Fig 1.
Treatment with ICG-001 inhibits canonical Wnt signaling in MM cells.
(A) MM1.S cells were transiently transfected with TOPFlash or FOPFlash reporter plasmids and co-transfected with pRL-TK vector. Cells were untreated (control) or treated for 24 hours with ICG-001 (10 μM), Wnt3a (100 ng/mL) or Wnt3a and ICG-001 followed by dual luciferase reporter assay. Combined data from 2 independent experiments are shown. Statistically significant differences are indicated. (B, C) MM cell lines (B) or primary CD138+ MM cells isolated from BM of patients with MM (C) were treated with vehicle control or ICG-001 (2.5 μM for RPMI-8226 cells and 10 μM for other cells) for 24 hours. Expression of indicated genes was determined by real-time PCR. Gene expression in vehicle-treated control cells is indicated by the horizontal line. Combined data from 4 independent experiments (B) and combined results for primary MM cells obtained from 4 MM patients (C) are shown.
Fig 2.
Treatment with ICG-001 decreases viability and induces growth arrest of MM cells.
(A) MM cells were treated with vehicle control or indicated concentrations of ICG-001 for 24 hours. Cell viability was determined by MTT assay. (B, C) MM H929 cells were treated with indicated concentrations of ICG-001 for 24 or 48 hours. Cells were then collected, fixed, and stained with propidium iodide. Cell cycle distribution (B) and the proportion of cells in subG1 phase (C) were determined. Experiment was performed twice. Results of one experiment performed in triplicates are shown. *P<0.05, **p<0.01, and +p<0.005, as compared to vehicle control group. (D, E) Apoptosis of MM cell lines (D) or primary CD138+ MM cells (E) treated with ICG-001 for 24 hours (10 μM for RPMI-8226 and 40 μM for other cell lines) was evaluated using Annexin V binding assay by flow cytometry. In D, experiment was repeated 3 times with similar results, with results shown from one representative experiment performed in triplicate. *P<0.000001. In E, combined results for primary MM cells obtained from 4 MM patients are shown.
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.
Fig 4.
Involvement of Wnt signaling in ICG-001-induced apoptosis in MM cells.
(A, B) MM MM.1S cells were transfected with siRNA against β-catenin or control non-targeting pool siRNA. (A) The efficiency of knockdown was evaluated by Western blotting with antibody against β-catenin. Equal loading was confirmed by re-probing the membrane with antibody against β-actin. (B) Cells were then either left untreated (control) or were treated with indicated concentration of doxorubicin for 24 hours. Apoptosis of MM cells was detected by Annexin V/DAPI staining by flow cytometry. Three experiments were performed with similar results. Results of 1 representative experiment are shown. (C, D) MM.1S cells were transfected with pcDNA/Myc TCF4 or control empty pcDNA3 plasmid followed by selection of stably transfected clones with G418. (C) Overexpression of TCF4 was evaluated by Western blotting using anti-Myc-Tag antibody. Equal loading was confirmed by re-probing the membrane with antibody against β-actin. (D) MM.1S cells transfected with pcDNA/Myc TCF4 or empty pcDNA3 vectors were treated with ICG-001 (10 μM) for 24 hours followed by detection of apoptosis by Annexin V binding assay. Combined results of 2 independent experiments are shown.
Fig 5.
Treatment with ICG-001 overcomes BMS-mediated MM cell chemoresistance.
(A) MM cells were cultured with or without BMS for 24 hrs. MM cells were then collected and the expression of β-catenin was determined by Western blotting (left panel). BMS cultured alone or with MM cells (indicated in the parenthesis) for 24 hrs were collected and subjected to Western blotting with antibody against β-catenin (right panel). H929 MM cells were used as a positive control for β-catenin expression. (B) MM cells cultured on the monolayer of BMS or kept in suspension (without BMS) were treated with ICG-001 or vehicle control for 24 hours. Apoptosis of MM cells was evaluated by Annexin V binding assay. Combined data from 4 independent experiments performed with BMS derived from BM of 4 patients with MM are shown. (C) MM H929 or U266 cells were cultured on the top of BMS or kept in suspension (without BMS) with or without 15 μM ICG-001 overnight followed by 24-hour treatment with doxorubicin or melphalan. Apoptosis of MM cells was evaluated using Annexin V binding assay. Shown are values of specific drug-induced apoptosis calculated by subtracting background values (cells treated with vehicle control) from values of drug-induced apoptosis. Combined results from 4 independent experiments performed with BMS derived from BM of 4 patients with MM are shown. *P<0.05, **p<0.005, and ***p<0.001 between ICG-001+chemotherapy combination group and both chemotherapy and ICG-001groups. #p<0.001 between drug combination and doxorubicin group and p<0.05 between drug combination and ICG-001 group.
Fig 6.
In vivo effect of ICG-001 on MM tumor burden.
(A) MM tumors were established in SCID-beige mice by subcutaneous inoculation of RPMI-8226 cells. In ~ 3 weeks, mice were split into 2 groups (4 mice/group) and treated with either vehicle control (PBS) or 100 mg/kg ICG-001 twice a day. Tumor growth was monitored by measuring tumor size twice a week. Two-way ANOVA was used to analyze the difference between the 2 groups. (B, C) MM tumors were established in NSG mice by intravenous inoculation of RPMI-8226-dsRed2 cells. In 2 weeks mice were randomly assigned to 2 groups and treated with either vehicle control (vc; n = 3) or ICG-001 (n = 4). (B) Tumor burden was evaluated by measuring dsRed2 protein fluorescence using IVIS 200 In Vivo Imaging System 3 weeks after start of the treatment. (C) Onset of hunched posture and hind leg paralysis has been evaluated and compared between groups.