Figure 1.
The effect of niclosamide on breast cancer cells viability.
(A) Proliferation of MDA-MB-231, MCF-7, MDA-MB-468 cells treated with various concentrations (0–10 µM) of niclosamide for 72 hours, respectively. Cell viability was detected by MTT assay. The data are expressed as the means ± SD from three independent experiments. (B) MTT assays showed niclosamide inhibited 4T1 breast cancer cells proliferation concentration- and time-dependently. Values represented means ± SD from three experiments (*p<0.05; **p<0.01; ***p<0.001). (C) The effects of niclosamide (a-f:0–1.25 µM) on colony formation in 4T1 cells 12 days, the statistic results of colony formation assays presented as surviving colonies. Data are expressed as means ± SD from three experiments (*p<0.05; **p<0.01). (D) The fluorescence microscopic appearance of Hoechst 33342 staining nuclei of 4T1 cells with various concentration niclosamide for 24 h (40×). Data are the representative from three parallel experiments.
Figure 2.
Niclosamid induces 4T1 breast cancer cells apoptosis.
(A) 4T1 cells were treated with niclosamide at indicated doses for 24 hours, and the level of apoptosis was evaluated using the Annexin V/PI dual-labeling technique, as determined by FCM. Data shown are representative of three independent experiments. (B) Statistic results of apoptosis assays, 4T1 cells positive for both Annexin V and PI were considered apoptotic. Data are expressed as means ± SD from three independent experiments (*p<0.05; **p<0.01). (C) Western blot analyses of 4T1 cells treated (24 h) with different concentrations of niclosamide to evaluate protein expression of Bcl-2, Mcl-1, Cleaved caspase-3, Survivin and β-actin was employed as a standard.
Figure 3.
Niclosamide inhibits breast cancer cell 4T1 and MDA-MB-231 migration and invasion and inhibits FAK-involved pathway.
(A) A total of 5×104 4T1 cells were seeded in the top chamber of transwell with serum-free medium, and treated with different concentration of niclosamide. After 20 hours, migrated cells were stained, photographed (10×) and quantified. (B) A total of 5×104 4T1 cells were treated with various concentration of niclosamide and allowed to invade through Matrigel and Transwell membrance, Invaded cell number was stained, photographed (20×) and counted. (C) Niclosamide inhibited MDA-MB-231 migation and invasion. The number of migrated cells and invaded cells was counted, respectively. Data represent means ±SD. (n = 3, in triplicate; *p<0.05; **p<0.01; ***p<0.001). (D) 4T1 cells were treated with different concentration of niclosamide (0–5 µM). After 24 hours, cell lysates were blotted with spectfic antibodies (anti-phospho-STAT3, anti-phospho-FAK, anti-phospho-Src, STAT3, FAK and Src), β-actin was the loading control.
Figure 4.
Effect of niclosamide treatment on primary tumor growth and pulmonary metastasis.
(A) 4T1 tumor-bearing female BALB/c mice were treated as described with vehicle, niclosamide at 10 and 20 mg/kg, the mean tumor volumes ± SD of six mice per every group. (B) After 28 days of tumor cell inoculation, the body weight of the niclosamide treatment and vehicle groups were statisticed, and there were no significant difference among the groups. (C) Lung metastatic nodules were visualized to show the inhibitory effect of niclosamide on 4T1 tumor 21 days after treatment. Arrow indicated metastatic nodules (up), The H&E staining of lungs from each group (10×). (D) The mean lung metastasis nodules of each group, the treatment with niclosamide at 20 mg/kg resulted in significant inhibition of lung metastasis versus vehicle control. Bars showed means ± SD (n = 6; *P<0.05).
Figure 5.
Effect of niclosamide treatment on tumor-associated Gr1+ CD11b+ MDSCs.
Gr1+ CD11b+ cells were gated and analyzed by FCM for the expression of MDSCs. MDSCs isolated from the tumor tissue of 4T1 tumor-bearing mice were treated with vehicle (A); or treated with niclosamide at 10 mg/kg (B); or treated with niclosamide at 20 mg/kg (C). (D) Statistic results of each group. Treatment of niclosamide significantly reduces the number of MDSCs compared with vehicle group. Values represented means ± SD (n = 6; *p<0.05; **p<0.01).
Figure 6.
Niclosamide reduces tumor cell proliferation, induces tumor apoptosis and inhibits tumor angiogenesis in vivo.
(A) Tumor cell proliferation was evaluated on paraffin-embedded 4T1 tumor sections by Ki67 immunohistochemical staining (40×), 4T1 tumor tissues removed after 21 days treatment. The treatment with niclosamide resulted in markedly reduced proliferation versus vehicle group (n = 5; **p<0.01). (B) Apoptosis was measured on paraffin-embedded 4T1 tumor sections by Cleaved caspase-3 (CC3) immunohistochemical staining (40×). The apoptosis index was calculated by dividing the number of CC3-positive cells by the total number of cells. The treatment with niclosamide significantly increased apoptosis in a dose-dependent manner compared with vehicle group (n = 5; *p<0.05; **p<0.01). (C) Immunohistochemistry was performed to measure the expression of VEGF in tumor tissues isolated from vehicle and niclosamide-treated mice (40×). The treatment with niclosamide markedly reduced VEGF-positive cells versus vehicle group (n = 5; **p<0.01). (D) Niclosamide significantly inhibited tumor vessels in 4T1 tumor. Paraffin-embedded of 4T1 tumor sections were tested by immunohistochemical analysis with anti-CD31 antibody. Representative tumor vasculature from vehicle- and niclosamide-treated mice was shown (40×). The density of microvessel was calculated in each group (n = 5; *p<0.05; **p<0.01).
Figure 7.
Evaluation of side effects of niclosamide in mice.
There were no significant different among the vehicele-treated group and the niclosamide-treated groups in blood routine analysis of toxicity test, (A) Red blood cell. (B) White blood cell. (C) Platelet. (D) Niclosamide did not casue obvious pathologic abnormalities in normal tissues. H&E staining of paraffin-embedded sections of the heart, liver, spleen and kidney (20×).