Effect of butorphanol on the proliferation,apoptosis,and angiogenesis of lung cancer cells by regulating the sonic hedgehog/glioma-associated oncogene homolog 1 signaling pathway
Objective To investigate the effect of butorphanol on the proliferation,apoptosis and angiogenesis of lung cancer cells by regulating the sonic hedgehog(SHH)/glioma-associated oncogene homolog 1(GLI1)signaling pathway.Methods The effect of 0.5,1.0,2.0,4.0,8.0,and 16.0 µmol/L butorphanol on the proliferation of lung cancer A549 cells was determined by 2,5-diphe-nyl-2H-tetrazolium bromide(MTT)assay to determine the optimal intervention concentration.According to the completely random method,lung cancer A549 cells were divided into five groups(6 wells per group):a control group,a cyclopamine group,a butorphanol group,a butorphanol+no-loaded plasmid(pcDNA-NC)group,and a butorphanol+overexpressed SHH plasmid(pc-SHH)group.There was no treatment in the control group,while 10 μmol/L cyclopamine was added to the cyclopamine group,8 μmol/L of butorphanol was added to the butorphanol group.Meanwhile,8 μmol/L butorphanol was added to the pcDNA-NC group followed by transfection of pcDNA-NC;8 µmol/L butorphanol was added to the butorphanol+pc-SHH group followed by transfection of pc-SHH.The optical densi-ties at 490 nm D490(24 h and 48 h),proliferation rate,apoptosis rate,and the levels of SHH messenger RNA(mRNA)and GLI1 mRNA were detected by MTT assay,5 acetyli-2'deoxyuridine(Edu)staining,flow cytometry,and real-time fluorescence quantitative poly-merase chain reaction(FQ-PCR),respectively.The cell lumen of each group was observed by three-dimensional culture using a Matri-gel matrix.The levels of vascular endothelial growth factor(VEGF-A),vascular endothelial cadherin(VE-cadherin),B-cell lymphoma-2(Bcl-2),B-cell lymphoma-2-associated X protein(Bax),SHH,and GLI1 were measured by Western blot.Results Butorphanol at 0.5-16.0 μmol/L were able to inhibit the proliferation activity of A549 cells in a concentration-dependent manner,with a half maximal in-hibitory concentration of about 8.0 µmol/L.The A549 cells in the control group presented a relatively complete lumen structure.In con-trast,the lumen structures of A549 cells in the cyclophosphamide group,the butorphanol group,and the butorphanol+pcDNA-NC group were incomplete and obviously damaged,while D490(24 h and 48 h),the proliferation rate,the levels of SHH mRNA and GLI1 mRNA,VEGF-A,VE cadherin,Bcl-2,SHH,and GLI1 decreased(all P＜0.05),but the apoptotic rate and Bax protein expression in-creased(all P＜0.05).There was no statistical difference in each detection indicators of A549 cells between the cyclophosphamide group and the butorphanol group(all P＞0.05).Compared with the butorphanol+pcDNA-NC group,the luminal structure of A549 cells in the butorphanol+pc-SHH group was relatively intact,with obviously less damage,while D490(24 h and 48 h),the proliferation rate,the levels of SHH mRNA and GLI1 mRNA,VEGF-A,VE cadherin,Bcl-2,SHH,GLI1 protein increased(all P＜0.05),but the apoptotic rate and Bax protein expression decreased(all P＜0.05).Conclusion Butorphanol inhibits the proliferation and angiogenesis of lung can-cer A549 cells and promotes their apoptosis by blocking the SHH/GLI1 signaling pathway.
ButorphanolSonic hedgehogGlioma-associated oncogene homolog 1Lung cancer cellsAngiogenesis
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