Matrix metalloproteinase-activated anthrax lethal toxin demonstrates high potency in targeting tumor vasculature - PubMed (original) (raw)

. 2008 Jan 4;283(1):529-540.

doi: 10.1074/jbc.M707419200. Epub 2007 Nov 1.

Hailun Wang 1, Brooke M Currie 2, Alfredo Molinolo 2, Howard J Leung 1, Mahtab Moayeri 1, John R Basile 2, Randall W Alfano 3, J Silvio Gutkind 2, Arthur E Frankel 3, Thomas H Bugge 4, Stephen H Leppla 5

Affiliations

Matrix metalloproteinase-activated anthrax lethal toxin demonstrates high potency in targeting tumor vasculature

Shihui Liu et al. J Biol Chem. 2008.

Abstract

Anthrax lethal toxin (LT), a virulence factor secreted by Bacillus anthracis, is selectively toxic to human melanomas with the BRAF V600E activating mutation because of its proteolytic activities toward the mitogen-activated protein kinase kinases (MEKs). To develop LT variants with lower in vivo toxicity and high tumor specificity, and therefore greater potential for clinical use, we generated a mutated LT that requires activation by matrix metalloproteinases (MMPs). This engineered toxin was less toxic than wild-type LT to mice because of the limited expression of MMPs by normal cells. Moreover, the systemically administered toxin produced greater anti-tumor effects than wild-type LT toward human xenografted tumors. This was shown to result from its greater bioavailability, a consequence of the limited uptake and clearance of the modified toxin by normal cells. Furthermore, the MMP-activated LT had very potent anti-tumor activity not only to human melanomas containing the BRAF mutation but also to other tumor types, including lung and colon carcinomas regardless of their BRAF status. Tumor histology and in vivo angiogenesis assays showed that this anti-tumor activity is due largely to the indirect targeting of tumor vasculature and angiogenic processes. Thus, even tumors genetically deficient in anthrax toxin receptors were still susceptible to the toxin therapy in vivo. Moreover, the modified toxin also displayed lower immunogenicity compared with the wild-type toxin. All these properties suggest that this MMP-activated anti-tumor toxin has potential for use in cancer therapy.

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Figures

Fig. 1

Fig. 1

Cytotoxicity of the anthrax lethal toxins to human tumor cells. (A) Ten different NCI60 cell lines were incubated with various concentrations of PA or PA-L1 in the presence of 5 nM LF for 72 h, and the cell viability was measured as described in Experimental Procedures. Note that all the cells having the BRAF mutation (indicated in red) were sensitive to the lethal toxins, whereas cells without the mutation (except MDA-MB-231 cells) were resistant to the toxins. (B) The same set of cell lines were also treated with PA or PA-L1 in the presence of 1.9 nM FP59 as described in (A). All the cells were sensitive to the toxins, demonstrating that the cells express MMP activities.

Fig. 2

Fig. 2

PA-L1/LF displays broad and potent anti-tumor activity regardless of the BRAF mutation status of the tumor. (A) Nude mice bearing human C32 melanoma (left), HT144 melanoma (middle), or A549/ATCC lung carcinoma (right) were injected (i.p.) with 6 doses of PBS, PA/LF, or PA-L1/LF as indicated by red arrows (n=10 for each group). Weights of tumors in this and the following experiments are expressed as mean tumor weight ± s.e.m. (B) PA-L1/LF causes extensive necrosis of A549/ATCC tumors. A549/ATCC tumor-bearing nude mice were treated with 4 doses of 30/10 μg of PA-L1/LF or PBS (at days 0, 2, 4, and 7). Two hours after injection of BrdU, tumors were dissected and subjected to histological analysis. H&E staining shows extensive toxin-dependent necrosis of a representative tumor treated with PA-L1/LF, which is observed in all the toxin-treated A549/ATCC tumors. (C) BrdU incorporation assay reveals remarkable DNA synthesis cessation in PA-L1/LF-treated but not PBS-treated A549/ATCC tumors. The tumor sections analyzed in (D-E) were stained with an antibody against BrdU 2 h after systemic administration of BrdU. Note, BrdU positive cells are easily detected in PBS-treated tumors, but hardly detected in viable areas of the toxin-treated tumors. (D) C57BL mice bearing mouse B16-BL6 melanomas or LL3 Lewis lung carcinomas were treated (i.p.) with 5 doses of PBS or PA-L1/LF as indicated (n=10 for each group). (E) PA-L1/LF displays much stronger anti-tumor activity than PA/LF. Nude mice bearing Colo205 colon carcinoma were treated (i.p.) with 6 doses of PBA, PA/LF, or PA-L1/LF as indicated (n=10 for each group). A significant difference (*, p<0.05; **, p<0.01) is shown between 15/5 μg of PA-L1/LF and 15/5 μg of PA/LF treated tumors.

Fig. 3

Fig. 3

Increased plasma half-life and decreased immunogenicity of the MMP-activated protective antigen. (A) PA-L1 has a longer plasma half-life than PA. Mice were injected (i.v.) with 100 μg of PA or PA-L1, euthanized at 2 h or 6 h, blood samples were collected, and PA protein concentrations were measured using ELISA. There is a significant difference (*, p<0.05; **, p<0.01) between PA and PA-L1. (B) C57BL/6 mice were injected i.p. with 6 doses of 5 or 15 μg of wild-type PA or PA-L1, respectively within a period of two weeks. Ten days later, the mice were bled, and the titers of the serum neutralizing antibodies against PA measured in a cytotoxicity assay using mouse macrophage RAW264.7 cells challenged with LT (75 ng/ml each of PA and LF). The titers of the PA neutralizing antibodies were expressed as mean of fold dilution ± s.e.m of the sera that could protect 50% of RAW264.7 cells from LT treatment. Note that the neutralizing activities from the mice treated with wild-type PA were approximately 6-fold higher that those from PA-L1 treated mice: PA vs. PA-L1 (6×5 μg): 1097 ± 272 vs. 178 ± 36, p=0.0002; PA vs. PA-L1 (6×30 μg): 1081 ± 142 vs. 162 ± 31, p=0.0004.

Fig. 4

Fig. 4

The potent anti-tumor activity of PA-L1/LF is not solely dependent on its inhibitory effects on IL8. (A) Angiogenic factor profiling RT-PCR analysis reveals that the expression of IL8 by tumor cells is down-regulated by anthrax lethal toxin. Colo205, A549/ATCC, HT144, and HT29 cells were treated with or without PA/LF (10/3.3 nM) for 8 h, then the total RNA was isolated, and subjected to the angiogenic factor RT-PCR profiling analyses following the recommendations of the manufacturer. Note that IL8 is consistently down-regulated by PA/LF in all four cancer cell lines. ANGP1, angiopoietin 1; CSF3, colony stimulating factor 3; ECGF1, endothelial cell growth factor 1; FGF1 and FGF2, fibroblast growth factor 1 and 2; FST, follistatin; HGF, hepatocyte growth factor; LEP, leptin; PDGFB, platelet derived growth factor B; PGF, placental growth factor. (B-C) Both A549/ATCC carcinomas (B) and C32 melanomas (C) transfected with lethal LT ‘resistant’ IL8 retain susceptibility to PA-L1/LF. Nude mice bearing tumors transfected with IL8 or the empty vector were treated with 6 doses of 30/10 μg of PA-L1/LF or PBS. PA- L1/LF shows potent anti-tumor activity against the tumors transfected with either IL8 or the empty vector.

Fig. 5

Fig. 5

PA-L1/LF impairs the function of primary human endothelial cells. (A) PA protein-dependent translocation of LF into the cytosol of HMVEC and HUVEC cells. HUVEC and HMVEC cells were incubated with either PA-L1/LF (6 nM/6 nM) or PA/LF (6 nM/6 nM) for 2 or 4 h. The binding and proteolytic processing of PA proteins, the binding and translocation of LF, and the MEKs cleavages were detected by Western blotting using the corresponding antibodies. The non-specific bands, indicated by the arrow heads left of images, served as protein loading controls in these experiments. (B-C) Cytotoxicity of PA-L1/FP59 (B) and PA-L1/LF (C) to human primary vascular endothelial cells. HUVEC and HMVEC were treated with the indicated toxins as described in Fig. 1. The expression of MMPs by the endothelial cells was evidenced by their high sensitivity to PA-L1/FP59. (D) PA-L1/LF can efficiently inhibit the migration of vascular endothelial cells toward angiogenic factors-containing endothelial cell growth medium (GM). The experiments were performed as described in the Materials and Methods section. SFM, serum and angiogenic factors free medium.

Fig. 6

Fig. 6

PA-L1/LF demonstrates potent anti-tumor vasculature and angiogenesis activities. (A) Sections of A549/ATCC tumors treated with PBS or PA-L1/LF, as described inFig. 2Bwere stained with an antibody against the endothelial cell marker CD31. CD31-positive structures were quantified using the Northern Eclipse Image Analysis Software (Empix Imaging, North Tonawanda, NY). In inserts, black arrows point to the examples of CD31 positive endothelial cells; dash line, the boundary between the tumor and its surrounding normal tissues. N, necrotic area; V, area with viable cancer cells. (B) Directed in vivo angiogenesis analysis demonstrates that PA-L1/LF can inhibit tumor cell independent in vivo angiogenesis. There is a significant difference (**, p<0.01) between the angioreactors treated with PBS (n=8) and treated with PA-L1/LF (15/5 μg, n=8; 30/10 μg, n=10). (C) Anthrax toxin receptors-deficient CHO tumors are susceptible to PA-L1/LF. CHO PR230 tumor-bearing nude mice were injected (i.p.) with 6 doses of 30/10 μg of PA-L1/LF as indicated (n=6 for each group). There is a significant difference (*, p<0.05) between the tumors treated with PA and PA-L1.

Fig. 7

Fig. 7

PA-L1/LF delays, but does not prevent, incisional skin wound healing. C57BL/6 mice with the incisional skin wounds were treated with either PA-L1/LF (30/10 μg) (n=7) or PBS (n=8) three times per week until all the wounds were healed. The average wound healing time was delayed for the toxin-treated mice compared to the mock-treated group (14.5 days vs. 10 days, p<0.001, Mann-Whitney U-test, two-tailed). Inserts, representative examples of the appearance of skin wounds from mice treated with PA-L1/LF (left) or PBS (right) at days 5-9.

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