Abstract

BACKGROUND: TGFalphaL3-SEB chimeric protein is a synthetic protein, which is produced by combining the third loop (L3) of transforming growth factor-alpha (TGF-alpha) with staphylococcal enterotoxin type B. To the best of our knowledge, anti-cancer activity of this chimeric protein against colon cancer that overexpresses epidermal growth factor receptor (EGFR) has not yet been studied. Thus, in the present study, the anti-tumor effects of TGFalphaL3-SEB chimeric protein on HT-29 colon cancer cells were evaluated. MATERIALS AND METHODS: The TGFalphaL3-SEB chimeric protein was previously designed and cloned in Escherichia coli (E. coli) 1,2. The level of expression and the purity of this novel protein were examined for further analysis. For this purpose, the cells were treated with different concentrations (25, 50 and 75 mug/ml) of TGFalphaL3-SEB and then the proliferation was detected using the MTT assay. The apoptosis-inducing potential of TGFalphaL3-SEB in HT-29 and HEK-293 cells was evaluated by flow cytometry using Annexin V/PI double staining method; in addition, bax/bcl2 mRNA ratio, caspase-3 and caspase-9 activity were also assessed. RESULTS: In the present study, TGFalphaL3-SEB chimeric protein was produced in E. coli. After effective purification, its growth inhibitory effect was evaluated. Our results indicated that the incubation of HT-29 colon cancer cell with 25, 50 and 75 mug/ml of TGFalphaL3-SEB for 24 h leads to significant reduction of proliferation in a dose-dependent manner (P < 0.05). Further analysis indicated that exposure of EGFR expressing HT-29 cells to TGFalphaL3-SEB leads to significant increase of the caspase-3 and caspase-9 activity in a concentration-dependent manner (P < 0.05). Bax/bcl-2 ratio also confirmed that TGFalphaL3-SEB has the pro-apoptotic effect. Flow cytometry analysis of TGFalphaL3-SEB treated cells showed that in addition to apoptotic cells, necrotic cells were also increased significantly at the concentration of 25, 50 and 75 mug/ml (P < 0.05). CONCLUSION: In conclusion, our results demonstrated that TGFalphaL3-SEB chimeric protein induced cell death through both mechanisms of apoptosis and necrosis in HT-29 colon cancer cells. This paper has highlighted that TGFalphaL3-SEB has the potential to target EGFR expressing cancer cell.