Serum deprivation increases NB4 sensitivity to zaragozic acid that is specifically due to reduced import of LDL cholesterol.

(A) NB4 cells cultured in serum-depleted medium (RPMI 1640 supplemented with 0.2% bovine calf serum [BCS]) were treated with zaragozic acid A (ZGA; 64 or 256 μM) for 24 hours with or without low-density lipoprotein cholesterol (LDL; 2.6 mM [100 mg/dL]) and assayed for cellular cholesterol using the Amplex Red assay. Treatment with 64 μM ZGA produced significant cellular cholesterol decrements in NB4 cells cultured in serum-depleted medium, while the addition of LDL produced cellular cholesterol increments. Cholesterol levels could not be determined for cells treated with 256 μM ZGA in serum-depleted medium because too few cells survived this treatment. LDL “add-back” to cells treated with ZGA increased cholesterol levels beyond untreated levels. Cellular cholesterol measures are expressed relative to that of untreated NB4 cells cultured in serum-replete medium (RPMI 1640 supplemented with 5% BCS). Data are presented with standard errors and represent the mean of 2 to 6 independent replicates (except +64ZGA+LDL, performed once with other treatments). (B) NB4 cells treated as described for panel A were assayed for cytotoxicity by trypan blue uptake (TB+). As can be seen, serum deprivation markedly increased the cytotoxicity of ZGA (compare with Figure 4A), while the addition of LDL reversed this cytotoxicity. Data are presented with standard errors and represent the mean of 2 to 6 independent replicates (except +64ZGA+LDL, performed once).