melitten and Melanoma

Melanoma has the highest mortality rate among all the types of skin cancer. In melanoma, M2-like tumor-associated macrophages (TAMs) are associated with the invasiveness of tumor cells and a poor prognosis. Hence, the depletion or reduction of M2-TAMs is a therapeutic strategy for the inhibition of tumor progression. The aim of this study was to evaluate the therapeutic effects of M-DM1, which is a conjugation of melittin (M), as a carrier for M2-like TAMs, and mertansine (DM1), as a payload to induce apoptosis of TAMs, in a mouse model of melanoma.. M-DM1 was found to specifically reduce M2-like TAMs in melanoma, which potentially leads to the suppression of tumor growth, migration, and invasion. In addition, we also found that M-DM1 improved the survival rates in a mouse model of melanoma compared to M or DM1 treatment alone. Flow cytometric analysis revealed that M-DM1 enhanced the infiltration of CD8+ cytotoxic T cells and natural killer cells (NK cells) in the tumor microenvironment.. Taken together, our findings highlight that M-DM1 is a prospective agent with enhanced anti-tumor effects.

Topics: Animals; Female; Macrophages; Melanoma; Melitten; Mice; Mice, Inbred C57BL; Prospective Studies; Tumor Microenvironment; Tumor-Associated Macrophages

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.. 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.. 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.. 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.

Topics: Animals; Immunotherapy; Melanoma; Melitten; Mice; Mice, Inbred C57BL; Oncolytic Viruses; Tumor Microenvironment

Melanoma is an immunogenic tumor and a serious type of skin cancer. Tumor-associated macrophages (TAMs) express an M2-like phenotype and are involved in all stages of melanomagenesis; it is hence a promising target for cancer immunotherapy. We herein investigated whether melittin-dKLA inhibits the growth of melanoma by inducing apoptosis of M2-like macrophages. For the in vitro study, a conditioned medium of macrophages was prepared from M0, M1, or M2-differentiated THP-1 cells with and without melittin-dKLA. The affinity of melittin for M2 macrophages was studied with FITC (fluorescein isothiocyanate)-conjugated melittin. For the in vivo study, murine melanoma cells were inoculated subcutaneously in the right flank of mice, melittin-dKLA was intraperitoneally injected at 200 nmol/kg every three days, and flow cytometry analysis of TAMs was performed. Since melittin binds preferentially to M2-like macrophages, melittin-dKLA induced more caspase 3 expression and cell death in M2 macrophages compared with M0 and M1 macrophages and melanoma cells. Melittin-dKLA significantly inhibited the proliferation and migration of M2 macrophages, resulting in a decrease in melanoma tumor growth in vivo. The CD206

Topics: Animals; Cell Line, Tumor; Immunotherapy; Macrophages; Melanoma; Melitten; Mice; Tumor-Associated Macrophages

Malignant melanoma is the deadliest form of skin cancer and highly chemoresistant. Melittin, an amphiphilic peptide containing 26 amino acid residues, is the major active ingredient from bee venom (BV). Although melittin is known to have several biological activities such as anti-inflammatory, antibacterial and anticancer effects, its antimelanoma effect and underlying molecular mechanism have not been fully elucidated. In the current study, we investigated the inhibitory effect and action mechanism of BV and melittin against various melanoma cells including B16F10, A375SM and SK-MEL-28. BV and melittin potently suppressed the growth, clonogenic survival, migration and invasion of melanoma cells. They also reduced the melanin formation in α-melanocyte-stimulating hormone (MSH)-stimulated melanoma cells. Furthermore, BV and melittin induced the apoptosis of melanoma cells by enhancing the activities of caspase-3 and -9. In addition, we demonstrated that the antimelanoma effect of BV and melittin is associated with the downregulation of PI3K/AKT/mTOR and MAPK signaling pathways. We also found that the combination of melittin with the chemotherapeutic agent temozolomide (TMZ) significantly increases the inhibition of growth as well as invasion in melanoma cells compared to melittin or TMZ alone. Taken together, these results suggest that melittin could be potentially applied for the prevention and treatment of malignant melanoma.

Topics: Bee Venoms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Melanoma; Melitten; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Temozolomide; TOR Serine-Threonine Kinases

The cytolytic peptide melittin is a potential anticancer candidate that may be able to overcome tumor drug resistance due to its lytic properties. However, in vivo applications of melittin are limited due to its main side effect, hemolysis, which is especially pronounced following intravenous administration. Here, we designed a hybrid cytolytic peptide, α-melittin, in which the N-terminus of melittin is linked to the C-terminus of an amphipathic α-helical peptide (α-peptide) via a GSG linker. The strong α-helical configuration allows α-melittin to interact with phospholipids and self-assemble into lipid nanoparticles, with a high efficiency for α-melittin encapsulation (>80%) and a strong ability to control the structure of the nanoparticle (~20 nm). This α-melittin-based lipid nanoparticle (α-melittin-NP) efficiently shields the positive charge of melittin (18.70 ± 0.90 mV) within the phospholipid monolayer, resulting in the generation of a neutral nanoparticle (2.45 ± 0.56 mV) with reduced cytotoxicity and a widened safe dosage range. Confocal imaging data confirmed that α-melittin peptides were efficiently released from the nanoparticles and were cytotoxic to the melanoma cells. Finally, α-melittin-NPs were administered to melanoma-bearing mice via intravenous injection. The growth of the melanoma cells was blocked by the α-melittin-NPs, with an 82.8% inhibition rate relative to the PBS-treated control group. No side effects of treatment were found in this study. Thus, the excellent properties of α-melittin-NP give it potential clinical applications in solid tumor therapeutics through intravenous administration.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Diffusion; Lipids; Materials Testing; Melanoma; Melitten; Mice; Nanocapsules; Treatment Outcome

Topics: Animals; Antineoplastic Agents; Bee Venoms; Carcinoma, Squamous Cell; Drug Carriers; Humans; Melanoma; Melitten; Mice; Nanospheres; Neoplasms; Precancerous Conditions; Skin Neoplasms; Xenograft Model Antitumor Assays

Honeybee (Apis mellifera) venom (BV) has been reported to exhibit anticancer effects, but its mode of action at the cellular and molecular levels remains largely unknown. We found that honeybee venom induced apoptosis in human melanoma A2058 cells but not in normal skin fibroblast Detroit 551 cells. The BV-induced apoptosis was accompanied by generation of reactive oxygen species and alteration of mitochondrial membrane potential transition. Treatment with antioxidants significantly attenuated BV-induced apoptosis. Although caspase-2 and -3 were slightly activated by BV, inhibitors of caspase-2 and -3 could not block BV-induced apoptosis in A2058 cells. Data from immunostaining indicated that EndoG and AIF were translocated from mitochondria to the cytosol or nucleus, suggesting that BV induces apoptosis in A2058 cells via a caspase-independent pathway. In addition, cJun N-terminal kinases (JNK) and ERK were rapidly activated after a 5 min incubation with BV, while p38 and AKT were inactivated after 30 min administration of BV. Inhibition of JNK significantly attenuated BV-triggered apoptotic death. Moreover, BV induced a rapid and marked increase in cytosolic calcium ion. Incubation of cells under calcium-free conditions effectively diminished BV-induced apoptosis. Furthermore, when the calcium-free treatment was combined with ouabain, the recovery of cellular calcium fluctuation protected A2058 cells against BV-induced apoptosis. Finally, treatment of A2058 cells with melittin, the major component of BV, resulted in similar elevation of calcium levels and cell killing effects, suggesting that melittin is the major determinant in BV-triggered cell death. These observations provide a molecular explanation for the antiproliferative properties of BV, and suggest that this agent may be useful in treating melanoma.

Topics: Animals; Apoptosis; Bee Venoms; Bees; Calcium; Caspases; Cell Line, Tumor; Cell Survival; Cytosol; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Formazans; Humans; MAP Kinase Kinase 4; Melanoma; Melitten; Membrane Potential, Mitochondrial; Ouabain; Reactive Oxygen Species; Tetrazolium Salts

Melanotropin (MSH) receptor activity in the M2R mouse melanoma cell line is tightly controlled by calcium by an unknown mechanism. The possibility that calcium regulation is mediated by calmodulin or a calmodulin-related calcium binding protein has been addressed in this report by studying the effects of two known calmodulin antagonists, fluphenazine and melittin, on MSH receptor function. Stimulation of adenylate cyclase (AC) in M2R plasma membranes by beta MSH was strongly inhibited by both antagonists. The concentrations of fluphenazine and melittin yielding half-maximal inhibition (IC50) of AC were 16 microM and 2.4 microM, respectively. Both fluphenazine and melittin also inhibit prostaglandin E1-, GTP gamma S, and forskolin-stimulated AC activity, as well as that of unstimulated enzyme, although inhibition is shown to occur at significantly higher concentrations of antagonist. We have shown that the calcium-dependent rate-limiting step in MSH stimulation of adenylate cyclase, that of hormone binding, is strongly inhibited by these antagonists at concentrations identical to, if not lower than, those required for the inhibition of AC activity (fluphenazine-IC50, 14 microM; melittin-IC50, 0.7 microM). The actions of these antagonists, furthermore, appear to be calcium insensitive, as melittin affects the stability of both the high affinity (calcium containing) and low affinity (calcium depleted) receptor-MSH complexes. The sensitivity of the MSH receptor to inhibition by calmodulin antagonists resembles that described for purified calmodulin-sensitive enzyme systems, which suggests a possible role for calmodulin in MSH receptor function. Among peptide hormone receptors, this effect by calmodulin antagonists appears to be unique for the MSH receptor.

Topics: Adenylyl Cyclases; Bee Venoms; Cell Line; Cell Membrane; Fluphenazine; Kinetics; Melanocyte-Stimulating Hormones; Melanoma; Melitten; Receptors, Pituitary Hormone

Topics: Animals; Arachidonic Acid; Bee Venoms; Bees; Cell Differentiation; Cell Division; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Melanoma; Melitten; Mice; Prostaglandins; Tetradecanoylphorbol Acetate