Apoptotic activity of betulinic acid derivatives on murine melanoma B16 cell line
Introduction
Betulinic acid [3β-hydroxy-lup-20(19)lupaen-28-carbonic acid] is a lupane-type triterpene which was first isolated from the stem bark of an East African evergreen tree, Ziziphus mauritiana Lam. (Rhamnaceae) and which is abundant in the white birch (Pisha et al., 1995). It possesses selective apoptotic activity toward melanoma cells (Pisha et al., 1995, Wick et al., 1999, Fulda and Debatin, 2000) and also toward tumor cells of neuroectodermal origin (Schmidt et al., 1997, Fulda et al., 1999). Betulinic acid-induced apoptosis is not exerted through a ligand/receptor system but is mediated by a decrease in mitochondrial permeability (Fulda et al., 1998), the release of mitochondrial cytochrome c into the cytosol (Fulda and Debatin, 2000), the formation of reactive oxidative species and the activation of crm-A-insensitive caspase activity (Wick et al., 1999). Recently, betulinic acid was also found to inhibit the replication of human immunodeficiency virus (HIV) (Hashimoto et al., 1997, Vlietinck et al., 1998, Holz-Smith et al., 2001). Because of its potential therapeutic action against melanoma and human immunodeficiency virus replication (De Clercq et al., 1996, De Clercq, 2000), a number of modified betulinic acid compounds were synthesized for clinical trial (Soler et al., 1996, Hashimoto et al., 1997, Jeong et al., 1999).
Apoptosis is a process of cell death which is characterized by chromatin condensation, DNA fragmentation and formation of apoptotic bodies (Kerr et al., 1972). It is generally believed that betulinic acid induces loss of the mitochondrial transmembrane potential and interferes with the permeability transition pore complex, by which mitochondrial apoptotic factors are released into the cytosol for cleavage of caspases and activation of the apoptotic machinery (Fulda et al., 1998). In the present study, we compared the cytotoxicity of five natural betulinic acid derivatives and revealed that 3-oxo-23-hydroxybetulinic acid and 23-hydroxybetulinic acid exhibited more potent apoptotic activity by increasing the formation of intracellular reactive oxidative species and reducing the mitochondrial membrane potential of murine melanoma B16 cells.
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Cell cultures
The murine melanoma B16 cell line (NBL6323) and murine connective tissue fibroblast L929 cell line (CCL1) were obtained from the American Type Culture Collection and maintained in F10 medium and RPMI medium, respectively, supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin and 100 IU/ml penicillin. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 for 48 h for comparison of cytotoxicity between betulinic acid and its derivatives. In order to compare the cell
Cytotoxicity of betulinic acid and its derivatives
Fig. 2A shows the cytotoxicity of the five compounds, with 3-oxo-23-hydroxybetulinic acid being the most toxic compound (IC50=22.5 μg/ml), followed by 23-hydroxybetulinic acid, betulinic acid, betulin and lupeol (IC50=32, 76, 100 and >100 μg/ml, respectively). Hydroxylation and oxidation at positions R1, R2 and R3 interfered with the cytotoxicity of this lupane-type triterpene. The hydroxylation of the methyl group at R4 did not affect cytotoxicity, as indicated by lupeol and betulin (both IC50
Discussion
The mitochondrion plays an essential role in the process of apoptosis (Fesik, 2000, Gottlieb, 2000). When cells receive intracellular or extracellular signals leading to apoptosis, prominent mitochondrial alterations, including changes in membrane permeability, decrease in membrane potential, swelling, disruption and the release of caspases and endonucleases, are observed and result eventually in DNA fragmentation and cell death. The mitochondrion is thus a novel target for the development of
Acknowledgments
The authors thank Miss NLY Wong for her excellent technical assistance.
References (27)
- et al.
Chemically de-acetylated 2′,7′-dichlorodihydrofluorescein diacetate as a probe of respiratory burst activity in mononuclear phagocytes
J. Immunol. Methods
(2001) - et al.
NADPH-oxidase activation in murine neutrophils via formyl peptide receptors
Exp. Cell Res.
(2003) Insights into programmed cell death through structural biology
Cell
(2000)- et al.
Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid
J. Biol. Chem.
(1998) Mitochondria: execution central
FEBS Lett.
(2000)- et al.
Anti-AIDS agents—XXVII. Synthesis and anti-HIV activity of betulinic acid and dihydrobetulinic acid derivatives
Bioorg. Med. Chem.
(1997) - et al.
Preparation of amino acid conjugates of betulinic acid with activity against human melanoma
Bioorg. Med. Chem. Lett.
(1999) - et al.
Synthesis of betulinic acid derivatives with activity against human melanoma
Bioorg. Med. Chem. Lett.
(1998) - et al.
Betulinic acid induces apoptosis in human neuroblastoma cell lines
Eur. J. Cancer
(1997) - et al.
Betulinic acid, a potent inhibitor of eukaryotic topoisomerase I: identification of the inhibitory step, the major functional group responsible and development of more potent derivatives
Med. Sci. Monit.
(2002)
Mitochondrion as a novel target of anticancer chemotherapy
J. Natl. Cancer Inst.
Novel compounds in preclinical/early clinical development for the treatment of HIV infections
Rev. Med. Virol.
Betulinic acid derivatives: a new class of specific inhibitors of human immunodeficiency virus type 1 entry
J. Med. Chem.
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