ASSOCIATION OF MMP-2 ACTIVATION POTENTIAL WITH METASTATIC PROGRESSION IN HUMAN BREAST-CANCER CELL-LINES INDEPENDENT OF MMP-2 PRODUCTION
ASSOCIATION OF MMP-2 ACTIVATION POTENTIAL WITH METASTATIC PROGRESSION IN HUMAN BREAST-CANCER CELL-LINES INDEPENDENT OF MMP-2 PRODUCTION
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DOI：
10.1093/jnci/85.21.1758
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发表时间：
1993-11-03
期刊：
影响因子：
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通讯作者：
THOMPSON, EW
中科院分区：
文献类型：
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作者：
AZZAM, HS;ARAND, G;THOMPSON, EW
背景资料：癌细胞表达基质金属蛋白酶-2（MMP-2），即72 kd的IV型胶原酶/明胶酶，与通过IV型胶原降解介导的癌细胞侵袭基底膜的转移有关。然而，这种潜在的酶原在正常组织和液体中的丰度表明，MMP-2酶原的利用受到其生理活化的限制，而不是单独表达。我们以前报道激活这种酶原的正常和恶性成纤维细胞培养的胶原蛋白I（vitrogen）凝胶。目的：我们在这项研究中的目的是1）确定MMP-2激活是否仅限于更具侵袭性的人乳腺癌细胞系和2）定位激活机制。研究方法：酶谱法用于通过检测潜在MMP-2（72 kd）和较小分子量的成熟物质（59或62 kd）来监测MMP-2活化。因此，筛选在塑料、vitrogen和其他基质上培养的人乳腺癌细胞系的MMP-2活化。胶原蛋白I培养的细胞暴露于放线菌酮，蛋白质合成抑制剂，或蛋白酶抑制剂，以确定MMP-2激活机制的性质。Triton X-114（TX-114）洗涤剂提取物培养的胶原蛋白I或塑料上的细胞与潜伏的MMP-2孵育，并通过酶谱分析定位MMP-2激活剂。结果如下：MMP-2活化仅在更具侵袭性、高度侵袭性的雌激素受体阴性、波形蛋白阳性的人乳腺癌细胞系（Hs 578 T、MDA-MB-436、BT549、MDA-MB-231、MDA-MB-435、MCF-7ADR）中由胶原I培养物诱导，并且不依赖于MMP-2的产生。MMP-2活化检测到在胶原蛋白I凝胶上培养的细胞中，但在明胶凝胶、Matrigel或胶原蛋白I或IV、明胶或纤连蛋白薄层上培养的细胞中未检测到。胶原蛋白诱导的激活是特定的酶物种MMP-2，因为MMP-9，92 kd的IV型胶原酶/明胶酶，在类似的条件下是不能激活的。MMP-2的活化被放线菌酮抑制，对金属蛋白酶抑制剂敏感，但对乙酰氨基酚，丝氨酸或半胱氨酸蛋白酶抑制剂不敏感。MMP-2激活检测到的疏水性，质膜富集，TX-114提取物从侵袭性胶原蛋白I培养的细胞。结论：胶原蛋白I诱导的MMP-2活化仅限于高度侵袭性的雌激素受体阴性、波形蛋白阳性的人乳腺癌细胞系，不依赖于MMP-2的产生，并且与转移潜能相关。我们的研究结果是一致的质膜定位的激活剂。提示：MMP-2激活机制可能成为乳腺癌诊断、预后和治疗的新靶点。
Background: Expression of matrix metalloproteinase-2 (MMP-2), the 72-kd type IV collagenase/gelatinase, by cancer cells has been implicated in metastasis through cancer cell invasion of basement membranes mediated by degradation of collagen IV. However, the abundance of this latent proenzyme in normal tissues and fluids suggests that MMP-2 pro-enzyme utilization is limited by its physiological activation rather than expression alone. We previously reported activation of this proenzyme by normal and malignant fibroblastoid cells cultured on collagen I (vitrogen) gels. Purpose: Our purposes in this study were 1) to determine whether MMP-2 activation is restricted to the more invasive human breast cancer cell lines and 2) to localize the activating mechanism. Methods: Zymography was used to monitor MMP-2 activation through detection of latent MMP-2 (72 kd) and mature species of smaller molecular weight (59 or 62 kd). Human breast cancer cell lines cultured on plastic, vitrogen, and other matrices were thus screened for MMP-2 activation. Collagen I-cultured cells were exposed to cycloheximide, a protein synthesis inhibitor, or to protease inhibitors to determine the nature of the MMP-2-activating mechanism. Triton X-114 (TX-114) detergent extracts from cells cultured on collagen I or plastic were incubated with latent MMP-2 and analyzed by zymography to localize the MMP-2 activator. Results: MMP-2 activation was only induced by collagen I culture in the more aggressive, highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines (Hs578T, MDA-MB-436, BT549, MDA-MB-231, MDA-MB-435, MCF-7ADR) and was independent of MMP-2 production. MMP-2 activation was detected in cells cultured on collagen I gels but not in those cultured on gelatin gels, Matrigel, or thin layers of collagen I or IV, gelatin, or fibronectin. Collagen-induced activation was specific for the enzyme species MMP-2, since MMP-9, the 92-kd type IV collagenase/gelatinase, was not activatable under similar conditions. MMP-2 activation was inhibited by cycloheximide and was sensitive to a metalloproteinase inhibitor but not to aspartyl, serine, or cysteinyl protease inhibitors. MMP-2 activation was detected in the hydrophobic, plasma membrane-enriched, TX-114 extracts from invasive collagen I-cultured cells. Conclusion: Collagen I-induced MMP-2 activation is restricted to highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines, is independent of MMP-2 production, and is associated with metastatic potential. Our findings are consistent with plasma membrane localization of the activator. Implications: The MMP-2 activation mechanism may represent a new target for diagnosis, prognosis, and treatment of human breast cancer.