Gastric cancer is the fourth leading cause of cancer deaths worldwide. Most patients are diagnosed in the advanced stage, and inadequate therapeutic strategies and the high recurrence rate leads to the poor 5-year survival rate. Therefore, effective chemoprevention drugs for gastric cancer are urgently needed. Repurposing clinical drugs is an effective strategy for cancer chemoprevention. In this study, we found vortioxetine hydrobromide, an FDA-approved drug, is a dual JAK2/SRC inhibitor, and has inhibitory effects on cell proliferation of gastric cancer. Computational docking analysis, pull-down assay, cellular thermal shift assay (CETSA) and in vitro kinase assays were used to illustrate whether vortioxetine hydrobromide directly bound to JAK2 and SRC kinases and inhibited their kinase activities. The results of non-reducing SDS-PAGE and Western blotting indicated that vortioxetine hydrobromide suppressed STAT3 dimerization, nuclear translocation and transcriptional activity. Furthermore, vortioxetine hydrobromide inhibited the cell proliferation dependent on JAK2 and SRC gene expression and suppressed the growth of gastric cancer PDX model in vivo. These data demonstrated that vortioxetine hydrobromide, as a novel dual JAK2/SRC inhibitor, curbed the growth of gastric cancer in vitro and in vivo by JAK2/SRC-STAT3 signaling pathways. It highlights that vortioxetine hydrobromide has the potential application in the chemoprevention of gastric cancer.
Article
Vortioxetine hydrobromide inhibits the growth of gastric cancer cells in vivo and in vitro by targeting JAK2 and SRC
https://doi.org/10.21203/rs.3.rs-1628199/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 05 May, 2023
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Gastric cancer (GC) requires over one million new cancer cases in 2020 and an estimated 769,000 cancer-related deaths (1). Most patients (> 70%) developed to an advanced stage due to a lack of specific signs (2). Chemoprevention is a promising strategy to improve GC patient 5-year survival rate. At present, accumulating evidence has illustrated that antipsychotics, anti-inflammatory drugs and antidepressants execute chemopreventive activities in GC (3, 4). NSAIDs, particularly aspirin, have been investigated extensively in gastric cancer chemoprevention, by inhibiting COX-1 and COX-2 production (3). Fluoxetine induced apoptosis and autophagy in gastric cancer cells (5). Therefore, screening effective chemoprevention drugs is of great significance for gastric cancer chemoprevention.
Tyrosine-protein kinase 2 (JAK2) and Proto-oncogene tyrosine-protein kinase (SRC) are non-receptor protein tyrosine kinases. JAK2, as a signaling hub, integrates extracellular signals from oncogenic receptor tyrosine kinases and interleukin receptors to signal transducer and activator of transcription 3 (STAT3) (6). SRC promotes cell proliferation, differentiation, survival, migration, invasion and angiogenesis by regulating Ras/Raf/ERK1/2, PI3K/Akt and STAT3 signaling pathways (7, 8). JAK2 and SRC have emerged as important therapeutic targets for several cancers including head and neck squamous cell carcinoma tumor, breast cancer, lung cancer and gastric cancer (7, 9–13). Therefore, JAK2/SRC-based targeted therapies would have broad therapeutic potential for GC treatment or chemoprevention.
Repurposing clinical drugs is a time-saving way of developing chemoprevention drugs with higher efficacy and fewer side effects. Our research group's previous studies have proved that repurposed FDA-approved drugs can be used for the chemoprevention of esophageal squamous cell carcinoma (ESCC) (14–16). Nuplazid suppresses ESCC growth by targeting PAK4. Tegaserod Maleate inhibits the ESCC cell proliferation through suppressing the peroxisome pathway. Cloperastine constrains ESCC growth via mitochondrial oxidative phosphorylation.
In this study, we found that vortioxetine hydrobromide, is a dual JAK2/SRC inhibitor and suppresses the growth of gastric cancer cells in vivo and in vitro.
Vortioxetine hydrobromide inhibited GC cell proliferation in vitro
The chemical structure of vortioxetine hydrobromide is shown in Fig. 1A. The half maximal inhibitory concentration (IC50) values of vortioxetine hydrobromide in HGC27 and AGS cells were 5.901 µM and 9.401 µM at 24 h, and 4.961 µM and 6.563 µM at 48 h (Fig. 1B). To determine the effects of vortioxetine hydrobromide on GC cell proliferation, HGC 27 and AGS cells were treated with different doses of vortioxetine hydrobromide (0, 0.5, 1, 2 and 4 µM). The proliferation inhibition rates of HGC27 and AGS were 66% and 45% after treatment with 4 µM vortioxetine hydrobromide for 96 h (Fig. 1C). Next, we investigated whether vortioxetine hydrobromide could inhibit anchorage-independent growth and clone formation of GC cells. The results of soft-agar assay and clone formation assay illustrated that vortioxetine hydrobromide significantly inhibited anchorage-independent growth (Fig. 1D, S1A) and anchorage-dependent growth (Fig. 1E, S1B) of GC cells in a dose-dependent manner. Together, these results indicated that vortioxetine hydrobromide inhibited GC cell growth in vitro.
Vortioxetine hydrobromide directly bound with JAK2 and SRC
To further investigate the molecular mechanism of vortioxetine hydrobromide on gastric cancer, we used Maestro 11.5 software to conduct computational docking models. We found JAK2 and SRC were potential targets of vortioxetine hydrobromide (Fig. 2A, B). Then pull-down assays were performed using recombinant JAK2 or SRC proteins, HGC27 and AGS cell lysates. The results demonstrated that the vortioxetine hydrobromide directly binds to JAK2 or SRC proteins (Fig. 2C-E). To further verify the interactions between vortioxetine hydrobromide and JAK2 or SRC kinases in intact cells, we performed cellular thermal shift assay (CETSA). The melting temperature (Tm) values of JAK2 and SRC in AGS cells were 51.2 ℃ and 62.6 ℃, which were higher than vehicle groups 47.3 ℃ and 57 ℃ (Fig. 2F). Similarly, the Tm values of JAK2 and SRC in HGC27 cell after vortioxetine hydrobromide treatment were 54.5 ℃ and 66.8 ℃, while the vehicle group were 51.8 ℃ and 59.8 ℃ (Fig. 2G). These results confirmed that vortioxetine hydrobromide bound with JAK2 and SRC in intact cells. Taken together, vortioxetine hydrobromide directly bound with JAK2 and SRC kinases.
Vortioxetine hydrobromide inhibited STAT3 signaling pathway by targeting JAK2 and SRC kinases
Previous studies have illustrated that STAT3 proteins can be activated by JAK2 and SRC kinases (6, 8). In vitro kinase assay was performed to detect the inhibitory effects of vortioxetine hydrobromide on JAK2 and SRC kinase activities. Results demonstrated that vortioxetine hydrobromide inhibited the activities of JAK2 and SRC kinases (Fig. 3A, B). Next, we determined whether the JAK2/SRC-STAT3 signaling pathways in GC cells could be regulated by vortioxetine hydrobromide. Western blotting analysis confirmed that vortioxetine hydrobromide suppressed STAT3 phosphorylation in a dose-dependent manner (Fig. 3C). Immunofluorescence assays also demonstrated that phosphorylation STAT3 were inhibited by 40% and 34% in HGC27 and AGS cells at 4 µM vortioxetine hydrobromide, respectively (Fig. 3D), whereas STAT3 remained unchanged (Fig. 3E, S3A). It has been reported that STAT3 protein is phosphorylated at the conserved tyrosine residue (Tyr705), resulting in STAT3 dimerization and nuclear translocation (18, 19). Non-reducing SDS-PAGE was performed to evaluate the change in STAT3 dimerization. Results demonstrated that STAT3 dimerization was decreased in GC cells after vortioxetine hydrobromide treatment (Fig. 3F). Furthermore, after the vortioxetine hydrobromide treatment, the expression levels of p-STAT3 and STAT3 in HGC27 cell nucleus (Fig. 3G) and AGS cell nucleus (Figure S3B) were decreased. Subsequently, the expression levels of STAT3-mediated downstream genes including Bcl-2, Mcl-1 and c-Myc were impaired by vortioxetine hydrobromide in HGC27 (Fig. 3H) and AGS cell (Figure S3C). Bcl-2 and Mcl-1 are anti-apoptotic genes, and c-Myc regulates cellular processes. Vortioxetine hydrobromide decreased Bcl-2, Mcl-1 and c-Myc transcription, reducing GC cell proliferation and survival. Thus, the above results suggested that vortioxetine hydrobromide inhibited GC by blocking the JAK2/SRC-STAT3 pathways.
JAK2 or SRC knockout suppressed GC cell growth and reduced the sensitivity of GC to vortioxetine hydrobromide
In order to assess the prognostic and potential therapeutic value of JAK2 and SRC in GC patients, the mRNA expressions of JAK2 and SRC were analyzed by UALCAN. The bioinformatic analysis confirmed that JAK2 and SRC mRNA levels were up-regulated in a variety of cancers (Figure S4A, B). It was observed that JAK2 and SRC expression levels were higher in GC tissues compared to normal tissues (Figure S4C, E), and higher in G1, G2 and G3 compared to normal tissues (Figure S4D, F). These data suggested that JAK2 and SRC are up-regulated in GC and their expressions are positively correlated with poor clinical grades.
To investigate the role of JAK2 or SRC in GC growth, we established the knockout cell lines by CRISPR/Cas9 system. We verified the knockout effect of JAK2 or SRC by Western blotting, respectively. Results showed that the expressions of JAK2 or SRC in sgJAK2#2, sgJAK2#3, sgSRC#2 and sgSRC#5 cells were decreased (Fig. 4A, 5A). After knockout JAK2 or SRC, the growths of GC cells were suppressed (Fig. 4B, 5B). Similarly, the cloning abilities of sgJAK2 and sgSRC cells were significantly attenuated (Fig. 4C, 5C, S5A). Hence, the phosphorylation of STAT3 in sgJAK2 and sgSRC cells were also decreased (Fig. 4D, 5D). To evaluate whether the effects of vortioxetine hydrobromide are dependent upon JAK2 and SRC expression, we performed cell proliferation assay and colony formation assay in sgJAK2 and sgSRC cells treated with vortioxetine hydrobromide. The results confirmed the inhibitory effect of vortioxetine hydrobromide on the proliferation and colony formation of sgJAK2 and sgSRC cells were decreased compared to the sgControl cells (Fig. 4E-F, 5E-F, S5B). Together, these results illustrated that vortioxetine hydrobromide suppressed GC growth through JAK2 and SRC kinases. and clone formation assay
Vortioxetine hydrobromide inhibited GC patient-derived xenograft (PDX) growth
In order to evaluate the antitumor activity of vortioxetine hydrobromide in vivo, we established LSG85 and HSG288 PDX models in mice. The information on patients is described in Fig. 6A. Vortioxetine hydrobromide inhibited the growth of GC PDX models LSG85 and HSG288 cases following 33-day and 18-day of treatment with 1.5 mg/kg or 12 mg/kg by gavage every day (Fig. 6B). The tumor volumes and weights of vortioxetine hydrobromide groups in both cases were decreased when compared with the vehicle groups (Fig. 6C, D). The tumor size of individual mice in LSG85 and HSG288 cases were shown in Figure S6A. We calculated tumor weight and found the average tumor growth inhibition rate of 12 mg/kg groups in the two cases were 50% and 49% (Fig. 6E). Next, the effect of vortioxetine hydrobromide on the expression of Ki67 and pY705-STAT3 in tumor tissues were determined by immunohistochemistry (IHC). The analysis illustrated vortioxetine hydrobromide significantly inhibited the expression of Ki67 and pY705-STAT3 compared with the vehicle groups in LSG85 case (Fig. 6F) and HSG288 case (Fig. 6G, S6B). These data indicated that vortioxetine hydrobromide inhibited tumor proliferation and progression. Western blotting results confirmed that vortioxetine hydrobromide reduced the expression levels of pY705-STAT3, c-Myc, Bcl-2 and Mcl-1 in PDX tumor xenografts (Fig. 6H). Meanwhile, there were no obvious toxic effects on the mice organs (heart, liver, spleen, lung and kidney) (Figure S6C), no significant changes in body weights (Figure S6D). All together, these data suggested that vortioxetine hydrobromide inhibited GC patient-derived xenograft (PDX) growth in vivo.
Currently, the main treatment methods for gastric cancer patients include surgery, systemic chemotherapy, radiotherapy and immunotherapy (20–22). However, the high recurrence and metastasis after surgery lead to the poor 5-year survival rate, and the toxic effects of chemoradiotherapy limit the use of these regimens for GC treatment (23, 24). Therefore, it is necessary to find effective chemopreventive drugs for gastric cancer recurrence prevention. Repurposing FDA-approved drugs is an effective way to prevent cancer, because the drugs have established safety, pharmacokinetics, tolerability and toxicity profile(14). In this study, we found that vortioxetine hydrobromide, an FDA-approved drug, had anticancer effects on GC cell proliferation in vitro and GC PDX growth in vivo. Recently, it has been reported that vortioxetine induced apoptosis and autophagy of GC AGS cells by PI3K/AKT pathway (25). This evidence further suggests that vortioxetine hydrobromide is a promising drug for GC chemoprevention.
JAK2 and SRC overexpression or overactivation are closely related to the development, maintenance and progression of cancers, suggesting that JAK2 and SRC are effective targets for cancer therapy(7, 11),(26). JAK2 mediates multiple aspects of cytokine signaling, leading to supporting survival and promoting proliferation in breast cancer and B-cell leukemias and lymphomas (27, 28). Excessive activation of the SRC increases cell adhesion, invasion, epithelial to mesenchymal transition (EMT), and migration in colon cancer, pancreatic cancer and breast cancer (7, 29, 30). Our data showed that knockout JAK2 or SRC could inhibit the proliferation of GC cells, and reduced the sensitivity of GC to vortioxetine hydrobromide. Furthermore, vortioxetine hydrobromide bound with JAK2 and SRC. Therefore, vortioxetine hydrobromide is a dual JAK2/SRC inhibitor. It has been reported that STAT3 could be activated by JAK2 and SRC kinases(6, 8). STAT3 is regarded as a therapeutic target in many cancers, it regulates cyclinD1, c-Myc, Bcl-2, survivin and ICAM-1, related to cell proliferation, survival, migration, invasion, stemness, immunosuppression and angiogenesis (6, 19, 31–33). In this study, vortioxetine hydrobromide inhibited the activities of JAK2 and SRC kinases, through reducing STAT3 dimerization, nuclear translocation and transcriptional activity. This evidence illustrated that vortioxetine hydrobromide suppressed JAK2/SRC-STAT3 signaling pathways is of great significance for GC chemoprevention.
In the past 5 years, the use of dual or multi-target inhibitors as new therapeutic ways for antitumor studies has been steadily increasing (34). Dual kinase inhibitors, Dasatinib and Lapatinib, have shown increased efficacy for inhibiting SRC/ABL and EGFR/HER2, respectively (35, 36). Indirubin derivative (IRD) E738, NS-018 and SKLB-850, dual JAK2/SRC inhibitors, showed inhibitory effects in pancreatic cancer, multiple myeloma and B-cell lymphoma (37–39). These inhibitors are newly developed small molecules and are still in preclinical research. Of note, (IRD) E738, NS-018 and SKLB-850 are not approved by FDA yet. While, vortioxetine hydrobromide is approved by FDA, and acts as an antidepressant by inhibiting serotonin transporter (SERT) and modulating 5-HT receptor activity (40). Our data showed that there were no obvious toxic effects on the mice organs and no significant changes in body weights (Figure S7), its safety and stability are guaranteed. In addition, the incidence of depression in cancer patients is high (41). Vortioxetine hydrobromide may have a better clinical therapeutic effect for gastric cancer patients with depression or anxiety. Therefore, our research is of great significance for the treatment and prevention of gastric cancer.
In conclusion, we identified vortioxetine hydrobromide, a novel dual JAK2/SRC inhibitor, inhibited gastric cancer cell proliferation, through reducing STAT3 dimerization, nuclear translocation and transcriptional activity in vivo and in vitro.
Cell culture
Human GC cell lines HGC27 and AGS cells were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). All the cell lines were cytogenetically by STR analysis. HGC27 cells were cultured in RPMI-1640 medium and AGS cells were cultured in F12k containing 10% fetal bovine serum (FBS) at 37°C in 5% CO2.
Chemicals and reagents
Vortioxetine hydrobromide (CAS number 960203-27-4) was purchased from Beijing Bailing wei Technology Co. Ltd. The primary antibodies pY705-STAT3(9145S), pS727-STAT3(9134S), STAT3 (9139S), JAK2(3230S), pY1007/1008-JAK2(3771S), SRC (2108S) were purchased from Cell Signaling Technology (Danvers, MA, USA). pY419-SRC (ab185617), Recombinant JAK2 protein (ab42619), Ki67(ab16667) were purchased from Abcam (Cambridge, MA, USA). STAT3 (sc-8019), pY705-STAT3 (sc-8059), Mcl-1 (sc-12756) and Bcl-2 (sc-7382) were from Santa Cruz (Dallas, TX, USA); GAPDH was from Good Here Biological Technology Co. Ltd. (Hangzhou, Zhejiang, China). The secondary antibodies anti-rabbit IgG (ZB-2301) and anti-mouse IgG (ZB-2305) were from Zhongshan Jinqiao (Beijing, China).
Cytotoxicity assay
HGC27 (6000 cells/well) and AGS (8000 cells/well) were plated on 96-well plates and treated with different doses of vortioxetine hydrobromide (0, 3.125, 6.25, 12.5, 25, 50 µM). After 24, 48 h, the cells were fixed with 4% paraformaldehyde for 30 min in a room temperature. Then the cells were stained with DAPI (1:10000) in the dark and counted using IN Cell Analyzer 6000 software.
MTT assay
GC cells (3000 cells/well) were plated on 96-well plates and treated with different doses of vortioxetine hydrobromide (0, 0.5, 1, 2, 4 µM). Plates were taken out at 0, 24, 48, 72 or 96 h. 10 µL of MTT (5 mg/mL) was added to each well and cells were incubated at 37 ℃ for 2–3 h. The medium was then aspirated from the plates and replaced with 100 µL of DMSO. The absorbance at 490 nm was measured using a microplate reader.
Anchorage-independent cell growth
Eagle’s Basal Medium (BME) with 0.4% agar was added to each well of a 6-well plate and solidified at room temperature for 1.5 h. HGC 27 and AGS cells (8000 /well) were suspended in Eagle’s Basal Medium (BME) containing 0.2% agar with different doses of vortioxetine hydrobromide (0, 0.5, 1, 2 and 4 µM). After incubation for 2 h at room temperature, cells were maintained in a 37°C, 5% CO2 incubator for 9 days, and then the clones were counted and scanned using IN Cell Analyzer 6000 software.
Colony formation assay
GC cells (300/well) were seeded into 6-well plates and treated with different doses of vortioxetine hydrobromide (0, 0.5, 1, 2 and 4 µM). The medium was replenished with fresh medium containing vortioxetine hydrobromide every 3 days. After incubation for 10 days, the medium was then aspirated from the plates. The clones were fixed and then stained with 0.3% crystal violet. Then the clones were imaged and counted.
Computational docking analysis
In silico docking was performed using the Maestro 11.5 software program. The crystal structures of JAK2 (PBD: 3E63) and SRC (PBD: EKSW) were obtained from the Protein Data Bank (PDB) (https://www.rcsb.org/). The vortioxetine hydrobromide chemical structure (PubChem CID: 9966051) was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). All water molecules and small molecule ligands were deleted and hydrogen atoms were added. The PyMOL (PyMOL Molecular Graphics System, Version 2.3.4) program was utilized to prepare the vortioxetine hydrobromide for the docking study.
Western blotting
Cells (4.5 × 106) were plated on a 15 cm dish and treated with various concentrations of vortioxetine hydrobromide (0, 0.5, 1, 2 and 4 µM) for 24 h. Then cells were collected and lysed with RIPA buffer. The protein concentration was detected using the BCA kit (BCA Protein Assay Kit, Beyotime Biotechnology, Shanghai, China). Protein samples were heated with protein loading buffer at 100°C for 5 min and then were separated by SDS–PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% nonfat milk for 1.5 h, incubated with primary antibodies overnight at 4°C. Next, the membrane was incubated with corresponding secondary antibodies for 1.5 h. Protein bindings were detected using a chemiluminescence reagent (ECL, Meilunbio).
Pull-down assay
Cell lysates (500 µg) or recombination proteins were incubated with vortioxetine hydrobromide-Sepharose 4 B beads or DMSO-Sepharose 4 B beads in reaction buffer at 4°C overnight. The beads were washed three times with buffer at 4 ℃. The proteins were eluted from the beads through boiling in loading buffer at 95°C for 5 min. Protein binding was assessed using Western blotting after elution.
Cellular thermal shift assay (CETSA)
Cells (4.5 × 106) were seeded into a 15 cm dish and treated with vortioxetine hydrobromide (0, 4 µM) for 24 h. Then they were harvested and resuspended in PBS. The cells were divided into 12 tubes equally. The control group and vortioxetine hydrobromide treatment group were heated at 37 ℃, 40 ℃, 43°C, 46°C, 49°C, 52°C, 55°C, 58°C, 61°C, 64°C, 67 ℃ or 70 ℃ for 3 min. Then the samples were quickly frozen twice in liquid nitrogen and centrifuged at 12000 rpm for 20 min at 4 ℃. The proteins were transferred to new tubes and then analyzed by Western blotting (17).
In vitro kinase assay
Active JAK2 (50 ng) or SRC (100 ng) recombination proteins were incubated with inactive STAT3 proteins (250 ng) for in vitro kinase assays separately. The reactions were run in kinase buffer and contained 100 µM ATP. Different doses of vortioxetine hydrobromide were added and the mixtures were incubated at 30°C for 30 min. The reactions were terminated by adding 5 µL protein loading buffer and boiling for 5 min at 95°C. The proteins were analyzed by using Western blotting.
Cell immunofluorescence assay
The slides were put in a 24-well plate, and then 2×104 GC cells were seeded in each well. After 16–18 h, cells were treated with various concentrations of vortioxetine hydrobromide for 24 h. The medium was then aspirated from the plate and the cells were fixed with 4% paraformaldehyde in PBS for 30 min. The cells were washed three times with PBS and incubated primary antibody (p-STAT3, STAT3 1:50) overnight at 4 ℃. Next, slides were incubated with the fluorescent secondary antibody (1:50) for 1.5-2 h in the dark. After several washes, slides were stained with DAPI (1:10000) for 5 min at 37 ℃. The images were captured and analyzed by using IN Cell Analyzer 6000 software.
Nuclear and cytoplasmic protein extraction
2 × 106 cells were seeded into a 10 cm dish and treated with different doses of vortioxetine hydrobromide (0, 0.5, 1, 2 and 4 µM) for 24 h. After harvesting the cells, the Nuclear and Cytoplasmic Protein Extraction kit (Cat#R0050, Solarbio) was used to extract nuclear proteins and cytoplasm proteins. The proteins were assessed by Western blotting.
CRISPR/Cas 9 knockout cell lines
To knockout the JAK2 or SRC gene in GC cells by using the CRISPR/Cas9 system, the lentiCRISPRv2 vector was used. Following the manufacturer’s suggested protocols, sgJAK2 or sgSRC plasmids were transfected into HEK293T cells by using Jet Primer (Thermo Fisher Scientific, Waltham, MA, USA). Two days after transfection, viral particles were harvested and filtered using a 0.22-µm filter. The GC cells were infected with 8 µg/mL polybrene and selected with 2 mg/mL puromycin for 72 h. Knockout efficiency was verified by Western blotting. The oligonucleotide sequences of JAK2 and SRC single guide (sg) RNA were designed in an online CRISPR tool (https://chopchop.cbu. uib.no/) and were listed as Table 1
Table 1 The oligonucleotide sequences of JAK2 and SRC single guide (sg) RNA
PDX mouse model
The study was approved by the Research Ethics Committee of Zhengzhou University. Five-to Six-week-old SCID/CB17 female mice were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). The tissues were cut into pieces of 8–10 mm3 and inoculated under the skin of the mice using forceps. Mice were randomly divided into three groups of LSG85 (n = 8) and HSG288 (n = 9) as follows: (1) vehicle; (2) vortioxetine hydrobromide (1.5 mg/kg); (3) vortioxetine hydrobromide (12 mg/kg). Mice were given vortioxetine hydrobromide or vehicle by gavage every day. When the tumor volume reached 1000 mm3, the mice were anesthetized and tumor tissues were excised. Tumor volume was calculated as follows: tumor volume (mm3) = (length × width2)/2.
Immunohistochemical analysis
Formalin-fixed tumor tissues were paraffin embedded, cut into 4-µm sections and then placed on slides. The paraffin tissue sections were incubated at 65°C for 2 h, deparaffinized, rehydrated, and processed for antigen retrieval. Next, following by incubation with 3% H2O2 for 10 min to inactivate endogenous peroxidase, then the tissues were incubated overnight with the Ki67 and pY705-STAT3 primary antibody (1:50) at 4°C. The next day, all tissue sections were washed three times, and then incubated with the secondary antibody followed by 3, 3′-diaminobenzidine (DAB) staining. Then the sections were counterstained with hematoxylin, dehydration, and mounted with neutral resin. The Tissue Faxes (Tissue Gnostics) and Image Pro Plus software program (Media Cybernetics, Rockville, MD) were utilized to scan all sections and calculate positive cells.
Statistical analysis
The Statistical Product and Service Solutions (SPSS 21.0, IBM, Inc. Armonk, NY, USA.) was used to calculate P values. The significance of differences was calculated by one-way ANOVA and data were expressed as the mean values ± SD. p < 0.05 was considered statistically significant.
Acknowledgements
This study was supported by the National Natural Science Foundations of China (No. 81872335), National Natural Science Youth Foundation (No. 81902486), The Central Plains Science and Technology Innovation Leading Talents (No. 224200510015), the Science and Technology Project of Henan Province (No. 212102310187).
Author contributions
Conceptualization: Zigang Dong, Kangdong Liu and Yanan Jiang; Data curation: Mingzhu Li; Funding acquisition: Kangdong Liu and Yanan Jiang; Investigation: Mingzhu Li, Lina Duan, Wenjie Wu; Methodology: Mingzhu Li, Lina Duan; Project administration: Zigang Dong, Kangdong Liu and Yanan Jiang; Resources: Zigang Dong, Kangdong Liu and Yanan Jiang; Supervision: Kangdong Liu and Yanan Jiang; Roles/Writing - original draft: Mingzhu Li and Yanan Jiang; Writing - review & editing: Kangdong Liu and Yanan Jiang.
Competing interests
The authors have declared that no competing interest exists.
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There is NO conflict of interest to disclose.