An 85-amino-acid polypeptide from Myrmeleon bore larvae (antlions) homologous to heat shock factor binding protein 1 with antiproliferative activity against MG-63 osteosarcoma cells in vitro

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 ×g for 10 min ×2 times at 4 °C to pellet out the cell debris, and lipids were removed by filtration through qualitative filter paper at medium speed to obtain a proteinaceous extract.

Protein concentration was determined by the method of Bradford [12]. 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 [15]. 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 × g for 20 min at 4 °C to remove cell debris. All crude extracts were adjusted to the same protein concentration using the Bradford method, and protein components were separated by electrophoresis in a 12% polyacrylamide gel containing SDS and transferred to polyvinylidene difluoride (PVDF) membranes. Primary antibodies, including rabbit polyclonal antibodies to β-actin (anti-ACTC1, catalog No. D224905), heat shock transcription factor 1 (anti-HSF1, catalog No. D220782), heat shock protein 90 kDa alpha (cytosolic), class A member 1 (anti-HSP90AA1, catalog No. D220009), cyclin-dependent kinase 4 (anti-CDK4, catalog No. D120396), and serine-threonine protein kinase encoded by AKT1 (anti-AKT1(Ab-129), catalog No. D151616), each with immunoreactivity for human and mouse homologs of the proteins, and secondary antibody of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (catalog No. D110058), were purchased from Sangon Biotech. The ECL chemiluminescence HRP substrate for western blotting (catalog No. T7101A) was purchased from TaKaRa and used in accordance with the protocols specified by the manufacturer. The expression of the various proteins was calculated relative to β-actin.

All data are presented as mean and standard deviation. Assays were conducted in triplicate. Significant difference at the level of P < 0.05 after a Student t test was used to compare the values of parameters tested.

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 (Figure 1A). The corresponding relationship between the 7 peaks and the number of the fractions was as follows: Peak 1 (Fraction 13), Peak 2 (Fractions 20–22), Peak 3 (Fractions 26–27), Peak 4 (Fractions 29–30), Peak 5 (Fractions 35–37), Peak 6 (Fractions 50–51), and Peak 7 (Fractions 56–58). Antiproliferative activity against MG-63 osteosarcoma cells was mainly detected in Peak 1 and Peak 6. Peak 1, which mainly consisted of proteins with >100 kDa molecular weight, was not considered for further study due to the low stability and poor cell permeability of these proteins compared with smaller polypeptides. Only the 2 aliquots of the summit of Peak 6 with a total of 4 mL were selected for further study. The pooled fractions with antiproliferative activity from Peak 6 of the gel filtration chromatography were concentrated and the buffer was desalted against 50 mM Tris-HCl (pH 7.6) using ultrafiltration. The Peak 6 fraction pool in the new low-salt buffer was applied to a column of Q-Sepharose-FF. A polypeptide with antiproliferative activity against MG-63 osteosarcoma cells was eluted with 50 mM Tris-HCl (pH 7.6) containing 0.15 M NaCl and named antlion antiproliferative polypeptide (ALAPP) (Figure 1B).

Figure 1

After the 3 steps of purification, a polypeptide with antiproliferative activity against MG-63 osteosarcoma cells (ALAPP) was purified from the crude antlion extract (Table 1). The final degree of purification was 60.5-fold with a total yield of 0.14%. The yield of each purification step was relatively low because the strategy employed was to obtain high purity, and not high recovery of the proteins, as its objective. The molecular weight of the ALAPP was estimated by Tricine–SDS–PAGE using a 14% polyacrylamide gel with 0.1% SDS in the electrode buffer and gel buffer. The relative molecular mass of ALAPP was estimated at 9.6 kDa (Figure 2). The band containing the ALAPP was excised from the gel and sent to Sangon Biotech for N-terminal Edman sequencing. The complete sequence obtained was:

MTDVKTTELNNEDVQNFTVSSSNDPKNMQELTQYVQTLLQTMQDKFQTMSDQIINRIDEMGNRIDDLEKNIADLMTQAGVEGPDK with a theoretical molecular weight of 9.714 kDa and pI 4.11 as calculated using the tool on the Expasy website (https://web.expasy.org/compute_pi/) operated by the SIB Swiss Institute of Bioinformatics [16].

Figure 2

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 (Figure 3A). The activity of ALAPP remained >95% at pH <5.5, and had the highest activity between pH 3 and 4. Although the activity of ALAPP decreased continuously when the pH was >6, >75% of activity remained at pH 11. Like pH, the temperature had a relatively small effect on the anti-proliferative activity of ALAPP (Figure 3B). The activity of ALAPP increased from 20 to 40 °C. Although the activity of ALAPP decreased continuously >40 °C, >50% activity remained at 70 °C.

Figure 3

The antiproliferative activity of ALAPP was not sensitive to any of the metal ions tested (Figure 4), except that Ca²⁺ showed an inhibitory effect on ALAPP activity at high concentration (>25 mM). The effect of other metal ions on the activity of ALAPP was not significant.

Figure 4

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 (Figures 5A–C). Nevertheless, as their ability to adhere to the well wall was reduced, aging and dying cells no longer formed spindles and contracted to form nearly round cells. However, the MG-63 osteosarcoma cells showed substantial changes in their morphology and features after being treated with ALAPP (Figures 5D–F). Their cell size was reduced substantially, and the ability of the 2 polar segments of the spindle to elongate was weakened. At a high concentration of ALAPP (50 μg/mL), the surviving cell morphology became very thin, and the cytoplasm became transparent and granular. The cells, which clumped together in large numbers, had lost their fusiform character.

Figure 5

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 (Figure 6). When ALAPP reached about 30 μg/mL, the relative viability of MG-63 osteosarcoma was inhibited by 50%. When ALAPP reached 50 μg/mL, the relative viability of MG-63 was only 37.8%. ALAPP had a clear concentration-dependent inhibitory effect on MG-63 osteosarcoma cell viability and, therefore, proliferation.

Figure 6

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 (https://www.uniprot.org/align) [17] and 56.8% similarity using the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) Emboss Needle tool (https://www.ebi.ac.uk/Tools/psa/emboss_needle/) [18], and 94% identity (83/85 or 97% positives) with the heat shock factor-binding protein 1 isoform X1 from Chrysoperla carnea, a relative of antlion in the order Neuroptera (NCBI sequence ID: XP_044742752.1) using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) [19]. The human form HSBP1 has an inhibitory activity on a variety of tumors because it binds to HSF1 to inhibit the expression of heat shock proteins (HSPs). Heat shock protein 90 (HSP90) is associated with a variety of signaling molecules, including CDK4 and AKT1, which are widely involved in the occurrence and development of tumors [20, 21]. The levels of expression of HSF1, HSP90, CDK4, and AKT1 and their mouse homologs were analyzed by western blotting. Without ALAPP treatment, the level of HSF1 expression in MG-63 osteosarcoma was about 2-fold the expression of Hsf1 in MC3T3 osteoblasts. After treatment with 50 μg/mL ALAPP, the level of Hsf1 expression was upregulated by about 50% in MC3T3 osteoblasts, but HSF1 expression remained almost unchanged in MG-63 osteosarcoma cells compared with untreated MG-63 cells (Figure 7A). HSF1 is a transcriptional regulator of HSP90, and positively regulates the expression of HSP90 and other HSPs. Without ALAPP treatment, the HSP90 expression level in MG-63 osteosarcoma was about 82% higher than Hsp90 in MC3T3 osteoblasts. After treatment with 50 μg/mL ALAPP, the level of Hsp90 expression was unchanged in MC3T3 osteoblasts, but compared with the level in untreated cells, HSP90 was downregulated by about 37% in MG-63 osteosarcoma cells (Figure 7B). The levels of expression of CDK4 and AKT1, which are client proteins of HSP90, are associated with HSP90. Without ALAPP treatment, the levels of CDK4 and AKT1 expression in MG-63 osteosarcoma cells were about 92.1% and 86% higher, respectively, than those of Cdk4 and Akt1 expression in MC3T3 osteoblasts. After treatment with 50 μg/mL ALAPP, the levels of expression of Cdk4 and Akt1 were unchanged in MC3T3 osteoblasts, but compared with levels in untreated cells, the levels of CDK4 and AKT1 expression were downregulated by about 42% and 43%, respectively, in MG-63 osteosarcoma cells (Figures 7C and 7D). Overall, in MC3T3 osteoblasts, ALAPP upregulated Hsf1, but failed to upregulate Hsp90, Cdk4, and Akt1. However, in MG-63 osteosarcoma cells, ALAPP failed to upregulate HSF1, and HSP90, CDK4, and AKT1 were downregulated to about the level seen in osteoblasts. These findings suggest that the levels of HSP90, CDK4, and AKT1 expression are comparable to those of their homologs in osteoblasts, but could not maintain the survival of the osteosarcoma cells.

Figure 7