In vivo antimyeloma efficacy of FK866. (A-B) MM.1S cells (3 × 106 in 100 μL of serum free RPMI-1640 medium) were implanted subcutaneously in CB17-SCID mice. After detection of tumor, mice were treated intraperitoneally with FK866 (30 mg/kg body weight) or vehicle twice a day for 4 days in 3 weeks. The treatment significantly inhibited MM tumor growth (P = .0061 in the first week and P = .0067 at the end of treatment) and increased survival (P = .0014) compared with control. Survival was evaluated from the first day of treatment until death using Kaplan-Meier curves. Error bars represent mean ± SD. (C) Tumor tissues from mice treated with vehicle or with FK866 were harvested; whole-tissue lysates were subjected to Western blotting using indicated antibodies. β-actin was used as loading control. Relative intensity of LC3 was calculated by normalizing the LC3-II intensity to β-actin using ImageJ Version 1.46 analysis software. (D) Schematic model of FK866-induced autophagy in MM cells. FK866 inhibits MAPK and induces nuclear translocation of TFEB, thereby up-regulating autophagy-related genes (transcriptional-dependent mechanism), as occurs in starvation conditions. In addition, FK866 directly inhibits PI3K/mTOR activity (nontranscriptional-dependent mechanism), thereby increasing autophagy in MM cells.
