Bottlebrush Polymer-Conjugated Melittin Exhibits Enhanced Antitumor Activity and Better Safety Profile.

Despite potency against a variety of cancers in preclinical systems, melittin (MEL), a major peptide in bee venom, exhibits non-specific toxicity, severe hemolytic activity, and poor pharmacological properties. Therefore, its advancement in the clinical translation system has been limited to early-stage trials. Herein, we report a biohybrid involving a bottlebrush-architectured poly(ethylene glycol) (PEG) and MEL. Termed pacMEL, the conjugate consists of a high-density PEG arrangement, which provides MEL with steric inhibition against protein access, while the high molecular weight of pacMEL substantially enhances plasma pharmacokinetics with a ∼10-fold increase in the area under the curve (AUC∞) compared to free MEL. pacMEL also significantly reduces hepatic damage and unwanted innate immune response and all but eliminated hemolytic activities of MEL. Importantly, pacMEL passively accumulates at subcutaneously inoculated tumor sites and exhibits stronger tumor-suppressive activity than molecular MEL. Collectively, pacMEL makes MEL a safer and more appealing drug candidate.

Molecular characterization of the anticancer properties associated with bee venom and its components in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a frequent form of malignant glioma. Strategic therapeutic approaches to treat this type of brain tumor currently involves a combination of surgery, radiotherapy and chemotherapy. Nevertheless, survival of GBM patients remains in the 12-15 months range following diagnosis. Development of novel therapeutic approaches for this malignancy is therefore of utmost importance. Interestingly, bee venom and its components have shown promising anti-cancer activities in various types of cancer even though information pertaining to GBMs have been limited. The current work was thus undertaken to better characterize the anti-cancer properties of bee venom and its components in Hs683, T98G and U373 human glioma cells. MTT-based cell viability assays revealed IC50 values of 7.12, 15.35 and 7.60 µg/mL for cell lines Hs683, T98G and U373 treated with bee venom, respectively. Furthermore, melittin treatment of these cell lines resulted in IC50 values of 7.77, 31.53 and 12.34 µg/mL, respectively. Cell viability assessment by flow cytometry analysis confirmed signs of late apoptosis and necrosis after only 1 h of treatment with either bee venom or melittin in all three cell lines. Immunoblotting-based quantification of apoptotic markers demonstrated increased expression of Bak and Bax, while Caspsase-3 levels were significantly lower when compared to control cells. Quantification by qRT-PCR showed increased expression levels of long non-coding RNAs RP11-838N2.4 and XIST in glioma cells treated with either bee venom or melittin. Overall, this study provides preliminary insight on molecular mechanisms via which bee venom and its main components can impact viability of glioma cells and warrants further investigation of its anticancer potential in gliomas.

GA-Hecate antiviral properties on HCV whole cycle represent a new antiviral class and open the door for the development of broad spectrum antivirals.

In recent years, synthetic peptides have been considered promising targets for drug development that possess low side-effects, are cost-effective and are susceptible to rational design. Hecate was initially described as a potent bacterial inhibitor and subsequently as an anticancer drug with functions related to its lipid interaction property. Viruses, such as hepatitis C virus (HCV), have a lipid-dependent life cycle and could be affected by Hecate in many ways. Here, we assessed modifications on Hecate's N-terminus region and its effects on HCV and hepatotoxicity. Gallic acid-conjugated Hecate was the most efficient Hecate-derivative, presenting high potential as an antiviral and inhibiting between 50 to 99% of all major steps within the HCV infectious cycle. However, the most promising aspect was GA-Hecate's mechanism of action, which was associated with a balanced lipid interaction with the viral envelope and lipid droplets, as well as dsRNA intercalation, allowing for the possibility to affect other ssRNA viruses and those with a lipid-dependent cycle.

A novel melittin nano-liposome exerted excellent anti-hepatocellular carcinoma efficacy with better biological safety.

Melittin is the main effective component of bee venom and has extensive biological functions; however, serious side effects have restricted its clinical application. Preclinical and clinical studies showed that the main adverse events were allergic reaction and pain at the administration site. To decrease the toxicity, we prepared melittin nano-liposomes by encapsulating melittin with poloxamer 188 and explored the inhibitory activities on liver cancer together with biological safety. Here, we showed that melittin nano-liposomes significantly inhibited the survival of hepatocellular carcinoma (HCC) cells in vitro and prominently suppressed the growth of subcutaneous and orthotopic HCC transplantation tumors in vivo. It was important that it induced less inflammation and allergy in mice compared with melittin. Overall, melittin nano-liposomes would have a better application in HCC therapy due to its significant anti-tumor activity and better biological safety.

Melittin, the Major Pain-Producing Substance of Bee Venom.

Melittin is a basic 26-amino-acid polypeptide that constitutes 40-60% of dry honeybee (Apis mellifera) venom. Although much is known about its strong surface activity on lipid membranes, less is known about its pain-producing effects in the nervous system. In this review, we provide lines of accumulating evidence to support the hypothesis that melittin is the major pain-producing substance of bee venom. At the psychophysical and behavioral levels, subcutaneous injection of melittin causes tonic pain sensation and pain-related behaviors in both humans and animals. At the cellular level, melittin activates primary nociceptor cells through direct and indirect effects. On one hand, melittin can selectively open thermal nociceptor transient receptor potential vanilloid receptor channels via phospholipase A2-lipoxygenase/cyclooxygenase metabolites, leading to depolarization of primary nociceptor cells. On the other hand, algogens and inflammatory/pro-inflammatory mediators released from the tissue matrix by melittin's pore-forming effects can activate primary nociceptor cells through both ligand-gated receptor channels and the G-protein-coupled receptor-mediated opening of transient receptor potential canonical channels. Moreover, subcutaneous melittin up-regulates Nav1.8 and Nav1.9 subunits, resulting in the enhancement of tetrodotoxin-resistant Na(+) currents and the generation of long-term action potential firing. These nociceptive responses in the periphery finally activate and sensitize the spinal dorsal horn pain-signaling neurons, resulting in spontaneous nociceptive paw flinches and pain hypersensitivity to thermal and mechanical stimuli. Taken together, it is concluded that melittin is the major pain-producing substance of bee venom, by which peripheral persistent pain and hyperalgesia (or allodynia), primary nociceptive neuronal sensitization, and CNS synaptic plasticity (or metaplasticity) can be readily induced and the molecular and cellular mechanisms underlying naturally-occurring venomous biotoxins can be experimentally unraveled.

Melittin induced cytogenetic damage, oxidative stress and changes in gene expression in human peripheral blood lymphocytes.

Melittin (MEL) is the main constituent and principal toxin of bee venom. It is a small basic peptide, consisting of a known amino acid sequence, with powerful haemolytic activity. Since MEL is a nonspecific cytolytic peptide that attacks lipid membranes thus leading to toxicity, the presumption is that it could have significant therapeutic benefits. The aim was to evaluate the cyto/genotoxic effects of MEL in human peripheral blood lymphocytes (HPBLs) and the molecular mechanisms involved using a multi-biomarker approach. We found that MEL was cytotoxic for HPBLs in a dose- and time-dependent manner. It also induced morphological changes in the cell membrane, granulation and lysis of exposed cells. After treating HPBLs with non-cytotoxic concentrations of MEL, we observed increased DNA damage including oxidative DNA damage as well as increased formation of micronuclei and nuclear buds, and decreased lymphocyte proliferation determined by comet and micronucleus assays. The observed genotoxicity coincided with increased formation of reactive oxygen species, reduction of glutathione level, increased lipid peroxidation and phospholipase C activity, showing the induction of oxidative stress. MEL also modulated the expression of selected genes involved in DNA damage response (TP53, CDKN1A, GADD45α, MDM), oxidative stress (CAT, SOD1, GPX1, GSR and GCLC) and apoptosis (BAX, BCL-2, CAS-3 and CAS-7). Results indicate that MEL is genotoxic to HPBLs and provide evidence that oxidative stress is involved in its DNA damaging effects. MEL toxicity towards normal cells has to be considered if used for potential therapeutic purposes.

IL-4 and IL-13 induce protection from complement and melittin in endothelial cells despite initial loss of cytoplasmic proteins: membrane resealing impairs quantifying cytotoxicity with the lactate dehydrogenase permeability assay.

Endothelial cell activation and injury by the terminal pathway of complement is important in various pathobiological processes, including xenograft rejection. Protection against injury by human complement can be induced in porcine endothelial cells (ECs) with IL-4 and IL-13 through metabolic activation. However, despite this resistance, the complement-treated ECs were found to lose membrane permeability control assessed with the small molecule calcein. Therefore, to define the apparent discrepancy of permeability changes vis-à-vis the protection from killing, we now investigated whether IL-4 and IL-13 influence the release of the large cytoplasmic protein lactate dehydrogenase (LDH) in ECs incubated with complement or the pore-forming protein melittin. Primary cultures of ECs were pre-treated with IL-4 or IL-13 and then incubated with human serum as source of antibody and complement or melittin. Cell death was assessed using neutral red. Membrane permeability was quantitated measuring LDH release. We found that IL-4-/IL-13-induced protection of ECs from killing by complement or melittin despite loss of LDH in amounts similar to control ECs. However, the cytokine-treated ECs that were protected from killing rapidly regained effective control of membrane permeability. Moreover, the viability of the protected ECs was maintained for at least 2 days. We conclude that the protection induced by IL-4/IL-13 in ECs against lethal attack by complement or melittin is effective and durable despite severe initial impairment of membrane permeability. The metabolic changes responsible for protection allow the cells to repair the membrane injury caused by complement or melittin.

The anti-cancer potency and mechanism of a novel tumor-activated fused toxin, DLM.

Melittin, which acts as a membrane-disrupting lytic peptide, is not only cytotoxic to tumors, but also vital to normal cells. Melittin had low toxicity when coupled with target peptides. Despite significant research development with the fused toxin, a new fused toxin is needed which has a cleavable linker such that the fused toxin can release melittin after protease cleavage on the tumor cell surface. We describe a novel fused toxin, composed of disintegrin, uPA (urokinase-type plasminogen activator)-cleavable linker, and melittin. Disintegrin is a single strand peptide (73 aa) isolated from Gloydius Ussuriensis venom. The RGD (Arg-Gly-Asp) site of disintegrin dominates its interaction with integrins on the surface of the tumor cells. uPA is over-expressed and plays an important role in tumor cell invasiveness and metastatic progression. The DLM (disintegrin-linker-melittin) linker is uPA-cleavable, enabling DLM to release melittin. We compared binding activity of our synthesized disintegrin with native disintegrin and report that DLM had less binding activity than the native form. uPA-cleavage was evaluated in vitro and the uPA-cleavable linker released melittin. Treating tumors expressing uPA with DLM enhanced tumor cell killing as well as reduced toxicity to erythrocytes and other non-cancerous normal cells. The mechanism behind DLM tumor cell killing was tested using a DNA ladder assay, fluorescent microscopy, flow cytometry, and transmission electron microscopy. Data revealed tumor cell necrosis as the mechanism of cell death, and the fused DLM toxin with an uPA-cleavable linker enhanced tumor selectivity and killing ability.

The membrane-lytic peptides K8L9 and melittin enter cancer cells via receptor endocytosis following subcytotoxic exposure.

We investigate the cell entry mechanism of the membrane-lytic peptides K8L9 and melittin in cancer cell lines. K8L9 and melittin interacted with the highly expressed endocytic receptors neuropilin-1, low-density lipoprotein-related protein receptor 1 (LRP1), and transferrin receptor. Silencing of these receptors by small interfering RNAs (siRNAs) attenuated the cytotoxic activity of K8L9 in four cancer cell lines. Intracellular K8L9 and melittin triggered enlargement of the lysosomal compartments and cytosolic translocation of cathepsin B. Hsc70 was identified as a melittin-interactive molecule using coimmunoprecipitation and mass spectrometry, and Hsc70-siRNA attenuated the cellular uptake of K8L9 and cytotoxic activity by K8L9 and melittin. These findings suggest that K8L9 and melittin can enter cancer cells via receptor endocytosis following subcytotoxic treatment and subsequently affect lysosomal compartments.

Melittin: a lytic peptide with anticancer properties.

Melittin (MEL) is a major peptide constituent of bee venom that has been proposed as one of the upcoming possibilities for anticancer therapy. Recent reports point to several mechanisms of MEL cytotoxicity in different types of cancer cells such as cell cycle alterations, effect on proliferation and/or growth inhibition, and induction of apoptotic and necrotic cell death trough several cancer cell death mechanisms, including the activation of caspases and matrix metalloproteinases. Although cytotoxic to a broad spectrum of tumour cells, the peptide is also toxic to normal cells. Therefore its therapeutic potential cannot be achieved without a proper delivery vehicle which could be overcome by MEL nanoparticles that possess the ability to safely deliver significant amount of MEL intravenously, and to target and kill tumours. This review paper summarizes the current knowledge and brings latest research findings on the anticancer potential of this lytic peptide with diverse functions.

Role of the inflammasome in defense against venoms.

Venoms consist of a complex mixture of toxic components that are used by a variety of animal species for defense and predation. Envenomation of mammalian species leads to an acute inflammatory response and can lead to the development of IgE-dependent venom allergy. However, the mechanisms by which the innate immune system detects envenomation and initiates inflammatory and allergic responses to venoms remain largely unknown. Here we show that bee venom is detected by the NOD-like receptor family, pyrin domain-containing 3 inflammasome and can trigger activation of caspase-1 and the subsequent processing and unconventional secretion of the leaderless proinflammatory cytokine IL-1ß in macrophages. Whereas activation of the inflammasome by bee venom induces a caspase-1-dependent inflammatory response, characterized by recruitment of neutrophils to the site or envenomation, the inflammasome is dispensable for the allergic response to bee venom. Finally, we find that caspase-1-deficient mice are more susceptible to the noxious effects of bee and snake venoms, suggesting that a caspase-1-dependent immune response can protect against the damaging effects of envenomation.

Cytolytic nanoparticles attenuate HIV-1 infectivity.

BACKGROUND: We investigated whether cytolytic melittin peptides could inhibit HIV-1 infectivity when carried in a nanoparticle construct that might be used as a topical vaginal virucide. Free melittin and melittin-loaded nanoparticles were prepared and compared for cytotoxicity and their ability to inhibit infectivity by CXCR4 and CCR5 tropic HIV-1 strains. METHODS: TZM-bl reporter cells expressing luciferase under the control of the HIV-1 promoter were incubated with HIV-1 NLHX (CXCR4) or HIV-1 NLYU2 (CCR5) viral strains and different doses of soluble CD4 (positive control) or free melittin to determine infectivity and viability. Melittin-loaded nanoparticles were formulated and different doses tested against VK2 vaginal epithelial cells to determine cell viability. Based on VK2 viability, melittin nanoparticles were tested for prevention of CXCR4 and CCR5 tropic HIV-1 infectivity and viability of TZM-bl reporter cells. Low-speed centrifugation was used to compare the ability of blank non-melittin nanoparticles and melittin nanoparticles to capture CCR5 tropic HIV-1. RESULTS: As expected, the soluble CD4 positive control inhibited CXCR4 (50% inhibitory concentration [IC50] 3.7 µg/ml) and CCR5 (IC50 0.03 µg/ml) tropic HIV-1 infectivity. Free melittin doses <2 µM were not cytotoxic and were highly effective in reducing HIV-1 infectivity for both CXCR4 and CCR5 strains in TZM-bl reporter cells, while VK2 vaginal cell viability was adversely affected at all free melittin doses tested. However, VK2 cell viability was not affected at any dose of melittin-loaded nanoparticles. Melittin nanoparticles safely and significantly decreased CXCR4 (IC50 2.4 µM and IC90 6.9 µM) and CCR5 (IC50 3.6 µM and IC90 11.4 µM) strain infectivity of TZM-bl reporter cells. Furthermore, melittin nanoparticles captured more HIV-1 than blank nanoparticles. CONCLUSIONS: These data illustrate the first proof-of-concept for therapeutic and safe nanoparticle-mediated inhibition of HIV-1 infectivity. Future investigations appear warranted to explore the antiviral prophylactic potential of melittin nanoparticles to capture, disrupt and prevent initial infection with HIV-1 or potentially other enveloped viruses.

Multifunctional CPP polymer system for tumor-targeted pDNA and siRNA delivery.

Cell-penetrating peptides (CPPs) are a very interesting class of molecules to be introduced in gene and siRNA vectors. They can be used to overcome one of the biggest hurdles in gene and siRNA delivery in vitro and in vivo, the transfer across cell membranes. This chapter describes protocols for the synthesis and biological evaluation of a polylysine-based polymer. In this carrier system, melittin is used as CPP with a high activity to disrupt membranes. pH-Labile masking is applied to render the lytic activity specific for intracellular acidic endolysosomal organelles.

A dimethylmaleic acid-melittin-polylysine conjugate with reduced toxicity, pH-triggered endosomolytic activity and enhanced gene transfer potential.

BACKGROUND: Poor endosomal release is one major barrier of gene delivery. Endosomolytic polyethylenimine-melittin conjugates have shown to enhance gene transfer efficiency; however, cytotoxicity due to their general membrane-destabilizing properties limits their application. To overcome this drawback we grafted a polycation with a masked pH-responsive melittin derivate and investigated lytic activity, gene transfer efficiency and cytotoxicity of the resulting conjugate. METHODS: Melittin (Mel) was modified with dimethylmaleic anhydride (DMMAn) and covalently coupled to poly-L-lysine (PLL). The membrane lytic activity was analyzed after incubation at neutral or endosomal pH. PLL-DMMAn-Mel polyplexes were generated in HEPES-buffered glucose and tested in transfection experiments using luciferase as reporter gene. Cellular cytotoxicity was analyzed by measurement of membrane integrity and metabolic activity. RESULTS: Covalent attachment of DMMAn-modified melittin to PLL resulted in a pH-responsive conjugate. No lytic activity was observed at neutral pH; after acidic cleavage of the protecting groups at pH 5 lytic activity was regained. Acute toxicity was greatly reduced (as compared to PLL-Mel or even unmodified PLL) and high gene expression levels (up to 1800-fold higher than unmodified PLL) were obtained. CONCLUSIONS: Modification of the polycationic carrier PLL with DMMAn-masked melittin not only enhances gene transfer efficiency, but also strongly reduces the acute toxicity of melittin and PLL. Hence this modification might be useful for optimizing polycationic gene carriers.

Investigation of the cytotoxicity of eukaryotic and prokaryotic antimicrobial peptides in intestinal epithelial cells in vitro.

Antimicrobial peptides (AMPs) are a diverse group of proteinaceous compounds ranging in size, complexity and antimicrobial spectrum. The activity of AMPs against gut pathogens warrants the study of the interaction of AMPs with the mammalian gastrointestinal tract. In particular, the investigation of the in vitro cytotoxicity of these peptides is critical before they can be considered in clinical infections. The cytotoxicity of gallidermin, nisin A, natural magainin peptides, and melittin was investigated in two gastrointestinal cell models (HT29 and Caco-2) with the MTT conversion assay, neutral red dye uptake assay and compared with that of vancomycin. The hemolytic activities were also investigated in sheep erythrocytes and the effect of AMPs on paracellular permeability was examined by transepithelial resistance (TEER) and TEM. Gallidermin was the least cytotoxic AMP followed by nisin A, magainin I, magainin II and melittin. Melittin and nisin were the only peptides to result in significant hemolysis. However, while nisin caused hemolysis at concentrations which were 1000-fold higher than those required for antimicrobial activity, melittin was hemolytic at concentrations in the same order of magnitude as its antimicrobial activity. Melittin was the only AMP to affect paracellular permeability. Long term melittin treatment also resulted in loss of microvilli, an increase in cell debris and destruction of intestinal tight junctions and cell-cell adhesion. Gallidermin shows most promise as a therapeutic agent, with relatively low cytotoxicity and potent antimicrobial activities. Melittin, while showing little potential as an antimicrobial agent, may have potential in delivery of poorly bioavailable drugs.

[Effect of melittin on apoptosis and necrosis of U2 OS cells].

OBJECTIVE: To study the effect of melittin on apoptsis and necrosis of osteosarcoma cell line U2 OS in vitro. METHODS: Osteosarcoma cell line U2 OS was treated with melittin. The growth and proliferation was observed by MTT assay and cell counting, and the necrosis was estimated by Trypan blue staining. The cell apoptsis, Fas and Apo2. 7 expression were detected by cytometer. RESULTS: The data showed that melittin could inhibit the proliferation of U2 OS dose-dependently at 16 and 64 mg/L. Cell apoptsis was detected by cytometer, when the cells were treated by 16 mg/L and 32 mg/L of melittin respectively, and the percentages of Fas and Apo2. 7 positive cells were increased. CONCLUSION: Melittin inhibits the proliferation of osterosarcoma cell line through up-regulating Fas expression and inducing apoptsis.

A novel approach of targeted ablation of mammary carcinoma cells through luteinizing hormone receptors using Hecate-CGbeta conjugate.

Recent studies have shown that human and animal mammary gland carcinoma cell line express luteinizing hormone receptors (LHRs). We have examined the cytotoxic effect of Hecate-CGbeta conjugate, that is, fusion of a lytic peptide (Hecate) and a 15-amino acid fragment of the CGbeta-chain in vitro. To test the hypothesis that the Hecate-CGbeta conjugate selectively abolishes cells possessing LHR, estrogen dependent and independent human breast cancer cell lines (MCF-7; MDA-MB-231) and a mouse Leydig tumor cell line (BLT-1) were treated in vitro with Hecate-CGbeta conjugate and Hecate alone. Cytotoxic effects of the Hecate-CGbeta conjugate and the Hecate alone was measured by lactate dehydrogenase (LDH) release immediately after treatment. We observed that the Hecate-CGbeta conjugate selectively, in dose-dependent manner destroys cells possessing LHR in lower concentrations of preparate comparing to the Hecate alone and that the cytotoxic effect is strongly correlated with the number of LHR. Using Western blot analysis we characterized the LHR on membranes of MDA-MB-231, MCF-7 and BLT-1 tumor cell lines. In addition, we showed the evaluation of inhibition potential of the Hecate-CGbeta conjugate to LHR. At a concentration of 33 microM the conjugate inhibited (50%; IC50) the binding of CG to LHR. We suggest further development of this novel approach for the treatment of breast cancer by the Hecate-CGbeta for in vivo trials.

Sequence requirements for the activity of membrane-active peptides.

Synthetic peptides were constructed with the sequence of the first 20 residues of melittin and terminating with a range of different amino acid amides. These were found to have haemolytic and cytolytic activity similar to that of melittin, provided that certain charge constraints were observed. The nature of the 21st residue was not critical except when the residue introduced a negative charge. The presence of at least two positive charges in the molecule was found to be essential for activity. One of these charges could be the amino-terminal amine. Peptides could be inactivated by the addition of a non-acidic presequence which was acetylated at the N-terminus. Introducing a protease cleavable sequence into an N-terminal extension of the peptides produced analogues with low haemolytic activity that could be activated by proteolytic action. A peptide with extra positive charges introduced on the hydrophilic face of the helix possessed a haemolytic activity that was greater than that of melittin.

Soluble expression of a functional recombinant cytolytic immunotoxin in insect cells.

We have previously described the production of a recombinant melittin-based cytolytic immunotoxin (IT), scFv-mel-FLAG, in bacterial cells. While the IT exhibited specific cytotoxicity for a human lymphoblastoid cell line, HMy2, yields from expression were low. Here, we describe a baculovirus expression system for the overexpression and secretion of scFv-mel-FLAG. A novel snake phospholipase A2 inhibitor signal peptide was used to aid in the secretion of the immunotoxin. Sf21 insect cells infected with the recombinant virus secreted soluble scFv-mel-FLAG into the culture medium from which it was purified directly on an affinity column. The final yield of scFv-mel-FLAG was estimated at 3-5 mg/L, which was an improvement of 30-fold compared to expression in the prokaryotic system. The cell binding characteristics of the recombinant IT were assessed by flow cytometry using the antigen expressing cell line HMy2. ScFv-mel-FLAG bound specifically to HMy2 cells in direct binding assays and this binding was completely inhibited in the presence of an excess of soluble antigen. Significant cytotoxicity for HMy2 cells, measured by leakage of cytosolic LDH, was also observed for the IT at a concentration of 60 pmol/10(4) cells. Cytotoxicity was concentration dependent and was specific for antigen-positive cells. Thus the baculovirus expression system, under the control of a novel secretion signal, can be used for the production of soluble and functional recombinant cytolytic immunotoxins. To our knowledge, this is the first report of expression of a recombinant immunotoxin in the baculovirus expression vector system.