The cockroach, which is a household insect, is an established model organism in research. Periplanetasin-2, derived from the American cockroach Periplaneta americana, exerted potent antifungal effect against pathogenic fungi without causing hemolysis. Periplanetasin-2 induced oxidative stress by generation of reactive oxygen species (ROS) and lipid peroxidation. Periplanetasin-2 also caused apoptosis by exposure of phosphatidylserine and fragmentation of DNA, exerted in a concentration-dependent manner. Hence, we investigated the mitochondrial apoptotic mechanism of periplanetasin-2 in Candida albicans. After treatment with periplanetasin-2, we observed mitochondrial depolarization and calcium accumulation. Moreover, we observed a decrease in cytosolic glutathione, and an increase in mitochondrial glutathione, indicating that periplanetasin-2 induced oxidative stress and high ROS production in the mitochondria. Because of this mitochondrial dysfunction, cytochrome c was released from the mitochondria into the cytosol, and caspase was activated in a time-dependent manner. In summary, the antifungal peptide periplanetasin-2 activates apoptotic signals in the mitochondria by induction of oxidative stress.

Introduction

The principal domestic cockroach species are Blattella germanica and Periplaneta americana. P. americana is the American cockroach, ∼2 inches in length; it requires higher temperatures and humidity for growth [1]. Asthma and allergy are the most common diseases attributable to cockroach infestation of housing worldwide. The cockroach produces several potent allergens, making it a serious public health problem [2]. However, a new interest in the cockroach has emerged in microbiology. Lee et al. [3] has suggested that the cockroach is a good source of antimicrobial agents, and cockroach brain tissues, from P. americana, show broad-spectrum antimicrobial activity. Kim et al. [4] performed de novo transcriptome analysis of P. americana that were immunized, or non-immunized, with Escherichia coli, and obtained potent antimicrobial peptide candidates.

The Candida species are common fungal pathogens in humans that cause oral and vaginal candidiasis, resulting in severe worldwide morbidity. Candida albicans is the most pathogenic fungi [5], particularly when it opportunistically infects patients in hospitals. This opportunistic pathogen causes systemic and fatal infections in immunocompromised patients, those with AIDS, patients undergoing chemotherapy, and recipients of organ transplants [6]. Because of these clinical considerations, there is a critical need for the development of new antifungal agents and strategies. Apoptosis is programmed cell death, used for homeostasis and maintenance of an organism. Recent studies reveal the existence of apoptosis in fungal cells [7]. Several apoptotic hallmarks, such as phosphatidylserine (PS) exposure, DNA fragmentation, cytochrome c release, and caspase activation, are observed in fungal cells undergoing apoptosis [8,9].

Mitochondria are important for the energetic functioning of the cell; they are also the fatal organelle, controlling cellular life and death [10]. Mitochondria are the richest source of reactive oxygen species (ROS) in the cell. Inhibition of the mitochondrial electron transport chain, which results in the subsequent release of ROS, leads to apoptotic cell death. Cytochrome c, which activates apoptotic caspases, is abundant in the mitochondria and is released into the cytosol during mitochondrial dysfunction [11]. Additionally, the mitochondria are the main site of ROS production in fungal cells. Numerous studies have identified the involvement of the mitochondria in fungal apoptosis and show that the accumulation of ROS in the mitochondria and release of cytochrome c are observed in apoptosis of fungal cells [12,13]. Herein, we report on mitochondrial involvement in the concentration- and time-dependent apoptotic effect of periplanetasin-2.

Materials and methods

Materials

2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), fura-2 acetoxymethyl ester (Fura-2AM), rhod-2 acetoxymethyl ester (Rhod-2AM), and MitoSOX Red were obtained from Molecular Probes (Eugene, OR, U.S.A.). 5-Sulfosalicylic acid (SSA), amphotericin B, and fluconazole were obtained from Sigma (St. Louis, MO, U.S.A.). CaspACE FITC-VAD-FMK was obtained from Promega (Madison, WI, U.S.A.).

Synthesis of periplanetasin-2

Periplanetasin-2 was synthesized by Anygen Co. (Gwangju, Korea). The peptides were assembled using a 60-min cycle for each residue, at an ambient temperature, using the following method. 2-Chlorotrityl (or 4-methylbenzhydrylamine amide) resin was charged using a reactor and then washed sequentially with dichloromethane and N,N-dimethylformamide (DMF). Then, a coupling step was performed with vigorous shaking using a 0.14 mM solution of standard and 60-min-preactivated Fmoc-l-amino acids with a 0.1 mM solution of 0.5 M 1-hydroxybenzotriazole/diisopropylcarbodiimide in DMF. Finally, the peptide was cleaved from the resin using a trifluoroacetic acid cocktail solution at an ambient temperature. Mass spectrometry was performed using an AXIMA CFR MALDI-TOF mass spectrometer (Kratos/Shimadzu, Manchester, U.K.) [14,15].

Minimum inhibitory concentration

C. albicans (90028) and Candida parapsilosis (22019) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Trichosporon beigelii (7707), Malassezia furfur (7744), Aspergillus flavus (6905), and Aspergillus fumigatus (6145) were obtained from the Korean Collection for Type Cultures (KCTC). Fungal strains were routinely grown in yeast extract peptone dextrose (YPD, Difco, Sparks, MD, U.S.A.) agar plates at 28°C, and M. furfur was grown in modified yeast malt (YM, Difco, Sparks, MD, U.S.A.) agar plates containing 1% olive oil at 32°C. Cells, cultured overnight in YPD or YM with 1% olive oil, were dispensed into 96-well plates at a volume of 200 µl/well. The minimum inhibitory concentration (MIC) was determined using a standard microdilution method. After incubation for 24 h, the growth was measured using a microtiter ELISA Reader (Molecular Devices Emax, CA) with absorbance at 600 nm [16].

Measurement of hemoglobin released from human erythrocytes

An ELISA reader, with absorbance at 414 nm, was used to determine the hemolytic effect of periplanetasin-2 by the percentage of hemoglobin released from an 8% suspension of human erythrocytes. The absorbance (Abs414 nm) in phosphate-buffered saline (PBS) and Abs414 nm in 0.1% Triton X-100 were used to calculate 0 and 100% release of hemoglobin. The percentage of hemolysis was calculated using the following equation: hemolysis (%) = 100[(Abs414 nm in the peptide solution − Abs414 nm in PBS)/(Abs414 nm in 0.1% Triton X-100 − Abs414 nm in PBS)] [17].

Cell culture and treatment with periplanetasin-2

C. albicans cells were cultured in YPD broth at 28°C. Cells (3 × 105/ml) were washed with PBS and incubated with 5, 10, 20, and 40 µM periplanetasin-2 for 3 h or 10 µM periplanetasin-2 at time intervals.

Measuring the levels of ROS

After incubation, the cells were washed and incubated with 1 µg/ml H2DCFDA for 1 h. H2DCFDA is an indicator of ROS; the fluorescence intensity of H2DCFDA was measured using a spectrofluorometer (Shimadzu RF-5301PC, Shimadzu, Kyoto, Japan) at an excitation wavelength of 495 nm and emission of 525 nm [18].

Measurement of lipid peroxidation

After incubation, the cells were washed and sonicated for 1 min in lysis buffer (10 mM Tris–HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 2% Triton X-100, and 1% SDS). The suspension was centrifuged at 12 000 rpm for 10 min, and 5% trichloroacetic acid was added to the collected supernatant to precipitate the proteins. Next, the supernatant was collected and mixed with thiobarbituric acid (TBA). The mixture was incubated at 95°C for 40 min and cooled down for 5 min at room temperature. The level of lipid peroxides was measured using a spectrophotometer (DU530, Beckman, Fullerton, CA, U.S.A.) with absorbance at 532 nm [19].

Detection of potassium release

After incubation in 1 h intervals, the cells were centrifuged, and each supernatant was transferred into 24-well plates. Next, an ionic strength adjuster for potassium was added to the wells, and the voltage was measured using an ion-selective electrode meter (Orion Star A214; Thermo Scientific, Singapore). Potassium release was calculated using the following equation: potassium release (%) = 100 × ([K+]m − [K+]i)/([K+]t − [K+]i). [K+]m represents the potassium voltage of compound-treated samples; the initial (Ki) and total (Kt) of potassium represents the voltage of the medium and that of sonicated samples, respectively [20].

Double-staining with Annexin V and propidium iodide

Protoplasts were prepared before the procedures. Cells were digested for 4 h at 28°C with 0.1 M potassium phosphate buffer (PPB, pH 6.0) containing 1 M sorbitol and 20 mg/ml lysing enzyme. Protoplasts were washed with 0.1 M PPB (pH 6.0) containing 1 M sorbitol and were then treated with periplanetasi-2. After incubation, the cells were washed twice with 0.1 M PPB (pH 6.0) containing 1 M sorbitol and stained with Annexin V and propidium iodide (PI), supplied in the Apoptosis Detection Kit, for 15 min at 28°C. Annexin V is an indicator of PS externalization; the fluorescence of Annexin V was analyzed using a FACSVerse flow cytometer (Becton Dickinson, NJ, U.S.A.) [21].

Terminal deoxynucleotidyl transferase dUTP nick end labeling

After incubation, the cells were fixed with 2% paraformaldehyde for 1 h and incubated with a permeabilization solution (0.1% Triton X-100 and 0.1% sodium citrate) for 2 min on ice. Next, the cells were stained for 1 h at 37°C with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction mixture provided in the In Situ Cell Death Detection Kit. TUNEL is an indicator of DNA fragmentation; the fluorescence intensity of TUNEL was measured using a spectrofluorometer at an excitation wavelength of 495 nm and emission of 519 nm [21].

Detection of mitochondrial depolarization

After incubation, the cells were resuspended in warm PBS and incubated with 2.5 µg/ml JC-1. JC-1 is an indicator of mitochondrial membrane potential; the fluorescence of JC-1 was analyzed using a FACSVerse flow cytometer [22].

Detecting the levels of cytosolic and mitochondrial calcium

After incubation in 1 h intervals, the cells were washed twice with Krebs buffer (pH 7.2) and resuspended in the Krebs buffer containing 1% bovine serum albumin and 0.01% pluronic acid F-127. Next, the cells were incubated with 5 µM Fura-2AM for 40 min or 10 µM Rhod-2AM for 30 min, and washed thrice with calcium-free Krebs buffer (pH 7.2). Fura-2AM and Rhod-2AM are indicators of calcium levels in the cytosol and mitochondria; the fluorescence intensity of Fura-2AM and Rhod-2AM was measured using a spectrofluorometer at an excitation wavelength of 340 or 552 nm, and an emission wavelength of 510 or 581 nm [23].

Preparation of mitochondria

The mitochondria were separated before being used in the procedures. After incubation, the cells were homogenized in a homogenization medium (50 mM Tris [pH 7.5], 2 mM EDTA, and 1 mM phenylmethylsulphonyl fluoride [PMSF]) and 2% glucose was added. After centrifugation for 45 min, the supernatants (cytosol) and pellets (mitochondria) were transferred into a new Eppendorf tube. Next, the pellets were resuspended in the buffer appropriate for each procedure [7].

Measuring the levels of intracellular glutathione

After the cells were incubated in 1 h intervals, the mitochondria were separated. The volumes of the cellular supernatants (cytosol) and cell pellets (mitochondria) were measured, and resuspended in 3 volumes of 5% SSA solution, exposed to three freeze-thaw cycles for cell lysis. Next, the cells were centrifuged at 12 000 rpm for 10 min, and the supernatant was collected and used to measure the levels of glutathione. The measurement of glutathione levels was based on the method for glutathione reductase enzymatic recycling, and the levels of glutathione were measured using a microtiter ELISA Reader with absorbance at 415 nm [24].

Measurement of mitochondrial ROS levels

After incubation, the cells were washed and incubated with 5 µM MitoSOX Red for 30 min at 37°C. Next, the cells were washed thrice with PBS. MitoSOX Red is an indicator of the levels of mitochondrial ROS; the fluorescent intensity of MitoSOX Red was measured using a spectrofluorometer at an excitation wavelength of 510 and an emission of 580 nm [25].

Detection of cytochrome c release

After the cells were incubated in 1 h intervals, and the mitochondria were separated, the supernatants (cytosol) were used to measure the levels of cytochrome c in the cytosol; the pellets (mitochondria) were resuspended in 50 mM Tris (pH 5.0) with 2 mM EDTA to measure the levels of cytochrome c in the mitochondria. Next, ascorbic acid (1 g/ml), which is a reducing agent for cytochrome c, was added, and the levels of cytochrome c were measured using a spectrophotometer with absorbance at 550 nm [7].

Detecting the levels of activated caspase

After incubation in 1 h intervals, the cells were washed twice with PBS and incubated with 2.5 µM CaspACE FITC-VAD-FMK for 20 min. CaspACE FITC-VAD-FMK is a pan-caspase inhibitor; the fluorescence of CaspACE FITC-VAD-FMK was analyzed using a FACSVerse flow cytometer [26].

Fractional inhibitory concentration

The MICs of antibiotics were determined using a standard microdilution method as described above. The fractional inhibitory concentration (FIC) was determined using a method. Cells were cultured overnight in YPD and dispensed into 96-well plates at a volume of 100 µl/well. A checkerboard, with two-fold dilutions of each agent, was used to determine the effect of combining periplanetasin-2 with the antibiotics fluconazole and amphotericin B. After incubation for 24 h, the growth was measured using a microtiter ELISA Reader with absorbance at 600 nm. The FIC index (FICI) was calculated by the following equation: FICI = (MICagent A in combination/MICagent A alone) + (MICagent B in combination/MICagent B alone). The FICI was interpreted as follows: FICI < 0.5, synergistic; 0.5 ≤ FICI < 1, additive; 2 ≤ FICI < 4, indifferent; FICI > 4, antagonistic [27,28].

Statistical analyses

The results are expressed as the mean ± standard deviation of at least three independent experiments. The differences and significance of results were analyzed using Student's t-test.

Results

Property of periplanetasin-2

Periplanetasin-2 is derived from the E. coli-immunized cockroach

A comparison of E. coli-immunized and non-immunized cockroaches yielded total sequences, which were assembled into contigs and functionally annotated using the Basic Local Alignment Search Tool (BLAST), Gene Ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database terms. The 86 antimicrobial peptide candidates were predicted from the transcriptome by the silico antimicrobial peptide prediction method; 21 of these peptides were experimentally validated as antimicrobial peptides [4]. In the interests of cost efficiency, the active regions, including periplanetasin-2 (YPCKLNLKLGKVPFH-NH2), were synthesized as antimicrobial peptides based on the AMPA stretch (Table 1).

Table 1
Amino acid sequence and physicochemical features of periplanetasin-2
Peptide Sequence Purity (%) Molecular mass Net charge 
Periplanetasin-2 YPCKLNLKLGKVPFH-NH2 98.0 1756.2 +4 
Peptide Sequence Purity (%) Molecular mass Net charge 
Periplanetasin-2 YPCKLNLKLGKVPFH-NH2 98.0 1756.2 +4 

Periplanetasin-2 has an antifungal effect on pathogenic fungi

To evaluate the antifungal effect of periplanetasi-2, we conducted an MIC test against pathogenic fungi; the potent antimicrobial peptide melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2) was used to compare the antifungal effects [29]. Similar to melittin, periplanetasin-2 showed an antifungal effect against C. albicans, C. parapsilosis, T. beigelii, M. furfur, A. flavus, and A. fumigatus (Table 2). In the test strain C. albicans, the MIC of periplanetasin-2 was 10 µM. These results indicate that the periplanetasin-2 possesses antifungal activity against pathogenic fungi.

Table 2
Antifungal effect of periplanetasin-2

The MIC was determined using a standard microdilution method. After incubation for 24 h, the growth was measured using a microtiter ELISA Reader with absorbance at 600 nm.

Fungal strains MIC (μM) 
Periplanetasin-2 Melittin 
C. albicans ATCC 90028 10 2.5 
C. parapsilosis ATCC 22019 1.3 
T. beigelii KCTC 7707 1.3 
M. furfur KCTC 7744 2.5 
A. flavus KCTC 6905 10 2.5 
A. fumigatus KCTC 6145 10 1.3 
Fungal strains MIC (μM) 
Periplanetasin-2 Melittin 
C. albicans ATCC 90028 10 2.5 
C. parapsilosis ATCC 22019 1.3 
T. beigelii KCTC 7707 1.3 
M. furfur KCTC 7744 2.5 
A. flavus KCTC 6905 10 2.5 
A. fumigatus KCTC 6145 10 1.3 

Periplanetasin-2 has no hemolytic effect on human erythrocytes

To assess the cytotoxicity of periplanetasin, we evaluated human erythrocytes for hemolysis. Melittin is highly hemolytic and toxic, but also exerts a potent antimicrobial effect [29]. At the concentrations ranging from 1.3 to 80 µM, periplanetasin-2 showed no hemolytic activity (Table 3). However, melittin caused 99.0% hemolysis at 80 µM and 13.3% hemolysis at 2.5 µM value. These results indicate that periplanetasin-2 is not cytotoxic to human erythrocytes.

Table 3
Hemolytic effect of periplanetasin-2

Hemoglobin, released from an 8% suspension of human erythrocytes, was measured at 414 nm with an ELISA reader. The percentage of hemolysis was calculated using the following equation: hemolysis (%) = 100[(Abs414 nm in the peptide solution − Abs414 nm in PBS)/(Abs414 nm in 0.1% Triton X-100 − Abs414 nm in PBS)].

Peptide Hemolysis (%) 
80.0 μM 40.0 μM 20.0 μM 10.0 μM 5.0 μM 2.5 μM 1.3 μM 
Periplanetasin-2 
Melittin 99.0 91.1 70.2 54.3 23.8 13.3 1.9 
Peptide Hemolysis (%) 
80.0 μM 40.0 μM 20.0 μM 10.0 μM 5.0 μM 2.5 μM 1.3 μM 
Periplanetasin-2 
Melittin 99.0 91.1 70.2 54.3 23.8 13.3 1.9 

Intracellular oxidative stress caused by periplanetasin-2

Periplanetasin-2 generates intracellular ROS

ROS, including hydrogen peroxide, hydroxyl, and peroxyl radicals, are important for the induction of apoptosis in inflammatory, tumor, and fungal cells [30]. H2DCFDA is an oxidant-specific probe that undergoes oxidation and cleavage of its acetate groups by esterases; H2DCFDA fluoresces when it reacts with ROS [31]. Hydrogen peroxide was used as a positive control for examining the generation of ROS. In cells treated with periplanetasin-2, the increases in the intensity of fluorescence H2DCFDA were dependent on the concentration of periplanetasin-2 (Figure 1A). These results indicate that periplanetasin-2 generates intracellular ROS, leading to intracellular oxidative damage.

Intracellular oxidative stress induced by periplanetasin-2.
Figure 1.
Intracellular oxidative stress induced by periplanetasin-2.

C. albicans cells were treated with 5, 10, 20, and 40 µM periplanetasin-2 for 3 h at 28°C. (A) Cells were washed and incubated with 1 µg/ml H2DCFDA for 1 h. The bars indicate the fluorescent intensity of H2DCFDA, which was measured using a spectrofluorometer at an excitation wavelength of 495 and emission of 525 nm. (B) Cells were sonicated in lysis buffer, and 5% trichloroacetic acid was added for protein precipitation. Next, the supernatant was mixed with 2-TBA. The bars indicate the level of lipid peroxidation, which was measured using a spectrophotometer with absorbance at 532 nm. *P < 0.05, **P < 0.01, and ***P < 0.001 for the significance and difference.

Figure 1.
Intracellular oxidative stress induced by periplanetasin-2.

C. albicans cells were treated with 5, 10, 20, and 40 µM periplanetasin-2 for 3 h at 28°C. (A) Cells were washed and incubated with 1 µg/ml H2DCFDA for 1 h. The bars indicate the fluorescent intensity of H2DCFDA, which was measured using a spectrofluorometer at an excitation wavelength of 495 and emission of 525 nm. (B) Cells were sonicated in lysis buffer, and 5% trichloroacetic acid was added for protein precipitation. Next, the supernatant was mixed with 2-TBA. The bars indicate the level of lipid peroxidation, which was measured using a spectrophotometer with absorbance at 532 nm. *P < 0.05, **P < 0.01, and ***P < 0.001 for the significance and difference.

Periplanetasin-2 induces lipid peroxidation

Free radicals induce oxidation and degradation of membrane lipids, resulting in cell damage [32]. The TBA test is a common method for the detection of lipid peroxidation. Several aldehydes, such as malonaldehyde (MDA), are formed as by-products of lipid peroxidation; TBA reacts with these by-products [33]. In periplanetasin-2-treated cells, the level of MDA (nmole/mg protein) was increased compared with that in control cells (Figure 1B). Similar to the production of ROS, the increase in the level of MDA is dependent on concentration. These results indicate that periplanetasin-2 induces cellular lipid peroxidation and increases by-products, such as MDA.

Potassium release induced by periplanetasin-2

Potassium is a predominant ion in cells. Reduced concentrations in intracellular potassium lead to the induction of apoptosis and enhance the activation of the cell death programme [34]. After treatment with periplanetasin-2, the increase in the release of potassium was observed during the time intervals (Figure 2). At MIC, periplanetasin-2 induced ∼60% of potassium release, whereas hydrogen peroxide induced ∼80% of potassium release in 1 h. However, potassium release increased gradually, and ∼80% of potassium release was observed in 3 h. These results indicated that periplanetasin-2 caused cellular damage by releasing intracellular potassium.

Potassium release induced by periplanetasin-2.

Figure 2.
Potassium release induced by periplanetasin-2.

C. albicans cells were treated with 5, 10, 20, and 40 µM periplanetasin-2 for 3 h. The supernatants were transferred to 24-well plates and potassium voltage was measured using an ion-selective electrode meter. *P < 0.05, **P < 0.01, and ***P < 0.001 for the significance and difference.

Figure 2.
Potassium release induced by periplanetasin-2.

C. albicans cells were treated with 5, 10, 20, and 40 µM periplanetasin-2 for 3 h. The supernatants were transferred to 24-well plates and potassium voltage was measured using an ion-selective electrode meter. *P < 0.05, **P < 0.01, and ***P < 0.001 for the significance and difference.

Concentration-dependent apoptotic effect of periplanetasin-2

Periplanetasin-2 induces PS exposure

The translocation of PS from the inner plasma membrane to the outer plasma membrane, and exposure of PS at the external surface of the cell, is a marker of early apoptosis. Annexin V is a Ca2+-dependent phospholipid-binding probe, showing high affinity for PS; therefore, Annexin V is used as a marker for early apoptosis [35]. Dual-staining with fluorescent Annexin V and PI, a membrane-impermeable, DNA-specific probe, is used to discriminate necrosis [36]. In periplanetasin-2-treated cells, the number of Annexin V-stained cells was increased compared with that of PI-stained cells (Figure 3A). These results indicate that periplanetasin-2 induced apoptosis by causing translocation and exposure of PS.

Concentration-dependent apoptotic effect of periplanetasin-2.
Figure 3.
Concentration-dependent apoptotic effect of periplanetasin-2.

C. albicans cells were treated with 5, 10, 20, and 40 µM periplanetasin-2 for 3 h at 28°C. (A) Cells were washed twice with 0.1 M PPB (pH 6.0), containing 1 M sorbitol, and stained using the Apoptosis Detection Kit for 15 min. The fluorescence intensity of Annexin V was analyzed by a FACSVerse flow cytometer. (B) Cells were fixed with 2% paraformaldehyde and permeabilized on ice. Next, the cells were stained using the In Situ Cell Death Detection Kit for 1 h. The bars indicate the fluorescence intensity of TUNEL staining, which was measured using a spectrofluorometer at an excitation wavelength of 495 and an emission wavelength of 519 nm. *P < 0.05, **P < 0.01, and ***P < 0.001 for the significance and difference.

Figure 3.
Concentration-dependent apoptotic effect of periplanetasin-2.

C. albicans cells were treated with 5, 10, 20, and 40 µM periplanetasin-2 for 3 h at 28°C. (A) Cells were washed twice with 0.1 M PPB (pH 6.0), containing 1 M sorbitol, and stained using the Apoptosis Detection Kit for 15 min. The fluorescence intensity of Annexin V was analyzed by a FACSVerse flow cytometer. (B) Cells were fixed with 2% paraformaldehyde and permeabilized on ice. Next, the cells were stained using the In Situ Cell Death Detection Kit for 1 h. The bars indicate the fluorescence intensity of TUNEL staining, which was measured using a spectrofluorometer at an excitation wavelength of 495 and an emission wavelength of 519 nm. *P < 0.05, **P < 0.01, and ***P < 0.001 for the significance and difference.

Periplanetasin-2 induces DNA fragmentation

Degradation and fragmentation of DNA, occurring as single- or double-strand breaks, is a marker of late apoptosis. DNA fragmentation is detectable using the TUNEL assay, which end-labels the single- or double-strand breaks in the DNA using terminal deoxyribonucleotidyl transferase [37]. In periplanetasin-2-treated cells, the increase in the fluorescence intensity of the TUNEL was dependent on the concentration of periplanetasi-2 (Figure 3B). These results indicate that periplanetasin-2 induces apoptosis, and that DNA fragmentation and degradation were dependent on the concentration of periplanetasin-2.

Mitochondrial dysfunction induced by periplanetasin-2

Periplanetasin-2 depolarizes mitochondrial membrane potential

Mitochondrial transmembrane potential is lost in response to oxidative stress [38]. JC-1 is a lipophilic cationic probe that forms aggregates from monomers, depending on the state of membrane potential. The color of JC-1 is changed reversibly from green (indicating JC-1 monomers) to orange (indicating JC-1 aggregates) as the mitochondrial membrane becomes depolarized [39]. In periplanetasin-2-treated cells, the mean values of JC-1 were decreased compared with those in control cells (Figure 4A). These results indicate that periplanetasin-2 caused damage to the mitochondria, resulting in depolarization of the mitochondrial membrane potential.

Mitochondrial dysfunction induced by periplanetasin-2.
Figure 4.
Mitochondrial dysfunction induced by periplanetasin-2.

(A) C. albicans cells were treated with 5, 10, 20, and 40 µM periplanetasin-2 for 3 h at 28°C. Cells were resuspended in warm PBS and incubated with 2.5 µg/ml JC-1. The fluorescence of JC-1 was analyzed by a FACSVerse flow cytometer. (B) C. albicans cells were treated with 10 µM periplanetasin-2 for 3 h. For measurements of cytosolic and mitochondrial levels of calcium, cells were incubated with 5 µM Fura-2AM for 40 min or with 10 µM Rhod-2AM for 30 min. The bars indicate the fluorescence intensity of Fura-2AM and Rhod-2AM, which was measured using a spectrofluorometer at an excitation wavelength of 340 or 552 nm, and an emission wavelength of 510 or 581 nm. *P < 0.05, **P < 0.01, and ***P < 0.001 for the significance and difference.

Figure 4.
Mitochondrial dysfunction induced by periplanetasin-2.

(A) C. albicans cells were treated with 5, 10, 20, and 40 µM periplanetasin-2 for 3 h at 28°C. Cells were resuspended in warm PBS and incubated with 2.5 µg/ml JC-1. The fluorescence of JC-1 was analyzed by a FACSVerse flow cytometer. (B) C. albicans cells were treated with 10 µM periplanetasin-2 for 3 h. For measurements of cytosolic and mitochondrial levels of calcium, cells were incubated with 5 µM Fura-2AM for 40 min or with 10 µM Rhod-2AM for 30 min. The bars indicate the fluorescence intensity of Fura-2AM and Rhod-2AM, which was measured using a spectrofluorometer at an excitation wavelength of 340 or 552 nm, and an emission wavelength of 510 or 581 nm. *P < 0.05, **P < 0.01, and ***P < 0.001 for the significance and difference.

Periplanetasin-2 causes the accumulation of calcium in cytosol and mitochondria

The accumulation of calcium in the cytosol and mitochondrial matrix is critically important to cellular function [39]. The calcium-specific probes, Fura-2AM and Rhod-2AM, are commonly used to assess the changes in the level of calcium in the cytosol and mitochondria, respectively [40]. After treatment with periplanetasin-2, the increase in the fluorescence intensity of Fura-2AM and Rhod-2AM was similar to those caused by treatment with hydrogen peroxide (Figure 4B). These results indicate that periplanetasin-2 caused an accumulation of calcium in the cytosol and mitochondria, leading to mitochondrial and cellular dysfunction.

Mitochondrial oxidative stress induced by periplanetasin-2

Periplanetasin-2 decreases the levels of glutathione in the cytosol, leading to increased levels of glutathione in the mitochondria

Glutathione plays an important role in antioxidant processes and is involved in first and second lines of defense against the ROS [41]. In periplanetasin-2-treated cells, the level of cytosolic glutathione (nmol/mg protein) was decreased, while that of mitochondrial glutathione was increased (Figure 5A). A change in the levels of glutathione was observed after 1 h; in particular, the level of mitochondrial glutathione increased rapidly after 2 h of treatment. These results indicate that periplanetasin-2 induced an excess of ROS production, which overwhelmed the cellular antioxidant system, leading to a decreased level of cytosolic glutathione. This oxidative damage extended to the mitochondria, leading to the increased levels of mitochondrial glutathione.

Mitochondrial oxidative stress induced by periplanetasin-2.
Figure 5.
Mitochondrial oxidative stress induced by periplanetasin-2.

(A) C. albicans cells were treated with 10 µM periplanetasin-2 for 3 h. After separation of the mitochondria, cell supernatants (cytosol) and pellets (mitochondria) were resuspended in 5% SSA and exposed to three freeze–thaw cycles for cell lysis. The bars indicate the levels of glutathione, which were measured using a microtiter ELISA Reader with absorbance at 415 nm. (B) C. albicans cells were treated with 5, 10, 20, and 40 µM periplanetasin-2 for 3 h at 28°C. Cells were incubated with 5 µM MitoSOX Red for 30 min and washed thrice with PBS. The bars indicate the fluorescence intensity of MitoSOX Red, which was measured using a spectrofluorometer at an excitation wavelength of 510 nm and an emission wavelength of 580 nm. *P < 0.05, **P < 0.01, and ***P < 0.001 for the significance and difference.

Figure 5.
Mitochondrial oxidative stress induced by periplanetasin-2.

(A) C. albicans cells were treated with 10 µM periplanetasin-2 for 3 h. After separation of the mitochondria, cell supernatants (cytosol) and pellets (mitochondria) were resuspended in 5% SSA and exposed to three freeze–thaw cycles for cell lysis. The bars indicate the levels of glutathione, which were measured using a microtiter ELISA Reader with absorbance at 415 nm. (B) C. albicans cells were treated with 5, 10, 20, and 40 µM periplanetasin-2 for 3 h at 28°C. Cells were incubated with 5 µM MitoSOX Red for 30 min and washed thrice with PBS. The bars indicate the fluorescence intensity of MitoSOX Red, which was measured using a spectrofluorometer at an excitation wavelength of 510 nm and an emission wavelength of 580 nm. *P < 0.05, **P < 0.01, and ***P < 0.001 for the significance and difference.

Periplanetasin-2 generates mitochondrial ROS

Mitochondrial ROS are toxic by-products of metabolism because of their potential to cause damage to lipids, proteins, and the DNA [42]. MitoSOX Red is a mitochondrial oxidation-specific probe, detecting the production of superoxide in the mitochondria [43]. In periplanetasin-2-treated cells, the increase in the fluorescence intensity of MitoSOX Red was dependent on the concentration of periplanetasin-2 (Figure 5B). These results indicate that periplanetasin-2 generated mitochondrial ROS, changing the mitochondria into fatal organelles and triggering apoptosis.

Time-dependent apoptotic effect of periplanetasin-2

Periplanetasin-2 induces the release of cytochrome c from the mitochondria into cytosol

Cytochrome c is a proapoptotic factor; the release of cytochrome c from the mitochondria triggers activation of caspases and apoptotic cell death [44]. After treatment with periplanetasin-2, the level of cytosolic cytochrome c was increased, while that of mitochondrial cytochrome c was decreased (Figure 6A). The changes in the levels of cytochrome c in periplanetasin-2-treated cells peaked in 3 h. These results indicate that periplanetasin-2 induced the release of cytochrome c from the mitochondria into the cytosol, leading to the activation of apoptotic proteins.

Time-dependent apoptotic effect of periplanetasin-2.
Figure 6.
Time-dependent apoptotic effect of periplanetasin-2.

C. albicans cells were treated with 10 µM periplanetasin-2 for 3 h. (A) After separation of the mitochondria, cell supernatants (cytosol) and pellets (mitochondria) were used to measure the levels of cytochrome c in the cytosol and mitochondria. Ascorbic acid (1 g/ml) was added to reduce cytochrome c. The bars indicate the levels of cytochrome c, which were measured using a spectrophotometer with absorbance at 550 nm. (B) Cells were washed twice and incubated with 2.5 µM CaspACE FITC-VAD-FMK for 20 min. The fluorescence of CaspACE FITC-VAD-FMK was analyzed by a FACSVerse flow cytometer. *P < 0.05, **P < 0.01, and ***P < 0.001 for the significance and difference.

Figure 6.
Time-dependent apoptotic effect of periplanetasin-2.

C. albicans cells were treated with 10 µM periplanetasin-2 for 3 h. (A) After separation of the mitochondria, cell supernatants (cytosol) and pellets (mitochondria) were used to measure the levels of cytochrome c in the cytosol and mitochondria. Ascorbic acid (1 g/ml) was added to reduce cytochrome c. The bars indicate the levels of cytochrome c, which were measured using a spectrophotometer with absorbance at 550 nm. (B) Cells were washed twice and incubated with 2.5 µM CaspACE FITC-VAD-FMK for 20 min. The fluorescence of CaspACE FITC-VAD-FMK was analyzed by a FACSVerse flow cytometer. *P < 0.05, **P < 0.01, and ***P < 0.001 for the significance and difference.

Periplanetasin-2 induces caspase activation

The apoptotic signal is accompanied by activation of caspases, which selectively cleave specific cellular substrates [45]. CaspACE FITC-VAD-FMK is a FITC-conjugated broad-spectrum inhibitor of caspases that irreversibly binds to activated caspases [46]. After treatment with periplanetasin-2, we observed caspase activation (Figure 6B). Compared with the percentage of activated caspase in the control cells, 13.17% of caspase was activated after 2 h, and 26.27% was activated after 3 h in cells treated with periplanetasi-2. The results indicate that periplanetasin-2 induced caspase activation, triggering apoptotic events in a time-dependent manner.

Synergistic effect of periplanetasin-2

Periplanetasin-2 acts synergistically with antibiotics

To evaluate the synergistic effect of periplanetasin-2 combined with antibiotics, we used the antibiotics fluconazole and amphotericin B. Fluconazole is fungistatic, and amphotericin B is fungicidal [47]. The MICs of periplanetasin-2, fluconazole, and amphotericin B were 10, 30, and 3 µM, respectively, in C. albicans (Table 4). However, when periplanetasin-2 was combined with fluconazole or amphotericin B, the FICs of the combined treatments were 1.25/7.50 and 1.25/0.06 µM, respectively. The FICI of periplanetasin-2 + fluconazole was 0.38, and that of periplanetasin-2 + amphotericin B was 0.15, indicating synergistic effects.

Table 4
Synergistic effect of periplanetasin-2
Strain MIC (μM) 
Drug alone Drug in combination FICI Drug alone Drug in combination FICI 
PER-2 FLC PER-2 FLC PER-2/FLC PER-2 AMB PER-2 AMB PER-2/AMB 
C. albicans ATCC90028 10 30 1.25 7.50 0.38 (S) 10 1.25 0.06 0.15 (S) 
Strain MIC (μM) 
Drug alone Drug in combination FICI Drug alone Drug in combination FICI 
PER-2 FLC PER-2 FLC PER-2/FLC PER-2 AMB PER-2 AMB PER-2/AMB 
C. albicans ATCC90028 10 30 1.25 7.50 0.38 (S) 10 1.25 0.06 0.15 (S) 

The FICI was calculated using the following equation: FICI = (MICagentA in combination/MICagent A alone) + (MICagent B in combination/MICagent B alone). FICI < 0.5, synergistic; 0.5 ≤ FICI < 1, additive; 2 ≤ FICI < 4, indifferent; FICI > 4, antagonistic. Abbreviations: PER-2, periplanetasin-2; FLC, fluconazole; AMB, amphotericin B; S, synergistic.

Combined treatment with periplanetasin-2 and antibiotics induces oxidative stress

The effectiveness of the compounds, used for treatment, was evaluated based on the results of the FIC test. The fluorescence intensity of H2DCFDA in 7.50 µM fluconazole-treated cells was the same as that in the control cells (Figure 7). A slight increase in fluorescence was observed in 1.25 µM periplanetasin-2- and 0.06 µM amphotericin B-treated cells. However, rapid increases in fluorescence were observed in treatments combining periplanetasin-2 + fluconazole and periplanetasin-2 + amphotericin B. These results indicate that the synergistic effect of periplanetasin-2 and fluconazole, or periplanetasin-2 and amphotericin B, resulted from the induction of oxidative stress leading to excessive production of ROS.

Synergistic oxidative stress induced by combining periplanetasin-2 with antibiotics.
Figure 7.
Synergistic oxidative stress induced by combining periplanetasin-2 with antibiotics.

A checkerboard, with two-fold dilutions of each agent, was used to determine the effect of combining periplanetasin-2 with fluconazole and amphotericin B. C. albicans cells were treated with 1.25 µM periplanetasin-2, 7.5 µM fluconazole, and 0.06 µM amphotericin B for 3 h at 28°C. Cells were washed and incubated with 1 µg/ml H2DCFDA for 1 h. The bars indicate the fluorescence intensity of H2DCFDA, which was measured by a spectrofluorometer at an excitation wavelength of 495 nm and an emission wavelength of 525 nm. *P < 0.05, **P < 0.01, and ***P < 0.001 for the significance and difference.

Figure 7.
Synergistic oxidative stress induced by combining periplanetasin-2 with antibiotics.

A checkerboard, with two-fold dilutions of each agent, was used to determine the effect of combining periplanetasin-2 with fluconazole and amphotericin B. C. albicans cells were treated with 1.25 µM periplanetasin-2, 7.5 µM fluconazole, and 0.06 µM amphotericin B for 3 h at 28°C. Cells were washed and incubated with 1 µg/ml H2DCFDA for 1 h. The bars indicate the fluorescence intensity of H2DCFDA, which was measured by a spectrofluorometer at an excitation wavelength of 495 nm and an emission wavelength of 525 nm. *P < 0.05, **P < 0.01, and ***P < 0.001 for the significance and difference.

Discussion

Allergens, produced by cockroaches, are important causes of asthma and several allergic diseases. The principal domiciliary species of cockroach are the German (B. germanica) and American (P. americana) cockroach. P. americana is the dominant species found in houses [48]. However, numerous studies suggest that the cockroach is a good source of antimicrobial agents [3]. Periplanetasin-2 is derived from E. coli-immunized P. americana and a cationic peptide, resulting in a positive net charge of +4 [4]. The MIC test indicated that similar to melittin, the cationic and antimicrobial peptide periplanetasin-2 had an antifungal effect on pathogenic fungi, raising the possibility of using periplanetasin-2 as a new antifungal agent. Additionally, periplanetasin-2 has shown no hemolytic effect on human erythrocytes and is non-cytotoxic in humans. In recent research, the new antifungal peptides were discovered and their modes of action were investigated. RsAFP2 induced a signaling cascade, which involves ROS generation leading to cell growth arrest of C. albicans, and eryngin inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola [49,50]. Therefore, we investigated the antifungal mechanism of periplanetasin-2.

ROS are continually produced as by-products of aerobic metabolism. Because of their high toxicity, ROS are rapidly detoxified by various antioxidant systems. However, ROS are also signaling molecules that control various processes including defense against pathogens and programmed cell death [51]. The ROS assay, conducted with H2DCFDA, showed that periplanetasin-2 generated intracellular ROS; the excess levels of ROS caused cellular damage, leading to apoptosis. Lipid peroxidation involves the formation and propagation of lipid radicals, oxygen uptake, rearrangement of double bonds in unsaturated lipids, and eventual destruction of membrane lipids, producing a variety of breakdown products, including alcohols, ketones, aldehydes, and ethers [52]. The TBARS assay showed that periplanetasin-2 induced the peroxidation of the intracellular membrane; the increase in by-products, resulting from lipid peroxidation, was detected by TBA. The oxidative effect of periplanetasin-2 created an oxidative intracellular environment, inducing cell damage and leading to apoptosis.

Potassium is the dominant positive ion within cells and contributes to a variety of cellular functions such as cellular signaling and regulation of cellular volume. Potassium efflux has been shown in damaged cells, and cellular apoptosis can result from potassium deprivation [53]. The increased release of potassium, induced by periplanetasin-2 in this study, was dependent on the time and concentration of the periplanetasin-2 treatment. Depletion of intracellular potassium, caused by periplanetasin-2, damaged cellular functions. Moreover, periplanetasin-2 showed two apoptotic hallmarks in a concentration-dependent manner. PS is constitutively present in the inner leaflet, whereas phosphatidylcholine and sphingomyelin are constitutively present in the outer leaflet of the plasma membrane. However, when cell death occurs, PS is translocated to the outer layer of the plasma membrane, while the cell membrane remains intact [54]. The exposure of PS on the outer layer of the plasma membrane, induced by periplanetasin-2, was confirmed with Annexin V and PI double-staining. The exposure of PS, an early apoptotic hallmark, was increased when the concentration of periplanetasin-2 was increased. The TUNEL assay, which detects the formation of breaks in the DNA strand, present in late apoptosis, indicated that DNA fragmentation occurred after treatment with periplanetasin-2 [55]. This was also increased when the concentration of periplanetasin-2 was increased. Taken together, these results suggest that the apoptotic effect of periplanetasin-2 was dependent on concentration.

Mitochondria are important for energy production and determination of cellular life and death [56]. Mitochondrial membrane potential is essential for the maintenance of mitochondrial functions. Mitochondrial membrane depolarization induces changes in the mitochondrial structures, which result in the translocation of cytochrome c from the cristae to the intermembrane space [57]. Depolarization of the mitochondrial membrane, induced by periplanetasin-2, was detected using JC-1 staining, which indicated the involvement of the mitochondria in the apoptotic process. The change in the levels of calcium supported this notion. In malfunctioning cells, excessive calcium is accumulated in the cytosol. Rapidly elevated calcium levels in the cytosol activate the mitochondrial calcium uniporter, which mediates the uptake of calcium by the mitochondria, leading to mitochondrial calcium overload and dysfunction [58]. Measurements, using Fura-2AM and Rhod-2AM, indicated that the levels of cytosolic and mitochondrial calcium increased after treatment with periplanetasin-2. Excessive calcium accumulation in the cytosol led to an accumulation of calcium in the mitochondria, causing severe mitochondrial damage.

Glutathione is a cellular antioxidant that detoxifies H2O2 and lipid hydroperoxides. The depletion of glutathione is an early event in apoptosis [59]. Moreover, mitochondrial oxidative stress induces the import of glutathione from cytosol into the mitochondria [60]. In this study, the glutathione assay indicated that the levels of glutathione decreased after treatment with periplanetasin-2, because periplanetasin-2 generated excessive levels of ROS; thus, the intracellular antioxidant system failed to protect the cells. This oxidative damage extended to the mitochondria, causing a rapid increase in the mitochondrial levels of glutathione and inducing the mitochondria to trigger apoptotic signaling. Mitochondrial ROS are the toxic by-products of metabolism and can potentially cause damage to lipids, proteins, and the DNA [42]. MitoSOX Red staining indicated a rapid increase in the levels of mitochondrial ROS after treatment with periplanetasin-2. Taken together, these results suggest that the oxidative damage, induced by periplanetasin-2, led to mitochondrial dysfunction and involvement of the mitochondria in periplanetasin-2-induced apoptosis.

The release of cytochrome c is an important signal in the mitochondrial pathway of apoptosis. When cytochrome c is released into the cytosol, it reacts with a variety of cytoplasmic components, inducing apoptotic signals and activating caspases [61]. Measurements of the levels of cytochrome c indicated that periplanetasin-2 induced the release of cytochrome c from the mitochondria into the cytosol. Although scant release of cytochrome c was shown 1 h post-treatment, the release proceeded more rapidly after 2 h. These results indicate that the mitochondrial pathway of apoptosis became progressively more involved after 2 h. Our hypothesis, with respect to time dependence of the apoptotic function of the mitochondria, was confirmed by the caspase activation assay. Caspases are a family of cysteine proteases and are key components of the apoptotic machinery. Several biochemical and morphological events of apoptosis are associated with caspase-mediated cleavage of specific substrates [62]. Using CaspACE FITC-VAD-FMK staining, we detected caspase activation after treatment with periplanetasin-2. Similar to the release of cytochrome c, caspase was activated in a time-dependent manner. Caspase activation was triggered robustly 3 h after cytochrome c was released from the mitochondria. Taken together, these results suggest that the apoptotic effect of periplanetasin-2 was time-dependent, and the involvement of the mitochondria in the apoptotic events was apparent after 2 h.

Combined antibiotic therapy can broaden the antimicrobial spectrum and lead to effective therapy and reduced therapeutic doses, adverse effects, and duration of treatment [63]. Synergy implies the rapid increase in the antimicrobial abilities of two agents acting together. The synergistic effect, resulting from combining antibiotics, is highlighted as a novel antifungal strategy [64]. The checkerboard method is used for the determination of the combined effect of two agents. There are four types of combined effect: synergistic, additive, indifferent, and antagonistic [27]. The conventional antibiotics fluconazole and amphotericin B have been previously assessed for combined therapy; therefore, we aimed to determine the effect of combining fluconazole and amphotericin B with periplanetasin-2. The FIC test indicated that periplanetasin-2 had a synergistic effect when combined with fluconazole and amphotericin B. All the agents showed an antifungal effect at the concentrations decreased to one quarter of their respective MICs.

Recent studies have demonstrated that the synergistic effect, which occurs in therapies combining conventional antibiotics, results from the production of highly deleterious hydroxyl radicals [65]. Because periplanetasin-2 induced severe oxidative damage, leading to apoptosis, we investigated the synergistic effect of combining periplanetasin-2, fluconazole, and amphotericin B on the generation of ROS. Similar to the ROS assay, H2DCFDA staining was used to detect the intracellular levels of ROS. After treatment at each FIC, no ROS generation was detected in fluconazole-treated cells, whereas scant ROS generation was detected in periplanetasin-2- and amphotericin B-treated cells. However, all the combined treatments produced a robust generation of ROS, which was similar to that produced by hydrogen peroxide. Stronger ROS generation was induced by the combination of periplanetasin-2 and amphotericin B. Numerous studies demonstrate that fluconazole is a fungistatic agent; it does not induce oxidative stress, or exert fungicidal effects, unless it is used at high concentrations. Amphotericin B exerts its fungicidal effect by forming pores in the cell membrane and triggering apoptosis by generating ROS. The results indicate that the synergistic effect was caused by the generation of ROS and the resultant oxidative damage, which enhanced the antifungal mechanisms of the antibiotics.

Conclusion

Periplanetasin-2 is an antifungal peptide, derived from P. Americana, which demonstrates a robust antifungal effect on pathogenic fungi and does not cause hemolysis. Periplanetasin-2 induces oxidative damage via excessive production of ROS and lipid peroxidation. Hence, we investigated the apoptotic effect and mechanism of periplanetasin-2-induced apoptosis in C. albicans. The apoptotic effect, induced by periplanetasin-2, resulted in the exposure of PS and DNA fragmentation, which were increased in a concentration-dependent manner. Furthermore, mitochondria were involved in periplanetasin-2-induced apoptosis. Calcium accumulation and mitochondrial ROS production induced mitochondrial dysfunction. Consequently, mitochondrial toxicity led to apoptosis via the release of cytochrome c and activation of caspases, which occurred in a time-dependent manner. In conclusion, periplanetasin-2 induced apoptosis in C. albicans by activating mitochondrial apoptotic signaling.

Abbreviations

     
  • DMF

    N,N-dimethylformamide

  •  
  • FIC

    fractional inhibitory concentration

  •  
  • FICI

    FIC index

  •  
  • Fura-2AM

    fura-2 acetoxymethyl ester

  •  
  • H2DCFDA

    2′,7′-dichlorodihydrofluorescein diacetate

  •  
  • JC-1

    5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide

  •  
  • MDA

    malonaldehyde

  •  
  • MIC

    minimum inhibitory concentration

  •  
  • PBS

    phosphate-buffered saline

  •  
  • PI

    propidium iodide

  •  
  • PPB

    potassium phosphate buffer

  •  
  • PS

    phosphatidylserine

  •  
  • Rhod-2AM

    rhod-2 acetoxymethyl ester

  •  
  • ROS

    reactive oxygen species

  •  
  • SSA

    5-sulfosalicylic acid

  •  
  • TBA

    thiobarbituric acid

  •  
  • TUNEL

    terminal deoxynucleotidyl transferase dUTP nick end labeling

Author Contribution

J. Yun and D.G. Lee planned experiments. J.S. Hwang contributed the peptide. J. Yun performed experiments. J. Yun and D.G. Lee analyzed data. J. Yun wrote the paper.

Funding

This work was supported by a grant from the Next-Generation BioGreen 21 Program [Project No. PJ01104303], Rural Development Administration, Republic of Korea.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Author notes

*

These authors contributed equally to this work and should be considered co-first authors.