Nutr Res Pract.  2020 Oct;14(5):463-477. 10.4162/nrp.2020.14.5.463.

Rhus verniciflua Stokes extract suppresses migration and invasion in human gastric adenocarcinoma AGS cells

• Lee HS
• Jung JI
• Kim KH
• Park SJ
• Kim EJ

• KMID: 2506884
• DOI: http://doi.org/10.4162/nrp.2020.14.5.463

Abstract

BACKGROUND/OBJECTIVES
Many studies have suggested that Rhus verniciflua Stokes (RVS) and its extract are anticancer agents. However, RVS had limited use because it contains urushiol, an allergenic toxin. By improving an existing allergen-removal extraction method, we developed a new allergen-free Rhus verniciflua Stokes extract (RVSE) with higher flavonoid content. In this study, we examined whether RVSE inhibits the ability of AGS gastric cancer cells to migrate and invade.
MATERIALS/METHODS
The flavonoids content of RVSE was analyzed by HPLC. The effects of RVSE on migration and invasion in AGS cells were analyzed by each assay kit. Matrix metalloproteinase (MMP)-9, plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) protein expression was analyzed by protein antibody array. The Phosphorylation of signal transducer and activator of transcription (STAT) 3 were assayed by Western blot analysis.
RESULTS
RVSE treatment with 0–100 μg/mL dose-dependently reduced the ability of AGS cells to migrate and invade. Notably, treatment with RVSE strongly inhibited the expression of MMP-9 and uPA and the phosphorylation of STAT3. In contrast, RVSE treatment dramatically increased the expression of PAI-1. These results indicate that the inhibition of MMP-9 and uPA expression and STAT3 phosphorylation and the stimulation of PAI-1 expression contributed to the decreased migration and invasion of AGS cells treated with RVSE.
CONCLUSIONS
These results suggest that RVSE may be used as a natural herbal agent to reduce gastric cancer metastasis.

Keyword

Gastric cancer; metastasis; matrix metalloproteinases; urokinase-type plasminogen activator; plasminogen activator inhibitors

Figure

• 

  Fig. 1 High-performance liquid chromatography chromatograms of pRVSE and RVSE.(A) 1 mg/mL pRVSE. (B) 1 mg/mL RVSE.pRVSE, pre-Rhus verniciflua Stokes extract; RVSE, Rhus verniciflua Stokes extract; PDA, photodiode array; RT, retention time.

• 

  Fig. 2 Effect of pRVSE and RVSE on the viability of AGS cells.Cells were plated in 24-well plates at 5 × 104 cells/well in DMEM supplemented with 100 ml/L FBS. After 24 h, the cells were serum-deprived with DMEM supplemented with 10 mL/L charcoal stripped FBS (SDM) for 24 h. After serum deprivation, the cells were incubated in serum-deprivation medium at varying concentration (0, 25, 50, 100, 150 µg/mL) of pRVSE or RVSE. Viable cell numbers were estimated by MTT assays. Each bar represents the mean ± SEM (n = 4). Means without a common letter differ, P < 0.05.pRVSE, pre-Rhus verniciflua Stokes extract; RVSE, Rhus verniciflua Stokes extract; DMEM, Dulbecco's Modified Eagle's Medium; FBS, fetal bovine serum; SDM, serum-deprivation medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

• 

  Fig. 3 Effect of pRVSE and RVSE on the migration of AGS cells.The migration ability of AGS cells was measured using the CytoSelect™ 24-well Cell Migration Assay kit. The cells were serum-deprived in DMEM supplemented with 10 mL/L charcoal-stripped FBS for 24 h. Serum-deprived cells were plated in trans-well inserts in 24-well plates at 1.5 × 105 cells/filter and treated with 0–100 µg/mL pRVSE or RVSE. The lower chamber of the well was filled with SDM supplemented with 1g/L BSA as a chemoattractant. The cells were then incubated for 4 h. (A) The migrated cells were stained with hematoxylin and eosin. (B) The migrated cells were stained with cell stain solution and quantified by measuring the absorbance at 560 nm after dye extraction. Each bar represents the mean ± SEM (n = 4). Means without a common letter differ, P < 0.05.pRVSE, pre-Rhus verniciflua Stokes extract; RVSE, Rhus verniciflua Stokes extract; DMEM, Dulbecco's Modified Eagle's Medium; FBS, fetal bovine serum; SDM, serum-deprivation medium; BSA, bovine serum albumin.

• 

  Fig. 4 Effect of RVSE on the invasion of AGS cells.The invasion ability of AGS cells was measured using the CytoSelect™ 24-well Cell Invasion Assay kit. Serum-deprived AGS cells were plated in trans-well inserts in 24-well plates at 1.5 × 105 cells/filter and treated with 0–100 µg/mL RVSE. The lower chamber of the well was filled with SDM supplemented with 1g/L BSA as a chemoattractant. The cells were then incubated for 6 h. The invaded cells were stained with cell stain solution and quantified by measuring the absorbance at 560 nm after dye extraction. Each bar represents the mean ± SEM (n = 4). Means without a common letter differ, P < 0.05.RVSE, Rhus verniciflua Stokes extract; SDM, serum-deprivation medium; BSA, bovine serum albumin.

• 

  Fig. 5 Effect of RVSE on MMP-9 protein expression in AGS cells.Cells were plated in 100 mm dishes at 1 × 106 cells/dish in DMEM containing 100 mL/L FBS. After 24 h, the cells were serum-deprived with SDM for 24 h. After serum-deprivation, the cells were incubated with 0–100 µg/mL RVSE for 24 h. The cell lysates were subjected to Western blotting with MMP-9 antibody. (A) Photographs of chemiluminescent detection of the blots, which are representative of 3 independent experiments, are shown. (B) Quantitative analysis of the blots. The relative abundance of each band to its own β-actin was quantified, and the control levels were set at 1. Each bar represents the adjusted mean ± SEM (n = 4). Means without a common letter differ, P < 0.05.RVSE, Rhus verniciflua Stokes extract; MMP, matrix metalloproteinase; DMEM, Dulbecco's Modified Eagle's Medium; FBS, fetal bovine serum; SDM, serum-deprivation medium.

• 

  Fig. 6 Effect of RVSE on uPA and PAI-1 protein levels in AGS cells.The cells were plated and treated as described in Figure 5. The cell lysates were subjected to Western blotting with uPA and PAI-1 antibodies. (A) Photographs of chemiluminescent detection of the blots, which are representative of 3 independent experiments, are shown. (B) Quantitative analysis of the blots. The relative abundance of each band to its own β-actin was quantified, and the control levels were set at 1. Each bar represents the adjusted mean ± SEM (n = 4). Means without a common letter differ, P < 0.05.RVSE, Rhus verniciflua Stokes extract; uPA, urokinase-type plasminogen activator; PAI-1 plasminogen activator inhibitor-1.

• 

  Fig. 7 Effect of RVSE on the phosphorylation of STAT3 in AGS cells.The cells were plated and treated as described in Fig. 5. The cell lysates were subjected to Western blotting with pY705 STAT3 and STAT3 antibodies. (A) Photographs of chemiluminescent detection of the blots, which are representative of 3 independent experiments, are shown. (B) Quantitative analysis of the blots. The relative fold-change in pSTAT3 to its own STAT3 was quantified, and the control levels were set at 1. Each bar represents the adjusted mean ± SEM (n = 4). Means without a common letter differ, P < 0.05.RVSE, Rhus verniciflua Stokes extract; STAT, signal transducer and activator of transcription.

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