Most anti-cancer agents induce apoptosis, however, a development of multidrug resistance in cancer cells and
defects in apoptosis contribute often to treatment failure. Here, the mechanism of curcumin-induced apoptosis
was investigated in human leukemia HL60 cells and their HL60/VCR multidrug-resistant counterparts. In both
cell lines curcumin induced a bi-phasic ceramide generation with a slow phase until 6 h followed by a more
rapid one. The level of the ceramide accumulation correlated inversely with the cell viability. We found that
the ceramide elevation resulted from multifarious changes of the activity of sphingolipid-modifying enzymes.
In both cell lines curcumin induced relatively fast activation of neutral sphingomyelinase (nSMase), which
peaked at 3 h, and was followed by inhibition of sphingomyelin synthase activity. In addition, in HL60/VCR
cells the glucosylceramide synthase activity was diminished by curcumin. This process was probably due to
curcumin-induced down-regulation of P-gp drug transporter, since cyclosporine A, a P-gp blocker, also inhibited
the glucosylceramide synthase activity. Inhibition of nSMase activity with GW4869 or silencing of SMPD3 gene
encoding nSMase2 reversed the curcumin-induced inhibition of sphingomyelin synthase without affecting the
glucosylceramide synthase activity. The early ceramide generation by nSMase was indispensable for the later
lipid accumulation, modulation of Bax, Bcl-2 and caspase 3 levels, and for reduction of cell viability in
curcumin-treated cells, as all these events were inhibited byGW4869 or nSMase2 depletion. These data indicate
that the early ceramide generation by nSMase2 induced by curcumin intensifies the later ceramide accumulation
via inhibition of sphingomyelin synthase, and controls pro-apoptotic signaling.