Canonical Wnt Pathway Inhibitor ICG-001 Induces Cytotoxicity of Multiple Myeloma Cells in Wnt-Independent Manner
Fig 3
Mechanism of ICG-001-induced apoptosis in MM cells.
(A) MM cells were treated with ICG-001 (5 μM for RPMI-8226 cells, and 10 μM for H929 and MM1.S cells) 24 hours. Cells were then collected, lysed, and subjected to Western blotting with antibody against cleaved caspase 3. Equal loading was confirmed by re-probing the membrane with β-actin antibody. (B) Mononuclear cells were isolated from BM aspirates obtained from patients with MM and were cultured in vitro for 24 hours with or without ICG-001. Cells were then collected, labeled with anti-CD138 and anti-λ/κ light chain immunoglobulin antibodies, and stained with antibody against cleaved caspase 3. Level of cleaved caspase 3 was detected by flow cytometry in gated MM and non-MM cell populations. Shown are combined results obtained from BM cells isolated from 9 patients with MM. *P<0.05. (C) MM RPMI-8226 cells were treated with 5 μM ICG-001 with or without 100 μM z-VAD for 24 hours. Apoptosis of MM cells was evaluated by Annexin V binding assay using flow cytometry. Experiment was performed twice with similar results. (D-G) MM cells were treated with ICG-001 or vehicle control (D) overnight or (E-G) for 24 hours. (D) Expression of indicated genes was determined by real-time PCR and normalized to the expression of β-actin. Shown is fold increase in expression of indicated genes in ICG-001-treated cells over cells treated with vehicle control. Combined results of 3 independent experiments are shown. *P<0.05; **p<0.01. (E-G) Western blotting with indicated antibodies was performed. Equal loading was confirmed by re-probing the membranes with antibody against β-actin.