Fusion of apoptosis-related protein Cytochrome c with anti-HER-2 single-chain antibody targets the suppression of HER-2+ breast cancer

Abstract

Cancer treatment has gradually developed from toxic chemotherapy to targeted therapy with fewer side effects. Approximately 30% of breast cancer patients overexpress human epidermal growth factor receptor 2 (HER-2). Previous studies have successfully produced single-chain antibodies (scFv) targeting HER-2+ breast cancer; however, scFv have poor stability, easy aggregation and a shorter half-life, which have no significant effect on targeting therapy. Moreover, scFv has been considered as a drug delivery platform that can kill target cells by effector molecules. However, the functional killing domains of immunotoxins are mainly derived from plant or bacterial toxins, which have a large molecular weight, low tissue permeability and severe side effects. To address these concerns, we designed several apoptotic immune molecules to replace exogenous toxins using endogenous apoptosis-related protein DNA fragmentation factor 40 (DFF40) and tandem-repeat Cytochrome c base on caspase-3 responsive peptide (DEVD). Our results suggest that DFF40 or Cytc fusion scFv specifically targets HER-2 overexpressing breast cancer cells (SK-BR-3 and BT-474) rather than HER-2 negative cells (MDA-MB-231 and MCF-7). Following cellular internalization, apoptosis-related proteins inhibited tumour activity by initiating endogenous apoptosis pathways, which significantly reduced immunogenicity and toxic side effects. Therefore, we suggest that immunoapoptotic molecules may become potential drugs for targeted immunotherapy of breast cancer.

Keywords: HER-2; breast cancer; immunoapoptotic molecules; scFv; targeted therapy.

FIGURE 1

Genetic engineering technology: construction of recombinant proteins and verification of expression. (A–D) pET‐32a(+) recombinant plasmid maps were inserted with scFv, DFF40‐scFv, Cytc‐scFv and 3Cytc‐scFv gene sequences, respectively. All structural units were marked. (E) Two‐dimensional structural diagrams of the four protein constructs. It was mainly divided into two parts: (1) tumour‐targeting area and (2) apoptosis‐inducing area. (F) SDS‐PAGE electrophoresis results of four protein constructs purified by affinity chromatography. M denotes the Marker lane, followed by the 100 mM imidazole eluate of each protein construct. (G) Western blot results that further confirm the expression of all protein constructs. An anti‐6His mAb antibody was used

FIGURE 2

Targeting activity of the scFvs. Normal cells (HEK‐293T), high HER‐2 expressing cells (SK‐BR‐3 and BT‐474) and HER‐2 negative cells (MDA‐MB‐231 and MCF‐7) were incubated with scFv and three other fusion proteins, respectively at 37°C for 30 min. All treated cells were observed and imaged by confocal microscopy. Green denotes the target proteins labelled with a FITC fluorescent secondary antibody, and blue denotes the nucleus stained with DAPI. Scale bar = 25 µm

FIGURE 3

Cytotoxicity and apoptosis‐inducing activity of immunoapoptotic molecules in different cell lines. (A) Cytotoxicity data of normal cells (HEK‐293T), high HER‐2 expressing cells (SK‐BR‐3 and BT‐474) and HER‐2 negative cells (MDA‐MB‐231 and MCF‐7) treated with different concentrations of the protein constructs. (B) Crystal violet–stained images of cells stimulated with the different protein constructs. The proliferation activity was reflected by the number of cells. (C) The degree of caspase‐3 activation in the cells stimulated with the different protein constructs. (D) The level of expression of the apoptosis‐related proteins, Bcl‐2 and Bax, as detected by Western blot. α‐tubulin was used as an internal reference. (E) Statistical graph obtained by a quantitative analysis of protein bands in (D), reflecting the ratio of Bcl‐2/Bax. n ≥ 3 compared with the control group; **p < 0.01; ***p < 0.001; ns, not significant

FIGURE 4

Flow cytometry detection of apoptosis induced by immunoapoptotic molecules. (A) The flow graphs of different cell lines treated with four kinds of proteins for 48 h. The abscissa is represented by Annexin V‐FITC and the ordinate is represented by PI. (B) Statistical graphs were obtained by performing a quantitative analysis of the apoptosis data in (A). n = 3, compared with the control group; ***p < 0.001; ns, not significant

FIGURE 5

Morphology of the apoptotic cells. (A) The nuclei of SK‐BR‐3 cells incubated with different protein constructs showed varying degrees of shrinkage following Hoechst staining. (B) The morphological changes of SK‐BR‐3 cells at 0, 24 and 48 h under a long‐term imager. Bubbling positions were marked with white arrows. (C–F) Statistical graphs of the percentage of apoptosis in the SK‐BR‐3, MDA‐MB‐231 and MCF‐7 cell lines incubated with four protein constructs for 48 h. n = 3, compared with the control group; ***p < 0.001; ns, not significant

FIGURE 6

Anti‐tumour activity of immunoapoptotic molecules in vivo. (A) Schematic diagram showing the establishment of the tumour xenograft model in nude mice and the associated treatment plan. (B) Changes in the tumour volume were recorded every three days. (C) Tumour tissue was isolated at the end of the experiment. (D) Final quality of the tumour tissue. (E) Apoptosis rate in the tumour paraffin sections by TUNEL staining. Apoptotic cells appeared brown under a light microscope. n = 5, compared with the control group, **p < 0.01; ***p < 0.001