Amphipathic tail-anchoring peptide is a promising therapeutic agent for prostate cancer treatment

Abstract

Amphipathic tail-anchoring peptide (ATAP) derived from the human anti-apoptotic protein Bfl-1 is a potent inducer of apoptosis by targeting mitochondria permeability transition. By linking ATAP to an internalizing RGD peptide (iRGD), selective targeting for ATAP to tumor cell was achieved. Confocal fluorescence microscopy showed that ATAP-iRGD could penetrate into cancer cells and distribute along the mitochondria network. ATAP-iRGD triggered mitochondria-dependent cell death through release of cytochrome c. In an effort to promote ATAP-iRGD physiochemical properties to approach clinic application, amino acid substitution and chemical modification were made with ATAP-iRGD to improve its bioactivity. One of these modified peptides, ATAP-iRGD-M8, was with improved stability and aqueous solubility without compromising in vitro cytotoxicity in cultured cancer cells. In vivo xenograft studies with multiple prostate cancer cell lines showed that intravenous administration of ATAP-iRGD-M8 suppressed tumor growth. Toxicological studies revealed that repetitive intravenous administration of ATAP-iRGD-M8 did not produce significant toxicity in the SV129 mice. Our data suggest that ATAP-iRGD-M8 is a promising agent with high selectivity and limited systemic toxicity for prostate cancer treatment.

Figure 1. ATAP-iRGD fusion peptides maintain pro-apoptotic function

(A) ATAP is coupled with the disulfide-based cyclic RGD moiety (iRGD) to form a chimeric peptide ATAP-iRGD. (B) Schematic representation of iRGD to illustrate exposure of the [CRGDK] element after proteolytic cleavage. (C) ATAP-iRGD peptide, related mutations ATAP^C19A-iRGD, and the cleavage products ATAP-CRGDK, ATAP^C19A-CRGDK are compared with the wild-type sequence (mutation is highlighted by red color). (D) HeLa cells were transfected with plasmids expressing GFP-ATAP and GFP-ATAP related plasmids. At 24 hours after transfection, cells were treated with DAPI and observed under fluorescence microscope. Representative photographs showing GFP- or DAPI-positive cells in the same field. The scale bar represents 20 μm. (E) Percentage of surviving cells was determined by the ratio of GFP-positive cells without DAPI staining to total GFP-positive cells. About 200 cells from three different fields were scored. Data are expressed as the means ± SEM.

Figure 2. ATAP-iRGD targets mitochondria to cause apoptotic effects on tumor cells

(A) MTT assay for ATAP and ATAP-iRGD in DU145 cells. (B) When applied externally to DU145 cells, the Dylight488-labeled ATAP-iRGD (Green, left) can enter into cells and associate with mitochondria labeled with Mito Tracker Red 633 (Red, center). Overlapping patterns for ATAP-iRGD and Mito Tracker was observed (right). These experiments were performed in the presence of 50 μM Q-VD-OPh to allow for observation of co-localization before induction of cell death. The scale bar represents 20 μm. (C) Western blots showing effect of ATAP-iRGD on cytochrome c release in DU145 cells. Incubation with ATAP-iRGD induces release of cytochrome c from mitochondria into the cytosol. (D) ATAP-iRGD inhibits DU145 cells colony formation, whereas no significant difference was observed between cells treated with control and BH3-iRGD peptide. Results were means of triplicates ± SEM from three independent experiments. (E) Representative pictures of colony formation assay with different treatments.

Figure 3. ATAP-iRGD-M8 displays more favorable property than ATAP-iRGD for treatment of prostate cancer cells

(A) 200 μM indicated peptides were added to DU145 cells grown in a normal culture medium at 37 °C. ATAP-iRGD, ATAP-iRGD-M6 and M7 peptides all precipitate during 24 hours incubation, whereas the ATAP-iRGD-M8 shows no precipitation. The scale bar represents 20 μm. (B) Establishment of IC₅₀ of ATAP-iRGD and ATAP-iRGD-M8 in DU145 cells by MTT assay. (C) Typical mitochondria localization pattern of ATAP-iRGD-M8 following incubation in DU145 cells. When applied externally to cells the Dylight488-labeled ATAP-iRGD-M8 (Green, left) can enter into cells and co-localized with mitochondria labeled with Mito Tracker Deep Red 633 (Red, center) as shown by the overlay image (right). The scale bar represents 20 μm.

Figure 4. ATAP-iRGD-M8 suppresses prostate tumor growth with no toxic effects in xenograft model

(A) DU145 cells were subcutaneously injected into both flanks of nude mice and allowed to establish xenografts for two weeks. 21 mg/kg of ATAP-iRGD-M8 or saline were administered into the mice via tail vein injection. Picture was taken at 16 days post treatment. (B) The DU145 xenograft volumes from animals treated with ATAP-iRGD-M8 were significantly smaller than those from animals treated with the control saline (ATAP-iRGD-M8 treatment group n=6, saline treatment group n=9). (C) Nude mice implemented with PC-3 cells also show significant suppression of tumor growth following treatment with ATAP-iRGD-M8. (D) Morphological details were investigated using H&E staining. No significant pathological changes in kidney, heart, liver, lung, and spleen were observed in nude mice following treatment with ATAP-iRGD. The scale bar represents 50 μm.

Figure 5. ATAP-iRGD has low immunogenicity in mouse model with minimum effect on hemolysis

(A) Treatment of mouse red blood cells (RBCs) with 20 μM ATAP-iRGD did not cause hemolysis. PBS and 1%Triton-100 sample were used as negative and positive controls, respectively. Results were means of triplicates ±SEM. (B) Repetitive intravenous delivery of ATAP-iRGD did not produce obvious toxicity to vital organs in the SV129 mice. H&E staining of the ATAP-iRGD and control saline treatment mice show no significant pathological changes in kidney, heart, liver, lung and spleen. The scale bar represents 50 μm. (C) SV129 mice were immunized with 100 μg ATAP-iRGD-M8 daily for five weeks. Blood samples were collected 1, 2, 3, and 5 weeks after first immunization. Antibodies were detected on ATAP-iRGD-M8 coated plates, respectively, using ELISA against the mouse whole IgG. Error bars indicate ±SD (n=5).