Suppression Effects Of Crotoxin On Human Lung Adenocarcinoma Cell Line A549

Part1 In vitro inhibition of Crotoxin on human lung adenocarcinoma cell line A549 and the related mechanismsObjective To observe the anti-tumor effect of Crotoxin in vitro and explore its related mechanisms.Methods A549 cells, a cell line of human lung adenicarinoma (wild-type p53), was used as the experimental materials. MTT colorimetry was used to test the growth inhibition ratio, and the flat colony experiment reflected the colony formation of cells. Both of the experiments were used to estimate the effects of Crotoxin and its combination with Iressa. The flow cytometry was used to detect the apoptosis rate and the cell cycle of A549 cells treated with Cortoxin . Additionally, we also used Western Blot to detect the expression of some proteins, such as wt p53, p17, the active subunit of caspase-3, phospho-P38MAPK. Futhermore, we used SB203580 to inhibit the activity of P38MAPK to observe whether A549 cells have some changes after treated by Crotoxin.Results Crotoxin can not only inhibit the growth of A549 but also has synergetic or additive effect when it is combined with Iressa.Cortoxin can induce A549 cells to apoptosis and block them in G1 period, and it can also up-regulate the expression of wt p53, p17, phospho-P38MAPK. When using SB203580 to inhibit the activity of P38MAPK, apoptosis was almost disappeared and there was no expression of p17, but the blockage of G1 period remained unchanged, wt p53 was still upregulated.Conclusion Crotoxin can inhibit the growth of A549 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 P38MAPK, which further activate Caspase-3.The G1 blockage possibly due to up-regulation of wt p53.Part2 Growth--inhibiting efects of Crotoxin on A549 cells xenografts in nude miceObjective To observe the inhibitory efects of Crotoxin on A549 cells xenografts in nude mice and the influence of angiogenesis.Methods A549 cells were incubated and were inoculated subcutaneous1y in athymic nude mice to establish xenograft models.The mice were divided into control group and Crotoxin group. The inhibitory rate was calculated according to the weights of xenografts.The intratumoral microvessel density(MVD) was evaluated by immunohistochemical staining. The electron microscop was used to observe the morphological change of microvess endothelium.Results Inhibited the xenografts growth significantly and the inhibitory rate was 36.3%. Immunohistochemical results revealed that the MVD in Crotoxin group(13.56±3.27)was signifieanfly lower than those in control group(34.29±8.64),(P<0.05). Decrease of microvessels in the xenograft tumor and morphological change of endothelium were observed under electron microscop.Conclusion Crotoxin can inhibit the growth of A549 cells xenografts in nude mice, which may be due to the inhibit tumor angiogenesis.