Bullatacin, a potential antitumor Annonaceous acetogenin (AA), is isolated from the seed of the Formosa Annona atemoya. We reported previously that
bullatacin inhibits the secretion of
hepatitis B surface antigen from 2.2.15 cells (human
hepatoma HepG2 cells transfected with hepatitis B virus
DNA plasmid). In the present study, we determined cell apoptosis by using double-
dye staining with
fluorescein-
isothiocyanate-labeled
annexin V and
propidium iodide. We found that
bullatacin induced apoptosis in 2.2.15 cells in a time-dependent manner; the most significant apoptotic change appeared at 16 hr. Moreover, different concentrations (10(-3) to 1.0 microM) of
bullatacin induced apoptosis in a concentration-dependent manner at 16 hr. The determination of intracellular
cyclic AMP (cAMP) and
cyclic GMP (cGMP) levels in 2.2.15 cells after exposure to
bullatacin demonstrated that
bullatacin caused both to decrease in a time- and concentration-dependent manner. A time course (0.33, 1, 6, 16, 24hr) study indicated that while both cAMP and cGMP levels decreased early (at 0.33 hr), the most dramatic decline appeared at 6 hr. Meanwhile, the inhibitory effect on cAMP and cGMP levels reached a maximum at 16 hr (90.5+/-3.2 and 47.3+/-12.8%, respectively). The concentration-dependent decrease of both cAMP and cGMP induced by
bullatacin was parallel with the magnitude of apoptosis induced by various concentrations (10(-3) to 1.0 microM) of
bullatacin. Additionally, the
bullatacin-induced apoptosis was inhibited by the addition of cAMP and cGMP elevating agents (
forskolin and
S-nitrosoglutathione). Our results suggest that a decrease of both cAMP and cGMP levels may play a crucial role in
bullatacin-induced apoptosis in 2.2.15 cells.