L-
leucyl-leucine methyl ester (LLOMe) is a lysosomotropic
detergent, which was evaluated in clinical trials in
graft-vs-host disease because it very efficiently killed monocytic cell lines. It was also shown to efficiently trigger apoptosis in
cancer cells, suggesting that the drug might have potential in anticancer
therapy. Using U-937 and THP-1 promonocytes as models for monocytic cells, U-87-MG and HeLa cells as models for
cancer cells, and noncancerous HEK293 cells, we show that the drug triggers rapid
cathepsin C-dependent lysosomal membrane permeabilization, followed by the release of other
cysteine cathepsins into the cytosol and subsequent apoptosis. However, monocytes were found to be far more sensitive to the drug than the
cancer and noncancer cells, which is most likely a consequence of the much higher intracellular levels of
cathepsin C-the most upstream molecule in the pathway-in monocytic cell lines as compared to
cancer cells. Overexpression of
cathepsin C in HEK293 cells substantially enhances their sensitivity to the drug, consistent with the crucial role of
cathepsin C. Major involvement of
cysteine cathepsins B, S, and L in the downstream signaling pathway to mitochondrial cell death was confirmed in two gene ablation models, including the ablation of the major cytosolic inhibitor of
cysteine cathepsins,
stefin B, in primary mouse
cancer cells, and simultaneous ablation of two major
cathepsins, B and L, in mouse embryonic fibroblasts (MEFs). Deletion of
stefin B resulted in sensitizing primary murine
breast cancer cells to cell death without affecting the release of
cathepsins, whereas simultaneous ablation of
cathepsins B and L largely protected MEFs against cell death. However, due to the extreme sensitivity of monocytes to LLOMe, it appears that the drug may not be suitable for anticancer
therapy due to risk of systemic toxicity.