Growth inhibitory effects and molecular mechanisms of crotoxin treatment in esophageal Eca-109 cells and transplanted tumors in nude mice

摘要

Aim: 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.Conclusion: 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.