Study On The Mechanism Of NCI-H661 Cells Apoptosis Induced By Crotoxin

Objective To oberve anti tumor effect of crotoxin on human large cell lung cancer cell line Which cultured in vitro and the effect of crotoxin on human large cell lung cancer cells xenografts in nude mice tumor inhibition,and to explore its related mechanism of action.Methods The CCK-8 colorimetry was used to test the growth inhibition rate of crotoxin,gefinitib and the two drugs combination on human large cell lung cancer NCI-H661 cell line,and the flat colony experiment oberved the colony formation of cells.To study the effect of crotoxin,gefinitib and the two drugs combination on NCI-H661 cell cycle and apoptosis rate by flow cytometry,and the changed of cell cycle and apoptosis rate of NCI-H661 cells, after inhibited p38 activity by SB203580 or inhibited p53 activity with PFT-α; It was used to detect the expression of p38 MAPK, phospho-p38 MAPK, wtp53, Bax, Bcl-2 and the activity of Caspase-3 subunit p17 protein by the Western blot in NCI-H661 cells,which was treated by crotoxin,gefinitib and the two drug combination, and the changed of these proteins in NCI-H661 cells,after inhibited p38 activity by SB203580 or inhibited p53 activity with PFT-α. To adjust human large cell lung cancer NCI-H661 cells into nude mice axilla to establish tumor bearing nude mice model. The tumor bearing mice were randomly divided into 6 groups,which were crotoxin, gefinitib, crotoxin+ gefiniti, crotoxin + SB203580, crotoxin + PFT-αgroup and negative control group. The experimental mice were administered by intraperitoneal injection, ministered once every three days, at the same time, the negative control group was injected with normal saline. To removal completely of subcutaneous transplantation tumor nodules after 4 weeks, compare different intervention group with control group in tumor weight, calculate tumor inhibitory rate.Results Crotoxin can inhibit the growth of NCI-H661 cells, and it can enhance the anti-tumor effect of gefinitib combined with Crotoxin and gefinitib,it was a significant difference compared with the control group(P<0.05); Crotoxin can inhibit NCI-H661 cells colony, combined with gefinitib can enhance the inhibitory effect of gefinitib on NCI-H661 cell colony, there was a significant difference compared with the control group(P<0.05). Crotoxin can induce the apoptosis of human large cell lung cancer cell line NCI-H661,and block NCI-H661 cells in the G1 phase of the cell cycle; the function of crotoxin on inducding apoptosis and cell cycle arrest in G1 phase can be suppressed after inhibited p38 activity by SB203580 or inhibited p53 activity with PFT-α. In the Western blot experiment, the crotoxin, gefinitib, crotoxin+gefinitib group expression level of phospho-P38 MAPK, wt P53, the activity of Caspase3 subunit P17 and bax protein increased compared with the control group,and the expression of crotoxin group closed to gefinitib group, the crotoxin+gefinitib group expression was higher than that of the former two; The expression level of Bcl-2 protein decreased which compared with the control group, and the crotoxin+gefinitib group was lower than that of crotoxin, the gefinitib group, the difference was statistically significant. In group crotoxin+SB203580, the expression level of phospho-P38 MAPK, wt P53, Caspase3 activity subunit P17, bax, bcl-2 protein is similar to the control group, the difference was not statistically significant. The protein expression level of phospho-P38 MAPK, wt P53 was higher than that of control group, the difference was statistically significant; The Caspase3 activity subunit P17, bax, bcl-2 protein expression was not significantly changed compared with the control group, the difference was not statistically significant. Close to the level of control group the expression of P protein in 38 MAPK group, no significant difference.It was closed to the level of control group the expression of p38 MAPK protein in each group, there was no significant difference. Crotoxin, gefinitib caninhibit growth of human lung cancer NCI-H661 cells transplanted tumor, the combination of the two can significantly improve the tumor growth inhibition effect, the inhibition rate were 39.5%, 43.7% and 75.1%, it was statistically significant difference compared with the negative control group(P<0.05).Conclusion Crotoxin can inhibit the growth of human large cell lung cancer cell line NCI-H661 in vitro, combined with gefinitib can enhance the anti-tumor effect of gefinitib, it was related with the induction of apoptosis and G1 phase arrest, Induction of apoptosis may be associated with that crotoxin activated P38 MAPK signal transduction pathway then activated caspase3 protein, G1 phase arrest may be caused by the higher expression of wt P53 protein in cells. Crotoxin on human lung cancer NCI-H661 cells transplanted tumor growth inhibition, and crotoxin can enhance the inhibition effect of gefinitib on NCI-H661 cells xenografts in nude mice.