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A sensitive and specific RP-HPLC assay was developed to measure the levels of polymorphonuclear elastase (PMN-E) activity in growing cell cultures. By combining a preincubation of the cells with a relatively nontoxic, PMN-E-specific inhibitor, MeOSuc Ala-Ala-Pro-Val-chloromethylketone (MAAPVCK), the pnitroaniline formed by the hydrolysis of the substrate MeOSuc-Ala-Ala-Pro-Val-p-NA by PMN-E is quantified. Elastase-like activity was measured in 14 human cells lines: 13 cancer cell lines (HL-60, U-937, A-427, LCLC-103H, YAPC, DANG, PATU 8902, KYSE-70, -510, -520, 5637, SISO and MCF-7) and one immortalized epithelial cell line (hTert-RPE1). Activity was detected in all lines; the lowest was found in hTert-RPE1 cells while the highest was detected in a pancreas adenocarcinoma line (PA-TU-8902). When the results were normalized according to cell volume instead of cell number, the leukemia line HL-60 had the highest activity and PA-TU-8902 ranked second. A 1 h pre-incubation with 9.0 uM of the irreversible PMN-E inhibitor MAAPVCK led to varying degrees of enzyme inhibition depending on the cell line; the strongest inhibition was observed with the PA-TU-8902 pancreatic cancer cell line (90% inhibition) while the weakest was seen with the A-427 lung cancer cell line (52%). These results indicate that PATU-8902 is a suitable in vitro model for testing the efficacy of PMN-E-activated prodrugs of antitumor agents.

Quantification of Elastase-Like Activity in 13 Human Cancer Cell Lines and in an Immortalized Human Epithelial Cell Line by RP-HPLC