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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

Rui Ding
Rui Ding
, 
Ming He
Ming He
, 
Huoying Huang
Huoying Huang
, 
Jing Chen
Jing Chen
, 
Mingxing Huang
Mingxing Huang
 and 
Yonghui Su
Yonghui Su
   | Aug 31, 2022
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Article Category: Brief communication (original)
Published Online: Aug 31, 2022
Page range: 201 - 211
DOI: https://doi.org/10.2478/abm-2022-0024
Keywords
© 2022 Rui Ding et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1

Purification of ALAPP by gel filtration and ion exchange chromatography. (A) Gel filtration in Sephacryl-S100-HR. The protein concentration in the eluate was estimated from its absorbance at 280 nm (solid line). Antiproliferative activity against MG-63 osteosarcoma cells (dashed line) was measured by MTT cell viability assay. Peak 1 (Fraction 13), Peak 2 (Fraction 20–22), Peak 3 (Fraction 26–27), Peak 4 (Fraction 29–30), Peak 5 (Fraction 35–37), Peak 6 (Fraction 50–51), and Peak 7 (Fraction 56–58). (B) Ion exchange chromatography on Q-Sepharose-FF. After elution with about 5 column volumes of equilibration buffer (50 mM Tris-HCl, pH 7.4) until absorbance reached baseline, bound proteins were eluted with equilibration buffer containing a stepwise gradient of NaCl (0–0.5 M) (dashed line), and the polypeptide with antiproliferative activity (ALAPP) was in the fraction eluted with the equilibration buffer containing 0.15 M NaCl. ALAPP, antlion antiproliferative polypeptide.
Purification of ALAPP by gel filtration and ion exchange chromatography. (A) Gel filtration in Sephacryl-S100-HR. The protein concentration in the eluate was estimated from its absorbance at 280 nm (solid line). Antiproliferative activity against MG-63 osteosarcoma cells (dashed line) was measured by MTT cell viability assay. Peak 1 (Fraction 13), Peak 2 (Fraction 20–22), Peak 3 (Fraction 26–27), Peak 4 (Fraction 29–30), Peak 5 (Fraction 35–37), Peak 6 (Fraction 50–51), and Peak 7 (Fraction 56–58). (B) Ion exchange chromatography on Q-Sepharose-FF. After elution with about 5 column volumes of equilibration buffer (50 mM Tris-HCl, pH 7.4) until absorbance reached baseline, bound proteins were eluted with equilibration buffer containing a stepwise gradient of NaCl (0–0.5 M) (dashed line), and the polypeptide with antiproliferative activity (ALAPP) was in the fraction eluted with the equilibration buffer containing 0.15 M NaCl. ALAPP, antlion antiproliferative polypeptide.

Figure 2

Purification profiles of ALAPP from antlion. Proteins in samples following each purification step were separated by Tricine–SDS–PAGE (14% polyacrylamide, 3.3% crosslinked) and stained with Coomassie Brilliant Blue R. Lane 1, protein molecular weight markers (catalog No. C600201; Sangon Biotech). Lane 2, crude protein extract. Lane 3, proteins of Peak 6 pool from Sephacryl-S100-HR. Lane 4, proteins eluted from Q-Sepharose-FF, including the ALAPP with antiproliferative activity. ALAPP, antlion antiproliferative peptide; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.
Purification profiles of ALAPP from antlion. Proteins in samples following each purification step were separated by Tricine–SDS–PAGE (14% polyacrylamide, 3.3% crosslinked) and stained with Coomassie Brilliant Blue R. Lane 1, protein molecular weight markers (catalog No. C600201; Sangon Biotech). Lane 2, crude protein extract. Lane 3, proteins of Peak 6 pool from Sephacryl-S100-HR. Lane 4, proteins eluted from Q-Sepharose-FF, including the ALAPP with antiproliferative activity. ALAPP, antlion antiproliferative peptide; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.

Figure 3

Effect of pH and temperature on the antiproliferative activity of ALAPP. Relative antiproliferative activity of the treated ALAPP against MG-63 osteosarcoma cells was measured using an MTT cell viability assay. (A) Effect of pH. ALAPP was incubated at various pH for 60 min at 4 °C. The pH was adjusted to 7.2 before measuring the anti-proliferative activity of the treated ALAPP. (B) Effect of temperature. ALAPP was incubated at various temperatures for 60 min followed by cooling in ice bath before measuring the antiproliferative activity of the treated ALAPP. Values are shown as mean (n = 3); error bars indicate standard deviation. ALAPP, antlion antiproliferative polypeptide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Effect of pH and temperature on the antiproliferative activity of ALAPP. Relative antiproliferative activity of the treated ALAPP against MG-63 osteosarcoma cells was measured using an MTT cell viability assay. (A) Effect of pH. ALAPP was incubated at various pH for 60 min at 4 °C. The pH was adjusted to 7.2 before measuring the anti-proliferative activity of the treated ALAPP. (B) Effect of temperature. ALAPP was incubated at various temperatures for 60 min followed by cooling in ice bath before measuring the antiproliferative activity of the treated ALAPP. Values are shown as mean (n = 3); error bars indicate standard deviation. ALAPP, antlion antiproliferative polypeptide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 4

Effect of metal ions on the antiproliferative activity of ALAPP. The mixture of purified ALAPP and metal ion was incubated at 25 °C for 30 min, and the relative antiproliferative activity against MG-63 osteosarcoma cells was measured using an MTT cell viability assay against a control culture without any metal ion. Values are shown as mean (n = 3); error bars indicate standard deviation. ALAPP, antlion antiproliferative polypeptide.
Effect of metal ions on the antiproliferative activity of ALAPP. The mixture of purified ALAPP and metal ion was incubated at 25 °C for 30 min, and the relative antiproliferative activity against MG-63 osteosarcoma cells was measured using an MTT cell viability assay against a control culture without any metal ion. Values are shown as mean (n = 3); error bars indicate standard deviation. ALAPP, antlion antiproliferative polypeptide.

Figure 5

Morphological characteristics and changes in MG-63 osteosarcoma cells and non-neoplastic MC3T3-E1 subclone 14 osteoblast controls effected by ALAPP. Osteosarcoma and osteoblasts were treated with ALAPP at various concentrations or with PBS as a control for 48 h. The morphological characteristics and changes were evaluated using an inverted phase contrast microscope EVOS FL Auto Cell Imaging System 20× LPlan FH objective (catalog No. AMEP-4682; Thermo Fisher Scientific). Untreated cells (A and D), and the effect of only 2 concentrations, 20 μg/mL ALAPP (B and E) and 50 μg/mL ALAPP (C and F), are shown to highlight the substantial differences in cell morphology. A, B, and C. Mouse osteoblasts MC3T3-E1 subclone 14 derived from osteoblast precursors of the parietal calvaria (ATCC CRL-2594); D, E, and F. Human osteosarcoma cells MG-63 (ATCC CRL-1427). Scale bars 100 μm. ALAPP, antlion antiproliferative polypeptide; PBS, phosphate-buffered saline.
Morphological characteristics and changes in MG-63 osteosarcoma cells and non-neoplastic MC3T3-E1 subclone 14 osteoblast controls effected by ALAPP. Osteosarcoma and osteoblasts were treated with ALAPP at various concentrations or with PBS as a control for 48 h. The morphological characteristics and changes were evaluated using an inverted phase contrast microscope EVOS FL Auto Cell Imaging System 20× LPlan FH objective (catalog No. AMEP-4682; Thermo Fisher Scientific). Untreated cells (A and D), and the effect of only 2 concentrations, 20 μg/mL ALAPP (B and E) and 50 μg/mL ALAPP (C and F), are shown to highlight the substantial differences in cell morphology. A, B, and C. Mouse osteoblasts MC3T3-E1 subclone 14 derived from osteoblast precursors of the parietal calvaria (ATCC CRL-2594); D, E, and F. Human osteosarcoma cells MG-63 (ATCC CRL-1427). Scale bars 100 μm. ALAPP, antlion antiproliferative polypeptide; PBS, phosphate-buffered saline.

Figure 6

Antiproliferative activity of ALAPP. MG-63 osteosarcoma cells and MC3T3 osteoblasts were treated with ALAPP for 48 h, and antiproliferative activity against the cells was determined using an MTT cell viability assay. Each bar represents the average of n = 3 and error bars indicate the standard deviation. Student t tests were conducted to determine differences in antiproliferative activity. *P < 0.05, **P < 0.005, ***P < 0.001 vs control. ALAPP, antlion antiproliferative polypeptide.
Antiproliferative activity of ALAPP. MG-63 osteosarcoma cells and MC3T3 osteoblasts were treated with ALAPP for 48 h, and antiproliferative activity against the cells was determined using an MTT cell viability assay. Each bar represents the average of n = 3 and error bars indicate the standard deviation. Student t tests were conducted to determine differences in antiproliferative activity. *P < 0.05, **P < 0.005, ***P < 0.001 vs control. ALAPP, antlion antiproliferative polypeptide.

Figure 7

Effect of ALAPP on the levels of HSF1, HSP90, CDK4, and AKT1 expression by MG-63 osteosarcoma cells and level of Hsf1, Hsp90, Cdk4, and Akt1 expression by MC3T3 osteoblasts. The levels of protein expression were determined by western blotting. After treatment with 50 μg/mL ALAPP for 48 h, MC3T3 osteoblasts and MG-63 osteosarcoma cells were collected and mixed with 100 μL xTractor Buffer (Clontech). Total cell protein extracts were adjusted to the same protein concentration using the Bradford method, and their protein components were separated by electrophoresis in a 12% polyacrylamide gel containing SDS and transferred to PVDF membranes. Primary antibodies, each with immunoreactivity for human and mouse homologs of the proteins, were purchased from Sangon Biotech, including the rabbit polyclonal antibodies anti-ACTC1 (catalog No. D224905), anti-HSF1 (catalog No. D220782), anti-HSP90AA1 (catalog No. D220009), anti-CDK4 (catalog No. D120396), and anti-AKT1(Ab-129) (catalog No. D151616). Immunoreactivity was detected with secondary antibody HRP-conjugated goat anti-rabbit IgG (catalog No. D110058, Sangon) using an ECL chemiluminescence substrate (catalog No. T7101A, TaKaRa). Expression of the various proteins was calculated relative to β-actin used as a control. Each bar represents the average of n = 3 and error bars indicate standard deviation. Student t tests were conducted to determine differences in expression. *P < 0.05, **P < 0.005 vs untreated control cells. A. HSF1. B. HSP90. C. CDK4, D. AKT1. ACTC1, β-actin; AKT1, serine-threonine protein kinase encoded by AKT1; ALAPP, antlion antiproliferative polypeptide; CDK4, cyclin-dependent kinase 4; HRP, horseradish peroxidase; HSF1, heat shock transcription factor 1; HSP90, heat shock protein 90 kDa alpha (cytosolic), class A member 1 (HSP90AA1); PVDF, polyvinylidene difluoride.
Effect of ALAPP on the levels of HSF1, HSP90, CDK4, and AKT1 expression by MG-63 osteosarcoma cells and level of Hsf1, Hsp90, Cdk4, and Akt1 expression by MC3T3 osteoblasts. The levels of protein expression were determined by western blotting. After treatment with 50 μg/mL ALAPP for 48 h, MC3T3 osteoblasts and MG-63 osteosarcoma cells were collected and mixed with 100 μL xTractor Buffer (Clontech). Total cell protein extracts were adjusted to the same protein concentration using the Bradford method, and their protein components were separated by electrophoresis in a 12% polyacrylamide gel containing SDS and transferred to PVDF membranes. Primary antibodies, each with immunoreactivity for human and mouse homologs of the proteins, were purchased from Sangon Biotech, including the rabbit polyclonal antibodies anti-ACTC1 (catalog No. D224905), anti-HSF1 (catalog No. D220782), anti-HSP90AA1 (catalog No. D220009), anti-CDK4 (catalog No. D120396), and anti-AKT1(Ab-129) (catalog No. D151616). Immunoreactivity was detected with secondary antibody HRP-conjugated goat anti-rabbit IgG (catalog No. D110058, Sangon) using an ECL chemiluminescence substrate (catalog No. T7101A, TaKaRa). Expression of the various proteins was calculated relative to β-actin used as a control. Each bar represents the average of n = 3 and error bars indicate standard deviation. Student t tests were conducted to determine differences in expression. *P < 0.05, **P < 0.005 vs untreated control cells. A. HSF1. B. HSP90. C. CDK4, D. AKT1. ACTC1, β-actin; AKT1, serine-threonine protein kinase encoded by AKT1; ALAPP, antlion antiproliferative polypeptide; CDK4, cyclin-dependent kinase 4; HRP, horseradish peroxidase; HSF1, heat shock transcription factor 1; HSP90, heat shock protein 90 kDa alpha (cytosolic), class A member 1 (HSP90AA1); PVDF, polyvinylidene difluoride.

Purification of ALAPP from Myrmeleon bore larvae (antlions)

Purification procedure Total protein (mg) IC50 (μg/mL)† Purification degree (fold) Yield (%)
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
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