In vitro and in vivo anti-tumor activities of anti-EGFR single-chain variable fragment fused with recombinant gelonin toxin

Abstract

Purpose: Epidermal growth factor receptor (EGFR) plays an important role in the growth and metastasis of many solid tumors. Strategies that target EGFR hold promising therapeutic potential for the treatment for non-small cell lung cancer (NSCLC), as EGFR is normally overexpressed in these tumors. This study was designed to determine whether an anti-EGFR immunotoxin has anti-tumor activity against NSCLC, and if so, to further investigate the possible mechanisms of cytotoxicity.

Methods: A fusion protein of anti-EGFR single-chain variable fragment (anti-EGFR scFv) and the plant toxin gelonin (rGel) was constructed, expressed in bacteria, and purified to homogeneity. Cytotoxicity of anti-EGFR scFv/rGel (E/rG) immunotoxin was assessed on A549, HCC827, and H1975 cells (EGFR-overexpressing NSCLC-derived cell lines) and A549 xenografts in nude mice.

Results: Cytotoxicity experiments using E/rG on A549, HCC827, and H1975 cells demonstrated that E/rG can specifically inhibit proliferation of these cells, whereas it did not affect the proliferation of Raji cells that do not express EGFR. Treatment for A549 xenografts in nude mice with E/rG resulted in significant suppression of tumor growth compared to controls. Immunofluorescence in frozen tissue sections confirmed that E/rG could specifically bind to tumor tissues in nude mice bearing A549 tumor xenografts, while rGel alone showed no binding activity. Furthermore, E/rG inhibited the growth of A549 cells by cytotoxic effects that blocked tumor proliferation, and the immunotoxin-induced cell death may be mediated by autophagy.

Conclusions: These results showed that E/rG might have significant potential as a novel clinical therapeutic agent against human NSCLC.

Fig. 1

Preparation of the E/rG immunotoxin. a Construction of the recombinant fusion toxin expression vector pET32a-E/rG. b, c SDS-PAGE analysis of the expression and purification of the E/rG immunotoxin. Lane M is the ladder of molecular weight standards; lane 1 is the whole cell lysate from uninduced cells; lane 2 is the whole cell lysate from induced cells; lane 3 is the supernatant of the cell lysates; lane 4 is the sediments of the cell lysate; and lane 5 is the purified immunotoxin E/rG

Fig. 2

Characterization of E/rG immunotoxin in vitro. a, b The binding ability of E/rG immunotoxin on A549 cells. The cells were analyzed by flow cytometric analysis and immunofluorescent staining. Cells were stained for rGel by using a rabbit anti-rGel serum followed by FITC-coupled anti-rabbit IgG as detection reagent. Images were captured using a fluorescence microscope at ×400 amplification, and the nuclei were counterstained with propidium iodine during immunofluorescent staining. c Cytotoxicity of the E/rG immunotoxin to Raji, A549, HCC827, and H1975 cells. Cell viability indicates the percentage of cells in drug-treated wells compared with that of control (untreated) wells. Points, mean of 3 replicates; bars, SD

Fig. 3

In vivo anti-tumor activity of E/rG immunotoxin against A549 tumor xenografts in nude mice. a Treatment for A549 tumors with E/rG fusions at doses of 20 mg/kg. Points, mean tumor volume versus time; bars, SD. b Immunofluorescence staining of tumor samples with anti-rGel serum (green) after IV injection of E/rG and rGel. Two hours after injection, the animal was killed and frozen tumor sections were prepared and detected with anti-rGel serum. Sections were counterstained with Hoechst (blue) to visualize the cell nuclei

Fig. 4

Effect of E/rG on cell proliferation and angiogenesis in vivo. a The number of strongly PCNA-positive cancer cell nuclei was counted as a ratio of the immunoreactive-positive cells to the total number of cells counted. Dramatic reduction of PCNA expression was noted in the E/rG group compared with 2 control groups (*P < 0.05) (magnification, ×200). b Angiogenesis within tumors was detected by CD34 staining of microvessels. The average number of microvessels per vascular hot spot was significantly decreased in E/rG-treated tissues compared with those in the 2 control groups (*P < 0.05)

Fig. 5

Cell-killing mechanism of E/rG immunotoxin. a Western blot analysis cell extract and medium for HMGB1 protein of A549, HCC827, and H1975 cells after E/rG treatment for 48 h. b Analysis of PARP cleavage and LC3-II of A549, HCC827, and H1975 cells after 48 h of E/rG immunotoxin treatment. c Induction of apoptosis was quantified by TUNEL assay. TUNEL-positive cells display dark green nuclei when observed under a fluorescence microscope (×200 magnification), and the percentage of apoptotic cells was determined as described in “Materials and methods” section. (magnification, ×200, *P > 0.05). d Analysis of PARP cleavage and LC3-II of A549 tumor tissues after E/rG immunotoxin treatment