Glycyl-alanyl-histidine protects PC12 cells against hydrogen peroxide toxicity
© The Author(s). 2017
Received: 1 March 2017
Accepted: 9 November 2017
Published: 22 November 2017
Peptides with cytoprotective functions, including antioxidants and anti-infectives, could be useful therapeutics. Carnosine, β-alanine-histidine, is a dipeptide with anti-oxidant properties. Tripeptides of Ala-His-Lys, Pro-His-His, or Tyr-His-Tyr are also of interest in this respect.
We synthesized several histidine-containing peptides including glycine or alanine, and tested their cytoprotective effects on hydrogen peroxide toxicity for PC12 cells. Of all these peptides (Gly-His-His, Ala-His-His, Ala-His-Ala, Ala-Ala-His, Ala-Gly-His, Gly-Ala-His (GAH), Ala-His-Gly, His-Ala-Gly, His-His-His, Gly-His-Ala, and Gly-Gly-His), GAH was found to have the strongest cytoprotective activity. GAH decreased lactate dehydrogenase (LDH) leakage, apoptosis, morphological changes, and nuclear membrane permeability changes against hydrogen peroxide toxicity in PC12 cells. The cytoprotective activity of GAH was superior to that of carnosine against hydrogen peroxide toxicity in PC12 cells. GAH also protected PC12 cells against damage caused by actinomycin D and staurosporine. Additionally, it was found that GAH also protected SH-SY5Y and Jurkat cells from damage caused by hydrogen peroxide, as assessed by LDH leakage.
Thus, a novel tripeptide, GAH, has been identified as having broad cytoprotective effects against hydrogen peroxide-induced cell damage.
More than 7000 peptides have been identified as playing crucial roles in human physiology, including those acting as hormones, neurotransmitters, growth factors, ion channel ligands, or having anti- microbial activity [1–4]. Peptides are selective and efficacious signaling molecules that bind to specific cell surface receptors where they induce intracellular effects. Peptides represent an excellent starting point for the design of novel therapeutics. Even small peptides, such as dipeptides and tripeptides, may also have potent functions [5–8]. Some have cytoprotective functions and have been used in clinical trials for human disease . Carnosine is a well-characterized antioxidant dipeptide composed of β-alanine and histidine. It has cytoprotective activity against various stresses as determined in both in vitro and in vivo models . The imidazole ring of histidine is reported to have an important role in antioxidant cell protection . Carnosine is a more effective singlet oxygen scavenger than L-histidine, although both compounds have been shown to protect against oxidative DNA damage and against liposome oxidation induced experimentally in vitro .
Histidine is a scavenger of hydroxyl radicals , and may interact chemically with toxic oxygen species through at least two distinct mechanisms: (1) by interfering with the redox reactions involving metal ions that produce the hydroxyl radical, and (2) by direct interactions of the histidine imidazole ring with singlet oxygen . The imidazole ring of L-histidine has been shown to be responsible for the antioxidant activity of several biologically important dipeptides, including carnosine (β-alanyl-L-histidine), anserine (β-alanyl-3-methyl-L-histidine), and homocarnosine (l-aminobutyryl-L-histidine) . Ala-His-Lys, Pro-His-His, and Tyr-His-Tyr were also reported to have antioxidant properties [15–17].
We hypothesized that histidine-containing tripeptides might also have antioxidant activity. In the present study, we synthesized and determined the antioxidant activities of tripeptides containing histidine and the small amino acids alanine and glycine.
The histidine-containing tripeptides Gly-His-His (GHH), Ala-His-His (AHH), Ala-His-Ala (AHA), Ala-Ala-His (AAH), Ala-Gly-His (AGH), Gly-Ala-His (GAH), Ala-His-Gly (AHG), His-Ala-Gly (HAG), His-His-His (HHH), Gly-His-Ala (GHA), and Gly-Gly-His (GGH) were synthesized by and purchased from Biogate (Gifu, Japan). Carnosine was purchased from Sigma-Aldrich (St. Louis, MO, USA). GAH at 1 μg/μl = 3530 μM.
PC12 (CRL-1721), SH-SY5Y (CRL-2266) and Jurkat cells (CRL-1990) were purchased from ATCC. PC12, Jurkat, and SH-SY5Y cells were grown at 37 °C (5% CO2 atmosphere) in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL of penicillin, and 100 mg/mL of streptomycin. Cell culture medium was changed three times per week, and when confluent, cells were split 1:6. For the experiments reported here, subconfluent cells were treated with different concentrations of 100–10,000 μM hydrogen peroxide, 10 μM staurosporine, or 500 μg/mL of actinomycin D.
Lactate dehydrogenase assay
To assess cytotoxicity, lactate dehydrogenase (LDH) activity was measured using LDH cytotoxicity detection kits (Takara, Otsu, Shiga Japan). PC-12 cells were seeded into a 96-well plate at 2 × 106 cells/mL with assay medium, for a period of 18 h at 37 °C in a 5% CO2 humidified incubator. The culture medium was then removed and replaced with serum-free medium and 1% bovine serum albumin (BSA) was added. The plates were treated with 100–5000 μM hydrogen peroxide for 1–24 h. After incubation, the samples were centrifuged for 10 min at 250 g. One hundred μL/well of supernatant was removed, without disturbing the cell pellet, and transferred into corresponding wells of a new 96-well plate. Solution C (100 μL, the reaction mixture) was added to each well and incubated for 30 min at room temperature. The 96-well plate was protected from light during this time. The absorbance of the samples was measured at 490 nm using the ARVO SX 1420 Multilabel Counter (PerkinElmer Wallac Inc., Turku, Finland).
Flow cytometry analysis
Cells were washed twice with buffer (140 mM NaCl, 10 mM HEPES, 2.5 mM CaCI2, pH 7.4), resuspended in 1 mL of the same buffer, and incubated on ice for 30 min with 5 μL of propidium iodide (50 μg/mL H2O stock solution) added to each sample. They were then analyzed by flow cytometry (Becton Dickinson, San Jose, CA, USA) .
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
The TUNEL assay of PC12 cells was conducted using the Click-iT TUNEL Alexa Fluor 594 Imaging Assay Kit (Molecular Probes™, Eugene, OR, USA). DNase I was used to generate strand breaks in the DNA to provide a positive TUNEL reaction control. The number of Alexa Fluor 594-positive cells was counted using BZ-II Analyzer software (Keyence, Japan). Nuclear was stained with 4′, 6-diamidino-2-phenylindole (DAPI).
Dead cell images
PC12 cells were incubated for 30 min at 37 °C with 4 μM of the EthD-1 (LIVE/DEAD viability/cytotoxicity kit reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA). EthD-1 enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (excitation/emission, ~495 nm/~635 nm, respectively). The number of Alexa Fluor 594-positive cells was counted using BZ-II Analyzer software (Keyence).
Cell viability analysis
Cell growth was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assay. The cells (2 × 104) were plated in 100 μL of media and added to 100 μL of hydrogen peroxide with or without GAH or carnosine in each well of a 96-well flat-bottomed microtiter plate. Assays were done in triplicate cultures and incubated at 37 °C in an incubator with 5% CO2. Ten μL of the CCK8 solution was added to each well after 24 h of treatment, and the cells were cultured for another 2 h at 37 °C. The absorbance was measured using a microplate reader (Nanoquant Plate™; Tecan, Männedorf, Switzerland), at 450 nm with 600 nm used as the reference wavelength. The cell viability was expressed as a percentage of absorbance in cells with indicated treatments to that of the control cells.
All values are expressed as mean ± SEM. One-way analysis of variance and post hoc Fisher’s protected least significant difference tests were used to determine the significance of differences between the groups. P values < 0.05 indicated significant difference.
We screened newly synthesized histidine-containing tripeptides for their radical scavenging activity. GHH, AHH, AHA, AAH, AGH, GAH, AHG, HAG, HHH, GHA, and GGH were screened for their ability to decrease LDH leakage from PC12 cells treated with hydrogen peroxide. Of these peptides, GAH at 1 μg/μL had the strongest protective effect against cell damage as assessed by LDH leakage, by ethidium bromide staining, cell morphology, TUNEL assays, CCK-8 assays, and PI assays. GAH also protected Jurkat cells and SH-SY5Y cells; it may therefore have a protective effect against many different types of cells. GAH was not effective for SH-SY5Y cells compared to PC12 cells suggesting that effectiveness of GAH might depends on cell type.
Several histidine-containing dipeptides or tripeptides with antioxidant activity have been identified. Hartman et al.  have shown that carnosine is an efficient singlet-oxygen scavenger, quenching singlet oxygen more effectively than histidine. They also reported that carnosine, anserine, and histidine protect phages against gamma-irradiation, which gives rise to oxidative DNA damage. Tsuge et al. reported the isolation of a potent antioxidative peptide, Ala-His-Lys, from an egg white albumin hydrolysate . Chen et al. reported that Pro-His-His was the most active antioxidant among the 28 synthetic peptides that were structurally related to Leu-Leu-Pro-His-His . Saito et al. reported that Tyr-His-Tyr had a strong synergistic effect with phenolic antioxidants . As reported here, GAH attenuated cell damage by hydrogen peroxide, suggesting that it might be an efficient singlet oxygen scavenger, similar to carnosine, Pro-His-His, and Tyr-His-Tyr.
GAH protected cells not only against hydrogen peroxide damage, but also prevented apoptosis induced by staurosporine and actinomycin D. Staurosporine is a broad-spectrum inhibitor of protein kinases, and has been widely used for the induction of apoptosis in diverse cellular models [22, 23]. Staurosporine preferentially activates the mitochondrial apoptotic pathway, relying on caspase activation to cause cell death. Actinomycin D, on the other hand, is a widely-used intercalating transcription inhibitor. . Protection by GAH against hydrogen peroxide, staurosporine, and actinomycin D suggests that it might not only be a radical scavenger but could also protect by other mechanisms. The protective effects of carnosine include actions on glycolytic enzymes, metabolic regulatory activities, redox biology, protein glycation, glyoxalase activity, apoptosis, gene expression, and cancer cell metastasis .
In this study, we did not address the mechanism of how GAH attenuated cell death. Further studies will be needed to clarify the mechanism of GAH cytoprotection.
The present studies showed that GAH has protective effect against cell damage determined by LDH leakage, by ethidium bromide staining, cell morphology, TUNEL assays, CCK-8 assays, and PI assays. GAH also protected Jurkat cells and SH-SY5Y cells. GAH might has a potential for cytoprotective agents.
We acknowledge R Ishikawa for performing experiments.
This study was partly supported by a High Technology Research Center grant and a Grant-in-Aid for exploratory research from the Ministry of Education, Culture, Sports, Science and Technology in Japan and by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (21,700,395, H. Shimura;) and from the Takeda Science Foundation (H. Shimura). This study was supported (in part) by a Grant-in-Aid (S1311011) from the Foundation of Strategic Research, Projects in Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.
HS designed and performed the experiments, participated in the interpretation of data, and wrote manuscript. RT, YS, KY performed the biological experiments. NH, TU designed the experiments, interpreted the data, and wrote the manuscript. All the authors read and approved the final manuscript.
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