Identification and Cloning of an inhibitor of Cancer Metastasis and Invasion

正常腹膜、子宫肌层和卵巢组织的条件培养基以浓度依赖性方式增加卵巢癌（NOM 1）细胞MMP-2和MMP-9的分泌。特别是MMP-9的显着增加，通过添加正常腹膜组织条件培养基。为了评估MMP-9刺激因子，将正常腹膜组织的条件培养基分级，并通过酶谱法检测每个级分对MMP-9分泌的影响。大于100 kD的组分主要增加NOM 1细胞MMP-9的分泌。将细胞外基质、层粘连蛋白、纤连蛋白和IV型胶原加入NOM 1细胞中以鉴定超过100 kD的因子。纤连蛋白可浓度依赖性地增加NOM 1细胞MMP-9的分泌。此外，通过用RGD肽（纤连蛋白受体）和针对整合素α 5受体的抗体预处理来抑制纤连蛋白诱导的MMP-9分泌。正常腹膜组织培养24小时后纤维连接蛋白浓度为164 ng/ml。由于100 ng/ml纤连蛋白足以刺激卵巢癌细胞分泌MMP-9，纤连蛋白被认为是一个重要的刺激因子。

Conditioned media of normal peritoneal, myometrial, and ovarian tissues increased MMP-2 and -9 secretion from ovarian cancer (NOM1) cells in a concentration dependent manner. Especially, MMP-9 was markedly augmented by addition of normal peritoneal tissue conditioned medium. To estimate MMP-9 stimulating factors, conditioned medium of normal peritoneal tissue was fractionated and the influence of each fraction on MMP-9 secretion was examined by zymography. More than 100 kD fraction increased mostly MMP-9 secretion from NOM1 cells. Extracellular matrix, laminine, fibronectin, and type IV collagen, were added to NOM1 cells to identify the more than 100 kD factors. Fibronectin could increase MMP-9 secretion from NOM1 cells in a concentration dependent manner. Furthermore, fibronectin induced secretion of MMP-9 was inhibited by pretreatment with RGD peptides (fibronectin receptor) and antibody against integrin alpha5 receptor. Concentration of fibronectin was 164 ng/ml after 24 hours-culture of normal peritoneal tissue. Since 100 ng/ml fibronectin was enough to stimulate MMP-9 secretion from ovarian cancer cells, fibronectin was suggested to be an important stimulating factor.