Stonelake P S, Jones C E, Neoptolemos J P, Baker P R
Department of Surgery, University of Birmingham, UK.
Br J Cancer. 1997;75(7):951-9. doi: 10.1038/bjc.1997.166.
The relative importance of different proteinases, and their inhibition, in the breakdown of human endothelial basement membrane (BM) by MDA-MB-231 and MCF7ADR human breast cancer cell lines has been studied using 35S-labelled BM-coated 96-well culture plates. Basement membrane degradation (BMD) was independent of cell proliferation above the seeding density. Inhibitors of aspartic (pepstatin and PD 134678-0073) and cysteine proteinases (E64) had little effect on BMD under normal culture conditions, suggesting that cathepsins D, B and L have only a minor role. In contrast, inhibitors of urokinase-type plasminogen activator (uPA) and/or plasminogen activation to plasmin (aprotinin, amiloride, EACA, tranexamic acid, anti-uPA antibody) all reduced BMD by MDA-MB-231 cells by approximately 30-40%, but only in the presence of serum or plasminogen. BB94, an inhibitor of matrix metalloproteinases (MMPs), also reduced BMD by about 30% under these conditions but was similarly effective in serum-free medium. Combinations of BB94 with any of the uPA/plasminogen activation inhibitors in serum-containing medium had additive effects, while BB94 with pepstatin and E64 under serum-free conditions reduced BMD to 16% of control. Serum-containing conditioned medium exhibited appreciable BMD, largely due to aprotinin-inhibitable activity. Although small reductions in cell proliferation were seen with some inhibitors, the combination of BB94 with E64 or E64d reduced the cell population by about 60% under serum-containing conditions. These in vitro observations suggest that combinations of proteinase inhibitors, particularly of uPA/plasminogen activation and MMPs, may merit clinical evaluation as potential antimetastatic therapy for breast cancer.
利用35S标记的包被有基底膜的96孔培养板，研究了不同蛋白酶及其抑制作用在MDA - MB - 231和MCF7ADR人乳腺癌细胞系对人内皮基底膜（BM）降解过程中的相对重要性。在接种密度以上，基底膜降解（BMD）与细胞增殖无关。在正常培养条件下，天冬氨酸蛋白酶抑制剂（胃蛋白酶抑素和PD 134678 - 0073）和半胱氨酸蛋白酶抑制剂（E64）对BMD影响很小，这表明组织蛋白酶D、B和L作用较小。相反，尿激酶型纤溶酶原激活剂（uPA）抑制剂和/或纤溶酶原激活为纤溶酶的抑制剂（抑肽酶、阿米洛利、6 -氨基己酸、氨甲环酸、抗uPA抗体）均使MDA - MB - 231细胞的BMD降低约30 - 40%，但仅在有血清或纤溶酶原存在时有效。基质金属蛋白酶（MMPs）抑制剂BB94在这些条件下也使BMD降低约30%，但在无血清培养基中同样有效。在含血清培养基中，BB94与任何一种uPA/纤溶酶原激活抑制剂联合使用具有相加作用，而在无血清条件下，BB94与胃蛋白酶抑素和E64联合使用可使BMD降至对照的16%。含血清的条件培养基表现出明显的BMD，这主要归因于抑肽酶可抑制的活性。尽管一些抑制剂使细胞增殖略有减少，但在含血清条件下，BB94与E64或E64d联合使用可使细胞数量减少约60%。这些体外观察结果表明，蛋白酶抑制剂组合，特别是uPA/纤溶酶原激活抑制剂和MMPs抑制剂组合，作为乳腺癌潜在的抗转移治疗方法可能值得进行临床评估。
