The role of lysosomal enzymes in killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazole.

Abstract

Abstract. The sensitivity of cultured human and hamster fibroblast cells to killing by the lysosomotropic detergent N-dodecylimidazole (Ct2-Im) was investigated as a function of cellular levels of general lysosomal hydrolase activity, and specifically of cysteine cathepsin activity. Fibroblasts from patients with mucolipidosis II (I-cell disease) lack mannose-6-phosphate-containing proteins, and therefore possess only 10-15 % of the normal level of most lysosomal hydrolases. I-cell fibroblasts are about one-half as sensitive to killing by C~2-Im as are normal human fibroblasts. Overall lysosomal enzyme levels of CHO cells were experimentally manipulated in several ways without affecting cell viability: (a) Growth in the presence of 10 mM ammonium chloride resulted in a gradual decrease in lysosomal enzyme content to 10-20% of control values within 3 d. Subsequent removal of ammonium chloride from the growth medium resulted in an increase in lysosomal enzymes, to ~125% of control values within 24 h. (b) Treatment with 80 mM sucrose caused extensive vacuolization within 2 h; lysosomal enzyme levels remained at control levels for at least 6 h, but increased 15-fold after 24 h of treatment. (c) Treatment with concanavalin A (50 ~tg/ml) also caused rapid (within 2 h) vacuolation with a sevenfold rise in lysosomal enzyme levels occurring only after 24 h. The sensitivity of these experimentally manipulated cells to killing by Ci2-Im always paralleled the measured intracellular lysosomal enzyme levels: lower levels were associated with decreased sensitivity while higher levels were associated with increased sensitivity, regardless of the degree of vacuolization of the cells. The cytotoxicity of the cysteine proteases (chiefly cathepsin L in our cells) was tested by inactivating them with the irreversible inhibitor E-64 (100 ~tg/ml). Cell viability, protein levels, and other lysosomal enzymes were unaffected, but cysteine cathepsin activity was reduced to <20% of control values. E-64-treated cells were almost completely resistant to C12-Im treatment, although lysosomal disruption appeared normal by fluorescent visualization of Lucifer Yellow CHloaded cells. It is concluded that cysteine cathepsins are the major or sole cytotoxic agents released from lysosomes by Ci2-Im. These observations also confirm the previous conclusions that C~2-Im kills cells as a consequence of lysosomal disruption.

Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation

“…Finally, the characteristics of siramesine-induced cell death, e.g., an early lysosome damage occurring prior to cell rounding and death, dependence on cysteine cathepsin activity and cell density as well as a highly sigmoidal dose-response curve, 10 closely resemble those of other lysosomotropic detergents. 36,38 It should, however, be noted that whereas other lysosomotropic detergents have been reported to kill all lysosome-containing cells, 37 siramesine-mediated killing displays selectivity to transformed cells both in vitro and in vivo. 10,18 Our earlier data showing that siramesine induces oxidative stress and that lipophilic but not hydrophilic anti-oxidants effectively inhibit siramesine-induced cell death have suggested that lipid peroxidation may be involved in siramesine-induced lysosomal leakage and cytotoxicity.…”

Mucolipidosis IV consists of one complementation group

“…Initially, we speculated that MLIV fibroblasts bearing abnormal lysosomes would be more sensitive to lysosomotropic drugs than normal cells. Dodecylimidazol was previously found to be more toxic to normal cells than mucolipidosis II cells (I cell disease) (14). Other lysosomotropic drugs, which were found to inhibit lysosomal enzymatic processing (15)(16)(17), were also tested.…”

Effect of Phospholipidosis on the Cellular Pharmacokinetics of Chloroquine

“…Therefore, clinically relevant urine concentrations (0 -200 M) of CQ were used to mimic the physiological conditions of the distal tubule and used as input parameters to mathematically model transport behavior of CQ. Simulation results were compared with experimental measurements of intracellular CQ mass in dose-response and time course experiments, under conditions that either enhanced the phospholipidosis effect [cotreatment with sucrose (Wilson et al, 1987;Helip-Wooley and Thoene, 2004)] or inhibited vacuolation [cotreatment with bafilomycin A1 ].…”