title: MHP-1 inhibits cancer metastasis and restores topotecan sensitivity via regulating epithelial-mesenchymal transition and TGF-β signaling in human breast cancer cells.
abstract: BACKGROUND
  Cordyceps has long been used to treat cancer. However, its pharmacologically active components as well as the molecular mechanisms underlying its effects are still unclear.
  
  
  OBJECTIVE
  To investigate the effect of MHP-1, a newly isolated polysaccharide from Mortierella hepialid (the asexual structure of C. sinensis), on breast cancer metastasis.
  
  
  METHODS
  The effect of MHP-1 on breast cancer cell migration, epithelial-mesenchymal transition (EMT) and TGF-β signaling were investigated in vitro and in vivo. The effect of MHP-1 against topotecan-resistant MCF-7 cells that developed an EMT-like phenotype was also examined.
  
  
  METHODS
  The in vitro effect of MHP-1 on breast cancer cell proliferation and migration was evaluated by CCK8 and transwell assay. Morphological changes were observed and EMT markers were detected by western blot. The production of MMPs was measured by quantitative PCR and ELISA assay. To further investigate the mechanism that MHP-1 inhibited breast cancer EMT, western blot, ELISA, luciferase reporter gene assay, siRNA, quantitative PCR, immunohistochemistry, and xenograft tumor model were performed.
  
  
  RESULTS
  MHP-1 inhibited breast cancer cell migration but did not cause any cytotoxicity. MHP-1 significantly surpressed breast cancer EMT, and slightly decreased MMP-9 secretion. TGF-β signaling was selectively inhibited after MHP-1 treatment, and other EMT-related pathways, like Wnt and Notch, were not affected. MHP-1 reduced the secretion of TGF-β1, but rarely affected other EMT-induced cytokines. Dual luciferase assay and Smad2/3 phosphorylation analysis indicated that MHP-1 suppressed TGF-β signaling. We further showed that MHP-1 restored sensitivity in topotecan (TPT)-resistant MCF-7 cells that developed an EMT-like phenotype. Similarly, the effect of TPT on resistant MCF-7 cells was also increased either by ALK5 (TGFβRI) siRNA or by a small molecular inhibitor of ALK5, SB-431542. MHP-1 inhibited breast cancer metastasis in the MDA-MB-231 xenograft model, and the immunohistochemical staining showed dramatically decreased expression of ALK5 and vimentin, and increased expression of E-cadherin.
  
  
  CONCLUSIONS
  MHP-1 significantly inhibited breast cancer metastasis and restored drug sensitivity in TPT-resistant cells via down-regulation of TGF-β signaling and EMT program. The combination of non-toxic agents like MHP-1 and current anti-cancer drugs should be considered in the future treatment of cancer.
authors.affiliation: Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Sensen
authors.initials: S
authors.lastname: Lin
authors.affiliation: Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Lyu
authors.affiliation: Jiangsu Cancer Hospital, Nanjing 210009, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Yu
authors.affiliation: Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Li
authors.initials: L
authors.lastname: Sun
authors.affiliation: Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Danyu
authors.initials: D
authors.lastname: Du
authors.affiliation: Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Yanqi
authors.initials: Y
authors.lastname: Lai
authors.affiliation: Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Hongyang
authors.initials: H
authors.lastname: Li
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Luyong
authors.initials: L
authors.lastname: Zhang
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China. Electronic address: yinhongping63@hotmail.com.
authors.forename: Hongping
authors.initials: H
authors.lastname: Yin
authors.affiliation: Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China. Electronic address: 13914798635@163.com.
authors.forename: Shengtao
authors.initials: S
authors.lastname: Yuan
mesh_terms: D001943:Breast Neoplasms / Q000188:drug therapy* / Q000503:physiopathology; D045744:Cell Line, Tumor / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects*; D032661:Cordyceps / Q000737:chemistry; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D005260:Female; D006801:Humans; D061986:MCF-7 Cells / Q000187:drug effects*; D009362:Neoplasm Metastasis / Q000188:drug therapy*; D010936:Plant Extracts / Q000494:pharmacology* / Q000627:therapeutic use; D011134:Polysaccharides / Q000494:pharmacology* / Q000627:therapeutic use; D019772:Topotecan / Q000627:therapeutic use*
chemical_list: D010936:Plant Extracts; D011134:Polysaccharides; D019772:Topotecan
keywords: Cancer metastasis; EMT; MHP-1; TGF-β; Topotecan resistance