Molecular Cloning and Activity Study of the Novel Antimicrobial Peptide Brevinin-1EG From Pelophylax nigromaculatus

Abstract

Objective/Background: Cancer is one of the leading causes of death in the world. Therefore, there is an urgent need to develop new drugs for fighting this disease. Recent studies have reported that antimicrobial peptides (AMPs) can be used as anticancer drugs. Methods: We discovered brevinin-1EG, a novel antimicrobial peptide from Pelophylax nigromaculatus, using gene cloning techniques. Brevinin-1EG was then investigated for its structure and biological activity. Results: Brevinin-1EG, with an alpha-helical structure, showed broad-spectrum anti-proliferative activity against several cancer cell lines, including U-2OS, HepG2, HT-29, A375, HeLa, and A549 cells, with IC₅₀ values ranging from 15 to 22 μg/mL. These same concentrations were insufficient to induce hemolysis in erythrocytes, indicating the safety of this molecule. Brevinin-1EG also exhibited antioxidant activity, scavenging ABTS^·+ and DPPH free radicals. Conclusion: Brevinin-1EG has good safety and potential use as an anticancer therapeutic agent. It provides support for the research of new peptide drugs and templates for structural modification.

Introduction

Cancer poses a serious present threat to human health. According to statistics from the World Health Organization, in 2020 there were 19.29 million new cancer cases and 9.96 million deaths worldwide. ¹ Cancer is considered the second largest cause of death globally. ² Although anticancer drugs have made remarkable progress, many cancers remain resistant or even virtually intractable. ³ Many anti-cancer drugs also feature serious adverse reactions and high prices, both of which take a heavy toll on patients and their families. Therefore, the development of anticancer agents is an urgent need.

Bioactive peptides have been widely demonstrated to exert anticancer effects through multiple pathways.^4,5 Antimicrobial peptides (AMPs) are a subset of biological peptides derived from both prokaryotes and eukaryotes. True to their namesake, they boast antibacterial, antiviral, and immunomodulatory properties.^6‐8 However, they have also been considered as potential anticancer drugs. Numerous AMPs are found in amphibians, which are inseparably related to their living environment.^9,10 AMPs are usually composed of 12 to 50 amino acid residues and have an overall positive charge. ¹¹ This positive charge is one of the key features of AMPs. Maria Magana ¹² and Jaspreet Kaur Boparai ¹³ found that AMPs often achieve their antibacterial effects by destroying the membrane structure through electrostatic interaction with bacterial membranes. The precise mechanisms of action include electroporation, barrel staves, disordered toroidal pores, toroidal pores, membrane thinning or thickening, and many others. Other properties of AMPs, which include structure and amphiphilicity, are also involved. Some AMPs achieve cell necrosis or apoptosis by interfering with mitochondrial performance, cell cycle, and other pathways. ⁶ This has been documented for a variety of malignancies, including bladder, hepatocellular, gastric, skin, and ovarian cancer.^14‐16

In this study, we discovered a novel antimicrobial peptide, named brevinin-1EG, from the skin tissue of Pelophylax nigromaculatus. We extracted total RNA from the skin tissues and obtained the sequence of the gene encoding brevinin-1EG by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Curiously, the PCR product's sequence differed from that cataloged in existing gene and protein databases. We analyzed this new AMP sequence using online tools to assess their features and tested their antioxidant and hemolytic activity. We then further evaluated brevinin-1EG for anti-proliferative activity against several common human cancer cells and cytotoxicity in normal human cells. Finally, we assessed the effect of brevinin-1EG on tumor membranes.

Materials and Methods

Materials

U-2OS, HepG2, HT-29, A375, HeLa, and A549 cells used in this study were preserved by our laboratory. RPMI 1640 medium was used to grow HT-29, A375, and A549 cells. The rest of the cells were grown in Dulbecco's modified Eagle medium (DMEM).

Experimental Animals

Pelophylax nigromaculatus was obtained from Kunming, Yunnan Province, China, and raised at 25 °C. Experiments on live animals were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals.

Extraction of Skin Tissue RNA and Construction of cDNA Library

Frogs were executed under anesthesia. A small section of dorsal skin was removed and minced in 1 mL of TRIzol. Total RNA was extracted according to the instructions that came with the TRIzol reagent. A cDNA library was subsequently constructed by reverse transcription using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme). Oligo (dT)18 in the kit was replaced with specific primers of our own design (5'-ATTGACTCGAGTCGACATCGATTTTTTTTTTTTTTTTTT-3ʹ). The specific operation refers to the instruction manual.

PCR and Original TA Cloning Kit

Using the synthetic cDNA as our template, we amplified a polymerase chain reaction (PCR) product using the upstream primer (5'-ATTGACTCGAGTCGACATCGA-3ʹ) and the downstream primer (5'-ATGTTCACCACAAAGAAATCCA-3ʹ). The PCR cycle program was as follows: predenaturation at 95 °C for 2 min; 30 cycles of denaturation at 95 ^oC for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 1 min. PCR products were detected using agarose gel electrophoresis and recovered using gel recovery kit (Zomanbio). The recovered products were ligated with pMD-19T, and the ligated products were transformed into Escherichia coli DH5α receptor cells, which were then sent to Tsingke Biotechnology Company.

BLAST Alignment

The sequencing results were compared on the NCBI website by Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and the amino acid sequences of the mature peptides were analyzed.

Bioinformatic Characterization of Brevinin-1EG

Bioinformatic characterization was performed with online analysis tools Heli Quest and I-TASSER, which predicted the basic properties and structural features of brevinin-1EG.

Synthesis of Brevinin-1EG

Brevinin-1EG was synthesized by GL Biochem (Shanghai) Limited using a solid-phase chemical synthesis method. Its molecular weight was calculated to be 2637.25 Da, and its purity was found to be > 95% by mass spectrometry and high-performance liquid chromatography.

Structural Testing

The structure of samples in different solutions was detected by translational circular dichroism. ¹⁷ We dissolved brevinin-1EG in 10 mM phosphate buffered saline (PBS), 30 mM sodium dodecyl sulfate (SDS), or 50% trifluoroethanol (TFE) solution and formulated it into a loading sample with a concentration of 100 μg/mL. The circular dichroism parameters were as follows: wavelength range 190 to 300 nm; bandwidth 1 nm; reaction time 1 s; scanning speed 100 nm/min. Finally, these data were analyzed using Colabfold online tools.

Hemolytic Activity Assay

Hemolysis experiments were slightly modified from existing methods. ¹⁸ Briefly, 200 μL of 4% (v/v) sheep red blood cells (Solarbio) were placed into an Eppendorf tube, along with either peptide solutions of different concentrations or Triton X-100. These tubes were allowed to incubate at 37 °C for 30 min. The above solution was centrifuged at 1000 rpm for 5 min, and the absorbance of the supernatant was measured at 540 nm. PBS and 1% TritonX-100 were used as the negative and positive control, respectively. Hemolysis was calculated using the following formula:

$$\mathrm{Hemolytic}\mathrm{rate}(\text{\%})=[({\mathrm{A}}_{\mathrm{sample}}-{\mathrm{A}}_{\mathrm{blank}})/{\mathrm{A}}_{\mathrm{Triton}\mathrm{X}}-{\mathrm{A}}_{\mathrm{blank}})]\times 100$$

Antioxidant Assays

We examined the ability of brevinin-1EG to scavenge ABTS^·+ and DPPH radical cations, which have demonstrated antioxidant activity. The specific experimental operation method can be found in the reference. ¹⁹ Each experiment consisted of 3 independent replicates. The following equation was used to measure the radical scavenging rate:

$$\mathrm{Radical}\mathrm{scavenging}\mathrm{rate}(\text{\%})=[({\mathrm{A}}_{\mathrm{blank}}-{\mathrm{A}}_{\mathrm{sample}})/{\mathrm{A}}_{\mathrm{blank}}]\times 100$$

ABTS^·+ Radical Scavenging Ability

A stock solution, prepared by mixing 0.2 mL of 7.4 M ABTS (Aladdin) and 0.2 mL of 2.6 M K₂S₂O, was left to incubate in the dark for 12 h. We then diluted it with PBS buffer to ensure that the absorbance at 734 nm was 0.7 ± 0.02. We mixed 190 μL of ABTS^·+ working solution with different concentrations of 10 μL brevinin-1EG in a 96-well plate, which was incubated at 37 °C for 30 min in the dark. Finally, we detected the absorbance of the mixed solution at 734 nm.

DPPH Radical Scavenging Ability

We prepared a 0.1 mM DPPH (MedChemExpress) solution, diluted with absolute ethanol to an absorbance of 1.0 at 517 nm. DPPH solution (190 mL) and 10 μL of brevinin-1EG solution were added to each well of a 96-well plate and allowed to incubate in the dark at 37 °C for 30 min. After the reaction was complete, the absorbance of the solution at 517 nm was detected.

Antiproliferative Activity Assay

The antiproliferative activity of brevinin on human HeLa, melanoma, and liver cancer cells was measured by the CCK-8 kit (Bioss). ²⁰ We seeded 5 × 10³ cells into each well of a 96-well plate and cultured them in a medium containing 10% serum for 24 h. Different concentrations of brevinin-1EG were then added to each well and allowed to incubate for a further 24 h. We then added 10 µL of CCK-8 to each sample and incubated it for 2 h. Finally, the absorbance at 450 nm of each sample was measured using an ELISA plate reader.

Lactate Dehydrogenase (LDH) Detection

The content of total intracellular LDH was detected by the LDH cytotoxicity detection kit (Beyotime), following the manufacturer's instructions. We seeded 5 × 10³ cells into each well of a 96-well plate. Once the cells had reached 80% to 90% confluency, different concentrations of brevinin-1EG were added to the treated cells for 24 h. In addition, we set background blank control wells, sample control wells, and sample maximum enzyme activity control wells. One hour before the scheduled detection time point, we added 20 μL LDH release reagent provided by the kit to the sample maximum enzyme activity control well.

Statistical Analysis

We analyzed the data using Student t-tests or one-way ANOVA in GraphPad Prism 9.0. The error bars in the graph represent the standard error of the mean (SEM). By convention, we considered P < .05 to be statistically significant. Experiments were performed on more than 3 replicates.

Results

Cloning of cDNA Encoding the Premaster of Brevinin-1EG

The full-length cDNA encoding brevinin-1EG was successfully cloned from the skin tissue cDNA library. The DNA sequence consists of 216 nucleotides in total and the brevinin-1EG precursor of 71 amino acid residues (Figure 1). These include a signal peptide region, an acidic amino acid-rich spacer region, a translocase cleavage site, and a maturation region (consisting of 24 amino acids), whose sequence is FLPLLAGLAANFLPTIFCKISRKC.

Figure 1.

The cDNA and deduced amino acid sequence of brevinin-1EG.

We discovered a peptide that had a “Rana box” (Cys18-(Xaa)4-Lys-Cys24) and 4 invariant residues, identified as Ala9, Cys18, Lys23, and Cys24. These are hallmark features of the brevinin family. ²¹ Thus, we introduce brevinin-1EG as the most newly discovered member of the brevinin-1 family.

Bioinformatics Analysis of Brevinin-1EG

To understand brevinin-1EG further, bioinformatics and secondary structure were further analyzed using Heli Quest, I-TASSER, and Colabfold online tools. The results show that brevinin-1EG carries a + 3 net charge, a hydrophobicity of 0.815, a hydrophobic moment of 0.279, and a hydrophobic surface composed of I, A, L, I, L, A, C, F, A (Figure 2A and B). What is more, the secondary structure of brevinin-1EG contains a large number of α-helix structures (Figure 2A to D).

Figure 2.

Bioinformatics and structure analyses of brevinin-1EG. (A) Structural parameters of brevinin-1EG. (B-C) The α-helix-dominated structure prediction of brevinin-1EG and the arrow indicates the hydrophobic surface. (D) Secondary structure prediction of brevinin-1EG. (E) Circular dichroism analysis of brevinin-1EG in 10 mM PBS, 30 mM SDS, or 50% TFE solution.

Structure Prediction of Brevinin-1EG

The molecular weight of the synthetic peptide brevinin-1EG was 2637.25 Da, and its purity was greater than 95% (Figure 3). To confirm the online analysis results, circular dichroism was used to test the structure of brevinin-1EG dissolved in either 10 mM PBS, 30 mM SDS, or 50% TFE (Figure 2E). When brevinin-1EG was dissolved in 10 mM PBS, it exhibited a randomly coiled structure. In a 30 mM SDS and 50% TFE solution of brevinin-1EG, there were 2 negative Cotton effects at >200 nm and one positive Cotton effect at 192 nm, which is characteristic of α-helices. Thus, brevinin-1EG appears to be mainly dominated by an α-helical structure, which is consistent with the predicted structure.

Figure 3.

High-performance liquid chromatogram (HPLC) and mass spectra (ms) of brevinin-1EG. (A) The purity of brevinine-1EG was >95%. (B) The retention time and peak area information of brevinin-1EG. (C) The molecular mass of brevinin-1EG is 2637.25.

Antiproliferative Activity

To detect the antiproliferative activities of the synthesized peptides, we treated U-2OS, HepG2, HT-29, A375, HeLa, and A549 cells with brevinin-1EG. Brevinin-1EG exhibited strong anti-proliferative activity against all 6 tested tumor cells, particularly against HepG2 cells, with an IC₅₀ value of 15.88 μg/mL (Figure 4 and Table 1). The IC₅₀ values of brevinin-1EG on U-2OS, HT-29, A375, HeLa, and A549 cells were 19.30, 19.75, 19.33, 21.21, and 21.46 μg/mL, respectively (Table 1).

Figure 4.

The antproliferative activity of brevinin-1EG and its effect on lactate dehydrogenase (LDH) release in cancer cells. (A) U-2OS, HepG2, HT-29. (B) A375, HeLa, A549. (C) and (D) Brevinin-1EG induced the release of LDH in HepG2 and HeLa cells. Compared to the negative control group, *P < .05, **P < .01, and ***P < .001 represent a significant difference between the 2 groups. Results are expressed as mean ± SD (n = 3).

Table 1.

The IC₅₀ Value of Brevinin-1EG Calculated From Figure 4A and B.

Cells	IC50 (μg/mL)	95% CI
U-2OS	19.30	18.26-20.41
HT-29	19.75	19.22-20.30
A375	19.33	18.47-20.19
HeLa	21.21	20.73-21.73
A549	21.46	20.97-21.98
HepG2	15.88	15.44-16.38

LDH Detection

LDH released by cells can assess cell membrane integrity. We detected the release of LDH when different concentrations of brevinin-1EG acted on HepG2 and HeLa cells. The results indicate that brevinin can exert an anti-proliferative effect by destroying the membrane of cancer cells (Figure 4C and D).

Hemolytic Activity

The hemolytic activity of brevinin-1EG was tested using 4% (v/v) sheep red blood cell suspension. Brevinin-1EG has a certain hemolytic activity with an HC₅₀ of 138.6 μg/mL (Figure 5A and B). However, when added to the blood cell suspension at the IC₅₀ effective against tumor cells, brevinin-1EG caused hardly any hemolysis. Only about 34.44% hemolysis was observed at 5 times the IC₅₀, supporting the safety of brevinin-1EG.

Figure 5.

Hemolytic and antioxidant activities of brevinin-1EG. (A) and (B) Effects of 6.25, 12.5, 25, 50, 100, 200, and 400 μg/mL concentrations of brevinin-1EG on erythrocytes. (C) Brevinin-1EG on ABTS⁺ radical scavenging activity. (D) Brevinin-1EG on DPPH radical scavenging activity. *P < .05, **P < .01, ***P < .001 in comparison to the control group denotes a significant difference between the 2 groups. Values are expressed as mean ± SD (n = 3).

Antioxidant Activity In Vitro

Figure 5C and D shows the ABTS^·+ and DPPH free radical scavenging kinetics of brevinin-1EG, which exhibits potent antioxidant activity in a concentration-dependent manner. Among these 2 free radicals, the ability of brevinin-1EG to scavenge ABTS^·+ was stronger than that of DPPH. When the concentration of brevinin-1EG was 200 μg/mL, 89.25% of ABTS^·+ and 42.37% of DPPH were removed after 5 min.

Discussion

Frogs are an important source of AMPs, which are an important part of their innate immunity and defense against microbial infections. ²² AMPs can be divided into cathelicidin, brevinin, and other families. ²³ The brevinin family consists of brevinin-1 and brevinin−2, both of which have a “Rana box” and α-helical structure. We confirmed the antimicrobial peptide in this article as brevinin-1 and named it brevinin-1EG. Both the online tool and the circular dichroism test indicated the structure of brevinin-1EG to be an α-helix (Figure 2).

Curing cancer is one of the biggest challenges in biomedicine. At present, although a variety of drugs have been marketed, there is still a long way to go toward achieving a cure, and present treatments exact a heavy toll in terms of adverse reactions. A considerable number of research institutions and pharmaceutical companies are devoted to the research and development of new drugs. AMPs have been repeatedly shown to have utility as anticancer agents^24,25 as a result of their ability to induce apoptosis and membrane lysis or pore formation. The strength of this anti-proliferative activity is related to their amino acids, charge, and structure. AMPs are able to target cancer cells effectively in comparison to nonmalignant cells due to the differences between healthy and cancer cell membranes, which include membrane electrical properties, and membrane flow velocity. ²⁶ Some peptides can also hydrophobically interact with healthy cell membranes, resulting in toxicity.

In this study, we tested the anti-proliferative activity of brevinin-1EG on U-2OS, HepG2, HT-29, A375, HeLa, and A549 cells. We also tested LDH release in HepG2 and HeLa cells. Our results show that brevinin-1EG can kill a variety of cancer cells by attacking their membrane structure. Brevinin-1EG possesses a similar amino acid sequence and structure to brevinin-1RL1. Brevinin-1RL1 exerts anti-proliferative effects by inducing apoptosis and necrosis. ²⁰ Therefore, we speculate that the anti-proliferative mechanism of brevinin-1EG is similar to that of brevinin-1RL1, though this, of course, requires confirmation.

Hemolytic activity is often an important indicator to measure the safety of peptides. ²⁷ This study shows that the hemolytic activity of brevinin-1EG is concentration-dependent and has good safety at concentrations effective to kill cancer cells. This hemolytic activity can be further reduced by structural modifications. Studies have found that the hemolytic activity of brevinin-1 is related to its hydrophobicity, hydrophobic surface, and α-helix length. In addition, brevinin-1EG also has an antioxidant capacity, which can scavenge ABTS^·+ and DPPH free radicals, with the former being greater than the latter. This may be affected by the absolute ethanol solution.

To summarize, we discovered a new member of the brevinin family in Pelophylax nigromaculatus. This new antimicrobial peptide, which named brevinin-1EG, consists of 24 amino acid residues with an α-helical structure. It can inhibit the growth of several cancer cell lines by disrupting their cell membrane. Although it has some hemolytic activity, at IC₅₀, the hemolytic activity is almost absent. We feel that brevinin-1EG is a promising novel anticancer drug candidate. Hopefully, these findings may provide new insights into the prototyping of natural anticancer drugs.