Nothing Special   »   [go: up one dir, main page]
Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Jul 10.
Published in final edited form as: Cancer Lett. 2014 Apr 13;349(1):8–14. doi: 10.1016/j.canlet.2014.04.003

Multi-targeted therapy of cancer by niclosamide: a new application for an old drug

Yonghe Li 1,*, Pui Kai Li 2, Michael J Roberts 3, Rebecca Arend 4, Rajeev S Samant 5, Donald J Buchsbaum 6
PMCID: PMC4166407  NIHMSID: NIHMS585642  PMID: 24732808

Abstract

The rapid development of new anticancer drugs that are safe and effective is a common goal shared by basic scientists, clinicians and patients. The current review discusses one such agent, namely niclosamide, which has been used in the clinic for the treatment of intestinal parasite infections. Recent studies repeatedly identified niclosamide as a potential anticancer agent by various high-throughput screening campaigns. Niclosamide not only inhibits the Wnt/β-catenin, mTORC1, STAT3, NF-κB and Notch signaling pathways, but also targets mitochondria in cancer cells to induce cell cycle arrest, growth inhibition and apoptosis. A number of studies have established the anticancer activities of niclosamide in both in vitro and in vivo models. Moreover, the inhibitory effects of niclosamide on cancer stem cells provide further evidence for its consideration as a promising drug for cancer therapy. This article reviews various aspects of niclosamide as they relate to its efficacy against cancer and associated molecular mechanisms.

Keywords: niclosamide, FDA-approved drug, multi-targeted therapy, drug discovery, cancer stem cells

1. Introduction

Niclosamide (trade name Niclocide), a teniacide in the anthelmintic family which is especially effective against cestodes, has been approved for use in humans for nearly 50 years (Fig. 1) [1, 2]. Niclosamide inhibits oxidative phosphorylation and stimulates adenosine triphosphatase activity in the mitochondria of cestodes (eg. tapeworm), killing the scolex and proximal segments of the tapeworm both in vitro and in vivo [2]. Niclosamide is well tolerated in humans. The treatment of T. saginata (beef tapeworm), D. latum (fish tapeworm) and Dipylidium caninum (dog tapeworm) in adult is 2 g as a single oral dose. For the treatment of H. nana (dwarf tapeworm), the same oral dose is used for 7 days [2].

Fig. 1.

Fig. 1

Open in a new tab

The chemical structure of niclosamide.

Drug development, from the initial lead discovery to the final medication, is an expensive, lengthy and incremental process [3]. Finding new uses for old or failed drugs is much faster and more economical than inventing a new drug from scratch, as existing drugs have known pharmacokinetics and safety profiles and have often been approved for human use, therefore any newly identified use(s) can be rapidly evaluated in clinical trials [4]. In the last 5 years niclosamide has been identified as a potential anticancer agent by various high-throughput screening campaigns. This article reviews the current studies regarding various aspects of niclosamide as they relate to its potential new use in cancer therapy.

2. Niclosamide – a multiple pathway inhibitor for anti-cancer efficacy

Recently, several studies reported the inhibitory effects of niclosamide on multiple intracellular signaling pathways. The signaling molecules in these pathways are either over-expressed, constitutively active or mutated in many cancer cells, and thus render niclosamide as a potential anticancer agent. The effects of niclosamide on these pathways are described below.

2.1. The Wnt/β-catenin pathway

The Wnt/β-catenin signaling pathway regulates cancer progression, including tumor initiation, tumor growth, cell senescence, cell death, differentiation and metastasis [5-7]. In the absence of Wnt, β-catenin is sequestered in a complex that consists of the adenomatous polyposis coli (APC) tumor suppressor, axin, glycogen synthase kinase-3β (GSK3β), and casein kinase 1 (CK1). This complex formation induces the phosphorylation of β-catenin by CK1 and GSK3β, which results in the ubiquitination and subsequent degradation of β-catenin by the 26S proteasome. Conversely, when Wnt proteins form a ternary complex with the cell surface receptors, low-density lipoprotein receptor-related protein5/6 (LRP5/6) and Frizzled (Fzd), signaling from Wnt receptors proceeds through the proteins dishevelled (Dvl) and axin, leading to the inhibition of GSK3β and the stabilization of cytosolic β-catenin. The β-catenin then translocates into the nucleus where it interacts with T-cell factor/lymphoid enhancing factor (TCF/LEF) to induce the expression of specific target genes [5-7] (Fig. 2A).

Fig. 2.

Fig. 2

Open in a new tab

The intracellular signaling pathways inhibited by niclosamide in cancer cells. (A) Niclosamide blocks Wnt/β-catenin signaling by enhancing Wnt receptor Fzd1 internalization, promoting Wnt co-receptor LRP6 degradation, suppressing Wnt signaling regulator Dvl2 expression, and inhibiting β-catenin/TCF complex formation. (B) mTORC1 inhibition by niclosamide is associated with cytosolic acidification and lysosomal dysfunction. (C) Niclosamide inhibits STAT3 transcriptional activity by blocking its phosphorylation and nuclear translocation. (D) Niclosamide ablates TNF-induced NF-κB activation at TAK1 and IKK steps. (E) Niclosamide inhibits Notch signaling by suppressing the expression of cleaved activated Notch1 receptor.

Chen et al. performed a high-throughput screening of a library containing approximately 1200 FDA-approved drugs and drug-like molecules with a primary imaged-based green fluorescent protein (GFP) fluorescence assay that used Fzd1 endocytosis as the readout in human osteosarcoma U2OS cells, and identified niclosamide as a small molecule inhibitor of Wnt/β-catenin signaling [8]. Niclosamide promoted Wnt receptor Fzd1 endocytosis, downregulated Dvl2 protein, and inhibited Wnt3A-stimulated β-catenin stabilization and TCF/LEF reporter activity in U2OS cells [8]. It also decreased the cytosolic expression of endogenous Dvl2 and β-catenin in human colorectal cancer cell lines and in human colorectal cancer cells isolated by surgical resection of metastatic disease, regardless of mutations in APC [9]. Moreover, we recently demonstrated that niclosamide was able to inhibit Wnt/β-catenin signaling by promoting Wnt co-receptor LRP6 degradation, but had no effect on Dvl2 expression in prostate and breast cancer cells [10, 11], suggesting that the mechanism underlying niclosamide-mediated inhibition of Wnt/β-catenin signaling could be cell type-dependent (Fig. 2A). In addition, the inhibitory effects of niclosamide on Wnt/β-catenin signaling were also demonstrated in primary human glioblastoma cells [12].

More than 90% of colorectal cancers bear mutations that result in the activation of the Wnt/β-catenin pathway [13]. S100 calcium binding protein A4 (S100A4) is a target gene of the Wnt/β-catenin pathway in colorectal cancers [14]. Sack et al. performed a high-throughput screening of the LOPAC chemical library containing 1280 compounds with a cell-based luminescence assay that used S100A4 promoter-driven luciferase activity as the readout in human colorectal cancer HCT116 cells, and identified niclosamide as an inhibitor of S100A4 through reduction of S100A4 mRNA and protein expression in colorectal cancer cells [15]. It was proposed that niclosamide inhibits β-catenin/TCF complex formation and thereby interrupts target gene transcription, an alternative model of Wnt/β-catenin inhibition by niclosamide in colorectal cancer cells [15] (Fig. 2A).

2.2. The mTORC1 pathway

The mammalian target of rapamycin complex 1 (mTORC1) is a heterotrimeric protein kinase that consists of the mTOR catalytic subunit and two associated proteins, raptor (regulatory associated protein of mTOR) and mLST8 (mammalian lethal with sec-13). The mTORC1 activity is regulated by upstream signals from growth factors, amino acids, stresses and energy state, and its activation induces the phosphorylation of p70S6 kinase (p70S6K) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), leading to the enhanced translation of a subset of mRNAs that are critical for cell growth and metabolism [16, 17] (Fig. 2B). Activation of either the serine/threonine protein kinase Akt (also known as protein kinase B or PKB) or the extracellular signal-regulated kinase (ERK) pathway, or inhibition of the adenosine monophosphate-activated protein kinase (AMPK) pathway, leads to activated mTORC1signaling. As a downstream effector of Akt, mTORC1 has been described as the most essential effector in driving cell proliferation and susceptibility to oncogenic transformation. This leads to the targeting of mTORC1 as a therapeutic strategy in many types of cancer [16, 17].

Balgi et al. performed a high-throughput screening of a collection of >3,500 chemicals with a primary imaged-based enhanced GFP (EGFP) fluorescence assay that used EGFP-LC3 punctate staining as the readout in human breast cancer MCF-7 cells, and identified niclosamide as an inhibitor of mTORC1 signaling in cells maintained in nutrient-rich conditions [18]. Niclosamide did not inhibit mTORC2, which also contains mTOR as a catalytic subunit, suggesting that niclosamide does not inhibit mTOR catalytic activity but rather inhibits signaling to mTORC1 [18]. Tuberous sclerosis complex (TSC2), a negative regulator of mTORC1, was not required for the inhibition of mTORC1 signaling by niclosamide, as niclosamide was able to suppress mTORC1 signaling in TSC2-deficient cells where mTORC1 activity was elevated [18]. Further studies demonstrated that niclosamide does not impair PI3K/Akt signaling, nor does it interfere with mTORC1 assembly and mTORC1 kinase activity [19]. It had been proposed that increased cytosolic acidification is responsible for the mTORC1 inhibition, and that the structural features of niclosamide required for protonophoric activity are essential for the mTORC1 inhibition [19]. Lysosome is the degrading machine for autophagy, but has also been linked to mTORC1 activation through the Rag/RRAG GTPase pathway [20]. More recently, Li et al. proposed that mTORC1 inhibition by niclosamide is caused by lysosomal dysfunction [21]. Niclosamide inhibits the lysosomal degradative function likely by altering lysosomal permeability and the pH gradient [21], which is consistent with the finding that niclosamide can cause dispersion of protons from the lysosomes to the cytosol, leading to cytosolic acidification [19] (Fig. 2B).

2.3. The STAT3 pathway

Signal transducers and activators of transcription 3 (STAT3), a member of a family of six different transcription factors, is one of the down-stream signaling proteins for cytokine and growth factor receptors [22]. Engagement of cell-surface cytokine or growth factor receptors activates the janus kinase (JAK) family of protein kinases, which in turn phosphorylates STAT3 at tyrosine residue 705, leading to the dimerization of two STAT3 monomers through the SH2 domains of the proteins [23, 24]. The activated STAT3 dimers then translocate into the nucleus and activate the transcription of a panel of genes that control cell proliferation, apoptosis, angiogenesis and other cell functions [25, 26]. This JAK2/STAT3 signaling pathway is one of the three major modules that play an essential role in transmitting external signals from the surface membrane to target genes in the nucleus, controlling processes such as growth, differentiation, senescence and apoptosis [27, 28]. Importantly, STAT3 is the major intrinsic pathway for cancer inflammation owing to its frequent activation in malignant cells and key role in regulating many genes crucial for cancer inflammation in the tumor microenvironment [22].

Ren et al. demonstrated that niclosamide is a potent STAT3 inhibitor that suppresses STAT3 transcriptional activity by blocking its phosphorylation and nuclear translocation in prostate cancer DU145 cells [29] (Fig. 2C). The inhibitory effect of niclosamide on STAT3 signaling was subsequently reported in multiple myeloma cells [30]. STAT3 can mediate cancer cell survival by activating antiapoptotic genes such as Bcl-2 and Bcl-XL. Recently, it was reported that inhibition of STAT3 by niclosamide blocked the erlotinib activated STAT3/Bcl-2/Bcl-XL pathway and reversed erlotinib resistance of human head and neck cancer cells and non-small cell lung cancer cells [31, 32]. Furthermore, niclosamide not only blocked the ionizing radiation-induced activation of the STAT3/Bcl-2/Bcl-XL pathway in both radiosensitive and radioresistant human lung cancer cells, but also reversed the radioresistance and restored the sensitivity of radioresistant cells to ionizing radiation [33].

2.4. The NF-κB pathway

Nuclear factor-kappaB (NF-κB) is an important transcription factor associated with cancer, and has been implicated in many hallmarks of cancer development, including growth factor independent proliferation, inhibition of apoptosis, tumor angiogenesis, limitless replicative potential and tissue invasion and metastasis [34, 35]. NF-κB is activated by several agents, including cytokines, oxidant free radicals, inhaled particles, ultraviolet irradiation, and bacterial or viral products. In response to these stimuli, IκB kinase (IKK) is activated and can phosphorylate the inhibitory IκBα subunit of the NF-κB·IκBα complex in the cytoplasm. This phosphorylation marks IκBα for degradation by the proteasome and releases NF-κB from the inhibitory complex, and the freed NF-κB proteins are then transported into the nucleus where they bind to their target sequences and activate gene transcription [36, 37].

Jin et al. demonstrated that niclosamide blocked tumor necrosis factor α (TNF-α)-induced IκBα phosphorylation, translocation of NF-κB p65 subunit, and expression of NF-κB-regulated genes in acute myelogenous leukemia cells [38]. The inhibitory effects of niclosamide on NF-κB signaling were also reported in multiple myeloma cells [30] and primary human glioblastoma cells [12]. Transforming growth factor-β (TGF-β) activated kinase1 (TAK1) mediates IKK activation in the TNF-α signaling pathway [39, 40]. It was demonstrated that niclosamide inhibited the steps of TAK1→IKK and IKK→IκBα of the NF-κB activation [38] (Fig. 2D). In addition, niclosamide inhibited the DNA binding of NF-κB to the promoter of its target genes [38].

2.5. The Notch pathway

The Notch signaling pathway represents a critical component in the molecular circuits that control cell fate during development. Under physiological conditions, the Notch ligand binds to its receptor to initiate Notch signaling by releasing the intracellular domain of the Notch receptor (Notch-IC) through a cascade of proteolytic cleavages by both α-secretase and γ-secretase. The released intracellular Notch-IC then translocates into the nucleus where it modulates gene expression primarily by binding to a ubiquitous transcription factor, CBF1, and suppressor of hairless, Lag-1 (CSL). In cancers, molecular genetic alterations cause increased levels of intracellular Notch-IC and subsequent constitutive activation of the Notch pathway. Aberrant activation of this pathway contributes to tumorigenesis, and Notch inhibitory agents, such as γ-secretase inhibitors, are being investigated as candidate cancer therapeutic agents [41-43].

Wang et al. developed a cell based luciferase reporter gene assay to measure the endogenous CBF1-dependent Notch signaling in human erythroleukemia K562 cells, and identified niclosamide as a potent inhibitor of Notch signaling [44]. Niclosamide inhibited the luciferase activity of CBF1-dependent reporter gene, and suppressed the expression of cleaved activated Notch1 receptor and Notch target Hes-1 in K562 cells [44] (Fig. 2E). In addition, the inhibitory effects of niclosamide on Notch signaling were also reported in primary human glioblastoma cells [12].

3. Niclosamide targeting of mitochondria

Mitochondria are vital for cellular bioenergetics and play a central role in determining the point-of-no-return of the apoptotic process. It has been proposed that targeting mitochondria is an efficient strategy for cancer chemotherapy [45]. Khanim et al. screened a panel of 100 off-patent licensed oral drugs for anti-myeloma activity, and identified niclosamide as a killer of multiple myeloma cell lines and primary multiple myeloma cells [30]. Interestingly, niclosamide anti-multiple myeloma activity could be mainly mediated through the mitochondria with rapid loss of mitochondrial membrane potential, uncoupling of oxidative phosphorylation and production of mitochondrial superoxide [30]. Moreover, Park et al. screened the LOPAC chemical library and identified niclosamide as a potent inducer of mitochondrial fission, which induced mitochondria fragmentation and promoted both apoptotic and autophagic cell death [46]. In addition, it was reported that inactivation of NF-κB by niclosamide caused mitochondrial damage and the generation of reactive oxygen species (ROS), leading to apoptosis of acute myelogenous leukemia cells [38]. Finally, Yo et al. performed a genome-wide gene expression array and demonstrated that niclosamide was able to disrupt multiple metabolic pathways affecting biogenetics, biogenesis, and redox regulation in ovarian-cancer-initiating cells. These disruptions presumably lead to the activation of the intrinsic mitochondrial apoptosis pathway, loss of tumor stemness, and growth inhibition [47].

4. Anti-cancer activity of niclosamide: in vitro studies

Anti-cancer activity of niclosamide has been demonstrated in human breast cancer [10, 11, 18, 48, 49], prostate cancer [10, 29], colon cancer [9, 15], ovarian cancer [47], multiple myeloma [30], acute myelogenous leukemia [38], glioblastoma [12], head and neck cancer [21] and lung cancer cells [31]. As summarized in the earlier sections, niclosamide is able to block the multiple signaling pathways that govern cancer initiation and progression, thus it is not surprising that niclosamide has a potent activity to induce cancer cell cycle arrest, growth inhibition and apoptotic death across multiple cancer types [50].

The NCI-60 human tumor cell line anticancer drug screen was developed in the late 1980s as an in vitro drug-discovery tool, and was rapidly recognized as a rich source of information about the mechanisms of growth inhibition and tumor-cell kill [51]. To further characterize the anticancer activities of niclosamide, we performed a 60 human tumor cell line anticancer drug screen with niclosamide. We found that niclosamide inhibited cell proliferation of all tested cancer cell lines, and that the IC50 values for most cell lines were less than 1 μM (Table 1). These results indicate that niclosamide has a significant broad-spectrum and potent in vitro anticancer activity.

Table 1.

Effects of niclosamide on growth of 60 human tumor cell lines

IC50 (μM) IC50 (μM) IC50 (μM)
Leukemia Renal Ovarian

  • CCRF-CEM

  • HL-60 (TB)

  • K562

  • MOLT-4

  • RPMI-8226

  • SR

  • 0.51

  • 0.14

  • 0.18

  • 0.22

  • 0.05

  • 0.13

  • 786-0

  • A498

  • ACHN

  • CAKI-1

  • RXF 393

  • SN12C

  • TK-10

  • UO-31

  • 1.01

  • 0.52

  • 0.54

  • 1.11

  • 0.86

  • 0.58

  • 0.51

  • 0.55

  • IGR-OV1

  • OVCAR-3

  • OVCAR-4

  • OVCAR-5

  • OVCAR-8

  • NCI/ADR-RES

  • SK-OV-3

  • 1.01

  • 0.35

  • 0.61

  • 0.76

  • 0.81

  • 0.38

  • 0.72
Non-Small Cell Lung Colon Melanoma

  • A549/ATCC

  • EKVX

  • HOP-62

  • HOP-92

  • NCI-H226

  • NCI-23

  • NCI-H322M

  • NCI-H460

  • NCI-H522

  • 0.96

  • 0.50

  • 0.41

  • 0.61

  • 0.73

  • 0.22

  • 0.61

  • 0.16

  • 0.26

  • COLO 205

  • HCC-2998

  • HCT-116

  • HCT-15

  • HT29

  • KM12

  • SW-620

  • 0.98

  • 1.25

  • 0.374

  • 1.06

  • 4.17

  • 1.07

  • 0.54

  • LOX IMVI

  • MALME-3M

  • M14

  • MDA-MB-435

  • SK-MEL-2

  • SK-MEL-28

  • SK-MEL-5

  • UACC-257

  • UACC-62

  • 0.60

  • 0.74

  • 0.74

  • 0.52

  • 1.32

  • 0.95

  • 0.40

  • 1.07

  • 0.22
Breast CNS Prostate

  • MCF-7

  • MDA-MB-231

  • MDA-MB-468

  • HS 578T

  • BT-549

  • T-47D

  • 0.38

  • 0.86

  • 0.24

  • 0.43

  • 0.86

  • 0.43

  • SF-268

  • SF-295

  • SF-539

  • SNB-19

  • SNB-75

  • U251

  • 0.53

  • 1.05

  • 2.25

  • 0.29

  • 1.14

  • 0.34

  • PC-3

  • DU-145

  • 0.62

  • 0.60
Open in a new tab

Cells were seeded into 96-well tissue culture treated microtiter plates at a density of 5,000-20,000 cells/well (depending on cell line) in a total volume of 50 μl. After overnight incubation, the cells were treated with niclosamide for 72 h in triplicate by adding 50 μl of 2X stock solutions to appropriate wells already containing 50 μl of cells and medium to expose cells to the final concentrations of niclosamide required. Cell viability was measured by the Cell Titer Glo Assay (Promega).

Niclosamide is able to potentiate the cytotoxicity towards tumor cells of different anticancer agents in several in vitro cancer models. It enhanced the anti-proliferative activity of oxaliplatin, a commonly used drug for colorectal cancer, in human colorectal cancer Caco2 cells and colorectal cancer explant cells [9]. Furthermore, inhibition of STAT3 by niclosamide blocked erlotinib-induced STAT3 phosphorylation and sensitized human head and neck cancer cells and non-small cell lung cancer cells to erlotinib [31, 32]. In addition, combinatorial drug testing established that a heterozygous deletion of the NFKBIA locus in glioblastoma samples could serve as a genomic biomarker for predicting synergistic activity of niclosamide with temozolomide, the current standard in glioblastoma therapy [12]. Finally, synergistic effects of niclosamide in combination with Ara-C, VP-16, and DNR, the frontline chemotherapeutic agents for acute myelogenous leukemia, have been reported in acute myelogenous leukemia stem cells [38].

5. Anti-cancer activity of niclosamide: in vivo studies

The efficacy of niclosamide has been shown against tumor growth and metastases in several xenograft models. Jin et al. demonstrated for the first time that niclosamide has in vivo anti-cancer activities [38]. As niclosamide has limited solubility in water, Jin et al. synthesized a niclosamide analog – phosphate of niclosamide (p-niclosamide), and found p-niclosamide showed significant inhibition of xenograft tumor growth of acute myeloid leukemia HL-60 cells by suppressing the NF-κB pathway [38]. Subsequently, Osada et al. reported that niclosamide inhibited the growth of colorectal cancer cells in NOD/SCID mice by down-regulating Dvl2 and β-catenin expression [9]. Moreover, niclosamide in combination with erlotinib potently repressed erlotinib-resistant head and neck cancer xenografts and lung cancer xenografts by inhibiting STAT3 signaling [31, 32]. In addition, niclosamide alone or in combination with radiation overcame radioresistance in lung cancer xenografts [33]. Niclosamide was also able to significantly diminish the malignant potential of primary human glioblastoma cells in vivo by suppressing intracellular Wnt/β-catenin-, NOTCH-, mTORC1-, and NF-kB signaling cascades [12]. TRA-8 is an agonistic monoclonal antibody (mAb) to TRAIL death receptor 5 (DR5) [52, 53]. We recently found that niclosamide in combination with TRA-8 suppressed the growth of basal-like breast cancer 2LMP orthotopic tumor xenografts by inhibiting Wnt/β-catenin and STAT3 signaling [11]. Importantly, niclosamide was able to inhibit the tumor formation of ovarian-cancer-initiating cells derived from cancer cell lines in vivo [47] and tumor growth of breast cancer stem-like cell subpopulations in vivo [49]. Finally, it was found that niclosamide significantly reduced liver metastasis formation in mice bearing xenografted intrasplenic colon tumors by suppressing the expression of the Wnt/β-catenin signaling target S100A4 [15], as S100A4 plays a critical role in metastasis formation in colon cancer [14, 54-56]. Overall, these studies reveal the in vivo anticancer activities of niclosamide, and provide a rationale for its use in clinical trials.

6. Niclosamide as a drug for targeting CSCs

Current cancer treatments such as chemotherapy, targeted therapy and radiotherapy are successful at destroying bulk cancer cells, but fail to eliminate cancer stem cells (CSCs). CSCs are characterized by tumorigenic properties and the ability to self-renew, form differentiated progeny, and develop resistance to therapy. The inability to eradicate CSCs is thought to be the reason for cancer relapse and chemo-resistance, the major obstacles in current cancer therapy [57-59]. The Wnt/β-catenin, Notch, and Hedgehog pathways are three major pathways which have been implicated in CSC tumorigenic properties and the ability to self-renew, form differentiated progeny, and develop resistance to therapy [43, 60-62]. However, many other pathways such as the mTORC1, STAT3 and NF-κB pathways are also involved in CSC self-renewal and tumor initiation [63].

Many studies have been performed on surface markers for potential identification and isolation of CSCs [59]. In some cases, stem-like cancer cells were identified using the flow cytometry-based side population (SP) technique [64, 65]. Wang et al. isolated the SP of breast cancer MCF-7 cells, generated SP spheres, and performed a high-throughput drug screening using these SP spheres and the LOPAC chemical library, and identified niclosamide as an inhibitor of breast cancer stem-like cells [49]. Niclosamide downregulated several stem cell signaling pathways including the Wnt/β-catenin, Notch, and Hedgehog pathways, inhibited the formation of spheroids, and induced apoptosis in breast cancer SP spheres. Animal studies also confirmed this therapeutic effect [49]. Using a similar approach, the same group further demonstrated that niclosamide was an inhibitor of stem-like ovarian-cancer-initiating cells, and suppressed the tumor formation of ovarian-cancer-initiating cells in vivo [47]. Moreover, it was reported that niclosamide was effective in killing acute myeloid leukemia (AML) stem cells (CD34+CD38−), but had minimal cytotoxicity against progenitor cells in normal bone marrow [38]. In addition, STAT3-mediated stem cell marker OCT-4 gene expression was effectively suppressed by treatment with niclosamide during mammosphere culture of breast cancer MDA-MB-231 cells [48]. Aldehyde dehydrogenase (ALDH) is a known marker of CSCs [66, 67]. Recently, we isolated a non-adherent population of cells that have high ALDH expression, and found that niclosamide suppressed Wnt/β-catenin and STAT3 signaling, and showed cytotoxicity against these non-adherent ALDH expressing cells in addition to the adherent cells from four basal-like breast cancer cell lines [11]. The inhibitory effects of niclosamide on CSCs provide compelling evidence for its consideration as a promising drug for cancer therapy.

Salinomycin, a polyether ionophore antibiotic isolated from Streptomyces albus, is used as an antibiotic in animal husbandry [68]. Recently, Gupta et al. performed a high throughput screening to discover agents with specific toxicity for epithelial CSCs, and identified salinomycin as a selective inhibitor of breast CSCs [69]. Promising results from preclinical trials in human xenograft mice and a few clinical pilot studies reveal that salinomycin could be a promising novel anti-cancer agent [70-72]. Ketola et al. performed connectivity map analysis to identify compounds with similar or opposite effects to salinomycin [73]. The differentially expressed genes in response to salinomycin exposure for 6 h in prostate carcinoma VCaP cells were compared with the 47,000 expression profiles representing drug responses to 41,309 compounds. Interestingly, niclosamide was one of the two top enriched compounds altering gene expression in the same direction as salinomycin [73], providing further evidence that niclosamide, like salinomycin, can work as a CSC killer.

7. Challenges to using niclosamide as an anti-cancer agent in humans

The oral dose of niclosamide for adult in cestocidal treatment is 2 g as a single dose, leading to maximal serum concentrations of 0.25 to 6.0 μg/ml (corresponding to 0.76 – 18.35 μM) [1], which is well within the anti-cancer active concentration range. The reason for this wide range of serum concentrations is considered to be due to intra-individual difference in absorption rate [1]. Niclosamide has poor water solubility, and the oral bioavailability of niclosamide was only 10% in male Sprague-Dawley rats [74]. The plasma half-life (t1/2) of niclosamide was 6.7 ± 2.0 hr in rats [74]. However, no specific data for pharmacokinetic parameters of niclosamide in humans is available in the literature [2]. Overall, the low oral bioavailability and wide range of serum concentrations of niclosamide could result in variation of its anti-cancer efficacy when it is used in clinical studies.

Studies in animals have suggested that niclosamide has no mutagenic, oncogenic, or embryotoxic activity [1]. Long term experiments in rats (1,000 or 2,500 mg/kg b.w. oral daily for 55 or 64 days, thereafter 10,000 and 25,000 mg/kg feed; total duration 365-381 days) and dogs (100 mg/kg b.w. orally in capsules for 366-393 days) found no indication of a higher incidence of tumor occurrence [1]. Niclosamide has been proven to be without effect on hematological and urinalysis parameters and not to influence liver and kidney function tests [1]. Recent studies have also indicated that no significant toxicity was shown against non-tumor cells in vitro and no obvious side effects were observed in niclosamide-treated mice [9, 30, 38]. The fate of niclosamide in humans, which closely resembles that in animals, and the low acute toxicity in humans and animals suggest that even a prolonged contact with niclosamide will not result in cumulative toxic effects in humans [1]. However, niclosamide is poorly absorbed from the intestinal tract, which explains its good tolerability in humans [1, 2]. Therefore, additional safety studies on niclosamide with a well absorbed formulation are required before definitive conclusions can be drawn for cancer therapy.

8. Future directions

While considerable high-throughput screening campaigns have identified niclosamide is a potent inhibitor of a number of biological signaling pathways that mediate niclosamide’s anticancer effects in vitro and in vivo, direct targets of niclosamide still remain unclear. Niclosamide was tested in vitro against a panel of 95 protein kinases, and it did not significantly inhibit any of these kinases at concentrations (1 or 10 μM) efficiently inhibiting the Wnt/β-catenin, mTORC1, STAT3, NF-κB and Notch signaling pathways in cancer cells, indicating that the mechanism of action of niclosamide is probably not direct kinase inhibition [19]. Among the techniques currently available, protein affinity isolation using suitable small-molecule probes (pulldown) and subsequent mass spectrometric analysis of the isolated proteins appears to be the most powerful and most frequently applied [75]. Therefore, the targets of niclosamide could be identified using affinity reagents – biotinylated derivatives of niclosamide in the future.

Although many studies have demonstrated that niclosamide has potent in vitro and in vivo anti-tumor growth activities, the cancer chemopreventive efficacy of niclosamide has not been studied yet. Therefore, there is a great need for further research to examine cancer chemopreventive activity of niclosamide in various animal carcinogenesis models and transgenic models.

Niclosamide is an FDA-approved drug that is given orally to helminthosis patients, however its applications in cancer therapy could be limited, as niclosamide is only partially absorbed from the gastrointestinal tract. Thus, there is great interest in conducting studies to elucidate the structure-activity relationship of niclosamide and to identify novel derivatives of niclosamide with improved bioavailability as additional clinical candidates for cancer therapy [76, 77].

Finally, intravenous administration of niclosamide to rats gave rise to a peak plasma concentration of 25 μM [74]. Thus, an intravenous injectable niclosamide formulation would be desirable. However, the safety and possibility of systemic intravenous application of niclosamide needs to be comprehensively investigated before niclosamide could be administered intravenously in the clinic.

Acknowledgments

We thank Meredith S. Plaxco and Tommie A. Gamble for running the 60 human tumor cell line anticancer drug screen. This work was completed with the support of grants from the National Institute of Health (R01CA124531 and R21CA182056 to Y. Li).

Footnotes

Conflict of Interest

The authors declare that they have no conflict of interest.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Andrews P, Thyssen J, Lorke D. The biology and toxicology of molluscicides. Bayluscide Pharmacol Ther. 1982;19:245–295. doi: 10.1016/0163-7258(82)90064-x. [DOI] [PubMed] [Google Scholar]
  • 2.Al-Hadiya BM. Niclosamide: comprehensive profile. Profiles Drug Subst Excip Relat Methodol. 2005;32:67–96. doi: 10.1016/S0099-5428(05)32002-8. [DOI] [PubMed] [Google Scholar]
  • 3.Kraljevic S, Stambrook PJ, Pavelic K. Accelerating drug discovery. EMBO Rep. 2004;5:837–842. doi: 10.1038/sj.embor.7400236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Chong CR, Sullivan DJ., Jr New uses for old drugs. Nature. 2007;448:645–646. doi: 10.1038/448645a. [DOI] [PubMed] [Google Scholar]
  • 5.Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell. 2012;149:1192–1205. doi: 10.1016/j.cell.2012.05.012. [DOI] [PubMed] [Google Scholar]
  • 6.Anastas JN, Moon RT. WNT signalling pathways as therapeutic targets in cancer. Nat Rev Cancer. 2013;13:11–26. doi: 10.1038/nrc3419. [DOI] [PubMed] [Google Scholar]
  • 7.Polakis P. Drugging Wnt signalling in cancer. EMBO J. 2012;31:2737–2746. doi: 10.1038/emboj.2012.126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Chen M, Wang J, Lu J, Bond MC, Ren XR, Lyerly HK, Barak LS, Chen W. The antihelminthic niclosamide inhibits Wnt/Frizzled1 signaling. Biochemistry. 2009;48:10267–10274. doi: 10.1021/bi9009677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Osada T, Chen M, Yang XY, Spasojevic I, Vandeusen JB, Hsu D, Clary BM, Clay TM, Chen W, Morse MA, Lyerly HK. Antihelminth compound niclosamide downregulates Wnt signaling and elicits antitumor responses in tumors with activating APC mutations. Cancer Res. 2011;71:4172–4182. doi: 10.1158/0008-5472.CAN-10-3978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Lu W, Lin C, Roberts MJ, Waud WR, Piazza GA, Li Y. Niclosamide suppresses cancer cell growth by inducing Wnt co-receptor LRP6 degradation and inhibiting the Wnt/beta-catenin pathway. PloS one. 2011;6:e29290. doi: 10.1371/journal.pone.0029290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Londoño-Joshi A, Arend AC, Aristizabal L, Lu W, Samant RS, Metge BJ, Hidalgo B, Grizzle WE, Conner M, Forero-Torres A, LoBuglio AF, Li Y, Buchsbaum DJ. Effect of niclosamide on basal-like breast cancers. Mol Cancer Ther. 2014 doi: 10.1158/1535-7163.MCT-13-0555. in press. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Wieland A, Trageser D, Gogolok S, Reinartz R, Hofer H, Keller M, Leinhaas A, Schelle R, Normann S, Klaas L, Waha A, Koch P, Fimmers R, Pietsch T, Yachnis AT, Pincus DW, Steindler DA, Brustle O, Simon M, Glas M, Scheffler B. Anticancer effects of niclosamide in human glioblastoma. Clin Cancer Res. 2013;19:4124–4136. doi: 10.1158/1078-0432.CCR-12-2895. [DOI] [PubMed] [Google Scholar]
  • 13.Bienz M, Clevers H. Linking colorectal cancer to Wnt signaling. Cell. 2000;103:311–320. doi: 10.1016/s0092-8674(00)00122-7. [DOI] [PubMed] [Google Scholar]
  • 14.Stein U, Arlt F, Walther W, Smith J, Waldman T, Harris ED, Mertins SD, Heizmann CW, Allard D, Birchmeier W, Schlag PM, Shoemaker RH. The metastasis-associated gene S100A4 is a novel target of beta-catenin/T-cell factor signaling in colon cancer. Gastroenterology. 2006;131:1486–1500. doi: 10.1053/j.gastro.2006.08.041. [DOI] [PubMed] [Google Scholar]
  • 15.Sack U, Walther W, Scudiero D, Selby M, Kobelt D, Lemm M, Fichtner I, Schlag PM, Shoemaker RH, Stein U. Novel effect of antihelminthic Niclosamide on S100A4-mediated metastatic progression in colon cancer. J Natl Cancer Inst. 2011;103:1018–1036. doi: 10.1093/jnci/djr190. [DOI] [PubMed] [Google Scholar]
  • 16.Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer cell. 2007;12:9–22. doi: 10.1016/j.ccr.2007.05.008. [DOI] [PubMed] [Google Scholar]
  • 17.Dancey J. mTOR signaling and drug development in cancer. Nat Rev Clin Oncol. 2010;7:209–219. doi: 10.1038/nrclinonc.2010.21. [DOI] [PubMed] [Google Scholar]
  • 18.Balgi AD, Fonseca BD, Donohue E, Tsang TC, Lajoie P, Proud CG, Nabi IR, Roberge M. Screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mTORC1 signaling. PloS one. 2009;4:e7124. doi: 10.1371/journal.pone.0007124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Fonseca BD, Diering GH, Bidinosti MA, Dalal K, Alain T, Balgi AD, Forestieri R, Nodwell M, Rajadurai CV, Gunaratnam C, Tee AR, Duong F, Andersen RJ, Orlowski J, Numata M, Sonenberg N, Roberge M. Structure-activity analysis of niclosamide reveals potential role for cytoplasmic pH in control of mammalian target of rapamycin complex 1 (mTORC1) signaling. J Biol Chem. 2012;287:17530–17545. doi: 10.1074/jbc.M112.359638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Jewell JL, Russell RC, Guan KL. Amino acid signalling upstream of mTOR. Nat Rev Mol Cell Biol. 2013;14:133–139. doi: 10.1038/nrm3522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Li M, Khambu B, Zhang H, Kang JH, Chen X, Chen D, Vollmer L, Liu PQ, Vogt A, Yin XM. Suppression of Lysosome Function Induces Autophagy via a Feedback Downregulation of MTORC1 Activity. J Biol Chem. 2013;288:35769–35780. doi: 10.1074/jbc.M113.511212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nat Rev Cancer. 2009;9:798–809. doi: 10.1038/nrc2734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Shuai K, Horvath CM, Huang LH, Qureshi SA, Cowburn D, Darnell JE., Jr Interferon activation of the transcription factor Stat91 involves dimerization through SH2-phosphotyrosyl peptide interactions. Cell. 1994;76:821–828. doi: 10.1016/0092-8674(94)90357-3. [DOI] [PubMed] [Google Scholar]
  • 24.Sasse J, Hemmann U, Schwartz C, Schniertshauer U, Heesel B, Landgraf C, Schneider-Mergener J, Heinrich PC, Horn F. Mutational analysis of acute-phase response factor/Stat3 activation and dimerization. Mol Cell Biol. 1997;17:4677–4686. doi: 10.1128/mcb.17.8.4677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Zhong Z, Wen Z, Darnell JE., Jr Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science. 1994;264:95–98. doi: 10.1126/science.8140422. [DOI] [PubMed] [Google Scholar]
  • 26.Darnell JE, Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science. 1994;264:1415–1421. doi: 10.1126/science.8197455. [DOI] [PubMed] [Google Scholar]
  • 27.Boudny V, Kovarik J. JAK/STAT signaling pathways and cancer. Janus kinases/signal transducers and activators of transcription. Neoplasma. 2002;49:349–355. [PubMed] [Google Scholar]
  • 28.Steelman LS, Abrams SL, Whelan J, Bertrand FE, Ludwig DE, Basecke J, Libra M, Stivala F, Milella M, Tafuri A, Lunghi P, Bonati A, Martelli AM, McCubrey JA. Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia. Leukemia. 2008;22:686–707. doi: 10.1038/leu.2008.26. [DOI] [PubMed] [Google Scholar]
  • 29.Ren R, Duan L, He Q, Zhang Z, Zhou Y, Wu D, Pan J, Pei D, Ding K. Identification of Niclosamide as a New Small-Molecule Inhibitor of the STAT3 Signaling Pathway. ACS Med Chem Lett. 2010;1:454–459. doi: 10.1021/ml100146z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Khanim FL, Merrick BA, Giles HV, Jankute M, Jackson JB, Giles LJ, Birtwistle J, Bunce CM, Drayson MT. Redeployment-based drug screening identifies the anti-helminthic niclosamide as anti-myeloma therapy that also reduces free light chain production. Blood Cancer J. 2011;1:e39. doi: 10.1038/bcj.2011.38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Li R, Hu Z, Sun SY, Chen ZG, Owonikoko TK, Sica GL, Ramalingam SS, Curran WJ, Khuri FR, Deng X. Niclosamide overcomes acquired resistance to erlotinib through suppression of STAT3 in non-small cell lung cancer. Mol Cancer Ther. 2013;12:2200–2212. doi: 10.1158/1535-7163.MCT-13-0095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Li R, You S, Hu Z, Chen ZG, Sica GL, Khuri FR, Curran WJ, Shin DM, Deng X. Inhibition of STAT3 by niclosamide synergizes with erlotinib against head and neck cancer. PloS one. 2013;8:e74670. doi: 10.1371/journal.pone.0074670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.You S, Li R, Park D, Xie M, Sica GL, Cao Y, Xiao ZQ, Deng X. Disruption of STAT3 by niclosamide reverses radioresistance of human lung cancer. Molecular cancer therapeutics. 2014 doi: 10.1158/1535-7163.MCT-13-0608. in press. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Naugler WE, Karin M. NF-kappaB and cancer-identifying targets and mechanisms. Curr Opin Genet Dev. 2008;18:19–26. doi: 10.1016/j.gde.2008.01.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Karin M. Nuclear factor-kappaB in cancer development and progression. Nature. 2006;441:431–436. doi: 10.1038/nature04870. [DOI] [PubMed] [Google Scholar]
  • 36.Chen F, Castranova V, Shi X, Demers LM. New insights into the role of nuclear factor-kappaB, a ubiquitous transcription factor in the initiation of diseases. Clin Chem. 1999;45:7–17. [PubMed] [Google Scholar]
  • 37.Napetschnig J, Wu H. Molecular basis of NF-kappaB signaling. Annu Rev Biophys. 2013;42:443–468. doi: 10.1146/annurev-biophys-083012-130338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Jin Y, Lu Z, Ding K, Li J, Du X, Chen C, Sun X, Wu Y, Zhou J, Pan J. Antineoplastic mechanisms of niclosamide in acute myelogenous leukemia stem cells: inactivation of the NF-kappaB pathway and generation of reactive oxygen species. Cancer Res. 2010;70:2516–2527. doi: 10.1158/0008-5472.CAN-09-3950. [DOI] [PubMed] [Google Scholar]
  • 39.Sakurai H, Miyoshi H, Toriumi W, Sugita T. Functional interactions of transforming growth factor beta-activated kinase 1 with IkappaB kinases to stimulate NF-kappaB activation. J Biol Chem. 1999;274:10641–10648. doi: 10.1074/jbc.274.15.10641. [DOI] [PubMed] [Google Scholar]
  • 40.Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature. 2001;412:346–351. doi: 10.1038/35085597. [DOI] [PubMed] [Google Scholar]
  • 41.Shih Ie M, Wang TL. Notch signaling, gamma-secretase inhibitors, and cancer therapy. Cancer Res. 2007;67:1879–1882. doi: 10.1158/0008-5472.CAN-06-3958. [DOI] [PubMed] [Google Scholar]
  • 42.Rizzo P, Osipo C, Foreman K, Golde T, Osborne B, Miele L. Rational targeting of Notch signaling in cancer. Oncogene. 2008;27:5124–5131. doi: 10.1038/onc.2008.226. [DOI] [PubMed] [Google Scholar]
  • 43.Pannuti A, Foreman K, Rizzo P, Osipo C, Golde T, Osborne B, Miele L. Targeting Notch to target cancer stem cells. Clin Cancer Res. 2010;16:3141–3152. doi: 10.1158/1078-0432.CCR-09-2823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Wang AM, Ku HH, Liang YC, Chen YC, Hwu YM, Yeh TS. The autonomous notch signal pathway is activated by baicalin and baicalein but is suppressed by niclosamide in K562 cells. J Cell Biochem. 2009;106:682–692. doi: 10.1002/jcb.22065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Galluzzi L, Larochette N, Zamzami N, Kroemer G. Mitochondria as therapeutic targets for cancer chemotherapy. Oncogene. 2006;25:4812–4830. doi: 10.1038/sj.onc.1209598. [DOI] [PubMed] [Google Scholar]
  • 46.Park SJ, Shin JH, Kang H, Hwang JJ, Cho DH. Niclosamide induces mitochondria fragmentation and promotes both apoptotic and autophagic cell death. BMB Rep. 2011;44:517–522. doi: 10.5483/bmbrep.2011.44.8.517. [DOI] [PubMed] [Google Scholar]
  • 47.Yo YT, Lin YW, Wang YC, Balch C, Huang RL, Chan MW, Sytwu HK, Chen CK, Chang CC, Nephew KP, Huang T, Yu MH, Lai HC. Growth inhibition of ovarian tumor-initiating cells by niclosamide. Mol Cancer Ther. 2012;11:1703–1712. doi: 10.1158/1535-7163.MCT-12-0002. [DOI] [PubMed] [Google Scholar]
  • 48.Kim SY, Kang JW, Song X, Kim BK, Yoo YD, Kwon YT, Lee YJ. Role of the IL-6-JAK1-STAT3-Oct-4 pathway in the conversion of non-stem cancer cells into cancer stem-like cells. Cell Signal. 2013;25:961–969. doi: 10.1016/j.cellsig.2013.01.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Wang YC, Chao TK, Chang CC, Yo YT, Yu MH, Lai HC. Drug screening identifies niclosamide as an inhibitor of breast cancer stem-like cells. PloS one. 2013;8:e74538. doi: 10.1371/journal.pone.0074538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Pan JX, Ding K, Wang CY. Niclosamide, an old antihelminthic agent, demonstrates antitumor activity by blocking multiple signaling pathways of cancer stem cells. Chin J Cancer. 2012;31:178–184. doi: 10.5732/cjc.011.10290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Shoemaker RH. The NCI60 human tumour cell line anticancer drug screen. Nat Rev Cancer. 2006;6:813–823. doi: 10.1038/nrc1951. [DOI] [PubMed] [Google Scholar]
  • 52.Ichikawa K, Liu W, Zhao L, Wang Z, Liu D, Ohtsuka T, Zhang H, Mountz JD, Koopman WJ, Kimberly RP, Zhou T. Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity. Nature Med. 2001;7:954–960. doi: 10.1038/91000. [DOI] [PubMed] [Google Scholar]
  • 53.Buchsbaum DJ, Zhou T, Grizzle WE, Oliver PG, Hammond CJ, Zhang S, Carpenter M, LoBuglio AF. Antitumor efficacy of TRA-8 anti-DR5 monoclonal antibody alone or in combination with chemotherapy and/or radiation therapy in a human breast cancer model. Clin Cancer Res. 2003;9:3731–3741. [PubMed] [Google Scholar]
  • 54.Gongoll S, Peters G, Mengel M, Piso P, Klempnauer J, Kreipe H, von Wasielewski R. Prognostic significance of calcium-binding protein S100A4 in colorectal cancer. Gastroenterology. 2002;123:1478–1484. doi: 10.1053/gast.2002.36606. [DOI] [PubMed] [Google Scholar]
  • 55.Sack U, Walther W, Scudiero D, Selby M, Aumann J, Lemos C, Fichtner I, Schlag PM, Shoemaker RH, Stein U. S100A4-induced cell motility and metastasis is restricted by the Wnt/beta-catenin pathway inhibitor calcimycin in colon cancer cells. Mol Biol Cell. 2011;22:3344–3354. doi: 10.1091/mbc.E10-09-0739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Takenaga K, Nakanishi H, Wada K, Suzuki M, Matsuzaki O, Matsuura A, Endo H. Increased expression of S100A4, a metastasis-associated gene, in human colorectal adenocarcinomas. Clin Cancer Res. 1997;3:2309–2316. [PubMed] [Google Scholar]
  • 57.Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001;414:105–111. doi: 10.1038/35102167. [DOI] [PubMed] [Google Scholar]
  • 58.Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nature reviews Cancer. 2008;8:755–768. doi: 10.1038/nrc2499. [DOI] [PubMed] [Google Scholar]
  • 59.Baccelli I, Trumpp A. The evolving concept of cancer and metastasis stem cells. J Cell Biol. 2012;198:281–293. doi: 10.1083/jcb.201202014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Katoh M, Katoh M. WNT signaling pathway and stem cell signaling network. Clin Cancer Res. 2007;13:4042–4045. doi: 10.1158/1078-0432.CCR-06-2316. [DOI] [PubMed] [Google Scholar]
  • 61.Merchant AA, Matsui W. Targeting Hedgehog--a cancer stem cell pathway. Clin Cancer Res. 2010;16:3130–3140. doi: 10.1158/1078-0432.CCR-09-2846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Takebe N, Harris PJ, Warren RQ, Ivy SP. Targeting cancer stem cells by inhibiting Wnt, Notch, and Hedgehog pathways. Nat Rev Clin Oncol. 2011;8:97–106. doi: 10.1038/nrclinonc.2010.196. [DOI] [PubMed] [Google Scholar]
  • 63.Alison MR, Lin WR, Lim SM, Nicholson LJ. Cancer stem cells: in the line of fire. Cancer Treat Rev. 2012;38:589–598. doi: 10.1016/j.ctrv.2012.03.003. [DOI] [PubMed] [Google Scholar]
  • 64.Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG. Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic. Cancer Res. 2005;65:6207–6219. doi: 10.1158/0008-5472.CAN-05-0592. [DOI] [PubMed] [Google Scholar]
  • 65.Engelmann K, Shen H, Finn OJ. MCF7 side population cells with characteristics of cancer stem/progenitor cells express the tumor antigen MUC1. Cancer Res. 2008;68:2419–2426. doi: 10.1158/0008-5472.CAN-07-2249. [DOI] [PubMed] [Google Scholar]
  • 66.Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell. 2007;1:555–567. doi: 10.1016/j.stem.2007.08.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Croker AK, Goodale D, Chu J, Postenka C, Hedley BD, Hess DA, Allan AL. High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability. J Cell Mol Med. 2009;13:2236–2252. doi: 10.1111/j.1582-4934.2008.00455.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Miyazaki Y, Shibuya M, Sugawara H, Kawaguchi O, Hirsoe C. Salinomycin, a new polyether antibiotic. J Antibiot. 1974;27:814–821. doi: 10.7164/antibiotics.27.814. [DOI] [PubMed] [Google Scholar]
  • 69.Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA, Lander ES. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell. 2009;138:645–659. doi: 10.1016/j.cell.2009.06.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Huczynski A. Salinomycin: a new cancer drug candidate. Chem Biol Drug Des. 2012;79:235–238. doi: 10.1111/j.1747-0285.2011.01287.x. [DOI] [PubMed] [Google Scholar]
  • 71.Naujokat C, Steinhart R. Salinomycin as a drug for targeting human cancer stem cells. J Biomed Biotechnol. 2012;2012:950658. doi: 10.1155/2012/950658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72.Zhou S, Wang F, Wong ET, Fonkem E, Hsieh TC, Wu JM, Wu E. Salinomycin: a novel anti-cancer agent with known anti-coccidial activities. Curr Med Chem. 2013;20:4095–4101. doi: 10.2174/15672050113109990199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73.Ketola K, Hilvo M, Hyotylainen T, Vuoristo A, Ruskeepaa AL, Oresic M, Kallioniemi O, Iljin K. Salinomycin inhibits prostate cancer growth and migration via induction of oxidative stress. Br J Cancer. 2012;106:99–106. doi: 10.1038/bjc.2011.530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 74.Chang YW, Yeh TK, Lin KT, Chen WC, Yao HT, Lan SJ, Wu YS, Hsieh HP, Chen CM, Chen CT. Pharmacokinetics of anti-SARS-CoV agent niclosamide and its analogs in rats. J Food Drug Anal. 2006;14:329–333. [Google Scholar]
  • 75.Ziegler S, Pries V, Hedberg C, Waldmann H. Target identification for small bioactive molecules: finding the needle in the haystack. Angew Chem Int Ed Engl. 2013;52:2744–2792. doi: 10.1002/anie.201208749. [DOI] [PubMed] [Google Scholar]
  • 76.Chen H, Yang Z, Ding C, Chu L, Zhang Y, Terry K, Liu H, Shen Q, Zhou J. Discovery of -Alkylamino Tethered Niclosamide Derivatives as Potent and Orally Bioavailable Anticancer Agents. ACS Med Chem Lett. 2013;4:180–185. doi: 10.1021/ml3003082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 77.Mook RA, Jr, Chen M, Lu J, Barak LS, Lyerly HK, Chen W. Small molecule modulators of Wnt/beta-catenin signaling. Bioorg Med Chem Lett. 2013;23:2187–2191. doi: 10.1016/j.bmcl.2013.01.101. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES