The Isoflavone Puerarin Exerts Anti-Tumor Activitiy in Pancreatic Ductal Adenocarcinoma by Suppressing Akt/mTOR Activity

The Isoflavone Puerarin Exerts Anti-Tumor Activitiy in Pancreatic Ductal Adenocarcinoma by Suppressing Akt/mTOR Activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research The Isoflavone Puerarin Exerts Anti-Tumor Activitiy in Pancreatic Ductal Adenocarcinoma by Suppressing Akt/mTOR Activity Hengyue Zhu, Hong Lu, Yanyi Xiao, Hangcheng Guo, Yangyang Guo, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-152446/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Puerarin (7,4’-dihydroxyisoflavone-8-β-glucopyranoside) is a natural flavonoid compound isolated from the traditional Chinese herb Radix puerariae . Recent studies have demonstrated that puerarin has potential anti-tumor effects via induction of apoptosis and inhibition of proliferation. However, the effect and molecular mechanism of puerarin in pancreatic ductal adenocarcinoma (PDAC) remains unknown. Methods: The effects of puerarin on the proliferation, apoptosis, migration and invasion of pancreatic cancer cells (PCCs), and tumor growth and metastasis in PDAC xenograft mouse model were performed. In addition, Akt/mTOR signaling activity was evaluated both in vivo and in vitro . Results: Puerarin treatment significantly repressed PCC proliferation in concentration- and time-dependent manners. Puerarin induced the mitochondrial-dependent apoptosis of PCCs by causing a Bcl-2/Bax imbalance. Moreover, puerarin inhibited PCC migration and invasion by antagonizing epithelial-mesenchymal transition (EMT). In nude mouse model, PDAC growth and metastasis were reduced by puerarin administration. Mechanistically, puerarin exerted its therapeutic effects on PDAC by suppressing Akt/mTOR signaling. Importantly, puerarin bound to the kinase domain of mTOR protein, affecting the activity of the surrounding amino acid residues associated with the binding of the ATP-Mg 2+ complex. Further studies showed that the inhibitory effects of puerarin on PCCs were abolished by a mTOR activator MHY1485, indicating a crucial role of mTOR in anti-tumor effects of puerarin in PDAC. As a result, puerarin hindered glucose uptake and metabolism by downregulating the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) dependent upon HIF-1α and glucose transporter GLUT1. Conclusion: Puerarin has therapeutic potential for the treatment of PDAC by suppressing Akt/mTOR activity. Cancer Biology Puerarin Pancreatic ductal adenocarcinoma Glucose metabolism Akt/mTOR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Pancreatic ductal adenocarcinoma (PDAC) is the most common exocrine pancreatic cancer seen clinically. It is the fourth-leading cause of cancer-related death in the United States, second only to colorectal cancer in gastrointestinal-related deaths [ 1 ]. According to the World Health Organization (WHO) GLOBOCAN database and the 2017 Global Burden of Disease Study, PDAC is the seventh leading cause of cancer death in men and women worldwide [ 2 ]. Surgical resection is the only possible cure. Unfortunately, due to the late discovery, only 15–20% of PDAC patients are eligible for a pancreatectomy. However, even after complete resection, the prognosis of PDAC patients is poor. After resection margin-negative (R0) pancreaticoduodenectomy, the five-year survival rate of PDAC patients is about 30% for lymph node-negative and 10% for lymph node-positive patients [ 3 , 4 ]. The median survival of patients with untreated and unresectable locally advanced PDAC is 8–12 months, while the median survival of patients with metastatic disease at presentation is only 3–6 months. Systemic chemotherapy can improve the survival rate of patients with locally advanced and metastatic PDAC. In today’s modern treatment era, the FOLF NO × regimen (fluorouracil + leucovorin, irinotecan, and oxaliplatin) has achieved the best outcome, but the median patient survival time is only 11.1 months [ 5 ]. New drugs, new drug targets, and new, more effective chemotherapy regimens are desperately needed in all settings. Puerarin is a white crystal extracted from the roots of the kudzu plant or the kudzu vine. Its chemical name is 7,4’-dihydroxyisoflavone-8-β-glucopyranoside, and its molecular formula is C 21 H 20 O 9 [ 6 ]. Puerarin is the most abundant secondary metabolite, which was isolated from the rhizome of Pueraria lobata in the 1950s and is known as Asian ginseng. Since then, extensive research has been conducted on its pharmacological properties. Puerarin has various pharmacological effects, such as enhancing circulatory system function, reducing myocardial oxygen consumption, decreasing blood sugar, and preventing hypertension and arteriosclerosis. Anti-liver toxicity, anti-inflammatory, expectorant, antipyretic, immunity-enhancing, antibacterial, and antiviral activities have also been demonstrated [ 7 – 9 ]. Its low toxicity and wide range of pharmacological effects have attracted the attention of domestic and foreign researchers. In recent years, the anti-cancer effect of puerarin has been widely studied. Many studies showed that puerarin had good anti-tumor activity in animal model and many cancer cell lines [ 10 ]. However, the role of puerarin in PDAC has not been studied in-depth and needs to be further explored. In this study, we used in vitro and in vivo experiments to investigate the anti-tumor effects of puerarin. Real-time cell analysis (RTCA), the Cell Counting Kit-8 (CCK-8) assay, colony formation, and flow cytometry analysis were used to analyze the effects of puerarin on the proliferation and apoptosis of PCCs. The transwell invasion assay, the wound healing assay, and immunocytochemical staining were used to evaluate the effects of puerarin on cell migration and invasion, and the epithelial-mesenchymal transition (EMT) of PCCs. Moreover, the effects of puerarin on the activity of Akt/mTOR and glucose metabolism were also investigated. We found that puerarin inhibited the proliferation of PCCs, induced mitochondrial-dependent apoptosis, and suppressed invasion and migration by reversing EMT. In nude mouse model, PDAC growth and metastasis were reduced by puerarin treatment. Mechanically, the activity of Akt/mTOR in PCCs and PDAC tissue was suppressed by puerarin treatment via binding to the kinase domain of mTOR protein, resulting in the inhibition of glucose metabolism by decreasing HIF-1α and GLUT1 expression. Further studies showed that the small molecule activator of mTOR, MHY1485, eliminated the puerarin-mediated inhibition of PCC proliferation and EMT induction. Thus, puerarin can be a therapeutic approach to PDAC. Methods Cell culture and drug treatment Human PCC lines PANC-1 and PATU-8988T were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (Invitrogen). The cultured cells at a density of 1 × 10 6 were initially plated in a 10-cm dish for 24 h. After incubation for 24 h, the culture medium was replaced with serum-free medium. PANC-1 and PATU-8988T cells were treated with 0.2 and 0.5 mM puerarin (Fig. 1 a, CAS#: 3681-99-0, Purity: ≥ 98% by HPLC, Yuanye Biotechnology, Shanghai, China) with or without MHY1485 (CAS#: 326914-06-1, MedChem Express, Monmouth Junction, NJ, USA). Cell counting Kit-8 (CCK-8) assay The CCK-8 assay kit (Dojindo, Shanghai, China) was used to detect the anti-tumor activity of puerarin in PANC-1 and PATU-8988T cells according to the manufacturer’s instructions. First, the cells were cultured in 6-cm dishes with fresh medium for 24 h. The cells in the logarithmic growth stage were inoculated into 96-well plates at a density of 5 × 10 3 cells/ml. Then, the cells were treated with different concentrations of puerarin for 24 h. After that, 10 µl of CCK-8 medium and 10 µl of CCK-8 were added and the plates were incubated for another 4 h. The absorbance was measured at a wavelength of 450 nm using a microplate reader. Statistical analyses were performed using Stata statistical software (StataCorp LP). Each experiment was repeated thrice and the average value was taken as the final result. Flow cytometry analysis The cells were serum-starved for 24 h and the medium was replaced with complete medium. PANC-1 and PATU-8988T cells were exposed to culture medium containing different concentrations of puerarin for 24 h, and cells in the standard control group were treated with dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA). After centrifugation to collect the cells, quantification of the apoptotic cells was performed using an Annexin V-FITC Apoptosis Detection Kit (Multisciences, Hangzhou, China) according to the manufacturer’s instructions. Cell apoptosis was assessed by flow cytometry (Ex = 488 nm; Em = 530 nm, BD FACSVerse™, BD Biosciences, San Jose, CA, USA), and the results were analyzed using FlowJo (TreeStar, Ashland, OR, USA). Real-time cellular analysis (RTCA) Cell proliferation was monitored by the xCELLigence RTCA MP System (ACEA Biosciences, San Diego, CA, USA) using 16-well E-Plates (ACEA Biosciences). The cells were seeded in triplicate at 5 × 10 3 cells/well in the plates. For the RTCA experiments, the cells were treated with puerarin after reaching steady growth (24 h). Impedance was measured every 15 min over 96 h and represented as the cell index by the RTCA-integrated software of the xCELLigence System. The cell index was normalized to 1 at the time point of drug administration. From this data, real-time cell growth curves were generated with GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA). Transwell invasion assay Transwell assays were performed using Transwell chambers (Costar, New York City, NY, USA) with Matrigel® (BD Biosciences). After treatment with various concentrations of puerarin for 24 h, cell suspensions were prepared using ethylenetetraacetic acid (EDTA) enzyme. The cells were resuspended in serum-free medium and transferred to the inner chamber (5×10 4 cells per chamber). Complete medium was added to the outer chamber, and the plate was incubated in a CO 2 incubator (37 ℃) for observation for 12 h. After carefully removing the non-migrating cells at the membrane site with a cotton swab, the cells were fixed with formaldehyde and stained with 0.1% crystal violet (Sigma), and quantification was performed by counting five random fields under the microscope (Leica Microsystems, Wetzlar, Germany). Each experiment was repeated three times. Colony formation assay The cells were seeded into 6-well plates at 1 × 10 3 cells per well and treated with puerarin 24 h later. After 24 h, the media was replaced with fresh media and cultured for 14 days. The colonies were then fixed with 2% formaldehyde and stained with 0.5% crystal violet. The number of colonies with ≥ 50 cells was counted under a microscope. Wound healing assay PANC-1 or PATU-8988T cells were seeded in 6 well plates and maintained at 37 ℃ for 24 h. The cells were scratched using a crystal pipette tip to make a linear gap. Next, the detached cells were washed away with phosphate-buffered saline (PBS) and different concentrations of puerarin were added. The cells were allowed to fill the gap, and after 24 h, images of the areas were captured using a microscope (Leica Microsystems). Immunocytochemical staining Immunofluorescence staining was performed based on established protocols. PANC-1 and PATU-8988T cells with different treatments were grown on glass coverslips for 24 h. The cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100 (Thermo Scientific, Waltham, MA, USA). Blocking was performed with 4% goat serum (Gibco, Thermo Fisher Scientific) in Dulbecco’s phosphate-buffered saline (DPBS; Invitrogen, Paisley, UK) for 1.5 h at 37 ℃, followed by incubation with the primary antibodies (Table S1): anti-Ki67 (1:200), anti-α-SMA (1:200), anti-E-cadherin (1:200), and anti-p-mTOR (1:200) at 4℃ overnight. Next, the membranes were incubated in the appropriate second antibodies for 1 h at room temperature. At least three independent experiments for immunofluorescence staining were conducted. Western blot analysis After treating the cells for 24 h, the cells in each group were collected and the total cellular protein was extracted. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated overnight at 4 ℃ with the primary antibodies (Table S1). The membranes were washed three times in Tris-buffered saline with 0.1% Tween 20 (TBST) the following day and incubated with the second antibody (anti-rabbit IgG) at room temperature for 1 h. After the membranes were rinsed, the protein expression levels were detected by enhanced chemiluminescence (ECL) and visualized by autoradiography. GAPDH was used as the internal reference protein. Glucose metabolism assay The intact cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA). Briefly, 1 ×10 4 PANC-1 and PATU-8988T cells were seeded into 96-well cell plates and incubated overnight at 37 ℃ at 5% CO 2 . Both cells were pretreated with or without different concentrations of puerarin for 24 h. Simultaneously, the calibration plates were incubated overnight at 37 ℃ in a non-CO 2 incubator, then the cell medium was replaced with assay medium. Once the probe calibration was completed, the cell plate replaced the probe plate. The analyzer plotted the OCR value, followed by the injection of the compounds sequentially as follows: oligomycin (an inhibitor of ATP synthase; 2.5 µM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, an uncoupler of OXPHOS; 2 µM), rotenone (an inhibitor of complex I; 0.25 µM), and anti-mildew A (an inhibitor of complex III; 0.25 µM) (n = 8). The ECAR was evaluated after the continuous injection of glucose (10 mM), oligomycin (1 µM), and 2-Deoxy-D-glucose (2-DG, 50 mM) (n = 8). After completing the test, the BCA Protein Assay Kit was used to determine the protein concentrations to normalize the OCR and the ECAR according to the manufacturer’s instructions. Nude mouse tumorigenicity Male BALB/c nude mice (6–8 weeks old) were obtained from the Wenzhou Medical University Experimental Animal Center (Wenzhou, China). All mice were housed under controlled conditions (temperature, 21–23 ℃; 12 h light/dark cycle; 55% humidity). PANC-1 cells (3 × 10 6 ) in 0.2 ml PBS were subcutaneously injected into the right thighs of 10 nude mice, which were randomly divided into two groups (n = 5 in each group). Mice in the experimental group received puerarin by intragastric gavage every three days for one month. The control group received DMSO injections for one month. Tumor formation in the nude mice was monitored for 30 days, with the length and width measured every three days. The tumor size was calculated according to the standard formula: tumor volumes (cm 3 ) = (the longest diameter) × (the shortest diameter) 2 × 0.5. The mice were deeply anesthetized with sodium pentobarbital and euthanized by cervical dislocation. 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 and institutional guidelines for animal welfare (National Institutes of Health Publications, NIH Publications No. 80 − 23) [ 11 ]. Histopathological analysis Tumor specimens from the animals were paraffin-embedded and cut into 4-µm-thick sections. Standard hematoxylin and eosin staining (HE, Yuanye Biotechnology) was performed on 4-µm sections from the paraffin-embedded tumor samples. Immunohistochemical (IHC) analysis was conducted under a microscope according to a previous method [ 11 ]. In brief, 4-µm-thick sections were dewaxed with xylene and rehydrated in a graded ethanol series. The 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, China). IHC staining was performed using the following primary antibodies: anti-Ki67 (1:200), anti-c-Myc (1:200), anti-α-SMA (1:200), anti-E-cadherin (1:200), anti-cleaved caspase-8 (1:100), anti-cytochrome C (1:100), and anti-HIF-1α (1:200). 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. Molecular docking Molecular docking was performed as previously described [ 12 ], Puerarin and mTOR were rigidly docked, and the docking results were analyzed by PyMOL software. The puerarin molecule was downloaded from Pubchem, and the molecular energy was optimized through Chem 3D Ultra Software (8.0.3 version, Cambridge-Soft, MA, USA). The crystal structure of mTOR was downloaded from the Protein Structure Database (Protein Data Bank, PDB) ( http://www.rcsb.org/pdb/ ), and the protein was processed by Autodock (MGLTools-1.5.6) to remove water molecules and hydrogenate and to add volume. Database analysis The correlation between AKT and mTOR expression and the activity of KRAS, TP53, CDKN2A, and SMAD4 was evaluated in the GEPIA 2 database website ( http://gepia2.cancer-pku.cn/#analysis ). Statistical analysis The data are expressed as the mean ± standard deviation for the in vitro and in vivo experiments. All statistical analyses were performed using GraphPad Prism statistical analysis software (version 8.0, GraphPad Software, Inc., LaJolla, CA, USA). Statistical comparisons were made with a two-sided t-test. One-way analysis of variance (ANOVA) with Bonferroni’s post-hoc test was used when more than two groups were present. Statistical significance was indicated by a P -value of < 0.05. Results Puerarin inhibits PCC proliferation and induces mitochondria-mediated apoptosis To investigate the effect of puerarin on PCC proliferation, the RTCA, CCK-8, and colony formation assays were performed. As shown in Fig. 1 b and c, as expected, puerarin treatment (0.2 and 0.5 mM) significantly inhibited the growth of PANC-1 and PATU-8988T cells in concentration- and time-dependent manners. The CCK-8 assay results of the PANC-1 and PATU-8988T cells confirmed the concentration-dependent inhibition of cell growth by puerarin (Fig. 1 b, c). Puerarin also significantly reduced colony formation in the PANC-1 and PATU-8988T cells (Fig. 1 d). To investigate the effect of puerarin on cell proliferation, we used immunofluorescence staining for the Ki67 marker expressed by proliferating cells. The level of Ki67 protein varied with the cell cycle and was higher in the G2/M phase and lower in the G0/G1 phase [ 13 ]. In the PANC-1 and PATU-8988T cell lines, we observed a decrease in Ki67 protein expression in both the PANC-1 and PATU-8988T cells treated with puerarin (Fig. 1 e) compared to the control cells. Therefore, the above results suggest that puerarin inhibited PCC proliferation in concentration- and time-dependent manners. Next, we evaluated the effects of puerarin on PCC apoptosis by flow cytometry analysis. Puerarin treatment significantly increased the proportion of apoptotic and necrotic cells (Fig. 1 g). Further studies showed an increase in caspase-8 in both the PANC-1 and PATU-8988T cells treated with puerarin (Fig. 1 h). Apoptosis in cancer cells depends upon the dynamic equilibrium of Bax and Bcl-2 expression [ 14 ]. Puerarin was observed to increase Bax expression and decrease Bcl-2 expression (Fig. 1 h). These results suggest that puerarin induced the death receptor- and mitochondrial-mediated apoptosis of PCCs. Puerarin inhibits the migration and invasion of PCCs by antagonizing epithelial-mesenchymal transition Enhanced cell migration and invasion abilities underlie PCC metastasis mechanisms, resulting in poor prognosis [ 15 ]. Here, puerarin reduced the migration rate of PANC-1 and PATU-8988T cells as determined by the scratch wound assay (Fig. 2 a, b) and the effect was concentration-dependent as well as time-dependent. Also, puerarin treatment significantly inhibited the numbers of invading PANC-1 and PATU-8988T cells detected by the transwell assay (Fig. 2 c, d). Further studies showed that puerarin decreased the protein level of α-SMA in PANC-1 and PATU-8988T cells and increased the E-cadherin protein level (Fig. 2 e). Immunofluorescence analysis revealed the downregulated expression of α-SMA and the increased expression of E-cadherin after puerarin treatment (Fig. 2 f). In general, these results suggest that puerarin inhibited PCC migration and invasion by inhibiting EMT and tumor mammosphere formation. Puerarin suppresses PDAC growth and metastasis in vivo To determine the anticancer effects of puerarin in vivo , nude mice were injected with PANC-1 cells and then administrated puerarin or saline as a control. Figure 3 a shows the morphology of tumor xenografts changes in the experimental group after puerarin treatment. We found that the administration of puerarin significantly reduced tumor volume and weight (Fig. 3 c, d). The pathological results in the PDAC model tissue were shown by HE staining. Further studies showed that puerarin administration upregulated the expression of cleaved caspase-8 (Fig. 3 g, h). Also, puerarin increased Bax expression and decreased Bcl-2 expression (Fig. 3 g). A decrease in the expression of Ki67 was observed in the tumor tissue (Fig. 3 e). Therefore, our in vivo findings suggest that puerarin induced PCC apoptosis through death receptor- and mitochondrial-mediated pathways. To assess whether puerarin inhibited PDAC migration, we examined the expression of EMT process-related proteins. The results showed that puerarin decreased α-SMA expression and increased E-cadherin expression (Fig. 3 i). It also reduced the expression of c-Myc, an oncoprotein associated with tumor progression and drug resistance (Fig. 3 f) [ 16 ]. Hypoxia is usually observed in PDAC and some other solid tumors. HIF-1α protein, a key regulator of the hypoxia response, was found to accumulate in PDAC tissues. Several studies have shown that hypoxia was an independent predictor of poor prognosis [ 17 ]. We observed the downregulation of HIF-1α protein after puerarin treatment (Fig. 3 j). In summary, these data suggest that puerarin inhibited the growth and metastasis of PDAC in a mouse xenograft model. Puerarin reduces the activity of Akt/mTOR signaling in vitro and in vivo Anticancer effects involve many mechanisms, including oxidative stress, intrinsic and extrinsic mechanisms, as well as the survivin, PI3K/Akt/mTOR, SHH [ 18 ], Nrf2/Keap1 [ 19 ], inflammation, and autophagy pathways [ 20 ]. Studies have shown that the signal transduction pathway mediated by phosphatidylinositol 3 kinase (PI3K) was closely related to cancer occurrence. Many downstream molecules make up the PI3K/Akt signal pathway, including mTOR, one of the more important targets of rapamycin. mTOR signaling plays a crucial role in cell growth, protein translation, autophagy, and metabolism [ 21 ]. The activation of mTOR contributes to the pathogenesis of various tumors. We also found that these PCCs exhibited heterogeneous PI3K/Akt/mTOR pathway activation at the protein level (Fig. 4 c). In this study, we investigated the effect of puerarin on mTOR activity in PANC-1 and PATU-8988T cells. We found that puerarin suppressed the mTOR signaling pathway (Fig. 4 d, e), suggesting that mTOR may be a target of puerarin. Puerarin-induced the downregulation of phosphorylated mTOR expression in PANC-1 and PATU-8988T cells (Fig. 4 f, g). The in vitro experiments confirmed that puerarin inhibited the overexpression of mTOR in PDAC tissues (Fig. 4 h, i). Puerarin binds to the kinase domain of mTOR protein to inhibit protein activity To further analyze the biochemical pathways of puerarin affecting mTOR protein, we used Autodock (MGLTools-1.5.6) to rigidly dock puerarin with the FAT domain (blue cartoon) and the kinase domain (KD, green cartoon) areas of mTOR (Fig. 5 a). We found that the possible binding sites of puerarin and mTOR included two structural regions i and ii (Fig. 5 b, c), with binding energies of -5.17 and − 7.0, respectively. Figure 5 d shows that there were many ATP-Mg complex binding-related amino acid residues around the i and ii binding sites. Once puerarin binds to the i and ii sites on mTOR protein, it may affect the activity of the above amino acid residues, and then affect mTOR activation activity. Activated mTOR signaling eliminates puerarin-mediated anti-tumor effects Given the anti-tumor effect of puerarin on PDAC by inhibiting mTOR signal transduction, we next investigated whether activated mTOR signal transduction influenced this effect of puerarin. In the PANC-1 and PATU-8988T cells, we used MHY1485, a significant cell permeability mTOR activator to activate the mTOR pathway, targeting the ATP domain mTOR. The activation of mTOR signaling eliminated the anti-proliferative effect of puerarin as determined by the colony formation test (Fig. 6 a, b). Using the transwell and wound healing assays, we demonstrated that MHY1485 treatment increased the invasion and migration rates of the PANC-1 and PATU-8988T cells (Fig. 6 d-g). Thus, activated mTOR signaling eliminated puerarin-mediated EMT suppression, as shown by the increased expression of α-SMA, vimentin, Snail1, and Slug (Fig. 6 h, i). These findings confirmed that mTOR signaling played a crucial role in the anti-tumor effect of puerarin in PDAC. Puerarin inhibits mTOR-mediated glucose metabolism in PCCs To satisfy the need for rapid proliferation, tumor cells need more energy, so the process of bioenergy metabolism targeting tumor cells is a new therapeutic strategy to inhibit the growth of tumor cells [ 22 ]. A bioenergy analyzer was used to measure the corresponding OCR and ECAR, and the effects of external factors on mitochondrial uptake and glycolysis were analyzed statistically. The primary respiration, ATP production, maximum respiration, and spare respiration of cells treated with puerarin decreased significantly (Fig. 7 a-d), indicating that puerarin inhibited the energy metabolism of the mitochondria. The glycolysis of the tumor cells treated with puerarin was significantly inhibited (Fig. 7 e, g). The results showed that the basal glycolysis rate and the compensatory glycolysis rate decreased significantly (Fig. 7 f, h). Further studies showed that GLUT1 and HIF-1α protein expression was inhibited (Fig. 7 i). Considering the close connection between puerarin and the mTOR pathway, our research results indicate that puerarin may regulate downstream GLUT1 through the mTOR pathway and affect tumor cell metabolism. Discussion Puerarin has certain anti-cancer effects in a variety of tumors. However, its role in PDAC is still poorly understood. In the present study, we showed that puerarin treatment significantly repressed the proliferation of PCCs in concentration- and time-dependent manners. In addition, puerarin induced the mitochondrial-dependent apoptosis of PCCs by causing a Bcl-2/Bax imbalance. Moreover, puerarin inhibited the migration and invasion of PCCs by antagonizing EMT. In the nude mouse model, PDAC growth and metastasis were also reduced by puerarin administration. Thus, these in vitro and in vivo results indicate that puerarin exerted effective protection against PDAC. Previous studies have shown that puerarin impeded cell growth, blocked the cell development in the G0/G1 cell cycle phase, induced apoptosis in bladder cancer cells through the mTOR/p70 S6K signaling pathway, and suppressed cell growth and migration in HPV-positive cervical cancer cells by inhibiting the PI3K/mTOR signaling pathway [ 23 , 24 ]. In addition, puerarin 6’-O-xyloside, an analog of puerarin, suppressed hepatocellular carcinoma by regulating proliferation, stemness, and apoptosis by inhibiting PI3K/Akt/mTOR [ 25 ]. However, the anti-tumor effect and molecular mechanism of puerarin in PDAC remains unknown. Here, we identified effective protection against PDAC by puerarin and showed that the Akt/mTOR signaling pathway played an important role in the anti-tumor effect of puerarin. mTOR protein kinase is involved in many major signaling pathways and plays a key role in organizing the cellular and body physiology of all eukaryotes. In the two and a half years since its discovery, mTOR has been shown to be the central node in the network that controls cell growth. In this way, it integrates information about the availability of energy and nutrients to coordinate the synthesis or decomposition of new cellular components. The dysregulation of this basic signal transduction pathway can disrupt cellular homeostasis and may aggravate the overgrowth of cancer and pathology related to aging and metabolic diseases [ 26 ]. Although mTOR kinase itself is rarely mutated in cancer, it is easily hijacked by upstream oncogenic nodes, including those in the PI3K/Akt pathway and the MAPK pathway driven by Ras. As a result, mTOR signaling is active in as many as 80% of human cancers. In this case, mTOR signaling plays a key role in maintaining the growth and survival of cancer cells [ 27 ]. Cancer patients with acquired drug resistance have a poor prognosis, which prompted us to explore the vulnerability of cancer cells that are resistant to chemotherapy. The mTOR pathway is located downstream of the phosphoinositide 3-kinase (PI3K) and Akt pathway regulated by the phosphatase and tensin homolog ( PTEN ) tumor suppressor gene [ 28 ]. Inhibition of the mTOR pathway can inhibit tumor progression at multiple levels. In terms of mechanism, puerarin exerts a therapeutic effect on PDAC by inhibiting Akt/mTOR signal transduction activity, as shown by a decrease in phosphorylation and nuclear transcription. Further studies showed that the small molecule activator of mTOR, MHY1485, eliminated the puerarin-mediated inhibition of PCC proliferation and apoptosis induction. Viewing mTOR as a widespread driver of therapeutic resistance suggests considerable hope for targeting cancer drug resistance using mTOR inhibitors [ 29 ]. Significantly, puerarin inhibited the phosphorylation of mTOR, the downstream expression of GLUT1 and HIF-1α, and the glucose metabolism of PCC. In PDAC, even under normoxia, glycolysis is the primary energy source for cancer cell proliferation, invasion, migration, and metastasis [ 30 ]. We found that puerarin hindered glucose uptake and metabolism by downregulating the OCR and ECAR levels that depend upon HIF-1α and the glucose transporter GLUT1. Therefore, these findings indicate that puerarin has the therapeutic potential to treat PDAC by inhibiting the energy metabolism of tumor cells. Puerarin inhibited glucose uptake and metabolism by reducing the OCR and the ECAR dependent upon HIF-1α and glucose transporter GLUT1. Further studies showed that the mTOR small molecule activator MHY1485 could eliminate the puerarin-mediated inhibition of PCC proliferation and induction of apoptosis. Therefore, these findings suggest that puerarin has therapeutic potential for PDAC by inhibiting Akt/mTOR activity. The limitation of our study was that we did not explore the specific target of puerarin in the mTOR signal pathway, which needs further study. In response to the increasing interest in drug development, researchers have actively tried to develop new treatment strategies, including neoadjuvant chemotherapy for patients with resectable or marginally resectable incremental cancers, multi-drug combination chemotherapy for patients with advanced PDAC, and new complex drugs or immuno-oncology drugs for PDAC patients with specific gene mutations. Bax and Bak are two pro-apoptotic proteins with similar functions in the Bcl-2 family. Because of their essential role as effectors of mitochondrial outer membrane permeability (MOMP), Bcl and Bak are the portals of apoptosis in mitochondria, an essential step in the process of dependent apoptosis [ 31 ]. We observed an imbalance in the Bcl-2/Bax ratio after puerarin treatment, which indicated that puerarin could induce the mitochondria-dependent apoptosis of PCCs. EMT is a cellular process in which epithelial cells acquire a mesenchymal phenotype and behavior after epithelial downregulation. The cells then exhibit fibroblast-like morphology and cellular structure and increase their ability to migrate. Also, these now-migrating cells are usually invasive [ 32 ]. Metastasis-related events are the leading cause of cancer-related death, and circulating tumor cells (CTCs) play a crucial role in metastatic recurrence. The EMT marker expressed in CTCs is closely related to poor clinical results. As mentioned in previous studies, puerarin inhibits migration and invasion by antagonizing EMT [ 33 ]. We studied the effects of puerarin on PCC proliferation, apoptosis, migration, and invasion, tumor growth, and metastasis in a PDAC xenograft mouse model. In the nude mouse model, the use of puerarin reduced the growth and metastasis of PDAC. The limitation was that this study did not thoroughly explore the specific targets of puerarin acting in the mTOR signaling pathway. At the same time, our study used two cell lines, PANC-1 and PATU-8988T, so they could not fully cover the entire range of the tumor. More importantly, a genetic approach to exploring the association between mTOR signaling and the ant-tumor effects of puerarin needs to be implemented. Conclusion In conclusion, our results revealed that puerarin had a clear function in pancreatic cancer. It inhibited tumor cell proliferation and migration. Interestingly, our results suggest that the mTOR signaling pathway may play an important role in the anti-tumor process of puerarin. The process also involves downregulation of the OCR and the ECAR dependent upon HIF-1α and the glucose transporter GLUT1 to inhibit glucose uptake and metabolism. In addition, puerarin inhibited the migration and invasion of PCCs by antagonizing the EMT. In the nude mouse model, puerarin inhibited the growth and metastasis of PDAC. Further studies showed that MHY1485, a small molecule activator of mTOR, could block the puerarin-mediated effect of inhibiting PCCs proliferation and inducing PCCs apoptosis (Fig. 8 ). Therefore, puerarin has the potential to treat PDAC by inhibiting Akt/mTOR activity. Abbreviations Bax, Bcl-2-associated X protein Bcl-2, B-cell lymphoma 2 CCK-8, cell counting kit 8 DMEM, Dulbecco modified Eagle medium ECAR, extracellular acidification rate EMT, epithelial-mesenchymal transition FBS, fetal bovine serum GAPDH, glyceraldehyde 3-phosphate dehydrogenase GLUT1, glucose transporter type 1 HIF-1α, hypoxia-inducible factor-1α OCR, oxygen consumption rate PCC, pancreatic cancer cell PDAC, pancreatic ductal adenocarcinoma RTCA, real-time cell analysis Declarations Acknowledgements Not applicable Authors’ contributions LX and BY designed the experiments; ZH and LH carried out most of the experiments; ZH, LH, XY, GY, HY, and GH analyzed the data and organized the Figures; ZH wrote the manuscript and LH reviewed it. LX and SY provided important support for the design and implementation of supplementary experiments. All authors read and approved the final manuscript. Funding This study was sponsored by Wenzhou Science and Technology Plan Project, China (Grant No. Y20180100) and Key Laboratory of Diagnosis and Treatment of Severe Hepato-Pancreatic Diseases of Zhejiang Province (2018E10008). Availability of data and materials All data generated or analyzed during this study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Animal experiments were approved by the Committee for Animal Experiments at Wenzhou Medical University. Consent for publication All authors agreed on the manuscript Competing interests The authors declare that they have no competing interests. Author details 1 Key Laboratory of Diagnosis and Treatment of Severe Hepato-Pancreatic Diseases of Zhejiang Province, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China 2 Department of Laboratory Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China 3 Department of Laboratory Medicine, Wenzhou Hospital of Traditional Chinese Medicine, Wenzhou 325000, China 4 Center for Health Assessment, Wenzhou Medical University, Wenzhou, 325000, China 5 Department of Hepato-Pancreato-Biliary Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China References Siegel RL, Miller KD, Jemal A. Cancer statistics. 2019. CA Cancer J Clin. 2019; 69(1):7–34. doi: 10.3322/caac.21551 . Collaborators GBDPC. 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A The chemical structure of puerarin. B The growth of PANC-1 and PATU-8988T cells with or without puerarin treatment was determined by real-time cellular analysis (RTCA). C The viability of PANC-1 and PATU-8988T cells with or without puerarin treatment was analyzed by the CCK-8 assay. D The proliferation of PANC-1 and PATU-8988T cells with or without puerarin treatment was analyzed by the colony formation assay. E Immunocytochemical staining of Ki67 in PANC-1 and PATU-8988T cells with or without puerarin treatment. Bar = 100 μm. F Western blot analysis showing the expression of c-Myc in PANC-1 and PATU-8988T cells with or without puerarin treatment. G Flow cytometry analysis of cell apoptosis in PANC-1 and PATU-8988T cells with or without puerarin treatment. H Western blot analysis showing the expression of cleaved caspase-8, Bcl-2, and Bax in PANC-1 and PATU-8988T cells with or without puerarin treatment. Data were presented as the mean ± standard deviation and were analyzed by one-way ANOVA with Bonferroni’s post-hoc test. *P \u003c 0.05, **P \u003c 0.01, and ***P \u003c 0.001.","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-152446/v1/806d5e2ad860e282610e44c2.png"},{"id":5259276,"identity":"b5aa92ed-30b4-4ee1-a969-7f23a872bda4","added_by":"auto","created_at":"2021-01-25 23:24:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":299696,"visible":true,"origin":"","legend":"Puerarin inhibits PCC invasion and migration by antagonizing the Slug-E-cadherin axis. A-B The effect of puerarin on the migrated rate of PANC-1 and PATU-8988T cells was determined by the wound healing assay. C-D The effects of puerarin on the invading number of PANC-1 and PATU-8988T cells were analyzed by the transwell chamber assay. E Western blot analysis showing the expression of E-cadherin and α-SMA in puerarin-treated PANC-1 and PATU-8988T cells. F Immunocytochemical staining of E-cadherin and α-SMA in puerarin-treated PANC-1 and PATU-8988T cells. Bar = 25 μm. The data are presented as the mean ± standard deviation and were analyzed by one-way ANOVA with Bonferroni’s post-hoc test. *P \u003c 0.05, **P \u003c 0.01, and ***P \u003c 0.001.","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-152446/v1/c6d9d4008eccd01a1ae0b3b6.png"},{"id":5259258,"identity":"a242e33e-57b1-4622-aaa5-84a877a5b528","added_by":"auto","created_at":"2021-01-25 23:21:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1080997,"visible":true,"origin":"","legend":"Puerarin inhibits the tumor growth and metastasis of PDAC in animal xenograft model. A Effects of puerarin on morphologic changes in the experimental groups. B Pathological results of HE staining for PDAC in tissues of the model group. Bar = 50 μm. C Effect of puerarin on the volume of tumors in the animal xenograft model. D Effects of puerarin on tumor weight. E Immunohistochemical (IHC) staining for Ki67 in the puerarin-treated model. Bar = 50 μm. F IHC staining for c-Myc in the puerarin-treated model. Bar = 50 μm. G Western blot analysis showing the expression of cleaved caspase-8, Bax and Bcl-2 in PANC-1 and PATU-8988T cells with or without puerarin treatment. H IHC staining for cleaved caspase-8 and cytochrome C in the puerarin-treated model. Bar = 50 μm. I IHC staining for E-cadherin and α-SMA in the puerarin-treated model. Bar = 50 μm. J IHC staining for HIF-1α in the puerarin-treated model. Bar = 50 μm. The data are presented as the mean ± standard deviation, and were analyzed by a two-sided Student’s t-test. *P \u003c 0.05 and ***P \u003c 0.001.","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-152446/v1/14ec01dd9fa6ea364d970163.png"},{"id":5259331,"identity":"855c9761-5bb2-4d44-87bc-e4ad3dc49fe6","added_by":"auto","created_at":"2021-01-25 23:27:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":271290,"visible":true,"origin":"","legend":"Puerarin inhibits the activation of Akt/mTOR signaling in vitro and in vivo. A The correlation between the expression of Akt and the activity of KRAS, TP53, CDKN2A, and SMAD4 in the GEPIA 2 database was evaluated. B The correlation between the expression of mTOR and the activity of KRAS, TP53, CDKN2A, and SMAD4 in the GEPIA 2 database was evaluated. C The expression and phosphorylation of mTOR in normal pancreatic ductal cells (HPDE6-C7) and PCCs (PANC-1, PATU-8988T, and BxPc-3). D-E Western blot analysis showing the expression and phosphorylation of Akt and mTOR in PANC-1 and PATU-8988T cells with or without puerarin treatment. F-G Immunocytochemical staining of mTOR in PANC-1 and PATU-8988T cells with or without puerarin treatment. Bar = 50 μm. H Western blot analysis showing the expression and phosphorylation of Akt and mTOR in the the puerarin-treated animal xenograft model. I IHC staining for mTOR in the puerarin-treated model. Bar = 50 μm. The data are presented as the mean ± standard deviation and were analyzed by one-way ANOVA with Bonferroni’s post-hoc test and two-sided Student’s t-test. *P \u003c 0.05, **P \u003c 0.01, and ***P \u003c 0.001.","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-152446/v1/a61b4d9bf7da15f8b8f32f92.png"},{"id":5259278,"identity":"5bb53fd4-01ac-47c8-bb98-e0cbe2f074f5","added_by":"auto","created_at":"2021-01-25 23:24:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":344936,"visible":true,"origin":"","legend":"Puerarin binds to the kinase domain of mTOR protein to inhibit protein activity. A The kinase domain (KD, green cartoon) areas of mTOR. B-C The possible binding sites of puerarin and mTOR including two structural regions i and ii, and the binding energy is -5.17 and -7.0, respectively. D There are many ATP-Mg complex binding-related amino acid residues around the i and ii binding sites.","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-152446/v1/5be20650f2ed90c4e6814a94.png"},{"id":5259274,"identity":"d75897d2-a15c-4d8d-982b-8cb3cccfda28","added_by":"auto","created_at":"2021-01-25 23:24:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":418703,"visible":true,"origin":"","legend":"Reactivation of mTOR reverses the anti-tumor effects of puerarin. A-B The proliferation of puerarin-treated PANC-1 and PATU-8988T cells with or without MHY1485 treatment was analyzed by the colony formation assay. C Western blot analysis showing the expression of c-Myc in puerarin-treated PANC-1 and PATU-8988T cells with or without MHY1485 treatment. D-E The migration ability of puerarin-treated PANC-1 and PATU-8988T cells with or without MHY1485 treatment was determined by the wound healing assay. F-G The invasion ability of puerarin-treated PANC-1 and PATU-8988T cells with or without MHY1485 treatment was analyzed by the transwell chamber assay. H Western blot analysis showing the expression of vimentin and α-SMA in puerarin-treated PANC-1 and PATU-8988T cells with or without MHY1485 treatment. I Western blot analysis showing the expression of Snail1 and Slug in puerarin-treated PANC-1 and PATU-8988T cells with or without MHY1485 treatment. The data are presented as the mean ± standard deviation and were analyzed by one-way ANOVA with Bonferroni’s post-hoc test and two-sided Student’s t-test. *P \u003c 0.05, **P \u003c 0.01, and ***P \u003c 0.001.","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-152446/v1/c136cce7d316ea3a647cb3a7.png"},{"id":5259330,"identity":"7465da1e-c3a8-48f4-b0bb-ce252c08e050","added_by":"auto","created_at":"2021-01-25 23:27:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":146018,"visible":true,"origin":"","legend":"Puerarin controls glucose metabolism in PCCs. A-D Glucose metabolism assay showing downregulated levels of OCR, basal respiration, spare respiration, maximal respiration, and ATP production in puerarin-treated PANC-1 and PATU-8988T cells. E-H Glucose metabolism assay showing reduced ECAR, basal glycolysis, and compensatory glycolysis in puerarin-treated PANC-1 and PATU-8988T cells. I Western blot analysis showing the expression of GLUT1 and HIF-1α in puerarin-treated PANC-1 and PATU-8988T cells. The data are presented as the mean ± standard deviation and analyzed by one-way ANOVA with Bonferroni’s post-hoc test and two-sided Student’s t-test. *P \u003c 0.05, **P \u003c 0.01, and ***P \u003c 0.001.","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-152446/v1/476b7bf2aef656e6016a69e9.png"},{"id":5259257,"identity":"88ae7671-6b7d-497d-bfa9-0bb42a58e63e","added_by":"auto","created_at":"2021-01-25 23:21:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":73102,"visible":true,"origin":"","legend":"Puerarin suppresses oncogenesis and progression of PDAC via suppressing Akt/mTOR activity.","description":"","filename":"OnlineFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-152446/v1/07ec78090fc0733c679fd71c.png"},{"id":13651422,"identity":"6a16bcd8-77a1-4246-8e4c-a602bfb9ab00","added_by":"auto","created_at":"2021-09-17 09:45:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6551788,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-152446/v1/51605a52-35df-4cd7-b61e-fbeb7983fc16.pdf"},{"id":5259279,"identity":"c10b76e5-1cbc-48fb-9f49-a8912d4d3acf","added_by":"auto","created_at":"2021-01-25 23:24:35","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":16710,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-152446/v1/dcfab7e5ae1fdb8e0ea3c8d3.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eThe Isoflavone Puerarin Exerts Anti-Tumor Activitiy in Pancreatic Ductal Adenocarcinoma by Suppressing Akt/mTOR Activity\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003ePancreatic ductal adenocarcinoma (PDAC) is the most common exocrine pancreatic cancer seen clinically. It is the fourth-leading cause of cancer-related death in the United States, second only to colorectal cancer in gastrointestinal-related deaths [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. According to the World Health Organization (WHO) GLOBOCAN database and the 2017 Global Burden of Disease Study, PDAC is the seventh leading cause of cancer death in men and women worldwide [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Surgical resection is the only possible cure. Unfortunately, due to the late discovery, only 15\u0026ndash;20% of PDAC patients are eligible for a pancreatectomy. However, even after complete resection, the prognosis of PDAC patients is poor. After resection margin-negative (R0) pancreaticoduodenectomy, the five-year survival rate of PDAC patients is about 30% for lymph node-negative and 10% for lymph node-positive patients [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The median survival of patients with untreated and unresectable locally advanced PDAC is 8\u0026ndash;12 months, while the median survival of patients with metastatic disease at presentation is only 3\u0026ndash;6 months. Systemic chemotherapy can improve the survival rate of patients with locally advanced and metastatic PDAC. In today\u0026rsquo;s modern treatment era, the FOLF NO \u0026times; regimen (fluorouracil\u0026thinsp;+\u0026thinsp;leucovorin, irinotecan, and oxaliplatin) has achieved the best outcome, but the median patient survival time is only 11.1 months [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. New drugs, new drug targets, and new, more effective chemotherapy regimens are desperately needed in all settings.\u003c/p\u003e\u003cp\u003ePuerarin is a white crystal extracted from the roots of the kudzu plant or the kudzu vine. Its chemical name is 7,4\u0026rsquo;-dihydroxyisoflavone-8-β-glucopyranoside, and its molecular formula is C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Puerarin is the most abundant secondary metabolite, which was isolated from the rhizome of \u003cem\u003ePueraria lobata\u003c/em\u003e in the 1950s and is known as Asian ginseng. Since then, extensive research has been conducted on its pharmacological properties. Puerarin has various pharmacological effects, such as enhancing circulatory system function, reducing myocardial oxygen consumption, decreasing blood sugar, and preventing hypertension and arteriosclerosis. Anti-liver toxicity, anti-inflammatory, expectorant, antipyretic, immunity-enhancing, antibacterial, and antiviral activities have also been demonstrated [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Its low toxicity and wide range of pharmacological effects have attracted the attention of domestic and foreign researchers. In recent years, the anti-cancer effect of puerarin has been widely studied. Many studies showed that puerarin had good anti-tumor activity in animal model and many cancer cell lines [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the role of puerarin in PDAC has not been studied in-depth and needs to be further explored.\u003c/p\u003e\u003cp\u003eIn this study, we used \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments to investigate the anti-tumor effects of puerarin. Real-time cell analysis (RTCA), the Cell Counting Kit-8 (CCK-8) assay, colony formation, and flow cytometry analysis were used to analyze the effects of puerarin on the proliferation and apoptosis of PCCs. The transwell invasion assay, the wound healing assay, and immunocytochemical staining were used to evaluate the effects of puerarin on cell migration and invasion, and the epithelial-mesenchymal transition (EMT) of PCCs. Moreover, the effects of puerarin on the activity of Akt/mTOR and glucose metabolism were also investigated. We found that puerarin inhibited the proliferation of PCCs, induced mitochondrial-dependent apoptosis, and suppressed invasion and migration by reversing EMT. In nude mouse model, PDAC growth and metastasis were reduced by puerarin treatment. Mechanically, the activity of Akt/mTOR in PCCs and PDAC tissue was suppressed by puerarin treatment via binding to the kinase domain of mTOR protein, resulting in the inhibition of glucose metabolism by decreasing HIF-1α and GLUT1 expression. Further studies showed that the small molecule activator of mTOR, MHY1485, eliminated the puerarin-mediated inhibition of PCC proliferation and EMT induction. Thus, puerarin can be a therapeutic approach to PDAC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture and drug treatment\u003c/h2\u003e\u003cp\u003eHuman PCC lines PANC-1 and PATU-8988T were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (Invitrogen). The cultured cells at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e were initially plated in a 10-cm dish for 24 h. After incubation for 24 h, the culture medium was replaced with serum-free medium. PANC-1 and PATU-8988T cells were treated with 0.2 and 0.5 mM puerarin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, CAS#: 3681-99-0, Purity: \u0026ge; 98% by HPLC, Yuanye Biotechnology, Shanghai, China) with or without MHY1485 (CAS#: 326914-06-1, MedChem Express, Monmouth Junction, NJ, USA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003eCell counting Kit-8 (CCK-8) assay\u003c/h2\u003e\u003cp\u003e The CCK-8 assay kit (Dojindo, Shanghai, China) was used to detect the anti-tumor activity of puerarin in PANC-1 and PATU-8988T cells according to the manufacturer\u0026rsquo;s instructions. First, the cells were cultured in 6-cm dishes with fresh medium for 24 h. The cells in the logarithmic growth stage were inoculated into 96-well plates at a density of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/ml. Then, the cells were treated with different concentrations of puerarin for 24 h. After that, 10 \u0026micro;l of CCK-8 medium and 10 \u0026micro;l of CCK-8 were added and the plates were incubated for another 4 h. The absorbance was measured at a wavelength of 450 nm using a microplate reader. Statistical analyses were performed using Stata statistical software (StataCorp LP). Each experiment was repeated thrice and the average value was taken as the final result.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e\u003cp\u003eThe cells were serum-starved for 24 h and the medium was replaced with complete medium. PANC-1 and PATU-8988T cells were exposed to culture medium containing different concentrations of puerarin for 24 h, and cells in the standard control group were treated with dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA). After centrifugation to collect the cells, quantification of the apoptotic cells was performed using an Annexin V-FITC Apoptosis Detection Kit (Multisciences, Hangzhou, China) according to the manufacturer\u0026rsquo;s instructions. Cell apoptosis was assessed by flow cytometry (Ex\u0026thinsp;=\u0026thinsp;488 nm; Em\u0026thinsp;=\u0026thinsp;530 nm, BD FACSVerse\u0026trade;, BD Biosciences, San Jose, CA, USA), and the results were analyzed using FlowJo (TreeStar, Ashland, OR, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eReal-time cellular analysis (RTCA)\u003c/h2\u003e\u003cp\u003eCell proliferation was monitored by the xCELLigence RTCA MP System (ACEA Biosciences, San Diego, CA, USA) using 16-well E-Plates (ACEA Biosciences). The cells were seeded in triplicate at 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well in the plates. For the RTCA experiments, the cells were treated with puerarin after reaching steady growth (24 h). Impedance was measured every 15 min over 96 h and represented as the cell index by the RTCA-integrated software of the xCELLigence System. The cell index was normalized to 1 at the time point of drug administration. From this data, real-time cell growth curves were generated with GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eTranswell invasion assay\u003c/h2\u003e\u003cp\u003eTranswell assays were performed using Transwell chambers (Costar, New York City, NY, USA) with Matrigel\u0026reg; (BD Biosciences). After treatment with various concentrations of puerarin for 24 h, cell suspensions were prepared using ethylenetetraacetic acid (EDTA) enzyme. The cells were resuspended in serum-free medium and transferred to the inner chamber (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per chamber). Complete medium was added to the outer chamber, and the plate was incubated in a CO\u003csub\u003e2\u003c/sub\u003e incubator (37 ℃) for observation for 12 h. After carefully removing the non-migrating cells at the membrane site with a cotton swab, the cells were fixed with formaldehyde and stained with 0.1% crystal violet (Sigma), and quantification was performed by counting five random fields under the microscope (Leica Microsystems, Wetzlar, Germany). Each experiment was repeated three times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eColony formation assay\u003c/h2\u003e\u003cp\u003eThe cells were seeded into 6-well plates at 1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well and treated with puerarin 24 h later. After 24 h, the media was replaced with fresh media and cultured for 14 days. The colonies were then fixed with 2% formaldehyde and stained with 0.5% crystal violet. The number of colonies with \u0026ge;\u0026thinsp;50 cells was counted under a microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eWound healing assay\u003c/h2\u003e\u003cp\u003ePANC-1 or PATU-8988T cells were seeded in 6 well plates and maintained at 37 ℃ for 24 h. The cells were scratched using a crystal pipette tip to make a linear gap. Next, the detached cells were washed away with phosphate-buffered saline (PBS) and different concentrations of puerarin were added. The cells were allowed to fill the gap, and after 24 h, images of the areas were captured using a microscope (Leica Microsystems).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eImmunocytochemical staining\u003c/h2\u003e\u003cp\u003eImmunofluorescence staining was performed based on established protocols. PANC-1 and PATU-8988T cells with different treatments were grown on glass coverslips for 24 h. The cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100 (Thermo Scientific, Waltham, MA, USA). Blocking was performed with 4% goat serum (Gibco, Thermo Fisher Scientific) in Dulbecco\u0026rsquo;s phosphate-buffered saline (DPBS; Invitrogen, Paisley, UK) for 1.5 h at 37 ℃, followed by incubation with the primary antibodies (Table S1): anti-Ki67 (1:200), anti-α-SMA (1:200), anti-E-cadherin (1:200), and anti-p-mTOR (1:200) at 4℃ overnight. Next, the membranes were incubated in the appropriate second antibodies for 1 h at room temperature. At least three independent experiments for immunofluorescence staining were conducted.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot analysis\u003c/h2\u003e\u003cp\u003eAfter treating the cells for 24 h, the cells in each group were collected and the total cellular protein was extracted. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated overnight at 4 ℃ with the primary antibodies (Table S1). The membranes were washed three times in Tris-buffered saline with 0.1% Tween 20 (TBST) the following day and incubated with the second antibody (anti-rabbit IgG) at room temperature for 1 h. After the membranes were rinsed, the protein expression levels were detected by enhanced chemiluminescence (ECL) and visualized by autoradiography. GAPDH was used as the internal reference protein.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eGlucose metabolism assay\u003c/h2\u003e\u003cp\u003eThe intact cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA). Briefly, 1 \u0026times;10\u003csup\u003e4\u003c/sup\u003e PANC-1 and PATU-8988T cells were seeded into 96-well cell plates and incubated overnight at 37 ℃ at 5% CO\u003csub\u003e2\u003c/sub\u003e. Both cells were pretreated with or without different concentrations of puerarin for 24 h. Simultaneously, the calibration plates were incubated overnight at 37 ℃ in a non-CO\u003csub\u003e2\u003c/sub\u003e incubator, then the cell medium was replaced with assay medium. Once the probe calibration was completed, the cell plate replaced the probe plate. The analyzer plotted the OCR value, followed by the injection of the compounds sequentially as follows: oligomycin (an inhibitor of ATP synthase; 2.5 \u0026micro;M), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, an uncoupler of OXPHOS; 2 \u0026micro;M), rotenone (an inhibitor of complex I; 0.25 \u0026micro;M), and anti-mildew A (an inhibitor of complex III; 0.25 \u0026micro;M) (n\u0026thinsp;=\u0026thinsp;8). The ECAR was evaluated after the continuous injection of glucose (10 mM), oligomycin (1 \u0026micro;M), and 2-Deoxy-D-glucose (2-DG, 50 mM) (n\u0026thinsp;=\u0026thinsp;8). After completing the test, the BCA Protein Assay Kit was used to determine the protein concentrations to normalize the OCR and the ECAR according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eNude mouse tumorigenicity\u003c/h2\u003e\u003cp\u003eMale BALB/c nude mice (6\u0026ndash;8 weeks old) were obtained from the Wenzhou Medical University Experimental Animal Center (Wenzhou, China). All mice were housed under controlled conditions (temperature, 21\u0026ndash;23 ℃; 12 h light/dark cycle; 55% humidity). PANC-1 cells (3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) in 0.2 ml PBS were subcutaneously injected into the right thighs of 10 nude mice, which were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;5 in each group). Mice in the experimental group received puerarin by intragastric gavage every three days for one month. The control group received DMSO injections for one month. Tumor formation in the nude mice was monitored for 30 days, with the length and width measured every three days. The tumor size was calculated according to the standard formula: tumor volumes (cm\u003csup\u003e3\u003c/sup\u003e) = (the longest diameter) \u0026times; (the shortest diameter)\u003csup\u003e2\u003c/sup\u003e \u0026times; 0.5. The mice were deeply anesthetized with sodium pentobarbital and euthanized by cervical dislocation.\u003c/p\u003e\u003cp\u003e 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 and institutional guidelines for animal welfare (National Institutes of Health Publications, NIH Publications No. 80\u0026thinsp;\u0026minus;\u0026thinsp;23) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eHistopathological analysis\u003c/h2\u003e\u003cp\u003eTumor specimens from the animals were paraffin-embedded and cut into 4-\u0026micro;m-thick sections. Standard hematoxylin and eosin staining (HE, Yuanye Biotechnology) was performed on 4-\u0026micro;m sections from the paraffin-embedded tumor samples. Immunohistochemical (IHC) analysis was conducted under a microscope according to a previous method [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In brief, 4-\u0026micro;m-thick sections were dewaxed with xylene and rehydrated in a graded ethanol series. The 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, China). IHC staining was performed using the following primary antibodies: anti-Ki67 (1:200), anti-c-Myc (1:200), anti-α-SMA (1:200), anti-E-cadherin (1:200), anti-cleaved caspase-8 (1:100), anti-cytochrome C (1:100), and anti-HIF-1α (1:200). 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.\u003c/p\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003eMolecular docking\u003c/h2\u003e\u003cp\u003eMolecular docking was performed as previously described [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], Puerarin and mTOR were rigidly docked, and the docking results were analyzed by PyMOL software. The puerarin molecule was downloaded from Pubchem, and the molecular energy was optimized through Chem 3D Ultra Software (8.0.3 version, Cambridge-Soft, MA, USA). The crystal structure of mTOR was downloaded from the Protein Structure Database (Protein Data Bank, PDB) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rcsb.org/pdb/\u003c/span\u003e\u003c/span\u003e), and the protein was processed by Autodock (MGLTools-1.5.6) to remove water molecules and hydrogenate and to add volume.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eDatabase analysis\u003c/h2\u003e\u003cp\u003eThe correlation between AKT and mTOR expression and the activity of KRAS, TP53, CDKN2A, and SMAD4 was evaluated in the GEPIA 2 database website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia2.cancer-pku.cn/#analysis\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation for the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments. All statistical analyses were performed using GraphPad Prism statistical analysis software (version 8.0, GraphPad Software, Inc., LaJolla, CA, USA). Statistical comparisons were made with a two-sided t-test. One-way analysis of variance (ANOVA) with Bonferroni\u0026rsquo;s post-hoc test was used when more than two groups were present. Statistical significance was indicated by a \u003cem\u003eP\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003ePuerarin inhibits PCC proliferation and induces mitochondria-mediated apoptosis\u003c/h2\u003e\u003cp\u003eTo investigate the effect of puerarin on PCC proliferation, the RTCA, CCK-8, and colony formation assays were performed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and c, as expected, puerarin treatment (0.2 and 0.5 mM) significantly inhibited the growth of PANC-1 and PATU-8988T cells in concentration- and time-dependent manners. The CCK-8 assay results of the PANC-1 and PATU-8988T cells confirmed the concentration-dependent inhibition of cell growth by puerarin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). Puerarin also significantly reduced colony formation in the PANC-1 and PATU-8988T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). To investigate the effect of puerarin on cell proliferation, we used immunofluorescence staining for the Ki67 marker expressed by proliferating cells. The level of Ki67 protein varied with the cell cycle and was higher in the G2/M phase and lower in the G0/G1 phase [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In the PANC-1 and PATU-8988T cell lines, we observed a decrease in Ki67 protein expression in both the PANC-1 and PATU-8988T cells treated with puerarin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) compared to the control cells. Therefore, the above results suggest that puerarin inhibited PCC proliferation in concentration- and time-dependent manners.\u003c/p\u003e\u003cp\u003eNext, we evaluated the effects of puerarin on PCC apoptosis by flow cytometry analysis. Puerarin treatment significantly increased the proportion of apoptotic and necrotic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Further studies showed an increase in caspase-8 in both the PANC-1 and PATU-8988T cells treated with puerarin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Apoptosis in cancer cells depends upon the dynamic equilibrium of Bax and Bcl-2 expression [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Puerarin was observed to increase Bax expression and decrease Bcl-2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). These results suggest that puerarin induced the death receptor- and mitochondrial-mediated apoptosis of PCCs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003ePuerarin inhibits the migration and invasion of PCCs by antagonizing epithelial-mesenchymal transition\u003c/h2\u003e\u003cp\u003eEnhanced cell migration and invasion abilities underlie PCC metastasis mechanisms, resulting in poor prognosis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Here, puerarin reduced the migration rate of PANC-1 and PATU-8988T cells as determined by the scratch wound assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b) and the effect was concentration-dependent as well as time-dependent. Also, puerarin treatment significantly inhibited the numbers of invading PANC-1 and PATU-8988T cells detected by the transwell assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d). Further studies showed that puerarin decreased the protein level of α-SMA in PANC-1 and PATU-8988T cells and increased the E-cadherin protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Immunofluorescence analysis revealed the downregulated expression of α-SMA and the increased expression of E-cadherin after puerarin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). In general, these results suggest that puerarin inhibited PCC migration and invasion by inhibiting EMT and tumor mammosphere formation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePuerarin suppresses PDAC growth and metastasis\u003c/b\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ein vivo\u003c/span\u003e\u003c/p\u003e\u003cp\u003eTo determine the anticancer effects of puerarin \u003cem\u003ein vivo\u003c/em\u003e, nude mice were injected with PANC-1 cells and then administrated puerarin or saline as a control. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea shows the morphology of tumor xenografts changes in the experimental group after puerarin treatment. We found that the administration of puerarin significantly reduced tumor volume and weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). The pathological results in the PDAC model tissue were shown by HE staining. Further studies showed that puerarin administration upregulated the expression of cleaved caspase-8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, h). Also, puerarin increased Bax expression and decreased Bcl-2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). A decrease in the expression of Ki67 was observed in the tumor tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Therefore, our \u003cem\u003ein vivo\u003c/em\u003e findings suggest that puerarin induced PCC apoptosis through death receptor- and mitochondrial-mediated pathways. To assess whether puerarin inhibited PDAC migration, we examined the expression of EMT process-related proteins. The results showed that puerarin decreased α-SMA expression and increased E-cadherin expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). It also reduced the expression of c-Myc, an oncoprotein associated with tumor progression and drug resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Hypoxia is usually observed in PDAC and some other solid tumors. HIF-1α protein, a key regulator of the hypoxia response, was found to accumulate in PDAC tissues. Several studies have shown that hypoxia was an independent predictor of poor prognosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We observed the downregulation of HIF-1α protein after puerarin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). In summary, these data suggest that puerarin inhibited the growth and metastasis of PDAC in a mouse xenograft model.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePuerarin reduces the activity of Akt/mTOR signaling\u003c/b\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e\u003cb\u003eand\u003c/b\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ein vivo\u003c/span\u003e\u003c/p\u003e\u003cp\u003eAnticancer effects involve many mechanisms, including oxidative stress, intrinsic and extrinsic mechanisms, as well as the survivin, PI3K/Akt/mTOR, SHH [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], Nrf2/Keap1 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], inflammation, and autophagy pathways [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Studies have shown that the signal transduction pathway mediated by phosphatidylinositol 3 kinase (PI3K) was closely related to cancer occurrence. Many downstream molecules make up the PI3K/Akt signal pathway, including mTOR, one of the more important targets of rapamycin. mTOR signaling plays a crucial role in cell growth, protein translation, autophagy, and metabolism [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The activation of mTOR contributes to the pathogenesis of various tumors. We also found that these PCCs exhibited heterogeneous PI3K/Akt/mTOR pathway activation at the protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). In this study, we investigated the effect of puerarin on mTOR activity in PANC-1 and PATU-8988T cells. We found that puerarin suppressed the mTOR signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e), suggesting that mTOR may be a target of puerarin. Puerarin-induced the downregulation of phosphorylated mTOR expression in PANC-1 and PATU-8988T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, g). The \u003cem\u003ein vitro\u003c/em\u003e experiments confirmed that puerarin inhibited the overexpression of mTOR in PDAC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh, i).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003ePuerarin binds to the kinase domain of mTOR protein to inhibit protein activity\u003c/h2\u003e\u003cp\u003eTo further analyze the biochemical pathways of puerarin affecting mTOR protein, we used Autodock (MGLTools-1.5.6) to rigidly dock puerarin with the FAT domain (blue cartoon) and the kinase domain (KD, green cartoon) areas of mTOR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). We found that the possible binding sites of puerarin and mTOR included two structural regions i and ii (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, c), with binding energies of -5.17 and \u0026minus;\u0026thinsp;7.0, respectively. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed shows that there were many ATP-Mg complex binding-related amino acid residues around the i and ii binding sites. Once puerarin binds to the i and ii sites on mTOR protein, it may affect the activity of the above amino acid residues, and then affect mTOR activation activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eActivated mTOR signaling eliminates puerarin-mediated anti-tumor effects\u003c/h2\u003e\u003cp\u003eGiven the anti-tumor effect of puerarin on PDAC by inhibiting mTOR signal transduction, we next investigated whether activated mTOR signal transduction influenced this effect of puerarin. In the PANC-1 and PATU-8988T cells, we used MHY1485, a significant cell permeability mTOR activator to activate the mTOR pathway, targeting the ATP domain mTOR. The activation of mTOR signaling eliminated the anti-proliferative effect of puerarin as determined by the colony formation test (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). Using the transwell and wound healing assays, we demonstrated that MHY1485 treatment increased the invasion and migration rates of the PANC-1 and PATU-8988T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed-g). Thus, activated mTOR signaling eliminated puerarin-mediated EMT suppression, as shown by the increased expression of α-SMA, vimentin, Snail1, and Slug (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh, i). These findings confirmed that mTOR signaling played a crucial role in the anti-tumor effect of puerarin in PDAC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003ePuerarin inhibits mTOR-mediated glucose metabolism in PCCs\u003c/h2\u003e\u003cp\u003eTo satisfy the need for rapid proliferation, tumor cells need more energy, so the process of bioenergy metabolism targeting tumor cells is a new therapeutic strategy to inhibit the growth of tumor cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A bioenergy analyzer was used to measure the corresponding OCR and ECAR, and the effects of external factors on mitochondrial uptake and glycolysis were analyzed statistically. The primary respiration, ATP production, maximum respiration, and spare respiration of cells treated with puerarin decreased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-d), indicating that puerarin inhibited the energy metabolism of the mitochondria. The glycolysis of the tumor cells treated with puerarin was significantly inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, g). The results showed that the basal glycolysis rate and the compensatory glycolysis rate decreased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef, h). Further studies showed that GLUT1 and HIF-1α protein expression was inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei). Considering the close connection between puerarin and the mTOR pathway, our research results indicate that puerarin may regulate downstream GLUT1 through the mTOR pathway and affect tumor cell metabolism.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePuerarin has certain anti-cancer effects in a variety of tumors. However, its role in PDAC is still poorly understood. In the present study, we showed that puerarin treatment significantly repressed the proliferation of PCCs in concentration- and time-dependent manners. In addition, puerarin induced the mitochondrial-dependent apoptosis of PCCs by causing a Bcl-2/Bax imbalance. Moreover, puerarin inhibited the migration and invasion of PCCs by antagonizing EMT. In the nude mouse model, PDAC growth and metastasis were also reduced by puerarin administration. Thus, these \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e results indicate that puerarin exerted effective protection against PDAC.\u003c/p\u003e\u003cp\u003ePrevious studies have shown that puerarin impeded cell growth, blocked the cell development in the G0/G1 cell cycle phase, induced apoptosis in bladder cancer cells through the mTOR/p70 S6K signaling pathway, and suppressed cell growth and migration in HPV-positive cervical cancer cells by inhibiting the PI3K/mTOR signaling pathway [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In addition, puerarin 6\u0026rsquo;-O-xyloside, an analog of puerarin, suppressed hepatocellular carcinoma by regulating proliferation, stemness, and apoptosis by inhibiting PI3K/Akt/mTOR [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the anti-tumor effect and molecular mechanism of puerarin in PDAC remains unknown. Here, we identified effective protection against PDAC by puerarin and showed that the Akt/mTOR signaling pathway played an important role in the anti-tumor effect of puerarin.\u003c/p\u003e\u003cp\u003emTOR protein kinase is involved in many major signaling pathways and plays a key role in organizing the cellular and body physiology of all eukaryotes. In the two and a half years since its discovery, mTOR has been shown to be the central node in the network that controls cell growth. In this way, it integrates information about the availability of energy and nutrients to coordinate the synthesis or decomposition of new cellular components. The dysregulation of this basic signal transduction pathway can disrupt cellular homeostasis and may aggravate the overgrowth of cancer and pathology related to aging and metabolic diseases [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Although mTOR kinase itself is rarely mutated in cancer, it is easily hijacked by upstream oncogenic nodes, including those in the PI3K/Akt pathway and the MAPK pathway driven by Ras. As a result, mTOR signaling is active in as many as 80% of human cancers. In this case, mTOR signaling plays a key role in maintaining the growth and survival of cancer cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Cancer patients with acquired drug resistance have a poor prognosis, which prompted us to explore the vulnerability of cancer cells that are resistant to chemotherapy. The mTOR pathway is located downstream of the phosphoinositide 3-kinase (PI3K) and Akt pathway regulated by the phosphatase and tensin homolog (\u003cem\u003ePTEN\u003c/em\u003e) tumor suppressor gene [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Inhibition of the mTOR pathway can inhibit tumor progression at multiple levels. In terms of mechanism, puerarin exerts a therapeutic effect on PDAC by inhibiting Akt/mTOR signal transduction activity, as shown by a decrease in phosphorylation and nuclear transcription. Further studies showed that the small molecule activator of mTOR, MHY1485, eliminated the puerarin-mediated inhibition of PCC proliferation and apoptosis induction. Viewing mTOR as a widespread driver of therapeutic resistance suggests considerable hope for targeting cancer drug resistance using mTOR inhibitors [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Significantly, puerarin inhibited the phosphorylation of mTOR, the downstream expression of GLUT1 and HIF-1α, and the glucose metabolism of PCC. In PDAC, even under normoxia, glycolysis is the primary energy source for cancer cell proliferation, invasion, migration, and metastasis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. We found that puerarin hindered glucose uptake and metabolism by downregulating the OCR and ECAR levels that depend upon HIF-1α and the glucose transporter GLUT1. Therefore, these findings indicate that puerarin has the therapeutic potential to treat PDAC by inhibiting the energy metabolism of tumor cells. Puerarin inhibited glucose uptake and metabolism by reducing the OCR and the ECAR dependent upon HIF-1α and glucose transporter GLUT1. Further studies showed that the mTOR small molecule activator MHY1485 could eliminate the puerarin-mediated inhibition of PCC proliferation and induction of apoptosis. Therefore, these findings suggest that puerarin has therapeutic potential for PDAC by inhibiting Akt/mTOR activity. The limitation of our study was that we did not explore the specific target of puerarin in the mTOR signal pathway, which needs further study.\u003c/p\u003e\u003cp\u003eIn response to the increasing interest in drug development, researchers have actively tried to develop new treatment strategies, including neoadjuvant chemotherapy for patients with resectable or marginally resectable incremental cancers, multi-drug combination chemotherapy for patients with advanced PDAC, and new complex drugs or immuno-oncology drugs for PDAC patients with specific gene mutations.\u003c/p\u003e\u003cp\u003eBax and Bak are two pro-apoptotic proteins with similar functions in the Bcl-2 family. Because of their essential role as effectors of mitochondrial outer membrane permeability (MOMP), Bcl and Bak are the portals of apoptosis in mitochondria, an essential step in the process of dependent apoptosis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. We observed an imbalance in the Bcl-2/Bax ratio after puerarin treatment, which indicated that puerarin could induce the mitochondria-dependent apoptosis of PCCs.\u003c/p\u003e\u003cp\u003eEMT is a cellular process in which epithelial cells acquire a mesenchymal phenotype and behavior after epithelial downregulation. The cells then exhibit fibroblast-like morphology and cellular structure and increase their ability to migrate. Also, these now-migrating cells are usually invasive [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Metastasis-related events are the leading cause of cancer-related death, and circulating tumor cells (CTCs) play a crucial role in metastatic recurrence. The EMT marker expressed in CTCs is closely related to poor clinical results. As mentioned in previous studies, puerarin inhibits migration and invasion by antagonizing EMT [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. We studied the effects of puerarin on PCC proliferation, apoptosis, migration, and invasion, tumor growth, and metastasis in a PDAC xenograft mouse model. In the nude mouse model, the use of puerarin reduced the growth and metastasis of PDAC.\u003c/p\u003e\u003cp\u003eThe limitation was that this study did not thoroughly explore the specific targets of puerarin acting in the mTOR signaling pathway. At the same time, our study used two cell lines, PANC-1 and PATU-8988T, so they could not fully cover the entire range of the tumor. More importantly, a genetic approach to exploring the association between mTOR signaling and the ant-tumor effects of puerarin needs to be implemented.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our results revealed that puerarin had a clear function in pancreatic cancer. It inhibited tumor cell proliferation and migration. Interestingly, our results suggest that the mTOR signaling pathway may play an important role in the anti-tumor process of puerarin. The process also involves downregulation of the OCR and the ECAR dependent upon HIF-1α and the glucose transporter GLUT1 to inhibit glucose uptake and metabolism. In addition, puerarin inhibited the migration and invasion of PCCs by antagonizing the EMT. In the nude mouse model, puerarin inhibited the growth and metastasis of PDAC. Further studies showed that MHY1485, a small molecule activator of mTOR, could block the puerarin-mediated effect of inhibiting PCCs proliferation and inducing PCCs apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Therefore, puerarin has the potential to treat PDAC by inhibiting Akt/mTOR activity.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBax, Bcl-2-associated X protein\u003c/p\u003e\u003cp\u003eBcl-2, B-cell lymphoma 2\u003c/p\u003e\u003cp\u003eCCK-8, cell counting kit 8\u003c/p\u003e\u003cp\u003eDMEM, Dulbecco modified Eagle medium\u003c/p\u003e\u003cp\u003eECAR, extracellular acidification rate\u003c/p\u003e\u003cp\u003eEMT, epithelial-mesenchymal transition\u003c/p\u003e\u003cp\u003eFBS, fetal bovine serum\u003c/p\u003e\u003cp\u003eGAPDH, glyceraldehyde 3-phosphate dehydrogenase\u003c/p\u003e\u003cp\u003eGLUT1, glucose transporter type 1\u003c/p\u003e\u003cp\u003eHIF-1α, hypoxia-inducible factor-1α\u003c/p\u003e\u003cp\u003eOCR, oxygen consumption rate\u003c/p\u003e\u003cp\u003ePCC, pancreatic cancer cell\u003c/p\u003e\u003cp\u003ePDAC, pancreatic ductal adenocarcinoma\u003c/p\u003e\u003cp\u003eRTCA, real-time cell analysis\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLX and BY designed the experiments; ZH and LH carried out most of the experiments; ZH, LH, XY, GY, HY, and GH analyzed the data and organized the Figures; ZH wrote the manuscript and LH reviewed it. LX and SY provided important support for the design and implementation of supplementary experiments. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was sponsored by Wenzhou Science and Technology Plan Project, China (Grant No. Y20180100) and Key Laboratory of Diagnosis and Treatment of Severe Hepato-Pancreatic Diseases of Zhejiang Province (2018E10008).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments were approved by the Committee for Animal Experiments at Wenzhou Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agreed on the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eKey Laboratory of Diagnosis and Treatment of Severe Hepato-Pancreatic Diseases of Zhejiang Province, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eDepartment of Laboratory Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eDepartment of Laboratory Medicine, Wenzhou Hospital of Traditional Chinese Medicine, Wenzhou 325000, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eCenter for Health Assessment, Wenzhou Medical University, Wenzhou, 325000, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e5\u003c/sup\u003eDepartment of Hepato-Pancreato-Biliary Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Jemal A. 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Nat Commun. 2020;11(1):3303. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41467-020-17150-3\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Puerarin, Pancreatic ductal adenocarcinoma, Glucose metabolism, Akt/mTOR","lastPublishedDoi":"10.21203/rs.3.rs-152446/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-152446/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003ePuerarin (7,4’-dihydroxyisoflavone-8-β-glucopyranoside) is a natural flavonoid compound isolated from the traditional Chinese herb \u003cem\u003eRadix puerariae\u003c/em\u003e. Recent studies have demonstrated that puerarin has potential anti-tumor effects via induction of apoptosis and inhibition of proliferation. However, the effect and molecular mechanism of puerarin in pancreatic ductal adenocarcinoma (PDAC) remains unknown.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThe effects of puerarin on the proliferation, apoptosis, migration and invasion of pancreatic cancer cells (PCCs), and tumor growth and metastasis in PDAC xenograft mouse model were performed. In addition, Akt/mTOR signaling activity was evaluated both\u003cem\u003e in vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003ePuerarin treatment significantly repressed PCC proliferation in concentration- and time-dependent manners. Puerarin induced the mitochondrial-dependent apoptosis of PCCs by causing a Bcl-2/Bax imbalance. Moreover, puerarin inhibited PCC migration and invasion by antagonizing epithelial-mesenchymal transition (EMT). In nude mouse model, PDAC growth and metastasis were reduced by puerarin administration. Mechanistically, puerarin exerted its therapeutic effects on PDAC by suppressing Akt/mTOR signaling. Importantly, puerarin bound to the kinase domain of mTOR protein, affecting the activity of the surrounding amino acid residues associated with the binding of the ATP-Mg\u003csup\u003e2+\u003c/sup\u003e complex. Further studies showed that the inhibitory effects of puerarin on PCCs were abolished by a mTOR activator MHY1485, indicating a crucial role of mTOR in anti-tumor effects of puerarin in PDAC. As a result, puerarin hindered glucose uptake and metabolism by downregulating the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) dependent upon HIF-1α and glucose transporter GLUT1.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003ePuerarin has therapeutic potential for the treatment of PDAC by suppressing Akt/mTOR activity.\t\u003c/p\u003e","manuscriptTitle":"The Isoflavone Puerarin Exerts Anti-Tumor Activitiy in Pancreatic Ductal Adenocarcinoma by Suppressing Akt/mTOR Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-01-25 23:21:32","doi":"10.21203/rs.3.rs-152446/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4c685fa1-8e90-46a5-991e-2e747d7101ff","owner":[],"postedDate":"January 25th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":2045295,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2021-01-29T14:26:10+00:00","versionOfRecord":[],"versionCreatedAt":"2021-01-25 23:21:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-152446","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-152446","identity":"rs-152446","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}