The Effect And Mechanism Of Selectively Inhibiting The NF-κB Activation On Survivin Expression In Leukemic K562 Cell Line

Objectives1 To investigate the effect of selectively inhibiting the NF-κB activation on K562 cell apoptosis.2 To investigate the effect of highly specific proteasomal inhibitor MG132 on NF-κB activation and Survivin expression.3 To explore the correlation between Survivin expression and NF-κB activation.4 To explore the potential mechanism of modulating the NF-κB signal transduction pathway on K562 leukemic cell apoptosis.MethodsHuman myelogenous leukemic K562 cell was cultured by serial subcultivation. The newly K562 cells of exponential growth phase were divided into one control group(no MG132) and four treatment groups(2 4 6 8 μmol/L MG132, respectively), then tested after 24 hours culture. Apoptosis is detected by cell morphology and flow cytometry. Survivin expression and NF-κB activation were detected with immunocytochemistry, the correlation between them is quantitatively analyzed by Western blotting. All tests were repeated for three times. The data were analyzed by statistics.Results1 MG132 of different concentration can induce K562 cell apoptosis. Cell chromatin pyknosis, nuclear fragmentation and typical apoptotic body were observed in K562 cell with microscope. The analysis of flow cytometry showed that apoptosis rates of treatment groups were 11.2% ± 2.5% 17.2% ± 2.3% 21.7% ± 1.8% and 24.3% ± 3.4% respectively, which was significantly different compared with control group (P<0.01) and showed the dose-dependent effect.2 Highly abnormal expression of Survivin and NF-κB was detected in K562 cell. The positive products of NF-κB activation showed the brown particles located in cytoplasm and/or nucleus, the ones of Survivin expression were mainly located in cytoplasm, which showed brown particles as well. Both were downregulated after MG132 treating for 24 hours.3 Western blotting analysis showed that, compared with the control group, K562 cell treated with MG132 at different concentration for 24 hours showed the decrease of NF-κB activation to 75.0% ± 3.7%, 59.9% ± 5.3%, 45.4% ± 5.7% and 25.0% ± 4.2% respectively, and Survivin expression to 90.9% ± 10.1%, 66.7% ± 5.2%, 45.4% ± 5.7% and 30.3% ± 6.6% respectively. Downregulation of Survivin expression was closely correlated with inhibition of NF-κB activation(Pearson correlation coefficient t = 0.989, P<0.01).Conclusions1 Proteasome inhibitor MG132 can effectively inhibit the NF-κB activation and induce K562 leukemic cell apoptosis, which shows the dose-dependent effect.2 Survivin expression will downregulate accompanied with the inhibition of NF-κB activation , which was closely correlated .3 Inhibition of NF-κB activation by MG132 may affect the Survivin expression through interfering the gene transcription action.