[2-methoxyestradiol disrupts aggresomes induction by bortezomib and potentiates apoptosis in multiple myeloma cells.]

To investigate the synergistic anti-multiple myeloma (MM) effect of 2-methoxyestradiol (2-ME2) and bortezomib, and explore the relationship between this effect and blockade of aggresomes formation by 2-ME2.

Four MM cell lines RPMI-8226, NCI-H929, U266 and SKO-007 were used for study. Immunoflourescent anti-ubiquitin and Hoechst 33342 staining were used to examine aggresome-positive cells and apoptotic cells, respectively. Isobolographic analysis was used for determination of synergy.

(1) Quantitative assay showed that in the absence of bortezomib, only 6.6% - 8.9% of MM cells were aggresome-positive, but the percentage was increased to 71.9%-83.4% after treatment with bortezomib at IC(20) concentration for 24 h. Aggresome-positive cells with immunoreactivity to anti-ubiquitin were detected in almost all non-apoptotic cells, but not in apoptotic cells. (2) Treatment in a definite range of concentrations bortezomib plus 2-ME2 led to MM cell apoptosis compared with each agent alone and the significantly synergistic effect confirmed by isobolographic analysis. (3) Combination of bortezomib and 2-ME2 increased the apoptotic cells aggresome-negative cells (ANK) and decreased the non-apoptotic cells in aggresome positive cells (APC). In RPMI8226 and U266 cells, the apoptotic cells in ANC increased from (14.5 +/- 2.0)% and (20.1 +/- 2.9)% to (80.7 +/- 6.9)% and (71.6 +/- 6.2)%, and the non-apoptotic cells in APC decreased from (75.3 +/- 5.7)% and (69.1 +/- 8.6)% to (13.8 +/- 3.8)% and (19.5 +/- 4.2)%, respectively, in combined group and bortezomib alone group.

Bortezomib-induced aggresomes have a protective function for MM cells and combination of bortezomib with 2-ME2 induced a synergistic cytotoxicity to the cells.