斯诺普利抑制循环肿瘤细胞粘附于脐静脉内皮细胞的作用研究
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摘要
目的:探索斯诺普利(Cap-NO)干预循环肿瘤细胞(CTCs)粘附于人脐静脉内皮细胞(HUVECs)的作用。方法:UV-Vis检测在不同时间点及不同浓度梯度下Cap-NO的特征吸收峰(300 nm)峰值;MTT法检测Cap-NO对HUVECs和HT-29的毒性;粘附实验检测Cap-NO抑制肿瘤细胞HT-29粘附到HUVECs的效率;流式检测Cap-NO对肿瘤细胞粘附分子及整合素活性的作用;qRT-PCR和三磷酸腺苷(ATP)检测Cap-NO对肿瘤细胞HK1、HK2、MMP2的mRNA表达和ATP水平。结果:Cap-NO的特征峰(300 nm)峰值,随着浓度的减小和时间的延长而减小;Cap-NO对2种细胞都表现为低毒性;Cap-NO可抑制肿瘤细胞粘附到人脐静脉内皮细胞,且与给药浓度成正比;对肿瘤细胞的分子CD44具有抑制作用,且与给药浓度成正比;Cap-NO也可抑制HK1、HK2、MMP2的mRNA表达并降低ATP的形成,抑制肿瘤能量代谢。结论:Cap-NO是一种有效的NO供体化合物,在低细胞毒性浓度范围内能有效干预肿瘤细胞粘附于血管内皮细胞,抑制肿瘤细胞能量代谢,可能具有预防肿瘤转移的作用。
Objective:To explore the effect of Cap-NO on the adhesion of circulating tumor cells(CTCs) to human umbilical vein endothelial cells(HUVECs).Methods:UV-Vis was used to detect the characteristic absorption peak(300 nm)of Cap-NO at different time points and different concentration gradients.The cytotoxicity of Cap-NO to HUVECs and HT-29 cells was detected by MTT assay.Adhesion assay was used to detect the Cap-NO adhesion inhibition efficiency of HT-29 tumor cells to HUVECs.Flow cytometric analysis was used to detect the effect of Cap-NO on tumor cells adhesion molecules and integrin activity.q RT-PCR and ATP experiment were used to detect the expression of tumor cells mRNA of HK1,HK2 and MMP2 and adenosine triphosphate(ATP) levels.Results:The characteristic peak of Cap-NO(300 nm)decreased with the decrease of concentration and the prolongation time.Cap-NO showed low toxicity of HT-29 cells and HUVECs.Cap-NO inhibited the adhesion of tumor cells to HUVECs with the concentration of it.Cap-NO inhibited the growth of CD44,which was proportional to the concentration of drug.Cap-NO inhibited the mRNA expression of HK1,HK2 and MMP2 and reduced the formation of ATP to inhibit tumor energy metabolism.Conclusion:The present studies proved that Cap-NO is an effective NO donor compound.It can effectively interfere with the adherence of tumor cells to vascular endothelial cells and inhibit the energy metabolism of tumor cells in a low cytotoxic concentration range.It may have the effect of preventing metastasis of cancer.
Objective:To explore the effect of Cap-NO on the adhesion of circulating tumor cells(CTCs) to human umbilical vein endothelial cells(HUVECs).Methods:UV-Vis was used to detect the characteristic absorption peak(300 nm)of Cap-NO at different time points and different concentration gradients.The cytotoxicity of Cap-NO to HUVECs and HT-29 cells was detected by MTT assay.Adhesion assay was used to detect the Cap-NO adhesion inhibition efficiency of HT-29 tumor cells to HUVECs.Flow cytometric analysis was used to detect the effect of Cap-NO on tumor cells adhesion molecules and integrin activity.q RT-PCR and ATP experiment were used to detect the expression of tumor cells mRNA of HK1,HK2 and MMP2 and adenosine triphosphate(ATP) levels.Results:The characteristic peak of Cap-NO(300 nm)decreased with the decrease of concentration and the prolongation time.Cap-NO showed low toxicity of HT-29 cells and HUVECs.Cap-NO inhibited the adhesion of tumor cells to HUVECs with the concentration of it.Cap-NO inhibited the growth of CD44,which was proportional to the concentration of drug.Cap-NO inhibited the mRNA expression of HK1,HK2 and MMP2 and reduced the formation of ATP to inhibit tumor energy metabolism.Conclusion:The present studies proved that Cap-NO is an effective NO donor compound.It can effectively interfere with the adherence of tumor cells to vascular endothelial cells and inhibit the energy metabolism of tumor cells in a low cytotoxic concentration range.It may have the effect of preventing metastasis of cancer.
引文
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[2]YANG J,WEINBERY RA.Epithelial-mesenchymal transition:at the crossroads of development and tumor metastasis[J].Dev cell,2008,14(6):818
[3]YABROFF KR,WARREN JL,BROWN ML.Costs of cancer care in the USA:a descriptive review[J].Nat Clin Pract Oncol,2007,4(11):643
[4]LOBERG RD,BRADLEY DA,TOMLINS SA,et al.The lethal phenotype of cancer:the molecular basis of death due to malignancy[J].CA Cancer J Clin,2007,57(4):225
[5]FURCHGOTTRF,ZAWADZKIJV.Theobligatoryroleof endothelial cells in the relaxation of arterial smooth muscle by acetylcholine[J].Nature,1980,288(5789):373
[6]FURCHGOTT RF.Role of endothelium in responses of vascular smooth muscle[J].Circ Res,1983,53(5):557
[7]LOSCALZO J,SMICK D,ANDON N,et al.S-nitrosocaptopril.Ⅰ.Molecular characterization and effects on the vasculature and on platelets[J].J Pharmacol Exp Ther,1989,249(3):726
[8]AMANO M,TAKAHASHI M,KOSAKA T,et al.Differential inhibition of platelet aggregation and calcium mobilization by nitroglycerin and stabilized nitric oxide[J].J Cardiovasc Pharmacol,1994,24(6):860
[9]LINMJ,YANGXP,WANGJ,etal.Inhibitoryeffectsof S-nitrosocaptopril on vasomotor tone[J].Acta Pharmacol Sin,1998,19(5):485
[10]RADOMSKI MW,PALMER RM,MONCADE S.An L-arginine/nitric oxide pathway present in human platelets regulates aggregation[J].Proc Natl Acad Sci U S A,1990,87(13):5193
[11]LIAN S,LU Y,CHENG Y,et al.S-nitrosocaptopril interrupts adhesion of cancer cells to vascular endothelium by suppressing cell adhesion molecules via inhibition of the NF-small ka,Cyrillic B and JAK/STAT signal pathways in endothelial cells[J].Eur J Pharmacol,2016,791:62
[12]LU Y,LIANG H,YU T,et al.Isolation and characterization of living circulating tumor cells in patients by immunomagnetic negative enrichment coupled with flow cytometry[J].Cancer,2015,121(17):3036
[13]JIAL,WUCC,GUOW,etal.Antiangiogeniceffectsof S-nitrosocaptopril crystals as a nitric oxide donor[J].Eur J Pharmacol,2000,391(1-2):137
[14]JIA L,YOUNG X,GUO W.Physicochemistry,pharmacokinetics,and pharmacodynamics of S-nitrosocaptopril crystals,a new nitric oxide donor[J].J Pharm Sci,1999,88(10):981
[15]LU Y,YU T,LIANG H,et al.Nitric oxide inhibits hetero-adhesion of cancer cells to endothelial cells:restraining circulating tumor cells from initiating metastatic cascade[J].Sci Rep,2014,4:4344
[16]FURCHGOTT RF,ROBERT F.Furchgott:a pharmacologist’s pharmacologist[J].Mol Interv,2004,4(2):74
[17]JIA L,FURCHGOTT RF.Inhibition by sulfhydryl compounds of vascular relaxation induced by nitric oxide and endothelium-derived relaxing factor[J].J Pharmacol Exp Ther,1993,267(1):371
[18]LI J,LIU XJ,FURCHGOTT RF.Blockade of nitric oxide-induced relaxation of rabbit aorta by cysteine and homocysteine[J].Acta Pharmacol Sin,1997,18(1):11
[19]JIA L,STAMLER JS.Dual actions of S-nitrosylated derivative of vasoactive intestinal peptide as a vasoactive intestinal peptide-like mediator and a nitric oxide carrier[J].Eur J Pharmacol,1999,366(1):79
[20]JIA L,WONG H.In vitro and in vivo assessment of cellular permeability and pharmacodynamics of S-nitrosylated captopril,a nitric oxide donor[J].Br J Pharmacol,2001,134(8):1697
[21]KHAN BV,HARRISON DG,OLBRYCH MT,et al.Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells[J].Proc Natl Acad Sci U S A,1996,93(17):9114
[22]TSAO PS,BUITRAGO R,CHAN JR,et al.Fluid flow inhibits endothelial adhesiveness.Nitric oxide and transcriptional regulation of VCAM-1[J].Circulation,1996,94(7):1682
[23]deCATERINAR,LIBBYP,PENGHB,etal.Nitricoxide decreases cytokine-induced endothelial activation.Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines[J].J Clin Invest,1995,96(1):60
[24]LOSCALZO J.Nitric oxide insufficiency,platelet activation,and arterial thrombosis[J].Circ Res,2001,88(8):756
[25]JIA L,SCHWEIZER J,WANG Y,et al.Effect of nitric oxide on cytotoxicity of Taxol:enhanced Taxol transcellular permeability[J].Biochem Pharmacol,2003,66(11):2193
[26]JORDAN BF,PEETERBROECK J,KARROUM O,et al.Captopril and S-nitrosocaptopril as potent radiosensitizers:comparative study and underlying mechanisms[J].Cancer Lett,2010,293(2):213
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