Effect of Proteasome Inhibitor (Bortezomib) on the Intracellular Level of NF-κBÃ, Â AIκB and P-Gp and the Rate of Apoptosis in Chemotherapy Resistant Myeloid Leukemia Cell Line K562 Induced by Daunorubicin

Background: NF-κb is a transcription factor in eukaryotic cells that binds to inhibitor protein κB (IκB) in the cytoplasm. Once stress responses occur, IκB is phosphorylated and degraded by the proteasome. Upon dissociation with IκB, activated NF-κB enters the nucleolus and induces the expression of mdr1. MDR-1 encodes the P-glycoprotein (P-gp), which is an ATP-dependent drug efflux pump which lowers intracellular drug concentration resulting in drug resistance. Bortezomib (VELCADE®) is an anti-cancer drug that is a first-in-class proteasome inhibitor and is indicated in treatment of multiple myeloma (MM) patients. However, its role in myeloid leukemia therapy, is still being evaluated. In this study, we observed the effect of bortezomib on the expression of NF-κB AIκB and P-gp in daunorubicin (DNR) resistant cell line K562/DNR, a myeloid cell line; we also discussed the molecular mechanism and functional characteristics of reversing resistance by bortezomib and provided experimental basis to overcome multi-drug resistance in myeloid leukemia.

Methods:

1. Resistance of cell line will be evaluated by MTT;

2. After treating K562/DNR for 36h with DNR (100‚•g/ml) alone or combined with bortezomib at different concentration (0.4 mg/L A4 mg/L or 40 mg/L), the expression levels of NF-κB AIκB and P-gp, the activity of NF-κB p65 and the apoptosis rates of cells will be measured by Western blot followed by ELISA and flow cytometry, respectively. After treating K562/DNR for 12h, 24h and 36h with DNR (100 mg/ml) alone or combined with bortezomib (4 mg/L), the same indexes will also be measured;

3. Statistical analysis: With SPSS software, the two group means were compared by t-test and the two sample rates were compared by χ²-test.

Results: After treating K562/DNR for 36h with DNR alone or combined with bortezomib at different concentrations, apoptosis rates were measured by flow cytometry. As a single agent, botezomib at concenration of 0.4 mg/L and 4 mg/L did not result in an increase in the rate of apoptosis. However, an increase in apoptosis rate was observed with bortezomib 40 mg/L. In contrast, a combination of DNR and bortezomib resulted in significant increases in apoptosis rate at all doses of bortezomib. Moreover, a dose response was observed. After treating K562/DNR for 12h A24h and 36h with DNR alone or combined with bortezomib, we utilized Western blot to assess the intracellular level of NF-κB AP-gp and IκB. Compared with negative control, DNR as a single agent increased the level of NF-κB AP-gp and decreased the level of IκB. After the addition of bortezomib, the level of NF-κB AP-gp decreased while IκB level increased; furthermore, the effect increased by prolonging the treating time.

Conclusion: Based on our observations bortezomib may reverse drug resistance of K562/DNR cell line to DNR. Under optimized conditions, the effect of reversing resistance follows a dose-dependent manner and a time-dependent manner and increases with increasing the concentrations of bortezomib and duration of cellular exposure.