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Nifuroxazide induces apoptosis and impairs pulmonary metastasis in breast cancer model.

Yang F, Hu M, Lei Q, Xia Y, Zhu Y, Song X, Li Y, Jie H, Liu C, Xiong Y, Zuo Z, Zeng A, Li Y, Yu L, Shen G, Wang D, Xie Y, Ye T, Wei Y - Cell Death Dis (2015)

Bottom Line: Here, we reported our finding with nifuroxazide, an antidiarrheal agent identified as a potent inhibitor of Stat3.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.Notably, nifuroxazide reduced the number of myeloid-derived suppressor cell in the lung.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China.

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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Nifuroxazide induces breast cancer cells apoptosis. (a) 4T1 and MDA-MB-231 cells were treated with nifuroxazide at indicated doses for 24 h, 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, LR represents early apoptotic cells (positive for Annexin V only) and LL represents live cells. Graphical presentation of data obtained by Annexin V/PI staining after nifuroxazide treatment was also shown. Data are expressed as mean±S.D. from three independent experiments (*P<0.05; **P<0.01; ***P<0.001). (c) Western blot analyses of 4T1 cells treated (24 h) with different concentrations of nifuroxazide to evaluate protein expression of Bcl-2, cleaved caspase-3 and β-actin was used as a standard
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fig2: Nifuroxazide induces breast cancer cells apoptosis. (a) 4T1 and MDA-MB-231 cells were treated with nifuroxazide at indicated doses for 24 h, 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, LR represents early apoptotic cells (positive for Annexin V only) and LL represents live cells. Graphical presentation of data obtained by Annexin V/PI staining after nifuroxazide treatment was also shown. Data are expressed as mean±S.D. from three independent experiments (*P<0.05; **P<0.01; ***P<0.001). (c) Western blot analyses of 4T1 cells treated (24 h) with different concentrations of nifuroxazide to evaluate protein expression of Bcl-2, cleaved caspase-3 and β-actin was used as a standard

Mentions: We next explored whether nifuroxazide induced breast cancer cells apoptosis. Hoechst 33258 staining assay showed that nifuroxazide treated induced apoptosis in 4T1, MCF-7 and MDA-MB-231 cells, with the features of a bright-blue fluorescent-condensed nuclei and nuclear fragmentation (Supplementary Figure S1c). To further confirm the induction of apoptosis in 4T1 and MDA-MB-231 cells with nifuroxazide treatment, we also investigated the levels of apoptosis by low cytometry (FCM) using the Annexin V-FITC/PI dual-labeling technique. As shown in Figures 2a and b, after nifuroxazide treatment for 24 h, the apoptosis induction effects were observed. When the 4T1 cells were treated with 1.25 μM nifuroxazide, the apoptosis rate was 7.0%, whereas the apoptosis cells increased to 10.3, 13.4, 16.2, 18.7 and 42.8% when cells were treated with 1.25, 2.5, 5, 10 and 20 μM nifuroxazide, respectively, indicating that nifuroxazide was able to induce apoptosis in a concentration-dependent manner. Similarly, in MDA-MB-231 cells, the percentage of apoptotic cells was increased from 10.7 to 16.3, 35.1, 38.9, 46.3 and 52.6% after treatment with various concentrations of nifuroxazide for 24 h.


Nifuroxazide induces apoptosis and impairs pulmonary metastasis in breast cancer model.

Yang F, Hu M, Lei Q, Xia Y, Zhu Y, Song X, Li Y, Jie H, Liu C, Xiong Y, Zuo Z, Zeng A, Li Y, Yu L, Shen G, Wang D, Xie Y, Ye T, Wei Y - Cell Death Dis (2015)

Nifuroxazide induces breast cancer cells apoptosis. (a) 4T1 and MDA-MB-231 cells were treated with nifuroxazide at indicated doses for 24 h, 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, LR represents early apoptotic cells (positive for Annexin V only) and LL represents live cells. Graphical presentation of data obtained by Annexin V/PI staining after nifuroxazide treatment was also shown. Data are expressed as mean±S.D. from three independent experiments (*P<0.05; **P<0.01; ***P<0.001). (c) Western blot analyses of 4T1 cells treated (24 h) with different concentrations of nifuroxazide to evaluate protein expression of Bcl-2, cleaved caspase-3 and β-actin was used as a standard
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4385941&req=5

fig2: Nifuroxazide induces breast cancer cells apoptosis. (a) 4T1 and MDA-MB-231 cells were treated with nifuroxazide at indicated doses for 24 h, 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, LR represents early apoptotic cells (positive for Annexin V only) and LL represents live cells. Graphical presentation of data obtained by Annexin V/PI staining after nifuroxazide treatment was also shown. Data are expressed as mean±S.D. from three independent experiments (*P<0.05; **P<0.01; ***P<0.001). (c) Western blot analyses of 4T1 cells treated (24 h) with different concentrations of nifuroxazide to evaluate protein expression of Bcl-2, cleaved caspase-3 and β-actin was used as a standard
Mentions: We next explored whether nifuroxazide induced breast cancer cells apoptosis. Hoechst 33258 staining assay showed that nifuroxazide treated induced apoptosis in 4T1, MCF-7 and MDA-MB-231 cells, with the features of a bright-blue fluorescent-condensed nuclei and nuclear fragmentation (Supplementary Figure S1c). To further confirm the induction of apoptosis in 4T1 and MDA-MB-231 cells with nifuroxazide treatment, we also investigated the levels of apoptosis by low cytometry (FCM) using the Annexin V-FITC/PI dual-labeling technique. As shown in Figures 2a and b, after nifuroxazide treatment for 24 h, the apoptosis induction effects were observed. When the 4T1 cells were treated with 1.25 μM nifuroxazide, the apoptosis rate was 7.0%, whereas the apoptosis cells increased to 10.3, 13.4, 16.2, 18.7 and 42.8% when cells were treated with 1.25, 2.5, 5, 10 and 20 μM nifuroxazide, respectively, indicating that nifuroxazide was able to induce apoptosis in a concentration-dependent manner. Similarly, in MDA-MB-231 cells, the percentage of apoptotic cells was increased from 10.7 to 16.3, 35.1, 38.9, 46.3 and 52.6% after treatment with various concentrations of nifuroxazide for 24 h.

Bottom Line: Here, we reported our finding with nifuroxazide, an antidiarrheal agent identified as a potent inhibitor of Stat3.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.Notably, nifuroxazide reduced the number of myeloid-derived suppressor cell in the lung.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, China.

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.

Show MeSH
Related in: MedlinePlus