Background & objective: Tumor necrosis factor alpha (TNF-alpha) is an important agent for tumor biotherapy because of its strong antitumor activity, but its clinical application is limited because of its severe fatal systemic toxicity. A new recombinant human (rh) mutein, mutant type (mt) 471rhTNF-alpha, was produced to decrease toxicity while keep antitumor activity of wild type (wt) TNF-alpha. This study was to compare the antitumor activities of mt-471rhTNF-alpha and wt-rhTNF-alpha, and analyze the mechanism of tumor cell apoptosis induced by mt-471rhTNF-alpha.
Methods: Breast cancer cell line ZR75-1 was treated with 100 microg/L of mt-471rhTNF-alpha or wt-rhTNF-alpha for 12 h. Cell apoptosis was analyzed with 2% agarose gel electrophoresis, and cell cycle distribution was analyzed by flow cytometry. The activation of nuclear factor-kappaB (NF-kappaB) p65 in ZR75-1 cells treated with 10 microg/L, 50 microg/L, and 100 microg/L of mt-471rhTNF-alpha or wt-rhTNF-alpha for 4 h were detected using Trans AMTM NF-kappaB p65 kit based on ELISA.
Results: ZR75-1 cells treated with mt-471rhTNF-alpha had typical ladders of DNA fragmentation, whereas ZR75-1 cells treated with wt-rhTNF-alpha only had weak ladders; the apoptosis rate of ZR75-1 cells was significantly higher in mt-471rhTNF-alpha group than in wt-rhTNF-alpha group [(44.6+/-3.6)% vs. (24.7+/-2.7)%, P<0.05]. The proportion of G1 phase cells was significantly higher in mt-471rhTNF-alpha group than in control group and wt-rhTNF-alpha group [(66.6+/-4.2)% vs. (34.8+/-3.1)% and (46.2+/-3.8)%, P<0.05]; the proportions of S phase and G2 phase cells were significantly lower in mt-471rhTNF-alpha group than in control group and wt-rhTNF-alpha group [(33.2+/-2.9)% vs. (50.1+/-3.8)% and (45.7+/-2.9)%, P<0.05; (0.2+/-0.0)% vs. (15.1+/-2.2)% and (8.1+/-3.1)%, P<0.05]. When treated with 50 microg/L rhTNF-alpha for 4 h, NF-kappaB activation was significantly higher in wt-rhTNF-alpha group than in mt-471rhTNF-alpha group (3.3+/-0.1 vs. 1.5+/-0.2, P<0.05 ).
Conclusions: The antitumor ability of mt-471rhTNF-alpha is more stronger than that of wt-rhTNF-alpha. The explanation for the enhanced apoptosis-inducible ability of mt-471rhTNF-alpha could be that NF-kappaB activation is inhibited in tumor cells treated with mt-471rhTNF-alpha.