Panax ginseng is one of the most popular natural tonics that have been used in oriental countries. Ginseng showed anti-tumor activities in slow growing tumors but not in rapidly growing tumors1). Ginseng also inhibited tumor angiogenesis and metastasis2,3). In an epidemiological study, the intake of ginseng reduced the incidence of human cancer4). However, its mechanisms of action have not been elucidated yet.

The canonical Wnt signaling pathway has a dominant role in regulating epithelial-mesenchymal cell interactions critical for morphogenesis5,6). In this signaling system, Wnt ligands bind to a coreceptor complex consisting of a seven-transmembranedomain, Frizzled receptor and one of the LDL receptor-related proteins (LRPs), LRP5 or LRP67). A signaling cascade is then engaged that leads to the formation of a bipartite complex between the adhesion molecule, β-catenin, and transcription factors of the TCF family. In turn, the β-catenin-T-cell factor(TCF) complex activates expression of a cohort of target genes hat impact on cellular function. Although the TCF complex as an important role in regulating normal cell function, increasing evidence indicates that canonical Wnt signaling can be disrupted in various cancerous states8,9).

Accumulating evidence indicates that hyperactive Wnt signaling occurs in association with the progression of human cancer. As a consequence of engaging the canonical Wnt pathway, a TCF transcriptional complex is generated, which has been postulated to trigger the epithelial-mesenchymal transition (EMT), which convert epithelial cells into migratory mesenchymal cells10). Recent study has demonstrated that canonical Wnt signaling engages tumor cell dedifferentiation and tissue-invasive activity through an AXIN2-dependent pathway that stabilizes the Snail1 zinc-transcription factor, a key regulator of normal and neoplastic epithelial-mesenchymal transition programmes11). Axin2 regulates EMT by acting as a nucleocytoplasmic chaperone for GSK3β, the dominant kinase responsible for controlling Snail1 protein turnover and activity. Thus, a β-catenin-TCF regulated Axin2-GSK3β- Snail1 axis plays important role in cancer-associated EMT programmes.

Based on the grown environment and the cultivate method, the commercial trade ginseng is classified into three grades of ginseng, cultivated ginseng( CG), mountain cultivated wild ginseng(MCWG) and mountain wild ginseng(WG). The CG is cultivated artificially in forms and contributes the major quantity of ginseng in the current market. The WG grows in the natural environments, vegetating in the deep mountains and MCWG can be considered as mimicry WG which is seeded and grown up in the forest and mountain.

In this study, we examined the effect of MCWG on the transcription of the human AXIN2 gene that plays key roles in neoplastic EMT process.

Cultivated ginsengs(CG) used in this experiment were 5 years of age(Fig. 1-a). Mountain cultivated wild ginseng(MCWG) used in this experiment were of 10 years old(seeded in 1999) grown at ChonBangNongSan in Choongnam, Korea, (Fig. 1- b). Wild ginseng(WG) used in this experiment were collected from Changbai Mt. in July, 2007. They are about 20-40 cm long, dried weighs about 10- 20g with the approximate age of 20~50 years(Fig. 1-c).

Selected dried ginseng, CG, MCWG and WG(5g) were added with 80% ethyl alcohol 500㎖ and refluxed for 3hours, 3times to obtain extraction. Then this extraction was suspended in a distilled water for using experimental studies.

Human cancer cell lines were obtained from the Korean Cell Line Bank (KCLB, Korea) and were maintained in the recommended medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25μg/μl amphotericin B at 37℃ under a humidified 5% CO2 atmosphere.

RNA was extracted from various ginsengs treated cells using Easy-spin RNA Extraction kit (iNtRON, Biotech, Korea) according to the manufacturer’s instructions.

The reverse transcription reaction was performed on 2μg of total RNA using a SuperScript II First-Strand Synthesis system (Invitrogen) with random primer. PCR was carried out in solution containing 1X PCR buffer (TaKaRa, Japan), 200μM each deoxynucleotide triphosphate, 2.5pmol of each primer, 1 unit of ExTaq polymerase (TaKaRa, Japan). The housekeeping gene β-actin served as an internal control to confirm the success of the reverse transcription reaction. The PCR products were subjected to 2% agaroseg elelectrophoresis.

Volume of the reaction mixture was made up to 20μl. Real-time quantitative PCR was performed using SYBR GreenER qPCR SuperMix reagents (Invitrogen) and a Bio-Rad iCycler. Relative transcript quantities were calculated using the ΔΔCt method with β-actin as the endogenous reference gene amplified from the samples(Table 1.).

Recent study11) has demonstrated that canonical Wnt signaling engages tumor cell dedifferentiation and tissue-invasive activity through an AXIN2- dependent pathway(Fig. 2). Thus, the effect of cultivated wild ginseng on the expression of the AXIN2 gene was examined as follows. MCWG extract was added at the time of plating, and after 3 days AXIN2 gene expression was determined using RT-PCR. Treatment of the LOVO cells and human gastric carcinoma SNU 601 cells with the increased concentrations of mountain cultivated wild ginseng extracts resulted in a gradual decrease in the AXIN2 gene expression(Fig. 3).

Quantitative real-time PCR results confirmed a dose-dependent decrease in AXIN2 gene expression3),which decreased 4-fold after treatment with 100μg/ml of cultivated wild ginseng extracts (Fig. 4). Taken together, theses results suggest that the cultivated wild ginseng repress the specific expression of the AXIN2 gene.

Both of MCWG and WG are considered superior to CG. We therefore effects of these three types of ginseng in the expression of AXIN2 gene. Treatment with either MCWG extracts or WG for 3 days induced a decrease in the mRNA levels of AXIN2 gene in human colorectal carcinoma LOVO cells. However, CG treatment of the LOVO cells had no significant effects on the transcription of the AXIN2 gene(Fig. 5). These results suggest that the MCWG or WG are similar in their inhibitory effects on the expression of AXIN2, which are key regulators of neoplastic EMT programmes, but are substantially stronger than CG.

Maspin (mammary serine protease inhibitor, 42 kDa) is a metastasis-suppressor gene first described by Zou et al in 1994 by subtraction of cultured breast cancer cell and normal breast tissue mRNAs12). Maspin has been thought to inhibit carcinoma invasion, metastasis, and angiogenesis. In addition, it has been reported that maspin induced apoptosis of neoplastic cells13) and expression of maspin are suppressed as the carcinoma progresses in breast carcinoma and prostatic carcinoma14,15).

Nm23 (nonmetastatic)23 is a metastasis suppressor gene16) and its over expression has been shown experimentally to inhibit the metastatic phenotype and/or promote differentiation in melanoma, breast carcinoma, and transformed neural cell lines17). The low expression of the protein has been linked to increased metastatic potential in human breast carcinoma, hepatoma, and gastric carcinoma18,19).

As shown in Fig. 6, RT-PCR analysis revealed that the expression of two metastasis-suppressor genes, maspin and nm23 was not affected by the treatment of ginseng extracts in LOVO cells.

Metastasis is the major cause of death for cancer patients with solid tumors, due mainly to the ineffectiveness of current therapies once metastases begin to form. Metastasis is defined as the formation of progressively growing secondary tumor foci at sites discontinuous from the primary lesion20). In cancer, it is believed that epithelial tumor cells may be able to convert differentiated epithelial cancer cells into de-differentiated cells that possess more mesenchymal characteristics21). This epithelial-mesenchymal transition (EMT) phenotype in cancer has been associated with a decrease in tumor growth, increased resistance to apoptosis, increased motility and invasiveness, and enhanced metastatic ability22). These phenotypic transitions are reversible, and it is hypothesized that once tumor cells have reached their destination, they may transform back into an epithelial phenotype in order to facilitate tumor growth in the secondary site23). EMT is an important process in tumor development, and several studies suggest that the Wnt/β-catenin signal pathway may play an important role in EMT .

The present study focuses on identifying the mechanism that underlies the anti-metastasis activity of ginseng. The study shows, for the first time, that ginseng can repress the expression of AXIN2 gene that play key roles in the downsream signaling of the neoplastic EMT process. These results showed us a novel mechanism of ginseng’s antimetastasis activity and could provide a molecular link between ginseng intake and its inhibitory effects on metastasis.

[Fig. 1] Cultivated ginseng(CG-a), mountain cultivated wild ginseng(MCWG-b) and mountainwild ginseng(WG-c) wereused for ginseng extracts.

[Fig. 2] Schematic diagram showing that β?-catenin,stabilized either by canonical Wnt signalling,triggers TCF-dependent signaling that inducesAxin2 expression. In turn, Snail1 protein half-lifeis increased, thereby engaging a Snail1-dependentEMT process.

[Fig. 3] Repression of AXIN2 mRNA after treating LOVO andSNU601 cells with MCWG. AXIN2 mRNA levels wereanalyzed by semi-quantitative RT-PCR. Human colorectalcarcinoma LOVO cells and human gastric carcinomaSNU601 cells were cultured in media containingmountain cultivated wild ginseng extracts (0 -100μg/ml) for 3 days, and the AXIN2 mRNA level wasevaluated by semi-quantitative RT-PCR using the AXIN2-specific primers. RT-PCR analysis revealedthe expected 325 bp AXIN2 band and β-actin being aninternal control.

[Fig. 4] Quantitative real-time RT-PCR analysis of AXIN2 transcripts.Total RNA extracted from the cells (2μg) wasreverse-transcribed to cDNA (40μl), and aliquots (1μl)were applied to real-time PCR(20μl) with eachprimer(0.4 mM). Values represented relative expression of AXIN2 gene (calculated with threshold cyclenumber, CT) of MCWG treated cells compared withthat of non-treated control cells. Each value wasadjusted with CT of internal control(β-actin).

[Fig. 5] RT-PCR analysis of AXIN2 mRNA expression inhuman colorectal carcinoma LOVO cell linestreated with the CG extracts, MCWG extracts, or WG extracts(100㎍/㎖) for 3 days. Lane (-), negativecontrol.

[Fig. 6] Expression of mRNA for maspin and nm23 on LOVO cells. Total RNA(2μg) from LOVO cells treated with the CG extracts, MCWG extracts, or WG extracts(100㎍/㎖) for 3 days was examined by RTPCR analysis. Lane (-), negative control.

[Table 1] Primer for RT-PCR