melitten and Neuroblastoma

Free radicals are involved in neuronal cell death in human neurodegenerative diseases. Since ancient times, honeybee venom has been used in a complementary medicine to treat various diseases and neurologic disorders. Melittin, the main component of honeybee venom, has various biologic effects, including anti-bacterial, anti-viral, and anti-inflammatory activities.. We investigated the neuroprotective effects of melittin against H2O2-induced apoptosis in the human neuroblastoma cell line SH-SY5Y. The neuroprotective effects of melittin on H2O2-induced apoptosis were investigated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide assay, caspase 3 activity, 4,6-diamidino-2-phenylindole staining, a lactate dehydrogenase release assay, Western blots, and reverse transcription-polymerase chain reaction.. The H2O2-treated cells had decreased cell viability with apoptotic features and increased production of caspase-3. On the other hand, melittin treatment increased cell viability and decreased apoptotic DNA fragmentation. Melittin attenuated the H2O2-induced decrease in mRNA and protein production of the anti-apoptotic factor Bcl-2. In addition, melittin inhibited both the H2O2-induced mRNA and protein expression of Bax-associated pro-apoptotic factor and caspase-3.. These findings suggest that melittin has potential therapeutic effects as an agent for the prevention of neurodegenerative diseases.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Survival; DNA Fragmentation; Humans; Hydrogen Peroxide; Melitten; Neuroblastoma; Neuroprotective Agents

Antimicrobial peptides are promising anti-cancer agents with a unique mode of action. We established the usage of a chip-based sensor to monitor the dynamic interplay between cells on the chip and peptides and compared it with endpoint tests. Human neuroblastoma cancer cells and spontaneously immortalized non-cancer keratinocytes were perfused with representative peptides (NK-2, NK11, and melittin). The sensor system enabled continuous recording of cell layer impedance (adhesion/confluence), oxygen consumption (respiration) and extracellular acidification (glycolysis) and provided insights in cell damage, stress response and recovery. Cells responded differentially to peptide treatment. During perfusion, peptides accumulated on the cell surface until they reached a critical concentration. Preceding to cell death, melittin triggered glycolysis, suggesting stress response. NK-2 induced no change in energy metabolism, but led to an increase in impedance, i.e. a temporarily altered morphology, which appeared to be an excellent parameter to detect subtle structural changes of cell layers.

Topics: Cell Line; Cell Line, Tumor; Humans; Hydrophobic and Hydrophilic Interactions; Keratinocytes; Melitten; Neuroblastoma; Peptides

Topics: Animals; Cell Line; Drug Carriers; Endosomes; Gene Transfer Techniques; Hydrogen-Ion Concentration; Maleic Anhydrides; Melitten; Mice; Molecular Structure; Neuroblastoma; Polyamines; Polyelectrolytes; Polyethylene Glycols; RNA Interference; RNA, Small Interfering

The cellular cGMP content increased in response to a variety of receptor agonists, which activate [e.g., prostaglandin (PG) E1, E2, and F2 alpha] or inhibit (e.g., alpha-adrenergic, muscarinic, and opiate agonists) adenylate cyclase in neuroblastoma X glioma hybrid NG108-15 cells. The responses were additive when PGF2 alpha and enkephalin were mixed. The inhibitory guanine nucleotide regulatory protein (Ni) is involved in adenylate cyclase inhibition; this function of Ni is lost when it is ADP-ribosylated by islet-activating protein (IAP), pertussis toxin [H. Kurose, T. Katada, T. Amano, and M. Ui (1983) J. Biol. Chem. 258, 4870-4875]. The cGMP rise induced by stimulation of the receptors linked to adenylate cyclase inhibition was also diminished by IAP; the time course and dose response for the IAP-induced diminution were the same between adenylate cyclase inhibition and cGMP generation. Ni thus appears to mediate guanylate cyclase activation as well as adenylate cyclase inhibition initiated via the same receptors. Melittin also increased cGMP. No additivity was shown when enkephalin and melittin were combined, suggesting that phospholipase A2 might play a role in Ni-mediated guanylate cyclase activation. On the other hand, the PGF2 alpha-induced cGMP rise was associated with increased incorporation of 32Pi into phosphatidylinositol; was not affected by cholera toxin, IAP or forskolin; and showed no additivity when combined with A23187, which increased cGMP by itself. PGs would occupy receptors linked to phosphatidylinositol breakdown, thereby increasing the availability of intracellular Ca2+, which is responsible for guanylate cyclase activation. Thus, dual pathways are proposed for a receptor-mediated cGMP rise in NG108-15 cells.

Topics: Adenylate Cyclase Toxin; Animals; Bacterial Toxins; Calcimycin; Cyclic AMP; Cyclic GMP; Dinoprost; Enkephalins; Glioma; Hybrid Cells; Melitten; Mice; Neuroblastoma; Pertussis Toxin; Phospholipids; Prostaglandins F; Rats; Receptors, Cell Surface; Virulence Factors, Bordetella

Evidence is presented that has led us to abandon the hypothesis that receptor-mediated cyclic GMP formation in cultured nerve cells occurs via the influx of extracellular calcium ions and an increase in the cytosolic free calcium ion concentration. While the cyclic GMP response is absolutely dependent on the presence of Ca2+, there is no increase in free intracellular Ca2+ subsequent to agonist stimulation. Instead, we have found that muscarinic or histamine H1 receptor stimulation elicits the release of arachidonic acid through a quinacrine-sensitive mechanism, possibly phospholipase A2. Inhibition of the release or metabolism of arachidonate by the lipoxygenase pathway prevents receptor-mediated cyclic GMP formation. We hypothesize that neurotransmitter receptors that mediate cyclic GMP synthesis function by releasing arachidonic acid and that an oxidative metabolite of arachidonic acid then stimulates soluble guanylate cyclase.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Cell Line; Cyclic GMP; Lipoxygenase; Melitten; Mice; Neuroblastoma; Receptors, Histamine; Receptors, Histamine H1; Receptors, Muscarinic