Novel compounds TAD-1822-7-F2 and F5 inhibited ...

Biomedicine & Pharmacotherapy 103 (2018) 118–126

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Novel compounds TAD-1822-7-F2 and F5 inhibited HeLa cells growth through the JAK/Stat signaling pathway

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Tianfeng Yang, Xianpeng Shi, Yuan Kang, Man Zhu, Mengying Fan, Dongdong Zhang , ⁎ Yanmin Zhang School of Pharmacy, Health Science Center, Xi’an Jiaotong University, No. 76, Yanta Weststreet, #54, Xi’an, Shaanxi Province 710061, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: HeLa TAD-1822-7-F2 and F5 Surface plasmon resonance Quartz crystal microbalance JAK/Stat

Cervical carcinoma remains the second most common malignancy with a high mortality rate among women worldwide. TAD-1822-7-F2 (F2) and TAD-1822-7-F5 (F5) are novel compounds synthesized on the chemical structure of taspine derivatives, and show an effective suppression for HeLa cells. Our study aims to confirm the potential targets of F2 and F5, and investigate the underlying mechanism of the inhibitory effect on HeLa cells. In this study, Real Time Cell Analysis and crystal violet staining assay were conducted to investigate the effect of F2 and F5 on HeLa cells proliferation. And the analytical methods of surface plasmon resonance and quartz crystal microbalance were established and employed to study the interaction between F2 and F5 and potential target protein JAK2, suggesting that both compounds have strong interaction with the JAK2 protein. Western blot analysis, immunofluorescence staining study and PCR was conducted to investigate the molecules of JAK/Stat signaling pathway. Interestingly, F2 and F5 showed diverse regulation for signaling molecules because of their different chemical structure. F2 increased the expression of JAK2 and downregulated the level of P-JAK1 and PJAK2, and decreased P-Stat3 (Ser727). While F5 could increase the expression of JAK2 and naturally decrease the phosphorylation of JAK1 and Tyk2, and decreased the expression of P-Stat6. Moreover, F2 and F5 showed the same downregulation on the P-Stat3 (Tyr705). Therefore, F2 and F5 could target the JAK2 protein and prevent the phosphorylation of JAKs to suppress the phosphorylation of the downstream effector Stats, which suggested that F2 and F5 have great potential to be the inhibitors of the JAK/Stat signaling pathway.

1. Introduction Cervical carcinoma is the second most common malignancy among women in the world and the age of onset is declining recent years [1]. The therapeutic methods for cervical cancer include surgery, medication, radiotherapy, heat treatment and gene therapy. Surgery combined with concurrent chemo-radiotherapy can cure 80%–95% early cervical cancer patients, but the treatment effect is not satisfactory for advanced and metastatic cervical cancers [2]. Therefore, new and efficient chemotherapeutics are in great need for the treatment of cervical cancer. The JAK/Stat signaling pathway is critical for signal transmission of a wide array of cytokines and growth factors and mediates a lot of biological reactions including differentiation, migration, apoptosis and immune regulation, etc. [3]. Over activation of the JAK/Stat signaling pathway will promote the occurrence and development of solid tumors, lymphoma, leukemia and inflammatory disorders [4]. When the ligand binds to the receptors in the JAK/Stat signaling pathway, the receptors

dimerized and be activated by the alternated phosphorylation of the tyrosine residue from link-coupled JAKs. The phosphorylation of the receptor recruits the STAT proteins which are phosphorylated by the JAKs [5], and then Stats combined with the receptors through the SH2 structural domain and transferred to the nucleus in a homo/hetero dimer manner. Afterwards, Stats bound to the promoter of the targeted gene and regulate the transcription and expression of the downstream molecules [6]. In mammals, 4 JAKs and 7 Stats were comprised and among them, Stat3 is closely related to tumor [7]. The unphosphorylated Stat3 as well as P-Stat3 can bind directly to DNA in order to play a role in gene regulation and can be a novel drug target in oncology [8], but most of Stat3 drug targeted to the phosphorylated Stat3 protein [9]. The activated Stat3 could promote tumor angiogenesis and accelerate the epithelial mesenchymal transition process through the JAK/Stat signaling pathway [10]. HPV16/18 positive cervical cancer patients are in high expression of Stat3, such as HeLa is positive of HPV18 [11]. Furthermore, endometrial and cervical cancer patients in

Abbreviations: RPMI, Roswell Park Memorial Institute; FBS, fetal bovine serum; IC50, 50%-growth inhibitory concentrations; RTCA, Real Time Cell Analysis; SPR, surface plasmon resonance; QCM, quartz crystal microbalance; KD, equilibrium dissociation constant ⁎ Corresponding authors. E-mail addresses: [email protected] (D. Zhang), [email protected] (Y. Zhang). https://doi.org/10.1016/j.biopha.2018.03.174 Received 29 January 2018; Received in revised form 27 March 2018; Accepted 28 March 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.


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Fig. 1. F2 and F5 inhibited HeLa cells proliferation. (A) The design route of F2 and F5. (B) Real time cell growth curve of F2 and F5 on viability of HeLa cells. Cells were cultured with or without F2 and F5 at indicated concentrations for 48 h. (C) Photographs of HeLa cells stained with 0.2% crystal violet with or without F2 and F5.

2. Materials and methods

the USA were reported to overexpress Stat3 according to a previous study [12], so preventing Stat3 might have great potential for the treatment of the two gynecological cancers. The feasibility of kinases as therapeutic drugs target have been confirmed, along with countless studies linking JAK/Stat to malignancies, a number of jakinibs including ruxolitinib, tofacitinib and oclacitinib have been approved by FDA [13]. Discovery of active ingredients from natural products and structural optimization is of great importance for the creation of new drugs. Taspine is a kind of apanthrene alkaloids which is isolated from Radix et Rhizoma Leonticsi [14]. In the present study, we designed and synthesized two novel isomers compounds TAD-1822-7-F2 (F2) and TAD-1822-7-F5 (F5) from the chemical structure of taspine and its derivatives TPD7 (Fig. 1A). In the present study, we investigated the potential targets of F2 and F5 for their inhibition on HeLa cells proliferation, and explored the underlying mechanism of F2 and F5 as a novel inhibitor of the JAK/Stat signaling pathway.

2.1. Chemicals and reagents F2 and F5 (purity > 98%) were synthesized in the Research and Engineering Center for Natural Medicine, Xi’an Jiaotong University. AZD1480 was obtained from TargetMol (Massachusetts, USA) and OSM was purchased from R&D system (Minnesota, USA). Human cervical cancer cell HeLa (TCHu187) was purchased from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences (Shanghai, China). Roswell Park Memorial Institute 1640 medium was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Excell Bio (Shanghai, China). Trypsin was obtained from Amresco (Solon, OH, USA). The penicillin was purchased from General Pharmaceutical Factory (Haerbin, China), and the streptomycin was purchased from North China Pharmaceutical (Shijiazhuang, China). Crystal violet was purchased from Beijing Chemical Plant (Beijing, China). Propidium iodide (PI) was purchased from SigmaAldrich. Rabbit anti-GAPDH, Rabbit anti-β-actin, goat anti rabbit IgG,

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Fig. 2. F2 and F5 interacted with JAK2 protein. (A, B) The binding curves of F2 and F5 on the JAK2-NTA sensor chip by SPR. (C, D) The binding curves of F2 and F5 on the JAK2-COOH sensor chip by QCM.

Fig. 3. F2 and F5 increased the accumulation and subcellular localization of JAK2 in HeLa cells. (A) The mRNA expression of JAK2 in HeLa cells after treated with F2 and F5. (B) The accumulation and quantification of JAK2 in HeLa cells. (C) The accumulation and quantification of P-JAK2 in HeLa cells. (D, E) JAK2 staining with immunofluorescence in HeLa cells. The cells were treated with different concentrations of F2 and F5 and the subcellular localization of JAK2 was visualized, JAK2 (green), DAPI (blue). Data were expressed as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 compared with untreated control cells.

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Fig. 4. Effect of F2 and F5 on accumulation of P-JAKs in HeLa cells. (A, D) Protein accumulation of P-JAK1 and JAK1 in JAK/Stat signaling pathway of HeLa cells by Western blot analysis. (B, E) Proteins accumulation of P-JAK3 and JAK3. (C, F) Proteins expressions of P-Tyk2 and Tyk2. (G) Quantification of A, B and C. (H) Quantification of D, E and F. Data were expressed as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 compared with untreated control cells.

before the addition of 100 μL of the cell suspension (4 × 104 cells/well). After incubation at room temperature for 30 min, the E-Plates were placed on the reader in an incubator, and the cell index recording continued. The cells were allowed to grow for 20–24 h and then treated with F2 and F5 at different concentrations. Cells were monitored every 15 min for 48 h after treatment to capture the RTCA S16.

FITC-goat anti rabbit IgG, BCA protein assay reagent kit and enhanced chemiluminescent (ECL) plus reagent kit were obtained from Pierce (Pierce Biotech, Rockford, IL, USA). Phospho-Jak Family Antibody Sampler Kit, Stat Antibody Sampler Kit, Phospho-Stat Antibody Sampler Kit were obtained from Cell Signaling (Boston, Massachusetts, USA). JAK2 6*his fusion protein was purchased from Protein technology Group (Chicago, Illinois, USA). Protease inhibitor cocktail and phosphatase inhibitor cocktail were purchased from Roche Technology (Basle, Switzerland, USA). The RNA fast 200 kit was purchased from Fastagen (Shanghai, China) and Prime Script RT Master Mix Perfect Real Time kit and SYBR Premix Ex Taq TM II were purchased from Takara biotechnology (Dalian, China). The JAK2 and Stat3 primer were obtained from Takara biotechnology (Dalian, China). Other reagents used were analytical grade.

2.4. Crystal violet staining assay Exponentially growing HeLa cells were seeded to a 12 well plate and cultivated overnight. Cells were treated with F2 and F5 at 0, 0.15, 0.3, 0.6 μmol/L and incubated for 48 h. Then cells were washed by PBS and fixed with 4% paraformaldehyde for 10 min. Thereafter, cells were washed with PBS again and stained with crystal violet for 15 min. Images were photographed under the inverted fluorescence microscope.

2.2. Cell culture HeLa cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin. Cells were maintained in an incubator with a humidified atmosphere of 5% CO2 at 37 ℃.

2.5. Surface plasmon resonance (SPR) For the analysis of SPR, JAK2 protein with 6*his tag (30 μg/mL) was fixed on the NTA sensor chip by capture-coupling, then recombinant F2 and F5 at concentrations of 1, 5, 10, 20 μmol/L were injected sequentially into the chamber in PBS running buffer, the interaction of JAK2 with the small molecules fixed was detected by OpenSPRTM (Nicoya Lifesciences, Waterloo, Canada) at 25 °C. The binding time and disassociation time were both 250 s, the flow rate was 20 μl/s, the chip was regenerated with Hydrochloric acid (pH 2.0). A one to one diffusion

2.3. Real time cell analysis Real Time Cell Analysis (RTCA) was conducted using the xCELLigence RTCA S16 (ACEA, San Diego, USA). Briefly, 50 μL of the medium was added to 16-well E-Plates to obtain background readings 121


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Fig. 5. F2 and F5 inhibited the accumulation and nucleus translocation of P-Stat3 (Tyr705) in HeLa cells. (A) The mRNA expression of Stat3 in HeLa cells after treated with F2 and F5. (B, C) The accumulation and quantification of P-Stat3 (Tyr705) in HeLa cells by F2 and F5. (D, E) The accumulation of P-Stat3 (Tyr705) in HeLa cells by immunofluorescence by F2 and F5. HeLa cells were treated with different concentrations of F2 and F5 and P-Stat3 (Tyr705) nuclear translocation was detected by immunofluorescence, P-Stat3 (green), DAPI (blue), and the merged image indicates the nuclear location of P-Stat3 (Tyr705) protein. Data were expressed as mean ± SEM (n = 3). *P < 0.5, **P < 0.01, ***P < 0.001 compared with untreated control cells.

Next, 500 μL of 1% BSA was used to block the nonspecific adsorption. Then 1 mL PBS buffer was added again to get the baseline. To determine the equilibrium dissociation constant (KD) of the F2 and F5, different concentrations of F2 and F5 solution (1, 5, 10 and 20 μmol/L) were used in the binding assays. Regeneration of the electrode was carried out by reacting with 100 μL of Hydrochloric acid (0.1 mol/L) for 30 s. A nonlinear regression curve was fitted to the frequency shifts corresponding to the varied drug concentration. And the KD value was estimated using the equation Y = Bmax × X/(KD + X) by GraphPad Prism 5.0 software.

corrected model was fitted to the wavelength shifts corresponding to the varied drug concentration. The data was retrieved and analyzed with TraceDrawer software. 2.6. Quartz crystal microbalance (QCM) QCM measurements were carried out using a QCM 200 instrument (Stanford Research Systems, California, USA). The sensor is comprised of a 5 MHz AT-cut gold-coated quartz crystal with gold electrodes. Before use, the crystal was rinsed with pure water and absolute ethanol for three times. After drying with a stream of nitrogen, the crystal was submerged in 10 mmol/L MUA (11- Mercaptoundecanoic acid) for 2 h at room temperature to form self-assembled monolayers. The unreacted MUA was removed by three washes with absolute ethanol and pure water. Then, 1 mL of the freshly prepared EDC/NHS (100 mmol/L/ 50 mmol/L, v/v, 1:1) solution was added and reacted for 30 min to convert the terminal carboxylic group to an amine-reactive NHS-ester. After that, 500 μL of JAK2 protein with 6*his tag (10 μg/mL) was added and incubated for 60 min to be immobilized on the electrode surface.

2.7. Immunofluorescence staining HeLa cells were grown into the 96-well plate and cultivated overnight. Then different concentrations of F2 and F5 were added for 48 h. For immunofluorescence staining, the cells were washed three times with PBS and fixed in 4% paraformaldehyde for 15 min at R.T. After treating with 0.1% TritonX-100 for 10 min at room temperature, cells were blocked with 1% BSA (in PBS) at 37 ℃ for 1 h to avoid the non122


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Fig. 6. Effect of F2 and F5 on accumulation of Stats in the JAK/Stat signaling pathway of HeLa cells. (A, E) Protein accumulation of P-Stat1 and Stat1 in HeLa cells after treatment with F2 and F5 at indicated concentrations for 48 h. (B, F) Protein accumulation of P-Stat3(Tyr705) and Stat3 in HeLa cells. (C, G) Protein accumulation of P-Stat5 and Stat5 in HeLa cells. (D, H) Protein accumulation of P-Stat6 and Stat6 in HeLa cells. (I) Quantification of A, B, C and D. (J) Quantification of E, F, G and H. Data were expressed as mean ± SEM (n = 3). *P < 0.5, **P < 0.01, ***P < 0.001 compared with untreated control cells.

specific interaction. The cells were incubated with monoclonal rabbit JAK2/P-Stat3 (Tyr705) (1:400 dilutions) antibody at 37 ℃ for 4 h. Then the cells were incubated with FITC-goat anti rabbit IgG (1:200 dilution) at R.T for 2 h in dark. The nuclei were stained with PI for 15–20 min simultaneously. The treated cells were examined and captured under the inverted fluorescence microscope.

Mix Perfect Real Time kit. The PCR reactions were performed with the Thermal Cycler Dice Real Time System (Takara, Japan) in 96-well reaction plates. The relative amount of mRNA for each gene was normalized and presented as the ratio of the mRNA value of a target gene to that of the GAPDH gene.

2.8. Western blot analyses

2.10. Statistical analysis

Cellular proteins were extracted using RIPA lysis buffer (50 m mol/L Tris, 150 m mol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate) containing protease and phosphatase inhibitor cocktail at 4 °C for 30 min. The cell lysates were centrifuged at 12,000 ×g at 4 °C for 10 min. An equivalent amount of protein was resolved on a 10% SDS gel and transferred to polyvinylidene difluoride (PVDF) membranes after electrophoresis. The membranes were blocked with Tris-buffered saline containing 0.05% Tween-20 (TBST) and 10% low-fat powdered milk for 2 h. Then the blot was incubated with the blocking solution containing the primary antibody overnight at 4 °C. After washing with TBST for 5 min four times, the blot was incubated with a HRP-conjugated secondary antibody. 1 h later, the blot was washed four times with TBST and detected with enhanced chemiluminescence (ECL) kit. The ImagePro plus software (Image-Pro Plus 5.1, Media Cybernetics, Inc., Rockville, MD, USA) was used to analyzed the gray-scale value of bands and the expression of the targeted proteins was calculated.

Data were expressed as mean ± SEM. Statistical analysis was performed using the Statistical software SPSS18.0, ANOVA was used to analyze Statistical differences between groups under different conditions, and independent sample T test was used to analyze the values between two groups. A P-value < 0.05 was considered Statistically significant.

3. Results 3.1. F2 and F5 suppressed HeLa cells proliferation in vitro In order to investigate the inhibitory effect of F2 and F5 on the proliferation of HeLa cells, RTCA was conducted to detect the cell growth and the results showed that both F2 and F5 could inhibit the proliferation of the cells at different concentrations (Fig. 1B). The IC50 values for F2 and F5 were 0.654 and 0.701 μmol/L after 48 h incubation, respectively. As shown in Fig. 1C, the result of crystal violet staining assay clarified that F2 and F5 could obviously inhibit the proliferation of HeLa cells in a dose-dependent manner. These results demonstrated that F2 and F5 possessed good anti-tumor effect in HeLa cells in vitro.

2.9. RNA isolation and RT-PCR The total RNA of HeLa cells treated with or without F2 and F5 were isolated using the RNA fast 200 kit. The total RNA was reversely transcribed in 30 μL reaction solution using the Prime Script RT Master 123


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Fig. 7. The effect of F2 and F5 on accumulation of Stats in the JAK/Stat signaling pathway of HeLa cells after treatment with OSM or AZD1480. (A, B) Real time cell growth curve of F2 and F5 combined with AZD1480. (C, D) Protein accumulation of JAK2, P-JAK2, Stat3 and P-Stat3 (Tyr705) after treated with F2 and F5 combined with AZD1480. (E, F) Protein accumulation of Stat1, P-Stat1, Stat3 and P-Stat3 (Tyr705) after treated with F2 and F5 combined with OSM. (G, H) Quantification of C and D. (I, J) Quantification of E and F. Data were expressed as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 compared with untreated control cells.

3.2. F2 and F5 had good interaction with JAK2 protein

3.3. Effect of F2 and F5 on the JAK2 molecules

The JAK/Stat pathway was closely associated with the occurrence and development of cancer. In order to investigate the interaction of F2 and F5 with the JAK2 protein, SPR analysis was conducted. The results showed that both F2 and F5 could bind the JAK2 protein. The KD values for F2 and F5 calculated by TraceDrawer™ were 6.05 ± 0.13e-8 mol/L and 4.67 ± 0.18e-7 mol/L (Fig. 2A and B). To further confirm whether F2 and F5 can bind JAK2, QCM assay was conducted, and the results were shown in Fig. 2C and D. The KD values for F2 and F5 were 7.114e10 mol/L and 1.29e-9 mol/L. The obtained data from two methods indicated there was good interaction between F2 and F5 and JAK2 protein, and F2 is the better binder.

To further demonstrate whether F2 and F5 could regulate the expression of JAK2 protein, we investigated the effect of F2 and F5 on the expression of the JAK2. As is shown in Fig. 3A and B, F2 and F5 could upregulate the expression of mRNA and protein levels of JAK2. Immunofluorescence staining study in Fig. 3D and E showed the same change that the fluorescence intensity strengthened with the increase of the concentrations of F2 and F5. Then the expression of P-JAK2 in HeLa cells was detected by western blot, the results showed that F2 could downregulate the level of P-JAK2, but F5 have no obvious effect on PJAK2 (Fig. 3C). Then AZD1480, a adenosine triphosphate competitive, small-molecule inhibitor of JAK2 kinase [15], was used to confirm the results. HeLa cells were treated with AZD1480 (3μmol/L) alone, F2 and F5, or pre-treated with AZD1480 for 24 h before exposure to F2 and F5

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Fig. 8. Potential schematic diagram of F2 and F5 of inhibiting HeLa cells proliferation. F2 and F5 inhibited the phosphorylation JAKs and P-Stat3 nuclear translocation.

mediated downregulation of P-Stat3(Tyr705) (Fig. 7C, D, G and H), and reverse the OSM-mediated increasing of P-Stat3(Tyr705) (Fig. 7E, F, I and J).

for 48 h. As shown in Fig. 7A and B, we found that F2 and F5 combined with AZD1480 could further inhibit cell proliferation, and F2 could strengthen AZD1480-induced downregulation of P-JAK2 (Fig. 7C and G).

4. Discussion 3.4. Effect of F2 and F5 on the JAK family molecules Nowadays, molecular targeted chemotherapeutic agents play an essential role in the treatment of malignant tumors [18]. At the same time, the corresponding jakinibs also used in the treatment of tumors, for instance, ruxolitinib, tofacitinib and oclacitinib. However, the treatment for cervical carcinoma is still of great challenge because of the unclear nosogenesis and the loss of productive drug targets [19]. It is of urgent need to find new targeted drugs for the treatment of cervical cancer. In this study, we analyzed the effect of F2 and F5 on the proliferation of HeLa cells, and investigated the action target and potential mechanism. Our data demonstrated that F2 and F5 inhibited the proliferation of HeLa cells obviously and affected the biological behavior of HeLa cells by regulating the JAK/Stat signaling pathway. JAK/Stat are strongly correlated with tumor development and progression in HPV-mediated cervical carcinogenesis [20], so we investigated the effect of F2 and F5 on the JAK/Stat signaling pathway in HPV18 positive HeLa cells. The results of the SPR and QCM assay confirmed that both F2 and F5 could bind to the JAK2 protein, which suggested that the two compounds had the good inhibition through the JAK2 protein to regulate the JAK/Stat signaling pathway. Then we verified the effect of F2 and F5 on the expression of JAK2 at the molecular level. Western blot analysis and immunofluorescence staining study all demonstrated that F2 and F5 could increase the JAK2 protein expression. But in further experiments, we found that only F2 can decrease the phosphorylation of JAK2, which was the active form of JAK2. This study implied that F2 and F5 may play their biological functions by regulating other homologous proteins. In order to verify the regulation of F2 and F5 on the other signal molecules in the JAK/ Stat signaling pathway, others molecules were studied and the results showed that F2 also reduced the expression of P-JAK1 and P-Stat3 (Ser727) while F5 markedly downregulated the level of P-JAK1, P-Tyk2 and P-Stat6.

The JAK family including JAK1, JAK2, JAK3 and Tyk2. Because of JAKs plays its biological function by itself phosphorylation, so the other family molecules were investigated by F2 and F5. The results were shown in Fig. 4, F2 decreased the phosphorylation of JAK1 (Fig. 4A and G) and F5 obviously decreased the phosphorylation of JAK1 and Tyk2 (Fig. 4D, F and H). 3.5. F2 and F5 regulated the P-Stats molecules The phosphorylation of Stats could further lead to the nucleus translocation of phosphorylated Stats, and activate transcription finally. So, we investigated the effect of F2 and F5 on the phosphorylation of Stat1, Stat3, Stat5 and Stat6. We found that F2 and F5 could increase the mRNA expression of Stat3 (Fig. 5A). As shown in Fig. 5B and C, F2 and F5 could evidently downregulate the expression of P-Stat3 (Tyr705). Immunofluorescence staining study was also conducted and the results demonstrated that both F2 and F5 could suppress the nucleus transcription of P-Stat3 (Tyr705) (Fig. 5D and E). In addition, we investigated the effect of F2 and F5 on some of the Stats family members including Stat1, Stat3 (Ser727), Stat5 and Stat6. F2 could significantly decrease the expression of P-Stat3 (Ser727) (Fig. 6B and I), and F5 obviously decreased the phosphorylation of Stat6 (Fig. 6H and J). To further confirmed the importance of P-Stats molecules in F2 and F5-mediated effects in HeLa cells, we used the OSM as a positive control for activation at a concentration of 20 ng/mL for 6 h, which is mediators for P-Stat1 and P-Stat3 (Tyr705) stimulation in HeLa cells [16]. Meanwhile we used the AZD1480 which inhibits JAK/Stat pathway by downregulating P-Stat3(Tyr705) as a negative control for activation [17]. We found that both F2 and F5 could strengthen the AZD1480125


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Stat3 could promote tumor angiogenesis and epithelial interstitial transformation [21], and the phosphorylation of the Tyr705 site was closely associated with the dimerization [22,23]. Furthermore, we studied the change of the expression and phosphorylation of the Stat3 protein after the treatment of F2 and F5. The results demonstrated that both F2 and F5 could suppress Stat3 (Tyr705) phosphorylation and also inhibited the nucleus translocation of P-Stat3, which could further prevent the transcription initiation of the targeted molecules in the nucleus. In conclusion, we revealed the potential mechanism of the inhibitory effect of F2 and F5 on HeLa cells. As shown in Fig. 8, F2 and F5 could target the JAK2 protein and prevent the phosphorylation of JAKs to suppress the phosphorylation of the downstream effector Stats, suggesting that F2 and F5 have great potential to be the inhibitors of the JAK/Stat signaling pathway. It's interesting that even though F2 and F5 could both regulate the JAK/Stat signaling pathway, their concrete mechanisms are partially different with each other. F2 and F5 are isomers in the replacement of two substituent groups, the prospective study of structure-activity relationships for cervical cancer is needed. Stat phosphorylation is not necessarily sufficient for transcriptional activity, other post-translational modifications have been identified [24]. It is much more important to analyze Stat transcriptional target genes such as SOCS3 [25], so the Stat transcriptional target genes of F2 and F5 in regulating JAK/Stat signaling pathway will be investigated in the future study.

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Conflict of interest

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The authors declare that they have no conflicts of interest to disclose.

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