ACTH(6-9)PGP Peptide Protects SH-SY5Y Cells from the H2O2, tert-Butyl Hydroperoxide, and Cyanide Cytotoxicity via Prolif- eration Stimulation and Antioxidant-related Genes Induction

Stabilized melanocortin analog peptide ACTH(6-9)PGP (FRWGPGP) possess a wide range of neuroprotective activities. However, its mechanism of action remains poorly understood. In this paper, we studied the pro-proliferative and cytoprotective activity of the adrenocorticotropic hormone fragment 6-9 (FRWG) linked with the peptide Prolyl-Glycyl-Proline on the SH-SY5Y cells in the model of oxidative stress-related toxicity. The peptide dose-dependently protected cells from H2O2, tert-butyl hydroperoxide, and KCN. The mechanism of its action was the modulation of proliferation-related (NF-B and Nrf-2) and antioxidant-related (HO-1, Nqo1, Gclc) genes and apoptosis decrease.


Introduction
One of the actively studied classes of endogenous peptide regulators is adrenocorticotropic hormone (ACTH)/melanocyte-stimulating hormones (MSH)-like peptides, which are currently combined under the term melanocortins (MC). Studies of the effects of these peptides over the past 40 years showed that the spectrum of the physiological activity of these peptides is very wide. MC are involved in the regulation of memory and attention, emotional status, sexual and eating behavior, pain sensitivity, and some other physiological functions [1]. The MC family includes ACTH and MSH (α-, β-and γ-MSH), as well as fragments of these hormones and their synthetic analogs. The common sequence for all MC is ACTH (6)(7)(8)(9) or FRWG, which is necessary for binding to all known types of MC receptors [2]. Of particular interest is the ability of ACTH and MSH, as well as their fragments lacking hormonal activity, to accelerate the training of animals and improve the preservation of skills. The most active of the studied peptides is ACTH (4)(5)(6)(7)(8)(9)(10). The smallest ACTH fragment retaining nootropic activity is ACTH(4-7). However, it was also shown that the ACTH(7-10) fragment has insignificant nootropic activity. Elongation of the molecule to ACTH(7-16) leads to an increase in activity to a level comparable to ACTH(4-7) and ACTH (4)(5)(6)(7)(8)(9)(10) [3,4].
The described wide range of physiological activity of melanocortins opens up opportunities for the use of drugs of this class in clinical practice in various pathological conditions. However, an obstacle to the use of these compounds in the clinic is low bioavailability and insufficient action duration. The duration of the neurotropic effects of the natural adrenocorticotropic hormone (ACTH) 4-10 fragment is 30-60 minutes [5]. The prolongation of the effects of the peptide and increase of its peptidase resistance was MTT test, mean±standard error, N=7 experiments. *, a statistically significant difference from the untreated control; **, a statistically significant difference from the control without peptide, ANOVA with the Holm-Sidak post-test, p≤0.05.

ACTH(6-9)PGP Decreased Apoptosis and Increased Cell Viability, but Did Not Affect Acute ROS Level
Three possible mechanisms of the protective action of ACTH(6-9)PGP could be proposed: proliferation stimulation, apoptosis inhibition, and ROS level decrease either as a direct ROS scavenging effect or via the appropriate cellular enzyme systems activation. The latter hypothesis is possible, as far as all the toxic agents used are linked to reactive oxygen species generation [11,12].
To test the first two hypotheses, we studied SH-SY5Y proliferation increase after ACTH(6-9)PGP treatment and apoptosis levels after the combined application of the peptide and H2O2. The peptide stimulated cell proliferation (Figure 3 A, B). On the other hand, it decreased apoptosis, but the effect was statistically significant only at 100 M of the peptide ( Figure 3A). No ROS scavenging effect was observed after a 1 h incubation ( Figure 3C). For the proliferation studies, the cells were incubated with the peptide for 7 days and analyzed using the MTT (A) or BrdU (B) assay. SH-SY5Y cells were treated with with 475 M H2O2 either alone, or together with the peptide for 1 h, after which ROS level and apoptotic cell counts were determined. Untreated cells were used as control. Mean±standard error, N=3 experiments, *, statistically significant difference from H2O2 alone for ROS and apoptosis, and from the untreated control for the proliferation, ANOVA with the Holm-Sidak post-test, p≤0.05.

ACTH(6-9)PGP Protection from the KCN Cytotoxicity is Inhibited by the MEK, PKC, PLC, and Ras Inhibitors
To evaluate the signaling pathways involved in the peptide protective activity, we used a panel of receptor and intracellular signal transduction components inhibitors. As far as extracellular oxidative stress inducers like H2O2 could oxidize these inhibitors, we used the KCN based toxicity model. Of the inhibitors tested, only those for the protein kinases C and A (PKC+PKA), phospholipase C (PLC), mitogen-activated protein kinase 1 and 2 (MEK1,2), and Ras removed the protective effect of the peptide (Figure 4), and thus these components participate in the peptide action. Overall, the observed inhibitor response points to the activation of the PLC->PKC->Ras->MEK signal transduction pathway [13][14][15]. MTT assay data, mean±standard error. *, statistically significant difference from the KCN+peptide without any inhibitor; **, statistically significant difference from the untreated control, p≤0.05, ANOVA with the Tukey post-test, N=3 experiments.

ACTH(6-9)PGP did not Alter Cellular cAMP Content
As far as the receptor for the full-length ACTH is a GPCR coupled to a Gs subunit, which in turn activates cAMP synthesis, and that one of the active inhibitors was the mixed PKA/PKC one, we decided to check whether the action of ACTH(6-9)PGP affects the concentration of this second messenger. We analyzed cellular cAMP content after a 20-minute incubation with the peptide, while prostaglandin PGE2 was used as a positive control ( Figure 5). We did not detect neither increase nor drop of the cAMP concentration, and thus the participating kinase is PKC, and not a cAMP-dependent PKA.

ACTH(6-9)PGP Decreased the Expression of the NF-B and Nrf-2 Related Genes, but not of the Antioxidant Enzymes
The downstream mechanism for the protection against the ROS cytotoxicity could be the induction of the cellular antioxidant genes.
To check this possibility and to further elaborate the pro-proliferative activity of the peptide, we analyzed the mRNA levels of a set of signaling pathways after ACTH(6-9)PGP application both alone and in combination with H2O2 for 24 h [16]: The mRNA levels of the Akt3, Catalase, SOD1 and Pp2a genes were below the non-specific amplification (no template control) threshold (data not illustrated). Peptide treatment alone activated NF-B pathway expression, stimulated the p38 protein kinase, and activated the expression of several Nrf-2 pathway components ( Figure 6). In the combination with H2O2, the stimulation of the NF-B pathway was somewhat decreased, and also a small decrease in the Ki-67 gene expression was observed. P38 expression was further enhanced. Two specific gene expression changes were observed for the combination of the peptide with H2O2: first, the addition of ACTH(6-9)PGP restored the levels of p53 and pp5 to the control level, and second, the expression of the Gclc gene was increased. These changes agree with the pro-survival and pro-proliferative action of the peptide.

Discussion
The aim of this work was to identify the intracellular systems, which could participate in the neuroprotective activity of the ACTH(6-9)PGP peptide. Such data could give an insight into the possible interactions with other neuroprotective drugs to produce a drug combination with an enhanced activity in the future. The obtained results point to the following two peptide properties: 1) the peptide evoked pro-proliferative effect; 2) the peptide stimulates the NF-B and Nrf-2 signaling pathway.
The peptide enhanced cell viability in the H2O2, tBH, and KCN cytotoxicity models, but not in the MPP + cytotoxicity model. The ability of the peptide to enhance cell viability increase in the sequence H2O2 < tBH ≤ KCN.
The increase of cell survival in the H2O2 model after the ACTH(6-9)PGP treatment was comparable to the peptide's pro-proliferative effect. To induce cell death, H2O2 is added to the cells in vast quantities. Therefore, the antioxidant systems of the cell are hardly possible to cope with the ROS from H2O2, even if they are stimulated by the peptide, and the net increase of the cell viability after ACTH(6-9)PGP treatment should be due to the proliferation increase. The cytotoxic concentration of tBH was more than 20 times lower, and thus it is quite expectable that the peptide was able to protect much more cells in this model.
The effect of the peptide in the KCN cytotoxicity model was somewhat different from H2O2 and tBH, as it was active only at quite high concentrations. This discrepancy could be explained by the fact that tBH and H2O2 due to their short lifetime in the culture medium produce a rather short stress [17], while KCN, which targets mitochondria [18], produces a long-term ROS production and thus may require a much higher activation of the antioxidant systems.
MPP + , a neurotoxin which plays dominant neurotoxic roles in selectively damaging catecholaminergic neurons including dopaminergic neurons, has widely been used in the experimental models of PD, and it can operate in extracellular or intracellular oxidation, yielding ROS that lead to toxic downstream molecules and result in neuronal damage [19]. It was demonstrated that the treatment of SH-SY5Y cells with MPP + results in a significant increase of ROS [11]. However, MPP + is quite unstable, and thus it causes only a temporary ROS concentration increase. In this condition, the antioxidant activity of a substance should play a substantial role, as it was shown, for example, for -lipoic acid [20]. ACTH(6-9)PGP failed to decrease the ROS concentration after an hour and thus does not act as a direct ROS scavenger. Its structure (His-Phe-Arg-Trp-Pro-Gly-Pro) does not entirely preclude such activity, as phenylalanine and tryptophan residues may react with ROS [21]. It could be proposed that the concentration of the peptide is not enough for this activity to manifest. The vast quantities of MPP + required to induce cytotoxicity also make it hardly possible for the cellular antioxidant machinery to be able to cope with such stress, and this is the most probable explanation for the lack of the peptide activity in this model.
The signaling behind the activity of ACTH(6-9)PGP requires further investigation; however, several hypotheses could be put forward to explain it. As far as the active peptide concentrations were much smaller than the toxic agent's ones, ACTH(6-9)PGP should interact with some cellular signaling machinery, rather than inactivate the added toxins or produced ROS directly. This agrees with the observed disappearance of the peptide's protective action after the inhibition of the PLC->PKC->Ras->MEK signal transduction pathway.
Downstream of these signaling components, after the peptide treatment, the mRNA expression changed according to several patterns.
Peptide treatment alone activated NF-B expression and decreased its contra partner IB expression; the latter change was also observed in the presence of H2O2. This change should activate the pro-proliferative NF-B pathway [22,23] and agrees with the observed pro-proliferative action of the peptide. The increase of the NF-B expression after the peptide treatment also agrees with the detected inhibitor activity, as this gene activity is activated by Ras [24]. ACTH(6-9)PGP treatment also stimulated the expression of the p38 protein kinase, and of several Nrf-2 pathway components. In the presence of H2O2, p38 expression was further enhanced. PKC, which was one of the components of the detected peptide signaling, is also known to stimulate the activity of the Nrf-2 transcription factor [25], and thus the observed activation of the expression of the Nrf-2 targets Nqo1 and Ho-1 after the peptide treatment seems quite logical.
Three specific gene expression changes were observed for the combination of the peptide with H2O2: first, the addition of ACTH(6-9)PGP restored the levels of p53 and pp5 to the control level, second, the expression of the Gclc gene was increased, and finally, a small decrease in the Ki-67 gene expression was observed. These changes mostly agree with the pro-survival and pro-proliferative action of the peptide with some shift toward the former one in the presence of cytotoxic agents. The activation of the gene of the rate-limiting enzyme in the glutathione biosynthesis Gclc [26] is of particular interest, as it presents an interesting way of the long-term defense against the oxidative stress, induced by the peptide.
The direct target of the peptide is not clear. Based on the fact that the peptide represents a part of the adrenocorticotropic hormone, its receptor could be the first candidate. ACTHR is coupled to a Gs subunit [27], and so its activation should lead to an in-crease of cAMP concentration. However, cAMP levels did not change after ACTH(6-9)PGP application ( Figure 5), and thus ACTHR could be excluded from the list of potential targets.
Recently deorphanized cannabinoid receptors GPR18 and GPR55 are known to stimulate proliferation [28,29]; recently, a possibility of GPR55 peptide modulation was demonstrated [30]. In our experimental setting, the inhibitors of these two receptors (ML-193 at 2 µM and PSB CB5 at 0.5 µM) prevented ACTH(6-9)PGP protection against KCN cytotoxicity (Supplementary figure S1). Thus, these receptors could be at least allosteric targets for this peptide; however, a more detailed study of this interaction is out of the scope of this paper.
Other possibilities for the ACTH(6-9)PGP target include receptors like GLP1R, FGFR, and heat shock proteins. GLP1R is the receptor for neuroprotective action of the peptide Glp-1 [31]. This receptor signaling is biased [32], so it possibly could interact with ACTH(6-9)PGP. However, it is also coupled to Gs, and thus does not fit the observed data. FGFR activation by a peptide representing the receptor-binding domain of bFGF was shown to elicit a neuroprotective response in SH-SY5Y cells [33]. FGFR is a receptor tyrosine kinase, and it is linked to the Akt signal transduction pathway [34]. Therefore, this or a similar receptor could be the ACTH(6-9)PGP target. Heat shock proteins were found to interact with other bioactive peptides [35] and increase cell proliferation [36], and this pathway should be kept in mind. A more detailed investigation of discussed pathways is necessary to elucidate mechanisms of cytoprotective activity of modified ACTH(6-9) peptide. The peptide was synthesized, as described earlier [8].

Cell Culture
SH-SY5Y cells (ATCC CRL-2266) were maintained in the 1:1 MEM: F12 medium supplemented with 10% of fetal bovine serum, 2 mM of L-glutamine, 0.5 mM of sodium pyruvate, 0.5% of non-essential amino acids, 100 U/mL penicillin, 100 g/mL streptomycin and 2.5 g/mL amphotericin B in a CO2 incubator with the atmosphere of 5% CO2 and 95% humidity at 37C. Cells were passaged every 72 to 96 h by washing with Versene's solution and treatment with 0.25% trypsin with 0.53 mM EDTA in Hank's balanced salt solution.

Oxidative Stress Induction
For cell viability experiments, the cells were seeded at a density of 15,000 per well of 96-well plate in 100 L of culture medium and incubated for 12 h. After that, the substance solution with or without the toxic agent in 100 L fresh cell culture medium was added to the medium present in the wells and incubated for 24 h. Cytotoxicity was induced by either 475 M of H2O2, 850 µM KCN, 1.3 mM MPP + (freshly prepared1) or 15 µM tert-butyl hydroperoxide (all with the addition of 10 mM of HEPES, pH 7.4).

Cell Viability Assay
Cell viability was analyzed using the MTT test [37]. In short, the culture medium was removed from the wells, and 75 l of the 0.5 mg/ml solution of MTT with 1 g/L D-glucose in Earle's salts was added to each well and incubated for 90 min in the CO2 incubator at 37C. After that, 75 L of 0.04 M HCl in isopropanol was added to the MTT solution in each well and incubated on a plate shaker at 37C for 30 min. The optical density of the solution was determined using a plate reader (Efos 9226, MZ Sapphir, Russia) at the wavelength of 570 nm with a reference wavelength of 620 nm.

cAMP Assay
cAMP levels were measured using a cAMP determination kit according to the manufacturer's instructions. The cells were seeded at a density of 60,000 per well of 96-well plate and grown for 12 h. The substances were added in 100 L fresh cell culture medium to the medium present in the wells and incubated for 20 min. After that, the cell culture medium was removed, and the cells were subjected to cAMP determination.

ROS Assay
ROS generation was measured using the DCFH-DA dye. The cells were seeded at a density of 60,000 per well of 96-well plate and grown for 12 h. After that, the medium was replaced with a fresh one with 25 M of the dye, and the cells were incubated in the CO2 incubator at 37C for 1 h. After the incubation, the cells were washed twice with the culture medium and treated with the substances in the culture medium for 1 h at 25C. Cells treated with medium without H2O2 and substances were used as a control. After the incubation, the cells were washed twice with Hank's balanced salts solution with 25 mM HEPES and 1 mg/ml fatty acid-free bovine serum albumin, pH 7.4, and the fluorescence was measured using the plate reader Hidex Sense Beta Plus (Hidex, Turku, Finland), ex=490 nm, em=535 nm.

Apoptosis Assay
Apoptosis level was analyzed using the Apoptosis/Necrosis detection kit. The cells were seeded at a density of 15,000 per well of 96-well plate and grown for 12 h. After that, 475 M of H2O2 alone or with the peptide was added in 100 L of the fresh medium to 100 L of the old medium in the wells and incubated for 1 h at 37C in a CO2 incubator. After that, the medium was removed, and the cells were stained according to the manufacturer's instructions. The stained cells were photographed using an inverted fluorescent microscope Nikon Ti-S using a Semrock GFP-3035D filter cube with magnification 100x. For each well, 5 non-intersecting view fields were captured, and apoptotic cells were counted.

mRNA Assay
mRNA levels were analyzed using RT-qPCR. The cells were seeded at the density 240,000 per well of a 24-well plate in 200 L of culture medium and incubated for 12 h. The substances were added in 200 L of fresh culture medium to the medium in the wells and incubated for 24 h. Total RNA was extracted using a Total RNA isolation kit according to the manufacturer's protocol. The isolated RNA was treated with DNase I according to the manufacturer's protocol. cDNA was synthesized using an oligo-dT primer using the MMLV reverse transcription kit. qPCR was performed using a SYBR Green containing master mix qPCRmix-HS SYBR with the following amplification protocol: 95C for 2 min, cyclic 95C for 10 s, 57C for 20 s, 72C for 15 s for 40 cycles using a Bio-Rad C1000 thermal cycler (Bio-Rad, Hercules, CA, USA). After the amplification, PCR product melting curve was recorded in the range from 65C to 95C. Primer sequences were generated using the IDT PrimerQuest service (https://eu.idtdna.com/PrimerQuest) and validated using the NCBI PrimerBLAST service [38], or taken from the paper by Jaafru et al. [16]. The primer sequences were as follows (5'-3'): AKT3 forward AGGTGACACTATAGAATAAGACATTAAATTTCCTCGAA, reverse GTACGACTCACTATAGGGAATCCTCATCATATTTTTCAGGT,

Proliferation Assay via the BrdU Incorporation
The stimulation of the cell proliferation by the ACTH(6-9)PGP peptide was validated using the BrdU cell proliferation kit (Abcam, Cambridge, MA, USA). The cells were seeded in 96-well plates at the density of 4000 per well and grown overnight. After that, peptide solution in the fresh culture medium was added to the cells with full medium replacement; the peptide addition was performed on days 1 and 4 after the seeding. On day 6, BrdU reagent was added to the cells for 24 h, and assayed according to the manufacturer's protocol.

Statistics
All experiments were performed at least in triplicate. Statistical analysis was performed with the GraphPad Prism 6.0 software using ANOVA with the Holm-Sidak post-test; p<0.05 was considered a statistically significant difference.