melitten and Necrosis

In a series of in vivo and in vitro experiments, it was shown that membrane disrupting lytic peptides (Hecate, Phor14, or Phor21) conjugated to a 15 amino acid segment of the beta chain of CG or to LHRH were able to target and destroy hormone dependent and independent human prostate cancer xenografts in nude mice. In vitro sensitivity of the cells to the drugs was directly related to LH/CG receptor expression, and pretreatment in vitro or in vivo with estrogens or FSH to enhance LH/CG receptor expression capacity and increased sensitivity to the drugs. Administration of unconjugated Hecate and LHRH was ineffective. Most importantly, all of the lytic peptide-betaCG conjugates tested were highly effective in destroying prostate cancer metastatic cells in lymph nodes, bones and lungs.

Topics: Animals; Carcinoma; Cell Survival; Chorionic Gonadotropin, beta Subunit, Human; Gonadotropin-Releasing Hormone; Humans; Male; Melitten; Necrosis; Neoplasm Metastasis; Prostatic Neoplasms

In this study, we aimed to investigate the testicular toxicity of two molecules derived from bee venom (BV): phospholipase A2 (PlA2) and melittin (Mlt). Ultrastructural effects of purified BV PlA2 and Mlt were assessed consecutive to repeated dose (30 days) and acute toxicity studies. For the subchronic treatment, PlA2 and Mlt were injected in daily doses equivalent to those released by a bee sting (105 μg PlA2/kg/day and 350 μg Mlt/kg/day), while in the acute treatment their doses corresponded to those released by 100 bee stings (9.3 mg PlA2/kg and 31 mg Mlt/kg). Both PlA2 and Mlt affected the Leydig cells and the cells in seminiferous tubules, the Sertoli cells first of all. PlA2 injection resulted in detachment of the Sertoli cells from the surrounding cells, and extracellular vacuolations, cytoplasmic vacuolations in their basal region and in branches as well, detachment of spermatids, residual bodies and sometimes even spermatocytes into the lumen, changes that had a higher magnitude after the acute treatment. Mlt injection induced similar ultrastructural alterations, but more severe, including degeneration of cellular organelles and cellular necrosis, resulting into rarefaction of the seminiferous epithelium; the ultrastructural changes had a higher magnitude after the 30 repeated dose treatment. We concluded that either of the two molecules tested here, PlA2 and Mlt, were Sertoli cells toxicants at the used doses, and they participated both in the BV testicular toxicity. We consider the observed changes as part of a preceding mechanism of the more severe alterations produced by the BV. It also remains possible that these early unspecific changes reported here could represent the response of the SCs not only to the components of bee venom, but to molecules of other venoms as well. The Sertoli cells were the primary target of PlA2 and Mlt in the spermatogenic epithelium, and their alteration led to further degenerative changes of the germ cells. Since the exposure to PlA2 and Mlt caused severe alteration, including cell death and detachment of immature germ cells into the lumen, we may also conclude that the bee venom molecules had a potential to interfere with normal progression of spermatogenesis. All the degenerative changes observed in the Sertoli cells were accompanied with changes of the Leydig cells.

Topics: Animals; Bee Venoms; Male; Melitten; Necrosis; Phospholipases A2; Rats, Wistar; Seminiferous Tubules; Sertoli Cells; Testis; Toxicity Tests, Acute; Toxicity Tests, Subchronic

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

Apoptosis and necrosis reflect the program of cell death employed by a dying cell and the final stage of death, respectively. Whereas apoptosis is defined as a physiological, highly organized cell death process, necrosis is commonly considered to be accidental and uncontrolled. Physiological and weak pathological death stimuli preferentially induce apoptosis, while harsh non-physiological insults often immediately instigate (primary) necrosis. If an apoptosing cell transits into a phase of plasma membrane disintegration, this stage of death is referred to as secondary or post-apoptotic necrosis.Here, we present several conditions that stimulate primary and/or secondary necrosis and show that necrosis displays considerably different time courses. For subclassification of necrotic phenotypes we employed a flow cytometric single-tube 4-color staining technique including annexin A5-FITC, propidium iodide, DiIC1(5), and Hoechst 33342.

Topics: Annexin A5; Apoptosis; Benzimidazoles; Cell Membrane; Cell Survival; DNA; DNA Damage; DNA Fragmentation; Flow Cytometry; Fluorescein-5-isothiocyanate; Freezing; Hot Temperature; Humans; Hydrogen Peroxide; Jurkat Cells; Kinetics; Melitten; Mercuric Chloride; Necrosis; Phenotype; Propidium; Protein Denaturation; Staining and Labeling; Ultraviolet Rays

Melittin, a cationic antimicrobial peptide isolated from the venom of Apis mellifera, has shown potential as a permeability enhancer, transiently increasing intestinal permeability and enhancing the absorption of paracellular markers. Although it is cytotoxic to eukaryotic cells, its cytotoxicity is significantly lower in polarised epithelia compared to non-polarised cells. The aim of this study was to explore the mechanism of melittin cytotoxicity in gastrointestinal cells and to determine whether cytotoxicity was mediated by a necrotic or an apoptotic pathway. The role of cholesterol in melittin cytotoxicity was also examined. Using four distinct assays for apoptosis, phosphatidylserine translocation, caspase activation, DNA ladder formation and cell cycle analysis, no evidence of apoptotic pathway for cell death was observed with any of these approaches. It can therefore be concluded that cytotoxicity was likely to be mediated by necrosis in gastrointestinal epithelial cells. However, at low concentrations of melittin (<1 microM), BRDU uptake was enhanced, demonstrating proliferative effects of melittin at sub-lethal concentrations. Furthermore, melittin cytotoxicity was further enhanced by depletion of cholesterol, using methyl-beta-cyclodextrin, indicating that cholesterol depleting agents could be contradictory to its potential as an enhancer. Overall, although melittin appears to stimulate necrosis, with careful dosage selection the peptide could be considered for the oral delivery of poorly bioavailable drugs.

Topics: Caco-2 Cells; Caspases; Cell Cycle; Cell Line, Tumor; Cell Survival; Cholesterol; DNA; DNA Fragmentation; Epithelial Cells; Gastrointestinal Tract; Humans; Melitten; Necrosis; Phosphatidylserines

Many alternative treatment approaches, originating from Asia, are becoming increasingly popular in the Western hemisphere. Recently, an article published in a renowned journal reported that venom of apis mellifera (bee venom (BV)) and melittin mediate immune-modulating effects by blocking the activation of the transcription factor NF-kappaB. Such a modus operandi would corroborate the many claims of beneficial effects of BV treatment and give immediate credit to this form of therapy. Fibroblast-like synoviocytes from rheumatoid arthritis patients and dermal fibroblast cells and white blood cells from healthy volunteers were used to study the effects of BV and melittin on the activation of NF-kappaB and a series of genes that are markers of inflammation. EMSAs demonstrate that neither BV nor melittin blocked IL-1beta-induced NF-kappaB activation; neither did they affect phosphorylation or degradation of IkappaB. Contrary to published data, even high concentrations of BV and melittin were without any effect on NF-kappaB-p50-DNA interactions. More importantly, in fibroblast-like synoviocytes, but also in dermal fibroblasts as well as in mononuclear cells exposed to BV or melittin, mRNA levels of several proinflammatory genes are significantly increased, and Western blot data show elevated cyclooxygenase-2 protein levels. Furthermore, exposure to BV higher than 10 mug/ml resulted in disintegration of all cell types tested. In addition, large quantities of oxygen radicals are produced in a dose-dependent manner in leukocytes exposed to BV. Taken together, data presented in this work do not corroborate an earlier report regarding the effectiveness of BV as an inhibitor of the transcription factor NF-kappaB.

Topics: Apoptosis; Bee Venoms; Cell Membrane; Cells, Cultured; Cyclooxygenase 2; DNA; Enzyme Activation; Fibroblasts; Gene Expression Regulation; Humans; I-kappa B Proteins; Interleukin-1beta; Melitten; Mitogen-Activated Protein Kinases; Necrosis; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Phosphorylation; Protein Binding; Reactive Oxygen Species; Synovial Membrane; Transcriptional Activation

A Hecate-CGbeta conjugate (lytic peptide and beta-chorionic gonadotropin) selectively destroyed cells possessing LH receptors. This study described functional characteristics of the conjugate and the molecular mechanism of the cell death pathway in prostate cancer cells. Based on in vitro studies, we conclude that the conjugate kills cells possessing luteinizing hormone receptors (LHR) faster than Hecate alone. Competitive studies have shown that blocking of LHR by preincubation with chorionic gonadotropin (100 ng/ml) reduced toxicity of the conjugate in low concentrations. Further studies have also shown that the conjugate in treated cells both did not induce internucleosomal DNA fragmentation and did not induce morphological changes in cells characterized as having apoptotic features. These results proved that cells died by necrosis rather than apoptosis after the conjugate treatment.

Topics: Animals; Cell Line, Tumor; Cell Membrane; Chorionic Gonadotropin; Chorionic Gonadotropin, beta Subunit, Human; Gene Expression; Humans; L-Lactate Dehydrogenase; Male; Melitten; Mice; Necrosis; Prostatic Neoplasms; Receptors, LH; RNA, Messenger

We investigated the antitumoral efficacy, endocrine consequences, and molecular mechanisms underlying cell death induced by the Hecate-chorionic gonadotropin (CG)beta conjugate, a fusion protein of a 23-amino acid lytic peptide Hecate with a 15-amino acid (81-95) fragment of the human CGbeta chain. Transgenic (TG) mice expressing the inhibin alpha-subunit promoter (inhalpha)/Simian Virus 40 T-antigen (Tag) transgene, developing luteinizing hormone (LH) receptor (R) expressing Leydig and granulosa cell tumors, and wild-type control littermates were treated either with vehicle, Hecate, or Hecate-CGbeta conjugate for 3 weeks. Hecate-CGbeta conjugate treatment reduced the testicular and ovarian tumor burden (P < .05), whereas a concomitant increase (testis; P < .05) or no change (ovary) in tumor volumes occured with Hectate treatment. A drop in serum progesterone, produced by the tumors, and an increase in LH levels occured in Hecate-CGbeta treated mice, in comparison with vehicle and Hecate groups, providing further support for the positive treatment response. Hecate-CGbeta conjugate induced a rapid and cell-specific membrane permeabilization of LHR-expressing cells in vitro, suggesting a necrotic mode of cell death without activation of apoptosis. These results prove the principle that the Hecate-CGbeta conjugate provides a novel specific lead into gonadal somatic cell cancer therapy by targeted destruction of LHR-expressing tumor cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blotting, Northern; Caspase 3; Caspases; Cell Death; Cell Line, Tumor; Cell Separation; Chorionic Gonadotropin, beta Subunit, Human; Disease Models, Animal; Enzyme Activation; Female; Flow Cytometry; Granulosa Cell Tumor; Humans; Leydig Cell Tumor; Male; Melitten; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Necrosis; Ovarian Neoplasms; Progesterone; Promoter Regions, Genetic; Protein Structure, Tertiary; Receptors, LH; Recombinant Fusion Proteins; Testicular Neoplasms; Time Factors

A conjugate of a lytic peptide, hecate, and a 15-amino acid segment of the beta-chain of chorionic gonadotropin (CG) destroyed human prostate xenografts in nude mice by targeting LH receptors. Since these xenografts also express LHRH receptors, we prepared a LHRH-hecate conjugate and tested its ability to destroy PC-3 cells in vitro and in vivo.. LHRH-hecate was added to cultures of PC-3, BRF 41 T, DU145, and LNCaP cells in the presence and absence of steroids. PC-3 xenografts were established in nude male mice, which were treated with LHRH-hecate.. Injections of LHRH-hecate resulted in tumor growth arrest and marked reduction of tumor burden (62.2 mg/g body weight in saline controls vs. 10.5 mg/g body weight in treated mice; P < 0.0001); unconjugated LHRH and hecate had no effect on tumor burden and tumor viability (48.5 mg/g body weight in LHRH treated animals vs. 63.2 mg/g body weight in hecate treated mice). Marked tumor necrosis occurred in conjugate treated mice. Removal of steroids from the culture media decreased the sensitivity of LNCaP and PC-3 cells to the LHRH-hecate; adding estrogen restored the sensitivity.. LHRH-hecate may be effective in treating hormone dependent and independent prostate cancers.

Topics: Animals; Body Weight; Cell Death; Culture Media; Gonadotropin-Releasing Hormone; Humans; Male; Melitten; Mice; Mice, Nude; Necrosis; Neoplasms, Experimental; Peptides; Prostatic Neoplasms; Receptors, LHRH; Steroids; Transplantation, Heterologous; Tumor Cells, Cultured

Melittin and phospholipase A2 (PLA2) from bee (Apis mellifera) venom were rested for their ability to induce necrosis of skeletal muscle cells after intramuscular injection into mice. Light and electron microscopic examination of tissue indicated that both melittin (4 micrograms/g) and bee venom PLA2 (4 micrograms/g) caused necrosis of skeletal muscle cells within 30 min after i.m. injection. Early changes in the cells consisted of delta lesions, indicating a ruptured plasma membrane, and hypercontraction of myofibrils. By 24 hr the affected cells appeared as an amorphous mass of disorganized and disrupted myofibrils contained in an intact basal lamina. To ensure that the myotoxic activity of the melittin preparation was not due to contaminating. PLA2 activity, the preparation was treated with p-bromophenacyl bromide (p-BPB), a known inhibitor of PLA2 activity. The p-BPB-treated melittin was determined to have no detectable PLA2 activity using a sensitive muscle cell culture assay, and it still induced myonecrosis, although to a lesser extent and of a slower onset. Additionally, p-BPB treatment of purified bee venom PLA2 completely inhibited its myotoxic activity. These results indicate that both melittin and bee venom PLA2 are capable of inducing necrosis of skeletal muscle cells upon i.m. injection, and that the catalytic and myotoxic activities of bee venom PLA2 are inihibited by p-BPB. Also, melittin and contaminating PLA2 in the melittin fraction may be acting synergistically to induce a stronger and more rapid myotoxic effect than occurs with either alone.

Topics: Acetophenones; Animals; Bee Venoms; Cells, Cultured; Enzyme Activation; Female; Melitten; Mice; Mice, Inbred Strains; Muscle, Skeletal; Necrosis; Neurotoxins; Phospholipases A; Phospholipases A2

Lipocortin I, also called annexin I, a calcium and phospholipid binding protein, protected rat thymocytes from H2O2-elicited necrosis and facilitated H2O2-induced apoptosis, while anti-lipocortin I antibody enhanced H202-elicited necrosis by blocking H202-induced apoptosis. Essentially similar results were obtained with phospholipase A2 inhibitors and activators such as 3,4-octyadecyl-benzylacrylic acid and melittin, respectively. Available evidence suggests that lipocortin I modulates signals for cell death pathways of H2O2-treated rat thymocytes to apoptosis and necrosis by regulating cellular phospholipase A2 activities but not by inhibiting membrane lipid peroxidation.

Topics: Animals; Annexin A1; Antibodies; Apoptosis; Arachidonic Acid; Cell Death; Cells, Cultured; DNA; Dose-Response Relationship, Drug; Hydrogen Peroxide; Lipid Peroxidation; Male; Melitten; Necrosis; Phospholipids; Rats; Rats, Sprague-Dawley; Thymus Gland

Although hepatocellular carcinoma (HCC) cells are more resistant to anoxic injury than normal hepatocytes, the mechanisms responsible for this differential sensitivity remain obscure. Because enhanced calpain protease activity contributes to hepatocyte necrosis, we tested the hypothesis that HCC cells resist anoxia by preventing calpain activation. Cell viability in two rat HCC cell lines (N1S1 and McA-RH7777 cells) was fourfold greater compared to rat hepatocytes after 4 h of anoxia. Although calpain activity increased twofold in rat hepatocytes during anoxia, no increase in calpain activity occurred in HCC cells. Western and Northern blot analysis revealed greater or equivalent expression of calpains and calpastatin in HCC cells compared to hepatocytes. Because increases in cytosolic free Ca++ (Cai++) and phospholipid degradation products regulate calpains in vitro, we measured Cai++ and phospholipid degradation. Ca++i did not change in any cell types during 60 min of anoxia. In contrast, phospholipid degradation was fourfold greater in hepatocytes compared to HCC cells. Melittin, a phospholipase A2 activator, increased calpain activity and cell necrosis in all cell types; melittin-induced cell necrosis was ameliorated by a calpain protease inhibitor. In summary, these data demonstrate for the first time 1) calpain activation without a measureable increase in Ca++i, 2) phospholipase-mediated calpain activation in hepatocytes and HCC cells, and 3) the adaptive mechanism responsible for the resistance of HCC cells to anoxia-an inhibition of phospholipid-mediated calpain activation. Interruption of phospholipase-mediated calpain activation may be a therapeutic strategy for preventing anoxic cell injury.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium-Binding Proteins; Calpain; Cell Hypoxia; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Enzyme Activation; Gene Expression Regulation, Neoplastic; Liver; Liver Neoplasms, Experimental; Male; Melitten; Necrosis; Phospholipases; Rats; Rats, Sprague-Dawley; Tumor Cells, Cultured