Resveratrol Suppresses the Growth and Metastatic Potential of Cervical Cancer through Inhibiting STAT3Tyr705 Phosphorylation


 Background : The aberrant STAT3 signaling promotes initiation and progression of human cancers by either inhibiting apoptosis or inducing cell proliferation, angiogenesis, invasion, and metastasis. This study aims to investigate the role of STAT3 and its phosphorylation status in resveratrol-mediated suppression of cervical cancer. Methods : The effects of resveratrol on cervical tumor growth were also determined by examining the tumor tissues and their histological changes and the volume and weight of tumor tissues grown from Hela cells injected in female athymic BALB/C nude mice following the pretreatment regimen and the treatment regimen. Resveratrol structure and targets interaction virtual screening was performed using molecular docking program Autodock Vina. The phosphorylated STAT3, EMT molecular markers and ECM degradation enzymes protein levels were determined using Western blotting. Results : Proliferation and the colony formation of Hela cells were inhibited after resveratrol treatment in both dose- and time- dependent manner. Resveratrol inhibited migration and invasion of Hela cells. Molecular docking analysis showed that resveratrol interacted with STAT3 at a pocket composed of 11 amino acidic residues. Treatment with resveratrol resulted in decreases in the level of phosphorylation of STAT3 at Tyr705 but not Ser727, and no obvious changes in the protein level of STAT3 in Hela cells and SiHa cells. Pretreatment or treatment with resveratrol resulted in decreases in the IL-6-induced phosphorylation level of STAT3Tyr705 in Hela cells and SiHa cells, compared with those of treatment with IL-6. Reduced STAT3Tyr705 phosphorylation after STAT3 inhibitors S3I201 enhanced inhibition of invasion potential of Hela cells and SiHa cells treated with Resveratrol. Resveratrol decreases the N-Cadherin, Vimentin, MMP-3, MMP-13 protein level and increases the E-Cadherin protein level in a dose-dependent manner. The tumor size, volume, and weight, and the N-Cadherin, Vimentin, MMP-3, and MMP-13 protein level were significantly decreased, and the E-Cadherin protein level was increased, and the tumors were histologically damaged as revealed by H&E staining in both the resveratrol pretreatment group and the resveratrol treatment group and the magnitude of changes was higher in the former than that of the latter. Conclusion : Resveratrol inhibits growth and metastatic potential of cervical cancer through blocking STAT3Tyr705 phosphorylation.


STAT3Tyr705 phosphorylation.
Background Cervical cancer is one of the most common malignant tumors in the world for woman. Adjuvant chemotherapy and adjuvant radiotherapy (chemoradiotherapy) after radical hysterectomy show similar survival outcomes in cervical cancer patients and also appear to reduce the risk of distant recurrence (1). Neoadjuvant chemotherapy followed by radical surgery also predicts a favorable prognosis for local advanced cervical cancer (2, 3). Platinum-based drugs and taxane are main regimens for chemotherapy of cervical cancer. New drugs are in need of development because of drug resistance and adverse reaction (4, 5).
Resveratrol is a natural stilbene and a non-flavonoid polyphenol. It possesses anti-oxidant, antiinflammatory, cardioprotective, and anti-cancer properties. It has been reported that resveratrol has anti-cancer effects on breast, cervical, blood, kidney, liver, bladder, thyroid, prostate, brain, lung, gastric, colon, head and neck, bone and cervical cancers (6-8). In addition, resveratrol can reverse multidrug resistance in cancer cells, and sensitize cancer cells to standard chemotherapeutic agents (6-8). Several studies have shown that resveratrol induces autophagy and apoptotic cell death in cervical cancer cells (9, 10) and suppresses migration and invasion of human cervical cancer cells (11). Resveratrol significantly inhibits the occurrence and development of cervical cancer by regulating phospholipid scramblase 1 (12, 13) and exhibits antitumor activity on HPV E6-positive cervical cancer (14, 15). These studies suggest that resveratrol is a potential chemotherapeutic drug for cervical cancer.
Signal transducers and activators of transcription (STATs) is a family of cytoplasmic transcription factors that mediate intracellular signaling from cell surface receptors to the nucleus and transactivates genes encoding apoptosis inhibitors, cell-cycle regulators and induces of angiogenesis.
ChemiDoc™ MP Imaging System with Image Lab™ Software (Bio-Rad Laboratories, Inc., Version 5.1, USA) was used as image acquisition tools and image processing software packages.

Molecular Docking
Structure-based virtual screening was employed by molecular docking program Autodock Vina (version 1.1.2) (32). The 3D schematic representation of protein-ligand macromolecule was generated by PyMol (version 2.3) (33). The 2D schematic representation of interaction between ligand and other amino acid residues was shown by LigPlus (version 2.1) (34). The structure file of STAT3 protein (PDB ID: 6QHD) was extracted from The Research Collaboratory for Structural Bioinformatics Protein Data Bank (http://www.rcsb.org/) (35,36), which was a X-ray crystal homodimer structure bound with a DNA at 2.85-Å resolution (37). All hetatoms including double-stranded DNA and crystallographic water were removed and we keep chain A as monomer STAT3. In order to examine weather resveratrol competitively binds to the binding pocket of the pTyr705 peptide, the native pTyr peptide in 6QHD crystal structure of the monomer ligand was removed. The tran-resveratrol molecular structure (PubChem CID: 445154) was obtained in PubChem database (38). The autodock vina tutorial was followed to convert the ligand and the receptor pdb to pdbqt file by AutoDock Tools (39). The grid size in XYZ was set with 96, 66, 118, which is large enough to containing all potential pockets in STAT3 monomer. The pocket with the lowest score that predicted highest binding affinity was chosen as the resveratrol binding site in STAT3.
Animal model and in vivo anti-tumor efficacy of RES Twenty-four female athymic BALB/C nude mice weighing 14-20 g (4-6 weeks) were purchased from Hunan SJA Laboratory Animal Co.,Ltd (Changsha, China). Mice were housed at 20-22℃ with a 50-60% relative humidity and fed with a standard laboratory chow and tap water ad libitum. All mice were randomly divided into two groups receiving the pretreatment regimen and the treatment regimen respectively. For the pretreatment regimen, mice were further randomly divided into the control group where mice were treated with vehicle (Normal saline contain 0.1% ethanol, 3 times/week), and the treatment group where mice were treated with resveratrol (30 mg/kg, 3 times/week). After two weeks of treatment, all mice were subcutaneously injected in the right flank with 200 µl Hela cell suspensions containing 5 × 10 6 cells in sterile saline. All mice were further treatment with vehicle and resveratrol respectively for 5 weeks. For the treatment regimen, all mice were subcutaneously injected in the right flank with 200 µl Hela cell suspensions containing 5 × 10 6 cells in sterile saline.
After 10 days of injection, all mice were randomly divided into the control group and the treatment group. Mice in the control group were treated with vehicle (Normal saline contain 0.1% ethonal, 3 times/week), and mice in the treatment group were treated with resveratrol (30 mg/kg, 3 times/week) for 5 weeks. Food and water intake as well as behavioral changes were monitored every day and body weight of animals recorded every three days over the course. Tumor volumes and body weight were measured every three days. Tumor volumes were calculated with the tumor lengths and widths which were measured using a caliper: tumor volume =(length) × (width) 2 ×0.5. At the end of treatment, all mice were sacrificed by cervical dislocation. Tumors were isolated, weighed, and aliquoted for Western blotting analysis, HE staining and immunohistochemistry staining assay. This study was approved by the Ethical Committee for Animal Experimentation of Xiangyang No.1 People's Hospital (NO. 2017DW006).

H&E Staining And Immunohistochemistry (IHC) Assay
Tumor tissues were harvested, fixed with 4% formaldehyde, embedded with paraffin and cut into 4 µm thick sections. For examination of histology of tumor tissues, the paraffin-embedded sections were subjected to H&E staining and examined under an inverted microscope (OLYMPUS IX73). For immunohistochemistry assay of the expressions of p-STAT3 Tyr705 , E-cadherin, N-cadherin and Vimentin, the paraffin-embedded sections were incubated with anti-human p-STAT3 Tyr705 , E-cadherin, N-cadherin, Vimentin primary antibodies, and a biotinylated goat anti-rabbit antibody was used as secondary antibody. Then the slides were washed with PBS and incubated with diaminobenzidine (DAB) chromogen for 3-5 min to yield a dark brown color. The sections were counter-stained with hematoxylin for microscopic observation (IX73P2F, OLYMPUS OPTICAL CO., LTD., Japan). Cells with moderate and strong brownish cytoplasmic staining were considered as positive, whereas cells with unstained or weakly stained cytoplasm were considered as negative. The expression levels of p-STAT3 Tyr705 , E-cadherin, N-cadherin, and Vimentin were determined by calculating the ratio of the number of positively stained cells to total number of cells in five randomly selected 400 × magnification microscopic field.

Statistical analysis
Data analysis was performed using Stata 7.0 (Stata Corp LP, College Station, TX, USA) and Microsoft Office Excel 2003. All the results are expressed as mean ± standard deviation (SD) for at least three independent experiments. Statistical comparisons between or among groups were performed using Student's t-test or one-way analysis of variance followed by the post hoc StudentNewmanKeuls test, respectively. P < 0.05 were considered statistically significant.

Resveratrol inhibits the proliferation of cervical cancer cells
To examine the effect of resveratrol on the proliferation of cervical cancer cells, we treated Hela cells with resveratrol and determined cell proliferation using CCK-8 assay and colony formation assay. The proliferation of Hela cells was inhibited by resveratrol in a dose-dependent manner (Fig. 1A). The IC50 of resveratrol on Hela cells was 291.3, 50.09, and 8.73 µΜ for 24, 48, 72 h treatment respectively ( Fig. 1B). The numbers of colonies of Hela cells were decreased with resveratrol treatment in a dosedependent manner ( Fig. 1C and 1D). These results supported that resveratrol inhibits the proliferation of cervical cancer cells in both dose-and time-dependent manner.

Resveratrol Suppresses Migration And Invasion Of Cervical Cancer Cells
To examine the effects of resveratrol on migration and invasion of cervical cancer cells, we treated Hela cells with resveratrol and determined the migration and invasion ability using Wound healing assay and Transwell assay. The results of Wound healing assay showed that treatment with resveratrol resulted in decreases in the width of scratches among Hela cell layers in a dosedependent manner ( Fig. 2A and 2B). The results of Transwell assay demonstrated that resveratrol decreased the number of Hela cells on the lower surface of the filters in the upper chambers of Transwells ( Fig. 2C and 2D). These data suggested that resveratrol inhibits migration and invasion of cervical cancer cells.
To examine the effects of resveratrol on EMT and ECM degradation enzymes of cervical cancer cells, To further examine the role of resveratrol in the regulation of STAT3 phosphorylation in cervical cancer cells, we treated Hela cells and SiHa cells with resveratrol and IL-6 or combination of both, and determined STAT3 Tyr705. As expected, IL-6 indeed increased the phosphorylation of STAT3 Tyr705 in Hela cells and SiHa cells (Fig. 4B). Pretreatment or treatment with resveratrol decreased the phosphorylation of STAT3 Tyr705 activated by IL-6 in Hela cells and SiHa cells (Fig. 4B).
These data demonstrated that resveratrol inhibits phosphorylation of STAT3 and potentially interacts with STAT3 in cervical cancer cells. and SiHa cells (Fig. 5). The results suggested that reduced STAT3 phosphorylation enhances the inhibition of invasion potential of cervical cancer cells by resveratrol.

Resveratrol Inhibits Cervical Tumor Growth In Mice Model
To determine the effects of resveratrol on cervical tumor growth in vivo, we examined the tumor tissues and their histological changes and the volume and weight of tumor tissues grown from Hela cells injected in female athymic BALB/C nude mice following the pretreatment regimen and the treatment regimen. The results showed that the size (Fig. 6A), the volume (Fig. 6B), and the weight (Fig. 6C) of tumor tissues were significantly decreased in both the resveratrol pretreatment group and the resveratrol treatment group, compared with their respective controls. Both the resveratrol pretreatment regimen and the treatment regimen resulted in damages of tumor mass, as revealed by H&E staining (Fig. 6D). The magnitude of changes in the volume (Fig. 6B), the weight (Fig. 6C), and the histology of tumor tissues (Fig. 6D) was higher in the resveratrol pretreatment regimen group than the resveratrol treatment regimen group. These results suggested that resveratrol inhibits cervical tumor growth in mice model.

Resveratrol inhibits phosphorylation of STAT3, EMT and invasion potential of cervical cancer in mice model
To examine the effects of resveratrol on phosphorylation status of STAT3 and EMT and invasion potential of cervical cancer, we tested the p-STAT3 (Tyr705), N-cadherin, E-cadherin, β-catenin, Vimentin, MMP-13, and MMP-3 protein level in the tumor tissues grown from Hela cells were determined using immunohistochemistry and/or Western blotting. The immunohistochemistry results showed that E-cadherin protein was increased and the level of p-STAT3 (Tyr705), N-cadherin, and Vimentin protein was decreased in the tumor tissues grown from Hela cells in both the resveratrol pretreatment group and the resveratrol treatment group, compared with their respective controls (Fig. 7A). The Western blotting results showed that the p-STAT3 (Tyr705), β-catenin, Vimentin, MMP-13, and MMP-3 were decreased in the tumor tissues grown from Hela cells in both the resveratrol pretreatment group and the resveratrol treatment group, compared with their respective controls (Fig. 7B). The magnitude of changes was higher in the resveratrol pretreatment regimen group than the resveratrol treatment regimen group. These results suggested that resveratrol inhibits phosphorylation of STAT3 and EMT and invasion potential of cervical cancer in mice model.

Discussion
In the current study, our data showed that resveratrol inhibits proliferation, migration and invasion of the cervical cancer cell line Hela and SiHa cells. Resveratrol inhibits Hela cells xenograft cervical tumor growth in mice model. This is consistent with previous studies (9-12, 14, 15). In addition, pretreatment with resveratrol also has inhibitory effect on Hela cells xenograft cervical tumor in mice model by even higher magnitude that that of resveratrol treatment regimen, suggesting that resveratrol has constant suppressive effects on cervical cancer independent on treatment regimen, and earlier treatment probably has better outcome.
Metastasis is a complicated biological process that involves primary tumor angiogenesis, cancer cell Compared with the treatment group. Further, our structure-based molecular docking study showed that there is a direct interaction between resveratrol and STAT3. Therefore, resveratrol inhibits phosphorylation of STAT3 at Tyr705 but not Ser727 in SiHa and Hela cells probably through direct interaction between resveratrol and STAT3.
In the current study, our data showed that treatment with resveratrol, S3I201, or AG490 resulted in correspondence between the level of phosphorylation of STAT3 Tyr705 and EMT and ECM enzyme biomarkers is observed in tumor tissues grown from Hela cells in mice model. Therefore, it is likely that resveratrol inhibits growth and metastatic potential of cervical cancer through inhibiting STAT3 Tyr705 phosphorylation.

Conclusion
The current study supports that resveratrol inhibits growth, epithelial mesenchymal transition and extracellular matrix degradation of cervical cancer through inhibiting STAT3 Tyr705 phosphorylation.
Resveratrol is a potential chemotherapeutic and chemopreventive natural compound for cervical cancer.     Reduced STAT3 phosphorylation enhanced inhibition of the expression of EMT molecular markers and ECM degradation enzymes of cervical cancer cells treated with resveratrol.

List Of Abbreviations
Hela and SiHa cells were seeded in 6-well plates at 1×106 cell/well and cultured for 24 h, and then treated with S3I201, AG490 in fresh media for 2 h. Afterwards, resveratrol was added at indicated concentrations. After 24 h culture, cells were collected and the protein levels were determined using Western blotting, the samples derive from the same experiment and that blots were processed in parallel (cropped blot images are combined into graph, full-length blots are presented in Supplementary Figure 4). Photoshop CC 2019(Adobe, v20, USA) was used as cropping and drawing tools. The results were quantitatively analyzed. *, compared with the control, P<0.05. #, compared with AG490 treatment, P<0.05. Δ, compared with S3I201 treatment P<0.05.

Figure 6
Resveratrol inhibited cervical tumor growth in mice model. Hela cells were injected into female athymic BALB/C nude mice which were treated with resveratrol following the pretreatment regimen and the treatment regimen (n=6/group). The size (A), the volume (B), and weight (C) of the tumor tissues grown from Hela cells were examined and measured.
The histology of the tumor tissues was examined using H&E staining (D). *, P<0.05.