Inhibition of human lung cancer cells by anti-p21Ras scFv mediated by the activatable cell-penetrating peptide

Abstract

Activatable cell-penetrating peptide (ACPP) is a tumour-targeting cell-penetrating peptide. Here, we used ACPP to carry anti-p21Ras scFv for Ras-driven cancer therapy. The ACPP-p21Ras scFv fusion protein was prepared by a prokaryotic expression system and Ni-NTA column purification. The human tumour cell lines A549, SW480, U251 and Huh7 and the normal cell line BEAS 2B were used to study the tumor-targeting and membrane-penetrating ability of ACPP-p21Ras scFv. The antitumour activity of ACPP-p21Ras scFv on A549 cells and H1299 cells in vitro was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, scratch wound healing, plate cloning and apoptosis assays. The penetration pathway of ACPP was determined by enhanced green fluorescent protein. The ACPP-p21Ras scFv fusion protein was successfully obtained at a concentration of 1.8 mg/ml. We found that ACPP-p21Ras scFv could penetrate tumour cell membranes with high expression of matrix metalloproteinase-2 (MMP-2), effectively inhibit the migration and proliferation of A549 cells and H1299 cells, and promote the apoptosis of A549 cells and H1299 cells. The membrane penetration experiment demonstrated that ACPP could enter A549 cells by direct penetration. The ability of ACPP to penetrate the membrane was affected by the addition of a membrane affinity inhibitor and a change in the potential difference across the cell membrane but not by the addition of endocytosis inhibitors and a change in temperature. The ACPP-p21Ras scFv fusion protein can penetrate tumour cells with MMP-2 expression and has antitumour activity against A549 cells and H1299 cells in vitro. This molecule is expected to become a potential antitumour drug for Ras gene-driven lung cancer.

Fig. 1

Construction and expression of the recombinant activatable cell-penetrating peptide (ACPP)-p21Ras scFv fusion protein. (a) Schematic structure of the ACPP-p21Ras scFv fusion protein. Cell-penetrating peptide (CPP): polycationic peptide with the cell-penetrating property. MMP-2 substrate: oligopeptide that binds to MMP-2. Polyanionic peptide: anionic peptide that blocks the membrane-penetrating function of CPP. Once the MMP-2-sensitive oligopeptide is cleaved, the polyanionic peptide drifts away, and the polycationic CPP is activated to carry p21Ras scFv into cells. (b) Schematic diagram of the recombinant plasmids design. A Flag tag was attached to the 3 ‘end of the p21Ras scFv, while the ACPP peptide was designed at the 5’ end of the p21Ras scFv. The sequence was inserted between BamH I and Hind III sites of plasmid. a, ACPP-p21Ras scFv; b, ACPP-L-p21Ras scFv; c, ACPP-L-EGFP. (c) PCR analysis showed that the sizes of ACPP-p21Ras scFv, ACPP-L-p21Ras scFv and ACPP-L-EGFP were 1185, 1230 and 1197 bp, respectively, which were consistent with the expected values. (d) The molecular weights of ACPP-p21Ras scFv, ACPP-L-p21Ras scFv and ACPP-L-EGFP was identified by SDS-PAGE, that is, 36, 37 and 37 KD, respectively. (e) The binding ability of the recombinant antibodies to K-p21Ras was identified by WB. After electrophoresis and membrane transfer, K-p21Ras was incubated with ACPP-p21Ras scFv, ACPP-L-p21Ras scFv or p21Ras scFv. All the antibodies could combine with K-p21Ras. EGFP, enhanced green fluorescent protein; MMP-2, matrix metalloproteinase-2; WB, western blot.

Fig. 2

Mechanism and targeting of activatable cell-penetrating peptide (ACPP) uptake. (a) The uptake of ACPP-L-EGFP was inhibited in A549 cells treated with heparin, but not by endocytosis inhibitors [50 µM amiloride (EIPA), 5 mM methyl-β-cyclodextrin (MβCD) and chlorpromazine]. (b) The uptake of ACPP-L-EGFP by A549 cells was not inhibited at 4 °C. (c) The uptake of ACPP-L-EGFP in A549 cells was significantly inhibited by weaken the potential difference across the cell membrane [PBS (K⁺)]. (d) The uptake of ACPP-L-EGFP was higher by tumour cells with high expression of MMP-2 (A549, SW480, Huh7 and U251) than by normal cells without MMP-2 expression (BEAS-2B). EGFP, enhanced green fluorescent protein; MMP-2, matrix metalloproteinase-2.

Fig. 3

The cell-penetrating ability of activatable cell-penetrating peptide (ACPP)-p21Ras scFv was detected. (a) ACPP-p21Ras scFv and ACPP-L-p21Ras scFv were found in the A549 cells (brown), while p21Ras scFv was not found in the A549 cells. Immunohistochemical staining, ×40. (b) ACPP-p21Ras scFv and ACPP-L-p21Ras scFv were observed by immunofluorescence assay in A549 cells, while p21Ras scFv was not observed in the cells. Red: Rho-stained of p21Ras scFv, ACPP-p21Ras scFv and ACPP-L-p21Ras scFv, blue: DAPI-stained nuclei (×1000). (c) ACPP-p21Ras scFv and ACPP-L-p21Ras scFv were detected by WB in A549, SW480, Huh7 and U251 cells, but not in Beas-2B cell. DAPI, 4’,6-diamidino-2-phenylindole; WB, western blot.

Fig. 4

Anti tumour effects of activatable cell-penetrating peptide (ACPP)-p21Ras scFv on A549 cells and H1299 cells in vitro. (a) The scratch experiment revealed that the healing of A549 cells and H1299 cells treated with ACPP-p21Ras scFv and ACPP-L-p21Ras scFv were slower than those treated with p21Ras scFv and PBS at 24 and 48 h, indicating that ACPP-p21Ras scFv can inhibit the migration of A549 cells and H1299 cells. (b) The MTT assay was used to investigate the killing effect of ACPP-p21Ras scFv to A549 cells and H1299 cells. The OD490 (living cell) of A549 cells and H1299 cells treated with ACPP-p21Ras scFv and ACPP-L-p21Ras scFv were lower than those treated with p21Ras scFv and PBS (P < 0.05) on 2 d and 3 d (P < 0.05). (c and d) The clone formation test demonstrated that the proliferation ability of A549 cells and H1299 cells treated with ACPP-p21Ras scFv and ACPP-L-p21Ras scFv were lower than that treated with p21Ras scFv and PBS (P < 0.05). MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Fig. 5

Activatable cell-penetrating peptide (ACPP)-p21Ras scFv induce apoptosis on A549 cells and H1299 cells in vitro. (a) The cells treated with ACPP-p21Ras scFv, ACPP-L-p21Ras scFv and p21Ras scFv, then were stained with TUNEL kit and then detected by microscope, and the apoptotic cells are indicated with arrows. (b and c) Apoptotic cells are stained with annexin V/propidium iodide, then detected by flow cytometry. Annexin V/propidium iodide assay showed that the apoptotic percentage of A549 cells and H1299 cells treated with ACPP-p21Ras scFv and ACPP-L-p21Ras scFv were higher than that with p21Ras scFv (P < 0.05). TUNEL, terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling.