A Stable Pep2-proapoptotic Peptide Inducing Apoptosis of Acute Myeloid Leukemia Cells by Down-Regulating EZH2

Abstract

Introduction: Proapoptotic peptide, (KLAKLAK)₂, exhibits strong anti-tumor effect with the help of cell-penetrating peptides such as Pep2, targeting TLR2 with high expression in acute myeloid leukemia (AML). However, the applications are limited due to the peptide's instability and high cost of synthesis. Recombinant PP7 bacteriophage-like particles (VLPs) can protect the peptides from degradation by proteases, based on their ability to display foreign peptides.

Methods: Here, we evaluated the feasibility of PP7 VLPs carrying Pep2 and (KLAKLAK)₂ (2PP7-Pep2-KLAK VLPs) expressed in E. coli. We further investigated the characteristics including size, toxicity, thermal stability, penetrating ability, anti-tumor activity, and potential anti-tumor mechanism of 2PP7-Pep2-KLAK VLPs.

Results: 2PP7-Pep2-KLAK VLPs was expressed in E. coli BL21(DE3) successfully with high yield and thermal stability. They penetrated the AML cells THP-1 rapidly after 30 min of incubation. Moreover, 2PP7-Pep2-KLAK VLPs were non-replicative, non-infectious, and non-toxic against normal cells, but inhibited the proliferation of THP-1 cells by inducing cell apoptosis after 24 h of exposure. This effect extends through 120 h of exposure, indicating their anti-proliferation effect was superior to that of synthetic peptides. In addition to the mitochondrial apoptotic pathway, the anti-tumor activity of 2PP7-Pep2-KLAK VLPs was also correlated with down-regulation of expression of enhancer of zeste homolog 2 (EZH2) and trimethylation of histone H3K27.

Conclusions: We identified the feasibility to prepare the stable, active Pep2-KLAK peptide by using PP7 bacteriophage as the vehicle. We revealed this peptide was an inhibitor of EZH2. 2PP7-Pep2-KLAK VLPs may have significant clinical implications in the treatment of MLL-AF9 AML as an epigenetic modulator.

Keywords: Acute myeloid leukemia; Enhancer of zeste homolog 2 (EZH2); PP7 bacteriophage; Proapoptotic peptide; Toll-like receptor 2 (TLR2); Virus-like particle (VLP).

Figure 1

Construction of the plasmid pETDuet-2PP7-Pep2-KLAK. (a) The inserted site of Pep2-KLAK peptide in the pETDuet-2PP7 and its DNA sequence. (b) The second structure of 2PP7-Pep2-KLAK protein and Pep2-KLAK peptide predicted using I-TASSER. The Pep2-KLAK peptide in the PP7 coat protein dimer was marked by red circle.

Figure 2

Identification of PP7 VLPs carrying Pep2-KLAK peptide. (a) Expression of 2PP7-Pep2-KLAK VLPs and 2PP7-Pep2 VLPs in the supernatant of BL21 (DE3) after sonication. The target proteins are marked by black arrows. (b) Purification chromatogram of 2PP7-Pep2-KLAK VLPs by gel filtration chromatography. The peaks of the target proteins are marked by black arrows. (c) Western blot of the pure VLPs. (d) Assembly of the PP7 coat protein dimer carrying Pep2-KLAK peptide into whole VLPs. The VLPs was observed using TEM at 60 kV and ×100,000 screen magnification. (e) Size analysis of VLPs by particle size analyzer.

Figure 3

Cytotoxicity and thermal stability of 2PP7-Pep2-KLAK VLPs. (a) Cytotoxicity of 2PP7-Pep2-KLAK VLPs. Human PBMCs (5 × 10³/well) were incubated with various concentrations (0, 2, 4, 8, 16, 32, 64, 128, 256 nM) of 2PP7-Pep2-KLAK VLPs for 24 h or 48 h in triplicate. The cytotoxicity was expressed as cell viability. (b) Thermal stability of 2PP7-Pep2-KLAK VLPs at 20, 37, 42, 49, 54, 59, 64, 70, 75, 80, 85, 90, 95, 100 °C. ***p < 0.001.

Figure 4

Cell-penetrating ability of 2PP7-Pep2 VLPs and 2PP7-Pep2-KLAK VLPs in THP-1 cells. (a) Strong cell-penetrating ability of both VLPs was observed in THP-1 cells by the fluorescence microscope. Pictures were taken under fluorescence (×400). (b) The cell-penetrating ability of both VLPs in THP-1 cells was evaluated by flow cytometry.

Figure 5

Anti-proliferation effect of 2PP7-Pep2-KLAK VLPs in THP-1 cells. (a) Cytotoxicity of 2PP7-Pep2-KLAK VLPs in THP-1 cells in different doses (0, 10, 20, 40, 80, 160, 200, 250 nM). (b) Cytotoxicity of 2PP7-Pep2-KLAK VLPs in THP-1 cells in different time (0, 24, 48, 72, 96, 120 h). The cytotoxicity was expressed as cell viability. (c) Cell apoptosis effect of the 2PP7-Pep2-KLAK VLPs on THP-1 cells. THP-1 cells were exposed to 80 nM of the Pep2-KLAK peptide, 2PP7-Pep2 VLPs or 2PP7-Pep2-KLAK VLPs for 48 h, stained with FITC-conjugated annexin-V and PI, and quantified by flow cytometric analysis. **p < 0.01; ***p < 0.001.

Figure 6

Effect of 2PP7-Pep2-KLAK VLPs on EZH2 in THP-1 cells. THP-1 cells were exposed to 80 nM of 2PP7-Pep2 VLPs or 2PP7-Pep2-KLAK VLPs. After 48 h, the cells were collected by centrifugation. The mRNA and protein levels of EZH2 were detected by RT-qPCR (a) and WB (b), respectively. Also, the expression of H3K27me3 was quantified by WB (b).