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. 2012 Jan;33(1):120-6.
doi: 10.1038/aps.2011.139. Epub 2011 Nov 21.

Epigallocatechin-3-gallate inhibits proliferation and migration of human colon cancer SW620 cells in vitro

Fang Zhou  1 , Hong Zhou, Ting Wang, Yuan Mu, Biao Wu, Dong-lin Guo, Xian-mei Zhang, Ying Wu
Affiliations

Epigallocatechin-3-gallate inhibits proliferation and migration of human colon cancer SW620 cells in vitro

Fang Zhou et al. Acta Pharmacol Sin. 2012 Jan.

Abstract

Aim: Epigallocatechin-3-gallate (EGCG) is the major polyphenolic constituent in green tea. The aim of this study is to investigate the effects of EGCG on proliferation and migration of the human colon cancer SW620 cells.

Methods: Proliferation and migration of SW620 cells were induced by the protease-activated receptor 2-agonist peptide (PAR2-AP, 100 μmol/L) or factor VIIa (10 nmol/L), and analyzed using MTT and Transwell assays, respectively. The cellular cytoskeleton was stained with rhodamine-conjugated phalloidin and examined with a laser scanning confocal fluorescence microscope. The expression of caspase-7, tissue factor (TF) and matrix metalloproteinase (MMP)-9 in the cells was examined using QT-PCR, ELISA and Western blot assays. The activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and nuclear factor-kappa B (NF-κB) signaling pathways was analyzed with Western blot.

Results: Both PAR2-AP and factor VIIa promoted SW620 cell proliferation and migration, and caused cytoskeleton reorganization (increased filopodia and pseudopodia). Pretreatment with EGCG (25, 50, 75, and 100 μg/mL) dose-dependently blocked the cell proliferation and migration induced by PAR2-AP or factor VIIa. EGCG (100 μg/mL) prevented the cytoskeleton changes induced by PAR2-AP or factor VIIa. EGCG (100 μg/mL) counteracted the down-regulation of caspase-7 expression and up-regulation of TF and MMP-9 expression in the cells treated with PAR2-AP or factor VIIa. Furthermore, it blocked the activation of ERK1/2 and NF-κB (p65/RelA) induced by PAR2-AP or factor VIIa.

Conclusion: EGCG blocks the proliferation and migration of SW620 cells induced by PAR2-AP and factor VIIa via inhibition of the ERK1/2 and NF-κB pathways. The compound may serve as a preventive and therapeutic agent for colon cancers.

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Figures

Figure 1
Figure 1
Effects of EGCG on PAR2-AP- and factor VIIa-induced proliferation and migration of SW620 cells. (A) SW620 cells were plated at 5×104 cells in 96-well plates and pretreated with different concentrations of EGCG (0, 25, 50, 75, and 100 μg/mL) for 15 min, then incubated with PAR2-AP (100 μmol/L) or factor VIIa (10 nmol/L) for 24 h. The above EGCG was kept in media. The cell proliferation was measured using MTT colorimetric assay. n=6. Mean±SEM. eP<0.05, fP<0.01 vs PAR2-AP- or factor VIIa-stimulated alone. (B) The cells were pretreated with or without EGCG (100 μg/mL) for 15 min as described above and then incubated with PAR2-AP (100 μmol/L) or factor VIIa (10 nmol/L) for 8 h. The cell migratory potential was measured by modified Boyden Chambers as described in Methods. n=3. Mean±SEM. aP>0.05, cP<0.01 vs media only; eP<0.05, fP<0.01 vs PAR2-AP- or factor VIIa-stimulated alone.
Figure 2
Figure 2
Fluorescence photomicrographs of SW620 cells with rhodamin-conjugated phalloidin and Hoechst 33342 staining (×400). SW620 cells were placed in 24-well culture dishes at 3×104 cells/well and treated with different conditions for 1 h. Then, the cells were fixed with 4% paraformaldehyde and washed with PBS three times. The DNA was stained with Hoechst 33342 (blue) and the actin cytoskeleton with rhodamin-conjugated phalloidin (red) which were observed under a laser scanning confocal fluorescence microscope. (A) Media only; (B) With 100 μmol/L of PAR2-AP; (C) With 10 nmol/L of factor VIIa; (D) With 100 μg/mL of EGCG; (E) With EGCG (100 μg/mL)/PAR2-AP (100 μmol/L); (F) With EGCG (100 μg/mL)/factor VIIa (10 nmol/L).
Figure 3
Figure 3
Effects of EGCG on caspase-7 expression in SW620 cells. SW620 cells (1×106–1×107) were stimulated with PAR2-AP (100 μmol/L) or factor VIIa (10 nmol/L) in the absence or presence of EGCG (100 μg/mL) for 2 h or 24 h. Then the total RNAs (2 h) and cell lysates (24 h) were collected for caspase-7 mRNA (A) and its protein (B) determination using QT-PCR and Western blot analysis, respectively. The caspase-7 mRNA levels were normalized to control values of β-actin and its protein levels were expressed as the ratio of caspase-7/β-actin bands density. n=3. Mean±SEM. aP>0.05, cP<0.01 vs media only; fP<0.01 vs PAR2-AP- or factor VIIa-stimulated alone.
Figure 4
Figure 4
Effect of EGCG on the expression of TF and MMP-9 in SW620 cells. The cells (1×106) were incubated with PAR2-AP (100 μmol/L) or factor VIIa (10 nmol/L) in the absence or presence of EGCG (100 μg/L) for 24 h. The cell lysates were prepared and the TF activity (A) as well as MMP-9 levels (B) was measured by the specific kits, respectively. n=3. Mean±SEM. aP>0.05, bP<0.05, cP<0.01 vs media only; fP<0.01 vs PAR2-AP- or factor VIIa -stimulated alone.
Figure 5
Figure 5
Effects of EGCG on activation of ERK1/2 and NF-κB pathway in SW620 cells. The cells were pretreated with or without the indicated concentrations of EGCG for 15 min and then incubated with PAR2-AP (100 μmol/L) or factor VIIa (10 nmol/L) for indicated time. The above EGCG was kept in media. The cytoplasmic and nuclear lysates of the cells were collected and subjected to Western analysis with antibodies to total and phosphorylated ERK1/2 (A) and to NF-κB/p65 as well as Histon H3 (B). n=3. Mean±SEM. aP>0.05, bP<0.05, cP<0.01 vs control; dP>0.05, eP<0.05, fP<0.01 vs PAR2-AP- or factor VIIa-stimulated alone.
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