title: Jacarelhyperol A induced apoptosis in leukaemia cancer cell through inhibition the activity of Bcl-2 proteins.
abstract: Hypericum japonicum Thunb. ex Murray is widely used as an herbal medicine for the treatment of hepatitis and tumours in China. However, the molecular mechanisms of its effects are unclear. Our previous research showed that extracts of H. japonicum can induce apoptosis in leukaemia cells. We also previously systematically analysed and isolated the chemical composition of H. japonicum.
  
  
  
  The fluorescence polarisation experiment was used to screen for inhibitors of Bcl-2 proteins which are proved as key proteins in apoptosis. The binding mode was modelled by molecular docking. We investigated the proliferation attenuating and apoptosis inducing effects of active compound on cancer cells by MTT assay and flow cytometry analysis. Activation of caspases were tested by Western blot. A broad-spectrum caspase inhibitor Z-VAD-FMK was used to investigate the caspases-dependence. In addition, co-immunoprecipitation was performed to analyse the inhibition of heterodimerization between anti-apoptotic Bcl-2 proteins with pro-apoptotic proteins. Moreover, in vivo activity was tested in a mouse xenograph tumour model.
  
  
  
  Jacarelhyperol A (Jac-A), a characteristic constituent of H. japonicum, was identified as a potential Bcl-2 inhibitor. Jac-A showed binding affinities to Bcl-xL, Bcl-2, and Mcl-1 with Ki values of 0.46 μM, 0.43 μM, and 1.69 μM, respectively. This is consistent with computational modelling results, which show that Jac-A presents a favorable binding mode with Bcl-xL in the BH3-binding pocket. In addition, Jac-A showed potential growth inhibitory activity in leukaemia cells with IC50 values from 1.52 to 6.92 μM and significantly induced apoptosis of K562 cells by promoting release of cytochrome c and activating the caspases. Jac-A also been proved that its effect is partly caspases-dependent and can disrupt the heterodimerization between anti-apoptotic Bcl-2 proteins with pro-apoptotic proteins. Moreover, Jac-A dose-dependently inhibited human K562 cell growth in a mouse xenograph tumour model with low toxicity.
  
  
  
  In this study, a characteristic constituent of H. japonicum, Jac-A, was shown to induce apoptosis in leukaemia cells by mediating the Bcl-2 proteins. Therefore, we propose a new lead compound for cancer therapy with a low toxicity, and have provided evidence for using H. japonicum as an anti-cancer herb.
authors.forename: Shoude
authors.initials: S
authors.lastname: Zhang
authors.forename: Jun
authors.initials: J
authors.lastname: Yin
authors.forename: Xia
authors.initials: X
authors.lastname: Li
authors.forename: Jigang
authors.initials: J
authors.lastname: Zhang
authors.forename: Rongcai
authors.initials: R
authors.lastname: Yue
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Diao
authors.forename: Honglin
authors.initials: H
authors.lastname: Li
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.affiliation: School of Pharmacy, Second Military Medical University, 325# Guohe Road, Shanghai 200433, China. shanleicn@126.com.
authors.forename: Lei
authors.initials: L
authors.lastname: Shan
authors.forename: Weidong
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001665:Binding Sites; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D004195:Disease Models, Animal; D004354:Drug Screening Assays, Antitumor; D005260:Female; D006801:Humans; D020128:Inhibitory Concentration 50; D020014:K562 Cells; D007938:Leukemia / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D055503:Protein Multimerization / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D014966:Xanthenes / Q000737:chemistry / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays; D051020:bcl-X Protein / Q000737:chemistry / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D014966:Xanthenes; D051020:bcl-X Protein; C458679:jacarelhyperol A; D045304:Cytochromes c; D020169:Caspases