Investigation of antitumor effects of synthetic epothilone analogs in human myeloma models in vitro and in vivo

Abstract

26-Trifluoro-(E)-9,10-dehydro-12,13-desoxyepothilone B [Fludelone (Flu)] has shown broad antitumor activity in solid tumor models. In the present study, we showed, in vitro, that Flu significantly inhibited multiple myeloma (MM) cell proliferation (with 1-15 nM IC50), whereas normal human bone marrow stromal cells (HS-27A and HS-5 lines) were relatively resistant (10- to 15-fold higher IC50). Cell-cycle analysis demonstrated that Flu caused G2/M phase arrest and induced cell apoptosis. After Flu treatment, caspase-3, -8, and -9 were activated, cytochrome c and second mitochondrial-derived activator of caspase were released to the cytosol, and c-Jun N-terminal kinase was activated, indicating that mitochondria were involved in the apoptosis. Flu toxicity to human hematopoietic stem cells was evaluated by CD34+ cell-apoptosis measurements and hematopoietic-progenitor assays. There was no significant toxicity to noncycling human CD34+ cells. We compared the efficacy of Flu with the epothilone analog 12,13-desoxyepothilone B (dEpoB) in xenograft nonobese diabetic/severe combined immunodeficient mouse models with subcutaneous or disseminated MM. Flu caused tumor disappearance in RPMI 8226 subcutaneous xenografts after only five doses of the drug (20 mg/kg of body weight), with no sign of relapse after 100 d of observation. In a disseminated CAG MM model, mice treated with Flu had a significantly decreased tumor burden, as determined by bioluminescence imaging, and prolonged overall survival vs. mice treated with dEpoB or vehicle control, indicating that Flu may be a promising agent for MM therapy.

Fig. 1.

Flu showed high potency in growth inhibition of MM cells in vitro. (A) Morphological changes of MM cells after treatment with Flu (10 × IC₅₀) for 24 h. (B) RPMI 8226 cells were pulse-exposed to either Flu or dEpoB at 10 × IC₅₀ for 1, 2, 4, 8, or 24 h, and the drug was washed out and the cells incubated for up to 48 h. (C) Effect of epothilones and paclitaxel on in vitro proliferation of purified bone marrow CD138⁺ cells (NPCs, normal plasma cells). The data are given as mean ± SD (n = 3). Note that significant inhibition is seen only with Flu on MM cells at 72 h (P < 0.01). (D) Morphology of primary MM cells treated with Flu at 48 and 72 h.

Fig. 2.

Epothilones induce MM cells to undergo cell-cycle arrest at the G₂/M phase, followed by apoptosis. (A) Cell-cycle profile of OPM-2 MM cells. (B) Influence of Flu dose on RPMI 8226 cell-cycle arrest. (C) Annexin-V-binding assay on RPMI 8226 MM cells. (D) DNA fragmentation assay on RPMI 8226 and CAG cells.

Fig. 3.

Flu-induced MM-cell apoptosis is associated with the activation of caspase pathway. (A) Caspase-3 was activated after epothilone treatment in CAG cells. The cells treated with tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) were used as a positive control. (B) Cleavage of PARP in CAG cells. (C and D) Caspase-8 and -9 activities were increased and inhibited by specific caspase inhibitors. FMK, fluoromethyl ketone. (E) Detection of cytosolic second mitochondrial-derived activator of caspase (SMAC) (Top) and cytochrome c (Middle) in CAG cells. (F) Activation of phosphorylated JNK in CAG cells (Top and Middle, mean ± SEM) and the total JNK (Bottom). All blots are representative of three independent experiments.

Fig. 4.

Therapy with Flu and dEpoB on NOD/SCID mouse models with subcutaneous or disseminated MM xenografts. (A) Suppression of growth of RPMI 8226 MM xenografts in an s.c. mouse model. Data plotted are the representative tumor volume of two rounds of experiments (n = 10 for each group). (B) BLI of mice with s.c. RPMI 8226 xenografts at day 50. (C) Quantification of dorsal and ventral tumor burden by BLI. (D) In vivo therapy in disseminated CAG MM xenografts. Shown is the representative BLI at day 40, and all images are displayed in the same scale. (E) Quantification of dorsal and ventral tumor burden in mice with disseminated CAG xenografts by BLI at day 40. Data plotted are the percentage of tumor burden (mean ± SD) of mice, relative to control (n = 10 in each group). (F) Kaplan-Meier survival curve of disseminated MM mice treated with Flu or dEpoB (log-rank test, ^*, P < 0.005 for Flu cohort vs. control or dEpoB cohort; n = 10 per treatment group).