title: Antiproliferative effects of cinobufacini on human hepatocellular carcinoma HepG2 cells detected by atomic force microscopy.
abstract: OBJECTIVE
  To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action.
  
  
  METHODS
  HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level.
  
  
  RESULTS
  MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P < 0.05). FCM analysis demonstrated cell cycle arrest at S phase induced by cinobufacini. The immunofluorescence studies of cytoskeletal and nuclear morphology showed that after cinobufacini treatment, the regular reorganization of actin filaments in HepG2 cells become chaotic, while the nuclei were not damaged seriously. Additionally, high-resolution AFM imaging revealed that cell morphology and ultrastructure changed a lot after treatment with cinobufacini. It appeared as significant shrinkage and deep pores in the cell membrane, with larger particles and a rougher cell surface.
  
  
  CONCLUSIONS
  Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity.
authors.affiliation: Qing Wu, Wei-Dong Lin, Guan-Qun Liao, Li-Guo Zhang, Shun-Qian Wen, Jia-Ying Lin, Department of General Surgery, the Affiliated Foshan Hospital of Southern Medical University, Foshan 528000, Guangdong Province, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Wu
authors.affiliation: Qing Wu, Wei-Dong Lin, Guan-Qun Liao, Li-Guo Zhang, Shun-Qian Wen, Jia-Ying Lin, Department of General Surgery, the Affiliated Foshan Hospital of Southern Medical University, Foshan 528000, Guangdong Province, China.
authors.forename: Wei-Dong
authors.initials: WD
authors.lastname: Lin
authors.affiliation: Qing Wu, Wei-Dong Lin, Guan-Qun Liao, Li-Guo Zhang, Shun-Qian Wen, Jia-Ying Lin, Department of General Surgery, the Affiliated Foshan Hospital of Southern Medical University, Foshan 528000, Guangdong Province, China.
authors.forename: Guan-Qun
authors.initials: GQ
authors.lastname: Liao
authors.affiliation: Qing Wu, Wei-Dong Lin, Guan-Qun Liao, Li-Guo Zhang, Shun-Qian Wen, Jia-Ying Lin, Department of General Surgery, the Affiliated Foshan Hospital of Southern Medical University, Foshan 528000, Guangdong Province, China.
authors.forename: Li-Guo
authors.initials: LG
authors.lastname: Zhang
authors.affiliation: Qing Wu, Wei-Dong Lin, Guan-Qun Liao, Li-Guo Zhang, Shun-Qian Wen, Jia-Ying Lin, Department of General Surgery, the Affiliated Foshan Hospital of Southern Medical University, Foshan 528000, Guangdong Province, China.
authors.forename: Shun-Qian
authors.initials: SQ
authors.lastname: Wen
authors.affiliation: Qing Wu, Wei-Dong Lin, Guan-Qun Liao, Li-Guo Zhang, Shun-Qian Wen, Jia-Ying Lin, Department of General Surgery, the Affiliated Foshan Hospital of Southern Medical University, Foshan 528000, Guangdong Province, China.
authors.forename: Jia-Ying
authors.initials: JY
authors.lastname: Lin
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D000664:Amphibian Venoms / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000648:ultrastructure*; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure; D049109:Cell Proliferation / Q000187:drug effects*; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force; D018613:Microscopy, Confocal; D059807:S Phase Cell Cycle Checkpoints / Q000187:drug effects
chemical_list: D000664:Amphibian Venoms; D000970:Antineoplastic Agents; C545813:huachansu
keywords: Atomic force microscopy; Cell viability; Cinobufacini; HepG2 cells; Hepatocarcinoma