Qigesan inhibits esophageal cancer cell invasion and migration by inhibiting Gas6/Axl-induced epithelial-mesenchymal transition

Gas6 and Axl expression in normal and carcinoma tissues from ESCC patients

HE staining was used to examine morphology and positioning differences between normal esophageal and ESCC tissues. Cancer cells had large, irregular nuclei and low cytoplasmic ratios (Figure 1A). Immunohistochemistry showed that Gas6 was highly expressed in ESCC carcinoma tissues, but minimally expressed in adjacent normal tissues (Figure 1B), which is consistent with findings in other tumor tissues. Similarly, immunofluorescence indicated that Axl was highly expressed in ESCC carcinoma tissues and minimally expressed in adjacent ESCC tissues (Figure 1C). These results confirmed that Gas6 and Axl are highly expressed in EC tissues.

Figure 1. Gas6 and Axl expression in ESCC tissues. (A) HE staining; NC: nontumor esophageal tissue, ESCC: esophageal squamous cell carcinoma tissue. (B) Immunohistochemical assessment of Gas6 expression in NC and ESCC tissues. (C) Immunofluorescence assessment of Axl expression in NC and ESCC tissues; DAPI-labeled nuclei are in blue, and the merge imaged shows Axl and nuclear overlap. Scale bars indicate 50 μm.

QGS inhibits Gas6/Axl binding

In order to examine the effects of QGS on cellular localization and expression of Gas6, Axl, and Gas6/Axl complexes, immunofluorescence assays were performed using the ESCC cell lines ECA109 and TE13. Gas6, Axl, and Gas6/Axl complexes were highly expressed on the cell membranes of control group cells. In comparison, cell membrane Gas6/Axl complex expression was significantly reduced in QGS-stimulated cells; Gas6 and Axl expression also decreased in these cells in a concentration-dependent manner. These effects were observed to a similar degree in both cell lines (Figure 2A, 2B). Western blotting was used to examine the effects of QGS on Gas6 and Axl protein expression. Again, QGS treatment significantly reduced the expression of Gas6 and Axl in a concentration-dependent manner, and this effect was similar in both cell types (Figure 2C, 2D).

Figure 2. QGS regulates the localization and expression of Gas6/Axl. Cells were stimulated with QGS (0, 100, or 200 μg/mL) for 24 h. (A, B) Immunofluorescence images of Gas6, Axl, and Gas6/Axl channels in ECA109 and TE13 cells. Scale bars indicate 10 μm. (C, D) Western immunoblots of Gas6 and Axl. Bar graphs show significant dose-dependent inhibition of relative Gas6 and Axl protein density (normalized to β-act) after QSG stimulation compared to the unstimulated control group. Results are from three independent experiments. *p<0.05, **p<0.01, Gas6 relative protein density compared to control; ^&p<0.05, ^&&p<0.01, Axl relative protein density compared to control.

QGS inhibits EMT in ESCC cells

Immunofluorescence assays and confocal laser microscopy showed that E-ca was mainly expressed at relatively low levels on cell membranes and in the cytoplasm in the control group. QGS treatment increased E-ca expression on the cell membrane in a concentration-dependent manner to a similar degree in both ECA109 and TE13 cells (Figure 3A, 3D). N-ca and Snail were also mainly expressed on the cell membrane and in the cytoplasm. However, N-ca and Snail were highly expressed in the control group, and membrane expression of both gradually decreased with increasing concentrations of QGS in both ESCC cell lines (Figure 3B, 3C, 3E, 3F).

Figure 3A-F. QGS inhibits EMT in esophageal cancer cells. (A–C) Immunofluorescence images showing E-ca, N-ca, and Snail1 localization in ECA109 cells. QSG dose-dependently increased E-ca expression and inhibited N-ca and Snail1 expression. (D–F) Immunofluorescence images of E-ca, N-ca, and Snail1 in TE13 showing the same trend after QSG stimulation. Scale bars indicate 10 μm.
(G, H) Representative Western immunoblots. Bar graphs show significant dose-dependent increases in E-ca and decreases in N-ca and Snail1 relative protein density (normalized to β-act) after QSG stimulation compared to the unstimulated control group. Results are from three independent experiments. **p<0.01, E-ca relative protein density compared to control; &p<0.05, &&p<0.01, N-ca relative protein density compared to control; #p<0.05, ##p<0.01, Snail relative protein density compared to control.

Western blotting was used to examine the effects of QGS on E-ca, N-ca, and Snail protein expression. As QGS concentrations increased, E-ca protein expression increased and N-ca and Snail decreased in a dose-dependent manner. Although QGS-induced inhibition of N-ca was stronger in TE13 cells than in ECA109 cells, the direction of the effects was similar in both cell lines (Figure 3G, 3H). This indicates that QGS regulates the expression of three important EMT-associated proteins in EC.

Effects of QGS on microfilament arrangement in ESCC cells

The effects of QGS on microfilament arrangement in ESCC cells were observed using a laser confocal microscope. Microfilaments were arranged more regularly in ESCC cells stimulated with QGS; as QGS concentration increased, the cellular microfilament skeletons became more isotropic and cell morphology became increasingly regular. In contrast, microfilament arrangement was more irregular in the control group, especially in the cell membrane, where small outward-protruding pseudopods were common (Figure 4).

Figure 4. ECA109 and TE13 cytoskeleton and microfilament structures. After QGS stimulation, isotropic arrangement of microfilaments was enhanced in ECA109 and TE13 cells; microfilament arrangement was disordered in the control group. In addition, microfilament arrangements were more regular and numbers of pseudopods were reduced after 200 μg/mL of QGS. Scale bars indicate 10 μm.

QGS inhibits ESCC cell migration

A cell scratch test was performed to determine whether QGS inhibited migration of ECA109 and TE13 ESCC cells. In both cell lines, increasing QGS concentrations inhibited rates of cell migration into the scratch area in a time- and concentration-dependent manner; 200 μg/mL QGS resulted in significantly stronger inhibition than the 100 μg/mL dose (Figure 5). These results show that QGS can significantly inhibit the migration of ESCC cells.

Figure 5. QGS inhibits ESCC cell migration. Scratch areas were analyzed after ECA109 and TE13 cells were stimulated with QGS (0, 100, or 200 μg/mL). (A) Representative photomicrographs of unstimulated and QGS-stimulated cells at 0 h and 24 h. Red lines indicate migrating edges. (B) Bar graphs showing the percentage relative wound density (RWD) in stimulated cells compared to controls. Results are from three independent experiments; *p<0.05.

QGS inhibits invasion of ESCC cells

Next, cell invasion experiments were performed with ECA109 and TE13 cells to examine whether QGS inhibited ESCC cell invasion. Compared to the control group, the QGS groups in both cell lines exhibited a gradual concentration-dependent decrease in the number of ESCC cells passing through the transwell membranes; this effect was significantly stronger in the high-dose group (200 μg/mL) than in the low-dose group (100 μg/mL) (Figure 6). These results showed that QGS inhibited the invasive ability of ESCC cells.

Figure 6. QGS inhibits ESCC cell invasion. (A) Invasion assays were performed by placing ECA109 and TE13 cells treated with QGS (0, 100, or 200 μg/mL) in the transwell system for 24 h; the control is shown on the left. (B) Bar graphs showing number of invading cells for each cell line after 0, 100, or 200 μg/mL QGS. Combined results from three independent experiments are shown. *p<0.05, **p<0.01, compared to control.

Preparation and composition of QGS

QGS Granule Prescription was obtained from Shineway Pharmaceutical Co., Ltd. (Shijiazhuang, Hebei, China); its composition is based on the work of the famous Qing Dynasty doctor Cheng Zhongling [17, 21]. The production of all Granule Prescriptions meets GMP standards and has been approved by the Hebei Food and Drug Administration, and all botanicals have been certified as compliant with the Traditional Chinese Medicines Integrated Database (TCMID, http://119.3.41.228:8000/tcmid/search/) and The Plan List (http://www.theplantlist.org/). All Granule Prescription medicines were diluted in 100 mL of RPMI-1640 medium and filtered through a 0.22 μm syringe filter before use. The QGS concentrations used in this study were chosen based on the results of our previous experiments [17, 21].

Gas6 and Axl expression in ESCC and adjacent tissues

Twenty ESCC patients who provided cancer and paracancerous tissue samples were randomly selected for in-patient and outpatient examination in our hospital. All tissues were confirmed by senior pathologists. Paraffin-embedded sections of selected ESCC and adjacent normal tissues were stained with HE and used for tissue and cell morphological observations. Gas6 and Axl expression were examined using immunohistochemistry. First, paraffin sections were dehydrated, antigen repair was performed, endogenous peroxidases were quenched, and serum was used for blocking. Tissues were then incubated with anti-GAS6 primary antibody (67202, Cell Signaling Technology (CST)) and secondary antibody (G23301, Servicebio), nuclei were counterstained with DAPI, and tissues were mounted on slides and dehydrated. The intelligent high-content cell imaging analysis system Invitrogen EVOS FL Auto2 (Thermo Fisher Scientific, Waltham, MA, USA) was used to observe and photograph the tissues [22]. Axl expression was detected using an immunofluorescence method [23] which involved tissue section dewaxing, antigen repair, autofluorescence quenching, incubation with primary antibody (bs-5180R, Bioss) and fluorescent secondary antibody (GB21303, Servicebio), and DAPI counterstaining of nuclei. After slides were coverslipped, EVOS FL Auto2 was used for observation and image collection.

Reagents and antibodies

Newborn calf serum (NCS, Biological Industries, Beit Haemek, Israel), phosphate buffered saline (PBS, Biological Industries, Beit Haemek, Israel), and trypsin-EDTA digestion solution (Solarbio, Beijing, China), RPMI 1640 cell culture medium (Corning, Steuben County, New York, USA), and DAPI (D9542-1MG, Sigma Life Science, MO, USA) were used.

Cell lines and cell culture

Human ECA109 and TE13 ESCC cell lines were obtained from Shanghai Cell Bank, Stem Cell Bank of the Chinese Academy of Sciences and Hebei Cancer Institute (Shijiazhuang, Hebei, China) and passed genotype identification. Cells were placed in RPMI-1640 culture medium containing 10% NCS and cultured in a cell incubator with 5% CO₂ at 37°C.

Immunofluorescence staining

ECA109 and TE13 cells were seeded on coverslips in a 24-well plate at 2 × 10⁴ and 5 × 10⁴ cells/well, respectively, treated with QGS (0, 100, or 200 μg/mL) for 24 hours, and fixed with 4% paraformaldehyde. Cell membranes were then permeabilized with 0.3% Triton X-100. After blocking with 10% BSA serum, cells were incubated with the following primary antibodies at 4°C overnight: anti-GAS6 (67020, CST), anti-mouseAXL (ab89224, Abcam), anti-E-cadherin (ET1607-75, Hangzhou HuaAn Biotechnology Co., Ltd), anti-N-Cadherin (ET1607-37, Hangzhou HuaAn Biotechnology Co., Ltd), and anti-Snail1 (ER1706-22, Hangzhou HuaAn Biotechnology Co., Ltd). Cells were then incubated with the appropriate FITC or TRITC-conjugated secondary antibody (KLP) at room temperature for 1 hour. Unbound antibody was washed away, and the cells were either mounted on glass slides with mounting medium containing DAPI or placed directly in a confocal laser scanning microscope system (Leica Microsystems, Heidelberg, Germany). Cells were observed and images captured using EVOS FL Auto2.

Western blotting analysis

ESCC cells were seeded into cell culture flasks, and stimulated with QGS (0, 100, or 200 μg/mL) for 24 hours. Cells were then washed twice with prechilled PBS, harvested by scraping, centrifuged at 10,000 × g for 5 minutes at 4°C, and lysed on ice for half an hour with whole cell lysis buffer. The lysate was then centrifuged at 12,000 × g for 20 minutes, and the supernatant was collected and stored at -80°C for later use. Protein samples were separated using 4% - 12% NuPAGE Novex SDS gels and electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% bovine serum albumin (BSA Sigma St. Louis, MO) and incubated at room temperature for 2 hours. The membranes were then incubated the with the following primary antibodies at 4°C overnight: anti-GAS6 (67202, CST), anti-rabbit AXL (8661, CST), anti-E-cadherin (ET1607-75, Hangzhou HuaAn Biotechnology Co., Ltd), anti-N-cadherin (ET1607-37, Hangzhou HuaAn Biotechnology Co., Ltd), anti-Snail1 (ER1706-22, Hangzhou HuaAn Biotechnology Co., Ltd), and anti-β-ACT (AC026, Abclonal Technology). A fluorescent dye-labeled secondary antibody was then added to the membrane and incubated at room temperature for 1.5 hours. The membrane was washed three times with Tris-buffered saline (Sigma) and Tween-20 (Sigma) for 10 minutes, and then imaged using the Odyssey infrared imaging system (LI-COR Biosciences). Protein levels were calculated as the ratio of protein band intensity to β-act intensity. Each experiment was repeated three times.

Cytoskeleton experiment

ECA109 and TE13 ESCC cells were seeded on round glass slides in a 24-well plate at 3 × 10⁴ cells/well. After the cells had adhered, they were treated with QGS (0, 100, or 200 μg/mL) for 24 hours, washed 3 times with PBS, fixed with 4% paraformaldehyde for 20 minutes at room temperature, and washed 3 times with 1 × PBS. Phalloidin (1:1000, P5282, Sigma, USA) was then added to each well, followed by incubation at 37°C in the dark for 1 hour. Finally, the slides were coverslipped using mounting medium containing DAPI (Prolong Gold Antifade Reagent with DAPI, 8961, CST). A Leica Microsystems (Heidelberg, Germany) microscope was used to observe and take photos.

In vitro cell scratch assay

ECA109 and TE13 cell migration and invasion were analyzed using IncuCyte ZOOM according to the manufacturer’s instructions (Essen Bioscience, Hertfordshire, UK). Briefly, 4 × 10³ ECA109 or TE13 cells/well were seeded in a 96-well culture plate at 37°C and placed in a 5% CO₂ incubator overnight. Scratches were then made with a scratcher (Essen Bioscience), and the ESCC cells were washed twice in medium and stimulated with QGS (0, 100, or 200 μg/mL) for 24 h. The cell culture plates were placed on the IncuCyte ZOOM system and images of the scratched areas (10 × magnification) were taken at 0 and 24 h. The relative scratch density (RWD) value provided by IncuCyte software represents the rate of wound closure; an RWD value of 0 indicates that cell confluence at the wound surface is equal to cell confluence outside the wound surface. Changes in cell density tended to be consistent between experiments.

Cell invasion assays

A transwell system (BD Biosciences, San Jose, CA, USA) was used to detect tumor cell invasiveness [24]. Cells were stimulated with QGS (0, 100, or 200 μg/mL) for 24 h. A 40 μL polycarbonate membrane was then coated with basement membrane matrix (1.5 mg/mL; BD Biosciences, San Jose, CA) at 37°C for 4 h to reconstitute the basement membrane. 100 μL of bovine serum-free cells (1 × 10⁶/mL) were added to the upper chamber of the Transwell chamber, and 0.5 mL of culture medium containing bovine serum was placed in the lower chamber. After incubation overnight at 37°C with 5% carbon dioxide, a cotton swab was used to gently scrape the cells from the upper chamber. The transwell chamber was placed in a new plate, and the upper and lower chambers with their attached contents were fixed with 4% paraformaldehyde for 20 minutes and stained with crystal violet for 30 minutes. After washing with PBS, cells were observed and photos were taken under a microscope.

Statistical analysis

SPSS 21.0 software (SPSS, Inc. Chicago, USA) was used for statistical analysis of experimental results. Data are represented as the means ± standard deviation, and differences between groups were analyzed using One Way ANOVAs and independent sample t-tests. P < 0.05 indicated statistically significant differences.