Chaetocin is rapidly and dramatically accumulated in cancer cells by means that require intact/unreduced chaetocin disulfide bonds. (A) HPLC tracing indicating results of assessment of levels of intracellular chaetocin in chaetocin-treated A549 cells as described in “Patients, materials, and methods.” Note that only a single new HPLC peak, corresponding to that of unaltered/unreduced chaetocin, resulted from treatment with chaetocin. (B) Assessment of intracellular (A549 cells) and extracellular (media) chaetocin concentrations as functions of time in response to addition of 10 μM chaetocin to culture media at time 0. (Inset) Expansion to better show changes in media chaetocin concentration over time. (C) Time course of intracellular accumulation of chaetocin in A549 cells. Results shown are presented to indicate the fold changes relative to concentrations of chaetocin applied to media at time = 0. (B-C) Chaetocin concentrations were determined as described in “Patients, materials, and methods” using HPLC. (D) Effects of dithiothreitol-reduced chaetocin or S-methyl chaetocin on colony formation in A549 cells, indicating loss of cytotoxicity upon modification of the chaetocin disulfide bond. Cells were exposed to all agents for 24 hours; error bars indicate 1 standard deviation.