title: Total coumarins of Hedyotis diffusa induces apoptosis of myelodysplastic syndrome SKM-1 cells by activation of caspases and inhibition of PI3K/Akt pathway proteins.
abstract: BACKGROUND
  Hedyotis diffusa is an ethno-medicine used for anti-cancer treatment in the clinic of traditional Chinese medicine (TCM). The total coumarins of Hedyotis diffusa (TCHD) was a selected extract with observed antiproliferative activity, which has not been tested in treatment of myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML).
  
  
  OBJECTIVE
  This study aimed to evaluate the apoptosis-inducing effect of TCHD on human MDS cell line (SKM-1) and explore its action mechanism in association with caspase family and PI3K/Akt signaling pathway.
  
  
  METHODS
  The chemical constituents and total coumarins content of TCHD were determined by High Performance Liquid Chromatography-tandem mass spectrometry (HPLC-MS/MS) and UV-vis spectrophotometry, respectively. MTT assay, Hoechst 33258 staining, and Annexin V-FITC/PI double labeling were applied to evaluate TCHD's efficacy on SKM-1 cells. Western blot analysis was also used to clarify the action mechanism of TCHD on protein expression level.
  
  
  RESULTS
  Two compounds, p-coumaric acid and E-6-O-p-coumaroyl scandoside methyl ester, were identified in TCHD, and its total coumarins content reached 87.4%. By MTT assay, apoptosis-inducing effect of TCHD on SKM-1 cells was found in a dose-dependent manner after 24-48h treatment, with IC50values of 104.48μg/ml and 100.66μg/ml, respectively. Morphological and flow cytometry observation also confirmed such effect of TCHD. Western blot analysis clarified its action mechanism associating with the activation of caspases and inhibition of PI3K/Akt pathway proteins.
  
  
  CONCLUSIONS
  This is the first report regarding the apoptosis-inducing efficacy and mechanism of TCHD on SKM-1 cells, providing a promising candidate of TCM for MDS and AML therapy with fewer side effects.
authors.affiliation: The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, PR China; Zhejiang Chinese Medical University, Hangzhou 310053, PR China.
authors.forename: Jianping
authors.initials: J
authors.lastname: Jiang
authors.affiliation: The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, PR China.
authors.forename: Bo
authors.initials: B
authors.lastname: Wang
authors.affiliation: The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, PR China.
authors.forename: Baodong
authors.initials: B
authors.lastname: Ye
authors.affiliation: The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, PR China. Electronic address: lsyww2012@163.com.
authors.forename: Shengyun
authors.initials: S
authors.lastname: Lin
authors.affiliation: The First Affiliated Hospital of Zhejiang University, Hangzhou 310003, PR China.
authors.forename: Wenbin
authors.initials: W
authors.lastname: Qian
authors.affiliation: Zhejiang Chinese Medical University, Hangzhou 310053, PR China. Electronic address: letian.shan@hotmail.com.
authors.forename: Letian
authors.initials: L
authors.lastname: Shan
authors.affiliation: University of Mainz, Mainz 55128, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Efferth
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003374:Coumarins / Q000494:pharmacology*; D032064:Hedyotis; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000187:drug effects; D009190:Myelodysplastic Syndromes / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism
chemical_list: D003374:Coumarins; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D020169:Caspases
keywords: Apoptosis; Hedyotis diffusa; Myelodysplastic syndromes; SKM-1; Total coumarins; Traditional Chinese medicine