Gastric cancer (GC) is one of the most common cancers. The rate of surgical removal of primary cancer is very high, but a great risk comes from the fact that many patients are diagnosed after the tumor has invaded and spread to other parts of the body. In order to eradicate cancer cells, a high dose of medication is required, which is often accompanied by toxic side effects. Inhibition of metastasis can be a more effective strategy to control the progression of cancer. Extensive studies have been conducted to understand the regulatory mechanisms involved in tumor motility, invasiveness, and metastasis. Recently, metabolic parameters of tumor tissue, such as extracellular acidity, have been suggested to be linked to these events [1
Due to active aerobic and anaerobic glycolysis, solid tumors inevitably form an acidic extracellular environment. Such extracellular acidity is a critical factor driving chemoresistance as well as malignant transformation of tumor cells [3
]. The role of extracellular acidity in promoting invasiveness and metastasis has been demonstrated in human glioma, melanoma, and prostate cancer cells [4
]. Acidity was shown to modulate the expression of multiple oncogenes and EMT-related transcription factors, such as NFkB, HIF2α, and COX2 [7
], causing the epithelial properties of tumor cells to switch to a more mesenchymal-like phenotype [9
]. A low-pH environment triggers the loss of E-cadherin expression in melanoma cells, and tumor cells primed at acidic pH have an increased adherence to the surface at normal pH in vitro and a higher metastatic potential in vivo [10
], implicating the role of acidity in the migration of tumor cells from the original acidic tumor tissue to another non-tumor site. Therefore, treatment of tumors exposed to an acidic environment appears to be an important issue. Considerable attempts have been made to overcome adverse effects caused by acidic environment with strategies such as the use of proton pump inhibitors that block release of cellular proton into extracellular spaces, but the results have not been as successful as expected. Thus, it is imperative to find agents that prevent acidity-mediated malignancy, the activity of which is not inhibited by extracellular acidity.
Recently, use of natural medicines has been attracting more attention because of long-established medicinal effects and widely recognized safety. Numerous natural compounds have been extensively examined over the past several decades for their potential in cancer prevention and treatment [12
]. Ellagic acid is one of naturally occurring phenolic compounds that has recently received considerable attention due to its diverse pharmacological activities, including antioxidant, anti-inflammatory, and anticancer effects [13
]. Ellagic acid is contained in ellagitannins, mainly present in vegetables, nuts, and fruits such as raspberries and pomegranates. Among the various beneficial pharmacological activities of ellagic acid, the capacity to prevent several types of cancers is of particular importance. Ellagic acid and ellagic acid-rich foods have shown preventive and therapeutic effects against multiple types of cancers including colorectal cancer, esophageal cancer, breast cancer, prostate cancer, leukemia, and lymphoma [13
]. The anticancer effect of ellagic acid has shown to be mainly mediated through its antiproliferative and pro-apoptotic actions; however, there are also studies indicating that EA inhibits migration and invasion of prostate cancer cells and bladder cancer cells by inhibiting protease activity or reducing the expression of PD-L1 and VEGFR-2 [16
Here, we examined the potential of ellagic acid as an anti-invasive agent for gastric cancer cells exposed to an acidic environment. We used AGS and SNU601 GC cell lines maintained under acidic pH culture condition and assessed the effect of ellagic acid on their acidity-promoted invasive activity and the mechanisms involved.
2. Materials and Methods
2.1. Cell Lines and Culture Conditions
SNU-601 and AGS human gastric cancer (GC) cells were obtained from the Korean Cell Line Bank (Seoul, Korea) and American Type Culture Collection (Manassas, VA, USA), respectively. Cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum and 1% PS at 37 °C in an atmosphere containing 5% CO2. Drugs were purchased from Calbiochem (San Diego, CA, USA). Acidity-conditioned cells were maintained in pH 6.5-adjusted medium for longer than 3 weeks and subcultured at regular intervals.
2.2. Invasion and Migration Assay
To assay cell invasiveness, Matrigel-coated transwell chambers (Corning Costar) were used. Equal numbers of cells maintained in pH 7.4 or pH 6.5 were suspended in RPMI of each pH condition containing 1% FBS. Approximately 200 μL of the cell suspension was added to the upper portion of the insert, and medium containing 5% FBS was added to the lower portion of the inset. After 8 h (for AGS) and 18 h (for SNU601) of incubation at 37 °C in 5% CO2, noninvasive cells were removed from the upper surface of the transwell membrane with a cotton swab, and the invaded cells on the lower layer surface were fixed in 4% formaldehyde and stained with crystal violet solution. The numbers of invaded cells were counted or imaged under high-power (×200) microscope magnification (Olympus). For the migration assay, cells incubated at pH 7.4 and pH 6.5 were sampled in the same manner as above and grown in 24-well plates in growth medium. After overnight culture, the middle of the cell surface was scraped with a micropipette tip to make a wound of constant width. Debris was washed out with PBS, and the wound closures were monitored and photographed at 6 h (AGS cells) and 18 h (SNU601 cells) under the microscope (× 100, Olympus). Migration distance was calculated applying the software program HMIAS-2000.
2.3. Cytotoxicity Assays
The EZ-cytox viability assay was performed following the manufacturer’s protocol. Briefly, cells were plated in wells of a 24-well plate at a density of 5–8 × 104 cells/well, cultured for 24 h, and then incubated in the growth medium with or without ellagic acid for 48 h. The EZ-cytox solution (Daeillab, Korea) was added to the wells and incubated at 37 °C in a CO2 incubator for the last 2 h of incubation, and the plates were read using an enzyme-linked immunosorbent assay plate reader at 450 nm. The absorbance of the untreated cells was set as 100%, and cell survival was expressed as a percentage of this value.
2.4. Western Blot Analysis
Treated cells were lysed in a whole-cell lysis buffer (50 mM Hepes, 150 mM NaCl, 1% Triton X-100, 5 mM EGTA, protease inhibitor cocktail), and equal amounts of protein extracts were electrophoretically separated using 10%–12% SDS-PAGE and transferred to a nitrocellulose membrane using standard techniques. The proteins were probed using antibodies for COX1 (ab695, abcam), COX2 (sc-376861, Santa Cruz Biotechnology), and α-tubulin (sc-5286) diluted in TBS solution containing 2% skim milk and incubated overnight at 4 °C. Signals were acquired using an Image Station 4000MM image analyzer (Kodak, NY, USA).
2.5. Real-Time Reverse Transcription-Polymerase Chain Reaction
Real-time PCR was performed with the Light Cycler 2.0 (Roche) using the Fast Start DNA Master SYBR Green I Kit (Roche). For verification of the correct amplification product, PCR products were analyzed on a 2% agarose gel stained with ethidium bromide. The sequences of the primers were designed as follows: for β-actin, 5′-GACTATGACTTAGTTGCGTTA-3′ and 5′-GCCTTCATACATCTCAAGTTG-3′, for snail, 5′-GGCTCCTTCGTCCTTCT-3′ and 5′-GGCTGAGGTATTCCTTGTT-3′, for twist1, 5′-CGGGAGTCCGCAGTCTTA-3′ and 5′-CTGGTAGAGGAAGTCGATGT-3′, for c-myc, 5′- GCTTTATCTAACTCGCTGTAGTAAT-3′ and 5′- GCTGCTATGGGCAAAGTTTC-3′. Primers of MMP7 (P310408) and MM9 (P323207) were purchased from Bioneer. PCR was conducted at 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 seconds, 60 °C for 5 seconds, and 72 °C for 7 seconds. Melt curve analysis was performed to confirm that a single product was present. Negative controls without template were included in each run. Data were analyzed using Light Cycler software version 4.0 (Roche, Switzerland). The 2ΔΔCt method was used for analysis of relative gene expression.
2.6. RNA Interference (RNAi)
For the RNAi experiment, siRNAs of snail, twist1, and c-myc and a scrambled siRNA control were purchased from Bioneer (Daejeon, Korea). Cells were individually transfected with siRNA oligonucleotides using an Amaxa™ Transfection System (Basel, Switzerland) and grown for 48 h in the acidic pH medium.
2.7. Statistical Analysis
All numerical data are presented as mean ± SE of three independent experiments. For statistical analysis, student’s t-test was used for simple comparisons, and one-way ANOVA with Tukey’s test was used for multiple comparison test. A p-value of 0.05 or less was considered statistically significant.
All stages of the progression of tumors and their responses to treatments are greatly influenced by the physical microenvironment surrounding the tumor. Tumor cells must adapt to a wide range of environmental changes within the tumor mass; this is a factor driving transformation of tumor cells to become more resistant or malignant. One of the metabolic parameters of tumor tissue, extracellular acidosis, has been recognized as an important metastatic factor involved in the invasion and metastasis of human glioma, melanoma, and prostate cancer cells [4
] through its influence on the expression of multiple oncogenes and epithelial-mesenchymal transition (EMT)-related genes [7
]. In line with previous studies on other cell types, our study showed that gastric cancer cells maintained in acidic conditions showed increased motility and invasiveness as well as elevated expression of MMP7 and MMP9, proteolytic enzymes that cleave extracellular matrix proteins, which are well-established biomarkers of invasion and metastasis in human cancers [19
We investigated whether ellagic acid can be a useful agent for metastatic alteration of gastric cancer cells caused by the acidosis. Ellagitanins and their derivatives from black raspberry or pomegranate have shown inhibitory effects on several types of malignancies, including breast, ovarian, and prostate cancer, in studies in vivo and in vitro [16
]. The majority of previous studies have focused on the cytotoxic and cytostatic activity of ellagic acid in various types of cancers. For example, ellagic acid was shown to inhibit cell cycle progression by modulating expression of p53, p21, cyclin D1, and cyclin E, and to induce apoptosis by altering the Bax/Bcl-2 ratio in ovarian carcinoma cell lines [24
]. Ellagic acid or ellagitanins from pomegranate juice also inhibited proliferation of prostate cancer cells by inhibiting the expression of cyclin D1 and cyclin B1, and triggered the intrinsic and extrinsic apoptosis pathways [25
]. In addition, ellagic acid prevented cell growth and triggered apoptosis in multiple types of cancer cells through inhibition of signal pathways such as PKC, AKT, or PI3K/PKB pathway or induction of the mitochondrial apoptotic pathway [28
]. Furthermore, ellagic acid also inhibited pancreatic cancer growth in xenografted mice [33
In our study of gastric cancer cells, high concentrations of ellagic acid also decreased cell viability. However, we focused on the impact of ellagic acid specifically on the invasion capacity of the acidity-exposed cells, since ellagic acid concentrations that affect the survival of cancer cells are relatively high and may cause noncompliance with chemotherapy. The concentrations of ellagic acid used in previous studies ranged from 0.1 to 100 μM, and sensitivity to ellagic acid was highly dependent on cell type. We selected concentrations under which the survival rate of gastric cancer cells was higher than 90% after 48 h incubation. This low dose of ellagic acid reduced acidity-promoted expression of MMP7 and MMP9 and inhibited the migrating and matrigel-infiltrating capability of gastric cancer cells, indicating the inhibitory action of ellagic acid against acidity-enhanced invasiveness. Consistently with our study, the inhibitory roles of ellagic acid in multiple metastatic signaling pathways such as wnt/b-catenin, TGF/smad3, HIF1a, and HIF2a have been also demonstrated in several types of cancer cells such as colon, bladder, and breast cancer cells [17
], although the effect of ellagic acid on malignancy caused by acidity has not been elucidated.
Physiological acidic pH is closely associated with the inflammatory response [35
]. Acidity was suggested to be associated with the induction of pro-inflammatory factors, such as TNF-α, IL-1β, and IL-6 [36
], and COX2-dependent inflammatory responses [37
]. Overexpression of COX2 has been suggested to be related to invasion in several types of cancer cells, including glioma and breast cancer cells [38
], and inhibition of COX2 was shown to reduce lymphatic metastasis of a human gastric cancer cell line in xenografts [40
]. Ellagic acid has been shown to exert anti-inflammatory activity in several disease models and decrease COX2-triggered exacerbation of inflammation [41
]. Thus, we hypothesized that the inhibitory effect of ellagic acid on acidity-promoted invasion might be mediated through inhibition of COX2. As expected, acidic culture conditions increased COX2 expression, which was decreased by ellagic acid. Interestingly, COX1 expression was also increased by acidic culture and decreased by ellagic acid. A general COX inhibitor but not a selective COX2 inhibitor reduced acidity-induced MMP7 and MMP9 expression and cell invasion. These results suggest that inhibition of COX2 alone may not be sufficient to block MMP7 and MMP9 expression, and additional suppression of COX1 is required to block acidity-mediated invasiveness. In fact, the basal and induced levels of COX1 appeared to be higher than those of COX2. Hence, the inhibitory activity of ellagic acid on both COX1 and COX2 may be associated with the anti-invasive activity in these systems. Although COX1 has long been considered to be constitutively expressed in all tissues and linked to normal physiological functions, not to pathological ones, several recent studies have reported that COX1 can also be upregulated in various pathological conditions and associated with inflammation and cancer [43
Next, we evaluated other genes potentially involved in anti-invasive activity of ellagic acid under acidic conditions. Ellagic acid inhibited extracellular acidity-induced expression of EMT-regulating transcription factors snail and twist1 and proto-oncogene c-myc in our experimental system. EMT is considered a crucial step for cancer metastasis, and expression of snail or twist1 has been shown to be linked to increased mesenchymal marker expression and high metastatic potential in several tumor cells and multiple mouse models. Recent reports have shown an impact of extracellular acidosis on the induction of several EMT-related genes including snail and twist1 [8
]. Due to loss of E-cadherin in AGS and SNU601 cells, which is a common event in gastric cancer cells, we could not observe acidity-mediated reduction of E-cadherin expression, which is a critical sign of EMT. However, the acidity-induced expression of snail, twist1, and c-myc genes suggests that acidic culture conditions trigger EMT-like gene expression, and thus the ability of ellagic acid to inhibit expression of these genes may be associated with the suppression of an overall shift to a malignant phenotype. However, in contrast with our results, a previous report showed that acidosis lowers c-myc expression in U937 lymphoma cells [45
]. c-Myc is a pleiotropic transcription factor that regulates a wide spectrum of cancer cell features, such as tumorigenesis and metastasis [46
]. The discrepancy may be due to differences in the acid treatment conditions or variance in the cellular context resulting from different cell types. For example, the previous study was performed under acute acidosis, whereas in the current study, cells were exposed to acidic pH medium for a longer period before exposure to ellagic acid because cell viability was significantly reduced in the early stages of acidic condition.