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

• Published in: European journal of pharmacology 2004-09, Vol.498 (1), p.71-78
• Main Authors: Liu, Wing-Keung, Ho, Joyce C.K., Cheung, Florence W.K., Liu, Bonnie P.L., Ye, Wen-Cai, Che, Chun-Tao
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis; Apoptosis - drug effects; Betulinic acid derivative; Biological and medical sciences; Cell Cycle - drug effects; Cell Line, Tumor; Cell Survival - drug effects; Dose-Response Relationship, Drug; Inhibitory Concentration 50; Intracellular Membranes - drug effects; Intracellular Membranes - physiology; Medical sciences; Melanoma; Melanoma, Experimental - drug therapy; Melanoma, Experimental - metabolism; Melanoma, Experimental - pathology; Membrane Potentials - drug effects; Mice; Mitochondria - drug effects; Mitochondria - physiology; Mitochondrial membrane potential; NADPH Oxidases - metabolism; Pentacyclic Triterpenes; Pharmacology. Drug treatments; Piperidines - chemistry; Piperidines - pharmacology; Piperidines - therapeutic use; Propionates - chemistry; Propionates - pharmacology; Propionates - therapeutic use; Reactive oxidative species; Reactive Oxygen Species - metabolism; Structure-Activity Relationship; Triterpenes - chemistry; Triterpenes - pharmacology
• ISSN: 0014-2999; 1879-0712
• DOI: 10.1016/j.ejphar.2004.07.103

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 (IC 50=22.5 μg/ml), followed by 23-hydroxybetulinic acid and betulinic acid (IC 50=32 and 76 μg/ml, respectively), with lupeol and betulin exhibiting the weakest cytotoxicity (IC 50≥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.