title: Bullatacin, a potent antitumor Annonaceous acetogenin, induces apoptosis through a reduction of intracellular cAMP and cGMP levels in human hepatoma 2.2.15 cells.
abstract: 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.
authors.affiliation: Department of Pharmacology, College of Medicine, Kaohsiung Medical University, 100 Shin-Chuan 1st Road, Kaohsiung 807, Taiwan, ROC. huifen@cc.kmu.edu.tw
authors.forename: Hui Fen
authors.initials: HF
authors.lastname: Chiu
authors.forename: Tsai Tung
authors.initials: TT
authors.lastname: Chih
authors.forename: Yun Mei
authors.initials: YM
authors.lastname: Hsian
authors.forename: Chih Hua
authors.initials: CH
authors.lastname: Tseng
authors.forename: Ming Jung
authors.initials: MJ
authors.lastname: Wu
authors.forename: Yang Chang
authors.initials: YC
authors.lastname: Wu
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D004347:Drug Interactions; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005663:Furans / Q000494:pharmacology*; D006801:Humans; D026422:S-Nitrosoglutathione / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D017304:Annexin A5; D000970:Antineoplastic Agents; D005663:Furans; C061022:bullatacin; D005576:Colforsin; D026422:S-Nitrosoglutathione; D000242:Cyclic AMP; D006152:Cyclic GMP; D016650:Fluorescein-5-isothiocyanate