The isoflavone puerarin exerts anti-tumor activity in pancreatic ductal adenocarcinoma by suppressing mTOR-mediated glucose metabolism

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). The medium contained penicillin (100 U/mL) and streptomycin (100 μg/mL). FBS, penicillin and streptomycin were from Invitrogen. The cultured cells at a density of 1 × 10⁶ were initially plated in a 10-cm dish for 24 h. The culture medium was replaced with serum-free medium after 24 h. PDACs were treated with 0.2 and 0.5 mM puerarin (Figure 1A, puerarin chemical structure, CAS#: 3681-99–0, Purity: ≥98% by High Performance Liquid Chromatography, Yuanye Biotechnology, Shanghai, China) with or without MHY1485 (CAS#: 326914-06-1, MedChem Express, Monmouth Junction, NJ, USA).

Figure 1. Puerarin inhibits cellular proliferation and induces mitochondrial-dependent apoptosis in PDACs. (A) The chemical structure of puerarin. (B) The growth of PDACs with or without puerarin treatment was determined by (RTCA). (C) The viability of PDACs was analyzed by the CCK-8 assay. (D) The proliferation of PDACs in different groups was analyzed by the colony formation assay. (E) Immunocytochemical staining of Ki67 in PDACs. Bar = 100 μm. (F) Protein expression of c-Myc in PDACs. (G) Flow cytometry analysis of cell apoptosis in PDACs with or without puerarin treatment. (H) Western blot analysis showing the expression of Cleaved caspase-8, Bcl-2, and Bax in PDACs. Data were presented as the mean ± standard deviation and were analyzed by one-way ANOVA with Bonferroni’s post-hoc test. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

Cell counting kit-8 (CCK-8) assay

According to the manufacturer’s instructions, the CCK-8 assay kit (Dojindo, Shanghai, China) was used to detect the cell proliferation of PDACs. 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 × 10³ 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. PDACs 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 (BD FACSVerse™, BD Biosciences, 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 × 10³ 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 ethylenediaminetetraacetic acid (EDTA) enzyme. The cells were resuspended in serum-free medium and transferred to the inner chamber (5 × 10⁴ cells per chamber). Complete medium was added to the outer chamber, and the plate was incubated in a CO₂ incubator (37°C) 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 × 10³ 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

PDACs were seeded in 6 well plates and maintained at 37°C 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. PDACs 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°C, followed by incubation with the primary antibodies (Supplementary Table 1) at 4°C 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°C with the primary antibodies (Supplementary Table 1). 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 oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in intact cells were measured using a Seahorse XF96 Analyzer (Agilent Technologies, Santa Clara, CA, USA). Concisely, 1 × 10⁴ PDACs were seeded into 96-well seahorse cell culture plates and incubated overnight at 37°C. Then the cells were pretreated with different concentrations of puerarin for 24 h. For OCR detection, the compounds were added as follows: oligomycin (2.5 μM), carbonyl cyanide 4-(trifluoromethoxy), phenylhydrazone (FCCP, 2 μM), rotenone (0.25 μM). The ECAR was evaluated after the sequential injection of glucose (10 mM), oligomycin (1 μM), and 2-Deoxy-D-glucose (2-DG, 50 mM). At the end of the experiment, overall OCR and ECAR curves were normalized to protein concentrations and plotted using Wave software (Agilent Technologies).

Nude mouse tumorigenicity

BALB/c nude mice (6–8 weeks old, Wenzhou Medical University Experimental Animal Center, Wenzhou, China) were used to establish the nude mouse xenograft model. All mice were housed under controlled conditions (temperature, 21–23°C; 12 h light/dark cycle; 55% humidity). PANC-1 cells (3 × 10⁶) in 0.2 ml PBS were subcutaneously injected into the right thighs of nude mice, and randomly divided twelve mice into two groups (n = 6 in each group). Puerarin was solubilized in normal saline buffer. Mice in the experimental group was injected puerarin (50 mg/kg) by oral gavage every three days for one month. The control group received normal saline injections by oral gavage for one month [11]. Tumor formation in the nude mice was monitored for 30 days. The mice were deeply anesthetized with sodium pentobarbital and euthanized by cervical dislocation [12–14]. The tumor size was calculated according to the standard formula: tumor volumes (cm³) = (the longest diameter) × (the shortest diameter)² × 0.5.

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) [15].

Molecular docking

Molecular docking was performed as previously described [16], 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.

Histopathological analysis

Tumor specimens from the animals were paraffin-embedded and cut into 4-μm-thick sections. Standard hematoxylin-eosin (H&E) staining (Biyuntian, Hangzhou, China) was performed. According to a previous method, immunohistochemical (IHC) analysis was conducted under a microscope [15]. IHC staining was performed using the following primary antibodies (Supplementary Table 1). Two independent investigators semi-quantitatively assessed all samples in a blinded manner.

Database analysis

The correlation between AKT and mTOR expression and the activity of KRAS, TP53, CDKN2A, and SMAD4 were 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.

Ethics statement

Animal experiments were approved by the Committee for Animal Experiments at Wenzhou Medical University.

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 Figure 1B, 1C, as expected, puerarin treatment (0.2 and 0.5 mM) significantly inhibited the growth of PDACs in concentration- and time-dependent manners. The CCK-8 assay results of the PDACs confirmed the concentration-dependent inhibition of cell growth by puerarin (Figure 1B, 1C). Puerarin also significantly reduced colony formation in the PDACs (Figure 1D). 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 [17]. In the PDACs, we observed a decrease in Ki67 protein expression in both the PANC-1 and PATU-8988T cells treated with puerarin (Figure 1E) compared to the control cells. In addition, puerarin reduced the expression of proliferation-related protein c-Myc (Figure 1F). Therefore, the above results suggest that puerarin inhibited PCC proliferation in concentration- and time-dependent manners.

Then, we evaluated the effects of puerarin on PCC apoptosis by flow cytometry analysis. Puerarin treatment significantly increased the proportion of apoptotic and necrotic cells (Figure 1G). Further studies showed increased caspase-8 in both the PANC-1 and PATU-8988T cells treated with puerarin (Figure 1H). Apoptosis in cancer cells depends upon the dynamic equilibrium of Bax and Bcl-2 expression [18]. Puerarin was observed to increase Bax expression and decrease Bcl-2 expression (Figure 1H). 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 [19]. Here, puerarin reduced the migration rate of PDACs as determined by the scratch wound assay (Figure 2A, 2B) and the effect was concentration-dependent. Also, puerarin treatment significantly inhibited the numbers of invading PDACs detected by the transwell assay (Figure 2C, 2D). Further studies showed that puerarin decreased the protein level of α-SMA in PDACs and increased the E-cadherin protein level (Figure 2E). Immunofluorescence analysis revealed the downregulated expression of α-SMA and the increased expression of E-cadherin after puerarin treatment (Figure 2F). In general, these results suggest that puerarin inhibited PCC migration and invasion by inhibiting EMT and tumor mammosphere formation.

Figure 2. Puerarin inhibits PCC invasion and migration by antagonizing the Slug-E-cadherin axis. (A, B) The effect of puerarin on the migrated rate of PDACs was determined by the wound healing assay. (C, D) The effects of puerarin on the invading number of PDACs were analyzed by the transwell chamber assay. (E) Western blot analysis showing the expression of E-cadherin and α-SMA in puerarin-treated PDACs. (F) Immunocytochemical staining of E-cadherin and α-SMA in puerarin-treated PDACs. 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 < 0.05, ^**p < 0.01, and ^***p < 0.001.

Puerarin suppresses PDAC growth in vivo

To determine the anti-cancer effects of puerarin in vivo, nude mice were injected with PANC-1 cells and then administrated puerarin or DMSO as a control. Figure 3A shows the morphology of tumor xenografts changes in the experimental group after puerarin treatment. The pathological results in the PDAC model tissue were shown by H&E staining (Figure 3B). We found that the administration of puerarin significantly reduced tumor volume and weight (Figure 3C, 3D). In addition, puerarin administration downregulated the expression of Ki67 and c-Myc (Figure 3E, 3F). Moreover, puerarin upregulated the expression of Cleaved caspase-8 and Bax, and decreased Bcl-2 expression (Figure 3G, 3H), suggesting that puerarin inhibited PCC proliferation and induced death receptor- and mitochondrial-mediated apoptosis. 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 (Figure 3I). It also reduced the expression of c-Myc, an oncoprotein associated with tumor progression and chemoresistance (Figure 3F) [20–22]. 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 [23]. We observed the downregulation of HIF-1α protein after puerarin treatment (Figure 3J). In summary, these data suggest that puerarin inhibited the growth of PDAC in a mouse xenograft model.

Figure 3. Puerarin inhibits the tumor growth and metastasis of PDAC in the animal xenograft model. (A) Effects of puerarin on morphologic changes in the experimental groups. (B) Pathological results of H&E 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) 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) Protein expression of Cleaved caspase-8, Bax and Bcl-2 in PDACs in different groups. (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 < 0.05 and ^***p < 0.001.

Puerarin reduces the activity of Akt/mTOR signaling in vitro and in vivo

Anti-cancer effects involve many mechanisms, including oxidative stress, intrinsic and extrinsic mechanisms, as well as the survivin, PI3K/Akt/mTOR, SHH [24], Nrf2/Keap1 [25], inflammation, and autophagy pathways [26]. 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 [27]. In Pancreatic tissue, including PAAD (Pancreatic adenocarcinoma) Tumor, PAAD normal and pancreas, the activation of mTOR was associated with gene mutations, including KRAS, TP53, CKDN2A, and SMAD4, resulting in tumor development (Figure 4A, 4B). We also found that these PCCs exhibited heterogeneous PI3K/Akt/mTOR pathway activation at the protein level (Figure 4C). In this study, we investigated the effect of puerarin on mTOR activity in PDACs. We found that puerarin suppressed the mTOR signaling pathway (Figure 4D, 4E), suggesting that mTOR may be a target of puerarin. Puerarin-induced the downregulation of phosphorylated mTOR expression in PDACs (Figure 4F, 4G). The in vitro experiments confirmed that puerarin inhibited the overexpression of mTOR in PDAC tissues (Figure 4H, 4I).

Figure 4. 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 PDACs with or without puerarin treatment. (F, G) Immunocytochemical staining of mTOR in PDACs. Bar = 50 μm. (H) Western blot analysis showing the expression and phosphorylation of Akt and mTOR in 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 < 0.05, ^**p < 0.01, and ^***p < 0.001.

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 (Figure 5A). We found that the possible binding sites of puerarin and mTOR included two structural regions i and ii (Figure 5B, 5C), with binding energies of −5.17 and −7.0, respectively. Figure 5D 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.

Figure 5. 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.

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 (Figure 6A–6C). Using the transwell and wound healing assays, we demonstrated that MHY1485 treatment increased the invasion and migration rates of the PDACs (Figure 6D–6G). Thus, activated mTOR signaling eliminated puerarin-mediated EMT suppression, as shown by the increased expression of α-SMA, vimentin, Snail1, and Slug (Figure 6H, 6I). These findings confirmed that mTOR signaling played a crucial role in the anti-tumor effect of puerarin in PDAC.

Figure 6. Reactivation of mTOR reverses the anti-tumor effects of puerarin. (A, B) The proliferation of puerarin-treated PDACs with or without MHY1485 treatment was analyzed by the colony formation assay. (C) Protein expression of c-Myc in puerarin-treated PDACs with or without MHY1485 treatment. (D, E) The migration ability of puerarin-treated PDACs in different groups was determined by the wound healing assay. (F, G) The invasion ability of puerarin-treated PDACs was analyzed by the transwell chamber assay. (H) Protein expression of vimentin and α-SMA in puerarin-treated PDACs. (I) Protein expression of Snail1 and Slug in puerarin-treated PDACs. 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 < 0.05, ^**p < 0.01, and ^***p < 0.001.

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 [28]. 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 (Figure 7A–7D), indicating that puerarin inhibited the energy metabolism of the mitochondria. The glycolysis of the tumor cells treated with puerarin was significantly inhibited (Figure 7E, 7G). The results showed that the basal glycolysis rate and the compensatory glycolysis rate decreased significantly (Figure 7F, 7H). Further studies showed that GLUT1 and HIF-1α protein expression was inhibited (Figure 7I). 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.

Figure 7. 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 PDACs. (E–H) Glucose metabolism assay showing reduced ECAR, basal glycolysis, and compensatory glycolysis in puerarin-treated PDACs. (I) Protein expression of GLUT1 and HIF-1α in puerarin-treated PDACs. 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 < 0.05, ^**P < 0.01, and ^***P < 0.001.