Fig 1.
GE inhibited proliferation of EJ cells through blocking cell cycle progression at G2/M-phase.
EJ cells were incubated in culture medium with or without GE for 24 h. Cell viability and cell proliferation was measured by both MTT (A) and viable cell counting assays (B). Results are represented as mean ± SE from three different triplicate experiments. (C) The morphology of EJ cells under different concentrations of GE. Cellular images were photographed by a phase contrast microscopy. (D-H) GE induced cell-cycle arrest at G2/M-phase in EJ cells. Cell cycle distribution of EJ cells treated with different concentrations of GE was determined by flow cytometry. (H) Percentage of cells in each cell-cycle phase induced by GE. Results in bar graphs are represented as mean ± SE from three different triplicate experiments. *P<0.05 compared with the control.
Fig 2.
Effect of GE on G2/M-phase associated cell-cycle regulators and signaling pathways in EJ cells.
(A) Changes in cell cycle regulators by treatment of different concentrations of GE. Immunoblots were performed using specific antibodies indicated. The bar graphs were presented as a fold ratio to the control. (B, C) Effect of GE on MAPK (ERK1/2, JNK1/2, and p38) and AKT signaling. Phosphorylation levels of each molecule were assessed by immunoblots. Bar graphs were presented as fold changes compared to the control. (D) EJ cells were pre-incubated with U0126 (0.5 μM), SB203580 (10 μM), SP600125 (10 μM), and LY 294002 (10 μM) for 40 min prior to treatment with GE (800 μg/ml). The ratio of phosphorylated to non-phosphorylated form was measured and presented as fold changes compared to the control. Results in bar graphs are represented as mean ± SE from three different triplicate experiments. *P<0.05 compared with the control.
Fig 3.
GE inhibited the migration and invasion of EJ cells through diminished MMP-9 activity by suppressing binding activity of transcription factor AP-1, Sp-1, and NF-κB.
(A) Changes of migratory potential were assessed by scratch wound-healing assays. The cells were pre-treated with mitocycin C (5 μg/ml) for 2 h. The surface area of migrating cells was photographed by a phase contrast microscope. The recovery rate was measured as fold changes compared with the control. (B) Invasive capacity was measured using Matrigel®-coated transwell plates in GE-treated EJ cells. The cells were incubated with mitocycin C (5 μg/ml) for 2 h before the invasion assays. Cellular images were taken by a phase contrast microscope. The amount of invading cells was presented as a fold change relative to the control. (C) Gelatinase activity of MMP-2 and -9 were assessed by different concentrations of GE using zymography. Bar graph was presented as a fold change contrasted with the control. (D) Binding activity of transcription factor AP-1, Sp-1, and NF-κB, was measured by EMSA in GE-treated EJ cells. Bar graph was presented as a fold change compared with the control. Results in bar graphs are represented as mean ± SE from three different triplicate experiments. *P<0.05 compared with the control.
Fig 4.
Gene expression patterns in EJ cells treated with GE.
(A) Clustergram of differentially expressed genes by GE treatment was produced using microarray analysis. The red and green colors indicate high and low expression of particular genes, respectively. (B, C) Differentially expressed genes upon GE treatments were categorized by their biological processes (BP) and molecular functions (MF) through GO term enrichment analysis. (D) The expression of HSPA6 in response to GE treatment at different time points was measured by immunoblots. (E) Transfection efficiency of HSPA6 cDNA gene was evaluated by transient transfection compared to an empty vector (EV), pCMV. (F) After transfection of the HSPA6 gene, expression levels of HSPA6 were confirmed by immunofluorescence.
Table 1.
The 10 most up-regulated genes found with BP analysis following GE treatment.
Table 2.
The 10 most down-regulated genes found with BP analysis following GE treatment.
Table 3.
The 10 most up-regulated genes found with MF analysis following GE treatment.
Table 4.
The 10 most down-regulated genes found with MF analysis following GE treatment.
Table 5.
Up-regulated genes following GE treatment (800 μg/ml).
Fig 5.
HSPA6 potentiated the suppression of proliferation, migration, and invasion and the accumulation at G2/M cell-cycle phase in GE-treated EJ cells.
After transfection of either HSPA6 or an EV, cells were incubated with or without GE (800 μg/ml). (A) Effect of HSPA6 gene in GE-mediated inhibition of proliferation of EJ cells. Relative cell number was expressed as a fold change compared with the control. (B) Images of cellular morphology were photographed by a phase contrast microscope. (C) Effect of HSPA6 gene on GE-mediated inhibition of migration. After incubation of mitocycin C (5 μg/ml) for 2 h, wound recovery capacity was evaluated by comparing migrated area after 24 h. (D) Effect of HSPA6 on GE-mediated inhibitory activity against invasive potential of EJ cells. The cells were incubated with mitocycin C (5 μg/ml) for 2 h before the invasion assay. Cellular images were photographed by a phase contrast microscope after staining with crystal violet. (E-N) Effects of HSPA6 gene in GE-mediated G2/M-phase cell cycle distribution. GE-mediated G2/M cell-cycle phase distribution was evaluated by flow cytometric analysis. Percentage of cell populations in each cell-cycle phase was presented by bar graphs. Results in bar graphs are shown as a mean ± SE from three different triplicate experiments.*P < 0.05, compared with the control and **P < 0.05, compared with GE treatment.
Fig 6.
HSPA6 gene enhanced regulatory proteins participated in GE-mediated G2/M-phase cell cycle and early signaling pathways in EJ cells.
EJ cells were transfected with an EV (A, C) or HSPA6 (B, D) followed by incubation in the culture medium with or without GE. Cell cycle regulators and signaling molecules were assessed by immunoblots using specific antibodies indicated. Expression levels of proteins were normalized by corresponding total forms or GAPDH. *P < 0.05, compared with the control and **P < 0.05, compared with GE treatment.
Fig 7.
HSPA6 intensified GE-mediated inhibitory effect of MMP-9 activity via suppression of binding activity of AP-1, Sp-1, and NF-κB in EJ cells.
EJ cells were transfected with an EV (A, C) or HSPA6 (B, D) followed by incubation either in the presence or absence of GE. (A, B) Proteolytic activity of MMP-9 was assessed by gelatin zymography. (C, D) EMSA was performed to detect the binding activity of AP-1, Sp-1, and NF-κB using radiolabeled oligonucleotide probes. Unlabeled AP-1, Sp-1, and NF-κB oligonucleotides were used as competitors. Relative fold changes were indicated to compare against the control versus GE treatment. In each bar graph, results are presented as the mean ± SE from three different triplicate experiments.*P < 0.05, compared with the control and **P < 0.05, compared with GE treatment.