Distribution Of MMP-2 Activity And The Activation Mechanism Of MMP-2 In The Human Breast Cancer

Tumour invasion and metastasis are the result of a multistep process thatincludes basement membrane disruption, stromal infiltration,intravasation,extravasation and invasion of a target organ by tumour cells. All theseprocesses require the degradation of basement membrane components and ofextracellular matrix macromolecule by proteolytic enzymes. Among theseproteinases, matrix metalloproteinases (MMPs) seem to play an importantrole in the tumour progress. Especially,gelatinase A(MMP-2) and gelatinaseB(MMP-9) with type IV collagenase activity are the important proteinase ininvasion and metastasis. Expression of MMP-2 and MMP-9 is increased inmany tumors compared with normal tissues, and activation of the proform ofMMP-2 is crucial for the metastatic capacity of primary tumors. MMPs issecreted by cells as proenzymes that must be cleaved in order to becomefunctional. By contrast to most MMPs, proMMP-2 possesses a propeptide thatis not susceptible to proteolytic cleavage by serine protinases. It has been nowwidely documented that proMMP-2 activation first necessitates the formationof a ternary complex between proMMP-2, tissue inhibitor ofmetalloproteinase-2 (TIMP-2), and membrane type 1-matrixmetalloproteinase (MT1-MMP) at the cell plasma membrane; enzymeactivation would then require the participation of another free MT1-MMPmolecule. Some reported MMP-2 protein localized in the tumour cells andmatrix cells. However, the distribution of MMP-2 activity and activationmechanism of MMP-2 is not sure.It was the aim of the present study to investigate t the distribution ofMMP-2 activity and the activation mechanism of MMP-2 in the human breastcancer. Gelatinolytic activity was localized with an in situ zymographytechnique using DQ-gelatin. DQ-gelatin was applied in the present study in anin situ assay. The principle of fluorescence production is based on thepresence of quenched FITC molecules in DQ-gelatin that are liberated andstart to fluoresce on proteolytic degradation of DQ-gelatin into peptides.Meanwhile, MCF-7, the human breast cancer cell lines, was used in vitroexpriment. MCF-7 cells were cultured on three dimensional (3-D) type Icollagen gel. To prepare serum-free conditioned medium (SFCM), weexamined the expression of MMP-2 by zymography, and the expression ofMT1-MMP mRNA were examined by reverse transcription polymerase chainreaction (RT-PCR). Results: 1. Tissue zymography results showed only MMP-2 exited the mode ofactivation, cues that gelatinolytic activity is produced by MMP-2 in the breastcancer. 2,Gelatinolytic activity is showed in the breast cancer cells. In situzymography using DQ-gelatin revealed that strong green fluorescence ispresent in the carcinoma cell nests. meanwhile, green fluorescence is presentin peri-carcinoma, but the intensity of fluorescence is not the same. There was22 cases whose expression of fluorescence is ++ in carcinoma, there was 12cases whose expression of fluorescence is ++ in peri-carcinoma, nofluorescence was recognized in the normal breast tissue. In peri-carcinoma,there is a statistically significant correlation between the intensity of...