The Effect And Mechanism Of Anticancer Biological Activity Peptide Inhibites Proliferation And Induces Apoptosis Of Lung Cancer Cells Through Hedgehog Signaling Pathway

Objective To investigate the anti-tumor effect of Anticancer biological activity peptide(ACBP)on hedgehog signaling pathway of human lung cancer NCI-H226?NCI-H460?NCI-H1299?A549 and A549/DDP cells.Methods Based on the culture of human lung cancer NCI-H226?NCI-H460?NCI-H1299?A549 and A549/DDP cells in vitro,the effect of ACBP on growth of this human lung cancer cells was evaluated by MTT.The cell morphology,cell membrane structure,apoptosis in the early,cell migration ability,and cell cycle were detected by Hematoxylin-eosin(HE)staining,Annexin-V/PI staining,cell migration assay and flow cytometry respectively.The expression of Gli2 and Gli3 protein and the expression of Gli2,Gli3 and miR-132/212 gene was obtained by Western-Blot and fluorescence quantitative PCRanalysis,respectively.Results(1)The results of MTT showed that ACBP had a significant antiproliferative effect on lung cancer cells in a dose-dependent manner.Compared with 5-Fu,the IC50 value of AC BP/5-Fu on lung cancer cells decreased.(2)HE staining showed morpholgical characteristic changes of apoptosis appeared after treatment with ACBP and AC BP/5-Fu for 24 h,such as small nuclei?chromatin condensation and apoptotic bodies,and there are no significant apoptosis characteristics after treatment with 5-Fu.(3)The cell migration and scratch test showed that after treatment of each group for 20 h.they all could inhibit the migration ability of lung cancer cells,ACBP and ACBP/5-Fu group was more significant than 5-Fu.(4)The Annexin-V/PI staining by flow cytometry showed that the apoptos is rates of lung cancer cells were increased after treatment with ACBP and AC BP / 5-Fu for 24h(P <0.01).Compared with the control group,the apoptosis of NCI-H226 cell did not changed,and the apoptosis of the other lung cells were increased after treatment with 5-Fu for 24 h.The results of cell cycle by flow cytometry showed that ACBP,5-Fu and ACBP/5-Fu could block the cell cycle of lung cancer in G0/G1 stage after treatment with each group for 24 h..(5)Western blot analysis indicated that the expression leve l of Gli2 protein was decreased(P <0.01),while the expression level of Gli3 protein was increased(P <0.01)after treatment with AC BP and ACBP/5-Fu for 24 h.And the expression levels of Gli2 and Gli3 did not change after treatment with 5-Fu for 24 h.(6)The expression of Gli3 gene in ACBP and ACBP / 5-Fu group was decreased by Fluorescence quantitative PCR analysis showed that the expression of Gli2 and the expression of miR-132 and miR-212 genes were decreased(P <0.01),while the expression level of Gli3 gene was increased(P <0.01)after treatment with ACBP and ACBP/5-Fu for 24 h.And the expression levels of Gli2?Gli3 and miR-132/212 did not change after treatment with 5-Fu for 24 h.Conclusion(1)ACBP had a significant antiproliferative effect on human lung ca ncer NCI-H226?NCI-H460?NCI-H1299?A549 and A549/DDP cells in a dose—dependent manner.This was proved The research confirmed that(1)AC BP inhibited the hedgehog signaling pathway of human lung cancer cell,and induced cell apoptosis and blocked cell cycle in G0/G1 phase(2)ACBP effectively inhibited the expression of miR-132/212 family of human lung cancer cells,acted on the hedgehog signaling pathway indirectly,and inhibited the invasion and metastasis of lung cancer cells.(2)The combination of AC BP and 5-Fu could increase the effect of decreasing the proliferation of NCI-H226,NCI-H460,NCI-H1299,A549 and A549 /DDP cells in differentiated lung cancer,and increase the 5-Fu antitumor activity...