ACTH(6-9)PGP Peptide Protects SH-SY5Y Cells from H2O2, tert-Butyl Hydroperoxide, and Cyanide Cytotoxicity via Stimulation of Proliferation and Induction of Prosurvival-Related Genes

Abstract

Stabilized melanocortin analog peptide ACTH(6-9)PGP (HFRWPGP) possesses a wide range of neuroprotective activities. However, its mechanism of action remains poorly understood. In this paper, we present a study of the proproliferative and cytoprotective activity of the adrenocorticotropic hormone fragment 6-9 (HFRW) linked with the peptide prolyine-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 and demonstrated proproliferative activity. The mechanism of its action was the modulation of proliferation-related NF-κB genes and stimulation of prosurvival NRF2-gene-related pathway, as well as a decrease in apoptosis.

Keywords: ACTH(6–9); H2O2; MPP+; cyanide; melanocortins; neuroprotection; oxidative stress; tert-butyl hydroperoxide.

Figure 1

Cytotoxicity of KCN (A) and tBH (B) for the SH-SY5Y cells. Incubation time 24 h, MTT assay data, mean ± standard error, N = 5 experiments.

Figure 2

ACTH(6–9)PGP effect on SH-SY5Y cell viability after H₂O₂, tBH, MPP⁺, and KCN treatment. The cells were treated with 475 μM H₂O₂ (A), 27 µM tBH (B), 90.6 µM KCN (C), or 1.3 mM MPP⁺ (D) with various peptide concentrations for 24 h in the case of tBH, H₂O₂, and KCN or 48 h in the case of MPP⁺. Untreated cells were used as control. MTT assay data, 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.

Figure 3

ACTH(6–9)PGP influence on SH-SY5Y cell proliferation (A,B), H₂O₂-induced apoptosis (C), and ROS level (D). For the proliferation studies, the cells were incubated with the peptide for 7 days and analyzed using the MTT (A) or BrdU (B) assay. For apoptosis and ROS generation studies, the cells were treated with 475 μM H₂O₂ either alone or together with the peptide for 1 h, after which ROS level (using the DCFH-DA dye) and apoptotic cell counts (using the phosphatidylserine-reactive dye combined with the 7-AAD cell-impermeable dye) 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. **, statistically significant difference from the control without H₂O₂, ANOVA with the Holm–Sidak post-test, p ≤ 0.05.

Figure 4

Participation of intracellular signal transduction components in ACTH(6–9)PGP protection against KCN cytotoxicity for the SH-SY5Y cells. Inhibitors for CREB (666-11, 1 µM), JNK (SP 600125, “SP”, 1 µM), p38 (SB 202190, “SB”, 1 µM), CaMKII + IV (KN-93, 4 µM), PKC + PKA (HA-1004, 10 µM), PLD (FIPI, 0.5 µM), PKA (KT-5720, 0.5 µM), PLC (U-73122, “U73”, 10 µM), MEK1/2 (U-0126, 0.2 µM), NOS (L-NAME, 25 µM), and Ras (salirasib, 10 µM) were added 1 h before the KCN and then together with KCN (85.0 µM) and peptide (50 µM). The cells were incubated with the inhibitors, KCN, and peptide for 24 h. MTT assay data, mean ± standard error. *, a statistically significant difference from the KCN + peptide without any inhibitor; **, a statistically significant difference from the untreated control; p ≤ 0.05, ANOVA with the Tukey post-test, N = 3 experiments.

Figure 5

cAMP production after the ACTH(6–9)PGP treatment of SH-SY5Y cells. The cells were treated with 80 or 100 μM of the peptide or with 40 μM of PGE₂ (positive control) for 20 min, after which the cAMP concentration was measured using a competitive ELISA kit. Untreated cells were used as control. Mean ± standard error, N = 3 experiments. *, a statistically significant difference from the control; ANOVA with the Holm–Sidak post-test, p ≤ 0.05.

Figure 6

Gene expression changes of NF-κB (A), MAPK (B), and NRF2 (C) pathways after the ACTH(6–9)PGP treatment with and without H₂O₂. SH-SY5Y cells were treated with 475 μM of H₂O₂ either alone or with 50 μM of the peptide for 24 h, after which mRNA levels were determined using RT-qPCR. Untreated cells were used as control. Data are normalized to B2M, RPII, and GPDH. Mean ± standard error, N = 3 experiments; each data point represents a biological replicate; technical replicates are averaged. *, a statistically significant difference from untreated control; **, a statistically significant difference from the H₂O₂ alone; ANOVA with the Holm–Sidak post-test, p ≤ 0.05.