Article Figures & Data
Figures
- Figure 1.
Characterization of NAAE cells. A, 2LMP and SUM159 cells were fluorescence-activated cell sorted (FACS) for ALDH+ cells and analyzed for ALDH expression at 0, 6, 12, 24, 48, and 72 hours after sorting (solid line) and compared with unsorted cells in ultralow attachment plates with serum-free media (dotted line). HCC1143 cells were FACS sorted for ALDH+ cells and analyzed for ALDH expression at 0, 6, 12, and 24 hours. Unsorted cells harvested at the 12-hour time point are called NAAE cells. B, 20,000 2LMP NAAE cells (circle line) and 20,000 2LMP cells cultured in identical nonadherent (NA) conditions for 72 hours (square line) were injected into MFP of NOD/SCID mice and tumor growth was measured over time (difference at 48 days, P = 0.01). C, adherent and NAAE 2LMP cells were analyzed by Western blot for expression of Wnt/β-catenin signaling proteins.
- Figure 2.
Niclosamide effect on Wnt/β-catenin and STAT3 signaling. A, activity of TCF/LEF reporter in the TOPflash assay was evaluated in 2LMP, HCC1187, SUM159, and HCC1143 NAAE cells treated with 0.25 μmol/L niclosamide for 24 hours. The experiment was performed in triplicate. Bars, means ± SE. Niclosamide treatment (solid black bars) was compared with untreated control cells (striped bars; *, P < 0.01). B, Western blot analysis of adherent and NAAE 2LMP cell lines after 24-hour treatment with niclosamide (0, 0.5, 0.25, and 0.125 μmol/L).
- Figure 3.
The effect of TRA-8, niclosamide, and the combination on tumorsphere formation and cytotoxicity in NAAE cells. A, NAAE cells were cultured with media alone, pretreated with niclosamide (0.25–1.0 μmol/L) for 24 hours, followed by TRA-8 (1 ng/mL 2LMP, 0.5 ng/mL SUM159, 25 ng/mL HCC1143, and 5 ng/mL HCC1187) for 24 hours, for a total of 48 hours treatment. Tumorsphere formation was determined in four replicates and three separate experiments. (P < 0.05 treatment vs. control; P < 0.05 combination vs. either agent alone). Additive drug interaction against tumorsphere inhibition was observed for all four cell lines (P = 0.03). B, cell viability of drug combination was also tested on 2LMP, SUM159, HCC1143, and HCC1187 NAAE cells. 2LMP and SUM159 cells were pretreated for 24 hours with (0.25–1.0 μmol/L) niclosamide, followed by TRA-8 (1 ng/mL 2LMP and 0.5 ng/mL SUM159 for 24 hours, for a total of 48 hours). HCC1143 and HCC1187 were treated concurrently with (0.25 μmol/L) niclosamide, and TRA-8 (25 ng/mL HCC1143 and 5 ng/mL HCC1187), for a total of 48 hours treatment.
- Figure 4.
Niclosamide in combination with TRA-8 inhibits Wnt/β-catenin signaling. A, activity of TCF/LEF plasmid reporter in the TOPflash assay was evaluated in 2LMP, SUM159, HCC1143, and HCC1187 adherent cells. All cell lines were treated with 0.25 μmol/L niclosamide for 48 hours and 0.25 ng/mL TRA-8 for 24 hours. The experiment was performed in triplicate. Bars, mean ± SE. Single or combination treatment compared with control (*, P < 0.05), combination treatment versus either single agent (#, P < 0.05). B, Western blot analysis of β-catenin degradation was performed after 2-hour treatment with TRA-8 on both adherent and NAAE cell populations of the 2LMP and HCC1187 cell lines (top blot, low exposure; bottom blot, high exposure). C, 2LMP parental, LRP6 knockdown, and shRNA control cells were treated with TRA-8 for 48 hours. The cytotoxicity was measured by ATPlite. The experiment was performed in triplicate. LRP6 knockdown compared with control shRNA (P = 0.01).
- Figure 5.
Effect of niclosamide and TRA-8 in vivo on 2LMP orthotopic tumor growth. Tumors were established in athymic nude mice by MFP implantation of 2 × 106 2LMP cells. The therapy started when tumors reached a size of 16 mm2. Niclosamide (30 mg/kg) was given intraperitoneally 5 days a week, TRA-8 (200 μg) was given intraperitoneally 2× weekly for 3 weeks. Tumor size was measured with calipers twice a week. Each point in the curve represents the mean ± SE (n = 5). Single-agent niclosamide or TRA-8 versus control (*, P < 0.05), combination treatment versus control (**, P < 0.01), combination treatment versus TRA-8 or niclosamide (#, P < 0.05).
- Figure 6.
Metastatic pleural effusion patient sample sensitivity to niclosamide alone and in combination with TRA-8. A, sensitivity of patient samples to 48-hour niclosamide-mediated cytotoxicity (1, 2, 4, and 8 μmol/L) compared with untreated controls. Individual experiments were assayed in quadruplicate and bars represent mean ± SE (P < 0.01). B, activity of TCF/LEF viral reporter in the TOPflash assay was evaluated for patient samples UAB03 and UAB05. Both samples were treated with 4 μmol/L niclosamide for 24 hours in triplicate. Bars, mean ± SE. Niclosamide compared with control (*, P < 0.05). C, Western blot analysis of patient sample UAB03 after 24-hour treatment with niclosamide (4 μmol/L). D, cytotoxicity of patient samples UAB03 and UAB05 pretreated with niclosamide (0.5, 1, 2, 4, and 8 μmol/L) for 24 hours followed by 24 hours with TRA-8 (50 or 500 ng/mL). Cell viability was analyzed using ATPlite assay.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure 1 - PDF file - 128KB, Supplementary Figure S1. Sensitivity of MF10A mammary epithelial cells to niclosamide and TRA-8 mediated cytotoxicity. (A) Attached MCF10A cell line was treated for 48 hours with niclosamide (0.08, 0.1, 0.25 and 0.5 microM) and analyzed for cell viability using ATPlite assay.
- Supplementary Figure 2 - PDF file - 209KB, Supplementary Figure S2. Niclosamide effect on Wnt/beta-catenin signaling. Activity of TCF/LEF plasmid reporter in the TOPflash assay was evaluated on adherent 2LMP, SUM159, HCC143 and HCC1187 cells. Cell lines were treated with 1 microM niclosamide for 24 hours (SUM159, HCC1143 and HCC1187) or with 0.25 microM niclosamide (2LMP). Each cell line was also treated with Wnt3A ligand (5 ng/mL) alone and in combination with niclosamide. The experiment was performed in triplicate. The bars represent means plus-minus SE. Treatment was compared to control or Wnt3A. Niclosamide vs. control (*P < 0.05), niclosamide vs. control (**P < 0.01), niclosamide vs. Wnt3A (#P < 0.05), niclosamide + Wnt3A vs. Wnt3A (##P < 0.01).
- Supplementary Figure 3 - PDF file - 99KB, Supplementary Figure S3. ATPlite viability of cells treated with niclosamide. 2LMP, SUM159, HCC1143 and HCC1187 adherent (A) or NAAE (B) cells were treated with niclosamide for 24 hours and analyzed for viability using ATPlite assay. Individual experiments were assayed in triplicate and values represent the mean plus-minus SE.
- Supplementary Figure 4 - PDF file - 96KB, Supplementary Figure S4. Wnt/beta-catenin signaling proteins in LRP6 KD cells. Western blot for expression of Wnt/beta-catenin signaling proteins in adherent 2LMP shRNA LRP6 knockdown cells compared to shRNA control cells.
- Supplementary Figure 5 - PDF file - 59KB, Supplementary Figure S5. In vivo toxicity study. Athymic nude mice were treated with niclosamide (20 mg/kg), TRA-8 (200 ng) or the combination for two weeks. Mice were weighed twice a week for two weeks.
- Supplementary Table 1 - PDF file - 89KB, Supplementary Table S1. Sensitivity of BLBC cell lines to niclosamide mediated cytotoxicity. SUM159, HCC1187, HCC1143 and 2LMP BLBC NAAE and adherent cell lines were treated with niclosamide for 48 hours and analyzed for viability using the ATPlite assay. Individual experiments were assayed in quadruplicate and values represent the mean and SD of three independent experiments. SUM159 P < 0.05, HCC1187 P < 0.05, HCC1143 P < 0.0005, 2LMP P > 0.05.
- Supplementary Table 2 - PDF file - 72KB, Supplementary Table S2. Immunohistochemistry of mouse tumors treated for two weeks with vehicle control, niclosamide (20 mg/kg), TRA-8 (200 micro-g) or the combination of both agents. Tumors were stained and analyzed (H-score) for p-STAT3, cytosolic beta-catenin, nuclear beta-catenin and cleaved caspase 3. The values represent an average of three tumors per group (plus-minus SE).
Volume 13, Issue 4
