NGR与iso DGR对抗菌肽在CD13~-/α_vβ_3~+乳腺癌中抗瘤活性影响
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摘要
目的比较携带NGR结构抗菌肽(CNAK)与携带iso DGR结构抗菌肽(CDAK)在CD13~-/α_vβ_3~+乳腺癌细胞(MDA-MB-231)中靶向识别与和诱导凋亡能力。方法 MTT法计算不同浓度CDAK与CNAK作用MDA-MB-231细胞24 h的细胞存活率与半抑制浓度(IC_(50))。CRLK(随机肽)、CDAK与CNAK作用MDA-MB-231和HFF细胞12 h,荧光显微镜观察识别能力,流式细胞仪分析结合能力。CRLK、CDAK与CNAK作用MDA-MB-231细胞24 h,流式细胞仪与Western-blot分析凋亡与Caspase-3蛋白表达。结果 CDAK与CNAK对MDA-MB-231细胞生长抑制呈剂量依赖性,IC_(50)分别为227μg/ml与210μg/ml。CDAK与CNAK仅进入MDA-MB-231细胞,CRLK未进入细胞内。CDAK与CNAK细胞结合能力分别为22±2.16与28±2.98(P<0.05)。CDAK组与CNAK组细胞凋亡率分别为23.56%±2.16%与25.33%±2.77%,Caspase-3相对蛋白表达量分别为1.6±0.14与1.87±0.21,差异均无统计学意义(P>0.05)。各指标中,CRLK与CDAK和CNAK之间差异均有统计学意义(P<0.05)。结论 NGR较iso DGR能够促进更多抗菌肽与CD13~-/α_vβ_3~+乳腺癌细胞结合,但抑制细胞生长与诱导凋亡能力无明显差异。
Objective To compare the abilities of targeting recognition and inducing apoptosis between antimicrobial peptide containing NGR(CNAK)and antimicrobial peptide containing iso DGR(CDAK)in CD13~-/α_vβ_3~+breast cancer cells.MethodsCell viability and half maximal inhibitory concentration(IC_(50))of MDA-MB-231 cells treated with different concentrations of CNAK and CDAK were calculated by MTT method.CRLK(random peptide),CDAK and CNAK were used to treat MDA-MB-231 and HFF cells for 12 h.The recognition ability was observed by fluorescence microscopy and the binding ability was analyzed by flow cytometry.After MDA-MB-231cells were treated with CRLK,CDAK and CNAK for 24 h,the apoptosis was analyzed by flow cytometry,and Caspase-3 protein expression was analyzed by Western blot.Results Cell growth of MDA-MB-231 was inhibited dose-dependently by CDAK and CNAK,with IC50 of 227μg/ml and 210μg/m,respectively.CDAK and CNAK only entered MDA-MB-231 cells,and CRLK failed to enter the cells.The binding ability of CDAK and CNAK was 22±2.16 and 28±2.98,respectively(P<0.05).The apoptotic rates of MDA-MB-231 cells in CDAK group and CNAK group were 23.56%±2.16%and 25.33%±2.77%,respectively,the relative protein expression of Caspase-3 was 1.6±0.14 and 1.87±0.21,respectively,and the difference was not statistically significant(P>0.05).The above indicators showed different between CRLK group and CDAK group or CNAK group(P<0.05).Conclusion NGR can promote more antibacterial peptides to bind CD13~-/α_vβ_3~+breast cancer cells than iso DGR,but there is no significant difference in antitumor activity.
Objective To compare the abilities of targeting recognition and inducing apoptosis between antimicrobial peptide containing NGR(CNAK)and antimicrobial peptide containing iso DGR(CDAK)in CD13~-/α_vβ_3~+breast cancer cells.MethodsCell viability and half maximal inhibitory concentration(IC_(50))of MDA-MB-231 cells treated with different concentrations of CNAK and CDAK were calculated by MTT method.CRLK(random peptide),CDAK and CNAK were used to treat MDA-MB-231 and HFF cells for 12 h.The recognition ability was observed by fluorescence microscopy and the binding ability was analyzed by flow cytometry.After MDA-MB-231cells were treated with CRLK,CDAK and CNAK for 24 h,the apoptosis was analyzed by flow cytometry,and Caspase-3 protein expression was analyzed by Western blot.Results Cell growth of MDA-MB-231 was inhibited dose-dependently by CDAK and CNAK,with IC50 of 227μg/ml and 210μg/m,respectively.CDAK and CNAK only entered MDA-MB-231 cells,and CRLK failed to enter the cells.The binding ability of CDAK and CNAK was 22±2.16 and 28±2.98,respectively(P<0.05).The apoptotic rates of MDA-MB-231 cells in CDAK group and CNAK group were 23.56%±2.16%and 25.33%±2.77%,respectively,the relative protein expression of Caspase-3 was 1.6±0.14 and 1.87±0.21,respectively,and the difference was not statistically significant(P>0.05).The above indicators showed different between CRLK group and CDAK group or CNAK group(P<0.05).Conclusion NGR can promote more antibacterial peptides to bind CD13~-/α_vβ_3~+breast cancer cells than iso DGR,but there is no significant difference in antitumor activity.
引文
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[11]侯磊,岳晨莉,何莉,等.连接链对包含CisoDGRC结构抗菌肽诱导乳腺癌细胞凋亡的影响[J].山西医科大学学报,2017,4(11):1149-1153.
[12]Hou L,Zhao X,Wang P,et al.Antitumor activity of antimicrobial peptides containing CisoDGRC in CD13 negative breast cancercells[J].PLo S One,2013,8(1):e53491.
[13]Zhang Z,Hou L,Feng L,et al.An antimicrobial peptide containing NGR motif has potent antitumor activity against CD13+and CD13-tumor cells[J].Tumour Biol,2015,36(10):8167-8175.
[14]罗俊茜,张帆,杨晓峰,等.利用体内噬菌体展示技术筛选膀胱癌特异性结合肽[J].中国免疫学杂志,2015,31(4):509-513.
[15]Curnis F,Fiocchi M,Sacchi,et al.A NGR-tagged nano-gold:a new CD13-selective carrier for cytokine delivery to tumor[J].Nano Res,2016,9(5):1393-1408.
[2]牛书俐,庞骁,周莉,等.RGD三肽的临床应用进展[J].基础医学与临床,2018,38(1):107-111.
[3]周敏,瞿春莹,徐雷鸣,等.量子点-RGD荧光探针在胰腺癌诊治中的实验研究[J].临床肿瘤学杂志,2018,23(4):289-297.
[4]赵慧云,刘妍,贾兵,等.双靶点分子探针68Ga-RGD-BBN用于乳腺癌的micro PET显像[J].生物物理学报,2011,27(4):335-343.
[5]Tripodi AAP,Tóth S,Enyedi KN,et al.Development of novel cyclic NGR peptide-daunomycin conjugates with dual targeting property[J].Beilstein J Org Chem,2018,25(14):911-918.
[6]张紫欣,孟颖,梁宇霆.双模态对比剂Fe3 O4-Cy5.5-NGR在卵巢癌的体外靶向效能研究[J].首都医科大学学报,2018,39(1):84-91.
[7]Gregorc V,Cavina R,Novello S,et al.NGR-hTNF and doxorubicin as second-line treatment of patients with small cell lung cancer[J].Oncologist,2018,23(10):1133-1138.
[8]解朋,黄建敏,刘晓梅,等.99Tcm-MAG3-isoDGR-2C在荷瘤鼠体内分布和显像的研究[J].中国临床医学影像杂志,2017,28(7):480-483.
[9]Corti A,Gasparri AM,Ghitti M,et al.Glycine N-methylation in NGR-tagged nanocarriers prevents isoaspartate formation and integrin binding without impairing CD13 recognition and tumor homing[J].Adv Funct Mater,2017,27(36)doi:10.1002.
[10]Burns KE,Mc Cleerey TP,Thévenin D.pH-Selective cytotoxicity of p HLIP-antimicrobial peptide conjugates[J].Sci Rep,2016,23(6):28465-28474.
[11]侯磊,岳晨莉,何莉,等.连接链对包含CisoDGRC结构抗菌肽诱导乳腺癌细胞凋亡的影响[J].山西医科大学学报,2017,4(11):1149-1153.
[12]Hou L,Zhao X,Wang P,et al.Antitumor activity of antimicrobial peptides containing CisoDGRC in CD13 negative breast cancercells[J].PLo S One,2013,8(1):e53491.
[13]Zhang Z,Hou L,Feng L,et al.An antimicrobial peptide containing NGR motif has potent antitumor activity against CD13+and CD13-tumor cells[J].Tumour Biol,2015,36(10):8167-8175.
[14]罗俊茜,张帆,杨晓峰,等.利用体内噬菌体展示技术筛选膀胱癌特异性结合肽[J].中国免疫学杂志,2015,31(4):509-513.
[15]Curnis F,Fiocchi M,Sacchi,et al.A NGR-tagged nano-gold:a new CD13-selective carrier for cytokine delivery to tumor[J].Nano Res,2016,9(5):1393-1408.
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