Bee Venom Components as Therapeutic Tools against Prostate Cancer.

Prostate cancer is one of the most common cancers in men. Despite the development of a variety of therapeutic agents to treat either metastatic hormone-sensitive prostate cancer, advanced prostate cancer, or nonmetastatic/metastatic castration-resistant prostate cancer, the progression or spread of the disease often cannot be avoided. Additionally, the development of resistance of prostate cancer cells to available therapeutic agents is a well-known problem. Despite extensive and cost-intensive research over decades, curative therapy for metastatic prostate cancer is still not available. Therefore, additional therapeutic agents are still needed. The animal kingdom offers a valuable source of natural substances used for the treatment of a variety of diseases. Bee venom of the honeybee is a mixture of many components. It contains proteins acting as enzymes such as phospholipase A2, smaller proteins and peptides such as melittin and apamin, phospholipids, and physiologically active amines such as histamine, dopamine, and noradrenaline. Melittin has been shown to induce apoptosis in different cancer cell lines, including prostate cancer cell lines. It also influences cell proliferation, angiogenesis, and necrosis as well as motility, migration, metastasis, and invasion of tumour cells. Hence, it represents an interesting anticancer agent. In this review article, studies about the effect of bee venom components on prostate cancer cells are discussed. An electronic literature research was performed utilising PubMed in February 2021. All scientific publications, which examine this interesting subject, are discussed. Furthermore, the different types of application of these promising substances are outlined. The studies clearly indicate that bee venom or melittin exhibited anticancer effects in various prostate cancer cell lines and in xenografts. In most of the studies, a combination of bee venom or the modified melittin with another molecule was utilised in order to avoid side effects and, additionally, to target selectively the prostate cancer cells or the surrounding tissue. The studies showed that systemic side effects and unwanted damage to healthy tissue and organs could be minimised when the anticancer drug was not activated until binding to the cancer cells or the surrounding tissue. Different targets were used, such as the matrix metalloproteinase 2, hormone receptors expressed by prostate cancer cells, the extracellular domain of PSMA, and the fibroblast activation protein occurring in the stroma of prostate cancer cells. Another approach used loaded phosphate micelles, which were cleaved by the enzyme secretory phospholipase A2 produced by prostate cancer cells. In a totally different approach, targeted nanoparticles containing the melittin gene were used for prostate cancer gene therapy. By the targeted nonviral gene delivery, the gene encoding melittin was delivered to the prostate cancer cells without systemic side effects. This review of the scientific literature reveals totally different approaches using bee venom, melittin, modified melittin, or protoxin as anticancer agents. The toxic agents acted through several different mechanisms to produce their anti-prostate cancer effects. These mechanisms are not fully understood yet and more experimental studies are necessary to reveal the complete mode of action. Nevertheless, the researchers have conducted pioneering work. Based on these results, further experimental and clinical studies about melittin and modifications of this interesting agent deriving from nature are necessary and could possibly lead to a complementary treatment option for prostate cancer.

Bee Venom and Its Sub-Components: Characterization, Pharmacology, and Therapeutics.

Bee venom, which is a complex substance produced by Apis mellifera, is widely used to treat various diseases, such as pain [...].

Apamin from bee venom suppresses inflammation in a murine model of gouty arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Bee venom (BV) has been used for the treatment of inflammatory diseases, such as rheumatoid arthritis, and for the relief of pain in traditional oriental medicine. AIM OF STUDY: The aim of this study was to determine the anti-inflammatory effect of BV on monosodium urate (MSU)-induced gouty arthritis in a mouse model. MATERIALS AND METHODS: To develop a mouse model of acute gouty arthritis, 4 mg 50 µL-1 of MSU crystal suspension was injected intradermally into the right paw. After MSU crystal injection, we evaluated inflammatory cytokine production in mice of the BV-treated (0.5 and 1 mg kg-1 body weight) and apamin (APM)-treated (0.5 and 1 mg kg-1 body weight) groups. The positive control group was administered a colchicine (1 mg kg-1 body weight) injection with MSU crystals. RESULTS: BV and APM treatment suppressed inflammatory paw edema in MSU-administered mice. It also exerted anti-inflammatory effects in mice with gouty arthritis by inhibiting proinflammatory cytokine production and inflammasome formation. Interestingly, MSU crystal formation was decreased by BV and APM treatment. CONCLUSIONS: These results suggest that the APM from BV might be useful for the treatment of gouty arthritis due to its anti-inflammatory activities.

Therapeutic Effects of Apamin as a Bee Venom Component for Non-Neoplastic Disease.

Bee venom is a natural toxin produced by honeybees and plays an important role in defending bee colonies. Bee venom has several kinds of peptides, including melittin, apamin, adolapamine, and mast cell degranulation peptides. Apamin accounts for about 2%-3% dry weight of bee venom and is a peptide neurotoxin that contains 18 amino acid residues that are tightly crosslinked by two disulfide bonds. It is well known for its pharmacological functions, which irreversibly block Ca2+-activated K+ (SK) channels. Apamin regulates gene expression in various signal transduction pathways involved in cell development. The aim of this study was to review the current understanding of apamin in the treatment of apoptosis, fibrosis, and central nervous system diseases, which are the pathological processes of various diseases. Apamin's potential therapeutic and pharmacological applications are also discussed.

Expanding the MiniAp-4 BBB-shuttle family: Evaluation of proline cis-trans ratio as tool to fine-tune transport.

Venoms have recently emerged as a promising field in drug discovery due to their good selectivity and affinity for a wide range of biological targets. Among their multiple potential applications, venoms are a rich source of blood-brain barrier (BBB) peptide shuttles. We previously described a short nontoxic derivative of apamin, MiniAp-4, which can transport a wide range of cargoes across the BBB. Here, we have studied the conformation of the proline residue of a range of MiniAp-4 analogues by high-field NMR techniques, with the aim to identify whether there is a direct relation between the cis/trans population and a range of features, such as the capacity to transport molecules across a human-based cellular model and stability in various media. The most promising candidate showed improved transport properties for a relevant small fluorophore.

Application of free-flow electrophoresis for isolation and purification of proteins and peptides.

Free-flow electrophoresis (FFE) has been applied to the separation and purification of a variety of proteins and polypeptides: bee venom, tumor necrosis factor, interleukin-1beta, interferon-gamma and superoxide dismutase. FFE at constant pH and conductivity of the carrying buffer is shown to be efficient at various separation schemes. In some cases, the method allows us to obtain proteins with a purity of more than 90% at a productivity of 20-30 mg/h. An electrophoretic apparatus with a new, multi-sectional construction of the electrophoretic chamber and a system for cross-displacement of carrying buffer in the chamber is described.

On-line liquid chromatography/electrospray tandem mass spectrometry to investigate acrylamide adducts with cysteine residues: implications for polyacrylamide gel electrophoresis separations of proteins.

Adduction between acrylamide and cysteine residues is a post-translational modification associated with proteins separated by gel electrophoresis. In the present article, three model peptides containing 2-4 cysteine residues were reduced with dithiothreitol, incubated with acrylamide monomers and examined by on-line liquid chromatography coupled to electrospray tandem mass spectrometry. Each of the solutions examined in this work revealed the presence of four distinct components: the free peptide, two different peptide-acrylamide 1:1 adducts involving two cysteine residues at different positions within the same sequence, and the peptide-acrylamide 1:2 adducts. The use of liquid chromatography allowed the separation of components which differed only by the site of complexation of acrylamide, while the application of tandem mass spectrometry furnished reliable sequencing information permitting the identification of most cysteine residues involved in such complexation.

Photolabile derivatives of 125I-apamin: defining the structural criteria required for labeling high and low molecular mass polypeptides associated with small conductance Ca(2+)-activated K+ channels.

The structure of apamin-sensitive Ca(2+)-activated K+ channels has been investigated using high-affinity, photolabile azidoaryl derivatives of 125I-[alpha-formyl-Cys1]apamin and 125I-[epsilon-formyl-Lys4]-apamin. Labeling patterns suggest that similar structural constraints are required for labeling analogous polypeptides associated with distinct channel subtypes. When photoprobes are coupled at the epsilon-amino-Lys4 position of apamin, comparable low molecular mass (approximately 30 kDa) polypeptides are efficiently labeled on either brain or liver plasma membranes, irrespective of the structure of the photoprobe. However, when photoprobes are coupled at the alpha-amino-Cys1 position of apamin, the pattern of labeling on both brain and liver plasma membranes varies, depending upon the length of the spacer arm incorporated into the photoprobe. Spacer arms of approximately 8-9 A efficiently label only high molecular mass polypeptides (86, 59 kDa), accompanied by weak, variable labeling of a 44-kDa component. A shorter spacer arm (5.7 A) results in feeble labeling of 86- and 59-kDa polypeptides and barely detectable labeling of 44- and approximately 30-kDa polypeptides. In contrast, a long spacer arm (12.8 A) efficiently labels only approximately 30-kDa polypeptides. These findings point to close similarities in the topography of the 125I-apamin binding site present on pharmacologically distinct subtypes of apamin-sensitive Ca2+-activated K+ channels and indicates that heterooligomeric association of high and low molecular mass polypeptide subunits may be a general structural feature of members belonging to this family of K+ channels.

Identification of two toxins from scorpion (Leiurus quinquestriatus) venom which block distinct classes of calcium-activated potassium channel.

Two polypeptide toxins from scorpion (Leiurus quinquestriatus) venom which block distinct classes of calcium-activated potassium channels have been identified and partially purified. One toxin, at 50-100 ng/ml, blocks apamin-sensitive potassium fluxes in hepatocytes and inhibits [125I]monoiodoapamin binding. The other, more basic, toxin blocks apamin-insensitive potassium fluxes in erythrocytes at 200 ng/ml and, to our knowledge, is the first toxin shown to block the erythrocyte calcium-activated potassium channel with high affinity. The possible co-identity of this latter toxin with charybdotoxin is discussed.

Molecular structure of rat brain apamin receptor: differential photoaffinity labeling of putative K+ channel subunits and target size analysis.

Two photoreactive apamin derivatives were prepared with an aryl azide [[(azidonitrophenyl)amino]acetate (ANPAA)] group coupled at different positions on the neurotoxin molecule. These ligands were used to identify membrane components in the environment of the neuronal binding site that is associated with a Ca2+-activated K+ channel. 125I-[alpha-ANPAA-Cys1] apamin labeled a single Mr 86 000 chain in cultured neurons whereas two bands corresponding to Mr 86 000 and 59,000 were detected in synaptic membrane preparations, suggesting that the Mr 59,000 polypeptide may be a degradation product. 125I-[epsilon-ANPAA-Lys4]apamin however incorporated uniquely into two smaller components with Mr 33,000 and 22,000 in both cultured neurons and synaptic membranes. Randomly modified 125I-ANPAA-apamin gave a cross-linking profile equivalent to the sum of those obtained with the two defined derivatives. The apamin binding site seems to be located at the frontier between three or more putative K+ channel subunits which are only accessible from limited regions of the receptor-associated photoprobe. Irradiation of frozen rat brain membranes with high-energy electrons led to a reduction in 125I-apamin receptor capacity, yielding a target size for the functional binding unit of Mr 84,000-115,000, which could be constituted by the Mr 86,000 subunit alone or by the Mr 86,000 subunit in conjuction with one of the two smaller subunits.

Solid phase synthesis of apamin, the principal neurotoxin in bee venom. Isolation and characterization of acetamidomethyl apamin.

The synthesis of apamin, the principal neurotoxin in bee venom, has been accomplished by the solid phase method on a benzhydrylamine resin, 2-Phenylisopropyloxycarbonyl amino acids were used throughout the synthesis except for the C-terminal histidine. Improved yields in the coupling steps in the N-terminal part of the molecule were obtained by coupling each amino acid both in dichloromethane and dimethylformamide. The use of acetamidomethyl as an S-protecting group for cysteine made it possible to isolate and purify the linear peptide. The deblocked and oxidized peptide was fractionated by ion-exchange chromatography (Bio-Rex 70) to obtain a highly purified apamin with full biological activity and with the same physical and chemical properties as the natural peptide. Circular dichroism (CD) spectra of the synthetic and natural apamin were identical.