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. 2006 Dec;97(12):1315-20.
doi: 10.1111/j.1349-7006.2006.00336.x. Epub 2006 Oct 9.

Anti-tumor effect in an in vivo model by human-derived pancreatic RNase with basic fibroblast growth factor insertional fusion protein through antiangiogenic properties

Hiroshi Yagi  1 , Masakazu Ueda, Hiromitsu Jinno, Koichi Aiura, Shuji Mikami, Hiroko Tada, Masaharu Seno, Hidenori Yamada, Masaki Kitajima
Affiliations

Anti-tumor effect in an in vivo model by human-derived pancreatic RNase with basic fibroblast growth factor insertional fusion protein through antiangiogenic properties

Hiroshi Yagi et al. Cancer Sci. 2006 Dec.

Abstract

It is thought that the export of angiogenic fibroblast growth factors (FGF) from tumors may be involved in the onset of tumor angiogenesis. To create a new active targeting drug that inhibits the tumor angiogenic process without toxicities to normal cells, human basic FGF (h-bFGF) was inserted genetically into the Gly89 position of cross-linked RNase1 (the ribonuclease inhibitor protein [RI] binding site of cross-linked human pancreatic RNase) to prevent stereospecific binding to RI. The resultant insertional-fusion protein (CL-RFN89) was active both as h-bFGF and as RNase1. Furthermore, it acquired an additional ability of evading RI through steric blockade of RI binding caused by the fused h-bFGF domain. In the present study, the effect of the resultant protein, CL-RFN89, on the antitumor response though its antiangiogenic properties was investigated in an in vivo model. Continuous systemic treatment with CL-RFN89 significantly inhibited the growth of human A431 squamous cell carcinomas in vivo. Seven days of treatment with CL-RFN89 resulted in a 58.2% inhibition of tumor growth compared with control mice (P < 0.0001). Furthermore, immunohistochemistry using a rat antimouse CD31 antibody showed that treatment with CL-RFN89 reduced tumor vascularization. These findings identify CL-RFN89 as a potent systemic inhibitor of tumor growth as a result of its antiangiogenic properties. This protein appears to be a new systemic antitumor agent.

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Figures

Figure 1
Figure 1
Systemic therapy with CL‐RFN89 inhibits tumor growth of A431 in vivo. One day after tumor cell inoculation, mice were injected intraperitoneally with CL‐RFN89, basic fibroblast growth factor (bFGF) and phosphate‐buffered saline (PBS). The pumps placed in each mouse after injection contained CL‐RFN89, bFGF alone or PBS (control), and they released the liquids continually for 1 week. (a) Systemic treatment with CL‐RFN89 significantly inhibited human A431 tumor growth compared with control mice (*P = 0.0003). Error bars show the SD. (b) The representative pictures show retarded tumor growth after CL‐RFN89 treatment (3) compared with PBS‐control treatment (1) and bFGF treatment (2). (c) The final mean weights of the tumors were 1018.8 ± 164.7 mg (n = 8) (1), 993.0 ± 96.5 mg (n = 4) (2) and 426.3 ± 86.3 mg (n = 8) (3). CL‐RFN89 treatment (3) showed 58.2% inhibition of tumor growth compared with control‐treated mice (1) (*P < 0.0001). Error bars show the SD.
Figure 2
Figure 2
Immunohistochemistry of A431 tumors. Tumor cell proliferation and microvessel density were assessed by CD31 immunostaining of A431 tumors excised on day 11 after the start of therapy. Mice were treated with phosphate‐buffered saline (PBS) (control), basic fibroblast growth factor (bFGF) alone or CL‐RFN89 as shown in Fig. 1. (1) The sections were incubated in the presence of a CD31 antibody. (2) Counterstaining was carried out using Carazzi's hematoxylin. (3) Corresponding frozen tumor sections were also stained with hematoxylin and eosin. (a) Control and (b) bFGF‐treated A431 squamous cell carcinoma showed remarkable heterogeneity in lumen diameter with numerous large‐caliber vessels throughout the viable parts of the tumors, as assessed by analysis of CD31‐positive vessels. (c) In contrast, the extent of tumor angiogenesis in tumors of mice treated with CL‐RFN89 was less pronounced and was characterized by more homogenous small‐caliber vessels.
Figure 3
Figure 3
Computer‐assisted image analysis of CD31‐positive vessels indicated that the inhibition of angiogenesis with CL‐RFN89 (n = 11) caused a further decrease in total vascular area to values 95% lower (*P < 0.0001) than those measured in control (n = 11) and basic fibroblast growth factor (bFGF)‐treated tumors (n = 12). Error bars show the SD.
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