Figure 5.

Lycorine enhances the effect of bortezomib by inhibiting autophagy. (A) ANBL6 and ARP-1 cells were grown and treated with lycorine, BTZ or lycorine plus BTZ, then cell viability was obtained using a CCK-8 assay. Data are presented as the mean±SD (n=3, **p<0.01). (B) Combination index of lycorine and BTZ. ANBL6 and ARP-1 cells were treated 24 h across a range of concentrations of lycorine, BTZ or lycorine plus BTZ, and assessed for cell viability using CCK-8 assay. Combination index (CI) values corresponding to the specified data points on the table were obtained using CompuSyn software program for non-constant drug ratio and plotted on the graph against fraction effect (Fa) to detect lycorine-BTZ synergy. A CI<1 indicates synergism (C) After ANBL6 and ARP-1 cells were treated with lycorine, BTZ or lycorine plus BTZ, the expression of HMGB1 and the autophagy-related proteins Beclin-1 and LC3B was analyzed by Western blotting. GAPDH was used as a loading control. (D) Activity of lycorine against ANBL6 and ARP-1 cells in the presence of bone marrow stromal cells (BMSC) was analyzed by cell viability assay using a CCK-8 kit. Data presented are mean±SD (n=3, **p<0.01). (E) Effect of lycorine on primary bone marrow cells. Human primary BM CD138⁺ cells were treated with lycorine, BTZ and lycorine plus BTZ for 24 h and cell viability was measured by CCK-8 assay. Data presented are mean±SD from triplicate samples (**p<0.01). (F) Western blotting analysis of basal HMGB1 expression in bortezomib-naïve and bortezomib-resistance ANBL6 cells. (G) Effect of lycorine, BTZ and combination on BTZ resistance ANBL6.BR cells was analyzed by cell viability assay. Data were generated from three independent experiments (**p<0.01).