terminus of melittin anchors rapidly into the hydrophilic headgroup region of KLA, while the flexible N-terminus makes contacts with the phosphate groups of KLA, supporting melittin penetration into the boundary between the hydrophilic and hydrophobic regions of the lipids. Electrochemical techniques confirmed the binding of melittin to the model membrane. To probe the peptide conformation and orientation during interaction with the membrane, polarization modulation infrared reflection absorption spectroscopy was used. The measurements revealed conformational changes in the peptide, accompanied by reorientation and translocation of the peptide at the membrane surface. The study suggests that melittin insertion into the outer membrane affects its permeability and capacitance but does not disturb the membrane's bilayer structure, indicating a distinct mechanism of the peptide action on the outer membrane of Gram-negative bacteria.

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.

Revamped mini-αA-crystallin showed improved skin permeation and therapeutic activity against melittin-induced toxicity.

Melittin is honey bee venom's primary and most toxic pharmacologically active component. Melittin causes haemolysis, lymphocyte lysis, long-term pain, localised inflammation followed by rhabdomyolysis, and severe renal failure. Renal failure or cardiovascular complications could lead to the victim's death. Severe honey bee bites are treated with general medication involving antihistaminic, anti-inflammatory, and analgesic drugs, as a specific treatment option is unavailable. An earlier study showed the anti-hemolysis and anti-lymphocyte lysis activity of mini- αA-crystallin (MAC), a peptide derived from human eye lens alpha-crystallin. MAC's use has often been restricted despite its high therapeutic potential due to its poor skin permeability. This study compared the skin permeation, anti-inflammatory and analgesic activities of natural peptide MAC and its modified version (MAC-GRD) formed by attaching cell-penetrating peptide (CPP) and GRD amino residues into MAC. Gel formulations were prepared for MAC and MAC-GRD peptides using carbopol (1% w/w), Tween 80 (1%), and ethanol (10%). An ex-vivo skin permeation study was performed using a vertical-type Franz diffusion apparatus. Preclinical in-vivo experiments were conducted to compare the native and modified peptide formulations against melittin-induced toxicity in Wistar rats. MAC gel, MAC-GRD gel and 1% hydrocortisone cream significantly reduced the melittin-induced writhing (20.16 ± 0.792) response in rats with 15.16 ± 0.47, 11.16 ± 0.477 and 12.66 ± 0.66 wriths, respectively. There was a significant reduction in melittin-induced inflammation when MAC-GRD gel was applied immediately after melittin administration. At 0.5, 1, 3, and 5 h, the MAC-GRD-treated rat paws were 0.9 ± 0.043 mm, 0.750 ± 0.037 mm, 0.167 ± 0.0070 mm, and 0.133 ± 0.031 mm thick. Administration of melittin resulted in reduced GSH (antioxidant) levels (47.33 ± 0.760 µg/mg). However, treatment with MAC-GRD gel (71.167 ± 0.601 µg/mg), MAC gel (65.167 ± 1.138 µg/mg), and 1% hydrocortisone (68.33 ± 0.667 µg/mg) significantly increased the antioxidant enzyme levels. MAC-GRD gel significantly reduced the elevated MDA levels (6.933 ± 0.049 nmol/mg) compared to the melittin group (12.533 ± 0.126 nmol/mg), followed by the 1% hydrocortisone (7.367 ± 0.049 nmol/mg) and MAC gel (7.917 ± 0.048 nmol/mg). MAC-GRD demonstrated more skin permeability and superior anti-inflammatory, analgesic, and antioxidant activities when compared to MAC gel. When compared to standard 1% hydrocortisone cream, MAC-GRD had better anti-inflammatory, analgesic, antioxidant, and comparable action in anti-oxidant restoration against melittin. These findings suggest that the developed MAC-GRD gel formulation could help to treat severe cases of honey bee stings.

Inhibition of proteinase-activated receptor 2 (PAR2) decreased the malignant progression of lung cancer cells and increased the sensitivity to chemotherapy.

OBJECTIVES: This study aimed to study the effect of protease-activated receptor 2 (PAR2) on the proliferation, invasion, and clone formation of lung cancer cells. It also aimed to evaluate the inhibitory effect of melittin on PAR2 and the anti-lung cancer effect of melittin combined with gefitinib. METHODS: The correlation between the co-expression of PAR2 and epithelial-mesenchymal transition (EMT) markers was analyzed. PAR2 in A549 and NCI-H1299 cells was knocked down using siRNA. MTT assay, Transwell assay, and colony formation assay were used to detect the effects of PAR2 on cell proliferation, invasion, and clone formation. The anti-cancer effect of PAR2 knockdown on gefitinib treatment was analyzed. The synergistic effect of melittin on gefitinib treatment by inhibiting PAR2 and the underlying molecular mechanism were further analyzed and tested. RESULTS: The expression of PAR2 was upregulated in lung cancer, which was associated with the poor prognosis of lung cancer. PAR2 knockdown inhibited the stemness and EMT of lung cancer cells. It also inhibited the proliferation, invasion, and colony formation of A549 and NCI-H1299 cells. Moreover, PAR2 knockdown increased the chemotherapeutic sensitivity of gefitinib in lung cancer. Melittin inhibited PAR2 and the malignant progression of lung cancer cells. Melittin increased the chemotherapeutic sensitivity of gefitinib in lung cancer by inhibiting PAR2. CONCLUSION: PAR2 may promote the proliferation, invasion, and colony formation of lung cancer cells by promoting EMT. Patients with a high expression of PAR2 have a poor prognosis. Inhibition of PAR2 increased the chemotherapeutic sensitivity of gefitinib. PAR2 may be a potential therapeutic target and diagnostic marker for lung cancer.

Glutamate affects self-assembly, protein corona, and anti-4 T1 tumor effects of melittin/vitamin E-succinic acid-(glutamate)n nanoparticles.

Melittin (M) has attracted increasing attention for its significant antitumor effects and various immunomodulatory effects. However, various obstacles such as the short plasma half-life and adverse reactions restrict its application. This study aimed to systematically investigate the self-assembly mechanism, components of the protein corona, targeting behavior, and anti-4 T1 tumor effect of vitamin E-succinic acid-(glutamate)n /melittin nanoparticles with varying amounts of glutamic acid. Here, we present a new vitamin E-succinic acid-(glutamate)5 (E5), vitamin E-succinic acid-(glutamate)10 (E10) or vitamin E-succinic acid-(glutamate)15 (E15), and their co-assembly system with positively charged melittin in water. The molecular dynamics simulations demonstrated that the electrostatic energy and van der Waals force in the system decreased significantly with the increase in the amount of glutamic acid. The melittin and E15 system exhibited the optimal stability for nanoparticle self-assembly. When nanoparticles derived from different self-assembly systems were co-incubated with plasma from patients with breast cancer, the protein corona showed heterogeneity. In vivo imaging demonstrated that an increase in the number of glutamic acid residues enhanced circulation duration and tumor-targeting effects. Both in vitro and in vivo antitumor evaluation indicated a significant increase in the antitumor effect with the addition of glutamic acid. According to our research findings, the number of glutamic acid residues plays a crucial role in the targeted delivery of melittin for immunomodulation and inhibition of 4 T1 breast cancer. Due to the self-assembly capabilities of vitamin E-succinic acid-(glutamate)n in water, these nanoparticles carry significant potential for delivering cationic peptides such as melittin.