Silibinin Suppresses TNF-α-Induced MMP-9 Expression in Gastric Cancer Cells through Inhibition of the MAPK Pathway

Tumor necrosis factor (TNF)-α is one of the pro-inflammatory cytokines highly expressed in Helicobacter pylori that inhibits gastric acid secretion. In this study we determined the effect of silibinin on TNF-α-induced MMP-9 expression in gastric cancer cell lines. MMP-9 mRNA and protein expression was dose-dependently increased by TNF-α in SNU216 and SNU668 gastric cancer cells. On the other hand, TNF-α-induced MMP-9 expression was dose-dependently suppressed by silibinin. To verify the regulatory mechanism of silibinin on TNF-α-induced MMP-9 expression, the gastric cancer cell lines were pretreated with silibinin prior to TNF-α. TNF-α-induced MMP-9 expression was inhibited by the MEK1/2 specific inhibitor, UO126. Finally, we investigated the effect of adenoviral constitutively active (CA)-MEK and CA-Akt on MMP-9 expression. The expression of MMP-9 was significantly increased by CA-MEK overexpression, but not by CA-Akt overexpression. Taken together, we suggest that silibinin down-regulates TNF-α-induced MMP-9 expression through inhibition of the MEK/ERK pathway in gastric cancer cells.


Introduction
Silibinin is a major bioactive flavanone that has been isolated from milk thistle seeds, and is used for the protection against various cancers, such as skin, lung, and breast cancers [1,2,3]. Silibinin modulates the imbalance between cell survival and apoptosis through the regulation of cell cycle regulators [4]. In addition, silibinin has anti-metastasis effects by modulating specific proteins, including matrix metalloproteinases (MMPs) [4]. In a previous study we reported that silibinin downregulates TPA-induced MMP-9 and VEGF expression through inactivation of the MAPK pathway in the MCF-7 breast cancer cell line [2]. However, the effect of silibinin has not been fully elucidated with respect to the treatment of gastric cancer. Compared with normal tissue the level of expression of tumor necrosis factor (TNF)-α is abnormally increased in pre-neoplastic lesions, such as Helicobacter pylori-infected gastric lesions and inflamed colonic mucosa [5,6]. TNF-α is a key cytokine involved in inflammation, immunity, and cancer development [5]. TNF-α is also able to promote angiogenesis through the induction of VEGF, VEGFR2, and bFGF [6,7]. In addition, TNF-α augments tumor remodeling with respect to tumor cell motility and tumor invasion via the induction of MMPs [8,9].
The MMPs are a major group of enzymes which can degrade nearly all components of the extracellular matrix (ECM), as well as components of basement membranes [10,11]. An elevated expression of MMPs contributes to various pathologic processes, including rheumatoid arthritis, osteoarthritis, angiogenesis, invasion, and metastasis in carcinoma [12,13,14]. Two members of the MMP family, MMP-2 and MMP-9, are highly expressed in various tumors, including breast and bladder cancer [15,16]. Serum levels of MMP-9, but not MMP-2, are significantly higher in colorectal and gastric cancer compared to controls [17]. Elevated plasma MMP-9 correlates significantly with lymph node metastasis, lymphatic invasion, venous invasion, and had poorer survival rates [18].
In the present study we determined the effect of silibinin on MMP-9 expression in gastric cancer cells. Our results showed that silibinin suppresses TNF-α-induced MMP-9 expression. In addition, TNF-α-induced ERK phosphorylation was significantly decreased in SNU216 gastric cancer cells by silibinin treatment. We suggest that silibinin may be an effective therapeutic drug for anti-cancer therapy by preventing cancer metastasis through the down-regulation of MMP-9 expression in gastric cancer.

Results and Discussion
The basal level of MMP-9 mRNA and protein expression was increased by TNF-α in SNU216 and SNU668 gastric cancer cells in a dose-dependent fashion To verify the effect of TNF-α on MMP-9 expression, we treated cells with TNF-α for 24 h at the indicated concentrations in SNU216 and SNU668 gastric cancer cells. The secreted MMP-9 protein and mRNA expression were measured by gelatin zymography and RT-PCR, respectively. The basal level of MMP-9 mRNA and protein expression was increased by TNF-α in a dose-dependent manner ( Figure 1). In SNU216 cells, the level of MMP-9 mRNA and protein expression was increased by 47.84 ± 4.72-and 24.14 ± 10.69-fold of the control level at 20 ng/ml of TNF-α, respectively ( Figure  1A and 1B). In addition, the level of MMP-9 mRNA and protein of SNU668 cells was increased by 13.37 ± 4.17-and 9.94 ± 0.28-fold of the control level following treatment with TNF-α (20 ng/ml), respectively ( Figures 1C and 1D). Therefore, we demonstrated that induction of TNF-α by H. pylori may augment tumor metastasis through up-regulation of MMP-9 expression in gastric cancer cells. shown are the mean ± SEM. * P < 0.05, **P < 0.01 vs. control. Con; control.

TNF-α-induced MMP-9 mRNA and protein expression was decreased by silibinin in SNU216 gastric cancer cells in a dose-dependent fashion
To determine the effect of silibinin on TNF-α-induced MMP-9 expression, we pretreated cells with 50 or 100 μM silibinin for 60 min prior to treatment with TNF-α (20 ng/mL). TNF-α-induced MMP-9 mRNA and protein expression was decreased by silibinin in a dose-dependent fashion (Figures 2A and   2B). The level of MMP-9 mRNA was increased by 89.22±27.21-fold of the control level by treatment with 20 ng/ml of TNF-α treatment ( Figure 2A). However, TNF-α-induced MMP-9 mRNA was decreased by 23.55 ± 9.42-and 8.47 ± 5.04-fold of the control level in a dose-dependent fashion following treatment with 50 and 100 μM silibinin, respectively ( Figure 2A). The level of MMP-9 protein was also increased by 90.0 ± 7.68-fold of the control level with TNF-α treatment at 20 ng/mL ( Figure 2B). However, TNF-α-induced MMP-9 protein expression was decreased by 10.93 ± 3.73-and 3.37 ± 0.39-fold of the control level in a dose-dependent fashion at 50 and 100 μM silibinin, respectively ( Figure 2B). We next examined the effect of silibinin on the proliferation of gastric cancer cells. Under serum-starved conditions, the cell cycle was not affected by TNF-α and/or silibinin treatment ( Figure 2C). Therefore, we demonstrated that silibinin may be a therapeutic drug for metastasis of gastric cancer through down-regulation of MMP-9 expression. The values shown are the mean ± SEM. * P < 0.05, **P < 0.01 vs. control, † P < 0.05, † † P < 0.01 vs. TNF-α-treated cells. Con; control.

TNF-α-induced MMP-9 expression was decreased by UO126 and LY294002 in SNU216 gastric cancer cells
The effect of selective MEK1/2 and PI-3 kinase inhibitors on TNF-α-induced MMP-9 expression was determined. We pretreated SNU216 cells with UO126 (a MEK1/2 inhibitor) and LY294002 (a PI-30 min, and then continued the incubation for an additional 24 h. TNF-α-induced MMP-9 expression was inhibited by UO126 in a dose-dependent manner ( Figure 3A). The level of MMP-9 expression was increased 117.93 ± 11.17-fold of the control level at 20 ng/ml of TNF-α ( Figure 3A). In contrast, TNFα-induced MMP-9 expression was decreased by 40.45 ± 15.43-and 7.01 ± 0.18-fold of the control level in a dose-dependent fashion following treatment with 10 and 20 μM UO126, respectively ( Figure 3A).

MEK), but not by CA-Akt, in SNU216 gastric cancer cells
Finally, we investigated the regulatory mechanism of MMP-9 expression by the MAPK and PI-3 kinase pathways. Then, we transfected cells with CA-MEK and CA-Akt for 24 h, followed by incubation for an additional 24 h in fresh serum-free media. After 24 h, we harvested the culture media and cell lysates. As shown in Figure 4A, MMP-9 expression was increased by CA-MEK in a dosedependent manner. In cell lysates, the phosphorylation of ERK, a downstream target of MEK, was also increased by CA-MEK overexpression ( Figure 4A). On the other hand, MMP-9 expression was not affected by CA-Akt overexpression ( Figure 4B). Therefore, we demonstrated that silibinin prevented TNF-α-induced MMP-9 expression via suppression of the MAPK pathway in gastric cancer cells. Silibinin is a major bioactive flavanone that has been isolated from milk thistle seeds, and has been used as a traditional medicine [1]. Silibinin also suppresses the invasion and motility of cancer cells by down-regulating MMP-2 and up-regulating TIMP-2 expression [19,20]. Silibinin exerts an inhibitory effect on the expression of TNF-α and other pro-inflammatory cytokines, including IFN-γ, IL-4, and IL-2, as well as TNF-α signaling [21,22]. Silibinin also inhibits the invasion, motility and migration of prostate cancer cell lines via down-regulation of vimentin and MMP-2 [23]. In addition, Lin et al.
reported that silibinin also inhibits cell growth and protein translation through suppression of mTOR activity in MCF7 breast cancer cells [24].
H. pylori has been shown to be a potent carcinogen in human gastric cancer by the International Agency for Research on Cancer (IARC) [25]. It has been reported that infection with H. pylori augments the inflammation associated with the induction of TNF-α and other inflammatory cytokines, such as IL-1β and IL-10, in the stomach [26]. In addition, TNF-α increases the risk for chronic atrophic gastritis and gastric carcinoma [27].
TNF-α is a key pro-inflammatory cytokine and is secreted by activated macrophages and monocytes [28]. TNF-α has been implicated in various human diseases, such as rheumatoid arthritis, septic shock, and tumorigenesis [29,30]. TNF-α is elevated in various human cancers and has a positive correlation with tumor grade and poor prognosis [31,32]. Therefore, we investigated the role of silibinin as a therapeutic drug for inhibition of TNF-α-induced MMP-9 expression, which is a hallmark of cancer metastasis and invasion in gastric cancer cells.
MMP-9 is known to specifically cleave type IV collagen, which is the major component of basement membranes [33]. The MMP-9 promoter has several transcription factor-binding motifs, including the AP-1, Sp-1, and NF-κB binding sites [33]. These sites are required for stimulation in response to TNF-α [34,35]. In a previous study we also reported that TNF-α-induced MMP-9, and cell invasion is significantly decreased by berberine through inhibition of AP-1 activity in MDA-MB-231 human breast cancer cells [34]. TNF-α-induced MMP-9 expression is also decreased via inhibition of the MAPK pathway, such as the ERK and p38 pathways in human urinary bladder cancer cells [36]. In agreement with these reports, our results showed that TNF-α-induced ERK phosphorylation and MMP-9 expression is decreased by silibinin treatment. The MEK1/2 inhibitor, UO126, also completely blocked TNF-α-induced MMP-9 expression in SNU216 gastric cancer cells. On the other hand, the basal level of MMP-9 expression was significantly increased by CA-MEK overexpression. However, the phosphorylation of p38 had no affect by TNF-α. Therefore, we suggest that TNF-α-induced MMP-9 expression is mediated through a MEK/ERK-dependent pathway. Then, silibinin suppresses TNF-αinduced MMP-9 expression through inhibition of the MEK/ERK pathway.
However, Hwang et al. recently reported that TNF-α-induced MMP-9 up-regulation and cell migration is associated with the PI3K/Akt pathway in the JB6 mouse epidermal cell line [38]. We also observed that TNF-α-induced MMP-9 expression is inhibited by another PI-3 kinase inhibitor, LY294002, in a dose-dependent manner. To confirm these results, we determined the effect of CA-Akt on the basal level of MMP-9 expression. As shown in Figure 4B, CA-Akt overexpression had no affected on the level of MMP-9 expression in SNU216 and SNU668 (data not shown) gastric cancer cells. Therefore, we suggest that Akt activity does not directly affect TNF-α-induced MMP-9 expression in gastric cancer cells.

Conclusion
In conclusion, we determined the effect of silibinin on TNF-α-induced MMP-9 expression in gastric cancer cells. We suggest that silibinin inhibits TNF-α-induced MMP-9 expression through inactivation of the MEK/ERK pathway. Silibinin may be an effective additive to anti-metastatic therapy by preventing cancer metastasis through the down-regulation of MMP-9 in gastric cancer.

Reagents and cell cultures
Cell culture media, antibiotics, and 10% Zymogram gel were purchased from Life Technologies Zymography: Zymography was performed in 10% polyacrylamide gels that had been cast in the presence of gelatin, as described previously [39]. Briefly, samples (culture media) were resuspended in loading buffer and run on a 10% SDS-PAGE gel containing 0.5 mg/mL gelatin without prior denaturation. After electrophoresis, gels were washed to remove SDS and incubated for 30 min at room temperature (RT) in a renaturing buffer (50 mM Tris, 5 mM CaCl 2 , 0.02% NaN 3 , and 1% Triton X-100). The gels were then incubated for 48 h at 37 °C in a developing buffer (50 mM Tris-HCl [pH 7.8] 5 mM CaCl 2 , 0.15 M NaCl, and 1% Triton X-100). The gels were subsequently stained with coomassie brilliant blue G-250 and destained in 30% methanol, and 10% acetic acid to detect gelatinase secretion. Signal densities were quantified using a densitometric program (Bio 1D; Vilber Lourmat, Marne La Vallec, France).
Western blotting: Cell lysates were used in immunoblot analysis for ERK1/2, Akt, and β-actin. Proteins were boiled for 5 min in Laemmli sample buffer and electrophoresed in 10% SDS-PAGE gels.
Proteins were transferred to PVDF membranes and the membranes were then blocked with 10% skim milk in TBS with 0.01% Tween-20 for 15 min. The blots were incubated with anti-phospho-ERK and Akt antibodies (1/1,000 dilution) in 1% TBS/T buffer (0.01% Tween 20 in TBS) at 4°C overnight.
Blots were washed 3 times in TBS with 0.01% Tween 20 and subsequently incubated in anti-rabbit peroxidase-conjugated antibody (1/2,000 dilution) in TBS/T buffer. After 1 h incubation at RT, the blots were washed three times and ECL reagents (Amersham Bioscience) were used for development.
Signal densities were quantified using a densitometric program (Bio 1D; Vilber Lourmat, Marne La Vallec, France). After Ad-CA-MEK and CA-Akt transfection, the media were replaced with serum-free media for 24 h.
The expression of construct was confirmed by Western blotting against phospho-ERK and phospho-Akt.
Statistical analysis: Statistical significance was determined using Student's t-test. The results are presented as the mean ± SEM. All p-values were two-tailed and the significance was accepted at a p < 0.05.