Nifuroxazide induces apoptosis and impairs pulmonary metastasis in breast cancer model

Yang, F; Hu, M; Lei, Q; **a, Y; Zhu, Y; Song, X; Li, Y; Jie, H; Liu, C; **ong, Y; Zuo, Z; Zeng, A; Li, Y; Yu, L; Shen, G; Wang, D; **e, Y; Ye, T; Wei, Y

doi:10.1038/cddis.2015.63

Nifuroxazide induces apoptosis and impairs pulmonary metastasis in breast cancer model

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Published: 26 March 2015

Volume 6, page e1701, (2015)
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Cell Death & Disease

Nifuroxazide induces apoptosis and impairs pulmonary metastasis in breast cancer model

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F Yang¹^na1,
M Hu¹^na1,
Q Lei¹^na1,
Y **a¹,
Y Zhu¹,
X Song¹,
Y Li¹,
H Jie¹,
C Liu¹,
Y **ong^1,2,
Z Zuo¹,
A Zeng¹,
Y Li¹,
L Yu¹,
G Shen¹,
D Wang³,
Y **e¹,
T Ye¹ &
…
Y Wei¹

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Abstract

Breast carcinoma is the most common female cancer with considerable metastatic potential. Signal transducers and activators of the transcription 3 (Stat3) signaling pathway is constitutively activated in many cancers including breast cancer and has been validated as a novel potential anticancer target. Here, we reported our finding with nifuroxazide, an antidiarrheal agent identified as a potent inhibitor of Stat3. The potency of nifuroxazide on breast cancer was assessed in vitro and in vivo. In this investigation, we found that nifuroxazide decreased the viability of three breast cancer cell lines and induced apoptosis of cancer cells in a dose-dependent manner. In addition, western blot analysis demonstrated that the occurrence of its apoptosis was associated with activation of cleaved caspases-3 and Bax, downregulation of Bcl-2. Moreover, nifuroxazide markedly blocked cancer cell migration and invasion, and the reduction of phosphorylated-Stat3^Tyr705, matrix metalloproteinase (MMP) MMP-2 and MMP-9 expression were also observed. Furthermore, in our animal experiments, intraperitoneal administration of 50 mg/kg/day nifuroxazide suppressed 4T1 tumor growth and blocked formation of pulmonary metastases without detectable toxicity. Meanwhile, histological and immunohistochemical analyses revealed a decrease in Ki-67-positive cells, MMP-9-positive cells and an increase in cleaved caspase-3-positive cells upon nifuroxazide. Notably, nifuroxazide reduced the number of myeloid-derived suppressor cell in the lung. Our data indicated that nifuroxazide may potentially be a therapeutic agent for growth and metastasis of breast cancer.

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Main

Breast cancer is the most common type of cancer among women, and its incidence is increasing worldwide. According to statistics, about 62 570 cases of breast cancer in situ are expected to be newly diagnosed in the United States in 2014.¹ Moreover, the incidence of breast cancer – like most cancers – is on the rise in develo** countries such as Brazil and China, as populations increasingly adopt western lifestyles.² Therefore, breast cancer ranks the second most common cause of cancer death among women worldwide, with about 1.4 million new cases annually.^{1, 3, 4} Despite significant improvement in survival rates of patients with breast cancer, the disease remains a huge threat to women’s health, and particularly patients with ‘triple-negative’ breast cancer (TNBC), referring to cancers that express neither the estrogen receptor or progesterone receptor nor display amplification of human epidermal growth factor receptor 2, are insensitive to hormonal therapy or HER2-targeted drugs.^{5, 6, 7} Advanced TNBC confer an aggressive clinical course with a poor prognosis compared with non-TNBC.⁸ Furthermore, breast cancer is highly malignant with considerable metastatic potential, and metastatic breast cancer is a principle cause of female mortality.⁹ Unfortunately, there is currently no effective therapy to control the recurrence and metastasis of breast cancer, and therefore the development of new therapies is essential.

Signal transducer and activator of transcription 3 (Stat3) has important roles in cancer and other disease, and presents tremendous therapeutic potential.¹⁰ Stat3 is a point of convergence for multiple oncogenic signaling pathways. Meanwhile, Stat3 as a proto-oncogene could mediate cellular and biological processes.¹⁰ In a variety of human cancers, constitutively active Stat3 signaling promotes tumorigenesis and tumor progression by dysregulating the expression of key genes that control cell apoptosis (such as Bcl-2, Bcl-xl and Mcl-1), proliferation (cyclin d1, c-Myc), angiogenesis (vascular endothelial growth factor), migration, invasion or metastasis (matrix metalloproteinase 1 (MMP1), MMP7 and MMP-9).^{11, 12, 13, Full size image}

We then performed a Matrigel invasion assay. Figure 3c shows that both 4T1 and MDA-MB-231 cells exhibit significantly decreased invasion in the presence of nifuroxazide than vehicle. Moreover, constitutive Stat3 also has an important role in controlling cell migration and invasion by regulating the expression of genes involved MMP-2, -9 and others.³⁰ Therefore, we also investigated whether phosphorylated-Stat3 ^(Tyr705), MMP-2, -9, which are considered to be related with cell migration and invasion, are involved in nifuroxazide-mediated migration and invasion. As Figure 3d indicates, nifuroxazide treatment decreased the expression of phosphorylated-Stat3 ^(Tyr705), MMP-2 and MMP-9 without affecting the total Stat3 expression level in 4T1 cells. Altogether, our results showed that nifuroxazide possessed a strong ability on breast cancer cell migration and invasion.

Retardation of mammary tumor growth in vivo

The remarkable inhibitory effects of nifuroxazide on 4T1 cells proliferation in vitro implied that it might inhibit tumor growth in vivo. To verify this hypothesis, we examine the antitumor activity of nifuroxazide in vivo. 4T1 tumor-bearing mice were dosed daily at the dose of 10 and 50 mg/kg for 24 days. Nonsignificant changes in body weight were observed in nifuroxazide-treated animals (Supplementary Figure S3a). Importantly, there was a significant reduction in tumor outgrowth and in tumor weight in the nifuroxazide-treated groups (Figures 4a and b; Supplementary Figure S3b), showing that nifuroxazide has strong antitumor activity.

To determine the mechanisms underlying nifuroxazide activity, 4T1-induced tumors collected on day 31 were examined for proliferation and apoptosis. Nifuroxazide treatment caused a significant reduction in proliferating cells stained positive for nuclear Ki-67 (Figure 4c). Moreover, as shown in Figure 4d, CC-3-positive cells were showing an increase in the nifuroxazide-treated sections versus sections of the untreated groups. Overall, these data suggest that nifuroxazide inhibits cell proliferation and induces apoptosis in breast tumor tissues through inhibition of Ki-67 and activation of caspase-3. In addition, there is increasing evidence to indicate that MMPs have important roles in tumor invasion and metastasis.³¹ We therefore measured the effects of nifuroxazide on the expression of MMP-9 by immunohistochemistry (IHC). As shown in Figure 4e, treatment of mice with nifuroxazide inhibited the expression of MMP-9 in 4T1 tumor tissues.

Nifuroxazide treatment decreases lung metastasis

Previous studies have showed that 4T1 murine breast tumor have a high metastatic potential and spontaneously metastasize to lung as early as 2 weeks after inoculation.^{32, 33, 34} To analyze the effects of nifuroxazide on metastasis, lungs of 4T1 tumor-bearing mice killed on day 31 were removed and metastatic nodules were quantified. In vehicle-treated groups, multiple large nodules were evident, whereas the extent of lung metastasis was markedly reduced in nifuroxazide-treated mice (50 mg/kg groups; Figures 5a and b). Moreover, there was a remarkable decrease in lung weight after nifuroxazide treatment compared with the untreated control (Figure 5c). Importantly, histological analyses demonstrated that the number of micrometastatic nodules per field in the nifuroxazide-treated group at 50 mg/kg was also significant fewer than the other groups (Supplementary Figure S4). Overall, these results further indicated that nifuroxazide could inhibit lung metastasis in breast cancer.

Nifuroxazide modulates the lung metastatic environment

MDSCs, as mainly characterized by CD11b⁺ and Gr1⁺ double-positive myeloid cells in mice have been observed to accumulate in highly metastatic breast carcinoma 4T1. Moreover, it has been shown that accumulation of MDSCs into the lung has a key role in the development of metastasis, meanwhile, MDSCs are closely related to a lung metastatic in patients with breast cancer.^{35, 36, 37} Therefore, we further investigated lung myeloid cells infiltration in 4T1 tumor-bearing mice by FCM after 24 days of treatment. As shown in Figures 6a and b, the data showed that the percentage of MDSCs decreased in the 10 mg/kg-treated group compared with the control group. Moreover, we found that ~22% reduction of MDSCs in the lung after 50 mg/kg nifuroxazide treatment (Figure 6c). The statistical analysis demonstrated that nifuroxazide treatment reduced the number of MDSCs in lung in a dose-dependent manner (Figure 6d). These results suggested that nifuroxazide potently reduced the infiltration of MDSCs into the lung, which could inhibit tumor cell distant colonization.

Safety profile of nifuroxazide

As mentioned above, during the treatment of 4T1 tumor-bearing mice, we did not observed adverse effects, such as toxic death, skin ulceration and body weight loss, in the nifuroxazide-treated groups. To further investigate the safety profile of nifuroxazide, we determined here whether the nifuroxazide could cause the blood system’s abnormality, we performed blood routine analysis and blood chemistry analysis assays. Hematological and serum biochemistry analysis of the mice did not show any pathological changes (Figure 7a). Furthermore, no pathologic changes after nifuroxazide treatment were observed in the heart, liver, spleen and kidney by microscopic examination compared with the vehicle-treatment group (Figure 7b).

Discussion

Breast cancer is highly malignant with considerable metastatic potential. Meanwhile, tumor metastasis poses a predominant threat to cancer-related mortality.²⁹ Despite breast cancer is the most frequently diagnosed cancer and recent advances in the treatment of breast cancer, there are patients for whom no targeted therapies are available.³⁸ Therefore, the discovery of novel potential drug candidate to prevent tumor metastasis is still needed. The association between breast cancer and Stat3 pathway was established about 20 years ago.³⁹ Recent studies have also reported that activated Stat3 and its overexpression were closely associated with the development of breast cancer.⁴⁰ Importantly, with Stat3 being activated in ~70% of breast tumors, particularly in the less treatable triple-negative tumors, suggesting that targeting Stat3 might be a potentially important new form of breast cancer therapy.¹⁴

In the present study, we reported our finding with nifuroxazide, an antidiarrheal agent. Our results showed that nifuroxazide could inhibit breast cancer cell vitality with low micromole. Proliferation inhibitory activity of nifuroxazide against cancer cells was confirmed by MTT and clonogenicity assays. Apoptosis has been accepted as a fundamental component in the pathogenesis of cancer, and it has an important role in breast cancer progression. Therefore, inducing apoptosis is a therapeutic approach to treat cancers.^{41, 42} Our data indicated that nifuroxazide induced apoptotic death in breast cancer cells in a concentration-dependent manner, which was confirmed by the downregulation of Bcl-2 and the upregulation of CC-3 and Bax.

Because not all compounds have potent antitumor activity in vitro could exhibit anticancer activity in vivo, we then examined the antitumor effects of nifuroxazide in our established 4T1 tumor model in BALB/c mice. The results showed that tumor growth was significantly inhibited by nifuroxazide administration (50 mg/kg/day) with an inhibitory rate by 65%. Meanwhile, reduced expression of Ki-67 and increased expression of CC-3 in tumor cells were observed after nifuroxazide treatment compared with the untreated groups.

Breast cancer starts as a local disease, but it can metastasize to the lymph node, lung and other organs.²⁹ The metastatic process is complex, and tumor cells need to enter into blood vessels as well as the extravasation into the secondary organs. Therefore, tumor cell migration and invasion is a key step in successful cancer metastasis, and inhibition of this step is a practical approach to antitumor treatment.⁴³ Nifuroxazide was shown to inhibit MDA-MB-231 and 4T1 cell migration and invasion. In addition, in our animal experiments, administrations of nifuroxazide at the dose of 50 mg/kg significantly inhibited breast cancer metastasis to lung. These results consist with in vitro experiment, suggesting that breast cancer metastasis inhibition by nifuroxazide could be mainly ascribed to the impediments of tumor cell migration and invasion. Moreover, it has been reported that focal adhesion kinase/MMP-involved pathway is critical for cancer invasion and metastasis.^{28, 44} MMP-2 and MMP-9 upregulation can particularly enhance tumor cell metastatic potential in breast cancer.⁴⁵ In addition, constitutive Stat3 also has an important role in controlling cell migration and invasion.³⁰ In this study, nifuroxazide not only decreased expression of Stat3 phosphorylation at tyrsione residue 705 but also downregulated MMP-2 and MMP-9 expression in 4T1 cells. Meanwhile, IHC assay results showed that nifuroxazide inhibited the expression of MMP-9 in vivo. The results suggested the efficacy of MMPs signaling pathway blockade by nifuroxazide for inhibition of breast cancer cell invasion and metastasis.

A large body of evidence suggests that the tumor microenvironment can promote tumor development, progression and immune evasion.^{46, 47} MDSCs are critical components of the tumor microenvironment. In addition, MDSCs are present in lots of patients and experimental animals with cancer that downregulate immune surveillance and antitumor immunity.⁴⁸ In this study, our results indicated that the treatment of mice with nifuroxazide caused a significant decrease in the number of MDSCs in lung compared with that of vehicle-treated group. It is therefore conceivable that nifuroxazide could potentiate the antitumor effects and suppress the lung metastasis by downregulating the number of MDSCs.

In summary, our present studies provide important information regarding the antitumor activities of nifuroxazide in breast cancer. To our knowledge, this is the first study, to demonstrate that nifuroxazide could inhibit breast cancer cell growth, induce cell apoptosis and block cell migration and invasion. Moreover, nifuroxazide suppressed the breast tumor growth without significant toxicity. Importantly, nifuroxazide could enhance antitumor immunity and inhibit lung metastasis by reducing the number of MDSCs in lung. Therefore, these results implied that nifuroxazide might be a potential therapeutic agent for blocking breast cancer growth and metastasis.

Materials and Methods

Reagents and antibodies

Nifuroxazide was purchased from (**yashiji Chemical Co., LTD, ChengDu, Sichuan, China). Purity (98%) was measured by high-performance liquid chromatograph analysis. For in vitro assays, nifuroxazide was prepared initially as a 20 mM stock solution in dimethyl sulfoxide (DMSO) and stored at −20 °C. Then the stock solution diluted in the relevant assay medium, and 0.1% DMSO served as a vehicle control. For in vivo studies, nifuroxazide was prepared in 40% (v/v) polyethylene glycol 400 containing 5% (v/v) propylene glycol and dosed at 0.1 ml/10 g of body weight.

MTT, DMSO and Hoechst 33258 were from Sigma Chemical Co. (St Louis, MO, USA). The primary antibodies against Stat3/P-Stat3^Tyr705, MMP-2, -9 (MMP-2, MMP-9), CC-3, Bcl-2 and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). FITC-CD11b, PE-Gr1 conjugated antibodies were obtained from BD Biosciences (San Diego, CA, USA). Mouse monoclonal anti Ki-67 was purchased from Merck-Millipore (Bill, MA, USA). The Annexin V-FITC Apoptosis Detection Kit was purchased from KeyGen Biotech (Nan-**g, China).

Cell culture

The human breast cancer cell lines, MDA-MB-231 and MCF-7, the mouse mammary carcinoma cell line 4T1, were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were propagated in DMEM or RPMI 1640 media containing 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% antibiotics (penicillin and streptomycin) in 5% CO₂ at 37 °C.

Cell viability assay

The cell viability of nifuroxazide-treated breast cancer cells was assessed by MTT assay. In brief, the exponentially growing cells (3~5 × 10³/well) were plated in 100 μl/well in 96-well plates. After 24 h incubation, the cells were treated with different concentrations of nifuroxazide. After treatment for 24, 48 and 72 h, respectively, the 20 μl of 5 mg/ml MTT was added to each well, and the plates were incubated at 37 °C for additional 2–4 h. The medium was subsequently removed, the purple-colored precipitates of formazan were dissolved in 150 μl of DMSO. The color absorbance was recorded at 570 nm using a Spectra MAX M5 microplate spectrophotometer (Molecular Devices, CA, USA). All experiments were performed in triplicate.

Colony formation assay

Colony formation assay was measured as previously described.⁴⁹ In brief, 4T1, MCF-7 and MDA-MB-231 cells were seeded in specified numbers (400–600 cells/well) in six-well plates. After 24 h incubation, the cells were treated with various concentrations of nifuroxazide and then incubated for additional 12 days. Then the cells were fixed with methanol and stained with a 0.5% crystal violet solution for 15 min, and the colonies (>50 cells) were counted under microscope. Data shown represent the average of three independent experiments.

Morphological analysis by Hoechst staining

An apoptotic cell has special morphologic characteristics such as cell body shrinkage, chromatin condensation and margination as well as emerging apoptotic bodies.⁴⁹ To identify whether the nifuroxazide-inducing reduction in cell viability was attributable to the apoptosis, we stained the 4T1, MCF-7 and MDA-MB-231 cells with Hoechst 33258 dye. In brief, 4T1, MCF-7 and MDA-MB-231 cells (1~2 × 10⁵cells/well) were plated onto 18-mm coverglass in a six-well plate for 24 h, respectively. After treatment with different concentrations for following 24 h, the cells were washed with cold phosphate-buffered saline (PBS) and fixed in methanol for 15 min. The cells were stained with the Hoechst 33258 solutions according to the manufacturer’s instructions. Then nuclear morphology of apoptotic cells was examined under a fluorescence microscopy (Leica, DM4000B, Wetzlar, Germany).

Apoptotic assay

To further confirm the apoptosis-inducing effects of nifuroxazide, we subsequently estimated the number of apoptotic cells by FCM. In brief, breast cancer cells (1~2 × 10⁵ cells/well) were seeded in six-well plate overnight. Then treated with various concentrations of nifuroxazide for 24 h, the cells were harvested, washed with cold PBS twice. The apoptosis levels were examined using an apoptosis detection kit according to manufacturer’s instructions by FCM. Then data were analyzed with FlowJo software.

Flow cytometry

Single-cell suspensions from lung were prepared as described previously.¹⁵ Then 1 × 10⁶ freshly prepared cells were stained with fluorochrome-conjugated CD11b and Gr1 antibodies. Data were collected by FCM and analyzed with FlowJo software.

Western blot analysis

The western blot analysis was performed as described previously, with minor modification.⁵⁰ in brief, 4T1 cells were treated with nifuroxazide in designed concentration for 24 h, then cells were washed twice with cold PBS and lysed in RIPA buffer. The protein concentrations were measured using the Lowry method and equalized before loading. Equal amounts of protein from each sample were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresi gels and transferred onto polyvinylidene difluoridemembranes (Amersham Bioscience, Piscataway, NJ, USA). Then, the membranes were blocked for 2 h at 37 °C and incubated with specific primary antibodies overnight at 4 °C. After incubation with the relevant secondary antibodies, the reactive bands were identified using an enhanced chemiluminescence kit (Amersham).

Wound-healing migration assay

Wound-healing migration assay was performed as described previously.²⁸ When cancer cells grew to 80% confluence, cell monolayer was scraped by sterile 0.1 ml pipette tips, and fresh medium was added containing different concentrations of nifuroxazide. After 24 h incubation, cells were fixed and photographed. Images were acquired using a microscope (Zeiss, Jena, Germany) and the percentage inhibition of migrated cells was expressed using 100% as the value assigned for untreated group.

Boyden chamber migration and invasion assay

Boyden chamber (8 μm pore size) migration assay was performed as previously described, with some modification.^{22, 28} In brief, a total of 1 × 10⁵ cells (for 4T1) or 5 × 10⁴ cells (for MDA-MB-231) in 100 μl serum-free medium were added in the upper chamber, and 600 μl of medium containing 10% FBS was added at the bottom. Different concentrations of nifuroxazide were added in both chambers. Cells were allowed to migrate for ~24 h. Non-migrated cells in the upper chamber were discarded using a cotton swab. The migrated cells were fixed in methanol and stained with 0.5% crystal violet. Migrated cells in six randomly selected fields were counted and photographed under a light microscope. Invasion assay was performed according to previous studies.^{22, 28} In brief, the upper surface of the transwell membrane were coated with serum-free medium diluted Matrigel (1 : 1, 60 μl/well, BD Biosciences) and the lower compartment of the chambers were filled with 600 μl medium with 10 % FBS. 1 × 10⁵ cells (for 4T1) or 5 × 10⁴ cells (for MDA-MB-231) in 100 μl serum-free medium were placed in the upper part of each transwell and treated with different concentrations of nifuroxazide. After incubation for 24 h, cells on the upper side of the filter were removed. Cells located on the underside of the filter were fixed with methanol and stained with 0.5% crystal violet, then, migrated cells were counted and photographed under a light microscope. The results were expressed as the percentage inhibition rate of migration compared with untreated group.

Mice and tumor model

All animal experiments were approved by the Institutional Animal Care and Treatment Committee of Sichuan University in China (New Permit Number: 20131205-3). Female BALB/c mice (6–8 weeks old) were obtained from HFK bioscience CO., LTD, Bei**g, China. We inoculated female mice with 4T1 cell (1.0 × 10⁶/100 μl/each). After ~4 days, the injection sites could appear tumors. When visible tumors (~70 mm³) had been developed at the injection sites, the tumor-bearing mice were randomized into three groups (eight mice per group), and received intraperitoneally injection of nifuroxazide 50 mg/kg, 10 mg/kg or vehicle, respectively, once daily for 24 days. Tumor volumes and body weight were measured every 3 days. The tumor size was calculated according to the formula: Tumor volume (mm³)=0.52 × L × W² where L is the length and W is the width. At the termination of the experiment, all animals were euthanized by cervical dislocation. At that time tumors and internal organs, such as the heart, livers, spleens, lungs and kidney were excised from animals.

Immunohistochemistry

IHC staining of tumor sections were described previously.^{22, 28} One part of paraffin tumor sections and lung sections were stained with hematoxylin and eosin (H&E). The other part paraffin tumor sections were stained with Ki-67, CC-3 and MMP-9 antibodies. Images were taken with Leica microscope (Leica, DM4000B).

Toxicity evaluation

To investigate potential side effects or toxicity on mice during the treatment, all the animals were observed continuously for relevant indexes such as body weight, diarrhea, anorexia and other clinical symptoms. On the 31st day, all animals were euthanized by cervical dislocation after taking blood from eyeball. Blood was obtained for blood routine analysis and blood chemistry analysis. The tissues of heart, liver, spleen, lung and kidney were stained with H&E for histopathologic examination.

Statistical analysis

Data were represented as mean±S.D. of three independent experiments. The two-tailed Student’s t-test was used for statistical analysis and statistically significant P-values were labeled as follows: *P<0.05; **P<0.01; ***P<0.001.

Abbreviations

Stat3:: Signal transducers and activators of transcription 3
MTT:: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
FCM:: flow cytometry
MMP:: matrix metalloproteinase
IHC:: immunohistochemical
MDCSs:: myeloid-derived suppressor cells
TNBC:: triple-negative breast cancer
ER:: estrogen receptor
PR:: progesterone receptor
HER2:: human epidermal growth factor receptor 2
VEGF:: vascular endothelial growth factor
HPLC:: high-performance liquid chromatography
DMSO:: dimethyl sulfoxide
CC-3:: cleaved caspase-3
FBS:: fetal bovine serum
PBS:: phosphate-buffered saline
SDS-PAGE:: sodium dodecyl sulfate-polyacrylamide gel electrophoresis
PVDF:: polyvinylidene difluoride
H&E:: hematoxylin and eosin
FAK:: focal adhesion kinase
i.p.:: Intraperitoneally

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Acknowledgements

This study was funded by the grants from the National Key Basic Research Program of China (2010CB 529900), the National Natural Science Foundation of China (81123003) and the National Natural Science Foundation of China (81301980).

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F Yang, M Hu and Q Lei: These authors contributed equally to this work.

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State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China
F Yang, M Hu, Q Lei, Y **a, Y Zhu, X Song, Y Li, H Jie, C Liu, Y **ong, Z Zuo, A Zeng, Y Li, L Yu, G Shen, Y **e, T Ye & Y Wei
Department of Pharmacy, **nqiao Hospital, Third Military Medical University, Chongqing, China
Y **ong
School of Medicine, Tsinghua University, Bei**g, China
D Wang

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F Yang
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M Hu
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Q Lei
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Y Zhu
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X Song
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Y Li
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H Jie
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C Liu
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Y **ong
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A Zeng
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Y Li
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L Yu
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D Wang
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Y **e
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T Ye
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Y Wei
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Yang, F., Hu, M., Lei, Q. et al. Nifuroxazide induces apoptosis and impairs pulmonary metastasis in breast cancer model. Cell Death Dis 6, e1701 (2015). https://doi.org/10.1038/cddis.2015.63

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Received: 20 October 2014
Revised: 01 February 2015
Accepted: 09 February 2015
Published: 26 March 2015
Issue Date: March 2015
DOI: https://doi.org/10.1038/cddis.2015.63
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Nifuroxazide induces apoptosis and impairs pulmonary metastasis in breast cancer model

Abstract

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Main

Retardation of mammary tumor growth in vivo

Nifuroxazide treatment decreases lung metastasis

Nifuroxazide modulates the lung metastatic environment

Safety profile of nifuroxazide

Discussion

Materials and Methods

Reagents and antibodies

Cell culture

Cell viability assay

Colony formation assay

Morphological analysis by Hoechst staining

Apoptotic assay

Flow cytometry

Western blot analysis

Wound-healing migration assay

Boyden chamber migration and invasion assay

Mice and tumor model

Immunohistochemistry

Toxicity evaluation

Statistical analysis

Abbreviations

References

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