The antitumor activity of hPRDX5 against pancreatic cancer and the possible mechanisms

Abstract

Recombinant human peroxiredoxin-5 (hPRDX5), isolated from anti-cancer bioactive peptide (ACBPs), shows a homology of 89% with goat peroxiredoxin-5 (gPRDX5) and is reported to display anti-tumor activity in vivo. Herein, we explored the effect of hPRDX5 and the responsible mechanism in treating pancreatic cancer. Tumor-bearing mice were randomly divided into normal PBS group and treatment group (n=5; 10 mg/kg hPRDX5). Flow cytometry was employed to examine lymphocytes, myeloid-derived suppressor cell subsets, and the function proteins of natural killer (NK) cells in peripheral blood, spleen, and tumor tissues of mice. Western blot was used to measure the protein expressions of the key nodes in TLR4-MAPK-NF-κB signaling pathway. The rate of tumor suppression was 57.6% at a 10 mg/kg dose in orthotopic transplanted tumor mice. Moreover, the population of CD3+CD4+T cells, NK cells, and CD3+CD8+T cells was significantly increased in the tumor tissue of the hPRDX5 group, while the proportion of granulocytic-myeloid-derived suppressor cells decreased slightly. In addition, after treatment with hPRDX5, the percentage of NK cells in blood increased more than 4-fold. Our findings indicated that hPRDX5 effectively suppressed pancreatic cancer possibly via the TLR4-MAPK-NF-κB signaling cascade; hence hPRDX5 could be a prospective immunotherapy candidate for treating pancreatic cancer.

Figure 1. Treatment with human peroxiredoxin-5 (hPRDX5) alone exhibited in vivo antitumor activity in a mouse orthotopic transplantation model. A, Images of tumor tissues from tumor-bearing C57BL/6 mice. The tumors in the hPRDX5-treated group were smaller than those in the control group. B, The tumor weights decreased in hPRDX5-treated mice. C, The survival period was prolonged in hPRDX5-treated mice. D, Representative images of H&E staining of mouse pancreatic tumor (scale bar 100 μm). Data are reported as means±SD. *P<0.05 compared with the control (t-test).

Figure 2. Effect of human peroxiredoxin-5 (hPRDX5) on the proportion of T cells in tumor and spleen tissues of mice. Proportion of CD3+CD4+ T cells (A) and CD3+CD8+ T cells (B) in tumor tissues and of CD3+CD4+ T cells (C) and CD3+CD8+ T cells (D) in spleen tissues in the control and hPRDX5 treatment groups. Data are reported as means±SD. **P<0.01 and ***P<0.001 compared with the control (t-test). ns: not significant.

Figure 3. Effect of human peroxiredoxin-5 (hPRDX5) on the proportion of natural killer (NK) cells in peripheral blood (A), spleen (B), and tumor (C) tissues of mice. Data are reported as means±SD. **P<0.01, ***P<0.001 compared with the control (t-test). ns: not significant.

Figure 4. Effect of human peroxiredoxin-5 (hPRDX5) on the proportion of MDSC cells in spleen and tumor tissues of mice. A, Proportion of monocytic myeloid-derived suppressor cells (M-MDSC) cells and granulocytic (G)-MDSC cells in tumor tissues (A) and in spleen tissues (B) in the control group and the hPRDX5 treatment group. Data are reported as means±SD. *P<0.05 compared with the control (t-test). ns: not significant.

Figure 5. Effect of human peroxiredoxin-5 (hPRDX5) on the expression of function proteins of natural killer (NK) cells in tumor tissues. The levels of IFN-γ (A), granzyme B (B), and perforin (C) in NK cells in the control and hPRDX5 treatment groups. Data are reported as means±SD. **P<0.01 and ***P<0.001 compared with the control (t-test).

Figure 6. Effect of human peroxiredoxin-5 (hPRDX5) on the expression of function proteins of natural killer (NK) cells in vitro. A, The levels of IFN-γ, granzyme B, and perforin in NK cells were assessed using flow cytometry. B, The mean fluorescent intensity (MFI) of IFN-γ, granzyme B, and perforin in NK cells was measured by flow cytometry. C, Proportion of NK cells in bone marrow mononuclear cells stimulated by hPRDX5 for 24 h in vitro. Data are reported as means±SD. *P<0.05, compared with the control (ANOVA).

Figure 7. NK92 cells were activated by human peroxiredoxin-5 (hPRDX5) through TLR4-MAPK-NF-κB signaling pathway in vitro. A and B, Levels of downstream protein expressions in NK-92 cells treated with hPRDX5 (5, 10, 50 μg/mL) for 24 h by western blot analysis. C and D, Levels of downstream protein expressions in NK-92 cells treated with hPRDX5 (50 μg/mL) in the presence or absence of TLR4 inhibitor (resatorvid) by western blotting. Data are reported as means±SD from triplicate wells. *P<0.05, compared with the control. ^#P<0.05, compared with the relative hPRDX5-treated group (ANOVA). ns: not significant.