melitten and Stomach-Neoplasms

The cytotoxic effects of melittin, a bee-venom peptide, have been widely studied towards cancer cells. Typically, these studies have examined the effect of melittin over extended-time courses (6-24 hours), meaning that immediate cellular interactions have been overlooked. In this work, we demonstrate the rapid effects of melittin on both gastric and colorectal cancer, specifically AGS, COLO205 and HCT-15 cell lines, over a period of 15 minutes. Melittin exhibited a dose dependent effect at 4 hours of treatment, with complete cellular death occurring at the highest dose of 20 μg/mL. Interestingly, when observed at shorter time points, melittin induced cellular changes within seconds; membrane damage was observed as swelling, breakage or blebbing. High-resolution imaging revealed treated cells to be compromised, showing clear change in cellular morphology. After 1 minute of melittin treatment, membrane changes were observed, and intracellular material could be seen expelled from the cells. Overall, these results enhance our understanding of the fast acting anti-cancer effects of melittin.

Topics: Antineoplastic Agents; Apoptosis; Bee Venoms; Cell Line, Tumor; Cell Membrane; Colorectal Neoplasms; Humans; Melitten; Microscopy, Atomic Force; Microscopy, Fluorescence; Stomach Neoplasms

Melittin is the peptide toxin found in bee venom and is effective against cancer cells. To enhance its activity, a branched dimeric form of melittin was designed. The monomeric form of the peptide was more cytotoxic against gastric cancer cells at low concentrations (1-5 μM) than the dimer form, while the cytotoxic effect was comparable at higher concentrations (10 μM). Confocal microscopy showed that both the monomer and dimer forms of melittin with fluorescent label at the C terminus penetrated the cytoplasm and localized at the cell nucleus and disrupted the cell membrane. The results indicated that both peptides localized in the nucleus and no significant difference in penetration was observed between monomer and dimer of melittin. Although the C and N termini are important for melittin activity, using C terminus for dimerization of the peptide resulted in similar activity for the monomer and dimer against bacteria and gastric cancer cells.

Topics: Animals; Anti-Bacterial Agents; Antineoplastic Agents; Bee Venoms; Cell Line, Tumor; Cell Movement; Cell Survival; Dimerization; Female; Gram-Negative Bacteria; Hemolysis; Humans; Melitten; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Confocal; Neoplasm Metastasis; Nuclear Envelope; Protein Conformation; Reactive Oxygen Species; Solid-Phase Synthesis Techniques; Stomach Neoplasms

To investigate the apoptotic effects of melittin on SGC-7901 cells via activation of the mitochondrial signaling pathway in vitro.. SGC-7901 cells were stimulated by melittin, and its effect on proliferation and apoptosis of was investigated by methyl thiazolyl tetrazolium assay, morphologic structure with transmission electron microscopy, annexin-V/propidium iodide double-staining assay, measuring mitochondrial membrane potential (MMP) levels, and analyzing reactive oxygen species (ROS) concentrations were analyzed by flow cytometry. Cytochrome C (Cyt C), apoptosis-inducing factor (AIF), endonuclease G (Endo G), second mitochondria-derived activator of caspases (Smac)/direct IAP binding protein with low isoelectric point (Diablo), and FAS were analyzed by western blot. The expression of caspase-3 and caspase-8 was measured using activity assay kits.. Melittin was incubated at 1.0, 2.0, 4.0, or 6.0 μg/mL for 1, 2, 4, 6, or 8 h and showed a time- and concentration-dependent inhibition of SGC-7901 cell growth. Melittin induced SGC-7901 cell apoptosis, which was confirmed by typical morphological changes. Treatment with 4 μg/mL melittin induced early apoptosis of SGC-7901 cells, and the early apoptosis rates were 39.97% ± 3.19%, 59.27% ± 3.94%, and 71.50% ± 2.87% vs 32.63% ± 2.75% for 1, 2, and 4 h vs 0 h (n = 3, P < 0.05); the ROS levels were 616.53% ± 79.78%, 974.81% ± 102.40%, and 1330.94% ± 93.09% vs 603.74% ± 71.99% (n = 3, P < 0.05); the MMP values were 2.07 ± 0.05, 1.78 ± 0.29, and 1.16 ± 0.25 vs 2.55 ± 0.42 (n = 3, P < 0.05); caspase-3 activity was significantly higher compared to the control (5492.3 ± 321.1, 6562.0 ± 381.3, and 8695.7 ± 449.1 vs 2330.0 ± 121.9), but the caspase activity of the non-tumor cell line L-O2 was not different from that of the control. With the addition of the caspase-3 inhibitor (Ac-DEVD-CHO), caspase-3 activity was significantly decreased compared to the control group (1067.0 ± 132.5 U/g vs 8695.7 ± 449.1 U/g). The expression of the Cyt C, Endo G, and AIF proteins in SGC-7901 cells was significantly higher than those in the control (P < 0.05), while the expression of the Smac/Diablo protein was significantly lower than the control group after melittin exposure (P < 0.01). Ac-DEVD-CHO did not, however, have any effect on the expression of caspase-8 and FAS in the SGC-7901 cells.. Melittin can induce apoptosis of human gastric cancer (GC) cells through the mitochondria pathways, and it may be a potent agent in the treatment of human GC.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Proliferation; Cell Shape; Dose-Response Relationship, Drug; Humans; Melitten; Mitochondria; Mitochondrial Membranes; Oxidative Stress; Reactive Oxygen Species; Signal Transduction; Stomach Neoplasms; Time Factors

It has been previously reported that melittin, the main ingredient of honey bee venom, has anticancer properties. However, there appears to be no earlier study focusing on the isolation of melittin from Iranian honey bee venom (Apis mellifera meda), and evaluation of its effect on cancerous cells.. We isolated melittin using reversed-phase high performance liquid chromatography, and its potential toxicity on gastric cancer AGS cells was determined with an MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay. Furthermore, to ascertain whether melittin induces apoptosis or necrosis in these cells, morphological evaluation, DNA fragmentation assay, propidium podide and annexin-V-FITC dual staining, and flow cytometric analysis were also conducted.. The results of our study suggested that melittin inhibited the proliferation of AGS cells in a dose and time-dependent trend. All of the above four distinct assays indicated that melittin induces necrosis in AGS cells at concentrations of ≥ 1 μg/mL.. The present study indicated that melittin has an anticancer effect on gastric cancer AGS cells and stimulates necrotic cell death in these cells.

Topics: Apoptosis; Bee Venoms; Cell Line, Tumor; Chromatography, High Pressure Liquid; Flow Cytometry; Hemolysis; Humans; Iran; Melitten; Necrosis; Stomach Neoplasms