Study On The Mechanism Of Human Primary Gastric Cancer Cell Apoptosis Induced By AHVAC-?

Objective:Isolate and purify human primary gastric cancer cells,and to investigate the effects of Agkis-trodon halys venom anti-tumor component-I?AHVAC-I?on human primary gastric cancer cells and its mechanism.Methods:Trypsin digestion and differential adherence were used to isolate and purify human primary gastric cancer cells as in vitro research subjects.Following the CCK8proliferation test evaluation,cell proliferation and the IC₅₀ for AHVAC-I were obtained.Cultured human primary gastric cancer cells were randomly divided into 4 groups,and then different concentration?0,4,8,16?g/ml?of AHVAC-I were added and incubated for 24h.Hematoxylin-eosin staining?HE?was used to observe the morphological changes of human primary gastric cancer cells.Flow cytometry and immunohistochemical staining were used to analyze the apoptosis effects of AHVAC-I on human primary gastric cancer cells.Then,the effect of AHVAC-I on the oxidative stress in human primary gastric cancer cells was detected and the downstream Bax,and Cyt-C,and Caspase-3,and Bcl-2 protein expression levels was detected by western blot assay.Results:1.Seven cases of human primary gastric cancer cells were successfully isolated and cultured.2.After 24 hours of AHVAC-I treatment of human primary gastric cancer cells,CCK-8experimental results show that AHVAC-I can effectively inhibit cell proliferation in a concentration-dependent manner and the IC₅₀0 value was 17.77457?g/ml.Then,set the AHVAC-I experimental concentration group to?0,4,8,16?g/ml?according to IC₅₀value.The flow cytometry and immunohistochemistry were used to detect the apoptosis effects at different concentrations.The results showed that the apoptosis rate of human primary gastric cancer cells increased with the concentration of AHVAC-I at 0,4,8,16?g/mL,showing a dose-dependent effect.3.After 24 hours of AHVAC-I effect on human primary gastric cancer cells,the ROS level and Mn-SOD activity were significantly increased with the increase of AHVAC-I concentration,and the total antioxidant capacity of the cells decreased with the increase of AHVAC-I concentration.Western blot results showed that AHVAC-I significantly up-regulated the expression levels of Bax,Cyt-c,Caspase-3,and decreased the expression levels of bcl-2 protein in human primary gastric cancer cells.Conclusion:In conclusion,our study demonstrated the toxicity of AHVAC-I in human primary gastric cancer cells,which inhibited the proliferation of human primary gastric cancer cells in a dose-dependent manner via activating the ROS-Bax pathway.These findings may contribute to better understanding the effects of AHVAC-I on gastric cancer.