Apoptosis of tumor cells induced by mutant and wild type TNF-α

AIM: To compare the apoptosis-induced ability of recombinant human mutant type 471 rhTNF-α (mt 471rhTNF-α) with that of wild type rhTNF-α (wt rhTNF-α) and to study the apoptotic mechanism induced by mt 471rhTNF-α. METHODS: The apoptosis of ZR75-1 cells, a breast cancer cell line, induced by mt 471rhTNF-α or wt rhTNF-α was analyzed and compared by 20 g/L agarose gel electrophoresis and flow cytometry techniques. The activation profiles of nuclear factor NF-κB in ZR75-1 cells untreated and treated by mt 471rhTNF-α or wt rhTNF-α were detected using Trans AMTM NF-κB p65 kit based on ELISA for further study of the apoptotic mechanism induced by mt 471rhTNF-α. RESULTS: The results from 20 g/L agarose gel electrophoresis of genomic DNA indicated that ZR75-1 cells treated by mt 471rhTNF-α provided a typical apoptotic ladder pattern of DNA fragmentation, whereas weakened ladders were observed in ZR75-1 cells treated by wt rhTNF-α. Flow cytometry showed that the cell apoptotic rate in mt 471rhTNF-α treated group was 43%, but 25% in wt rhTNF-α treated group. The mt 471rhTNF-α held a strong apoptosis-inducible ability. The results of DNA-binding activity of NF-κB showed that NF-κB activation induced by wt rhTNF-α was significantly higher than that of mt 471rhTNF-α when the protein concentration of mt 471rhTNF-α or wt rhTNF-α reached 50 μg/L (P=0.002). CONCLUSION: The apoptosis-induced ability of mt 471rhTNF-α is superior to that of wt rhTNF-α. One of the possible reasons for the enhanced apoptosis-induced ability may be that NF-κB activation is inhibited in tumor cells treated with mt 471rhTNF-α.