Shikonin, a component of antiinflammatory Chinese herbal medicine, selectively blocks chemokine binding to CC chemokine receptor-1

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Abstract

Shikonin is a chemically characterized component of traditional Chinese herbal medicine and has been shown to possess antiinflammatory activities. We ascertained that shikonin blocked radiolabelled Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) and macrophage inflammatory protein-1 (MIP-1α) binding to human monocytes with IC50 values of 3.58×10−6 and 2.57×10−6 M, respectively. In contrast, up to 1.7×10−5 M of shikonin failed to inhibit stromal cell-derived factor-1 (SDF-1α) binding to the cells. Additionally, shikonin blocked RANTES and MIP-1α binding to stable CC chemokine receptor-1 (CCR1) transfected human embryonic kidney (HEK)/293 cells with IC50 values of 2.63×10−6 and 2.57×10−6 M, respectively. However, shikonin inhibited neither RANTES nor MIP-1α binding to CCR5 transfected HEK/293 cells. Shikonin also did not inhibit monocyte chemoattractant protein-1 (MCP-1) binding to CCR2 cells, eotaxin binding to CCR3 cells, interferon-inducible T cell α-chemoattractant (I-TAC) binding to CXCR3 cells and SDF-1α binding to CXCR4 cells. Additionally, shikonin inhibited RANTES-induced CCR1 cell migration, but did not inhibit CCR1 cell migration induced by epidermal growth factor (EGF). Our study suggests shikonin may be a target for the future design of more potent, highly selective therapeutics that could be useful antiinflammatory agents for selectively blocking the binding of CCR1 ligands.

Introduction

Chemokines are a group of small proteins that play an important role in recruiting leukocytes during inflammatory and immune responses. The biological effects of chemokines are produced by interacting with specific G protein coupled receptors on target cells. To date, a total of 18 chemokine receptors, including 11 CC, five CXC, one CX3C, and one C chemokine receptor, have been identified [1]. Chemokines are involved in a number of diseases, ranging from HIV-1 infection and tumor growth to acute and chronic inflammation. Discovery and design of chemokine inhibitors and antagonists may yield drugs with significant therapeutic benefit [2].

In an effort to find naturally occurring chemokine antagonists, we screened a number of Chinese herbal medicinal components for activity. Zicao [purple gromwell, the dried root of Lithospermum erythrorhizon Sieb. et Zucc, Arnebia euchroma (Royle) Johnst. and Arnebia guttata Bunge.], is believed to possess the properties of removing heat from the blood and detoxicating, and have been used in China for thousands of years to treat macular eruption, measles, sore-throat, carbuncle and burn [3]. The major active components of Zicao have been identified as shikonin and its derivatives [4]. These chemically characterized components from Zicao have been reported to possess multiple biological properties including anti-inflammatory effects [5], [6], [7], [8], [9].

In this study, we found that shikonin selectively blocked radiolabelled regulated on activation, normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein-1 (MIP-1α) binding to human monocytes, but did not block stromal cell-derived factor-1 (SDF-1α) binding. We further showed that shikonin specifically blocked radiolabelled chemokine binding to cells stably transfected with CC chemokine receptor-1 (CCR1), but did not inhibit ligand binding to CCR2, CCR3, CCR5, CXCR3 or CXCR4 transfected cells. Additionally, shikonin inhibited CCR1 cell migration induced by RANTES, but did not inhibit epidermal growth factor (EGF)-induced migration.

Section snippets

Reagents and cells

Shikonin (Fig. 1) was obtained from TCI (Tokyo, Japan). 125I-labelled RANTES, MIP-1β, monocyte chemoattractant protein-1 (MCP-1), SDF-1α, eotaxin, and interferon-inducible T cell α-chemoattractant (I-TAC) were purchased from Dupont NEN (Boston, MA); EGF was from peprotech (Rocky Hill, NJ). Human blood enriched in mononuclear cells was obtained from normal donors by leukapheresis (Transfusion Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD, under approved human

Effect of shikonin on binding of chemokines to human monocytes

In our study of anti-inflammatory compounds from traditional Chinese medicine, we discovered that shikonin significantly blocked 125I RANTES binding to monocytic cell line THP-1; however, this compound failed to inhibit 125I SDF-1α binding, even up to 3.46×10−4 M (100 μg/ml) (data not shown). Shikonin also inhibited binding of RANTES and MIP-1α to human monocytes in a dose-dependent manner; the observed IC50 were 3.58×10−6 and 2.57×10−6 M, respectively (Fig. 2A and B). At concentrations over

Discussion

This report shows that shikonin selectively inhibits RANTES and MIP-1α, two high affinity ligands for CCR1, binding to THP-1 cells, human monocytes and CCR1-expressing cells. The most likely mechanism of shikonin's action is direct interaction with the CCR1 receptor. Shikonin was unable to inhibit MCP-1 binding to CCR2 cells, eotaxin binding to CCR3 cells, I-TAC binding to CXCR3 cells and SDF-1α binding to CXCR4 cells. Additionally, shikonin only inhibited RANTES-induced CCR1 cell migration,

Acknowledgements

This project has been funded in whole or in part with Federal funds from the National Cancer Institute, and Alternative Medicine Program of National Institutes of Health, under Contract no. Y2-AT-9002.

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