Apoptotic activity of betulinic acid derivatives on murine melanoma B16 cell line

https://doi.org/10.1016/j.ejphar.2004.07.103Get rights and content

Abstract

The mitochondrion plays a crucial role in the process of apoptosis and has thus become one of the targets for the search for potential chemotherapeutic agents. Betulinic acid [3β-hydroxy-lup-20(19)lupaen-28-carbonic acid], a lupane-type triterpene which is abundant in many plant species, has been shown to exert a direct effect on the mitochondria and subsequent apoptosis in melanoma cells. Chemical synthesis and modification of betulinic acid are being explored to develop more potent derivatives. We present here the apoptotic activity of several natural derivatives of betulinic acid which were isolated from the roots of a Chinese medicinal herb, Pulsatilla chinensis (Bge) Regel [Ye, W., Ji, N.N., Zhao, S.X., Liu, J.H., Ye, T., McKervey, M.A., Stevenson, P., 1996. Triterpenoids from Pulsatilla chinensis. Phytochemistry 42, 799–802]. Of the five compounds tested, 3-oxo-23-hydroxybetulinic acid was the most cytotoxic on murine melanoma B16 cells (IC50=22.5 μg/ml), followed by 23-hydroxybetulinic acid and betulinic acid (IC50=32 and 76 μg/ml, respectively), with lupeol and betulin exhibiting the weakest cytotoxicity (IC50≥100 μg/ml). Exposure of B16 cells to betulinic acid, 23-hydroxybetulinic acid and 3-oxo-23-hydroxybetulinic acid caused a rapid increase in reactive oxidative species production and a concomitant dissipation of mitochondrial membrane potential in a dose- and time-dependent manner, which resulted in cell apoptosis, as demonstrated by fluorescence microscopy, gel electrophoresis and flow-cytometric analysis. Cell cycle analysis further demonstrated that both 3-oxo-23-hydroxybetulinic acid and 23-hydroxybetulinic acid dramatically increased DNA fragmentation at the expense of G1 cells at doses as low as 12.5 and 25 μg/ml, respectively, thereby showing their potent apoptotic properties. Our results showed that hydroxylation at the C3 position of betulinic acid is likely to enhance the apoptotic activity of betulinic acid derivatives (23-hydroxybetulinic acid and 3-oxo-23-hydroxybetulinic acid) on murine melanoma B16 cells.

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.

Section snippets

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.

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