L-02细胞系特性

Pyrrolizidine alkaloid clivorine inhibits human normal liver L-02 cells growth and activates p38 mitogen-activated protein kinase in L-02 cells

Abstract

Clivorine, a pyrrolizidine alkaloid extracted from Chinese medicinal plant Ligularia hodgsonii Hook significantly inhibited human normal liver L-02 cells proliferation and decreased L-02 cells viability. The results of western blot showed that clivorine strongly evoked phosphorylation of p38 mitogen-activated protein (MAP) Kinase in L-02 cells, but had no effect on extracellular signal-related kinases MAP Kinase phosphorylation. Moreover, another pyrrolizidine alkaloid monocrotaline had no effect on phosphorylation of p38 MAP Kinase in L-02 cells. These studies document the effects of pyrrolizidine alkaloid clivorine on the MAPK cascade and on the growth of human normal liver L-02 cells for the first time, which may be a possible reason for the toxic effects observed in those plants containing pyrrolizidine alkaloids.

Keywords: Clivorine; Pyrrolizidine alkaloid; L-02 cell; p38 Mitogen-activated protein kinase

1.Introduction

Pyrrolizidine alkaloids (PAs) occur naturally in a wide variety of plant species such as Leguminosae, Boraginaceae and Asteraceae. Many PAs are hepatotoxic, genotoxic, cytotoxic and pneumotoxic (Mattocks, 1986; Huxtable,1989). Ligularia hodgsonii belonging to Family Asteraceae has been used for cough, hepatitis and inflammation in traditional Chinese medicine (TCM). L. hodgsonii contains many PAs of which clivorine is the most abundant component (0.151%) and has been reported to be a carcinogenic agent (Kuhara et al., 1980). Our early study has found that oral administration of clivorine to rat leads to death and that the major hurt is happened in the liver (unpublished data). However, there is no study of clivorine about its toxicity on human hepatocytes.

Many evidences have demonstrated that signal transduction pathways actively modulate the hepatocyte’s response to toxin-induced liver injury (Jones and Czaja, 1998).Mitogen-activated protein kinases (MAPKs) are evolutionary conserved enzymes in the intracellular signal transduction pathways connecting cell surface receptors to critical regulatory targets, and control many aspects of mammalian cellular physiology including cell growth, differentiation and apoptosis (Lewis et al., 1998; Gutkind, 2000; Chang and Karin, 2001). So far, at least four distinctly regulated groups of MAPKs: extracellular signal-related kinases (ERK1/2),jun amino-terminal kinases (JNK1/2/3), p38 proteins (p38a/b/g/d) and ERK5 have been found to be expressed in mammals (Gutkind, 2000; Chang and Karin, 2001).

This study is conducted to investigate the effects of clivorine on the growth of human normal liver L-02 cells and the potential effects on two major MAPKs: p38 and ERK1/2 kinases.

2.Materials and methods

2.1.Chemicals and reagents

Roswell Park Memorial Institute 1640 (RPMI 1640),modified Eagle’s medium (MEM), fetal bovine serum (FBS), trypan blue solution and horseradish peroxidaseconjugated goat anti-rabbit secondary antibody were purchased from GIBCO-BRL. Phosphospecific rabbit polyclonal antibodies against 180Thr and 182Tyr dual-phosphorylated p38, against total p38 (phosphorylation state independent), against 204Tyr-phosphorylated ERK1/2 and against total ERK1/2 (phosphorylation state independent) were purchased from New England Biolabs Inc. Nitrocellulose membranes (Hybond) and enhanced chemiluminescence detection system were obtained from Amersham Pharmacia. All other reagents unless indicated were from Sigma Chemical Co.

2.2.Treatment solutions

Clivorine isolated from L. hodgsonii was structurally elucidated by IR, NMR, MS spectra and its purity determined by HPLC was 99.5%. The structure of clivorine is shown in Fig. 1. The roots and rhizomes of L. hodgsonii were collected on September 19, 1993 in Emei County,Sichuan Province of People’s Republic of China. The material was authenticated and stored dry until extraction. A voucher specimen is deposited in the Department of Pharmacognosy, China Pharmaceutical University. Monocrotaline gifted from Prof. Zhi-Ben Tu (Wuhan Institute of Botany, Chinese Academy of Sciences) was isolated from Crotalaria sessiliflora (Roder et al., 1992).

2.3.Cell culture

Human normal liver L-02 cells and Hela cells (Cell Bank, Type Culture Collection of Chinese Academy of Sciences) were cultured in RPMI1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM glutamine. Human embryonic kidney (HEK) 293 cells (American Type Culture Collection) were cultured in MEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM glutamine. Cells were incubated at 37.8℃in a humidified atmosphere (5% CO2).

2.4.Cell growth

Cells were seeded in 96-well microplates at a density of 104 per well and were cultured in cell culture medium for 10–12 h. After treatment with clivorine for 24, 48 and 72 h,500 mg/ml 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) was added and was incubated with cells for 4 h in a CO2 incubator. The functional mitochondrial succinate dehydrogenases in cells can convert MTT to formazan that generates a blue color. Because the extent of this conversion is proportional to the number of survival cells, MTT assay is widely used to quantify viable cells (Hansen et al., 1989). At last the formazan was dissolved in 10% SDS–5% iso-butanol–0.01 M HCl in a CO2 incubator for 12 h. The intensity was measured using a reader (EL311,Bio-TEK instruments) for enzymelinked immunosorbent assay under an absorption wavelength of 570/630 nm.

2.5.Cell viability assay

Cells (2 £ 105 per well) were cultured in serum-free RPMI1640 with clivorine for 24 h in 12-well microplates before trypsinization. Then the cells were mixed with 0.4% trypan blue-PBS for 10 min, and the dead cells were stained blue by trypan blue. The numbers of stained and unstained cells were counted using a hemocytometer.

2.6.Western blot analysis

Cells were cultured in six-well microplates to <90% confluence. The growth medium was removed and replaced with serum-free RPMI 1640 containing 0.1% BSA for 10 h. The cells were treated with clivorine (100 mM) for different time at 37 8C, then lysed with the SDS sample buffer containing 50 mM Tris (pH 7.4), 2% SDS (wt/vol), 5% 2- mercaptoethanol and 10% glycerol. Samples were heated at 95.8℃ for 5 min and centrifuged (13,000g, 5 min) at 4 8C and the supernatant was analyzed by SDS–PAGE in a 10% acrylamide gel. Proteins were transferred to nitrocellulose membrane, and the membranes were blocked with 20% nonfat dry milk in TBST [20 mM Tris (pH 8.0), 150 mM NaCl and 0.1% Tween-20]. The membranes were blotted with the primary antibody (phospho-p38, total-p38)

phospho-ERK1/2 or total ERK1/2 MAP Kinases), then with horseradish peroxidase-conjugated second antibody and detected by enhanced chemiluminescence according to the manufacturer’s intructions. Molecular weights of proteins were estimated by using prestained SDS–PAGE markers (Bio-Rad). For repeated immunoblotting, membranes were stripped in 62.5 mM Tris (pH 6.7), 20% SDS and 0.1 M2-mercaptoethanol for 30 min at 50 8C.

2.7.Statistical analysis

All values are expressed as means ^ standard error of the mean (SEM). Student t-test was used to determine the significance of differences in multiple comparisons. Values of p # 0.05 were considered as statistically significant.

3.Results

3.1.Toxic effects of clivorine on human normal liver L-02 cells as determined by MTT and trypan blue staining

As shown in Fig. 2(A), clivorine significantly decreased MTT reduction in a time and dose-dependent manner, while another pyrrolizidine alkaloid monocrotaline (100 mM) showed no obvious toxic effect in the same condition.Further trypan blue staining assay showed that the percentage of survival cells also decreased in a dosedependent manner following treatment with clivorine but not with monocrotaline (Fig. 2(B)). This suggests that clivorine inhibits human normal liver L-02 cells growth and cells viability.

3.2.Clivorine inhibited HEK293 and Hela cells growth

To further confirm the toxic effects of clivorine, we used other two cell lines HEK293 and Hela cells. As shown in Fig. 3, clivorine strongly inhibited both HEK293 cells and Hela cells growth. All of the results indicate that pyrrolizidine alkaloid clivorine has strong toxic effects.

3.3.The effects of clivorine on phosphorylation of p38 and ERK1/2 MAP Kinase in L-02 cells

It has been established that p38 MAP Kinase is activated by dual phosphorylation of 180Thr and 182Tyr residues, and the phosphorylation of p38 MAP Kinase has been widely used to represent its activation (Raingeaud et al., 1995).Furthermore, it has been reported that the activation of p38 MAP Kinase is implicated in hepatocyte growth arrest (Awad et al., 2000), so we tested the effect of clivorine on the phosphorylation of p38 MAP Kinase in L-02 cells. As shown in Fig. 4(A), 100 mM clivorine evoked a rapid increase in p38 MAP Kinase phosphorylation, which was detectable after 3 min of clivorine treatment. The results from the same membrane reprobed with total-p38 antibody demonstrated that the total amount of p38 MAP Kinase in L-02 cells was not changed. We also tested the effect of another pyrrolizidine alkaloid monocrotaline on p38 MAP Kinase activation. As shown in Fig. 4(B), 100 mM monocrotaline had no effect on phosphorylation of p38 MAP Kinase in L-02 cells. As ERK1/2 MAP Kinase pathway is also associatd with cell survival and proliferation (Bonni et al., 1999), we tested the effect of clivorine on ERK1/2 phosphorylation in L-02 cells. As shown in Fig. 4(C), 100 mM clivorine had no effect on ERK1/2 phosphorylation. It has been reported that EGF can strongly activate ERK1/2 (Yu et al., 1997), so we used EGF (10211 M) as a positive control for ERK1/2 phosphorylation.

4.Discussion

Recently, there has been a growing interest in alternative medicine, particularly the use of herbal products such as TCM which is a new trend in the development of novel pharmaceuticals (Yuan and Lin, 2000). But some TCM contain components with toxic effects and the underling mechanism is not clear. In the present results, we firstly demonstrate that clivorine, one of PAs in L. hodgsonii inhibits a human normal liver cell line L-02 cells growth (Liao et al., 2000; Guo et al., 2001). Thus, our results provide evidence for the toxic effects of PAs at the level of cellular and molecular biology, which may be helpful for understanding the side effects of those TCM. It is commonly believed that PAs themselves are not toxic, but require metabolic activation by the liver to form reactive and highly toxic dehydroalkaloids (Mattocks, 1986) which induce formation of DNA or protein crosslink products and lead to DNA and protein damage (Pereira et al., 1998; Coulomber et al., 1999; Kim et al., 1999). This is supported byour experiments in which no toxic effect of monocrotaline, a kind of PAs, could be found on L-02 cells growth, while it is reported that one of its metabolic products monocrotaline pyrrole had toxic effects on pulmonary artery endothelial cells (Thomas et al., 1998).However, in our experiments we also found that clivorine could strongly inhibit cell proliferation and decrease cell viability in both HEK293 and Hela cells which lack hepatic metabolic enzymes. Therefore, we suspect that the cytotoxicity of clivorine is not only relied on hepatic monoxygenase-generated metabolites but also due to its direct actions, which is in consistent with the previous report on another PAs Irniine (Rakba et al., 2000).

p38 MAP Kinase has been implicated primarily in the induction of apoptosis and inflammation after exposure to different agents. (Jing et al., 1999; Ozaki et al., 1999; Galan et al., 2000; Nakagami et al., 2001; Namgung and Xia, 2001; She et al., 2001; Thrane et al., 2001). Further studies show that p38 MAP Kinase activation results in hepatocyte growth arrest (Awad et al., 2000). In the present study, clivorine strongly stimulated phosphorylation and activation of p38 MAP Kinase in L-02 cells, which suggests that the p38 MAP Kinase may be associated with clivorine-induced inhibition of cell proliferation and viability. In addition to p38 MAP Kinase, some other pathways such as cdc2 Kinase and Bax, Bcl-x protein have also been reported to regulate the toxic effects of PAs (Thomas et al., 1998; Gordon et al.2000). However, we have not observed the significant effect of clivorine on the phosporylation of ERK1/2 MAP Kinase suggesting that ERK1/2 MAP Kinase pathway is not associated with the toxic effects of cliv rine on human normal liver L-02 cells.All together, our data confirm that clivorine is involved in the toxicological manifestations observed in humans and animals when consumption of medicinal plants containing clivorine. Moreover, p38 MAP Kinase activation may be involved in the toxic effects of clivorine. Although the underling mechanism in p38 MAP Kinase activation remains to be studied, the present studies reinforce the need to prohibit the consumption of L. hodgsonii and other medicinal plants containing clivorine in order to prevent further intoxication.

Acknowledgments

The authors wish to thank Professor Gang Pei for his kind direction and Professor Zhi-Ben Tu for providing monocrotaline. We wish to thank PhD Nan-Jie Xu for his help in preparing this manuscript. We thank Wen-Bo Zhang, Yue Sun, Yu-Jie Li, Zhu Wang, Ping Wang, Guo-Dong Tie, Yong-Xin Yu, Sun-Mei Xin and Pei-Hua Wu for their helpful discussion and kind assistance. This work was financially supported by the National Foundation for Outstanding Young Scientists to Prof. Zheng-Tao Wang(No. 39825129).

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