基质金属蛋白酶MMP-2/9分子探针对临床结肠癌组织的检测
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摘要
研发基质金属蛋白酶MMP-2/9特异性识别的荧光分子探针并用于临床结肠癌组织的快速、准确性检测意义重大,但仍然具有一定的挑战性.本文基于基质金属蛋白酶MMP-2和MMP-9(MMP-2/9)特异性识别的多肽底物,利用一价铜离子催化的"点击"化学和酰胺化反应,分别将荧光素(FITC-N_3)和荧光淬灭基团Dabcyl-NHS偶联于多肽底物两端,制备得到MMP-2/9特异性识别的荧光能量共振转移(FRET)荧光分子探针Dab-GPLGVRGYFITC.通过将重组基质金属蛋白酶MMP-2/9与探针作用,跟踪检测溶液在520 nm处荧光信号变化的情况,对探针的特异性和检测灵敏度进行评估.此外,利用人源大肠癌细胞LoVo和30例临床结肠癌组织样本开展了结肠癌快速体外检测初步研究.体外重组酶检测实验表明,探针对MMP-2/9具有较强的特异性和选择性,对MMP-2的最低检测限可达14 ng/mL.共聚焦显微成像实验显示,相比于正常人胃黏膜上皮细胞GES-1,肠癌细胞LoVo表现出较强的绿色荧光信号,如预先加入抑制剂,细胞的荧光信号则很弱.另外,15例活性MMP-2高表达的临床结直肠癌组织裂解液与探针作用后荧光信号较胃炎组织增强5~9倍,而15例癌旁组织裂解液荧光则增强约0.9~3.0倍.值得注意的是,如果将探针喷洒到结肠癌组织和癌旁组织表面,相比于胃炎组织,荧光信号明显增强.总之,该探针对基质金属蛋白酶MMP-2/9过表达肠癌细胞和结肠癌组织表现出较好的特异性和检测灵敏度,具有应用于临床结肠癌早期诊断的极大潜力.
Developing a novel fluorogenic probe which can be specifically recognized by active matrix metalloproteinase-2 and matrix metalloproteinase-9(MMP-2/9) to rapidly and accurately detect the clinical colorectal cancer is significant but remains a great challenge. Herein, we have designed and synthesized a novel fluorescence resonance energy transfer(FRET)-based probe Dab-GPLGVRGY-FITC for specific detection of MMP-2/9 activity. The probe was prepared by conjugating fluorescein-N_3(FITC-N_3) and fluorescent quencher dabcyl-NHS to MMP-2/9-responsive peptide substrate GPLGVRGY through copper(I)-catalyzed "click" and amidation reactions, respectively. To assess the specificity and sensitivity of the probe for active MMP-2/9 in vitro, the enzymatic assays were firstly performed with recombinant MMP-2/9. Fluorescent signal change of the mixed solution of probe and MMP-2 with and without treatment of MMP-2 specific inhibitor was recorded at 520 nm with fluorescence spectrometer. The capability of probe for specific detection of endogenous MMP-2/9 activity was further evaluated with human colorectal cancer cell Lo Vo and 30 clinical colorectal cancer tissue samples. The in vitro enzymatic assay results indicated that the probe showed good specificity and sensitivity for recombinant MMP-2/9 comparing to other proteins. The detection limit for recombinant MMP-2 is estimated to be 14 ng m L-1. Besides, the confocal laser scanning microscopy(CLSM) imaging results showed that enhanced green fluorescence signals were collected for Lo Vo cells upon treatment with probe. However, most of the fluorescence was readily competed away by pretreatment of the probe with inhibitor. In sharp contrast, only weak fluorescence was observed for normal gastric mucosa epithelial GES-1 cells. Additionally, the lysates of 15 clinical colorectal tissues displayed ~5 to 9-fold fluorescence enhancements after treatment of the probe in comparison with normal gastritis tissues. However, the fluorescence intensities of 15 peficancerous tissue lysates was only ~0.9 to 3-fold higher than normal tissues. Most notably, in comparison with normal gastritis tissue, strong green fluorescence was observed for both colorectal tissue and peficancerous tissue once the probe was sprayed onto the tissue surface. In summary, this probe shows good specificity and sensitivity for the detection of active MMP-2 both in cancer cell and clinical cancer tissue, which may have great potential for early diagnosis of MMP-2/9 active colorectal cancer in clinic.
Developing a novel fluorogenic probe which can be specifically recognized by active matrix metalloproteinase-2 and matrix metalloproteinase-9(MMP-2/9) to rapidly and accurately detect the clinical colorectal cancer is significant but remains a great challenge. Herein, we have designed and synthesized a novel fluorescence resonance energy transfer(FRET)-based probe Dab-GPLGVRGY-FITC for specific detection of MMP-2/9 activity. The probe was prepared by conjugating fluorescein-N_3(FITC-N_3) and fluorescent quencher dabcyl-NHS to MMP-2/9-responsive peptide substrate GPLGVRGY through copper(I)-catalyzed "click" and amidation reactions, respectively. To assess the specificity and sensitivity of the probe for active MMP-2/9 in vitro, the enzymatic assays were firstly performed with recombinant MMP-2/9. Fluorescent signal change of the mixed solution of probe and MMP-2 with and without treatment of MMP-2 specific inhibitor was recorded at 520 nm with fluorescence spectrometer. The capability of probe for specific detection of endogenous MMP-2/9 activity was further evaluated with human colorectal cancer cell Lo Vo and 30 clinical colorectal cancer tissue samples. The in vitro enzymatic assay results indicated that the probe showed good specificity and sensitivity for recombinant MMP-2/9 comparing to other proteins. The detection limit for recombinant MMP-2 is estimated to be 14 ng m L-1. Besides, the confocal laser scanning microscopy(CLSM) imaging results showed that enhanced green fluorescence signals were collected for Lo Vo cells upon treatment with probe. However, most of the fluorescence was readily competed away by pretreatment of the probe with inhibitor. In sharp contrast, only weak fluorescence was observed for normal gastric mucosa epithelial GES-1 cells. Additionally, the lysates of 15 clinical colorectal tissues displayed ~5 to 9-fold fluorescence enhancements after treatment of the probe in comparison with normal gastritis tissues. However, the fluorescence intensities of 15 peficancerous tissue lysates was only ~0.9 to 3-fold higher than normal tissues. Most notably, in comparison with normal gastritis tissue, strong green fluorescence was observed for both colorectal tissue and peficancerous tissue once the probe was sprayed onto the tissue surface. In summary, this probe shows good specificity and sensitivity for the detection of active MMP-2 both in cancer cell and clinical cancer tissue, which may have great potential for early diagnosis of MMP-2/9 active colorectal cancer in clinic.
引文
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21 Wang Z,Li X H,Feng D,et al.Poly(m-phenylenediamine)-based fluorescent nanoprobe for ultrasensitive detection of matrix metalloproteinase.Anal Chem,2014,86:7719-7725
22 Zhang J,Yuan Z F,Wang Y,et al.Multifunctional envelope-type mesoporous silica nanoparticles for tumor-triggered targeting drug delivery.J Am Chem Soc,2013,135:5068-5073
23 Sun R,Yin L,Zhang S,et al.Simple light-triggered fluorescent labeling of silica nanoparticles for cellular imaging applications.Chem Eur J,2017,23:13893-13896
24 Cheng X,Sun R,Yin L,et al.Light-triggered assembly of gold nanoparticles for photothermal therapy and photoacoustic imaging of tumours in vivo.Adv Mater,2017,doi:10.1002/adma.201604894
25 Kwong G A,von Maltzahn G,Murugappan G,et al.Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease.Nat Biotechnol,2013,31:63-70
26 Xia X H,Yang M X,Oetjen L K,et al.An enzyme-sensitive probe for photoacoustic imaging and fluorescence detection of protease activity.Nanoscale,2011,3:950-953
27 Hou Y,Zhou J,Gao Z Y,et al.Protease-activated ratiometric fluorescent probe for p H mapping of malignant tumors.ACS Nano,2015,9:3199-3205
28 Zhang Y,So M K,Rao J.Protease-modulated cellular uptake of quantum dots.Nano Lett,2006,6:1988-1992
29 Zucker S,Vacirca J.Role of matrix metalloproteinases(MMPs)in colorectal cancer.Cancer Metastasis Rev,2004,23:101-117
30 Kostova E,Slaninka Miceska M,Labacevski N,et al.Expression of matrix metalloproteinases 2,7 and 9 in patients with colorectal cancer.Vojnosanit Pregl,2014,71:52-59
31 Langers A M J,Verspaget H W,Hawinkels L J A C,et al.MMP-2 and MMP-9 in normal mucosa are independently associated with outcome of colorectal cancer patients.Br J Cancer,2012,106:1495-1498
2 Zheng Z X,Zheng R S,Chen W Q,et al.Colorectal cancer incidence and mortality in China 2010.Asian Pac J Cancer Prev,2014,15:8455-8460
3 Siegel R,De Santis C,Virgo K,et al.Cancer treatment and survivorship statistics 2012.CA Cancer J Clin,2012,62:220-241
4 Wanders L K,van Doorn S C,Fockens P,et al.Quality of colonoscopy and advances in detection of colorectal lesions:A current overview.Expert Rev Gastroenterol Hepatol,2015,9:417-430
5 Bressler B,Paszat L F,Vinden C,et al.Colonoscopic miss rates for right-sided colon cancer:A population-based analysis.Gastroenterology,2004,127:452-456
6 Egeblad M,Werb Z.New functions for the matrix metalloproteinases in cancer progression.Nat Rev Cancer,2002,2:161-174
7 Li J,Zhao T F,Duan E K.Matrix metalloproteinases(MMPs)and trophoblast invasion.Chin Sci Bull,2005,50:1169-1173[李晶,赵田夫,段恩奎.基质金属蛋白酶(MMPs)在人滋养层细胞侵入中的作用.科学通报,2005,50:1167-1171]
8 Garripelli V K,Kim J K,Son S,et al.Matrix metalloproteinase-sensitive thermogelling polymer for bioresponsive local drug delivery.Acta Biomater,2011,7:1984-1992
9 Roy R,Yang J,Moses M A.Matrix metalloproteinases as novel biomarkers and potential therapeutic targets in human cancer.J Clin Oncol,2009,27:5287-5297
10 Kessenbrock K,Plaks V,Werb Z.Matrix metalloproteinases:Regulators of the tumor microenvironment.Cell,2010,141:52-67
11 Dai Z F.Applications,opportunities and challenges of molecular probes in the diagnosis and treatment of major diseases(in Chinese).Chin Sci Bull,2017,1:25-35[戴志飞.分子探针在重大疾病诊疗中的应用、机遇与挑战.科学通报,2017,1:25-35]
12 Lee S,Xie J,Chen X Y.Peptides and peptide hormones for molecular imaging and disease diagnosis.Chem Rev,2010,110:3087-3111
13 Hanaoka H,Mukai T,Habashita S,et al.Chemical design of a radiolabeled gelatinase inhibitor peptide for the imaging of gelatinase activity in tumors.Nucleic Med Biol,2007,34:503-510
14 Gallo J,Kamaly N,Lavdas I,et al.CXCR4-targeted and MMP-responsive iron oxide nanoparticles for enhanced magnetic resonance imaging.Angew Chem Int Ed,2014,53:9550-9554
15 Bremer C,Tung C H,Weissleder R.In vivo molecular target assessment of matrix metalloproteinase inhibition.Nat Med,2001,7:743-748
16 Shi H,Ryan K T K,Tang B Z,et al.Real-time monitoring of cell apoptosis and drug screening using fluorescent light-up probe with aggregation-induced emission characteristics.J Am Chem Soc,2012,134:17972-17981
17 Shi H,Liu J,Geng J,et al.Specific detection of integrinαVβ3 by light-up bioprobe with aggregation-induced emission characteristics.JAm Chem Soc,2012,134:9569-9572
18 Zhu L,Zhang F,Ma Y,et al.In vivo optical imaging of membrane-type matrix metalloproteinase(MT-MMP)activity.Mol Pharmaceutics,2011,8:2331-2338
19 Olsona E S,Jiang T,Aguileraa T A,et al.Activatable cell penetrating peptides linked to nanoparticles as dual probes for in vivo fluorescence and MR imaging of proteases.Proc Natl Acad Sci USA,2010,107:4311-4316
20 Le S,Ryu J H,Park K,et al.Polymeric nanoparticle-based activatable near-infrared nanosensor for protease determination in vivo.Nano Lett,2009,9:4412-4416
21 Wang Z,Li X H,Feng D,et al.Poly(m-phenylenediamine)-based fluorescent nanoprobe for ultrasensitive detection of matrix metalloproteinase.Anal Chem,2014,86:7719-7725
22 Zhang J,Yuan Z F,Wang Y,et al.Multifunctional envelope-type mesoporous silica nanoparticles for tumor-triggered targeting drug delivery.J Am Chem Soc,2013,135:5068-5073
23 Sun R,Yin L,Zhang S,et al.Simple light-triggered fluorescent labeling of silica nanoparticles for cellular imaging applications.Chem Eur J,2017,23:13893-13896
24 Cheng X,Sun R,Yin L,et al.Light-triggered assembly of gold nanoparticles for photothermal therapy and photoacoustic imaging of tumours in vivo.Adv Mater,2017,doi:10.1002/adma.201604894
25 Kwong G A,von Maltzahn G,Murugappan G,et al.Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease.Nat Biotechnol,2013,31:63-70
26 Xia X H,Yang M X,Oetjen L K,et al.An enzyme-sensitive probe for photoacoustic imaging and fluorescence detection of protease activity.Nanoscale,2011,3:950-953
27 Hou Y,Zhou J,Gao Z Y,et al.Protease-activated ratiometric fluorescent probe for p H mapping of malignant tumors.ACS Nano,2015,9:3199-3205
28 Zhang Y,So M K,Rao J.Protease-modulated cellular uptake of quantum dots.Nano Lett,2006,6:1988-1992
29 Zucker S,Vacirca J.Role of matrix metalloproteinases(MMPs)in colorectal cancer.Cancer Metastasis Rev,2004,23:101-117
30 Kostova E,Slaninka Miceska M,Labacevski N,et al.Expression of matrix metalloproteinases 2,7 and 9 in patients with colorectal cancer.Vojnosanit Pregl,2014,71:52-59
31 Langers A M J,Verspaget H W,Hawinkels L J A C,et al.MMP-2 and MMP-9 in normal mucosa are independently associated with outcome of colorectal cancer patients.Br J Cancer,2012,106:1495-1498