AP9-cd, a standardized
lignan composition from Cedrus deodara consisting of (-)-wikstromal, (-)-
matairesinol, and dibenzyl butyrolactol, showed cytotoxicity in several human
cancer cell lines reported earlier. An attempt was made in this study to investigate the mechanism of cell death in human
leukemia Molt-4 and HL-60 cells. It inhibited Molt-4 cell proliferation with 48-h IC(50) of approximately 15 microg/ml, increased sub-G0 cell fraction with no mitotic block, produced apoptotic bodies and induced
DNA ladder formation. Flow cytometric analysis of annexinV-
FITC/PI-stained cells showed time-related increase in apoptosis and post-apoptotic
necrosis. All these
biological end-points indicated cell death by apoptosis. Further, initial events involved massive
nitric oxide (NO) formation within 4 h with subsequent late appearance of
peroxides in cells; measured by flow cytometry using specific
fluorescent probes. Persistently high levels of NO and
peroxide appeared to decrease mitochondrial membrane potential (Psi(mt)) which was recovered by
cyclosporin A in Molt-4 cells. AP9-cd caused 2-fold activation of
caspase-3 in Molt-4 and 5-fold activation in HL-60 cells. Also caspases-8 and -9 were activated in HL-60 cells. Ascorbate suppressed the enhanced
caspases activities indicating a
pro-oxidant effect of AP9-cd. Further,
caspase-3 activation correlated with NO generation that was partially impaired by
nitric oxide synthase (NOS) inhibitors and ascorbate suggesting a role of
pro-oxidant species in
caspase-3 activation. AP9-cd produced no cytotoxicity in primary rat hepatocyte culture at the concentrations used. The studies indicated that AP9-cd mediated early NO formation leads to
caspases activation,
peroxide generation, and mitochondrial depolarization which may be responsible for mitochondrial-dependent and -independent apoptotic pathways involved in the killing of
leukemia cells by AP9-cd.