Figure 6

Apigenin inhibits proliferation, down-regulates CK2 activity, and depletes Hsp90/Cdc37 client proteins in primary MM cells. (A) Enriched CD138⁺ cells from 12 multiple myeloma patients were prepared by immunomagnetic bead selection. After treatment of apigenin for 24 h, cell viability was measured by the MTS assay as described in Material and Methods. Values represent the mean ± SD. *Significant difference from control. *p < 0.05, **p < 0.01. (B) PBMCs were treated with the indicated concentrations of apigenin for 24 h and the cell viability was measured by MTS assays. (C) After treatment with apigenin for 24 h, the expression of CK2α in CD138 negative and positive cells was analyzed by flow cytometry and quantified using the mean fluorescence intensity. (D) After treatment with or without 50 μM apigenin for 24 h, PBMCs and the enriched primary myeloma cells were examined by immunofluorescence microscopy to detect the levels of CK2α. The bar is equal to 5 μm. (E) The CD138⁺ cells were treated with apigenin for 24 h and were stained for CK2α (green), CD138 antigen (red) and DNA (blue). The bar, 5 μm. (F) The fluorescence intensity of each sample from (D) was analyzed using the softWoRx Explorer software. The average pixel intensities from 30 cells of three different fields in each group were measured, and background pixel intensities were subtracted. Values represent the mean ± SD of at least 3 measurements. * Significant difference from control cells, **p < 0.01. (G) After treatment of apigenin for 24 h, the CK2 kinase activity in primary MM cell purified from patient No. 8 and No. 9 was analyzed. *Significant difference from control cells, *p < 0.05, **p < 0.01. (H) Purified PBMC or enriched CD138⁺ cells from MM patients were treated with apigenin for 24 h, and the whole-cell lysates were subjected to western blotting to determine the levels of CK2α, Raf-1, Src, Cdk4 and β-actin.