A fusion protein containing murine vascular endothelial growth factor and tissue factor induces thrombogenesis and suppression of tumor growth in a colon carcinoma model

Abstract

Induction of tumor vasculature occlusion by targeting a thrombogen to newly formed blood vessels in tumor tissues represents an intriguing approach to the eradication of primary solid tumors. In the current study, we construct and express a fusion protein containing vascular endothelial growth factor (VEGF) and tissue factor (TF) to explore whether this fusion protein has the capability of inhibiting tumor growth in a colon carcinoma model. The murine cDNA of VEGF A and TF were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned into prokaryotic expression plasmid pQE30 with a linker. The expression product recombinant VEGF-TF (rVEGF-TF) was purified and proved to have comparable enzyme activity to a commercial TF and the capability of specific binding to tumor vessels. Significant decrease of tumor growth was found in the mice administered with rVEGF-TF on Day 6 after initiated rVEGF-TF treatment (P<0.05), and the tumor masses in 2 of 10 mice were almost disappeared on Day 14 after the first treatment. In addition, valid thrombogenesis and tumor necrosis were observed in the tumor tissues injected with rVEGF-TF. Our results demonstrate that occlusion of tumor vasculature with rVEGF-TF is potentially an effective approach for cancer therapy.

Fig. 1

Amplification of TF and VEGF A cDNA, and analysis of the corresponding recombinant plasmids Lanes 1 and 2: The cDNA of TF (about 900 bp) at different conditions of PCR; Lane 3: The cDNA of VEGF A (about 400 bp); Lane 4: The fragment of empty pQE30 plasmid digested by KpnI (about 3.4 kb); Lane 5: The fragments of recombinant pQE-TF plasmid digested by KpnI and HindIII [about (3.4+0.9) kb]; Lane 6: The fragments of recombinant pQE-VEGF plasmid digested by SphI and KpnI [about (3.4+0.4) kb]; Lane 7: The fragments of recombinant pQE-VEGF-TF plasmid digested by SphI and HindIII [about (3.4+1.4) kb]. All the digested fragments were as predicted

Fig. 2

Expression and purification of the recombinant proteins. (a) The rVEGF-TF was purified by Ni-NTA agarose affinity chromatography. Peak 1 shows the nonspecific binding protein and peak 2 shows the recombinant protein rVEGF-TF; (b) Pro-expression (Lane 1), expression (Lane 2) and purification (Lane 3) of rVEGF-TF by SDS-PAGF; (c) Pro-expression (Lane 1), expression (Lane 2) and purification (Lane 3) of rVEGF-TF by Western blotting. The data for rVEGF and rTF were not shown

Fig. 2

Expression and purification of the recombinant proteins. (a) The rVEGF-TF was purified by Ni-NTA agarose affinity chromatography. Peak 1 shows the nonspecific binding protein and peak 2 shows the recombinant protein rVEGF-TF; (b) Pro-expression (Lane 1), expression (Lane 2) and purification (Lane 3) of rVEGF-TF by SDS-PAGF; (c) Pro-expression (Lane 1), expression (Lane 2) and purification (Lane 3) of rVEGF-TF by Western blotting. The data for rVEGF and rTF were not shown

Fig. 2

Expression and purification of the recombinant proteins. (a) The rVEGF-TF was purified by Ni-NTA agarose affinity chromatography. Peak 1 shows the nonspecific binding protein and peak 2 shows the recombinant protein rVEGF-TF; (b) Pro-expression (Lane 1), expression (Lane 2) and purification (Lane 3) of rVEGF-TF by SDS-PAGF; (c) Pro-expression (Lane 1), expression (Lane 2) and purification (Lane 3) of rVEGF-TF by Western blotting. The data for rVEGF and rTF were not shown

Fig. 3

The enzyme activities of rVEGF-TF and rTF were comparable with the commercial TF (cTF) determined by the Spectozyme Factor Xa assay

Fig. 4

Specific targeting to the tumor vessels by rVEGF-TF. The tumor tissues from mice injected with rVEGF-TF (a) or rTF (b) showed florescent signals 5 min after the injection of the correspondent recombinant protein. The tumor tissues from mice injected with rVEGF were not found to have any fluorescence signals 5 min (c) and 1 h (f) after injection. However, 1 h after the injection, only tumor tissues from the mice injected with rVEGF-TF had fluorescent signals (d), and the florescent signals from the mice injected with rTF disappeared (e), suggesting that specific binding to the tumor vessels was highly related to the specific binding between VEGF and VEGFR

Fig. 4

Specific targeting to the tumor vessels by rVEGF-TF. The tumor tissues from mice injected with rVEGF-TF (a) or rTF (b) showed florescent signals 5 min after the injection of the correspondent recombinant protein. The tumor tissues from mice injected with rVEGF were not found to have any fluorescence signals 5 min (c) and 1 h (f) after injection. However, 1 h after the injection, only tumor tissues from the mice injected with rVEGF-TF had fluorescent signals (d), and the florescent signals from the mice injected with rTF disappeared (e), suggesting that specific binding to the tumor vessels was highly related to the specific binding between VEGF and VEGFR

Fig. 4

Specific targeting to the tumor vessels by rVEGF-TF. The tumor tissues from mice injected with rVEGF-TF (a) or rTF (b) showed florescent signals 5 min after the injection of the correspondent recombinant protein. The tumor tissues from mice injected with rVEGF were not found to have any fluorescence signals 5 min (c) and 1 h (f) after injection. However, 1 h after the injection, only tumor tissues from the mice injected with rVEGF-TF had fluorescent signals (d), and the florescent signals from the mice injected with rTF disappeared (e), suggesting that specific binding to the tumor vessels was highly related to the specific binding between VEGF and VEGFR

Fig. 4

Specific targeting to the tumor vessels by rVEGF-TF. The tumor tissues from mice injected with rVEGF-TF (a) or rTF (b) showed florescent signals 5 min after the injection of the correspondent recombinant protein. The tumor tissues from mice injected with rVEGF were not found to have any fluorescence signals 5 min (c) and 1 h (f) after injection. However, 1 h after the injection, only tumor tissues from the mice injected with rVEGF-TF had fluorescent signals (d), and the florescent signals from the mice injected with rTF disappeared (e), suggesting that specific binding to the tumor vessels was highly related to the specific binding between VEGF and VEGFR

Fig. 4

Specific targeting to the tumor vessels by rVEGF-TF. The tumor tissues from mice injected with rVEGF-TF (a) or rTF (b) showed florescent signals 5 min after the injection of the correspondent recombinant protein. The tumor tissues from mice injected with rVEGF were not found to have any fluorescence signals 5 min (c) and 1 h (f) after injection. However, 1 h after the injection, only tumor tissues from the mice injected with rVEGF-TF had fluorescent signals (d), and the florescent signals from the mice injected with rTF disappeared (e), suggesting that specific binding to the tumor vessels was highly related to the specific binding between VEGF and VEGFR

Fig. 4

Specific targeting to the tumor vessels by rVEGF-TF. The tumor tissues from mice injected with rVEGF-TF (a) or rTF (b) showed florescent signals 5 min after the injection of the correspondent recombinant protein. The tumor tissues from mice injected with rVEGF were not found to have any fluorescence signals 5 min (c) and 1 h (f) after injection. However, 1 h after the injection, only tumor tissues from the mice injected with rVEGF-TF had fluorescent signals (d), and the florescent signals from the mice injected with rTF disappeared (e), suggesting that specific binding to the tumor vessels was highly related to the specific binding between VEGF and VEGFR

Fig. 5

Tumor volumes at different time points after the first treatment. rVEGF-TF: Mice injected with the recombinant fusion protein rVEGF-TF; rVEGF: Mice injected with rVEGF; rTF: Mice injected with rTF; NS: Mice injected with NS. Arrows represent the time points of treatment. ^a P<0.05, ^b P<0.01. Data are shown as mean±SD (n=10 in each group)

Fig. 6

Histology of the tumor tissues. Significantly thrombosis and tumor necrosis are found in the tumor tissues from the mice injected with rVEGF-TF (a), but not in the mice injected with rVEGF (b) or rTF(c)

Fig. 6

Histology of the tumor tissues. Significantly thrombosis and tumor necrosis are found in the tumor tissues from the mice injected with rVEGF-TF (a), but not in the mice injected with rVEGF (b) or rTF(c)

Fig. 6

Histology of the tumor tissues. Significantly thrombosis and tumor necrosis are found in the tumor tissues from the mice injected with rVEGF-TF (a), but not in the mice injected with rVEGF (b) or rTF(c)