Zervos E E, Shafii A E, Rosemurgy A S
Department of Surgery, University of South Florida, and James A. Haley Veteran's Administration Medical Center, Tampa, Florida, USA.
J Surg Res. 1999 Jan;81(1):65-8. doi: 10.1006/jsre.1998.5447.
Basement membrane degradation is a critical component of tumor invasion and metastasis that is facilitated by the family of enzymes known as matrix metalloproteinases (MMPs). MMP-2 and MMP-9 are two subtypes that have specifically been identified in tumors of gastrointestinal origin. We have previously shown that broad inhibition of these enzymes with the MMP inhibitor BB-94 improves survival in a murine model of pancreatic cancer. The purpose of this study was to determine MMP-2 and MMP-9 activity in orthotopic tumors from mice treated with and without BB-94.
Ten million cells of a moderately differentiated pancreatic cancer cell line (HPAC) were implanted orthotopically into Balb/c nu/nu mice. The mice were treated with BB-94 or vehicle control for 70 days or until death. At necropsy, tumors were harvested, total protein was extracted, and MMPs were purified from 400 microgram of crude protein extract by gelatin-Sepharose affinity chromatography. Relative enzyme levels and activity of MMP-2 and MMP-9 were determined by Western blot and gelatin zymography.
Tumors from treated animals were significantly smaller than those from nontreated animals. MMP-2 was present in greater amounts in both treated and nontreated animals than MMP-9. Active MMP-2 was present in both groups but significantly decreased in animals treated with BB-94. Active MMP-9 was absent in both groups, whereas levels of latent MMP-9 appeared lower than those of MMP-2 in all samples.
Activated MMP-2 and not MMP-9 in HPAC cells grown in nude mice suggests that this MMP subtype is more critical in the phenotypic behavior of such tumors. Furthermore, attenuated levels of active MMP-2 in animals treated with the enzyme inhibitor BB-94 suggest that previously observed improvements in survival correlate with the level of MMP-2 activity.
基底膜降解是肿瘤侵袭和转移的关键环节，基质金属蛋白酶（MMPs）家族可促进这一过程。MMP-2和MMP-9是在胃肠道起源肿瘤中特别鉴定出的两种亚型。我们之前已表明，用MMP抑制剂BB-94广泛抑制这些酶可提高胰腺癌小鼠模型的生存率。本研究的目的是确定用BB-94处理和未处理的小鼠原位肿瘤中MMP-2和MMP-9的活性。
将1000万个中度分化的胰腺癌细胞系（HPAC）原位植入Balb/c裸鼠体内。小鼠用BB-94或溶剂对照处理70天或直至死亡。尸检时，收获肿瘤，提取总蛋白，并通过明胶-琼脂糖亲和层析从400微克粗蛋白提取物中纯化MMPs。通过蛋白质印迹法和明胶酶谱法测定MMP-2和MMP-9的相对酶水平和活性。
处理组动物的肿瘤明显小于未处理组动物的肿瘤。处理组和未处理组动物中MMP-2的含量均高于MMP-9。两组均存在活性MMP-2，但在用BB-94处理的动物中显著降低。两组均不存在活性MMP-9，而所有样品中潜伏MMP-9的水平似乎低于MMP-2。
在裸鼠体内生长的HPAC细胞中，活化的MMP-2而非MMP-9表明该MMP亚型在此类肿瘤的表型行为中更为关键。此外，用酶抑制剂BB-94处理的动物中活性MMP-2水平降低表明，先前观察到的生存率提高与MMP-2活性水平相关。
