The Anti-Dysenteric Drug Fraxetin Enhances Anti- Tumor E cacy of Gemcitabine and Suppresses Pancreatic Cancer Development by Antagonizing STAT3 Activation


 BackgroundFraxetin, a natural product isolated and purified from the bark of Fraxinus bungeana A.DC., has anti-inflammatory, analgesic and anti-dysenteric activities. This study aimed to investigate the anti-tumor effects of fraxetin in pancreatic ductal adenocarcinoma (PDA) and elucidate the underlying molecular mechanism. MethodsThe effects of quercetin on the proliferation, apoptosis, migration and invasion of pancreatic cancer cells (PCCs), and tumor growth and metastasis in PDA xenograft mouse models were evaluated. Besides, the effects of fraxetin on the sensitivity of PCCs to gemcitabine were evaluated. Moreover, angiogenesis, glucose metabolism, the epithelial-mesenchymal transition (EMT), reactive oxygen species (ROS), and STAT3 activity were analyzed.ResultsIn PCCs, fraxetin inhibited the proliferation, induced mitochondrial-dependent apoptosis, and suppressed the invasion and migration by reversing the EMT. In nude mouse models, PDA growth and metastasis were reduced by fraxetin treatment. Moreover, fraxetin enhanced the sensitivity of PCCs to the chemotherapy drug gemcitabine. Mechanically, oncogenic KRAS-triggered STAT3 activation in PCCs and PDA tissues was suppressed by fraxetin treatment. Fraxetin shows important interactions with STAT3 Src Homology 2 (SH2) domain residues to occupy the pTyr‐recognition site of its SH2 domain of another STAT3 monomer, thereby preventing its homo-dimer formation, which then blocks the activation of downstream signal pathways. The anti-tumor activity of fraxetin in PDA was functionally rescued by a STAT3 activator colivelin. As a result, fraxetin hindered hypoxia-induced angiogenesis by decreasing HIF-1α and VEGFA expression, controlled glucose metabolism by reducing GLUT1 expression, inhibited the EMT by blocking the Slug-E-cadherin axis, and drove ROS-mediated apoptosis by regulating STAT3-Ref1 axis.Conclusion﻿These findings indicate that fraxetin enhances anti-tumor activity of gemcitabine and suppresses pancreatic cancer development by antagonizing STAT3 activation.


Abstract Background
Fraxetin, a natural product isolated and puri ed from the bark of Fraxinus bungeana A.DC., has antiin ammatory, analgesic and anti-dysenteric activities. This study aimed to investigate the anti-tumor effects of fraxetin in pancreatic ductal adenocarcinoma (PDA) and elucidate the underlying molecular mechanism.

Methods
The effects of quercetin on the proliferation, apoptosis, migration and invasion of pancreatic cancer cells (PCCs), and tumor growth and metastasis in PDA xenograft mouse models were evaluated. Besides, the effects of fraxetin on the sensitivity of PCCs to gemcitabine were evaluated. Moreover, angiogenesis, glucose metabolism, the epithelial-mesenchymal transition (EMT), reactive oxygen species (ROS), and STAT3 activity were analyzed.

Results
In PCCs, fraxetin inhibited the proliferation, induced mitochondrial-dependent apoptosis, and suppressed the invasion and migration by reversing the EMT. In nude mouse models, PDA growth and metastasis were reduced by fraxetin treatment. Moreover, fraxetin enhanced the sensitivity of PCCs to the chemotherapy drug gemcitabine. Mechanically, oncogenic KRAS-triggered STAT3 activation in PCCs and PDA tissues was suppressed by fraxetin treatment. Fraxetin shows important interactions with STAT3 Src Homology 2 (SH2) domain residues to occupy the pTyr-recognition site of its SH2 domain of another STAT3 monomer, thereby preventing its homo-dimer formation, which then blocks the activation of downstream signal pathways. The anti-tumor activity of fraxetin in PDA was functionally rescued by a STAT3 activator colivelin. As a result, fraxetin hindered hypoxia-induced angiogenesis by decreasing HIF-1α and VEGFA expression, controlled glucose metabolism by reducing GLUT1 expression, inhibited the EMT by blocking the Slug-E-cadherin axis, and drove ROS-mediated apoptosis by regulating STAT3-Ref1 axis.

Conclusion
These ndings indicate that fraxetin enhances anti-tumor activity of gemcitabine and suppresses pancreatic cancer development by antagonizing STAT3 activation.

Background
Pancreatic ductal adenocarcinoma (PDA) is a highly-malignant tumor of the digestive system with a very poor prognosis and high mortality [1]. However, the underlying mechanism of PDA development remains unclear [2]. Surgery clinically is regarded as the main treatment for PDA. However, only a few PDA patients can receive surgery. Patients who are unable to undergo surgery presently receive a combination Colony formation assay PANC-1 or Patu8988 cells were plated in 6-well plates at a density of 1 × 10 3 cells/well for 24 h, and then were treated with fraxetin. After treatment for 24 h, the culture medium was placed with DMEM for 14 days. Colonies were xed with formaldehyde for 30 min and stained with crystal violet (Sigma-Aldrich).
Finally, cell colonies were counted using a microscope (Leica Microsystems).
Wound healing assay PANC-1 or Patu8988 cells were seeded in 6well plates and maintained at 37˚C for 24 h. The culture area was scratched using a crystal pipette tip to make a linear gap among the cells. Next, the detached cells washed away with PBS and then added different concentrations of fraxetin. Cells were allowed to ll the gap, and after 24 h, images of the culture area were captured using a microscope (Leica Microsystems).
Immunocytochemical staining Immuno uorescence staining was performed according to our previous report [19]. Firstly, PANC-1 and Patu8988 cells treated with fraxetin were grown on glass coverslips for 24 h, and then xed with 4% formaldehyde for 30 min. Secondly, cells were permeabilized with 0.1% Triton X-100, and blocked in 4% normal goat serum for 1.

Glucose metabolism assay
The intact cellular oxygen consumption rate (OCR) and extracellular acidi cation rate (ECAR) were measured using a Seahorse XF96 Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, MA, USA). In short, 1.0 × 10 4 of PANC-1 and Patu8988 cells were seeded into 96-well cell plates and incubated overnight at 37 ℃, 5% CO 2 . Both cells were pretreated with or without different concentrations of fraxetin for 24 hours. Simultaneously, the calibration plates were incubated overnight at 37 ℃ in a non-CO 2 incubator. Then both cell mediums were replaced with assay medium, once the probe calibration was completed, the cell plate replaced the probe plate. The analyzer plotted the value of OCR followed by injection of the compounds sequentially as follows: oligomycin (inhibitor of ATP synthase; 2.5 µM), Carbonyl cyanide 4-(tri uoromethoxy) phenylhydrazone (FCCP, an uncoupler of OXPHOS; 2 µM), rotenone (inhibitor of complex I; 0.25 µM) and anti-mildew A (inhibitor of complex III; 0.25 µM) (n = 8). ECAR was evaluated after continuous injection of glucose (10 mM), oligomycin (1 µM) and 2-Deoxyglucose 2-Deoxyglucose (2-DG, 50 mM) (n = 8). After completing the test, the BCA Protein Assay Kit was performed to determine protein concentration to normalize OCR and ECAR according to the manufacturer's instructions.

Nude mouse tumorigenicity
Male nude mice (BALB/c) weighing 20-22 g and 6-8 weeks old were obtained from the Experimental Animal Centre of Wenzhou Medical University (Wenzhou, China). All mice were housed in a humidity-, temperature-, and light-controlled environment. Mice were randomly divided into two groups and each group contained ve mice. Then the right thigh root of mice was injected subcutaneously with 3 × 10 6 PANC-1 cells. The experimental group received intragastric administration of fraxetin (25 mg/kg·d) every three days for a month. In contrast, the control group received intragastric administration of DMSO.
Tumor formation in mice was monitored for 30 days, and the length and width were measured every three days. Tumor volume was calculated based on the formula V = (length × width 2 )/2, where length was always the longest dimension of the tumor. At the end of the experiment, all the mice were euthanized by CO2 asphyxiation. This animal study was approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University, China. The animal experiments were conducted according to all regulatory institutional guidelines for animal welfare (National Institutes of Health Publications, NIH Publications No. 80 − 23) [20].

Histopathological analysis
Tumor specimens from animals embedded in para n were cut into 4-µm sections and stained with hematoxylin and eosin (HE, Yuanye Biotechnology, Shanghai, China). Immunohistochemical (IHC) analysis was performed according to a previous report [18]. In brief, 4-µm thick sections were dewaxed with xylene and rehydrated in sequential ethanol. Sections were incubated in 0.1% sodium citrate buffer (pH 6.0) for antigen retrieval, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide (Beyotime). IHC staining was performed using the following primary antibodies: anti-Ki67 (1:200, Abcam) and anti-E-cadherin (1:200, Abcam). The integrated optical density (IOD) was measured using Image-Pro Plus software (version 6.0, Media Cybernetics, Silver Spring, MD, USA). All samples were semi-quantitatively or quantitatively assessed by two independent investigators in a blinded manner.

Western blot analysis
Whole proteins from the normal pancreatic ductal cell (hTERT-HPEN) and PCCs (PANC-1, Patu8988 and BxPc-3) or PDA tissues treated with fraxetin for 24 h were collected and protein concentrations were determined by double star choline acid protein analysis kit (Beyotime). Total protein (20 µg) of each sample was separated by SDS-PAGE and then was transferred to a polyvinylidene di uoride membrane (PVDF, Solarbio, Beijing, China). After blocking the membranes for 1 h at room temperature with 5% skim nonfat milk, membranes were incubated with the primary antibodies (1:1000), including anti-caspase-3 Finally, the protein bands were visualized using chemiluminescence detection on autoradiographic lm and the GADPH antibody (1:8000, Proteintech) was used as the internal reference.
ROS level assay ROS level assay was performed according to the manufacturer's instructions (Beyotime). In brief, PANC-1 or Patu8988 cells were seeded in a 6-well plate with a density of about 5 × 10 4 /well. After the cells adhere to the wall, cells were treated with fraxetin for 24 h. On the second day, 2 ml of the dilution (DCFH-DA, 1:1000, Beyotime) was added to each well, incubated at 37 ℃ for 30 min, and then the cells were collected. Images were captured using a uorescence microscope (Leica Microsystems). In addition, the cells were also resuspended in PBS, and ROS level was detected by ow cytometry (BD Biosciences).

Database analysis
The expression of STAT3 in PDA tissues and adjacent normal tissues in the GEPIA 2 database website (http://gepia2.cancer-pku.cn/#analysis) was analyzed. Besides, the correlation between STAT3 expression and KRAS activity was also evaluated in the GEPIA 2 database. Moreover, the potential target candidates were analyzed using the PharmMapper Server (http://www.lilabecust.cn/pharmmapper/index.html) according to a previous report [21].

Molecular docking
Fraxetin structure was downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov) as SDF les and converted into mol2 format using Chimera. The STAT3 crystal structure was downloaded from PDB no. 6NJS as a model (http://www.rcsb.org). Compound and protein were converted into PDBQT format using AudoDockTools and docked using AutoDock Vina. Visualize conformations and interactions were prepared by PyMol, Chimera, and LigPlot+.

Statistical analysis
Data were presented as the means ± standard deviations. All statistical analyses were performed using GraphPad Prism software (version 8.0, GraphPad Software, Inc., La Jolla, CA, USA). A two-sided Student's t-test was used to analyze differences between the two groups. One-way analysis of variance (ANOVA) with Bonferroni's post-hoc test was used when more than two groups were present. P < 0.05 was considered statistically signi cant.

Fraxetin inhibits cellular proliferation and induces mitochondrial-dependent apoptosis in PCCs
To investigate the effects of fraxetin on PCC proliferation, unlabeled real-time cell analysis (RTCA), CCK-8 assay and colony formation assay were performed. As shown in Fig. 1B, the viability of PANC-1 cells incubated for 24 h gradually decreased when the concentration of fraxetin (0 ~ 200 µM) gradually increased in a concentration-dependent manner. Similarly, CCK-8 results of Patu8988 cells also con rmed the concentration-dependent effects of fraxetin (Fig. 1B), especially when the concentration of fraxetin is 25 ~ 100 µM. Thus, we selected 50 and 100 µM of fraxetin for subsequent anti-proliferative studies. Based on the RTCA results, the growth of PANC-1 and Patu8988 cells was markedly inhibited by fraxetin treatment, and it was time-dependent (Fig. 1C). Moreover, fraxetin signi cantly reduced the number of colony formation in PANC-1 and Patu8988 cells (Fig. 1D). These results supported the anti-proliferative activity of fraxetin in PCCs.
A previous study has shown that Ki67 (also known as MKI67) is a cellular marker for proliferation and is highly expressed in PDA tissues [22]. Ki67 is present during all active phases of the cell cycle (G1, S, G2 and M), but is absent in resting cells (G0) and the cellular content of Ki67 protein markedly increases during PCC progression through the S phase of the cell cycle [23]. In this study, the ratio of Ki67-positive cells in total cells was reduced in fraxetin-treated PANC-1 and Patu8988 cells compared with the control (Fig. 1E). Taken together, these results suggested that fraxetin has proliferation-inhibitory activities on PCCs and it is time-and concentration-dependent.
Given fraxetin has anti-proliferative activity in PCCs, whether fraxetin can induce cellular apoptosis. The apoptotic PCCs were examined by ow cytometry to detect labeled Annexin V-FITC/PI. As expected, we found that fraxetin concentration-dependently increased the ratio of early apoptotic cells but also enhanced the ratio of late apoptotic and necrotic cells (Fig. 1F). Caspases, a family of cysteine proteases, are the central regulators of apoptosis. As a key protease, caspase-3 is cleaved and activated during the early stage of apoptosis to execute apoptosis by cleaving targeted cellular proteins [24]. In this study, the expression of cleaved caspase-3 was upregulated by fraxetin treatment (Fig. 1G). Besides, fraxetin also increased the expression of cleaved caspase-8 ( Fig. 1G), which can be used as an apoptotic initiator to activate caspase-3 [25]. Moreover, enhanced Bax expression and reduced Bcl-2 expression in fraxetintreated PANC-1 and Patu8988 cells were observed (Fig. 1G). Collectively, these ndings indicated that fraxetin induces apoptosis of PCCs through a mitochondrial-dependent pathway.

Fraxetin inhibits PCC invasion and migration via Slug-Ecadherin-dependent EMT
Next, we evaluated the effects of fraxetin on the invasion and migration of PCCs by transwell invasion and wound healing assay. As shown in Fig. 2A, in PANC-1 and Patu8988 cells, fraxetin treatment signi cantly reduced the number of invasive cells in a concentration-dependent manner. In addition, the migration of PANC-1 and Patu8988 cells was inhibited by fraxetin, and it is time-and concentrationdependent (Fig. 2B). Further study showed that the suppressive effect of fraxetin on the invasion and migration of PCCs might be related to the inhibition of epithelial-mesenchymal transition (EMT). EMT is a developmental process that plays an important role in PDA progression and metastasis [26]. The EMT is characterized by the loss of cell-to-cell adhesion and is associated with the phenotypic conversion of epithelial cells into mesenchymal-like cells [27]. E-cadherin is the major adhesion protein associated with epithelial cells and is considered an active suppressor of invasion and growth of PDA [28]. Currently, EMT-associated transcription factor Slug (Snail2), a member of the Snail superfamily, is known to directly bind to the E-boxes of E-cadherin gene promoter and repress its transcription [29]. Thus, we evaluated the expression of E-cadherin and Slug in fraxetin-treated PCCs. E-cadherin expression was enhanced in PANC-1 and Patu8988 cells upon fraxetin treatment (Fig. 2C,D). In addition, fraxetin decreased the expression of matrix components, including α-SMA, N-cadherin and Vimentin (Fig. 2C,D). Moreover, fraxetin reduced Slug expression (Fig. 2C). Thus, these data reveal an inhibitory effect of fraxetin on the invasion and migration of PCCs by regulating the EMT process dependent on the Slug-E-cadherin axis.
Fraxetin inhibits tumor growth and metastasis of PDA in animal xenograft models Given fraxetin inhibited the proliferation, migration and invasion, and induced apoptosis of PANC-1 and Patu8988 cells in vitro, whether the similar anti-tumor mechanism of fraxetin occurred in nude mice of PDA xenografts in vivo. Fraxetin (25 mg/kg·d) was administered continuously for 30 days to the mice subjected to the injection of PCCs. Figure 3A showed morphologic changes in the experimental groups as a result of fraxetin treatment. We found that fraxetin administration signi cantly reduced the volume and weight of the tumor (Fig. 3B,C). Evidence from HE-staining showed PDA pathological results in tissues of the model group (Fig. 3D). In addition, the decreased proportion of Ki67-positive cells in total cells in fraxetin-treated models showed the inhibition of tumor cell proliferation (Fig. 3E). Further studies showed that fraxetin administration enhanced the expression of cleaved caspase-8 and cleaved caspase-3 ( Fig. 3F,G). Moreover, fraxetin upregulated Bax expression and downregulated Bcl-2 expression (Fig. 3F,G). Thus, our in vivo experiments showed that fraxetin induced PCC apoptosis in a mitochondrial-mediated manner.
To access whether fraxetin suppressed PDA metastasis, we examined the expression of EMT-related proteins. Immunohistochemical analysis showed that fraxetin signi cantly increased E-cadherin expression (Fig. 3H). The enhanced expression of E-cadherin was con rmed by Western Blot results Fraxetin enhanced the sensitivity of PCCs to gemcitabine Gemcitabine is currently the rst-line drug for PDA chemotherapy, but chemotherapy resistance is widespread and has become the main reason for the failure of PDA chemotherapy [30]. Therefore, it is crucial to inhibit gemcitabine resistance and promote its anti-PDA e cacy. Here, evidence from colony formation assay showed that gemcitabine has a certain inhibitory effect on the proliferation of PCCs, and this effect can be enhanced by fraxetin (Fig. 4A,B). Besides, fraxetin treatment also strengthens the inhibitory effects of gemcitabine on the in ltration and invasion of PCCs (Fig. 4C-F) by downregulating Ncadherin expression and upregulating Vimentin expression (Fig. 4G,H). Thus, these ndings indicated that fraxetin could enhance the sensitivity of PCCs to the chemotherapy drug gemcitabine. Fraxetin induced STAT3 inactivation by occupying its SH2 domain in vitro and in vivo A previous study has shown that STAT3 is involved in fraxetin-mediated inhibition in the proliferation of lung cancer cells [17]. In PDA, KRAS mutation, including G12D and G12V, drives STAT3 activation [13], which was consistent with our conclusion (Fig. 5A-C). Moreover, over-activation of STAT3 induces the chemoresistance of PCCs to the chemotherapy drug gemcitabine [31]. Thus, we hypothesized that fraxetin exerts its protective effects by targeting STAT3 activity in PANC-1 (KRAS G12D) and Patu8988 (KRAS G12V) cells. Although there was no signi cant difference in the expression of JAK2 and STAT3 protein between the fraxetin-treated group and the control group, the phosphorylation levels of JAK2 (Y1007) and STAT3 (Y705) were signi cantly suppressed by fraxetin treatment (Fig. 5D). Considering that the activation of STAT3 is due to the nuclear expression of phosphorylated STAT3, we detected the nuclear localization of STAT3 by immunocytochemical staining. The results showed that the nuclear expression of STAT3 in PANC-1 and Patu8988 cells was inhibited by fraxetin (Fig. 5E). Furthermore, in animal xenograft models, fraxetin administration suppressed the phosphorylation of JAK2 and STAT3 ( Fig. 5F). Taken together, our in vitro and in vivo experiments showed that inhibition of STAT3 activity might be critical for fraxetin-mediated anti-tumor effects in PDA.
As a result, fraxetin has placed at the centre SH2 domain to occupy the pTyr-recognition site of its SH2 domain of another STAT3 monomer, thereby preventing its homo-dimer formation, which then inhibits the phosphorylation of STAT3 and blocks the activation of STAT3-downstream signaling pathways.

Reactivation of STAT3 reverses the anti-tumor effects of fraxetin in PDA
Since fraxetin exerts its protective effects on PDA by targeting STAT3 activity, whether reactivation of STAT3 can rescue the inhibitory effects of fraxetin. In this study, colivelin, a neuroprotective peptide with brain permeability, was used to activate STAT3 [32]. Firstly, evidence from colony formation assay showed that colivelin increased the number of colonies, revealing that reactivated STAT3 relieved fraxetin-mediated inhibition of PCC proliferation (Fig. 6A). In addition, colivelin enhanced the number of invasive cells in fraxetin-treated PANC-1 and Patu8988 cells, indicating that colivelin-induced overactivation of STAT3 reversed fraxetin-mediated inhibition of PCC invasion (Fig. 6B). Furthermore, the ability of cell mobility in fraxetin-treated PANC-1 and Patu8988 cells was strengthened by colivelin treatment, suggesting that upregulated STAT3 activity by colivelin abolished the inhibition of PCC migration caused by fraxetin (Fig. 6C). Thus, these results con rmed again that reactivation of STAT3 by colivelin enhances the biological behavior of PCCs upon fraxetin treatment, and the STAT3 is crucial for the inhibitory effect of fraxetin on PDA.
Next, we evaluated the effects of STAT3 reactivation on the EMT. In fraxetin-treated PANC-1 and Patu8988 cells, colivelin reduced E-cadherin expression, as determined by Western blot and immunocytochemical staining (Fig. 6D,E), revealing that epithelial phenotypic features are suppressed by reactivation of STAT3. Further study showed that Slug expression in fraxetin-treated PANC-1 and Patu8988 cells was increased by colivelin treatment (Fig. 6D), thereby indicating that STAT3 reactivation abolished fraxetin-mediated EMT inhibition. Moreover, in fraxetin-treated PANC-1 and Patu8988 cells, colivelin upregulated the expression of N-cadherin and type I collagen (Fig. 6F), as well as vimentin (Fig. 6G), suggesting that STAT3 reactivation induced excessive accumulation of matrix. Collectively, STAT3 reactivation by colivelin reverses the anti-tumor effects of fraxetin on PDA.

Fraxetin inhibited angiogenesis, glucose metabolism and EMT in PCCs
To further clarify the mechanism by which fraxetin exerts its protective effects on PDA by inhibiting STAT3 activation, we analyzed the expression and release of downstream target molecules of this signaling. STAT3 downstream molecules, including Bcl-2, E-cadherin, hypoxia-inducible factor-1α (HIF-1α), and vascular endothelial growth factor-α (VEGFA), are involved in the regulation of cellular proliferation, apoptosis, angiogenesis, and metastasis [10,33]. As mentioned before, reduced Bcl-2 expression in fraxetin-treated PCCs indicated mitochondrial pathway-dependent apoptosis (Fig. 1G). Also, fraxetin inhibits the EMT process by blocking the Slug-E-cadherin axis (Fig. 2C,D). In addition to these, fraxetin-mediated downregulation in the expression levels of HIF-1α and VEGFA revealed the inhibition of hypoxia-induced angiogenesis (Fig. 7A-C). Moreover, the levels of oxygen consumption rate (OCR) in PANC-1 and Patu8988 cells were downregulated by fraxetin treatment by inhibiting basal respiration, spare respiration, maximal respiration, and ATP production ( Fig. 7D-G). The levels of extracellular acidi cation rate (ECAR) were decreased by fraxetin by antagonizing basal glycolysis and compensatory glycolysis (Fig. 7H-K). Importantly, reduced expression of glucose transporter type 1 (GLUT1), a uniporter protein that facilitates the transport of glucose across the plasma membranes of mammalian cells for metabolism [34], in fraxetin-treated PCCs was responsible for the reduction of glucose metabolism (Fig. 7L). Thus, these ndings indicated that in PDA fraxetin hinders hypoxia-induced angiogenesis by decreasing the expression levels of HIF-1α and VEGFA, controls glucose uptake and metabolism by reducing GLUT1 expression, and inhibits the EMT by blocking the Slug-E-cadherin axis.
Fraxetin drove ROS-mediated apoptosis by regulating STAT3-Ref1 axis As mentioned above, fraxetin induces cell apoptosis via Bcl-2-mediated mitochondrial pathway. In PDA, Bcl-2 expression can be regulated by ROS [35]. In this study, we found that the levels of ROS were signi cantly increased in fraxetin-treated PCCs (Fig. 8A-C), suggesting that fraxetin-induced apoptosis might be mediated by ROS. Further study revealed that downregulated Ref1 expression was involved in ROS-triggered apoptosis of PCCs when exposed to fraxetin (Fig. 8D,E). Ref1 is an important target molecule of STAT3 that can regulate ROS levels [36]. We also found that Ref1 expression was positively associated with STAT3 activity in PDA (Fig. 8F). Reactivation of STAT3 with colivelin abolished fraxetininduced downregulation of Ref1 expression in PDA (Fig. 8G). Thus, these results indicated that fraxetin drove ROS-mediated apoptosis by regulating the STAT3-Ref1 axis.

Discussion
In the present study, we investigated for the rst time the anti-tumor effects of fraxetin on pancreatic ductal adenocarcinoma (PDA) in vitro and in vivo. In pancreatic cancer cells (PCCs), fraxetin inhibited cell proliferation by downregulating Ki67 expression. In addition, fraxetin induced PCC apoptosis through the mitochondrial-mediated pathway. Moreover, fraxetin suppressed the invasion and migration of PCCs by regulating epithelial-mesenchymal transition (EMT) dependent on the Slug-E-cadherin axis. In nude mouse models, PDA growth and metastasis were reduced by fraxetin treatment. Thus, these evidence supported that fraxetin exerts effective protection against PDA.
PDA, which accounts for over 90% of pancreatic cancer, is an aggressive type of malignant tumor with an overall 5-year survival rate of less than 8% [1]. PDA can be hard to treat surgically due to the location of the organ, and because the disease has often spread in the body by the time it is diagnosed. Thus, developing effective drugs for treating PDA is still needed. Recently, multiple studies show that several natural products may become novel candidate agents for developing PDA therapeutics [37,38]. Fraxetin is a plant-derived coumarin primarily isolated from Fraxinus bungeana A.DC., and has been reported to exert potent anti-bacterial, neuroprotective and anti-in ammatory effects [14]. In breast cancer cells, fraxetin inhibits cellular proliferation, induces mitochondrial-dependent apoptosis by upregulating Bax expression and downregulating Bcl-2 expression [15]. Also, fraxetin treatment causes a signi cant cell cycle arrest and pro-apoptotic effects in non-small-cell lung cancer cells [17]. Similar to the above studies, our in vivo and in vitro experiments also identi ed anti-tumor effects of fraxetin in PDA by inducing PCC apoptosis, inhibiting proliferation, and reducing tumor growth. Moreover, fraxetin prevented PDA progression by suppressing the invasion and migration of PCCs. Thus, our results provide a rationale for the use of fraxetin as a potential supplemental treatment for PDA.
Interestingly, we found that fraxetin can enhance the sensitivity of PCCs to the chemotherapy drug gemcitabine. Gemcitabine is accepted as the standard treatment for patients with locally or metastatic advanced PDA, but the problem of its drug resistance has become increasingly prominent [39]. How to solve the weakening of its anti-tumor effect due to drug resistance is an important task now. For this reason, drugs such as human recombinant hyaluronidase (PEGPH20) or CD40 monoclonal antibody (CP-870,893) have entered clinical studies to increase the e cacy of gemcitabine against advanced PDA [40,41]. Our results identi ed fraxetin as an adjuvant drug to strengthen the anti-tumor effect of gemcitabine by inhibiting the EMT and PCC proliferation. Therefore, fraxetin may be a potential drug development candidate for PDA treatment.
Further studies have shown that STAT3 activity is responsible for fraxetin-mediated anti-tumor effects [11][12][13]. Although STAT3 is dispensable for the development of the pancreas, the majority of PDA show constitutive activation of STAT3 by phosphorylation at Y705 and induction into the nucleus [13]. Importantly, STAT3 activation is mainly driven by oncogenic KRAS mutation, which is found in ~ 95% of pancreatic intraepithelial neoplasias (PanINs), the earliest pre-neoplastic stages of PDA progression [13,42,43]. Thus, targeted inhibition of STAT3 might be a potential therapeutic strategy for PDA. AG-409, a tyrosine kinase inhibitor that inhibits STAT3 activation, decreased the invasion and metastasis ability of PDA [44]. In addition, novel small-molecule STAT3 inhibitors FLLL31 and FLLL32, which are derived from curcumin, inhibited multiple oncogenic processes and induced apoptosis in PCC lines, and reduced tumor growth and vascularity in PDA mouse xenografts [45]. Our ndings also supported STAT3 as a key target for the anti-tumor activity of fraxetin. Fraxetin treatment not only inhibited the phosphorylation (Y1007) of JAK2 in PCCs, but also antagonized the phosphorylation (Y705) and nuclear localization of STAT3.
Furthermore, reactivating STAT3 with colivelin relieved fraxetin-mediated induction of apoptosis, and inhibition of proliferation, invasion and migration of PCCs, thereby enhancing the biological behavior of PCCs upon fraxetin treatment.
Fraxetin binds to STAT3 SH2 domain residues by hydrogen bonding with Arg609, Ser611, glu612, Ser613, Thr620 and Val637 residues. It also creates a favorable group of a hydrophobic pocket formed by Ser636, Glu638 and Pro639. Thus, fraxetin occupies the pTyr-recognition site of STAT3 SH2 domain to prevent its homo-dimer formation, and thereby suppresses the phosphorylation of STAT3 and blocks the activation of STAT3-downstream signal pathways.
In PDA initiation and development, STAT3 exerts its important physiological function through a series of downstream target molecules. The STAT3-downstream molecules, including Bcl-2, E-cadherin, HIF-1α, and VEGFA, play key roles in the regulation of proliferation, apoptosis, angiogenesis, and metastasis of PCCs [10,33]. E-cadherin is a calcium-dependent cell adhesion protein involved in mechanisms regulating cell- In addition to the direct regulation of Bcl-2 expression by STAT3, we also found that fraxetin-induced apoptosis was mediated indirectly by ROS. ROS triggers mitochondrial apoptosis pathway by regulating Bcl-2 expression [35]. Further study revealed that reduced Ref1 expression contributed to ROS-triggered apoptosis of PCCs when exposed to fraxetin. As a target molecule of STAT3, Ref1 plays an important role in the regulation of ROS levels and oxidative stress [36]. Colivelin was used to reactivate STAT3 and thereby resulted in the abolishment of fraxetin-induced downregulation of Ref1 expression in PDA. These results indicated that fraxetin drove ROS-mediated apoptosis by regulating the STAT3-Ref1 axis.
However, there exist some apparent limitations. First, a genetic approach to evaluate the role of STAT3 in fraxetin-mediated anti-tumor effects needs to be presented. In addition, the mechanism of KRAS-mutation (G12D or G12V) affects the anti-tumor effects of fraxetin should be clari ed. It is also worth mentioning that the anti-tumor potential of fraxetin in clinical application, including pharmacokinetics and its side effects, needs to be further clari ed.

Conclusions
Fraxetin inhibited PCC proliferation, induced mitochondrial-mediated apoptosis, and suppressed the invasion and migration by regulating EMT. In nude mouse models, PDA growth and metastasis were reduced by fraxetin treatment. Moreover, fraxetin strengthens the anti-tumor effect of gemcitabine by inhibiting PCC proliferation and EMT. Mechanically, oncogenic KRAS-triggered STAT3 activation in PCCs and PDA tissues was suppressed by fraxetin via occupying STAT3 SH2 domain. Consequently, fraxetin hindered hypoxia-induced angiogenesis by decreasing HIF-1α and VEGFA expression, controlled glucose metabolism by reducing GLUT1 expression, inhibited the EMT by blocking the Slug-E-cadherin axis, and drove ROS-mediated apoptosis by regulating STAT3-Ref1 axis. Reactivation of STAT3 reverses the antitumor effects of fraxetin (Fig. 9). Thus, pharmacological inhibition of STAT3 activity with fraxetin suppresses oncogenesis and development of PDA by antagonizing STAT3 activation, and fraxetin can be a therapeutic approach to PDA.

Consent for publication
The authors declare that they agree to submit the article for publication.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Figure 9
Fraxetin enhances the anti-tumor e cacy of gemcitabine and suppresses oncogenesis and development of PDA via antagonizing STAT3 activation.

Figure 9
Fraxetin enhances the anti-tumor e cacy of gemcitabine and suppresses oncogenesis and development of PDA via antagonizing STAT3 activation.