Human ESCC cell lines KYSE30, KYSE520, KYSE140 and KYSE410 and immortalized HEEC human normal esophageal epithelial cells were purchased from the Qingqi (Shanghai) Biotechnology Development Co., Ltd. The cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Shanghai ExCell Biology, Inc.) and 1% penicillin-streptomycin mixture (Beijing Solarbio Science & Technology Co., Ltd.) in a 37°C, 5% CO₂ incubator (Zhejiang Jiemei Electronic & Technology Co., Ltd.). The medium was changed every two days and cells were allowed to grow to the exponential phase for use in subsequent experiments.

KYSE30 cells at exponential culture stage were taken out of the cell incubator. The medium was discarded and the cells were washed with PBS three times. Digestion was performed in T25 culture flasks by adding 1 ml of trypsin solution (Beijing Solarbio Science & Technology Co., Ltd.) for 2–3 min. Subsequently, 2 ml of RPMI-1640 complete medium was added to terminate the digestion. The cells were collected in a centrifuge tube and centrifuged at 1,200 × g for 3 min. RPMI-1640 complete medium was added to resuspend the cells, and 10 µl of cell suspension was aspirated to count the number of cells using a cell counter (Countess™ 3; cat. no. AMQAX2000; Invitrogen; Thermo Fisher Scientific, Inc.). The cells were transferred to 96-well plates at a concentration of 3,000 cells per well and incubated overnight in a cell incubator at 37°C and divided into four groups which were treated as follows: i) Control (DMSO); ii) DEX-HCl (25, 50, 100 and 200 nmol/l); iii) SFC (1.25, 2.5, 5 and 10 µmol/l); and iv) DEX-HCl (25 nmol/l) and SFC (1.25 µmol/l) combined treatment. The 96-well plate was then incubated for 48 h. A total of 10 µl of CCK-8 solution (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well and incubated for 3 h. The absorbance at 450 nm was measured using an enzyme immunoassay analyzer (ReadMax 1,200; Shanghai Shanpu Biotechnology Co., Ltd.). IC₅₀ values were calculated using GraphPad Prism 9.5.0 (Dotmatics). DEX-HCI (cat. no. 22022431) was purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd., and SFC (cat. no. 21A09311) was purchased from Yichang Renfu Pharmaceutical Co., Ltd.

KYSE30 cells at the exponential growth phase were subjected to cell digestion and cell counting as described previously for the CCK-8 assay. The cells were resuspended at 150 cells/ml. A total of 1 ml of cell suspension was mixed with 1 ml of RPMI-1640 complete medium, transferred to a six-well plate and incubated overnight in an incubator at 37°C with 5% CO₂. The medium was replenished every 2 days for 14 days. The medium was then discarded, the cells were rinsed with PBS solution three times and then stained using 0.1% crystal violet (Beijing Solarbio Science & Technology Co., Ltd.) for 15 min, 25°C. The cells were washed three times with PBS, air-dried naturally and imaged with an SLR camera (Nikon D850; Nikon Corporation). The images were analyzed using ImageJ version 1.52a (National Institutes of Health) bundled with Java 8.

KYSE30 cells at the exponential growth phase were subjected to cell digestion and cell counting as described previously for the CCK-8 assay. The cell suspension was inoculated into 6-well plates at 5×10⁵ cells per well and incubated overnight in a cell culture incubator (37°C and 5% CO₂). Three parallel vertical lines were drawn across the bottom of the six-well plate with a 10 µl pipette tip. After discarding the medium, the cells were washed three times with PBS, and serum-free RPMI-1640 medium containing DEX-HCl (25 nmol/l) and SFC (1.25 µmol/l) drug solution was added to a 6-well plate and incubated in an incubator. The six-well plate was imaged at 0, 24 and 48 h using an inverted fluorescence microscope [Sunny Optical Technology (Group) Co., Ltd.] at the intersection of the horizontal and vertical lines to ensure that images were obtained from the same location at different time points. The cell migration rate of each group was analyzed by ImageJ version 1.52a (National Institutes of Health) bundled with Java 8.

Matrix-Gel™ Matrigel (cat. no. C0371; Beyotime Institute of Biotechnology) was diluted 1:8 with serum-free RPMI-1640 medium and spread on the upper part of the Transwell chamber, and then left to set at 37°C for 2 h. Culture medium was then added to hydrate the gel for 30 min before being discarded. KYSE30 cells at the exponential phase were collected and counted as described previously. The cell concentration was diluted to 1×10⁶ cells/ml with serum-free RPMI-1640 medium and 50 µl of cell suspension was added to the upper chamber of the Transwell. The upper chamber of the Transwell was supplemented with 50 µl of serum-free RPMI 1640 medium containing DEX-HCl and SFC to give a final drug concentration of 25 nmol/l DEX-HCl and 1.25 µmol/l SFC. 500 ul of RPMI 1640 medium containing 20% FBS was added to the lower chamber of the Transwell. The Transwell chambers were then incubated in a cell culture incubator at 37°C for 12 h. Subsequently, 500 µl of cell fixation solution (cat. no. P0099; Beyotime Institute of Biotechnology) was added to each chamber for 25 min at room temperature, then washed with PBS. Finally, the cells were stained with 0.1% crystal violet solution for 25 min at room temperature before being imaged using a microscope.

After adding DEX-HCI (25 nmol/l) or SFC (1.25 µmol/l) alone or in combination, KYSE30 cells were incubated in a cell incubator at 37°C for 24 h. Cells were then collected and transferred to a 1.5 ml centrifuge tube with a sterile cell scraper. Cells were resuspended in 1 ml of distilled water and sonicated (cat. no. E0380; Beyotime Institute of Biotechnology) at room temperature for 1 min (40 Hz; ultrasound for 5 sec followed by 5 sec pauses). The cell lysate was incubated for 10 min in a water bath at 4°C and centrifuged at 12,000 × g for 15 min. The experiments were carried out according to the manufacturer's protocols of each kit using a UV spectrophotometer (Cary 60 UV–Vis; Agilent Technologies, Inc.) to determine the ATP, lactate and glucose content. ATP (cat. no. S0026), glucose (cat. no. S0201S) and lactate (cat. no. C0016) kits were purchased from Beyotime Institute of Biotechnology.

The RO8191 reverse validation experiment was divided into four groups and were treated as follows: i) Control (DMSO); ii) RO8191 (1 µmol/l); iii) DEX-HCl (25 µmol/l) combined with SFC (1.25 µmol/l); and iv) combined treatment with DEX-HCl (25 µmol/l), SFC (1.25 µmol/l) and RO8191 (1 µmol/l). Different drugs were added to KYSE30 cells according to the requirements of the different groups, and the cells were incubated in a cell incubator at 37°C for 48 h, the cells were digested, centrifuged and resuspended as previously described. The cell samples were incubated with 4% paraformaldehyde at room temperature for 20 min, then incubated with 0.5% Triton X-100 for 20 min. Subsequently, 5% bovine serum albumin (cat. no. SW3015; Beijing Solarbio Science & Technology Co., Ltd.) was added and the cells were incubated for 1 h at room temperature. The samples were incubated with primary antibodies at 4°C overnight and the cells were then washed with 1% Tween TBST for 3 min. The primary antibodies used were: E-cadherin (cat. no. ET1607-75; 1:100) and N-cadherin (cat. no. ET1607-37; 1:100), which were purchased from Hangzhou HuaAn Biotechnology Co., Ltd. Fluorescently labeled secondary antibodies (cat. no. ZF-0511; 1:500; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) were added and incubated in the dark at room temperature for 1 h. The slides were incubated with DAPI in the dark for 5 min, sealed with an anti-fluorescence quencher (cat. no. P0131; Beyotime Institute of Biotechnology), imaged using a confocal microscope and analyzed using ImageJ version 1.52a (National Institutes of Health) bundled with Java 8.

After treating KYSE30 cells with DEX-HCI (25 nmol/l) or SFC (1.25 µmol/l) alone or in combination for 24 h, the cells were collected with a sterile cell scraper and transferred to a 1.5 ml centrifuge tube. The RNA was extracted using TRIzol (Beijing Solarbio Science & Technology Co., Ltd.), according to the manufacturer's instructions. Briefly, cells were resuspended in 1 ml of TRIzol and incubated at room temperature for 5 min. The RNA sample was used immediately or stored at −80°C. RNA concentration was measured using a Q5000 UV–Vis Spectrophotometer (Pono-550; Prebo Instruments (Hangzhou) Co., Ltd.). RNA was reverse transcribed (at 42°C for 15 min) using the FastKing gDNA Dispelling RT SuperMix kit (Tiangen Biotech Co., Ltd.). The reverse transcribed complementary DNA (cDNA) was used immediately or stored in at −80°C. The cDNA concentration was measured using a Dalong Gradient Thermal Cycler TC1000-G (Shaying Scientific Instruments (Shanghai) Co., Ltd.) and amplified in a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.,) using FastKing One Step RT-qPCR kit (SYBR Green) (Tiangen Biotech Co., Ltd.). The primers used for RT-qPCR are listed in Table I. The thermal cycling conditions used were: 50°C for 30 min; 95°C for 3 min; followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. By comparing the target gene Cq with the internal reference Cq, ΔCq for each group, and the fold difference was calculated by 2^−ΔΔCq (27). GraphPad Prism version 9.5.0 (Dotmatics) was used for statistical analysis.

DEX-HC (25 nmol/l), SFC (1.25 µmol/l) and RO819 (1 µmol/l) were added to KYSE30 cells according to the aforementioned grouping requirements and incubated in a cell incubator at 37°C for 48 h. The cells were then digested and centrifuged according to the aforementioned method. The RIPA solution (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) was added to the cell precipitate and incubated on ice for 30 min. The lysate was cleared by centrifugation at 12,000 × g for 10 min at 4°C. Protein quantification was performed with bicinchoninic acid (BCA) protein quantification kit. Protein samples (30 µg) were loaded on a 10% polyacrylamide gel and separated by SDS-PAGE for 35 min at 80 V, then 120 V for 60 min. Proteins were transferred to a PVDF membrane using the Trans-Blot^® Turbo™ Transfer System (Bio-Rad Laboratories, Inc.) for 1 h, with a constant current of 260 mA. The PVDF membrane was blocked using 5% skimmed milk powder for 2 h at room temperature, and then washed with TBST (0.1% Tween) for 30 min. The membrane was then incubated with the corresponding primary antibodies at 4°C overnight. The PVDF membrane was washed 3 times with TBST (0.1% Tween) for 30 min, then blocked with 5% skim milk powder and incubated with secondary antibodies for 1 h at room temperature. Images were processed and analyzed using ImageJ version 1.52a (National Institutes of Health) bundled with Java 8. Antibodies used were as follows: β-actin (cat. no. EM21002; HUABIO; 1:2,000), anti-JAK2 (cat. no. M1501-8; HUABIO; 1:1,000), p-JAK2 (cat. no. ET1607-34; HUABIO; 1:1,000), STAT3 (cat. no. ET1607-38; HUABIO; 1:1,000), p-STAT3 (cat. no. ET1603-40; HUABIO; 1:1,000), HIF-1α (cat. no. R1510-5; HUABIO; 1:1,000), MMP2 (cat. no. bs-4605R; BIOSS; 1:1,000), MMP9 (cat. no. bs-4593R; BIOSS; 1:1,000), E-cadherin (cat. no. ET1607-75; Hangzhou HuaAn Biotechnology Co., Ltd.; 1:100), N-cadherin (cat. no. ET1607-37; Hangzhou HuaAn Biotechnology Co., Ltd.; 1:100), HK2 (cat. no. A0994; ABclonal Biotech Co., Ltd.; 1:1,000), lactate dehydrogenase A (LDHA; cat. no. A1146; ABclonal Biotech Co., Ltd.; 1:1,000), HRP Goat anti-rabbit (cat. no. AS014; ABclonal Biotech Co., Ltd.; 1:10,000) and HRP Goat anti-mouse (cat. no. AS003; ABclonal Biotech Co., Ltd.; 1:10,000).

Statistical analysis was performed using SPSS 22.0 software (IBM Corp.). Measurement data are presented as mean ± standard deviation. All data were analyzed using one-way ANOVA, followed by Tukey's post hoc analysis. All parallel experiments were repeated three or more times. P<0.05 was considered to indicate a statistically significant difference.

The relative molecular masses and structural formula of DEX-HCI and SFC are presented in Fig. 1A and B. Among the four esophageal squamous cell carcinoma cell lines (KYSE30, KYSE520, KYSE140 and KYSE410), p-STAT 3 and p-JAK were more significantly up-regulated in KYSE30 cells compared with normal human esophageal epithelial HEEC, and thus KYSE30 was chosen for use in further experiments (Fig. S1). CCK-8 and clonogenic assays of KYSE30 cells treated with DEX-HCl (25, 50, 100 and 200 nmol/l) and SFC (1.25, 2.5, 5 and 10 µmol/l) in vitro showed a significant and concentration-dependent decrease in cell viability compared with the control group after 48 h of treatment (Fig. 1C and D). Compared with DEX-HCL or SFC alone, the combination treatment demonstrated enhanced inhibition of KYSE30 cell viability (Fig. 1E). The IC₅₀ was calculated to be 83.96 nmol/l for DEX-HCl and 2.173 µmol/l for SFC. In subsequent experiments concentrations <IC₅₀ values were used, and thus, DEX-HCl was used at 25 nmol/l and SFC was used at 1.25 µmol/l. To evaluate the effect of the co-administration of DEX-HCl and SFC, the technique previously described by Chou (28) was used. The combination index (CI) theory provides quantitative definitions of additive effect (CI, 1), synergistic effect (CI, <1) and antagonistic effect (CI, >1) in drug combinations. The smaller the value of CI, the stronger the synergistic effect of drugs. The CI of DEX-HCl and SFC was 0.27, demonstrating a strong synergistic effect between DEX-HCl and SFC (Fig. 1E). The results of the clonogenic assay demonstrated that DEX-HCl and SFC were able to inhibit the proliferation of KYSE30 cells and demonstrated enhanced inhibition of KYSE30 cells in combination compared with single dosing, which suggested that DEX-HCl and SFC were more effective in combination (Fig. 1F and G).

Wound healing and Transwell assays were performed to assess the effects of DEX-HCl and SFC on cell migration and invasion. The wound healing assay results demonstrated that KYSE30 cells treated with DEX-HCl (25 nmol/l) or SFC (1.25 µmol/l) for 24 and 48 h demonstrated an inhibited migration, and demonstrated a significantly greater inhibition after co-administration for both 24 and 48 h after treatment. Cells that were treated with DEX-HCl and SFC in combination only healed 10% after 24 h of treatment, as shown in Fig 2A. The wound healing assay results demonstrated that DEX-HCI and SFC had an inhibitory effect on cell migration, while the DEX-HCI combined with SFC group had a stronger inhibitory effect on cell migration compared with the DEX-HCI (25 nmol/l) or SFC (1.25 µmol/l) only groups. In the DEX-HCI + SFC group there was also a time-dependent effect, and the longer the migration time, the stronger the inhibition ability of migration (Fig. 2A). The Transwell migration assay demonstrated that the cell invasion ability of KYSE30 cells treated with DEX-HCl (25 nmol/l) or SFC (1.25 µmol/l) was significantly inhibited after 12 h compared with the control group. The combination of DEX-HCl and SFC significantly increased the inhibitory effect on the invasive ability of KYSE30 cells compared with the single treatment groups (Fig. 2B).

Changes were observed in ATP levels, lactate production and glucose uptake in KYSE30 cells after treatment with DEX-HCl and SFC. The results showed that ATP levels, lactate production and glucose uptake were significantly reduced in KYSE30 cells in the DEX-HCl group, SFC group and the combination group compared with the control group (Fig. 2C). The combination group showed significantly decreased ATP levels, lactate production and glucose uptake in KYSE30 cells compared with the DEX-HCl and SFC alone groups (Fig. 2C).

To investigate the mechanism of action, the expression of JAK/STAT3/HIF-1α pathway-related, invasion-related marker proteins and glycolysis-related marker proteins were assessed using qPCR and western blotting. The results of the qPCR and western blotting experiments demonstrated that both DEX-HCI and SFC were able to significantly reduce the expression and protein levels of HIF-1α, HK2 and LDHA. Additionally, the combination group of DEX-HCI and SCF inhibited the expression and protein levels of HIF-1α, HK2 and LDHA more strongly compared with that of the drug alone (Fig. 3A and B). It was also demonstrated that DEX-HCl and SFC treatment significantly decreased the protein expression levels of p-STAT3 and p-JAK compared with the control group. The co-administration of DEX-HCl and SFC demonstrated a further significant decrease in the protein expression levels of p-STAT3 and p-JAK compared with both the control and single treatment groups alone (Fig. 3C), without affecting the expression levels of STAT3 and JAK proteins. The protein expression levels of E-cadherin and N-cadherin were examined by immunofluorescence assay and western blotting and the results showed that DEX-HCl and SFC were able to significantly upregulate E-cadherin and significantly downregulate N-cadherin protein expression compared with the negative control in KYSE30 cells. SFC significantly reduced the expression levels of MMP2 and MMP9 proteins in KYSE30 cells. DEX-HCI significantly reduced the protein expression level of MMP9, but the addition of DEX-HCI could not significantly reduce the protein expression level of MMP2. The DEX-HCI + SFC group significantly reduced the expression level of the MMP2 protein compared with the DEX-HCI group and SFC group, which further indicated that DEX-HCI and SFC combined have a drug synergistic effect (Fig. 4A and B).

To further assess whether DEX-HCl and SFC effect KYSE30 cell glucose metabolism and invasion through the JAK/STAT3/HIF-1α axis, reverse validation experiments were performed by adding RO8191 to induce STAT3/JAK to undergo phosphorylation.

RO8191 is an imidazolino pyridine compound that activates phosphorylation of JAK and STAT proteins thereby activating the JAK/STAT signaling pathway (29). Therefore, recovery of the invasion and migration ability of KYSE30 with the addition of RO8191 would suggest that DEX-HCl and SFC exhibit their function via the JAK/STAT pathway. The addition of RO8191 in the Transwell migration assay counteracted the inhibitory effect of DEX-HCl and SFC treatment on KYSE30 cells, significantly increasing the number of invaded cells compared with the DEX-HCl and SFC treatment group (Fig. 5A). DEX-HCL combined with SFC was demonstrated to reduce the expression levels of HIF-1α, HK2 and LDHA mRNA, whereas the addition of RO8191 was demonstrated to upregulate the expression levels of HIF-1α, HK2 and LDHA mRNA, counteracting the inhibitory effect of DEX-HCL combined with SFC (Fig. 5B).

Glucose uptake, ATP levels and lactate production of KEYSE30 cells significantly increased after treatment with RO8191 compared with the DEX-HCl and SFC co-administration group (Fig. 5C). DEX-HCI combined with SFC significantly inhibited the expression levels of HK2, LDHA, p-JAK, p-STAT3, and HIF-1α proteins, whereas RO8191 co-administration increased the expression levels of HK2, LDHA, p-JAK, p-STAT3 and HIF-1α proteins, counteracting the effect of DEX-HCI combined with SFC (Fig. 5D and E). Likewise, the addition of RO8191 in immunofluorescence experiments showed a significant decrease in E-cadherin fluorescence intensity compared with the DEX-HCl and SFC treatment group and a significant increase in N-cadherin fluorescence compared with the DEX-HCl and SFC treatment group (Fig. 6A). The addition of RO8191 was also demonstrated to increase the protein expression levels of MMP2, MMP9 and N-cadherin and decrease the protein expression levels of E-cadherin compared with the DEX-HCl and SFC treatment group (Fig. 6B). In summary, DEX-HCI in combination with SFC was able to inhibit KEYSE30 cell invasion and inhibit JAK/STAT3/HIF-1α axis activation, and RO8191, as a JAK/STAT3 pathway activator, was able to reverse this effect, which reinforces the conclusion that the inhibitory effect of DEX-HCI in combination with SFC on KEYSE30 cell invasion was mediated by the inhibition of JAK/STAT3/HIF-1α axis activation. STAT3/HIF-1α axis to exert pharmacological effects.