Down-regulation of STAT3 induces the apoptosis and G1 cell cycle arrest in esophageal carcinoma ECA109 cells

The human esophageal carcinoma ECA109 cell line was obtained from ATCC (Manassas, VA, USA). Plasmid pSi-STAT3 is a human STAT3-specific shRNA expression vector and pSi-Scramble is a negative control plasmid. The plasmids were kindly provided by Dr. Xuejian Zhao (Jilin University, Jilin, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from the Hyclone (Logan, UT, USA). Opti-MEM reduced serum medium and Lipofectamine 2000 transfection reagent were purchased from Invitrogen Life Technology (Carlsbad, CA, USA). Annexin-V-FLUOS staining kit and complete protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Indianapolis, IN, USA). TUNEL assay kit was from Keygen Biotechnology (Beijing, China). Mitochondrial membrane potential assay kit with JC-1 and DNA ladder extraction kit were purchased from Beyotime Institute of Biotechnology (Haimen, JiangSu, China). Antibodies to GAPDH, c-Myc and horseradish peroxidase-labelled conjugated secondary antibodies to rabbit, mouse and goat primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against STAT3 and Caspase-3 (32 kDa) were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies of Cyclin D1, cleaved Caspase-3 (17 kDa) and enhanced chemiluminescence reagent were purchased from Pierce Biotechnology (Rockford, IL, USA). Transwell chambers were purchased from Corning Incorporated (New York, USA). RT-PCR was performed using the TaKaRa RNA PCR kit from Takara Biotechnology (Dalian, Liaoning, China). All other chemicals were analytical reagent grade.

The ECA109 cell line was maintained in DMEM with 10% (v/v) FBS at 37 °C, 5% CO₂. The cells were passaged around every 3 days. When cells grew to 90% confluence, they were digested and plated into 6-well plates and cultured for 14 h to get the 75% confluence before transfection. The ECA109 cells were transfected with plasmids of pSi-STAT3 or pSi-Scramble, respectively, using Lipofectamine 2000 according to the manufacturer’s instructions for 24–72 h before being analysed.

The change of the mitochondrial membrane potential (∆ψm) implies the early stage apoptosis. In this study, the alterations in ∆ψm were monitored under fluorescence microscopy using JC-1 dye. After transfected with pSi-STAT3 and pSi-Scramble for 24 h, the ECA109 cells were incubated with JC-1 for 20 min at 37 °C, 5% CO₂. After the incubation, the dye was removed and the cells were washed twice with JC-1 buffer and examined with a fluorescent microscope using both red and green channels.

The ECA109 cells were transfected with pSi-STAT3 or pSi-Scramble, respectively, for 24 h, then harvested and washed with PBS buffer. The genomic DNA was extracted according to the manufacturer’s protocol of DNA ladder extraction kit. DNA samples were subsequently examined with 2% (w/v) agarose gel electrophoresis. The gel was visualized using ethidium bromide staining under ultraviolet light.

The ECA109 cells transfected with plasmids were processed for TUNEL assays with one step apoptosis in situ assay kit. Briefly, cells were plated onto the slides, fixed with 4% paraformaldehyde for 30 min, and then washed with PBS buffer, incubated in TdT buffer containing fluorescein-dUTP for 1 h at 37 °C. Then cells were checked under fluorescence microscope. The nuclei of normal cells are stained in blue, while the apoptotic cells are red.

The effect of pSi-STAT3 on ECA109 cell apoptosis was examined using annexin V-fluorescein and propidium iodide (PI) according to the manufacturer’s manual. Briefly, ECA109 cells transfected with or without plasmids were collected after trypsinized and washed with PBS twice, resuspended with 100 μl annexin V-FLUOS labeling solution and incubated for 15 min at 25 °C. A flow cytometry system was used to analyze the apoptotic ECA109 cells. The data were analyzed using Cell Quest data acquisition and analysis software (BD, Franklin Laked, NJ, USA).

Distribution of ECA109 cells in various cell cycle phases was estimated using a flow cytometer by quantitation of DNA content of cells stained with PI. The ECA109 cells were harvested by trypsinization after transfected with the plasmids, then washed with chilled PBS containing 4 mmol/l EDTA, and fixed with 70% cold ethanol at 4 °C for 30 min. After fixation, the cells were collected by centrifugation and washed with PBS. Then the pellets were resuspended and incubated in PBS containing PI, Triton X-100 and RNaseA at 4 °C for at least 30 min. The cell cycle phase distribution was assessed by flow cytometry.

Cell migration was determined using transwell chambers. The ECA109 cells were transfected with plasmids for 72 h, then harvested with trypsinization. 1 × 10⁵ cells were plated in 200 μl serum-free DMEM medium on the top chamber, while 400 μl DMEM containing 10% FBS were plated in the lower chamber. After 14 h of incubation, the cells on the top surface were removed. The chambers were washed with PBS three times and the cells on the bottom surface were fixed in 4% paraformaldehyde for 15 min. After fixing at room temperature, the chambers were rinsed in PBS and stained with 0.2% crystal violet for 15 min. Then, the cells on the top chamber were removed, and the cells on the lower chamber were counted under the microscope.

For RNA extraction, the ECA109 cells were transfected with plasmids for 48 or 72 h in the 12-well plates. Cells were washed with PBS before 500 μl Trizol reagent was added, and collected into the tubes then put on ice in the Trizol for 5 min. Subsequently, 100 μl chloroform was added, and the cells were heavily shaked for 30 s, put at room temperature for 10 min. Then the samples were centrifuged at 12,000g for 15 min resulting in two phases. Following centrifugation, the upper layer of supernatant was collected and added equal volume of isopropanol. The samples were stored on ice for 10 min and then centrifuged at 12,000g for 30 min at 4 °C. The RNA pellet was washed with 75% ethanol twice and centrifuged at 12,000g for 5 min. The isolated RNA was air-dried and dissolved in DEPC treated water, and reversely transcripted to cDNA using primescript™ RT reagent kit. Real-time PCR was performed with SYBR^®premix ex Taq™ II, ROX plus reagent kit, conducted in step one plus™ real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The PCR program was initiated at 94 °C for 10 min, followed by 40 cycles of 90 °C 5 s, 60 °C 30 s, products were verified by melting curve analysis. The results were normalized to GAPDH and were calculated from threshold cycle numbers. Fold-changes in target gene mRNA expression were determined using ΔΔCt method. The same calculation formula as determined in the microarray analysis. The fold induction = 2^−ΔΔCt, where Ct is the threshold cycle number, and ΔΔCt = [Ct gene of interest (unknown sample) − Ct GAPDH (unknown sample)] − [Ct gene of interest (calibrator sample) − Ct GAPDH (calibrator sample)]. Sequences of the primers used for the test were as follows: MMP-9: forward, 5′-ACCTGGGCAGATTCCAAACCT-3′; reverse, 5′-CGGCAAGTCTTCCGAGTAGT-3′. uPA: forward, 5′-GAGAATTCACCACCATCGA-3′; reverse, 5′-GCTGCCTCCACACACGTAG-3′. SUSD2: forward, 5′-TCACTGGACAACGGCCAC-3′; reverse, 5′-CGTAGTATTGCCAACGCGTC-3′. GAPDH: forward, 5′-GCACCACCAACTGCTTAG-3′; reverse, 5′-GCAGGGATGATGTTCTGG-3′.

Values are presented as the mean ± standard errors (SE). Data analysis for comparison between treated groups and corresponding controls was performed using SPSS software (IBM, Armonk, NY, USA), and the data were analyzed with two-sample Student’s t test and ANOVA followed by the LSD post hoc test. P < 0.05 was considered to be statistically significant.

To investigate the biological functions of STAT3 downregulation, we utilized recombinant plasmid of shRNA to inhibit endogenous STAT3 in ECA109 cells. The pSi-STAT3 plasmid was specifically against human STAT3 and pSi-Scramble was as a control plasmid expressing non-silencing shRNA sequence. ECA109 cells were transfected with pSi-STAT3 or pSi-Scramble, respectively, for 72 h, the gene silencing effect of pSi-STAT3 was assayed using Western blotting. As shown in Fig. 1, the protein levels of STAT3 were obviously reduced to ~ 30% in pSi-STAT3 transfected cells compared with the control cells.

Morphological changes of cells are the basic phenomena of cell apoptosis. To investigate the effects of STAT3 knockdown to the ECA109 cells, the morphological changes of cells transfected with plasmids were observed with phase-contrast microscopy. As shown in Fig. 2, we could see that the pSi-STAT3 plasmid induced apparent morphological changes like cell shrinkage, apoptotic vacuoles, membrane blebbing and forming the floating cells which appeared after transfection for 48 h and got worse after 72 h. These indicated that knockdown of STAT3 could induce morphological changes of the ECA109 cells in a time-dependent manner. Conversely, the control cells transfected with pSi-Scramble and the untreated cells displayed normal morphology with polygonal shape, distinct cell borders and intercellular tight junction all the while.

JC-1 is a cationic mitochondrial vital dye exhibiting potential-dependent accumulation in mitochondria, as represented by a shift of fluorescence emission from red in normal polarized mitochondria to green in abnormal depolarized mitochondria. The ECA109 cells in the pSi-Scramble group and untreated group exhibited red cell staining which suggested that they had normal high membrane potentials. While, pSi-STAT3 treatment caused a significant loss of JC-1 red fluorescence, that is, loss of mitochondrial membrane potential after transfected with pSi-STAT3 for 24 h, which correlated to apoptosis (Fig. 3). The mitochondrial depolarization of ECA109 cells implied that STAT3 down-regulation caused early stage apoptosis.

The agarose gel electrophoresis of genomic DNA ladder and TUNEL assay were also performed to determine whether STAT3 down-regulation is involved in apoptosis of ECA109 cells. As shown in Fig. 4A, there was a typical pattern of DNA fragmentation like ladder appeared in ECA109 cells transfected with pSi-STAT3 but not in the pSi-Scramble and untreated groups. Consistently, pSi-STAT3 transfection significantly increased the number of TUNEL-positive apoptotic cells, but few or no apoptotic cells were seen in the pSi-Scramble treated or untreated groups (Fig. 4B). To further investigate the apoptosis of ECA109 cells induced by pSi-STAT3, we quantified the apoptosis cells by a flow cytometer using annexin-V and propidium iodide (PI) double-staining. Annexin-V can be detected in both the early and late stages of apoptosis. PI enters the cells in late apoptosis or necrosis. As shown in Fig. 4C, the percentage of apoptotic cells in the pSi-STAT3 group exhibited a significant rise compare with the pSi-Scramble group from (2.63 ± 0.47)% to (21.5 ± 0.33)% after 24 h of transfection. Further, the percentage was increased to (35.75 ± 4.28)% in the pSi-STAT3 group compared with (5.45 ± 0.61)% in the pSi-Scramble after 48 h of transfection. The untreated group, meanwhile, exhibited a low percentage of apoptotic at both times which was (1.21 ± 0.02)% at 24 h and (2.25 ± 0.09)% at 48 h. Based on the results, we assured that STAT3 down-regulation induced the apoptosis of ECA109 cells.

Cell cycle progression is a highly-ordered and tightly-regulated process which involves multiple checkpoints that assess extracellular growth signals, cell size and DNA integrity, hence the dysregulation of the cell cycle is one of the most frequent alteration during tumor development. To explore whether STAT3 down-regulation induced inhibition of cell growth had relation to the abnormal control of cell cycle, we evaluated the distribution of cell cycle by flow cytometry. As shown in Fig. 5, the percentage of ECA109 cells at G0/G1 phase was significantly higher in the pSi-STAT3 group than that in the pSi-Scramble group both 24 h and 48 h after transfection, (64.76 ± 0.57)% vs. (56.75 ± 0.88)% and (67.02 ± 0.94)% vs. (48.51 ± 0.65)%, respectively (Fig. 5). While, the untreated group and pSi-Scramble group showed no significant difference. Consequently, we confirmed that the inhibition of STAT3 had impact on the cell cycles of ECA109 cells, with the cell cycles being arrested at the G1 phase.

To further identify if the STAT3 down-regulation had effect on the migration of ECA109 cells, we detected the cell migration ability using transwell system assay. It was found that the migration was also decreased in the pSi-STAT3 group compared to the pSi-Scramble group after the cells transfected for 72 h (Fig. 6A). The real-time PCR analysis revealed that the mRNA levels of matrix metallopeptidase 9 (MMP-9), plasminogen activator urokinase (uPA) and sushi domain containing 2 (SUSD2) that related to migration and cancer progression were significantly reduced in the pSi-STAT3 group, compared with the pSi-Scramble group after 72 h of transfection (Fig. 6B). The consequences of transwell system assay and the real-time PCR analysis of genes related with migration and invasion demonstrated that the decline of ECA109 cells migration ability may correlate to the lack of STAT3.

It has been found that cancer cells harboring aberrant STAT3 activity had elevated levels of anti-apoptotic and cell cycle regulating proteins like cyclin D1 and c-Myc. In this study, the protein levels of Caspase-3, cleaved Caspase-3, c-Myc which related to apoptosis were further assessed. As shown in Fig. 7, the levels of c-Myc and Caspase-3 were significantly decreased in ECA109 cell transfected with pSi-STAT3 contrast to that in the pSi-Scramble group. Conversely, the level of cleaved Caspase-3 was increased markedly in the pSi-STAT3 group than that in the pSi-Scramble group. Cyclin D1, a G0/G1 phase associated protein, was also decreased in the pSi-STAT3 group compared to the pSi-Scramble group, which was consistent with the cell cycle analysis.