A fluorogenic probe for sensitive detection of MMP-2/9 activities in clinical colorectal cancer tissues

Abstract

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-N3 (FITC-N3) 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 LoVo 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 mL−1. Besides, the confocal laser scanning microscopy (CLSM) imaging results showed that enhanced green fluorescence signals were collected for LoVo 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.