Characterization of a cathepsin L-like enzyme secreted from human pancreatic cancer cell line HPC-YP.

Spent culture medium from the human pancreatic carcinoma cell line HPC-YP, which can propagate in a protein-free, chemically defined medium without any other supplements, was analyzed for the presence of the cysteine protease, cathepsin L. The secreted form of cathepsin L was distinguished from the lysosomal form by its increased stability at alkaline pH, by its strong thermostability, and by its larger molecular size. HPC-YP cathepsin L was still stable at pH 7.4 and at 56 degrees C after 60-min preincubation. The molecular weight of this enzyme was estimated to be 68,000, compared with a molecular weight of 29,000 for normal liver cathepsin L. By Western blot analysis, HPC-YP enzyme was found to be composed of two components, one with a molecular weight of 37,000 and the other of 31,000. This result suggests that HPC-YP enzyme in the spent medium may be a complex of the proenzyme (in the case of liver proenzyme; Mr 39,000) and the mature enzyme (in the case of liver mature enzyme; Mr 29,000). Interestingly, an intrinsic inhibitor was also separated from the spent medium by gel filtration. The molecular weight of this inhibitor was estimated to be approximately 13,000. The cathepsin L of HPC-YP proved more resistant toward leupeptin than did liver cathepsin L. On the other hand, the former was more sensitive than the latter toward the diazomethane inhibitors, Z-Phe-Phe-CHN2 and Z-Phe-Ala-CHN2. These results indicate that cathepsin L secreted from cancer cell lines may play a role in the destruction of basal lamina, invasion of tissue, and formation of metastasis.