The reduction of NFkappaB by genistein in T lymphoma cell lines generated by mink cell focus-forming virus

We have tested the effect of genistein on marine T-cell lines derived from thymic lymphomas induced by a lymphomagenic murine leukemia virus. A study of cells treated with a range of genistein concentrations (15 μM–60 μM) indicated that the percentage of viable cells was significantly reduced over a 24 h period with genistein concentrations starting at 15 μM. Various assays for apoptosis, which included the detection of annexin V/PI stained cells, DNA fragmentation, and caspase-3 activation, demonstrated that cell killing was a result of apoptosis. The transcription factor NF-κB is elevated in murine T-cell lymphoma lines compared with normal thymic lymphocytes, and may play a role in the neoplastic transformation of these cells. To examine whether NF-κB is reduced by genistein, we analyzed NF-κB and its inhibitor, IκBα. We detected a decrease in nuclear levels of NF-κB. Furthermore, we detected a 34 kDa cleavage product ΔIκBα, which was induced by genistein before NF-κB decreases were evident. Our observation that a pan-caspase inhibitor could inhibit the induction of ΔIκBα by genistein suggested that caspase activity was responsible for this cleavage product. When the induction of ΔIκBα was prevented, we detected no reduction of NF-κB levels by genistein. These results support a direct role for ΔIκBα in the reduction of NF-κB by genistein. To determine the effect of genistein on some NF-κB target gene products, we examined the anti-apoptotic proteins Bcl-2, Bcl-X_L, A1, and clAP-1. Only changes in A1 and cIAP-1 levels were affected with significant reductions in response to genistein. To test if there was mitochondria involvement in the mechanism of genistein induced apoptosis, Western blot analysis revealed caspase-9 activation. This result correlated with immunofluorescent staining results for mitochondria depolarization. These findings identified the participation of mitochondria in the induction of apoptosis by genistein. The inhibition of depolarization by the mitochondria permeability transition pore (PTP) inhibitor, bongkrekic acid, implicated the PTP in genistein induced apoptosis. Our data demonstrate that genistein induces apoptosis in murine T lymphoma cells and the induction is a result of mitochondria depolarization via the permeability transition pore.