title: Growth inhibitory effects and molecular mechanisms of crotoxin treatment in esophageal Eca-109 cells and transplanted tumors in nude mice.
abstract: OBJECTIVE
  To investigate the antitumor actions of the Crotalus durissus neurotoxin (crotoxin) on human esophageal carcinoma (Eca-109) cells in vitro and transplanted esophageal Eca-109 tumors in nude mice.
  
  
  METHODS
  The growth-inhibitory effect was analyzed in Eca-109 cells using MTT assay. Cell morphology changes in nuclei were observed using Hoechst 33342 staining, while apoptosis and cell cycle distribution were examined by flow cytometry. RT-PCR was used to measure the Bcl-2, p15, and caspase-3 p17 gene expression levels. A tumor transplantation model was established by inoculation of Eca-109 cells were into female Balb/c nude mice. Crotoxin (25, 50, and 100 mg/kg) was subcutaneously injected into the transplanted tumors every 2 d for a total of 10 injections. Tumor size and weight were measured. Bcl-2, p15, and caspase-3 p17 protein expression in transplanted tumors was analyzed using Western blotting.
  
  
  RESULTS
  Crotoxin (25, 50, and 100 μg/mL) inhibited the growth of Eca-109 cells in a dose-dependent manner with inhibition rates of 22.9%, 35.8%, and 57.2%, respectively. Hoechst 33342 staining revealed apoptotic cells with pyknotic nuclear chromatin after crotoxin treatment. In Eca-109 cells, crotoxin induced apoptosis and G1 block, significantly upregulated the expression of p15 and caspase-3 p17 genes and downregulated the expression of Bcl-2 gene. Furthermore, crotoxin inhibited the growth of Eca-109 tumors in nude mice in a dose-dependent manner. Western blotting showed that crotoxin increased p15 and caspase-3 p17 protein levels and reduced Bcl-2 protein level in tumor specimens.
  
  
  CONCLUSIONS
  Crotoxin inhibits the growth of Eca-109 cells in vitro via apoptosis induction and G1 block. Local administration of crotoxin inhibits the growth of subcutaneously transplanted Eca-109 cells in nude mice, possibly via increasing p15 and caspase-3 p17 protein expression and reducing Bcl-2 protein expression.
authors.affiliation: Department of Cardiothoracic Surgery, the First Affiliated Hospital of Soochow University, Suzhou, China.
authors.forename: Jing-kang
authors.initials: JK
authors.lastname: He
authors.forename: Xiang-sheng
authors.initials: XS
authors.lastname: Wu
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Rong
authors.initials: R
authors.lastname: Han
authors.forename: Zheng-hong
authors.initials: ZH
authors.lastname: Qin
authors.forename: Yan
authors.initials: Y
authors.lastname: Xie
mesh_terms: D000818:Animals; D053148:Caspase 3 / Q000235:genetics; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D017839:Crotalus / Q000378:metabolism; D003439:Crotoxin / Q000494:pharmacology / Q000627:therapeutic use*; D004938:Esophageal Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D004947:Esophagus / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D019254:Genes, bcl-2 / Q000187:drug effects; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude
chemical_list: D003439:Crotoxin; D053148:Caspase 3