BAY-11-7082 induces apoptosis of multiple myeloma U266 cells through inhibiting NF-κB pathway

Y. Wang, X.-L. Zhang, C.-M. Sun

Department of Hematology, Yantai Yuhuangding Hospital, Yantai, China. wangyanyhd@sina.com

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• Hematology

OBJECTIVE: To study the effects of BAY-11-7082 on proliferation and apoptosis of U266 cells and its mechanism of action.

MATERIALS AND METHODS: Multiple myelomas U266 cells were cultured and divided into control group and gradient-concentration BAY-11-7082 groups (1 μmol/L, 2 μmol/L, 4 μmol/L and 8 μmol/L). Cells in BAY-11-7082 groups were treated with drugs in different concentrations for 4 h, while those in control group were added with an equal volume of solvent. The cell viability was detected via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and lactate dehydrogenase (LDH) release assay was used to detect the cytotoxicity. Furthermore, cells in low-concentration and high-concentration BAY-11-7082 groups were compared with those in control group. The cell proliferation level was evaluated via cell cycle assay, the interleukin-6 (IL-6) level in cells was detected via enzyme-linked immunosorbent assay (ELISA), and the β-catenin protein expression was detected via Western blotting. Moreover, flow cytometry and Hoechst staining were performed to detect the number of apoptotic cells, and the apoptosis level was detected via caspase3 activity and apoptosis-related protein expression. Finally, the levels of p65, p50 and inhibitor kappa B kinase β (IKKβ) were detected via polymerase chain reaction (PCR), and the expressions and changes of phosphorylated (p)-p65 and p-IKKβ were detected via Western blotting.

RESULTS: BAY-11-7082 could reduce the U266 cell viability and increase the cytotoxic effect. Based on gradient concentration, 2 μmol/L was selected as the low concentration, while 4 μmol/L was selected as the high concentration. Compared with those in control group, the number of cells in the S and G2/M phases in drug administration groups was significantly decreased, but that in the G0/G1 phase was significantly increased. Besides, the secretion of IL-6 in cells in drug administration groups was significantly decreased compared with that in control group. The β-catenin protein expression was decreased in drug administration groups, and there was also a difference between high concentration group and low concentration group. Flow cytometry and Hoechst staining showed that the proportion of apoptotic cells in drug administration groups was significantly increased. Western blotting and detection of caspase3 activity revealed that the expression and activation of apoptosis-related protein were increased in drug administration groups. It was found in the detection of nuclear factor-κB (NF-κB) pathway that the NF-κB pathway was inhibited in drug administration groups, and there was also a statistically significant difference between high concentration group and low concentration group.

CONCLUSIONS: BAY-11-7082 inhibits the proliferation and induces the apoptosis of U266 cells through inhibiting NF-κB pathway.

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