Anti-Metastasis Effects of Ginsenoside Rg3 in B16F10 Cells

Abstract

Ginsenoside Rg3 is a bioactive ginseng constituent that has been reported to have diverse pathological and physiological effects, including anti-inflammatory and anti-metastatic activities. Metastasis is one of the most important factors involved in patients with melanoma. However, the molecular mechanism underlying the anti-metastatic activities of Rg3 in malignant melanoma cancer has not been fully elucidated. In this study, we have evaluated that Rg3 effectively inhibits metastasis of B16F10 melanoma cancer cells. We found that Rg3 significantly suppresses the migration, invasion, wound healing, and colony-forming abilities of B16F10 cells in a dose-dependent manner. Mechanistically, we demonstrate that Rg3 suppresses B16F10 cell metastasis by inhibiting MMP-13 expression. These results indicate that Rg3 suppresses the metastasis of B16F10 mouse melanoma cancer cells via MMP-13 regulation. Importantly, MMP-13 downregulation may influence the migration and invasion capabilities of melanoma cells and has been correlated with melanoma progression. Therefore, Rg3 is a potential therapeutic candidate that could be used to treat patients with metastatic melanoma.

Introduction

Cutaneous malignant melanoma is an aggressive and the most deadly form of skin cancer [26]. The evolution of cancer cells into metastatic tumors is the major cause of death in patients with cancer [26,27]. In the United States, the percentage of death of patients with melanoma has more than doubled in the past 30 years [9]. Therefore, metastasis is one of the most important factors related to the efficacy of anticancer therapeutics [4].

The metastasis of malignant tumor cells from the primary tumor to distant sites is a complex process involving adhesion, migration, and proliferation [17]. The biological processes of metastasis require the destruction of the extracellular matrix (ECM) via proteolytic enzymes [6]. Matrix metalloproteinases (MMPs) are enzymes involved in degrading the extracellular matrix that are upregulated in malignant tumors during metastasis. Therefore, the inhibition of MMP activity is an attractive therapeutic target for drugs aimed at preventing cancer metastasis [4,8,13,28].

Previous studies have demonstrated that MMP-13 regulation in metastatic tumors is mediated by the p38 mitogen-activated protein kinase (MAPK) pathway [3,18,24]. We previously demonstrated that Rg3 significantly induced apoptosis of B16F10 melanoma cancer cells in a dose-dependent manner. In addition, we found overexpression of the pro-apoptotic protein p-p38 in apoptotic B16F10 cells following Rg3 treatment [15].

Panax ginseng has various pharmacological activities such as anti-inflammatory and anti-cancer effects, and has been mainly used as a medicinal plant in Asia regions for millennia [9-11]. Ginsenosides are the main bioactive components extracted from ginseng. Ginsenoside Rg3 produces a wide range of bioactive effects on the human body [23]. Recent studies reported that ginsenoside Rg3 inhibits metastasis of some types of cancer cells, including lung and ovarian carcinoma cells, suggesting that Rg3 has promise as a therapeutic agent for malignant tumor treatment [12,23]. Although Rg3 was known to inhibit metastasis in malignant melanoma tumors, the mechanisms of anti-metastasis effects are not well established.

In the present study, we investigated the anti-metastatic effects of ginsenoside Rg3 in B16F10 melanoma cancer cells with a focus on its potential mechanisms of action.

 

Materials and Methods

Chemicals and Cell Line

Ginsenoside Rg3, fibronectin (1 mg/ml), and 50% glutaraldehyde solution were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). The molecular formula for ginsenoside Rg3 is C42H72O13 and its molecular mass is 784.3 Da (Fig. 1A). A storage solution of Rg3 was dissolved in dimethyl sulfoxide (DMSO, 5 mg/ml) and stored at -80℃. Specific kinase inhibitor SB203580 was purchased from Calbiochem-Novabiochem (La Jolla, CA, USA). B16F10 melanoma cells were purchased from Korean Cell Line Bank (KCLB) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). The cell culture was performed under standard conditions: 37℃, 100% humidity, 5% CO2 /95% air atmosphere. The medium was replaced every 2 or 3 days.

Fig. 1.Rg3 suppresses the cell spreading ability of B16F10 melanoma cancer.

Cytotoxicity Assay

B16F10 melanoma cells were plated at a density of 3 × 103 cells/100 μl/well in 96-well plates and incubated overnight at 37℃. Cells were treated with various concentrations (25, 50, and 100 μM) of Rg3 for 12 and 24 h or were left untreated. At each time, MTT (3-(4,5)-dimethylthiazo (-2-y1)-3,5-diphenytetrazolium brromide) solution was added to each well and incubated for 3 h at 37ºC. Then, the formazan reaction product was dissolved with isopropyl alcohol (Duksan Pure Chemicals, Kyungkido, Korea). The absorbance of each sample was measured at 595 nm.

Cell Spreading Assay

Ninety-six-well plates were pre-coated overnight at 4℃ with 5 μg/ml fibronectin or 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The coated wells were washed with PBS and blocked with 2% BSA in PBS for 30 min. The cells (3 × 103 cells/100 μl /well) were pretreated with various concentrations (25, 50, and 100 μM) of Rg3 for 30 min or were left untreated. After incubation of the cells for 3 h, they were fixed in 6.0% glutaraldehyde (in PBS) and stained with 0.1% crystal violet (in 2% ethyl alcohol).

At a specific time point (3 h), the adherent cells were photographed under a microscope at 400× magnification. Images of three random fields from three replicate wells were acquired and the number of spreading cells was counted using Image J software (National Institutes of Health, Bethesda, MD, USA). The negative control 96-well plates were coated with 2% BSA, whereas the positive control 96-well plates were coated with fibronectin. The attached cells were fixed with 6% glutaraldehyde (Sigma; in PBS) and then stained with 0.1% crystal violet (Sigma; in 20% methanol). The cell area was measured using Image J software.

Transwell Migration Assay

Cell migration was assayed using trans-well chamber inserts (polycarbonate membrane, 8 μm pore size; Costar, MA, USA). The undersurface of the membrane was pre-coated overnight at 4℃ with 5 μg/ml fibronectin or 2% BSA in PBS. The coated wells were washed using PBS and blocked with 2% BSA for 30 min. The cells (3 × 103 cells/100 μl/well) were pretreated with Rg3 at various concentrations (25, 50, and 100 μM) for 30 min or left untreated. The cells were seeded onto the upper insert of the chamber and allowed to migrate for 24 h at 37℃. After the incubation, the inserts were cleaned with cotton swabs. The lower side of each insert was fixed with 6.0% glutaraldehyde and stained with 0.1% crystal violet. The migrated cells were photographed in a microscope at 200× magnification. Images of three random fields from three replicate wells were obtained and the number of migrated cells was counted using Image J software.

Matrigel Invasion Assay

Invasion assay was carried out using a cell invasion assay kit (Biolab Diagnostics, Midrand, South Africa). The cells (0.5 × 106/ml) were pretreated with Rg3 at concentrations of 50 and 100 μM for 30 min or were left untreated. The cells were seeded onto the upper insert of the chamber and were left to invade through matrigel for 24 h. Then, the inserts were washed with PBS and changed to serum-free media without Rg3 before being incubated for 24 h. After the incubation, the inserts were cleaned with cotton swabs and invasive cells were fixed and stained.

Wound Healing Assay

Cells (1 × 105/ml) were seeded into 6-well plates and cultured overnight, after which a scratch was made through the confluent cell monolayer using a 200 μl pipette tip. The perimeter of the area with a central cell-free zone was confirmed under a microscope and each well was rinsed using sterile PBS. Next, the cells were treated with Rg3 at concentrations of 25 or 50 μM or left untreated. At each time point (0, 12, and 24 h), the cells were photographed under a microscope at 100× magnification. Negative control cells were cultured in serum-free medium and positive control cells were cultured in medium containing 1% serum without Rg3. The migration rate was calculated using the following formula: migration rate (%) = (average wound distance - average no migration distance)/average wound distance × 100.

Colony Formation Assay

Cells (single cell suspension) were seeded in 24-well plates at a density of 50–100 cells/well and cultured overnight. On the second day of the experiment, the cells were treated with Rg3 at the concentration of 100 μM or left untreated. At every 3 days, the medium was replaced with fresh medium containing the same concentration of Rg3. After a 7-day treatment, the medium was removed and the cell colonies were fixed in 6.0% glutaraldehyde and stained with 0.1% crystal violet.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from B16F10 melanoma cells using RNAiso Plus reagent (TaKaRa Bio, Otsu, Shiga, Japan). Complimentary DNA (cDNA) was synthesized with the Prime-Script RT reagent Kit (TaKaRa Bio). PCR amplification of the cDNA products was performed with GT PCR Master Mix (TaKaRa Bio) and specific primer pairs (Macrogen, Seoul, Korea) for MMP-13 (NM_008607.2) (forward, 5’-ATG ATC TTT AAA GAC AGA TTC TTC TGC-3’; reverse, 5’-TGG GAT AAC CTT CCA GAA TGT CAT AA-3’) and beta-actin (GenBank: EF095208.1) (forward, 5’-GAC AAC GGC TCC GGC AG TGC AAA G-3’; reverse, 5’-TTC ACG GTT GGC CTT AGG GTT CAG-3’) [14,19]. Amplification consisted of 35 cycles as follows: denaturing at 95℃ for 30 sec, annealing at 60℃ for 30 sec, and extension at 72℃ for 45 sec, followed by a final 10 min extension at 72℃. The PCR products were separated by 1.2% agarose gel electrophoresis and the gel was stained with ethidium bromide. Beta-actin was used as the control gene.

Transient Transfection Assay

Confluent cells (60–70%) were transiently transfected with 60 nM of MMP-13-suppressing siRNA (siMMP-13) or control siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. After 16 h, the transfected cells were treated with Rg3 at 50 μM concentration for 24 or 48 h. To ascertain the efficacy of siRNAs, protein levels were investigated by western blot analysis.

Western Blot Analysis

Cell lysates were dissolved in radioimmune precipitation assay lysis buffer containing (Biosesang, Seongnam, Korea). 20 μg of each protein sample, separated by 10% SDS–PAGE, and transferred onto nitrocellulose membranes. The membranes were blocked using 5% non-fat skim milk in TBST (10 mmol/l Tris, pH 8.0, 150 mmol/l NaCl, and 0.05% Tween 20) and incubated with primary antibodies overnight at 4℃ and then with HRP-conjugated secondary antibodies during 1 h at room temperature. The proteins were detected using enhanced chemiluminescence reagent (GE Healthcare, Buckinghamshire, UK).

Statistical Analysis

Data were analyzed using SPSS ver. 21.0 (IBM Corporation, Armonk, NY, USA). Statistical comparisons between groups were performed using one-way ANOVA.

Values of p < 0.05 were considered statistically significant (*p < 0.05, **p < 0.01).

 

Results

Rg3 Inhibits Spreading of B16F10 Cells

Before investigating the effect of Rg3 in B16F10 cells, the cytotoxicity effect of Rg3 was examined by MTT assay. Cell viability assay showed no significant toxicity at various concentrations (25, 50, and 100 μM) of Rg3 for 12 and 24 h (Fig. 1B). In accordance with these results, Rg3 at 0-100 μM was used in subsequent experiments. Cellular adhesion to the ECM is an important determinant of cell migration. Fibronectin, an ECM protein, could promote spreading of B16F10 cells [2]. Therefore, we investigated B16F10 cell spreading following exposure to Rg3 after plating on fibronectin- or BSA-coated plates. The spreading assay showed that the positive control cells exhibited normal shape-change behavior in a time-dependent manner. However, cells pretreated with Rg3 exhibited a rounded morphology and slight changes in their shape at 3 h (Fig. 1C). After 3 h of Rg3 pretreatment, the cells were fixed and stained. Staining of the spreading cells showed that Rg3 significantly inhibited B16F10 cell spreading in a dose-dependent manner. When cells were pretreated with Rg3 at concentrations of 25, 50, and 100 μM, the ratios of cell spreading were reduced by 84.2%, 64.6%, and 50.1%, respectively (Fig. 1D). The inhibitory effect of Rg3 on cell spreading was evident in B16F10 cells at concentrations of 50 and 100 μM (p < 0.01).

Rg3 Inhibits Motility and Invasion of B16F10 Cells

Next, we tested the effects of Rg3 on cell migration and invasion of B16F10 by means of a wound healing, migration, and invasion assay. A wound through a confluent cell monolayer was created with a pipette tip. After incubation for 12 and 24 h, the cells migrated and covered the entire wounded area. The wound healing assay showed that the wound had almost completely filled in the cleared region in the negative and positive control cells (Fig. 2A). In contrast, cells treated with Rg3 exhibited notably slower recovery in the cleared region in comparison with control cells. Following incubation for 24 h with Rg3 at concentrations of 25 and 50 μM, the wound healing ratios in the wounded areas were reduced by 74.9% and 58.0%, respectively (Fig. 2B). These results demonstrate that Rg3 suppresses cell wound healing into the cleared wound region. In addition, the inhibitory effect of Rg3 on wound healing in B16F10 cells was evident at concentrations of 25 and 50 μM (p < 0.01). Furthermore, staining of the migrated cells showed that Rg3 significantly inhibited cell migration in a dose-dependent manner (Fig. 2C). When the cells were incubated with Rg3 at concentrations of 25, 50, and 100 μM, the ratios of migrated cells were reduced by 80.5%, 47.4%, and 36.2%, respectively (Fig. 2D). The inhibitory effect of Rg3 on B16F10 cell migration was evident at concentrations of 50 and 100 μM (p < 0.01). The inhibitory effect of Rg3 on cell wound healing was similar to its effect on migration. Furthermore, the cell invasion ability of B16F10 was suppressed by Rg3 at concentrations of 50 and 100 μM (Figs. 2E and 2F). These results suggest that Rg3 inhibits the motility and invasion of B16F10 cells in a dose-dependent manner.

Fig. 2.Rg3 suppresses the cell mobility and invasion abilities of B16F10.

Rg3 Inhibits Colony Formation of B16F10 Cells

Cell proliferation and colonization are the functions required in the metastatic progression of tumor cells [7]. The colony formation assay is used to confirm the ability of a single cell to form a growing colony, which is an important indicator of a cancer cell’s ability to attach, survive, and proliferate [20,21]. Therefore, we next investigated the effect of Rg3 on growing colonies of B16F10 cells. Rg3 suppressed the formation of B16F10 cell colonies (Fig. 3A). After Rg3 treatment for 7days, the colonies were fixed and stained (Fig. 3B). When B16F10 cells were treated with the Rg3 concentration of 100 μM, the colony size was reduced by 14.1% in comparison with the control cells (Fig. 3C). This result indicated that Rg3 strongly inhibits the ability of a single B16F10 cell to form a growing colony.

Fig. 3.Rg3 suppresses the cell colony formation ability of B16F10.

Rg3 Suppresses MMP-13 mRNA and Protein Expression in B16F10 Cells

MMPs are involved in degrading the ECM and are upregulated in cancer cells during metastasis. A previous study reported that MMP-13 was overexpressed in various tumor cells during metastasis [24]. Expression of MMP-13 is mediated through the p38 MAPK pathway [3].

We previously showed that pro-apoptotic protein p-p38 was overexpressed in B16F10 melanoma cells during apoptosis induced by Rg3 treatment [15]. To identify the mechanism through which Rg3 suppresses metastasis in B16F10 cells, we assessed MMP-13 expression by RT-PCR and western blotting. After treatment with Rg3 for 48 h, MMP-13 mRNA and protein expression was reduced in a dose-dependent manner (Fig. 4A). However, treatment with Rg3 for 24 h did not affect the mRNA expression level of MMP-13 (Fig. 4B). To determine whether Rg3 inhibits MMP-13 expression by affecting the p38 pathway, we treated the cells with Rg3 at the concentration of 100 μM alone or with both Rg3 and the specific p38 inhibitor SB203580. After 48 h treatment with Rg3 alone, MMP-13 mRNA expression was reduced in comparison with that of the control cells. However, cells treated with Rg3 in combination with SB203580 showed slightly increased MMP-13 expression (Fig. 4C). These results suggest that Rg3 inhibits MMP-13 expression by modulating the p38 signaling pathway.

Fig. 4.Rg3 reduced the MMP-13 expression.

MMP-13 Transient Silenced Cells Show Decreased Migration and Invasion Abilities

The above results showed that the metastasis ability of B16F10 cells may be inhibited by Rg3 and involved the MMP-13 gene in B16F10 cells. To evaluate the essential aspects of MMP-13 function in relation to the anti-metastatic effects of Rg3, MMP-13 siRNA was transfected into B16F10 cells with or without Rg3 treatment. The efficiency of RNA interference was confirmed by western blotting in the presence or absence of Rg3. MMP-13 protein expression in MMP-13 transient silenced cells with Rg3 treatment was suppressed compared with transfected with MMP-13 siRNA without Rg3 (Fig. 5A). Furthermore, we found that the cell migration and invasion abilities in MMP-13 transient silenced cells were reduced compared with cells transfected with control siRNA, although the difference was small and did not reach statistical significance (Figs. 5B-5E). MMP-13 transient silenced cells with treatment of Rg3 showed inhibited cell migration and invasion abilities, as compared with cells only transfected with MMP-13 siRNA.

Fig. 5.Knockdown of MMP-13 decreases the migration and invasion abilities of B16F10 cells.

Importantly, these results suggested that the B16F10 cell migration and invasion abilities may be suppressed by other molecular mechanisms as well as MMP-13 expression by modulating the p38 signaling pathway.

 

Discussion

The evolution of cancer cells into metastatic tumors is the major cause of death in patients suffering from cancer, but the mechanisms underlying this change remain elusive [1,7]. Metastasis is involved in multiple oncogenic processes, including survival, invasion, and anchorage-independent growth [19]. Among these essential processes, degradation of basement membranes and the stromal ECM is the key step for invasion and metastasis of malignant tumors [22]. MMPs are secreted as proenzymes and then activated through the cleavage of a specific peptide [13,22]. MMP-13 mediates cell proliferation in melanocytes and melanoma cells. The activation of p38 MAPK is critical for the regulation of expression of invasion-associated MMPs such as MMP-13 [5,16,25]. MMP induction is dependent on MAPK signaling; however, the effect of Rg3 on MMP-13 expression, which is involved in metastasis of B16F10 melanoma cells, is obscure.

In the present study, we showed that ginsenoside Rg3 inhibits metastasis of B16F10 mouse melanoma cancer cells through the regulation of MMP-13 expression. Accordingly, we observed the inhibitory effects of Rg3 on cell spreading, wound healing, migration, invasion, and colony formation of B16F10 cells. The results of our experiments showed that Rg3 significantly and dose-dependently suppresses the spread and migration of B16F10 cells, at concentrations of 25, 50, and 100 μM. In addition, when B16F10 cells were treated with Rg3 at the lowest toxic dose (25 μM), spreading, wound healing, and migration were inhibited. These results demonstrated that Rg3 produced strong inhibitory effects on metastatic processes in B16F10 cells. Furthermore, we examined MMP-13 expression after exposure to various concentrations of Rg3 and found that Rg3 downregulated MMP-13 expression through the upregulation of p-p38. Moreover, knockdown of MMP-13 using MMP-13 siRNA led to decreases in the migration and invasion abilities of B16F10. These results indicated that the MMP-13 gene may have played a role in the process of metastasis of B16F10 cells.

Overall, our findings suggested that the downregulation of MMP-13 expression by Rg3 may be related to its inhibitory effect on melanoma cell migratory capabilities and melanoma progression. The present study demonstrated that ginsenoside Rg3 inhibits the metastatic processes of B16F10 mouse melanoma cancer cells through the p38- dependent suppression of MMP-13 expression.

This is the first study to demonstrate that ginsenoside Rg3 suppresses metastasis in B16F10 mouse melanoma cancer cells through downregulation of MMP-13 expression in a dose-dependent manner. Therefore, we suggest that ginsenoside Rg3 may be useful as a chemotherapy agent because of its ability to inhibit melanoma cell metastasis. These results demonstrate that the anticancer effects of Rg3 merit further investigation and provide a foundation for the development of ginsenoside Rg3 as a chemotherapeutic agent.