Effects of (1R,9S)-β-hydrastine on l-DOPA-induced cytotoxicity in PC12 cells

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Abstract

(1R,9S)-β-Hydrastine in lower concentrations of 10–50 μM inhibits dopamine biosynthesis in PC12 cells (Planta Med. 57 (2001) 609). In this study, the effects of (1R,9S)-β-hydrastine on l-DOPA (l-3,4-dihydroxyphenylalanine)-induced cytotoxicity in PC12 cells were investigated. (1R,9S)-Hydrastine at concentrations up to 250 μM did not reduce cell viability. However, at concentrations higher than 500 μM it caused cytotoxicity in PC12 cells, as determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, TUNEL (terminal deoxynucleotidyltransferase dUTP nick-end labeling) method and flow cytometry. Exposure of PC12 cells to cytotoxic concentrations of (1R,9S)-β-hydrastine (500 and 750 μM) in combination with l-DOPA (20, 50 and 100 μM) after 24 or 48 h resulted in a significant decrease in cell viability compared with the effects of (1R,9S)-β-hydrastine or l-DOPA alone, and apoptotic cell death was observed. However, the decrease in cell viability induced by (1R,9S)-β-hydrastine was not prevented by the antioxidant N-acetyl-l-cysteine, indicating that it is not mediated by membrane-based oxygen free radical damage. These data suggest that (1R,9S)-β-hydrastine has a mild cytotoxic effect and at higher concentration ranges aggravates l-DOPA-induced cytotoxicity in PC12 cells.

Introduction

Hydrastine derivatives are composed of a phthalide and an isoquinoline alkaloid and exist in two configurations, such as (1R,9S)-β-hydrastine [(−)-β-hydrastine] and (1S,9R)-β-hydrastine [(+)-β-hydrastine] (Fig. 1). Concerning the pure base, the (1R,9S)-form is a well-known component of the roots of Hydrastis canadensis L. (Ranunculaceae) and Berberis laurina Billb. (Berberidaceae), and the (1S,9R)-form occurs in the roots of Corydalis stricta Steph. (Papaveraceae) Stanek and Manske, 1968, Tang and Eisenbrand, 1992. (1S,9R)-β-Hydrastine has an antagonistic effect on gamma-aminobutyric acid receptors (Huang and Johnston, 1990). (1R,9S)-β-Hydrastine has been found to inhibit competitively bovine adrenal tyrosine hydroxlase (EC 1.14.16.2; TH) with l-tyrosine as a substrate (Lee et al., 1997). Recently, (1R,9S)-β-hydrastine, but not (1S,9R)-β-hydrastine, at lower concentrations of 10–50 μM was found to inhibit dopamine biosynthesis, in part through the inhibition of TH activity in PC12 cells (Kim et al., 2001).

Many studies have confirmed that tetrahydroisoquinolines are linked to Parkinson's disease and brain damage due to chronic alcoholism Kotake et al., 1995, Maruyama et al., 1996. The underlying mechanisms of tetrahydroisoquinoline-induced apoptosis are mediated by oxidative stress and mitochondrial energy depletion Seaton et al., 1997, Morikawa et al., 1998. Hydrastine derivatives have a similar tetrahydroisoquinoline configuration, and so it is conceivable that hydrastine derivatives might cause neurodegeneration. However, the cytotoxicity of hydrastine derivatives, especially (1R,9S)-β-hydrastine, has not been examined even though in spite (1R,9S)-β-hydrastine has inhibitory activity on dopamine biosynthesis.

l-DOPA (l-3,4-dihydroxyphenylalanine) is the most frequently prescribed drug for controlling the symptoms of Parkinson's disease (Marsden, 1994), due to its ability to raise the dopamine level in the striatum (Hornykiewicz, 1994). However, some reports have suggested that l-DOPA may accelerate deterioration of the condition of Parkinsonian patients and that l-DOPA toxicity occurs in damaged dopamine neurons in vivo (Boyce et al., 1990). It is also reported that l-DOPA produces neurotoxic reactive oxygen species, leading to apoptosis due to autoxidation and enzymatic oxidation (Sandstrom et al., 1994).

The pheochromocytoma, PC12, cell lines were originally characterized from a catecholamine-secreting adrenal chromaffin tumor in rats (Greene and Tischler, 1976) and have been widely used as in vitro models to investigate dopaminergic toxicity, such as l-DOPA neurotoxicity, l-DOPA autoxidation, oxidative stress and mitochondrial impairment Itano et al., 1994, Basma et al., 1995, McNaught et al., 1996, Migheli et al., 1999.

In this study, therefore, the cytotoxic effects of (1R,9S)-β-hydrastine, alone or in combination with l-DOPA, in PC12 cells were investigated. In addition, we also examined the role of the antioxidant N-acetyl-l-cysteine in the cytotoxicity induced by (1R,9S)-β-hydrastine.

Section snippets

Chemicals

(1R,9S)-β-Hydrastine, l-DOPA, ribonuclease A (Rnase A), propidium iodide, ethylenediaminetetraacetic acid (EDTA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). The in situ cell death detection kit (TUNEL: terminal deoxynucleotidyltransferase dUTP nick-end labeling) was supplied by Boehringer Mannheim (Mannheim, Germany). All sera, antibiotics and RPMI 1640 for cell culture were obtained from the Gibco (Grand Island, NY,

Inhibition of cell viability by (1R,9S)-β-hydrastine and l-DOPA

(1R,9S)-β-Hydrastine at concentrations up to 250 μM did not significantly reduce cell viability in PC12 cells. However, when PC12 cells were treated with 500–750 μM (1R,9S)-β-hydrastine for 24 or 48 h, there was a concentration- and time-dependent reduction in cell viability, as determined with the MTT assay (Fig. 2). Cytotoxicity was greater when (1R,9S)-β-hydrastine was added to PC12 cells for 48 h instead of 24 h (Fig. 2).

l-DOPA at concentrations of 20 and 50 μM did not significantly

Discussion

Tetrahydroisoquinolines are inhibitors of TH activity, the rate-limiting enzyme of the catecholamine biosynthetic pathway (Nagatsu and Hirata, 1987), and have been reported to possess many of the cytotoxic characteristics of 1-methyl-4-phenyl-1,2,3,6-tetrahydrapyridine (MPTP), which causes a Parkinson-like syndrome in human and non-human primates Tasaki et al., 1991, Desole et al., 1996, McNaught et al., 1998, Ohta, 2002, Przedborski and Vila, 2001. l-DOPA itself has been shown to be toxic to

Acknowledgments

The authors sincerely thank the financial support of the Research Center for Bioresource and Health, KOSEF.

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