The samples were infiltrated in Spurr’s resin and polymerized at 60 C for 24 h. Ultrathin Astragaloside IV sections were cut on a Reichert-Jung Ultracut E ultramicrotome, collected on formvar-carbon coated copper grids, and stained with saturated methanolic uranyl acetate and Reynolds’ lead citrate [27]. at each time point. Cells treated with the non-inhibitory peptide, Fmoc-Tyr-Ala-OH, gave values similar to those of controls. The bar charts show percentage dead cells at each time point with (shaded) and without (open) FYAD. NIHMS176613-supplement-01.tif (420K) GUID:?45A100BE-778F-4D59-88FF-E9EF5B7B1976 Abstract A specific Astragaloside IV irreversible inhibitor of both cathepsins B and L, Fmoc-Tyr-Ala-CHN2 (FYAD) induced apoptosis of neuroblastoma cells but not other tumor cells. Cysteine protease inhibitors that were not efficient inhibitors of both proteases did not cause death of any cell line tested. Apoptosis was preceded by accumulation of large electron dense vesicles and multivesicular bodies in the cytoplasm. Exposure of cells to the cathepsin D inhibitor, pepstatin, failed to rescue cells from FYAD-induced death. These results indicate that inhibition of cathepsins B and L may provide a unique mechanism for selectively inducing death of neuroblastoma with limited toxicity to normal cells and tissues. aggressiveness of numerous tumors correlates with expression of lysosomal proteases [2; 3; 4; 5; 6]. Within the cell, release of lysosomal proteases into the cytosol is proposed to induce apoptosis [7; 8; 9; 10; 11; 12], indicating that cathepsin inhibition might prevent cell death and thus have a negative impact on cancer treatment. Conversely, broad based inhibitors such as E-64 and Z-Phe-Gly-NHO-Bz have been shown to induce apoptosis in a variety of cell types, indicating general cytotoxic effects of these compounds [5; 13]. Peptidyl diazomethylketones have been found to be remarkably specific inhibitors of cathepsins [14]. The diazomethylketone moiety allows covalent modification of the active-site cysteine of cathepsins. When a radio-iodinated form of Z-Tyr-Ala-CHN2 was incubated with live cells, the only reactive proteins identified were cathepsins B and L [15]; other cellular and extracellular proteins were not labeled. Treatment of a range of breast cancer cells with this specific inhibitor of cathepsins B and L was shown to effectively inactivate both enzymes and impair cell division [16; 17]. The inhibitor was shown to be cytostatic but not cytotoxic, and upon removal of inhibitor cells continued to divide. A recent study has shown that i.v. injection of a simple iodinated diazomethylketone inhibitor into mice labels only cathepsins B, L and S in whole tissue extracts [18]. This remarkable specificity and selectivity of the diazomethylketone inhibitors for cathepsins Astragaloside IV make them ideal tools to define biological functions of cathepsins. Treatment of rodents with inhibitors of cathepsins B and L does not have any significant toxic effects [19; 20], although inhibitors are teratogenic when administered to pregnant animals [21; 22; 23]. Major drawbacks of therapies to treat cancers arise when drug targets also perform critical functional roles in non-cancerous cells, so lack of general toxicity is a desirable feature of cathepsin inhibition. In this study we discovered that neuroblastoma cells are uniquely sensitive to inhibition of both cathepsins B and L, causing apoptotic cell death. 2. Materials and Methods 2. 1 Neuroblastoma cell lines Four different neuroblastoma cell lines were chosen for this study. IMR-32, TRA1 SK-N-SH and NB-1691 cells were obtained from ATCC (Manassas, VA) and GM11027 cells were from Coriell (Camden, NJ). SK-N-SH cells, representing less aggressive S-type Astragaloside IV tumors and IMR-32 cells from more aggressive N-type tumors are well-established neuroblastoma cell lines. NB-1691 cells (a gift from Peter Houghton, St Jude’s Children’s Hospital, Memphis, TN), like IMR-32 cells, are N-Myc amplified aggressive tumor cells. GM11027 is a less well established cell line derived from primary tumor tissue passaged in a nude mouse. These cell lines were chosen to give a wide variety of samples of neuroblastoma cells with different phenotypes. 2.2 Determination of effective targets of protease inhibitors For clear identification of targets of each of the cathepsin inhibitors, SK-N-SH and IMR-32 cells were incubated with Fmoc-Tyr-Ala-CHN2 (FYAD), Z-Phe-Tyr(OtBu)-CHN2 (ZFYD), Ca074Me or vehicle control for up to 48 h and then incubated with Fmoc-Tyr(I-125)-Ala-CHN2 (1 M) for an additional 2 h. Cells were then washed in serum-free medium and total proteins harvested by dissolving the cell pellets in SDS/PAGE sample buffer containing 8 M urea. Proteins were separated by SDS/PAGE and then the radioactive bands identified by phosphor image analysis of the dried gels. We have previously found that this live cell labeling technique is particularly valuable for detecting cathepsin L, an enzyme that is.