Mitochondrially Targeted Effects of Berberine Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a) quinolizinium on K1735-M2 Mouse Melanoma Cells: Comparison with Direct Effects on Isolated Mitochondrial Fractions 1. Gonalo C. Pereira, 2. Ana F. Branco, 3. Jlio A. C. Matos, 4. Sandro L. Pereira, 5. Donna Parke, 6. Edward L. Perkins, 7. Teresa L. Serafim, 8. Vilma A. Sardo, 9. Maria S. Santos, 10. Antonio J. M. Moreno, 11. Jon Holy and 12. Paulo J. Oliveira+ Author Affiliations1. Center for Neurosciences and Cell Biology (G.C.P., A.F.B., J.A.C.M., S.L.P., T.L.S., V.A.S., M.S.S., P.J.O.) and Institute for Marine Research (A.J.M.M.), Department of Zoology, University of Coimbra, Coimbra, Portugal; and Departments of Biochemistry and Molecular Biology (D.P., E.L.P.) and Anatomy, Microbiology and Pathology (J.H.), University of Minnesota Medical School, Duluth, Minnesota 1. Address correspondence to:Dr. Paulo J. Oliveira, Center for Neurosciences and Cell Biology, Department of Zoology, University of Coimbra, P-3004-517 Coimbra, Portugal. E-mail: paulolivci.uc.ptNext SectionAbstractBerberine Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a)quinolizinium is an alkaloid present in plant extracts and has a history of use in traditional Chinese and Native American medicine. Because of its ability to arrest the cell cycle and cause apoptosis of several malignant cell lines, it has received attention as a potential anticancer therapeutic agent. Previous studies suggest that mitochondria may be an important target of berberine, but relatively little is known about the extent or molecular mechanisms of berberine-mitochondrial interactions. The objective of the present work was to investigate the interaction of berberine with mitochondria, both in situ and in isolated mitochondrial fractions. The data show that berberine is selectively accumulated by mitochondria, which is accompanied by arrest of cell proliferation, mitochondrial fragmentation and depolarization, oxidative stress, and a decrease in ATP levels. Electron microscopy of berberine-treated cells shows a reduction in mitochondria-like structures, accompanied by a decrease in mitochondrial DNA copy number. Isolated mitochondrial fractions treated with berberine had slower mitochondrial respiration, especially when complex I substrates were used, and increased complex I-dependent oxidative stress. It is also demonstrated for the first time that berberine stimulates the mitochondrial permeability transition. Direct effects on ATPase activity were not detected. The present work demonstrates a number of previously unknown alterations of mitochondrial physiology induced by berberine, a potential chemotherapeutic agent, although it also suggests that high doses of berberine should not be used without a proper toxicology assessment. Previous SectionNext SectionBerberine (Fig. 1) is an isoquinoline alkaloid, usually extracted from rhizomes and roots from plants such as the Berbis, Coptis, and Hydrastis species. Such herbs have long been used in traditional Chinese and Native American medicine for the treatment of several maladies. Several pharmacologic properties have been attributed to berberine, including anti-inflammatory (Kuo et al., 2004), antimicrobial (Stermitz et al., 2000), antidiarrheal (Rabbani et al., 1987), antiproliferative (Letasiova et al., 2006), antioxidative (Shirwaikar et al., 2006), and vasorelaxant (Ko et al., 2000) actions. Different types of effects such as up-regulation of cyclooxygenase-2 (Fukuda et al., 1999) and hypoxia-inducible factor-1 (Lin et al., 2004) expression, modulation of multidrug resistance protein expression (Lin et al., 1999), reduction of amyloid- peptide levels (Asai et al., 2007), formation of DNA adducts (Kuo et al., 1995), and cell cycle arrest (Lin et al., 2006; Mantena et al., 2006b) were also previously reported. The antiproliferative properties of berberine raise the interesting possibility that the compound may be useful for anticancer therapeutics, and in fact, berberine has been shown to promote cell death in several transformed cell lines (Hwang et al., 2006; Lee et al., 2006; Letasiova et al., 2006; Lin et al., 2006; Mantena et al., 2006b). Apoptosis induced by berberine involves activation of caspases (Mantena et al., 2006b), increased Bax expression (Lin et al., 2006; Hwang et al., 2006; Lin et al., 2006; Mantena et al., 2006b), DNA fragmentation (Kuo et al., 1995; Hwang et al., 2006; Letasiova et al., 2006; Mantena et al., 2006b), and cytochrome c release (Hwang et al., 2006; Lin et al., 2006), suggesting a possible role of mitochondria in the process (Mantena et al., 2006a; Jantova et al., 2007). Mitochondria could be involved via release of cytochrome c, which can occur through Bax interaction with mitochondrial membranes, as well as through the formation of the mitochondrial permeability transition (