Polypeptides as therapeutic substances are regarded as being highly specific, efficacious, relatively safe, and well tolerated, and as a result there is growing interest in their therapeutic use in a wide range of disease areas such as antibiotic-resistant bacteria, cancer, and metabolic disorders . Polypeptide agents with potential antiproliferative activity are often small, with a cationic amphipathic structure that enables them to interact with anionic constituents on lipid membranes [2, 3]. The substances available from natural sources as the quintessence of natural evolutionary programs seem to be unlimited, and the value of natural products as sources of new drugs is highlighted by the large proportion of drugs in clinical use having a natural product origin. Nature produces an impressive myriad variety of biologically active peptides, and, therefore, presents one of the most promising sources for peptide drug discovery. Animals are a treasure trove of bioactive peptides that have evolved high affinity and selectivity for a diverse range of biological targets. Among the animals that produce pharmacologically active molecules are arthropods, such as scorpions, bees, wasps, spiders, and ants, whose venoms have been found to contain substances with great antiproliferative potential.
Antlions are the larvae of the
The present study included only
Antlions of about 3–4 mm in length were collected in Zhanjiang, Guangdong province, and washed clean with distilled water. The larvae were divided into 10 g aliquots and preserved at −80 °C. Antlions (about 250–300 larvae) were ground with liquid nitrogen and homogenized in 250 mL of 50 mM sodium phosphate buffer (pH 7.4) with 0.15 M NaCl. After leaching overnight at 4 °C, the mixture was centrifuged at 15,000 ×
Protein concentration was determined by the method of Bradford . Bovine serum albumin (BSA, 1 mg/mL) solution was used as standard. The absorbance of the dye–protein reaction mixtures in the wells of 96-well plates was measured at 570 nm using a microplate reading spectrophotometer (iMark, Bio-Rad).
The protein and polypeptide components of the antlion extract and purified fractions were separated, and the molecular weights of the components were determined by modified Tricine–sodium dodecyl (SDS)–polyacrylamide gel electrophoresis (PAGE) using a 14% gel (3.3% crosslinking) with 0.1% SDS in the electrode and gel buffers [13, 14]. Coomassie Brilliant Blue R-250 (0.1% w/v) was used to stain the protein bands. We used a LowRange Protein molecular weight marker set (catalog No. C600201; Sangon Biotech, Shanghai, China).
Sephacryl-S100-HR (GE Healthcare) was packed into a column (16 mm × 60 mm), which was equilibrated with 2 column volumes of 50 mM Tris-HCl buffer (pH 7.4) containing 0.15 M NaCl at 1 mL/min. After concentration by ultrafiltration using a Microsep centrifugal device (3 kDa molecular weight cut-off, Pall Corporation), 1 mL of the proteinaceous antlion extract was loaded onto the top of the gel. The column was eluted with the equilibration buffer at 0.5 mL/min, and the eluent was collected into 2 mL aliquots. The antiproliferative activity of each fraction against MG-63 human osteosarcoma cells was measured using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay.
Q-Sepharose-FF (GE Healthcare) was packed into a column (10 mm × 100 mm), which was equilibrated with 5 column volumes of 50 mM Tris-HCl (pH 7.6) at 5 mL/min. After gel filtration, fractions enriched in low-molecular-weight antiproliferative active polypeptide component were desalted against 50 mM Tris-HCl (pH 7.6) by ultrafiltration using a Microsep device (3kDa molecular weight cut-off), and the mixture loaded onto the ion exchange column at 2 mL/min. The column containing the loaded polypeptide mixture was then eluted with about 5 column volumes of equilibration buffer until absorbance had reached baseline, and the bound proteins were then eluted with 5 column volumes of 50 mM Tris-HCl (pH 7.6) containing a stepwise gradient of NaCl (0–0.5 M).
The human osteosarcoma cell line MG-63 (ATCC CRL-1427) was cultured in Eagle's Minimum Essential Medium (MEM), and the mouse osteoblast cell line MC3T3-E1 subclone 14 derived from osteoblast precursors of the parietal calvaria (ATCC CRL-2594) was cultured in alpha Minimum Essential Medium (α-MEM). Both media were supplemented with 10% fetal bovine serum, and the cells were incubated at 37 °C under an atmosphere of 5% CO2. Both cell lines were subcultured every 4–5 d after reaching 85%–95% confluence with medium renewal every 2–3 d. The cell cultures were trypsinized and adjusted to 5 × 104 cells/mL. Cell suspensions were seeded in 96-well plates at 5 × 103 cells per well (100 μL/well) and cultured overnight until cells had adhered to the well wall. The cells in the 96-well plate were treated for 48 h with purified antlion antiproliferative polypeptide (ALAPP) in a gradient of concentrations or with phosphate-buffered saline (PBS) as control. The morphological characteristics and changes in cells were evaluated using an inverted phase contrast microscope EVOS FL Auto Cell Imaging System fitted with a 20×LPlan FH objective (catalog No. AMEP-4682; Thermo Fisher Scientific) in monochrome transmission mode.
Cell viability and, therefore, antiproliferative activity was determined using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the conversion of pale yellow MTT tetrazole to purple formazan crystals by viable cells . The MTT reagent (10 μL) was added to each well of a 96-well plate containing cell culture and incubated for 4 h at 37 °C. After incubation, 100 μL of MTT solubilization buffer (equal to the volume of the original culture medium) was added to each well to dissolve the formazan crystals. Within 1 h, the absorbance of the well contents was measured at 490 nm on a microplate reading spectrophotometer (iMark).
To determine the polypeptide stability at various pH, 50 mM acetate buffer (for pH 2–5.5), 50 mM Tris-HCl buffer (for pH 6–8.5), and 20 mM sodium carbonate-NaOH buffer (for pH 9–11) were used for a pH gradient (with an interval of 0.5 pH units). The ALAPP was incubated at each pH for 60 min at 4 °C. The pH was adjusted to 7.2 before measuring the antiproliferative activity of the treated peptide against MG-63 osteosarcoma cells using an MTT assay. The effect of temperature on polypeptide stability was tested after incubation at 20–70 °C in a water bath for 60 min followed by cooling in an ice bath. The antiproliferative activity of the heat-treated ALAPP against MG-63 osteosarcoma cells was then measured using an MTT assay.
Metal ions were added to the solution of ALAPP in 50 mM Tris-HCl (pH 7.6) at final concentrations varying from 0 to 30 mM to test their effect on the antiproliferative activity of the peptide against MG-63 osteosarcoma cells. The metal ions tested included Na+, K+, Mg2+, and Ca2+. Mixtures of ALAPP and the individual types and concentrations of metal ions were incubated at 25 °C for 30 min, and the relative antiproliferative activity of the mixtures was measured using an MTT assay against a control without any metal ions.
About 5 mg of cell pellet was collected and mixed with 100 μL xTractor Buffer (Clontech). The crude lysate was centrifuged at 12,000 ×
All data are presented as mean and standard deviation. Assays were conducted in triplicate. Significant difference at the level of
About 232 mL of a crude proteinaceous extract of antlions with a concentration of 5.2 mg/mL was obtained (about 12% total yield protein). The crude protein extract was concentrated by ultrafiltration and was applied to a Sephacryl-S100-HR column eluted with 50 mM Tris-HCl buffer (pH 7.4) containing 0.15 M NaCl. The gel filtration resulted in the separation of 7 protein peaks (
After the 3 steps of purification, a polypeptide with antiproliferative activity against MG-63 osteosarcoma cells (ALAPP) was purified from the crude antlion extract ( MTDVKTTELNNEDVQNFTVSSSNDPKNMQELTQYVQTLLQTMQDKFQTMSDQIINRIDEMGNRIDDLEKNIADLMTQAGVEGPDK with a theoretical molecular weight of 9.714 kDa and pI 4.11 as calculated using the tool on the Expasy website (
MTDVKTTELNNEDVQNFTVSSSNDPKNMQELTQYVQTLLQTMQDKFQTMSDQIINRIDEMGNRIDDLEKNIADLMTQAGVEGPDK with a theoretical molecular weight of 9.714 kDa and pI 4.11 as calculated using the tool on the Expasy website (
Purification of ALAPP from
|Crude protein extract||1206||1845||1.0||12|
|Gel filtration chromatography||60.2||102||18.2||5|
|Ion exchange chromatography||1.67||30.5||3.33||2.8|
Against MG-63 osteosarcoma cells.
ALAPP, antlion antiproliferative polypeptide.
Antiproliferative activity of ALAPP at various pH and temperature was examined using an MTT assay of variously treated cell cultures. The effect of pH on the activity of ALAPP was relatively small. The activity of ALAPP was higher under acidic conditions than it was under alkaline conditions (
The antiproliferative activity of ALAPP was not sensitive to any of the metal ions tested (
Morphological characteristics and changes in MG-63 and MC3T3 cells were evaluated after being treated for 48 h by purified ALAPP at a gradient of concentrations (10–50 μg/mL) or with PBS as a control. MC3T3 osteoblasts showed a healthy and intact fusiform morphology at each concentration of ALAPP tested (
MG-63 and MC3T3 cell viability after 48 h treatment with ALAPP was determined using an MTT assay. As the concentration of ALAPP increased, the viability of MC3T3 osteoblasts showed a slight decrease (>90%), but MG-63 osteosarcoma cells showed a steady decline in viability (
The peptide sequence of ALAPP has a 56% identity with human heat shock factor binding protein 1 (HSBP1; NCBI sequence ID: NP_001528.1) using UniProt Align (
An 85-amino-acid polypeptide with a calculated molecular weight of about 9.7 kDa and remarkable antiproliferative activity against MG-63 osteosarcoma cells was purified from antlion, and identified as a homolog of HSBP1.
A point emphasized in TCM descriptions of formulae containing antlion recommended for treating tumors is that the antlion powder should be dissolved in warm water , although boiling water is used for most formulae in TCM. The active ingredients in TCM that cannot be continuously boiled in water are usually not resistant to high temperatures. Naturally occurring small molecules are usually the active components of botanically based TCM, while the active components of animal substance-based TCM are often peptides. Therefore, we screened the peptides found in antlion for their antiproliferative activity against osteosarcoma cells. Gel filtration chromatography indicated 2 peaks (Peak 1 and Peak 6) of antiproliferative activity. Peak 1 would mainly consist of proteins >100 kDa. The active component with inhibitory activity against osteosarcoma in Peak 1 is perhaps the 165 kDa protein toxin, whose paralytic activity against cockroaches was identified by others as about 130-fold higher than that of tetrodotoxin [22, 23]. Because large proteins have low stability and poor cell permeability compared with smaller polypeptides, Peak 6, comprised of low-molecular-weight polypeptides <10 kDa, was selected for further examination.
Microbial communities, which are symbiotic in the esophagus and intestines of insects, often play important roles in the nutrition, reproduction, development, and even behavior of their hosts. A wide variety of bacteria have been reported to be endosymbiotic in the gut and esophagus of antlion [6, 24, 25]. These endosymbiotic bacteria of antlion play important roles in their hunting behavior, because some toxins, which have been implicated as contributing to the paralyzation and death of prey, are proven to be produced by bacterial isolates. Yoshida et al.  reported an insecticidal 63 kDa protein purified from the saliva of antlion, as produced by the endosymbiont
Not only is ALAPP homologous to heat shock factor-binding protein 1 isoform X1 from
If ALAPP is a protein factor of the HSBP1 class, then the biochemical properties of ALAPP are well understood. The HSBP1 family members are comprised solely of a putative coiled-coil oligomerization domain without any other readily recognizable structural or functional motif . In the α-helix of HSBP1, all of residues are hydrophobic with an exception of Ser31, as it is energetically unfavorable to the structure stability, but likely bears some biological function [30, 31]. If the mode of action of ALAPP is to interact with the DNA-binding protein HSF1, as shown for HSBP1 located in the nucleus , it remains unclear how ALAPP incubated with cells can cross the cellular membrane and reach the nucleus when it apparently has an anionic amphipathic structure with a pI 4.11. Nevertheless, ALAPP has high stability to a wide range of temperature and pH, which makes it an ideal peptide-based drug, and reflects the advantages of peptide-based agents. ALAPP showed its strongest antiproliferative activity against the MG-63 osteosarcoma at lower pH (pH 3–4), suggesting that ALAPP could be developed as an oral drug with resistance to gastric acidity. The stability of the peptide warrants further investigation by techniques to investigate secondary structure, such as circular dichromism. However, in the present study we were limited by the low yield of the purified polypeptide.
The present study is limited in that we did not use HSBP1 or any other positive control. Nevertheless, the purified polypeptide was used for all cell viability and protein expression experiments. A limitation of many TCM is that only efficacy is known, but the specific substance in the mixtures that produce the effects is unknown. The substance that produces the effect may be one, or a combination or synergistic effect of the many substances in the mixture. Thus, the antitumor effect of TCM formulae containing antlion may also be attributable to another substance such as polysaccharides [8, 9], mercury , flavonoids , or cantharids .
The MC3T3-E1 subclone 14 cell line can exhibit high levels of osteoblast differentiation and is a good model for studying osteoblast differentiation in vitro, having behavior similar to primary calvarial osteoblasts, but it is a spontaneously transformed (immortalized) mouse cell line. As such, caution should be used when extrapolating these results to normal cells, and even more, to normal human cells.
ALAPP purified from antlion is an 85-aminoacid 9.7 kDa polypeptide with 56% sequence identity with HSBP1. ALAPP has significant antiproliferative activity against MG-63 osteosarcoma cells, but not non-neoplastic MC3T3-E1 osteoblasts. We speculate that non-neoplastic cells may evade the antiproliferative effect of ALAPP by upregulating HSF1 to maintain their HSP90, CDK4, and AKT1 expression at a relatively constant level.
Purification of ALAPP from Myrmeleon bore larvae (antlions)
|Crude protein extract||1206||1845||1.0||12|
|Gel filtration chromatography||60.2||102||18.2||5|
|Ion exchange chromatography||1.67||30.5||3.33||2.8|A One Health approach to antimicrobial resistance Sialic acid: an attractive biomarker with promising biomedical applications Antibiotic resistance, biofilm forming ability, and clonal profiling of clinical isolates of Staphylococcus aureusfrom southern and northeastern India An 85-amino-acid polypeptide from Myrmeleon borelarvae (antlions) homologous to heat shock factor binding protein 1 with antiproliferative activity against MG-63 osteosarcoma cells in vitro Long noncoding and micro-RNA expression in a model of articular chondrocyte degeneration induced by stromal cell-derived factor-1 Promoter methylation analysis of DKK2may be a potential biomarker for early detection of cervical cancer