Wogonin, an active ingredient of Chinese herb medicine Scutellaria baicalensis, inhibits the mobility and invasion of human gallbladder carcinoma GBC-SD cells by inducing the expression of maspin

Abstract

Ethnopharmacological relevance

A traditional Chinese medicine Scutellaria baicalensis is prescribed for the treatment of a variety of inflammatory diseases and tumors in clinic in China. However, the detailed mechanism of anti-metastasis effect of wogonin, a main active ingredient of Scutellaria baicalensis, remains elusive.

Aim of the study

The present study was designed to investigate the action and mechanism of wogonin on the mobility and invasion of human gallbladder carcinoma GBC-SD cells.

Materials and methods

Viability, apoptosis, mRNA and protein expression of tumor cells were analyzed by MTT, Annexin V staining, real-time PCR and Western blot, respectively. The migration and invasion assay was used to evaluate the anti-metastasis effect of wogonin. Knockdown of maspin was performed by specific small interference RNA.

Results

Wogonin at the dose of 1–10 μM, which did not induce apoptosis, significantly inhibited the mobility and invasion activity of human gallbladder carcinoma GBC-SD cells. In addition, the expressions of matrix metalloproteinase (MMP)-2, MMP-9 and phosphorylated extracellular regulated protein kinase 1/2 (ERK1/2) but not phosphorylated Akt were dramatically suppressed by wogonin in a concentration-dependent manner. Furthermore, the metastasis suppressor maspin was confirmed as the downstream target of wogonin. Both maspin mRNA and protein were upregulated by wogonin. Interestingly, the knockdown of maspin resulted in almost completely blocking of wogonin-induced inhibition of MMP-2, MMP-9 and phosphorylated ERK1/2 as well as the mobility and invasion activity of GBC-SD cells.

Conclusions

These findings suggest that wogonin inhibits cell mobility and invasion by upregulating the metastasis suppressor maspin. Together, these data provide novel insights into the chemoprotective effect of wogonin, a main active ingredient of Chinese medicine Scutellaria baicalensis.

Introduction

Tumor growth, invasion, and metastasis are multistep and complex processes that include cell division and proliferation, proteolytic digestion of the extracellular matrix, cell migration through basement membranes to reach the circulatory system, and remigration and growth of tumors at the metastatic sites (Klein, 2008). It is firmly established that matrix metalloproteinase (MMP)-2 and MMP-9 secretion from cancerous cells leads to hydrolysis of extracellular matrix, thus enabling cells to break out of their primary site into the circulation and form the secondary metastatic sites. Invasiveness inhibitors may act by down-modulating extracellular-barrier degrading proteinases such as MMP-2 and MMP-9 (Nozaki et al., 2003). Activation of extracellular regulated protein kinase 1/2 (ERK1/2) has been shown to be the mechanism for promoting the production of MMPs (Beshir et al., 2010), which are important for cell proliferation, invasion, and neovascularization. It has been shown that inhibition of ERK1/2 activation may lead to a reduction in the expression of MMP-2, as well as in the invasion of tumor cells (Lev et al., 2004).

Maspin, a member of the serine protease inhibitor family, has been shown to be a potent metastasis suppressor (Bailey et al., 2006, Khalkhali-Ellis, 2006, Lockett et al., 2006). The ectopic expression of maspin suppresses the invasive potential of breast cancer cells in vitro and the growth and metastasis of prostate and breast cancers in a mouse model (Zou et al., 1994, Shi et al., 2001). Maspin is expressed ubiquitously in multiple tissues and normal epithelial cells but reduced in tumor cells (Zou et al., 1994, Khalkhali-Ellis, 2006). Thus, inducing maspin expression in premalignant and malignant cells may be an effective strategy for cancer prevention and treatment.

Scutellaria (HUANG QIN, Lamiaceae), which includes about 350 species commonly known as skullcaps, is widespread in Europe, the United States and East Asia (Shang et al., 2010). A traditional Chinese medicine Scutellaria baicalensis is prescribed for the treatment of a variety of inflammatory diseases and tumors in clinic in China (Shang et al., 2010). The crude extract of Scutellaria baicalensis has been reported to exert anti-cancer effect against some human tumor cells (Ye et al., 2002, Kumagai et al., 2007). Wogonin, a main active ingredient of Chinese herb medicine Scutellaria baicalensis, exhibits a variety of pharmacological activities including antioxidant (Gao et al., 1999), anti-inflammatory (Chi et al., 2001), anxiolytic (Hui et al., 2002), anti-hepatitis (Guo et al., 2007), anticonvulsant (Park et al., 2007), anti-angiogenesis (Lu et al., 2008), neuroprotective (Lee et al., 2003, Lim et al., 2010), anticancer (Wang et al., 2006, Baumann et al., 2008, Li-Weber, 2009) activities. Recent study has demonstrated that wogonin at the dose of more than 20 μM could effectively inhibit the proliferation and induce the apoptosis in several cancer cell lines in vitro (Lee et al., 2008, Zhang et al., 2008). However, little is known about the action of wogonin on tumor cells at the sub-dose of apoptosis-inducing effect. Here we found that wogonin at the dose of 1–10 μM, which did not induce apoptosis, significantly inhibited the mobility and invasion activity of human gallbladder carcinoma GBC-SD cells. Furthermore, the metastasis suppressor maspin had been confirmed as the downstream target of wogonin. Our data provide novel insights into the chemoprotective effect of wogonin, a main active ingredient of Chinese medicine Scutellaria baicalensis, against highly metastatic tumor.