Figure 5

PPD exerted its cytotoxic effects on cancer cells through activation of neutral sphingomyelinase 2. (A) K562 cells were exposed to PPD at 0 (DMSO) and 25 μM for 24 hr. They were stained with BODIPY-Cholesterol ester or BODIPY-C12-SM, then examined using confocal microscopy. (B) K562 cells were exposed to PPD at 0, 12.5, 25 and 50 μM for 6 or 16 hr, then levels of different ceramides were analyzed using LC-MS/MS. K562 (C) and HT29 (D) cells were preincubated with neutral sphingomyelinase (nSMase) inhibitor GW4869 for 1 hr, exposed to PPD at 0, 25 and 50 μM for 48 hr and evaluated for cell viabilities using WST-1 assay. Inhibition studies with GW4869 were repeated three times with similar results. K562 (E) and HT29 (F) cells were transfected with nSMase 2 siRNA 1141725, exposed to PPD at 0, 6.25, 12.5, 25 and 50 μM for 48 hr and evaluated for cell viabilities using WST-1 assay. Inhibition studies with siRNA transfections were repeated twice with similar results. *, p < 0.05; **, p < 0.01; ***, p < 0.001 for control (vehicle) are considered as significant.