The antitumor activity and preliminary modeling on the potential mechanism of action of human peroxiredoxin-5

Abstract

Goat peroxiredoxin-5 (gPRDX5) was verified as a good anti-cancer bioactive peptide (ACBP) against different tumor cell lines. Considering the immunogenicity between species for further therapeutic application, it is necessary to similarly investigate the antitumor activity of human peroxiredoxin-5 (hPRDX5) with 89% similarity in sequence to gPRDX5. In order to evaluate its antitumor activity, the potential anti-neoplastic effect of hPRDX5 on a mouse model was observed directly. The results of its in vivo antitumor activity suggested that hPRDX5 could resist immunosuppression by promoting lymphocyte proliferation and up-regulating the levels of serum cytokines. Meanwhile, PD-L1 was speculated as one of the targets of hPRDX5 to inhibit tumor by enhancing the immune activity according to a preliminary molecular docking study on the interactions between hPRDX5 and PD-L1. The modeling provides a basis for structural modification on hPRDX5/PD-L1 for further biological and biochemical study on the pathway blocking mechanism of hPRDX5. In this work, the results demonstrate that hPRDX5 displays efficient antitumor and immunoregulatory properties in the colon cancer C26/BALB/c and melanoma B16/C57Bl/6 mice tumor models, and suggest the potential of developing peptides from hPRDX5 as low molecular weight drug candidates for corresponding cancer immunotherapy.

Keywords: PD-L1; anti-cancer bioactive peptide; antitumor activity; human peroxiredoxin-5; immunoregulation.

Figure 1. Coomassie stained SDS-PAGE gel of purified hPRDX5

The DNA fragment of hPRDX5 was constructed to expression vector pET-28a(+) and the plasmid was then transformed to BL21 (DE3) for heterogeneous expression. The hPRDX5 was purified by Affinity Chromatography and the molecular weight was about 17 kDa.

Figure 2. The anti-cancer bioactivity of hPRDX5 in C26-injected mice

(A) Tumors were harvested after treated with hPRDX5 and controls (TPT and PBS). (B) The inhibition rate of tumor was evaluated by measuring the tumor weight compared with solvent control. The tumor growth was decreased significantly in mice treated with hPRDX5 in a dose-dependent manner. The highest inhibition rate was 32.16 %, which demonstrated that hPRDX5 was likely to a potential tumor suppressor. (P < 0.05 compared to control group).

Figure 3. Effects of hPRDX5 on spleen and thymus indices in C26-injected mice

The spleen and thymus indices were increased significantly compared with control group when the mice were given intraperitoneal injection of hPRDX5 at 12.5, 25.0, 50.0, 75.0 or 150.0 mg/kg, respectively (P < 0.05 compared to control group).

Figure 4. Effect of hPRDX5 on mitogen-induced splenic lymphocyte proliferation

(A) LPS; (B) ConA. The proliferative responses of splenic lymphocytes to concanavalin A and LPS were enhanced significantly as compared with the control group. The proliferation of lymphocytes were enhanced by 75.0 mg/kg hPRDX5 with the stimulation index of 1.75, 2.53, 2.51 and 2.96 in the presence of LPS at 1, 5, 10, 20 μg/ml, respectively. Likewise, in the presence of concanavalin A at 5, 10, 20 μg/ml hPRDX5 elicited an increase in lymphocytes proliferation by 1.67, 1.91 and 1.87, respectively. (P < 0.05 compared to control group)

Figure 5. Effect of hPRDX5 on the levels of cytokines

The levels of IL-2, IL-4, IL-6, IL-10, TNF-α and TNF-β were increased by 75.0 mg/kg hPRDX5, but IFN-γ was reduced slightly. The secretion of IL-2 was promoted most significantly. Data are means ± SD of eight animals (P < 0.05 compared to control group).

Figure 6. Overall structure of the hPRDX5/PD-L1 complex (right)

hPRDX5 and PD-L1 are shown in green (ribbon diagram) and pink (surface representation), respectively; Close-up views of interfaces. Residues involved in hydrogen bonds (green dashes) are shown.

Figure 7. Schematic representation of de novo peptide design