Galla Rhois was purchased from Omniherb (Uiseong, Korea), which is a good manufacturing practices (GMP) certified company by the Korea Food and Drug Administration. To prepare GRWE, Galla Rhois (100 g) was boiled at 100°C for 3 h with 1 l of distilled water (DW). The extract was filtered through Whatman filter paper and lyophilized. The samples were used for the treatment of cells after dissolving in DW and filtering using a 0.22-µm syringe filter. The yield of the dried extract from the starting materials was about 12.03%.

The murine colorectal carcinoma cell line colon 26 (CT26) and human colorectal adenocarcinoma cell line (HT29) were obtained from Korean Cell Line Bank (Seoul, Korea). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (all from Gibco-BRL; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in an atmosphere of 5% CO₂.

The in vivo experiment was approved and performed in accordance with the internationally accepted principles for the care and use of laboratory animals by the Institutional Animal Care and Use Committee of Wonkwang University (WKU16-11). Twenty-four female BALB/c mice (4 weeks old, 17–18 g) were purchased from Samtako (Osan, Korea). The mice with ad libitum access to food and water were housed (8 mice/cage) in a laminar air-flow room with a controlled 12-h light/dark cycle at a constant temperature of 23±1°C and humidity of 55±1%.

Water-soluble tetrazolium salt-8 reagent (WST-8; Enzo Life Sciences, Farmingdale, NY, USA) was used for quantifying cell viability. CT26 cells (2×10³ cells/well) and HT29 cells (1×10⁴ cells/well) were seeded in 96-well plates and cultured overnight. The cells were treated with GRWE (20–100 µg/ml). After 24, 48 and 72 h of incubation, WST-8 reagent was mixed with new medium and added to each well. The absorbance was measured by microplate reader at 450 nm wavelength.

After GRWE (10–100 µg/ml) treatment for 24 h, the cells were collected and suspended in serum-containing medium. Cells (1×10⁵ cells/100 µl) were transferred to a new tube and mixed with Muse^™ Annexin V & Dead Cell Reagent (EMD Millipore, Billerica, MA, USA). Samples were incubated for 20 min in the dark and the apoptotic cells were measured by Muse^™ Cell Analyzer (EMD Millipore).

Anti-PARP (cat. no. 9532), caspase-3 (cat. no. 14220), cleaved caspase-3 (cat. no. 9664), caspase-8 (cat. no. 4790), caspase-9 (cat. no. 9508), Bcl-xL (cat. no. 2764), phospho-AMPK (cat. no. 2535), AMPK (cat. no. 2532), phospho-extracellular signal-regulated kinase (ERK) (cat. no. 4370), phospho-p38 (cat. no. 4511), E-cadherin (cat. no. 3195) and N-cadherin (cat. no. 13116) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Bcl-2 (cat. no. sc-7382), Bax (cat. no. sc-7480), ERK (cat. no. sc-94), p38 (cat. no. sc-7149), vimentin (cat. no. sc-6260), twist (cat. no. sc-81417), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cat. no. sc-47724) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-rabbit (cat. no. 111-035-003) and anti-mouse (cat. no. 115-035-062) secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (Pennsylvania, PA, USA). All antibodies were diluted 1:1,000 in 3% skim milk (BD Biosciences, San Diego, CA, USA).

CT26 cells (3×10⁵ cells/well) were seeded in a 6-well plate and treated with GRWE (10, 50 and 100 µg/ml). After treatment for 24 h, the cells were lysed with ice-cold lysis buffer (iNtRON Biotech, Seoul, Korea) for 1 h. Total lysates were centrifuged at 14,000 × g for 10 min, and the supernatants were collected for the determination of the extracted protein concentration using the Lowry method. Samples were mixed with 2X buffer, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 10% acrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with 5% skim milk for at least 1 h. After washing with 0.1% PBST (0.1% Tween-20 in PBS), the membranes were incubated with primary antibodies for 3 h, followed by their treatment with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The protein blots were detected using an enhanced chemiluminescence (ECL) system (Santa Cruz Biotechnology, Inc.). The density of western blot bands was quantified by densitometric analysis using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

RNA-spin™ extraction Kit (iNtRON Biotech) was used to extract total RNA in accordance with the manufacturer's protocol. First-strand cDNA synthesis was performed using High Capacity RNA-to-cDNA Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). Reverse transcription was conducted at 37°C for 60 min and then at 95°C for 5 min. Real-time RT-PCR was carried out using a Power SYBR^® Green PCR Master Mix with a StepOnePlus^™ Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR cycling condition was as follows: one cycle at 95°C for 10 min, then 40 cycles of 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec and melt curve at 60°C for 1 min, 95°C for 15 sec. Sequences of the primers used for murine genes were as follows: E-cadherin, 5′-AATGGCGGCAATGCAATCCCAAGA-3′ and 5′-TGCCACAGACCGATTGTGGAGATA-3′; N-cadherin, 5′-TGGAGAACCCCATTGACATT-3′ and 5′-TGATCCCTCAGGAACTGTCC-3′; vimentin, 5′-CGGAAAGTGGAATCCTTGCA-3′ and 5′-CACATCGATCTGGACATGCTG-3′; Twist, 5′-AGCTACGCCTTCTCCGTCT-3′ and 5′-TCCTTCTCTGGAAACAATGACA-3′; MMP-2, 5′-CCCCATGAAGCCTTGTTTACC-3′ and 5′-TTGTAGGAGGTGCCCTGGAA-3′; MMP-9, 5′-AGACCAAGGGTACAGCCTGTTC-3′ and 5′-GGCACGCTGGAATGATCTAAG-3′; GAPDH, 5′-GACATGCCGCCTGGAGAAAC-3′ and 5′-AGCCCAGGATGCCCTTTAGT-3′. The mRNA expression of genes was normalized to the expression of the gene encoding GAPDH. The real-time RT-PCR data was analyzed using comparative Cq method (29).

CT26 cells (3×10³ cells/well) were grown on an 8-well chamber slide (Thermo Fisher Scientific, Inc.) and treated with GRWE. The cells were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. After blocking with blocking buffer (3% bovine serum albumin and 0.3% Triton X-100 in PBS), the cells were incubated with the primary antibody at 4°C, overnight. Following incubation, the cells were treated with Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific, Inc.) for 2 h and nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Images were acquired using a fluorescence microscope (Zeiss Observer A1 microscope; Carl Zeiss AG, Oberkochen, Germany).

CT26 cells (5×10⁵ cells/well) were grown in a 6-well plate to form a confluent monolayer. A wound was created using a 200-µl micropipette tip and the detached cells were removed. The cells were treated with GRWE (1, 5 and 10 µg/ml) in the absence of FBS for 48 h. Images were captured under EVOS^® XL Core Imaging System (Thermo Fisher Scientific, Inc.).

Matrigel-coated BD Falcon cell culture chamber (BD Biosciences) was used to estimate the invasive activity of cancer cells. The lower wells were filled with 750 µl DMEM containing 10% FBS. Cells (5×10⁴ cells) were seeded in the upper part of the transwell chambers in 200 µl FBS-free DMEM with GRWE (1, 5 and 10 µg/ml) and chambers were placed on a 24-well plate for 24 h. The upper inserts were fixed with 3.7% formaldehyde in PBS and washed twice with PBS. The fixed cells were permeabilized with methanol for 25 min and stained with Giemsa solution (Sigma-Aldrich; Merck KGaA) for 25 min. The inner sides of the chambers were cleaned with a swab and dried. The stained cells were observed under EVOS^® XL Core Imaging System.

For gelatin zymography, CT26 cells (5×10⁵ cells/well) were seeded in a 6-well plate and treated with GRWE for 24 h. The supernatant from the GRWE-treated cells was mixed with 2X zymogram sample buffer (KOMA Biotech, Seoul, Korea) and the samples were electrophorized using SDS polyacrylamide gel containing 1% gelatin in the zymogram running buffer (KOMA Biotech). The gel was renatured in zymogram renaturing buffer for 30 min and incubated in zymogram developing buffer (both from KOMA Biotech) at 37°C overnight. The gel was stained with Brilliant Blue R staining solution (ELPIS Biotech, Daejeon, Korea) for 30 min.

CT26 cells (1×10⁵ cells/200 µl PBS/mouse) were injected into mice via the lateral tail vein. GRWE and DW were orally administrated 2 h prior to the injection of CT26 cells and then, every day. The mice were sacrificed by cervical dislocation after 14 days and lung tissues were stained and fixed with Bouin's solution (Sigma-Aldrich; Merck KGaA). The weight of the lungs was measured and the number of nodules in the lungs was counted to evaluate CRC metastasis.

During analysis of GT from GRWE, GT (molecular weight: 1701.20 g/mol; molecular formula: C₇₆H₅₂O₄₆; Santa Cruz Biotechnology, Inc.) was used as the standard. GRWE and the standard compound were dissolved in methanol (MeOH) and filtered through a 0.45-µm syringe filter before injection. An HPLC system comprised of an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) with a UV-vis detector was used. Separation was carried out on an YMC-Triart C₁₈ column (150×4.6 mm I.D., 5 µm,) maintained at 40°C. The mobile phase was comprised of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). The gradient condition of the mobile phase was as follows: 0–5 min, 15% A; 5–10 min, 15–20% A; 10–25 min, 20–35% A; 25–30 min, 35–15% A. Analysis was performed at a flow rate of 1.0 ml/min with the detection wavelength fixed at 280 nm (Fig. 1).

All data were presented as the mean ± standard deviation (SD). ANOVA with Tukey's post hoc test was used to determine the statistical significance. SPSS Statistics v18 (IBM Corp., Armonk, NY, USA) was used as the statistical analysis software. A value of P<0.05 was considered to indicate a statistically significant difference.

To investigate the cytotoxicity of GRWE on metastatic CRC cells, cell viability was determined by WST-8 assay. CT26 and HT29 cells were treated with GRWE at concentrations of 20–100 µg/ml. As shown in Fig. 2A and B, the viability of GRWE-treated cells decreased as compared with that of the control cells. Furthermore, treatment with GRWE for 72 h resulted in morphological changes in CT26 and HT29 cells (Fig. 2C).

Annexin V assay was performed after treatment of cells with GRWE (10, 50, and 100 µg/ml) for 24 h to determine whether GRWE-induced cell death was related to the apoptosis of cells. As shown in Fig. 3A and B, treatment with GRWE resulted in an increase in the apoptosis of CT26 cells in a dose-dependent manner. To further investigate the mechanisms underlying GRWE-induced apoptosis, the expression of apoptosis-related proteins was detected by western blotting. Cleavage of caspase-3 and PARP was induced following exposure of CT26 cells to GRWE for 24 h. In addition, GRWE treatment reduced the expression of caspase-8, caspase-9, Bcl-2, and Bcl-xL and increased the protein level of Bax in CT26 cells (Fig. 3C).

Activation of AMPK inhibits cell proliferation by promoting apoptosis of cancer cells. Various anticancer drugs and plant extracts can induce AMPK-mediated apoptosis in cancer cells (30). A previous study reported that PGG, a component of Galla Rhois, induced breast cancer cell death through phosphorylation of AMPK (31). Therefore, we hypothesized that apoptosis of CT26 cells by GRWE was related to activation of AMPK. As revealed in Fig. 4A, phosphorylation of AMPK was increased after GRWE treatment in a time-dependent manner. To confirm whether GRWE-induced AMPK phosphorylation was involved in apoptosis of CT26 cells, AMPK inhibitor compound C (20 µM) was pre-treated to CT26 cells for 4 h, and then treated with GRWE (20–100 µg/ml) for 72 h. Blockage of AMPK activation recovered GRWE-decreased viability of CT26 cells (Fig. 4B). To further investigate whether apoptosis-related factors were regulated by AMPK activation, the protein levels of cleaved caspase-3 and PARP were detected in compound C and GRWE-treated CT26 cells. Cleavage of caspase-3 and PARP was induced following treatment with GRWE, whereas AMPK inhibitor compound C suppressed this effect of GRWE (Fig. 4C). Several studies have shown that AMPK activators induce apoptosis via modulation of the MAPK pathway in human cancer cells (32). AMPK activation induces apoptosis via the downregulation of ERK in cancer cells, and AMPK activator AICAR inhibits p38 MAPK (33,34). Based on these studies, we confirmed whether GRWE could regulate the phosphorylation of ERK and p38. As expected, GRWE reduced the phosphorylation of ERK and p38 (Fig. 4D). These results indicated that GRWE-induced apoptosis occurred through the AMPK-ERK/p38 signaling pathway.

Gene expression analysis was conducted to elucidate whether GRWE regulated the expression of EMT markers, which are involved in metastatic properties of cancer cells. The mRNA level of epithelial phenotypic marker E-cadherin was upregulated after GRWE treatment (Fig. 5A). In addition, the expression of mesenchymal phenotypic markers N-cadherin, vimentin, and Twist was downregulated in GRWE-treated CT26 cells (Fig. 5B-D). Consistent with the mRNA expression levels, GRWE increased the protein levels of E-cadherin and reduced N-cadherin, vimentin, and Twist expression (Fig. 5E and F). We confirmed GRWE-mediated enhanced expression of E-cadherin by immunofluorescence (Fig. 5G).

After EMT, cancer cells acquire migratory and invasive properties, resulting in metastasis (35). We performed a wound healing assay to explore the effect of GRWE on the migration of CT26 cells. Following 48 h of incubation, the control cells were more proficient in repairing wounds as compared with the GRWE-treated cells. Treatment with GRWE (1, 5, and 10 µg/ml) resulted in the inhibition of the migratory ability of CT26 cells (Fig. 6A). An invasion assay was carried out using a Matrigel-coated Transwell chamber to further investigate the effect of GRWE on the invasion ability of CT26 cells. As revealed in Fig. 6B, the infiltration of CT26 cells in the Matrigel-coated membrane was decreased following GRWE treatment in a dose-dependent manner. In addition, we assessed the mRNA expression of matrix metalloproteinase (MMP)-2 and MMP-9 using real-time RT-PCR and determined that MMP-2 and MMP-9 expression was downregulated by GRWE treatment (Fig. 6C). We also analyzed the activity of MMP-2 and MMP-9 by gelatin zymography and found that GRWE reduced the activity of MMP-2 and MMP-9 in CT26 cells (Fig. 6D).

To investigate whether GRWE inhibited the lung metastatic ability of CRC cells in mice, we employed an experimental lung metastatic model. Body weight remained unaffected following treatment with GRWE (Fig. 7A). After intravenous injection of CT26 cells, the number of metastatic tumor nodules in the lungs increased initially but were reduced after the oral administration of GRWE (250 and 500 mg/kg) for 2 weeks (Fig. 7B and C).