Invasiveness in Cancer An Intracellular Form of Cathepsin B Contributes to

Cathepsin B is a lysosomal cysteine proteinase whose expression and trafficking are frequently altered in cancer, and plasma membrane and secreted forms are thought to contribute to the invasive and metastatic properties of malignant tumors. We have manipulated the expression of cathepsin B in several tumor cell lines and measured their capacity to invade through a reconstituted extracellular (Matrigel) matrix. Transient expression of human cathepsin B in a poorly metastatic B16F1 murine melanoma variant produced a 3–5-fold increase in cathepsin B activity and a comparable increase in invasiveness. Stable antisense cathepsin B-expressing clones of the highly metastatic human melanoma A375M and prostate carcinoma PC3M cell lines produced 40–50% less cathepsin B than control cells and were proportionately less invasive. In contrast, manipulating cathepsin B levels had no effect on cell migration across an uncoated membrane. The anionic cathepsin B inhibitor (L-3-trans-propylcarbamoyloxirane-2-carbony)-L-isoleucyl-L-proline (CA-074), at a concentration of 1 mM, caused a nearly quantitative inhibition of extracellular cathepsin B but had no effect on Matrigel invasion. In contrast, the equally potent but less selective inhibitor, trans-epoxysuccinyl-L-leucylamino(4-guanidino)butane (E-64) inhibited invasion by 75%. Surprisingly, at a concentration of 10 mM, CA-074 slowly permeated the cells, causing an 80–95% inhibition of intracellular cathepsin B after 12 h, the duration of the invasion assay. The membrane-permeant cathepsin B inhibitor, CA-074 methyl ester, and the higher concentration of CA-074 that inhibited intracellular cathepsin B both significantly reduced Matrigel invasion. Collectively, these results identify an intracellular role for cathepsin B in matrix degradation. They also indicate that caution should be exercised in assuming that CA-074 is unable to enter cells when it is used to inhibit biological processes of long duration.