Apatinib inhibits tumour progression and promotes antitumour efficacy of cytotoxic drugs in oesophageal squamous cell carcinoma

Abstract Apatinib, a highly selective inhibitor of vascular endothelial growth factor receptor‐2 (VEGFR‐2), inhibits the angiogenesis of tumours. The function and mechanism of apatinib in oesophageal squamous cell carcinoma (ESCC) remain unknown. In present study, we found that the development of ESCC in patients was controlled by treatment of combination of apatinib and a chemotherapeutic drug. Moreover, apatinib efficiently promotes cell apoptosis, inhibits cell proliferation, invasion, epithelial‐mesenchymal transition (EMT) and activity of the Akt/mTOR pathway in ESCC cells. Western blot analysis showed that apatinib significantly increased vimentin protein levels, decreased Bcl2, matrix metalloproteinase 9 (MMP9), E‐cadherin, p‐Akt and p‐mTOR protein levels in ESCC cells. Furthermore, apatinib enhanced chemosensitivity of cytotoxic drugs paclitaxel (TAX), 5‐fluorouracil (5‐FU) and cisplatin (DDP) by upregulating expression of vimentin protein, and downregulating expression of Bcl2, MMP9 and E‐cadherin protein in vitro. Compared with single‐agent groups, the combination of apatinib with each chemotherapeutic drug significantly repressed tumour growth and angiogenesis through blocking the expression of Ki67 and VEGFR‐2 in vivo. Taken together, apatinib efficiently inhibits cell growth through blocking Bcl2 and Akt/mTOR pathway, and suppresses metastasis via inhibiting MMP9 and EMT in ESCC cells. Apatinib promoted antitumour effect of chemotherapeutic agents through promoting cell apoptosis and inhibiting EMT and angiogenesis in ESCC.


| INTRODUC TI ON
Oesophageal carcinoma (ESCA) is one of the most common digestive tract malignant tumours in the world. 1 Oesophageal squamous cell carcinomas (ESCC) is the main pathological type in China, especially in Henan province where there is a higher incidence. ESCC in its early stages often affects its functions only slightly, with no obvious symptoms. When they have clinical manifestations, most of the patients are in the advanced stage and have missed the chance of surgical treatment. Chemotherapy is an essential method to advanced ESCC. However, the clinical efficacy of chemotherapy was limited, and the overall survival (OS) of patients with first-line treatment is only 5-10 months. Recurrence, metastasis and resistance to current chemotherapy are still the primary causes of death in ESCC patients.
Apatinib is a small molecule tyrosine kinase inhibitor of VEGFR-2. 2 It highly selective competes for the ATP binding site of VEGFR-2 so that blocks downstream signal transduction and inhibits the production of tyrosine kinase, which inhibits the process of tumour angiogenesis. 3 It has been reported that apatinib has antitumoral effect on a few malignancies. [4][5][6][7] Apatinib prolongs median overall survival (mOS) and progression-free survival (mPFS) of patients with advanced gastric cancer. 4 Lower daily doses of apatinib have greater benefits than higher daily doses in advanced gastric cancer patients. 8 Apatinib also has reliable efficacy in advanced non-small-cell lung cancer (NSCLC) patients and chemotherapyrefractory metastatic colorectal cancer. 6,9 Furthermore, apatinib enhances antitumour effect of chemotherapeutic drugs on several cancers. [10][11][12][13] Qin et al. found the synergistic effect between apatinib and the combination of the chemotherapy drugs TAX, oxaliplatin (L-OHP), 5-FU in gastric cancer. 14 Another study confirmed that apatinib inhibited tumour growth in cervical cancer and synergizes with TAX. 15 Moreover, it is reported that the combination of apatinib and docetaxel may prolong survival of patients with advanced oesophageal carcinoma. 16 However, the mechanism of apatinib in oesophageal cancer and the mechanism of synergistic antitumoral effect of apatinib combing with cytotoxic drugs in oesophageal cancer are not clear.
Previous studies have revealed that Akt/mTOR signalling pathway plays an important role in progression of cancers. Akt/mTOR pathway regulates cell growth and metastasis of cancer cells, including ESCC cells. Additionally, apatinib promotes apoptosis and autophagy through inactivating STAT3 pathway, and inhibits invasion and EMT in a few cancer. [17][18][19] In present study, we found that the development of ESCC in patients was controlled by treatment of combination of apatinib and a chemotherapeutic drug. Then, we explored the function and mechanism of apatinib in the treatment of oesophageal cancer. We assessed the effect of cytotoxic drugs TAX, 5-FU and DDP with or without apatinib on oesophageal cancer in vitro and in vivo. The results demonstrate that apatinib enhances antitumour efficacy of cytotoxic drugs through promoting cell apoptosis and inhibiting EMT in ESCC cells, suppresses tumour growth and angiogenesis via blocking Ki67 and VEGFR-2.

| Patients and treatment
Two patients were enrolled in our study. Case 1 was diagnosed with ESCC and retroperitoneal lymph node metastasis, and case 2 was diagnosed with ESCC and mediastinal lymph node metastasis. They never received chemotherapy, radiotherapy or surgery. All of the protocols used in this study were approved by the Medical Research Ethics Committee of our Hospital. The informed consent has been signed.

Patients received treatment of Nedaplatin and Gimeraciland
Oteracil Porassium Capsules. Nedaplatin 80 mg/m 2 (intravenously guttae, ivgtt) was given on Day 1; Gimeraciland Oteracil Porassium capsules were given 60 mg (P.O.) twice a day in first 14 days. If new lesions occur, they were treated with the combination of apatinib and docetaxel. Apatinib was administered 500 mg (P.O.) once a day continuously; docetaxel 75 mg/m 2 (ivgtt) was given on Day 1.

| Bioinformatic analysis
The expression of VEGFR-2 in patients with ESCA and normal was detected by bioinformatic algorithms from the publicly available databases TCGA and GEPIA. The cut-off was defined; the overall survival (OS) of high level and low level of VEGFR-2 group was predicted by GEPIA. The cut-off value was determined; the overall survival (OS) of the high and low levels of VEGFR-2 in ESCA patients was predicted by GEPIA.

| Agents
Apatinib, 5-FU, TAX and DDP were used in the present study. In vitro studies, the stock solution containing apatinib (Hengrui), 5-FU (Sigma), TAX (Sigma) and DDP (Sigma) were prepared in dimethyl sulfoxide (DMSO). In vivo assays, apatinib was dissolved in DMSO and diluted in physiological saline. 5-FU, TAX and DDP injection were diluted in physiological saline.

| Cell culture
Oesophageal squamous cell carcinomas cell lines (KYSE450, EC1) were preserved in our laboratory. They were cultured in RMPI 1640 medium (Hyclone) supplemented with 10% foetal bovine serum (Hyclone) and 1% penicillin-streptomycin at 37°C with 5% carbon dioxide. The cells were passed on every other day, and the cells of logarithmic growth phase were collected for subsequent experiments. Then, the experiment is divided into 8 groups: control group (DMSO), 5-FU group, TAX group, DDP group, apatinib group, Apatinib + 5-FU group, apatinib + TAX group and Apatinib + DDP group. ESCC cells (KYSE450 and EC1) were maintained in 96-well plates (5000 cells/well) and treated with agents. After 48 h, cell viability was determined by CCK-8 assay.

| Cell apoptosis assay
Flow cytometry was performed to detect cell apoptosis using an

| Western blotting
Protein was extracted from oesophageal cancer cells using RIPA lysis buffer supplemented with protease inhibitor and phosphatase inhibitor. Then, separating total proteins was performed using gel electrophoresis, and transferring proteins to a polyvinylidene fluoride membrane (PVDF; Beyotime) was performed using electroblotting. The membranes were immersed in 5% skim milk in TBST for 1 h and incubated in the following primary antibodies: rabbit anti-

| Wound-healing assay
Cell migration ability was measured by a wound-healing assay. Cells were planted in 6-well plate and treated with agents. We cultivated cells (KYSE450 and EC1) in 6-well plates until their density become 80% conflux. Then, cells were wounded with an aseptic 10 µl plastic pipette tip and treated with agents. Then, images were captured before and after culturing for 48 h.

| Transwell invasion assays
Cell invasive ability was measured by a 24-well transwell plate (Corning). Transwell chamber was placed in a 24-well plate and added with matrigel in a volumetric dilution of 1: 6. Cells (KYSE450 and EC1) were resuspended with serum-free medium contained agents and moved them into the upper chamber, added 600 µl culture medium with 20% FBS to the lower chamber. Cells were incubated at 37°C for 24 h, fixed with 4% paraformaldehyde for 20 min, washed with PBS and then stained with 1% crystal violet for 30 min.
Non-migrated cells on the upper surface of the film were erased by cotton swabs, and migrated cells on lower surface of the film were counted.

| Mouse xenograft model
EC1 cells (1 × 10 7 /100 μl/site) were injected into the right back side flanks of 5-week-old female BALB/c nude mice, and tumours were allowed to grow to 100 mm 3 . Then, the mice were randomly divided into 8 groups (n = 6 per group): control group, 5-FU group, TAX group, DDP group, apatinib group, 5-Fu + apatinib group, TAX + apatinib group and DDP + apatinib group. Mice were administered apatinib alone by oral gavage; 5-FU, TAX and DDP by intraperitoneal injection; or apatinib in combination with each cytotoxic drug at the indicated dose and schedule (Table 1). Tumour volumes were measured every 3 days. After 14 days treatment, the tumours were removed and measured.

| Tunel
Sections were dewaxed in xylene for 5-10 min and then dewaxed in fresh xylene for 5-10 min. Sections were placed in absolute ethanol for 5 min, 90% ethanol for 2 min, 70% ethanol for 2 min and distilled water for 2 min. 2% DNase-free proteinase K was added into tissues and incubated at 37°C for 20 min, then washed them with PBS three times. The samples were blocked with 5% serum at 37°C

| Immunohistochemistry
Tumour tissues were paraffin-embedded and sliced into 4μm thick

| Statistical analysis
SPSS (version 21.0; SPSS, Inc.) was used to analyses the data. The data among the groups were detected by one-way analysis of variance (ANOVA) and shown as the mean ± SEM. p < 0.05 was regarded as statistical significance.

Patient 1 received treatment of Nedaplatin and Gimeraciland
Oteracil Porassium Capsules for four cycles. New lesions were found in the patient's stomach. Then, the combination of apatinib and docetaxel was used to treat the patient for six cycles. We found the range of stomach lesions was decreased. Patient 2 received treatment of Nedaplatin and Gimeraciland Oteracil Porassium Capsules for two cycles. We found new lesions in right lung of the patient.
Then, the lower oesophageal lesion was reduced through the combination of apatinib and docetaxel for six cycles. Computed tomography (CT) was used to detect the lesions before and after treatment with combination of apatinib and chemotherapeutic drug (Figure 1).

| The ESCA patients with high level of VEGFR-2 have poor prognosis
Apatinib is a small molecule tyrosine kinase inhibitor of VEGFR-2.
To explore the function and mechanism of apatinib in ESCC, we analysed the expression of VEGFR-2 in the tissue database of ESCA patients. Although there is no statistical significance, we found that the levels of VEGFR-2 were upregulated in ESCA patients compared with normal in TCGA database through the bioinformatic analysis (Figure 2A). Moreover, the VEGFR-2 expression in stage Ⅲ and stage Ⅳ was higher than that in stage Ⅰ and stage Ⅱ ( Figure 2B). After defining the cut-off value, we analysed the OS of ESCA patients with VEGFR-2 overexpression or downregulation. The results showed that the OS of ESCA patients with low level of VEGFR-2 was longer than that of patients with high level of VEGFR-2 ( Figure 2C).

| Apatinib inhibits cell proliferation and promotes cell apoptosis in ESCC cells
To explore the role of apatinib in ESCC cell proliferation, we treated

| Apatinib inhibits cell migration, invasion and EMT in ESCC cells
KYSE450 and EC1 cells were wounded and treated with apatinib.
We calculated the healing rates and found that apatinib inhibited migration of KYSE450 and EC1 cells ( Figure 4A,B). Moreover, we detected invasion ability of KYSE450 and EC1 cells treated with apatinib or DMSO via transwell chamber assay. We found that the number of invasive cells treated with apatinib was less than control group ( Figure 4C,D). Western blot analysis showed that apatinib decreased the levels of MMP9 and vimentin, and increased the levels of E-cadherin in KYSE450 and EC1 cells ( Figure 3D).

| Apatinib reduces activity of the Akt/ mTOR pathway
A relevant research has shown that the activation of Akt/mTOR signalling pathway is vital to tumour development, progression and prognosis in ESCC cells. In this study, we investigated whether apatinib inhibits cell growth of ESCC by blocking Akt/mTOR signalling pathway.
Western blotting analysis showed that apatinib inactivated p-Akt and p-S6 protein expression in ESCC cells ( Figure 5A). To further confirm the results, we, respectively, treated ESCC cells with Akt pathway inhibitor (TCN-P)/mTOR pathway inhibitor (rapamycin), apatinib and both agents. Western blotting analysis showed that p-Akt and p-S6 protein expression was down-regulated in ESCC cells treated with TCN-P or apatinib, and apatinib enhanced the effect of Akt pathway inhibitor ( Figure 5B). Similarly, apatinib promoted the inhibition influence of the rapamycin ( Figure 5C). These results indicated that apatinib suppressed cell growth of ESCC by blocking Akt/mTOR signalling pathway.   Figure 7C,D).

| The combination of apatinib with cytotoxic drugs inhibits migration, invasion and EMT of ESCC cells
KYSE450 and EC1 cells were wounded and treated with apatinib combining with 5-FU or TAX, or DDP. After 48 h, we calculated the healing rates and found that apatinib or cytotoxic drugs alone inhibited the migration of KYSE450 and EC1 cells. Combining apatinib with each cytotoxic drug could more significantly inhibit migration of two cell lines ( Figure 7A). After treating KYSE450 and EC1 cells with apatinib and cytotoxic drugs for 24 h, invasion ability of cells was detected by transwell chamber assay. We found that the number of invasive cells treated with apatinib or each cytotoxic drug alone was less than the control group. The number of invasive cells treated with combination of apatinib and each cytotoxic drug was less than single treatment groups ( Figure 7B). Western blot analysis showed that the levels of MMP9 and vimentin in oesophageal cancer cells treated with apatinib and cytotoxic drugs were significantly decreased, and E-cadherin expression was increased ( Figure 7C,D).

| The combination of apatinib with cytotoxic drugs inhibits the growth of oesophageal tumour in vivo
To further access the antitumour function of combination of apatinib and cytotoxic drugs, we used 5-week-old female BALB/c nude mice to establish tumour xenografts model. Mice were treated with single agent, combination of apatinib and each cytotoxic drug. The results showed that each agent alone inhibited tumour growth compared with

F I G U R E 4 Apatinib inhibited migration and invasion of ESCC cells. (A and B)
Wound-healing assays showed that apatinib remarkably decreased KYSE450 and EC1 cells migration (×100). (C and D) Transwell assays showed that apatinib remarkably decreased KYSE450 and EC1 cells invasion (×400). Data are shown as mean ± SEM (*** p < 0.001) the control group ( Figure 8A-C). The combination of apatinib with each cytotoxic drug displayed synergistic inhibition effects on the growth of tumours compared with each treatment alone ( Figure 8A-C).
Additionally, Tunel results showed that apoptosis rates of oesophageal tumour increased after each agent treatment compared with control group, which were significantly enhanced in two agent treatment groups compared with each agent treatment groups ( Figure 8D).
Immunohistochemistry results showed that the Ki67 protein level was significantly reduced in the combination of apatinib and each cytotoxic drug groups compared with that in each agent groups ( Figure 8D). Moreover, the expression levels of VEGFR-2 in combination of apatinib with each cytotoxic drug groups were lower than that in one agent groups ( Figure 8D). These data demonstrated that apatinib markedly suppressed tumour angiogenesis, and the combination of apatinib with each cytotoxic drug had better effect on anti-angiogenesis in ESCC.

| DISCUSS ION
Local recurrence and distant metastasis after surgery are the main death causes in ESCC patients. Tumour angiogenesis plays an important role in tumour growth and metastasis, which is a complex process and predominantly regulated by vascular endothelial growth factor (VEGF) and VEGFR. 20 Bioinformatic analysis results showed that VEGFR-2 expression was upregulated in ESCC patients compared with normal. The expression of VEGFR-2 in advanced stage patients was higher than that in early stage patients. Additionally, high VEGFR-2 expression was a predictor of poor prognosis for patients with ESCC. Apatinib promotes tumour angiogenesis via binding to VEGF-A. 21 Apatinib competitively inhibits the binding of VEGF to VEGFR-2 and VEGFR-2 autophosphorylation by binding to VEGFR-2, which exerts significant antitumour effect. 22 In our study, the two ESCC patients were found to have distant metastases. Then, the data demonstrated that the combination of apatinib and chemotherapy can control the progression of ESCC.
Moreover, a retrospective study showed that apatinib combined with docetaxel can prolong the survival of patients with advanced ECA. 16 Therefore, apatinib is a potential target drug for ESCC.
Previous studies have reported that apatinib inhibited the progression of several malignancies. 23,24 Apatinib inhibited cell proliferation, migration and invasion in cancers, such as hepatocellular carcinoma (HCC), colon cancer, osteosarcoma and glioma. [17][18][19]25,26 To investigate the role of apatinib in ESCC cells, we treated ESCC cells with different density of apatinib. We found that apatinib EMT in osteosarcoma. 17 These results indicate that apatinib inhibited the progression of malignant neoplasms, including ESCC ( Figure 8E).
Akt/mTOR pathway is usually activated in a variety of malignancies. Emerging evidence suggests that Akt/mTOR signalling pathway affect tumour cells function, including cell proliferation, apoptosis, cell cycle, invasion, autophagy and angiogenesis. 25,27,28 In this study, we confirmed that apatinib inhibited cell growth of ESCC cells through suppressing p-Akt and p-S6 expression. Moreover, apatinib  of apoptotic cells of oesophageal tumour in two agent treatment groups was significantly higher than one agent treatment groups.
Immunohistochemistry analysis indicated that angiogenesis-related proteins (VEGFR-2) expression and prognostic biomarker (Ki67) were inactivated by apatinib combined with cytotoxic drugs compared with single agent. These data indicated that the combination of apatinib and cytotoxic drugs had synergistic antitumour efficacy on ESCC ( Figure 8E). However, the mechanism of the synergistic antitumour effect of apatinib combined with chemotherapy drugs on ESCC needed to be further explored.
In summary, we found that the combination of apatinib and a che-

ACK N OWLED G EM ENT
Not applicable.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.