Antitumor effect and mechanism of action of a tumor-targeting recombinant human tumor necrosis factor-α fusion protein mediated by urokinase.

Abstract

The aim of this study was to investigate the effect of the tumor‑targeting recombinant human tumor necrosis factor (rhTNF)‑α fusion protein mediated by urokinase on Sl80 tumor‑bearing mice, as well as to explore its mechanisms of action. Furthermore, the study aimed to observe the effect of the protein on liver and kidney function. rhTNF‑α fusion protein prokaryotic expression vectors were constructed using genetic engineering techniques, and were introduced into Escherichia coli. Expression of the fusion protein was induced, and it was then separated and purified in order to determine its cytotoxic activity on L929 cells. Kunming mice were randomly divided into four groups after being inoculated with S180 tumor cells. The groups were then injected with saline (control group, groupS), or saline with 0.1µg/ml fusion protein (low dose group, groupL), 0.2µg/ml fusion protein (middle dose group, groupM) or 0.3µg/ml (high dose group, groupH). The mice were sacrificed after 12 days and liver [mg/kg; (liver weight/body weight)x1,000] and kidney [mg/kg; (kidney weight/body weight)x1,000] indices, tumor weight, the percentage reduction in mean tumor size, and the levels of alanine transaminase (ALT), albumin (ALB), creatinine (Cr) and blood urea nitrogen (BUN) in each group of mice were determined. In addition, the levels of urokinase‑type plasminogen activator (uPA), the expression of bcl‑2, bax and vascular endothelial growth factor (VEGF), and the percentage of apoptotic cells were measured with an enzyme‑linked immunosorbent assay, streptavidin‑biotin complex of immunohistochemistry and terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling, respectively. The fusion protein significantly inhibited the growth of S180 tumor cells invivo in a dose‑dependent manner. With an increase in the dose of fusion protein, ALT, uPA, bcl‑2 and VEGF levels decreased, and ALB levels increased. However, liver and kidney indices and bax expression were not significantly altered. Cr and BUN levels did not change significantly in the low and middle dose groups, but did increase in the high dose group. Compared with the control group, the percentage of apoptotic cells in the high‑dose group was significantly higher. In conclusion, the fusion protein significantly inhibited S180 tumor growth in a mouse model, possibly by reducing the levels of uPA, bcl‑2 and VEGF. There was a mildly toxic effect on the kidneys with the high dose, but a protective effect in the liver.