Antihelminth compound niclosamide downregulates Wnt signaling and elicits antitumor responses in tumors with activating APC mutations

Abstract

Wnt/β-catenin pathway activation caused by adenomatous polyposis coli (APC) mutations occurs in approximately 80% of sporadic colorectal cancers (CRC). The antihelminth compound niclosamide downregulates components of the Wnt pathway, specifically Dishevelled-2 (Dvl2) expression, resulting in diminished downstream β-catenin signaling. In this study, we determined whether niclosamide could inhibit the Wnt/β-catenin pathway in human CRCs and whether its inhibition might elicit antitumor effects in the presence of APC mutations. We found that niclosamide inhibited Wnt/β-catenin pathway activation, downregulated Dvl2, decreased downstream β-catenin signaling, and exerted antiproliferative effects in human colon cancer cell lines and CRC cells isolated by surgical resection of metastatic disease, regardless of mutations in APC. In contrast, inhibition of NF-κB or mTOR did not exert similar antiproliferative effects in these CRC model systems. In mice implanted with human CRC xenografts, orally administered niclosamide was well tolerated, achieved plasma and tumor levels associated with biologic activity, and led to tumor control. Our findings support clinical explorations to reposition niclosamide for the treatment of CRC.

Figures

Figure 1. Niclosamide inhibits the proliferation of colorectal cancer cell Lines and has minimal toxicity on normal fibroblasts, PBMCs, or immortalized mammary epithelial cells

(A) Colorectal cancer cell lines, HT29, HCT116 and CaCO2, or (B) CRC explant cells growing in vitro (CRC039, CRC057, and CRC119) were cultured in 96-well flat-bottomed plates, and treated with several doses (0.4, 2, 10 μM) of niclosamide for 3 days. Oxaliplatin (10 μM) was used as a positive control. MTT assay was performed and the optical density (OD) at 562 nm was measured after cell lysis with DMSO. (C) HT29, HCT116, and CaCO2 cells were incubated with niclosamide (0.4, 2, 10 μM) for 24, 48, 72, and 96 h. Oxaliplatin (10, 20 μM) was used as a positive control. Percentages of proliferations (each OD 562nm value devided by OD 562nm value of the control (medium alone) of the same time point) are shown. (D) HCT116 and CRC119 tumor cells, fibroblasts, MCF-10A cells or PBMCs derived from normal donors, were incubated for 3 days with niclosamide at concentrations ranging from 0.2 μM to 20 μM. All cells were harvested, and labeled with annexin V. The percentage increase in annexin V-positive population (compared with untreated control) is shown.

Figure 2. Niclosamide inhibits Wnt/β-catenin signaling and downregulates dishevelled 2 and β-catenin expression by colorectal cancer cells in vitro

(A) CRC cell lines (CaCO2, HCT116) were transiently co-transfected with TOPflash or FOPflash plasmid DNA with pRL-TK Renilla Luciferase plasmid DNA. Cells were treated with different concentrations of niclosamide (0, 1, 5, 10 μM) for 24 h. Activity of Wnt/β-catenin signaling pathway was quantified by measuring relative firefly luciferase activity units (RLUs) normalized to Renilla luciferase. (B) CRC explants and colorectal cancer cell lines were treated with different concentrations (0, 1, 5, 10 μM) of niclosamide overnight (18 h). After washing cells with PBS, cell lysates were made with hypotonic lysis buffer. Cytosolic fractions of lysates were isolated and analyzed by Western Blot with anti-Dishevelled 2 (clone 10B5), anti-β-catenin (clone 7D11) and anti-β-actin (clone: C-11) monoclonal antibodies.

Figure 3. The mTOR and NF-κB pathway were not targets of niclosamide

(A) Colorectal cancer cell lines (HCT116, HT29, CaCO2) were treated with niclosamide (0-10 μM), everolimus (0-9 μM), or PS-1145 (0-50 μM) for 72 h, and cytotoxicity was analyzed in MTT assays. The relative ratio (percentages) of OD 562 nm values were calculated for each reagent by setting the OD 562 nm value at concentration 0 μM as 100%. (B, C) To examine if the NF-κB pathway could be perturbed by niclosamide, cancer cells were treated with niclosamide (1 μM) or PS-1145 (10 μM) for 24 h, and further incubated with TNF-α (10 ng/mL) for 1 h. Whole cell lysates were made and analyzed with anti-phospho-NF-κB p65 and anti-p65 antibodies by Western Blot (B). Cells were fixed, permeabilized and stained with FITC-conjugated anti-NF-κB p65 antibody (green) for 1 h. Nuclei were stained with DAPI (blue). Staining of HCT116 cells is shown. Unclear morphology of nuclei (blue) indicates nuclear translocation of p65 protein (C). Scale bars: 30 μm. (D) To determine whether niclosamide-induced cytotoxicity of colorectal cancers could be prevented by blocking the NF-κB pathway, cancer cells were treated with niclosamide (0-10 μM) in the presence/absence of PS-1145 (10, 20 μM) for 72 h and an MTT assay was performed.

Figure 4. Combination effect of niclosamide and oxaliplatin on colorectal cancer cell lines and explants

(A) Colorectal cancer cell line (CaCO2) and (B) CRC explant cells (CRC020) were cultured in 96 well plates at 5,000 cells or 10,000 cells per well, and treated with various combinations of niclosamide and oxaliplatin (CaCO2: niclosamide 0-2.0 μM, oxaliplatin 0-2.0 μM, CRC020: niclosamide 0-5.0 μM, oxaliplatin 0-10.0 μM). An MTT assay was performed after 72 h incubation. After cell lysis with DMSO, the optical density (OD) at 562 nm was measured.

Figure 5. Phamacokinetic analysis of niclosamide following oral administration in NOD/SCID mice

(A) NOD/SCID mice received oral administration of niclosamide (200 mg/kg of body weight). Blood samples were obtained at predose and at 0.25, 0.5, 0.75, 1, 1.5, 4, 8, 12, 24 h after drug administration. Quantification of niclosamide in mouse plasma was performed by LC/MS/MS method (19) and reported as ng/ml. (B) NOD/SCID mice were inoculated with HCT116 tumor cells (5 × 106 cells), and on day 4, oral gavage of niclosamide (200 mg/kg body weight) or control solvent was initiated. After 3 weeks of treatment, mice were sacrificed 24 h after the last oral administration, and blood and tumor tissue were collected simultaneously. Tumor tissue was cryo-crushed in liquid nitrogen and homogenated with three volumes of deionized water. Quantification of niclosamide in mouse plasma and tumor tissue was performed by LC/MS/MS. Circle: niclosamide-treated (n=5), Square: control-treated (n=5).

Figure 6. Niclosamide inhibits the growth of colorectal cancers in NOD/SCID mice and downregulates dishevelled 2 and β-catenin expression

(A) HCT116 colon cancer cells were harvested from flasks with 0.05% Trypsin/EDTA, resuspended with Hanks buffered solution at 5 × 106 cells/100 μL concentration. CRC explants (CRC039) cultured in vitro were harvested with the same procedure, and mixed with equal volume of Matrigel to make 1 × 106 cells/100 μL concentration. The cell suspension (100 μL) was inoculated into the flank of NOD/SCID mice 4 days before the start of treatment. Niclosamide (10, 100, 200 mg/kg body weight) was administered by gavage for 6 days /week for 2 (HCT116) or 3 weeks (CRC039). Tumor size was measured 3 times a week until mice were euthanized. * p < 0.05, ** p < 0.01. (B) HCT116 and CRC039 tumors grown in the flank of NOD/SCID mice were treated with/without niclosamide (200 mg/kg body weight) for 2 or 3 weeks. β-catenin and Dvl2 immunohistochemistry was performed for tumor sections as described in Materials and Methods. Scale bars: 100 μm. Inlets show higher magnification (Scale bars: 20 μm).