PLA2[1]

Melittin is a PLA2-stimulating peptide with immune-related properties that is extracted from bee venom and which raises the activity of high-molecular-weight enzymes [1]. A cytotoxic peptide called melettin is extracted from bee venom. A2780CR and A2780 cells both displayed toxicity to melittin, with IC50 values of 4.5 and 6.8 μg/mL, respectively. Natural antiviral, antibacterial, and anti-inflammatory qualities are present in melittin. In numerous cancer cell lines, including those from the stomach, breast, ovarian, liver, prostate, cervical, and lung malignancies, it has also been demonstrated to have variable anticancer effects. Melittin is an amphipathic hemolytic peptide that may have anti-cancer properties through preventing the growth of cells, triggering apoptosis, and causing immediate necrosis. By blocking the PI3K/Akt/mTOR signaling pathway, melittin can also stop EGF-induced cell invasion, albeit this effect is primarily shown in breast cancer cells [2].

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The role of the phospholipase A2 (PLA2) stimulating protein PLAP in the regulation of PLA2 activity was assessed by determination of the effects of PLAP on two purified PLA2s. An approx. 14 kDa enzyme was purified from mouse thymoma cells, EL-4 cells, by cation ion exchange HPLC and immunoaffinity HPLC (with antiserum to the N-terminal sequence of an inflammatory exudate PLA2). An approx. 110 kDa enzyme was purified from mouse mammary carcinoma derived cells by sequential hydrophobic, anion exchange, hydroxyapatite and gel filtration HPLC. Neither PLAP nor melittin, an immunologically related PLA2 stimulating peptide from bee venom, increased the activity of the high molecular weight enzyme. In contrast, there was more than a 20-fold stimulation of the low molecular weight PLA2 by PLAP and an approx. 5-fold stimulation by melittin. The stimulation of enzyme activity by PLAP was observed at a protein to phospholipid ratio of 1:10(6) while the ratio of melittin to phospholipid was 1:3. Thus, PLAP mediated stimulation of PLA2 activity may include an interaction between PLAP and the enzyme, in contrast to melittin stimulation, which involves interactions between melittin and phospholipid. [1]

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In the present study, liquid chromatography-mass spectrometry (LC-MS) was employed to characterise the metabolic profiles of two human ovarian cancer cell lines A2780 (cisplatin-sensitive) and A2780CR (cisplatin-resistant) in response to their exposure to melittin, a cytotoxic peptide from bee venom. In addition, the metabolomics data were supported by application of Biolog microarray technology to examine the utilisation of carbon sources by the two cell lines. Data extraction with MZmine 2.14 and database searching were applied to provide metabolite lists. Principal component analysis (PCA) gave clear separation between the cisplatin-sensitive and resistant strains and their respective controls. The cisplatin-resistant cells were slightly more sensitive to melittin than the sensitive cells with IC50 values of 4.5 and 6.8 μg/mL respectively, although the latter cell line exhibited the greatest metabolic perturbation upon treatment. The changes induced by melittin in the cisplatin-sensitive cells led mostly to reduced levels of amino acids in the proline/glutamine/arginine pathway, as well as to decreased levels of carnitines, polyamines, adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+). The effects on energy metabolism were supported by the data from the Biolog assays. The lipid compositions of the two cell lines were quite different with the A2780 cells having higher levels of several ether lipids than the A2780CR cells. Melittin also had some effect on the lipid composition of the cells. Overall, this study suggests that melittin might have some potential as an adjuvant therapy in cancer treatment. [2]

LDH Assay [2]
The cytotoxicity of the Melittin was determined by the lactate dehydrogenase (LDH) release assay on A2780 and A2780CR cells. LDH release into the medium is due to the loss of membrane integrity either due to apoptosis or necrosis. Briefly, A2780 and A2780CR cells were seeded at 1 × 104 cells/well in 96-well plates and incubated at 37 °C and 5% CO2 in a humidified atmosphere for 24 h. The cells were treated with different concentrations of the Melittin for 24 h. Then, the supernatant (50 μL) of the treated cells was transferred into 96-well flat-bottomed plates, and 50 μL of the LDH reaction mix (Lactate Dehydrogenase Activity Assay Kit, MAK066) was added for 30 min. Finally, the intensity of orange colour in the samples indicating the LDH activity was measured at 490 nm. LDH release increased in a dose-dependent manner in Melittin treated A2780 and A2780CR cells compared with untreated cells. The values are represented as the means ± SD of three separated experiments.

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Caspase Activity Assay [2]
Fluorometric assays of caspase activity were carried out by using the substrate Ac-DEVD-AMC for caspase-3. Both A2780 and A2780CR cells were seeded at 1 × 104 cells/well in costar 96-well black plates and incubated at 37 °C and 5% CO2 in a humidified atmosphere for 24 h. Then, the cells were treated for 6 and 24 h with different concentrations of Melittin to measure caspase-3 activity. Staurosporine was used to induce apoptosis at a concentration of 10 μM. The control cells were treated with media alone. The caspase-3 assay buffer was prepared as described previously. The caspase-3 assay buffer (3×) was added to each well and incubated at 37 °C in 5% CO2 for 1 h. Fluorescence was measured at 360 nm (excitation) and 460 nm (emission) using a Spectramax M3 microplate reader. The average fluorescence values of the background were subtracted from the fluorescence values of experimental wells. Statistical analysis was done using one-way ANOVA followed by Bonferroni’s Multiple Comparison test.

Cell Viability Assay against Melittin [2]
Melittin was purified from bee venom by reversed phase liquid chromatography and reconstituted in sterile water to form a stock solution of 1 mg/mL before storage at −20 °C until required for analysis. Cell viability was assessed by an Alamar® Blue (AB) cell viability reagent. Both A2780 and A2780CR cells were seeded at 1 × 104 cells/well in 96-well plates and incubated at 37 °C and 5% CO2 in a humidified atmosphere for 24 h. After this incubation period, the cells were treated with various concentrations of Melittin ranging from 0.5 to 14 µg/mL in 100 μL of medium, and re-incubated at 37 °C and 5% CO2 for a further 24 h. Triton X at 1% (v/v) and cell culture media were used as positive and negative controls, respectively. After this, AB was added at a final concentration of 10% (v/v) and the resultant mixture was incubated for a further 4 h at 37 °C and 5% CO2. Then, the plates were read at an excitation wavelength of 560 nm and the emission at 590 nm was recorded on a SpectraMax M3 microplate reader. Background-corrected fluorescence readings were converted to cell viability data for each test well by expressing them as percentages relative to the mean negative control value.

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Determination of Effect of Melittin on Cell Metabolomes [2]
The A2780 and A2780CR cell lines were separately treated with Melittin at concentrations of 6.8 and 4.5 μg/mL respectively for 24 h (n = 5). The cells were seeded at 75 × 104 cells/mL in T-25 cell culture flasks and incubated for 1 doubling time (48 h) before treatment with the Melittin and incubation for an additional 24 h. After the treatment, the medium was removed and the cells were washed twice with 3 mL of phosphate-buffered saline (PBS) at 37 °C before lysis. Cell lysates were prepared by extraction with ice cold methanol:acetonitrile:water (50:30:20) (1 mL per 2 × 106 cells). Lipids were extracted with isopropanol (4 °C). The cells were scraped and cell lysates mixed on a Thermo mixer at 1440 rotations per minute (r.p.m.) for 12 min at 4 °C, before being centrifuged at 13,500 r.p.m. for 15 min at 0 °C. The supernatants were collected and transferred into HPLC vials for LC-MS analysis. During the analysis, the temperature of the autosampler was maintained at 4 °C. Mixtures of authentic standard metabolites, prepared as previously described, and the pooled quality control (QC) sample, were injected in each analysis run in order to facilitate identification and to evaluate the stability and reproducibility of the analytical method, respectively. The pooled QC sample was obtained by taking equal aliquots from all the samples and placing them into the same HPLC vial.

Metabolism / Metabolites
Free toxin may be removed by opsonization via the reticuloendothelial system (primarily the liver and kidneys) or it may be degraded through cellular internalization via the lysosomes. Lysosomes are membrane-enclosed organelles that contain an array of digestive enzymes, including several proteases.

Toxicity Summary
Melittin has strong haemolytic activity. It integrates into cell membranes and has multiple effects, probably, as a result of its interaction with negatively charged phospholipids. Melittin inhibits well known transport pumps such as the Na+-K+-ATPase and the H+-K+-ATPase, and increases the permeability of cell membranes to ions, particularly Na+ and indirectly Ca2+, because of the Na+-Ca2+-exchange. (A608)

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Toxicity Data
LD50: 6.00 mg/kg (Intravenous, Mouse) (A686)

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16133648 rat LD50 intraperitoneal 17700 ug/kg Farmakologiya i Toksikologiya, 37(367), 1974
16133648 rat LDLo intravenous 1400 ug/kg Farmakologiya i Toksikologiya, 37(367), 1974
16133648 mouse LD50 intraperitoneal 7400 ug/kg Farmakologiya i Toksikologiya, 37(367), 1974
16133648 mouse LD50 intravenous 7040 ug/kg Farmakologiya i Toksikologiya, 37(367), 1974
16133648 guinea pig LD50 intraperitoneal 2750 ug/kg Farmakologiya i Toksikologiya, 37(367), 1974

[1]. Responses of purified phospholipases A2 to phospholipase A2 activating protein (PLAP) and Melittin. Biochim Biophys Acta. 1993 Feb 10;1166(1):124-30.

[2]. Metabolomic Profiling of the Effects of Melittin on Cisplatin Resistant and Cisplatin Sensitive Ovarian Cancer Cells Using Mass Spectrometry and Biolog Microarray Technology. Metabolites. 2016 Oct 13;6(4). pii: E35.

Melittin is a 26-membered polypeptide consisting of Gly, Ile, Gly, Ala, Val, Leu, Lys, Val, Leu, Thr, Thr, Gly, Leu, Pro, Ala, Leu, Ile, Ser, Trp, Ile, Lys, Arg, Lys, Arg, Gln and Gln-NH2 residues joined in sequence. It is the principal active component of bee venom. It has a role as an animal metabolite, a venom, an EC 2.7.11.13 (protein kinase C) inhibitor, a hepatoprotective agent, an apoptosis inducer, a neuroprotective agent and an antineoplastic agent. It is a polypeptide and a peptidyl amide.
Melittin has been reported in Apis mellifera with data available.
Basic polypeptide from the venom of the honey bee (Apis mellifera). It contains 26 amino acids, has cytolytic properties, causes contracture of muscle, releases histamine, and disrupts surface tension, probably due to lysis of cell and mitochondrial membranes.
See also: Melittin (annotation moved to).

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In conclusion, this study shows that the cisplatin sensitive A2780 cells contain relatively higher levels of ether lipids and polyamines, which might result in increased membrane stability and repair and thus resistance to the lytic action of melittin in comparison with the cisplatin resistant A2780CR cells. After exposure to melittin, the levels of most of the significantly altered metabolites, particularly amino acids and TCA cycle intermediates, were lower in A2780 compared to A2780CR cells, suggesting different metabolic responses in the two cell lines. The large increases in choline and glycerophosphocholine in A2780 cells may be related to increased de novo lipid synthesis and re-direction of cellular metabolism. Thus, analysis of the full lipidome could offer a more valuable insight. Given that melittin interacts with cell membranes, the observed effects might suggest that the membranes are less adaptable in the cisplatin resistant cells compared to the sensitive ones. Over all, this study shows that a LC-MS based metabolomics approach for the assessment of drug effects in vitro provides a powerful tool for obtaining insights into the mechanism of action of potential therapeutic agents, while offering the possibility to identify key metabolite markers for in vivo monitoring of tumour responsiveness to standard chemotherapy. Melittin might serve as a valuable adjuvant in cancer chemotherapy for overcoming chemoresistance. [2]

C132H230F3N39O33

2960.48

2958.74703

Melittin;20449-79-0

155977526

Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH2;
H-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH2.TFA;
glycyl-L-isoleucyl-glycyl-L-alanyl-L-valyl-L-leucyl-L-lysyl-L-valyl-L-leucyl-L-threonyl-L-threonyl-glycyl-L-leucyl-L-prolyl-L-alanyl-L-leucyl-L-isoleucyl-L-seryl-L-tryptophyl-L-isoleucyl-L-lysyl-L-arginyl-L-lysyl-L-arginyl-L-glutaminyl-L-glutaminamide trifluoroacetic acid

GIGAVLKVLTTGLPALISWIKRKRQQ

Typically exists as White to off-white solid at room temperature

42

42

99

208

6380

28

CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)N)NC(=O)CN.C(=O)(C(F)(F)F)O

ABMAJLCYJGUBMU-CTOWXMLOSA-N

InChI=1S/C131H229N39O31.C2HF3O2/c1-23-71(16)102(163-97(176)60-135)122(194)146-62-98(177)148-74(19)109(181)164-100(69(12)13)124(196)160-88(55-65(4)5)116(188)155-84(41-30-33-51-134)115(187)165-101(70(14)15)125(197)161-90(57-67(8)9)118(190)168-106(77(22)173)128(200)169-105(76(21)172)123(195)147-63-99(178)150-92(58-68(10)11)129(201)170-54-36-44-94(170)121(193)149-75(20)108(180)158-89(56-66(6)7)117(189)166-104(73(18)25-3)127(199)162-93(64-171)120(192)159-91(59-78-61-145-80-38-27-26-37-79(78)80)119(191)167-103(72(17)24-2)126(198)157-83(40-29-32-50-133)111(183)154-85(42-34-52-143-130(139)140)112(184)152-82(39-28-31-49-132)110(182)153-86(43-35-53-144-131(141)142)113(185)156-87(46-48-96(137)175)114(186)151-81(107(138)179)45-47-95(136)174;3-2(4,5)1(6)7/h26-27,37-38,61,65-77,81-94,100-106,145,171-173H,23-25,28-36,39-60,62-64,132-135H2,1-22H3,(H2,136,174)(H2,137,175)(H2,138,179)(H,146,194)(H,147,195)(H,148,177)(H,149,193)(H,150,178)(H,151,186)(H,152,184)(H,153,182)(H,154,183)(H,155,188)(H,156,185)(H,157,198)(H,158,180)(H,159,192)(H,160,196)(H,161,197)(H,162,199)(H,163,176)(H,164,181)(H,165,187)(H,166,189)(H,167,191)(H,168,190)(H,169,200)(H4,139,140,143)(H4,141,142,144);(H,6,7)/t71-,72-,73-,74-,75-,76+,77+,81-,82-,83-,84-,85-,86-,87-,88-,89-,90-,91-,92-,93-,94-,100-,101-,102-,103-,104-,105-,106-;/m0./s1

(2S)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[2-[[(2S,3R)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S,3S)-2-[(2-aminoacetyl)amino]-3-methylpentanoyl]amino]acetyl]amino]propanoyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxybutanoyl]amino]acetyl]amino]-4-methylpentanoyl]pyrrolidine-2-carbonyl]amino]propanoyl]amino]-4-methylpentanoyl]amino]-3-methylpentanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-3-methylpentanoyl]amino]hexanoyl]amino]-5-carbamimidamidopentanoyl]amino]hexanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-oxopentanoyl]amino]pentanediamide;2,2,2-trifluoroacetic acid

Melittin trifluoroacetate; Melittin (trifluoroacetate salt); TP1299L; AKOS040763958; ...; Melittin TFA(20449-79-0(free base))

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :≥ 50 mg/mL (~16.89 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)