Quantification of elastase-like activity in 13 human cancer cell lines and in an immortalized human epithelial cell line by RP-HPLC.
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 pre-incubation of the cells with a relatively non-toxic, PMN-E-specific inhibitor, MeOSuc-Ala-Ala-Pro-Val-chloromethylketone (MAAPVCK), the p-nitroaniline 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, DAN-G, PA-TU-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 microM 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 PA-TU-8902 is a suitable in vitro model for testing the efficacy of PMN-E-activated prodrugs of antitumor agents.
Achilles
Karin
K
Institute of Pharmacy, Ernst-Moritz-Arndt University, D-17487 Greifswald, Germany.
Bednarski
Patrick J
PJ
D000814:Aniline Compounds / Q000032:analysis; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D048429:Cell Size; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009369:Neoplasms / Q000201:enzymology*
D000814:Aniline Compounds; D004791:Enzyme Inhibitors; C019498:4-nitroaniline; D019272:Leukocyte Elastase