A Laboratory Study Of The Effect Of Crotoxin On Human Squamous Cell Carcinoma Of Esophagus

Objective: To observe the suppression effects of south america rattlesnake neurotoxin ( Crotoxin) on Eca-109 cells line of human carcinoma of esophagus , and observe the inhibitory efects of Crotoxin on Eca-109 cells xenografts in nude mice. To investigate the anti-tumor effect of the neurotoxin of south america rattlesnake ( Crotoxin) and explore its related mechanisms.Method: Eca-109 cells,a cell line of human carcinoma of esophagus,was used as the experimental materials. Eca-109 cells were cultured in vitro , use different saturation,s Crotoxin to Eca-109 cells, to set up negative group. MTT colorimetry was used to test the growth inhibition ratio of cells.The light microscope to be used to observe the morphology alter of Eca-109 cells,the fluorescence microscope to the alter of cell nucleus. The flow cytometry was used to detect the apoptosis rate and the cell cycle of Eca-109 cells treated with Cortoxin. Additionally, RT-PCR also be used to detect the gene amplification of BCL-2.P15 and P17. Eca-109cell suspension were implanted into the subcutaneous tissue of back,and establish nude mice ethotopic transplantation tumor model with human carcinoma of esophagus. Fractionate them to Crotoxin group and control group(Crotoxin group to be dilute to 25-100μg/kg by solution of 09%NcaI, nad control group were treated by the same way with equal volum solution of 09% NacI). The nude mices of both group were injected subcutancously to the perimeter of tumor once every other days,All animals were sacrificed at the next day after the 10th treatment. The inhibitory rate was calculated according to the weights of xenografts. Western blot to detect the expression of some proteins in the transplantation tumor,such asBcl-2.P15 and p17.Result: Crotoxin can inhibit the growth of Eca-109 cells,it is more effection than Cisplatin. Cortoxin can induce Eca-109 cells to apoptosis, The morphological chnages of apoptosis such as Cell shrinkage,chromatin condensation,nuclear fragmentation and apoptotic body were obeserved by both light and fluorescence microscope.Crotoxin can increases the gene amplification of P15 and P17, and grow downwards the Bcl-2. The flow cytometry can detect the apoptotic peak of Eca-109 cells ,and the higher concentration,the earer appearance of apoptotic peak,and the G1 stage cells were also grow in number. Crotoxin inhibited the xenografts growth significantly and the inhibitory rate was 35.7%.Western blot to be used to detect the expression of sproteins,it shows that P15 and P17 were increased,and Bcl-2 was decreased.Conclusion: Crotoxin can inhibit the growth of Eca-109 cells and the anti-tumor effect was closely related to the induction of apoptosis and cell cycle blockage. The mechanism of Crotoxin to induce apoptosis was contribute to the activation of Caspase-3. Crotoxin can inhibit the growth of Eca-109 cells xenografts in nude mice,and it,s inhibition relate to the dosage.