title: Naringenin prevents TGF-β1 secretion from breast cancer and suppresses pulmonary metastasis by inhibiting PKC activation.
abstract: BACKGROUND
  Targeting the TGF-β1 pathway for breast cancer metastasis therapy has become an attractive strategy. We have previously demonstrated that naringenin significantly reduced TGF-β1 levels in bleomycin-induced lung fibrosis and effectively prevented pulmonary metastases of tumors. This raised the question of whether naringenin can block TGF-β1 secretion from breast cancer cells and inhibit their pulmonary metastasis.
  
  
  METHODS
  We transduced a lentiviral vector encoding the mouse Tgf-β1 gene into mouse breast carcinoma (4T1-Luc2) cells and inoculated the transformant cells (4T1/TGF-β1) into the fourth primary fat pat of Balb/c mice. Pulmonary metastases derived from the primary tumors were monitored using bioluminescent imaging. Spleens, lungs and serum (n = 18-20 per treatment group) were analyzed for immune cell activity and TGF-β1 level. The mechanism whereby naringenin decreases TGF-β1 secretion from breast cancer cells was investigated at different levels, including Tgf-β1 transcription, mRNA stability, translation, and extracellular release.
  
  
  RESULTS
  In contrast to the null-vector control (4T1/RFP) tumors, extensive pulmonary metastases derived from 4T1/TGF-β1 tumors were observed. Administration of the TGF-β1 blocking antibody 1D11 or naringenin showed an inhibition of pulmonary metastasis for both 4T1/TGF-β1 tumors and 4T1/RFP tumors, resulting in increased survival of the mice. Compared with 4T1/RFP bearing mice, systemic immunosuppression in 4T1/TGF-β1 bearing mice was observed, represented by a higher proportion of regulatory T cells and myeloid-derived suppressor cells and a lower proportion of activated T cells and INFγ expression in CD8(+) T cells. These metrics were improved by administration of 1D11 or naringenin. However, compared with 1D11, which neutralized secreted TGF-β1 but did not affect intracellular TGF-β1 levels, naringenin reduced the secretion of TGF-β1 from the cells, leading to an accumulation of intracellular TGF-β1. Further experiments revealed that naringenin had no effect on Tgf-β1 transcription, mRNA decay or protein translation, but prevented TGF-β1 transport from the trans-Golgi network by inhibiting PKC activity.
  
  
  CONCLUSIONS
  Naringenin blocks TGF-β1 trafficking from the trans-Golgi network by suppressing PKC activity, resulting in a reduction of TGF-β1 secretion from breast cancer cells. This finding suggests that naringenin may be an attractive therapeutic candidate for TGF-β1 related diseases.
authors.affiliation: Protein & Peptide Pharmaceutical Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Fayun
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Protein & Peptide Pharmaceutical Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Dong
authors.affiliation: Protein & Peptide Pharmaceutical Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Wenfeng
authors.initials: W
authors.lastname: Zeng
authors.affiliation: Department of Gynecology, The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, 434000, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Protein & Peptide Pharmaceutical Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Gynecology, The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, 434000, China.
authors.forename: Yuqi
authors.initials: Y
authors.lastname: Qiu
authors.affiliation: Protein & Peptide Pharmaceutical Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Luoyang
authors.initials: L
authors.lastname: Wang
authors.affiliation: Protein & Peptide Pharmaceutical Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Xiaozhe
authors.initials: X
authors.lastname: Yin
authors.affiliation: Protein & Peptide Pharmaceutical Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. zhangcl@moon.ibp.ac.cn.
authors.forename: Chunling
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Protein & Peptide Pharmaceutical Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. weixx@ibp.ac.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Liang
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D005260:Female; D044950:Flavanones / Q000008:administration & dosage*; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000473:pathology / Q000556:secondary; D015674:Mammary Neoplasms, Animal / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D009362:Neoplasm Metastasis; D015398:Signal Transduction / Q000187:drug effects; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D044950:Flavanones; D053773:Transforming Growth Factor beta1; C005273:naringenin
keywords: Breast cancer metastasis; Naringenin; PKC activation; TGF-β1 secretion