Inhibitory effect of berberine on the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2
Introduction
Berberine is an isoquinoline derivative alkaloid isolated from many medicinal herbs, such as Hydrastis canadensis, Cortex phellodendri and Rhizoma coptidis. It is widely used in traditional Chinese medicine for the treatment of inflammation diseases and anti-microbial activities (Shvarev and Tsetlin, 1972, Ikram, 1975, Creasey, 1979, Ivanovska and Phillip, 1996). In recent years, berberine has been reported to have a wide range of pharmacological effects, including interaction with DNA to form complexes, inhibition of DNA and protein synthesis, arresting effect of cell cycle progress, inhibition of tumor cells proliferation and anti-cancer effect (Yang et al., 1996, Iizuka et al., 2000, Anis et al., 2001, Jantova et al., 2003, Tai and Luo, 2003, Kettmann et al., 2004, Kuo et al., 2004a, Kuo et al., 2004b). Recently, we have also reported the anti-oxidation effect of berberine in t-Butyl hydroperoxide (t-BHP)-induced liver injury model (Hwang et al., 2002).
The essential characteristics of cancer are the ability to invade surrounding tissues and metastasize to regional and remote sites. Metastatic spread is the critical factors for the mortality of cancer patients. During the metastatic process, tumor cells need to attach to other cells and ECM proteins. Translocation of neoplastic cells across ECM barriers is a part of the metastatic process; and lysis of matrix proteins by specific proteinases is also required for invasion (Woodhouse et al., 1997). Among these involved proteases, MMP2, MMP9 and urokinase-plasminogen activator (u-PA) u-PA receptor (uPAR) are the most vital ones for degradation of basemembrane and, therefore, deeply involved in cancer invasion and metastasis (Oka et al., 1991, Cockett et al., 1998, Kleiner and Stetler-Stevenson, 1999, Ohbayashi, 2002). u-PA converts the zymogen plasminogen to plasmin. The uPAR focuses u-PA activity on the cell membrane and also is involved in the regulation of cell adhesion and migration. It is capable of transmitting u-PA-mediated extracellular signals inside the cell probably through the association with different types of integrins and ECM component (Wei et al., 1996).
Lung cancer is the major cause of malignancy-related deaths worldwide, and its incidence is rising in many countries. The high mortality of this disease is attributable to difficulty in early diagnosis. In many cases, local invasion or metastasis to distant organs has already occurred by the time of the diagnosis. In many types of neoplasm, including lung cancer, higher levels of activated MMPs have been demonstrated in more invasive and/or metastatic tumors and may give prognostic information independent of stage (Murrer et al., 1991, Kanayama et al., 1998). Gelatinases (MMP2 and MMP9) are the major proteases in lung cancer and are closely correlated with invasive and metastatic potentials (Herbst et al., 2000, Ohbayashi, 2002).
Although it is quite clear that berberine is able to inhibit the growth of various cancers by inducing cancer cells toward cell cycle arrest, little is known about the molecular mechanisms of its anti-metastatic effects. Because cancer metastasis and invasion are highly related to the degradation of ECM, intercellular adhesion and cellular motility, in this study, the effect of berberine on the invasion and motility was investigated by looking at the impact of berberine on several relevant proteases, including MMP2, MMP9, u-PA and TIMP-2, in A549, a human non-small cell lung cancer cells with a highly metastatic capability.
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Cell culture and berberine treatment
A549, a human lung cancer cell line obtained from ATCC (Manassas, VA), was cultured in Dulbecco's modified Eagle's medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml of penicillin and 100 mg/ml streptomycin sulfate, 0.1 mM non-essential amino acid and 1 mM sodium pyruvate. All cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2. For berberine treatment, appropriate amounts of stock solution (0.01 M in DMSO) of
The cytotoxicity of berberine to A549 cells
Berberine is an isoquinoline derivative alkaloid with anti-oxidative activities and against cancer growth. In this study, we first determined the cytotoxicity of berberine by treating A549 cells with berberine at various concentrations for 24 h and 48 h followed by MTT assay. Compared to that of control (DMSO was treated alone), the cell viability of the samples treated with berberine at a concentration between 2.5 to 40 μM was not significantly altered (Fig. 1), indicating that berberine was
Discussion
Berberine is one of the major components of Coptis chinesis, which was frequently used in proprietary herbal medicine to treat inflammation in Europe and Asia (Ivanovska and Phillip, 1996, Kuo et al., 2004b). Berberine exhibits broad spectrum of anti-microbial activity by inhibiting fungal, yeast and bacterial proliferation with no toxicity. Studies have shown that berberine exerts anti-cancer, preventive, anti-carcinogenic and anti-proliferative effects in various in vivo and in vitro models (
Acknowledgments
This work was supported by the grants from the National Science Council of Taiwan (NSC 93-2320-B-040-058) and Chung Shan Medical University (CSMC 91-OM-B-023), Taichung, Taiwan.
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