Inhibition of in vitro Human Bladder Cancer Cell Invasion by Proteinase Inhibitor

Kim, Choung Soo; Kim, Si Whang

Abstract

The invasion of basement membrane is one of the most important steps for tumor dissemination. Invasive human bladder tumor cells have the ability to degrade basement membrane, especially laminin with cysteine proteinase, cathepsin B which is mainly located in plasma membrane of tumor cell. Cathepsin B was assayed by spectrofluorometer in plasma membrane of human invasive bladder tumor cell line J82. The activities of cathepsin B were 0.98+/-0.14 nmol/ mg/min in plasma membrane fraction. E-64, a synthetic cysteine proteinase inhibitor decrease the activities of cathepsin B to 0.45+/-0.06 nmol/mg/min, 0.30+/-0.06 nmol/mg/min, 0.17+/-0.04 nmol/mg/min, 0.12+/-0.02 nmol/mg/min and 0.09+/-0.01 nmol/mg/min at the concentrations of 20pM, 40pM, 60pM, 80pM and 100pM, respectively. E-64 could prevent the degradation of human basement membrane laminin by the plasma membrane fraction of J82 which was shown in Western blot using polyclonal rabbit antilaminin antibody. In an in vitro model of tumor invasion, membrane invasion culture system (MICS), the numbers of J82 cells which had invaded Matrigel were 4.7+/-2.2 cells/field, 13.1+/-5.6 cells/field an 28.0+/-5.3 cells/field with 25ug Matrigel after 2 hours, 4 hours and 6 hours of incubation period. Migrated tumor cells were 31.2+/-7.3 cells/field. 28.0+/-5.3 cells/field, 18.2+/-3.1 cells/field and 10.6+/-4.6 cells/field with 0ug, 25ug, 50ug and 100ug of the amount of Matrigel, respectively. The number of migrated tumor cells was decreased proportionally to the increase of Matrigel amount. When 25ug Matrigel was used for 6 hours in MICS the invaded tumor cells were 28.0+/-5.3 cells/field, 18.3+/-6.0cells/field, 13.1+/-6.0 cells/field, 12.4+/-3.9 cells/field and 5.5+/-2.3 cells/ field after adding E-64 at the concentration of 0uM, 50uM, 100uM, 200uM and 400uM, respectively. The findings suggest that cathepsin B, cysteine proteinase may play an important role in the progression of superficial bladder tumor into invasive bladder tumor and E-64, cysteine proteinase inhibitor, may inhibit the invasion of superficial bladder tumor.