Elsevier

Toxicology in Vitro

Volume 18, Issue 6, December 2004, Pages 755-763
Toxicology in Vitro

Glycoprotein isolated from Solanum nigrum L. inhibits the DNA-binding activities of NF-κB and AP-1, and increases the production of nitric oxide in TPA-stimulated MCF-7 cells

https://doi.org/10.1016/j.tiv.2004.03.003Get rights and content

Abstract

Solanum nigrum L. (SNL) has been used in traditional folk medicine to treat numerous cancers. We isolated a glycoprotein (150 kDa) from SNL and tested its effect on the modulation of transcriptional factors (NF-kappa B and AP-1) and iNO production in TPA induced-MCF-7 cells, which are part of the human breast cancer cell line, without estrogen receptors. However, the mechanism of SNL glycoprotein in pharmacological and biochemical actions in cancer cells has not been studied. To test the effect of SNL glycoprotein on the DNA-binding activities of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1), and nitric oxide (NO) production, these experiments were carried out using electrophoretic mobility shift assays (EMSA), western blot analysis, and the Griess method. Results in this experiment showed that SNL glycoprotein inhibits 12-O-Tetra decanoylphorbol-13-acetate (TPA; 100 nM)-induced DNA-binding activities of NF-κB and AP-1, and enhances NO production in MCF-7 cells. That is, our results indicated that SNL glycoprotein has the capacity to modulate the TPA-induced DNA-binding activities of transcription factors and NO production, which play a critical role with respect to cytotoxicity in MCF-7 cells. Therefore, SNL glycoprotein might be one of the agents that blocks TPA-mediated signal responses in tumor cells.

Introduction

Tumor promotion is an important process in carcinogenesis. Particularly, 12-O-tetradecanoyl phorbol-13-acetate (TPA) is one of the tumor promoters in several cell lines. TPA-induced tumor promotion in multistage carcinogenesis is closely related to biochemical and molecular events, such as increased activities of transcription factors [e.g. nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1)] and the reduction of reactive oxygen species [e.g. superoxide anion, hydrogen peroxide, nitric oxide (NO), etc.] (Shi et al., 2000). It has also been reported that TPA modulates diverse cellular responses such as gene transcription, cellular growth and differentiation, programmed cell death, the immune responses and receptor desensitization (Nomura et al., 2000).

Many scientists believe that blocking of the signal transduction pathways leads to the activation of important transcription factors, such as NF-κB and AP-1, and might be the best strategy for anti-promoting effects on tumor growth (Lin and Lee, 1995; Dong et al., 1997). In general, NF-κB and AP-1 are known eukaryotic transcription factors that are involved in an incredible diversity of molecular biological reactions (Meyer et al., 1993; Henkler and Koshy, 1996; Li et al., 1998; Handel and Girgis, 2001). NF-κB is a ubiquitously expressed transcription factor that regulates several vital functions such as apoptosis, cell proliferation and differentiation. Under normal conditions, NF-κB is retained in the cytoplasm of the cell, where it is bound by inhibitory proteins known as IκBs. It has also been documented that NF-κB, during carcinogenesis, has the potential to mediate several events associated with multi-step processes, including the acquisition of features such as the promotion of cell survival and deregulation of the normal control of proliferation, metastasis and angiogenesis (De Martin et al., 1999; Gilmore et al., 1996). On the other hand, AP-1 transcription factors can modulate proliferation, differentiation or apoptosis, depending on the cell type. In fibroblasts and breast epithelial cells, AP-1 activity is induced in response to multiple growth/mitogenic factors, resulting in the stimulation of cell proliferation, transformation and tumoral progression (Yang et al., 1997). The AP-1 factor consists of protein products of members of the Fos [v-fos, c-fos, fos B, fra-1/2 (fos-related antigen)] and Jun (v-jun, c-jun, jun B, jun D) families (Kaminska et al., 2000). It was shown in various experimental systems that strong proliferation, malignant transformation, and enhanced aggressiveness are accompanied by a change in AP-1 complex composition (Yang et al., 1997; Ahn et al., 2002). Such transcription factors play an important role in cell signaling, which relates to cell death. It has been reported that there exists a close relationship between the activation of transcriptional factors and the cytotoxicity of certain cell lines (Han et al., 2002; Hu et al., 2002; Keum et al., 2003). Therefore, oncologists are attempting to modulate cancer cell death through the regulation of NF-κB and AP-1 as a valuable strategy.

One of the free radicals, NO, is produced by inducible nitric oxide synthase (iNOS) and plays an important role in the cytotoxicity of cancer cells. Also, it has been shown that NO is a secondary messenger having diverse physiological activities, including its ability to kill microorganisms and cancer cells (Nguyen et al., 1992; Moncada et al., 1991). In addition, NO-mediated cytotoxicity affects the inhibition of mitochondrial respiration and DNA synthesis in cell targets, including breast cancer cells (Adami et al., 1998). In the relationship between NF-κB and iNOS, the inhibition of the NF-κB mediated induction of iNOS in epithelial cells leads to the blockage of important anti-apoptotic and anti-microbial survival mechanisms. NO serves in a negative feedback loop to antagonize the prolonged activation of NF-κB, thereby limiting cancer cell survival (Dijkstra et al., 2002).

Solanum nigrum L. (SNL) is a common herb that grows wildly and abundantly in open fields. It has been used in traditional folk medicine because of its diuretic and antipyretic effects. More specifically, it has been used to cure inflammation, edema, mastitis and hepatic cancer for a long time in oriental medicine (Sultana et al., 1995; Prashanth Kumar et al., 2001). Fruit extracts of SNL have been reported to have a strong dose-dependent cytotoxicity, to induce significant DNA damage in human lymphocytes (Yen et al., 2001), and to have significant hydroxyl radical scavenging potential (Prashanth Kumar et al., 2001). It has been shown that SNL contains steroidal glycosides, steroidal alkaroids, steroidal oligoglycosides, solamargine, and solasonine (Saijo et al., 1982; Ikeda et al., 2000). However, not many studies have addressed the function of the glycoprotein unit, one of the many components of the SNL. Generally speaking, most plant glycoproteins have a high hygroscopic character in air, because of the high viscosity. Glycoproteins with a high molecular weight have a more biologically active function than those with a low molecular weight. The reason for this phenomenon is that the glycoproteins with a large carbohydrate component such as lactose, fructose, galactose and mannose bind to proteins. Such glycoproteins provide biological functions to the cells, because the carbohydrate component supports the three-dimensional structure in the whole glycoprotein (Kimura et al., 2000; Bourne et al., 2002).

It has been reported that SNL glycoprotein has a cytotoxic effect on MCF-7 cells (Lee and Lim, 2003) and that SNL ethanol extract has anti-proliferative, apoptotic and cytotoxic effects on MCF-7 cells (Son et al., 2003). However, the mechanism of the cytotoxic effect of SNL glycoprotein in the signal pathway remains to be elucidated. Therefore, the aim of this study was to further examine the molecular events leading to the SNL glycoprotein-induced cytotoxic effect in MCF-7 cells. This experiment was carried out using TPA-induced MCF-7 cells, even though MCF-7 cells are part of the human breast cancer cell line and TPA is a cancer promoter. The reason for using TPA in MCF-7 cells is that an induction of distinct signal responses for the study of the signal pathway should be conducted in a positive control system. We have found that NF-κB and AP-1 activities are very important in mediating this cytotoxic process and that other markers, such as intracellular NO production, are also involved in this process.

Section snippets

Materials

All chemicals were received from Sigma/Aldrich and were of a highly purified grade. TPA, Griess reagent, and other chemicals were obtained from the Sigma Chemical Co. (St. Louis, MO, USA). Cell culture reagents, including Dulbecco's modified Eagle's medium (DMEM), RPMI-1640, fetal bovine serum (FBS), Phosphate buffered saline (PBS) and antibiotics were purchased from Gibco BRL Life Technologies (Grand Island, NY, USA). Proteinase K and oligonucleotides, containing NF-κB and AP-1 consensus

Activities of NF-κB and AP-1

We investigated the DNA-binding activities of NF-κB and AP-1 using EMSA with nuclear extracts prepared from MCF-7 cells. The DNA-binding activities of NF-κB and AP-1 in the presence of TPA (100 nM) for various times (0.5–4 h) or various doses of TPA (10–200 nM) for 4 h were investigated, as shown by the data in Fig. 1. In a time- and dose-dependent manner, the DNA-binding activity of NF-κB was remarkably activated at 40 nM TPA (Fig. 1A and B), whereas the AP-1 binding activity was rapidly

Discussion

To further understand the biological function of the cytotoxic effect of SNL glycoprotein, we evaluated the inhibitory effects of the SNL glycoprotein on DNA-binding activities of NF-κB and AP-1 in TPA-stimulated MCF-7 cells. The results in this experiment showed that TPA-stimulates both the DNA-binding activities of NF-κB and AP-1 in a time- and a dose-dependent manner (Fig. 1). Data indicate that the increasing DNA-binding activities of NF-κB and AP-1 by TPA treatment were dramatically

Acknowledgements

This work was supported by grant no. (R05-2002-000-00189-0) from the Basic Research Program of the Korea Science and Engineering Foundation.

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