Abstract

Background

   Pancreatic cancer is a malignant tumor of the digestive tract with a
   high mortality rate. Erianin has antitumor activity, but the regulatory
   targets and mechanism of action in pancreatic cancer are unclear. The
   objective of this study was to evaluate the anti-pancreatic cancer
   activity of Erianin and explore its underlying mechanisms.

Methods

   A network pharmacology approach was used to investigate the mechanism
   of action of Erianin in pancreatic cancer cells. Cell proliferation was
   analyzed using CCK8, colony-formation, and EdU proliferation assays.
   Cell migration was evaluated through wound healing and transwell
   assays, as well as determination of the protein expression levels of
   EMT markers and β-catenin. Apoptosis and the cell cycle were measured
   using flow cytometry and JC-1 staining, respectively. The protein
   expression levels of p-Rb, CyclinB1, P21, Cleaved-PARP, and
   Cleaved-Caspase3 were assessed using western blotting. RNA sequencing
   (RNA-seq) and bioinformatics analyses were performed to elucidate the
   mechanism underlying the action of Erianin in pancreatic cancer.
   Western blotting was used to examine the expression levels of key
   proteins in the AKT, JNK, and p38 MAPK signaling pathways. Molecular
   docking and CETSA were used to test hypotheses. The tumor-suppressive
   ability of Erianin in vivo was assessed using a tumor-bearing assay in
   nude mice.

Results

   Network pharmacology revealed that Erianin inhibited pancreatic cancer
   through multiple pathways. Erianin significantly inhibited pancreatic
   cancer cell proliferation and migration while promoting intracellular
   ROS and inducing apoptosis. Mechanistically, Erianin inhibited
   pancreatic cancer cell proliferation by regulating the AKT/FOXO1 and
   ASK1/JNK/p38 MAPK signaling pathways. In vivo experiments showed that
   Erianin inhibited subcutaneous tumor growth and promoted tumor tissue
   apoptosis in nude mice.

Conclusions

   The component-target-pathway network revealed that Erianin exerted
   anti-cancer effects through multiple components, targets, and pathways.
   Erianin inhibited the proliferation and migration of pancreatic cancer
   cells and induced apoptosis through the AKT/FOXO1 and ASK1/JNK/p38 MAPK
   signaling pathways. These results indicate that Erianin is a promising
   agent for pancreatic cancer treatment.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12935-024-03533-9.

   Keywords: Pancreatic cancer, Erianin, AKT signaling pathway, JNK/p38
   signaling pathway, Molecular mechanism

Background

   Pancreatic cancer is a common digestive tract tumor with a high degree
   of malignancy [[46]1]. The disease progresses without typical symptoms,
   which makes early diagnosis difficult. Pancreatic cancer has an
   extremely high mortality rate, with a 5-year survival rate of less than
   10%, and is expected to become the second deadliest cancer worldwide by
   2030 [[47]2, [48]3]. Moreover, pancreatic cancer is resistant to
   chemoradiotherapy and has limited treatment options, posing great
   challenges to clinical treatment [[49]4]. Consequently, the
   identification of more effective molecular therapeutic targets for
   pancreatic cancer and development of novel drug candidates are
   essential.

   An imbalance in the PI3K/AKT signaling pathway is a critical regulator
   of tumor aggressiveness. Angiogenesis provides oxygen and nutrients for
   tumor growth, and inhibition of AKT phosphorylation can inhibit
   angiogenesis in pancreatic cancer [[50]5]. Furthermore, forkhead box
   protein O1 (FOXO1) functions as a downstream molecular effector of the
   AKT pathway and is an important cell cycle-associated transcription
   factor [[51]6]. P38 and JNK are two major subfamily members of the
   mitogen-activated protein kinase (MAPK) signaling pathway and play
   essential roles in the regulation of intracellular metabolism, gene
   expression, cell growth, differentiation, and apoptosis [[52]7]. JNK
   and p38 play important roles in the malignant phenotype of various
   cancers [[53]8]. JNK is highly activated in pancreatic cancer cells
   [[54]9] and ASK1 overexpression plays a crucial role in pancreatic
   cancer development by promoting cell proliferation [[55]10]. Therefore,
   it is vital to evaluate whether the AKT/FOXO1 and JNK/p38 MAPK
   signaling pathways play a role in anti-pancreatic cancer effects.

   Erianin, a natural biphenyl compound derived from the Chinese herbal
   medicine Dendrobium nobile, is a prominent chemical component. Initial
   reports have shown that Erianin can be used as an antipyretic and
   analgesic drug [[56]11]. Recent studies have demonstrated remarkable
   anticancer activity of Erianin. For instance, Erianin can increase the
   expression of BAX and Caspase-3, inhibit tumor cell proliferation, and
   promote apoptosis [[57]12]. Additionally, Erianin exerts its anticancer
   effects by activating Ca2+/CaM signaling, increasing Ca2 + and
   Fe2 + levels, inducing ferroptosis, and inhibiting cell migration in
   lung cancer cells [[58]13]. While the pharmacological effects of
   Erianin and the molecular mechanisms of its antitumor activity and
   effects on natural immunity have been reviewed and systematically
   described [[59]14], the effect of Erianin and its molecular mechanism
   of action on pancreatic cancer remain to be explored.

   In this study, we investigated the effects of Erianin on pancreatic
   cancer cells and its potential mechanism of action. Our data revealed
   that Erianin significantly inhibited the proliferation and migration of
   pancreatic cancer cells by inducing cell cycle arrest and apoptosis.
   Mechanistically, Erianin directly bound to AKT and ASK1 and inhibited
   the progression of pancreatic cancer by regulating the AKT/FOXO1 and
   JNK/p38 MAPK signaling pathways. In conclusion, Erianin exhibited
   potent anticancer efficacy both in vivo and in vitro and has great
   potential as an effective therapeutic agent for pancreatic cancer
   patients.

Materials and methods

Prediction of drug targets for Erianin

   The SMILES chemical structure of Erianin was obtained using PubChem
   ([60]https://pubchem.ncbi.nlm.nih.gov/). Subsequently, the SMILES
   structure was uploaded to the Swiss Target Prediction database
   ([61]http://www.swisstargetprediction.ch/) to identify Erianin targets.

Prediction of targets for pancreatic cancer

   Pancreatic cancer-related targets were searched using the GeneCards
   ([62]https://www.genecards.org/), OMIM ([63]https://www.omim.org/), TTD
   ([64]https://db.idrblab.net/ttd/), and DisGeNET
   ([65]https://www.disgenet.org/home/) databases.

Construction of a protein-protein interaction (PPI) network and gene ontology
(GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses

   A PPI network of intersecting targets between Erianin and pancreatic
   cancer cells was constructed using the STRING database
   ([66]https://string-db.org/). The minimum required interaction score,
   ranging from 0 to 1, was set to > 0.9. The PPI network was further
   calculated, and hub genes were identified and visually analyzed using
   Cytoscape 3.7.2 software. The ClusterProfiler package of R3.18 was used
   to perform KEGG enrichment analyses.

Molecular docking and cellular thermal shift assay (CETSA)

   The 3D structure of Erianin was obtained from PubChem
   ([67]https://pubchem.ncbi.nlm.nih.gov). AutoDock Vina was used to dock
   the target proteins with Erianin. After exposure to DMSO or Erianin for
   48 h, cells were collected, washed with PBS containing protease
   inhibitors, aliquoted into 7 PCR tubes, and heated in a thermal cycler
   (T100, BioRad, Hercules, CA, USA) from 37 °C to 55 °C for 4 min to
   denature proteins. The cells were then subjected to three freeze-thaw
   cycles with liquid nitrogen, and centrifuged at 12,500 × g for 20 min
   at 4 °C. The supernatant was boiled in a loading buffer for western
   blot analysis.

Cell lines and cell culture

   Human pancreatic cancer cell lines (SW1990 and L3.7) were kindly
   donated by Tianjin Medical University Cancer Institute & Hospital,
   Tianjin, China. Cell lines were cultured in DMEM and RPMI 1640
   supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a
   humidified atmosphere containing 5% CO[2].

Cell viability analysis

   Pancreatic cancer cells were seeded at a density of 5,000 cells per
   well in 96-well plates and treated with different concentrations of
   Erianin (Yuanye Biotechnology, Shanghai, China) at various time points.
   Following treatment, 10 µL CCK8 solution (#C0039, Beyotime
   Biotechnology, Shanghai, China) was added to each well and the plates
   were placed in the dark at 37 °C for 2 h. Absorbance was measured at
   450 nm using a microplate reader.

Clone formation assay

   SW1990 and L3.7 cells (500 cells/well) were seeded in 6-well plates and
   maintained overnight. The cells were exposed to DMSO or Erianin for
   48 h, and then cultured in fresh medium for 14 d. The cell colonies
   were fixed with 4% paraformaldehyde and stained with 0.1% crystal
   violet. Colonies containing more than 50 cells were manually counted.

5-ethynyl-29-deoxyuridine (EdU) assay

   EdU assay was performed according to the EdU cell proliferation kit
   instructions (#C0078S, Beyotime Biotechnology, Shanghai, China). Cells
   (2 × 10^4 cells/well) were seeded in 24-well plates and exposed to
   Erianin for 48 h. Cells were incubated with 200 µL EdU reagent (10 µM)
   per well for 1 h, fixed with 4% paraformaldehyde for 15 min, and
   perforated with 0.3% TritonX-100 for 15 min. The Click Additive
   Solution and DAPI were added to the cells and incubated in the dark for
   30 min. The results were captured using a fluorescence microscope.

Tumor cell migration assays

   For the wound-healing assays, pancreatic cancer cells were seeded in
   6-well plates and scratched with a sterile plastic pipette tip (200 µL)
   when the monolayer reached approximately 90% confluence. The cells were
   then washed thrice with PBS and treated with Erianin for 48 h. Images
   were captured using a light microscope at 0 and 48 h after scratching.
   The results were quantitatively analyzed using ImageJ software.

   For the transwell assay, 1 × 10^4 cells in 200 µL serum-free medium
   were placed in the upper chambers, and the lower chambers were filled
   with a medium containing 20% FBS (total volume 600 µL). The cells were
   treated with Erianin for 48 h, fixed with 4% paraformaldehyde for
   30 min, and stained with 0.1% crystal violet for 30 min. Transwells
   were photographed and analyzed under a microscope. The migration
   ability of the pancreatic cancer cells was quantified as the mean
   number of cells in three randomly selected fields.

Flow cytometry analysis of apoptosis and cell cycle

   The proportion of apoptotic cells was assessed using the Annexin
   V-FITC/PI Apoptosis Detection Kit (#An0050, Zeta life, USA). SW1990 and
   L3.7 cells were treated with Erianin for 48 h. Treated cells (1 × 10^6)
   were centrifuged and re-suspended in 1× cold binding buffer.
   Subsequently, 5 µL each of Annexin V-FITC and PI were added and
   incubated for 15 min at room temperature in the dark.

   The cell cycle was assessed using the Cell Cycle Assay Kit (#E-CK-A351,
   Elabscience Biotechnology, Wuhan, China). Treated cells (5 × 10^5) were
   fixed with absolute ethanol and stored at -20 °C overnight. RNase A
   Reagent (100 µL) was added, the cells were re-suspended in a 37 °C
   water bath for 30 min, and 400 µL PI Reagent (500 µg/mL) was added and
   mixed thoroughly. Cells were incubated at 4 °C in the dark for 30 min.
   Stained cells were detected using an flow cytometer and analyzed using
   FlowJo 10.8 software.

JC-1 staining

   Cells (2 × 10^4 cells/well) were seeded in 12-well plates and exposed
   to Erianin for 48 h. The mitochondrial membrane potential in SW1990 and
   L3.7 cells was determined via a JC-1 fluorescent probe (#C2006,
   Beyotime Biotechnology, Shanghai, China). Cells were incubated with
   JC-1 working solution for 20 min at 37 °C, followed by at least three
   washes with JC-1 buffer solution. Cells were photographed using
   fluorescence microscopy; the red fluorescence intensity represents the
   level of mitochondrial destruction.

ROS detection

   For detection of total ROS in cells, SW1990 and L3.7 cells were treated
   with Erianin for 48 h and then incubated with the DCFH-DA (#S0033S,
   Beyotime Biotechnology, Shanghai, China) for 30 min at 37 °C. The cells
   were then washed thrice with FBS-free medium. Finally, fluorescence was
   observed and analyzed.

Western blotting

   Total protein was extracted using RIPA buffer and quantified using the
   BCA Protein Assay Kit (#PC0020, Solarbio, Beijing, China). Each lane
   was loaded with 20 µg of protein, and the proteins were separated using
   SDS-PAGE and transferred onto 0.22 µM PVDF membranes. The membrane was
   blocked using a 5% skim milk solution for 1 h at room temperature,
   followed by incubation with different primary antibodies and
   corresponding HRP-labelled secondary antibodies. Target protein
   expression was detected using an enhanced chemiluminescence reagent.
   The primary antibodies used were: N-cadherin (#22018-1-AP), E-cadherin
   (#20874-1-AP), β-catenin (#51067-2-AP), Vimentin (#10366-1-AP), β-ACTIN
   (#81115-1-RR), BCL2 (#68103-1-Ig), BAX (#50599-2-Ig), ASK1
   (#28201-1-AP), p-ASK1 (#28846-1-AP), P38 (#14064-1-AP), p-P38
   (Thr180/Tyr182, #28796-1-AP), these antibodies were purchased from
   Proteintech (Wuhan, China). Cleaved-Caspase3 (Asp175, #9664), AKT
   (#9272), p-AKT (#4060), Foxo1 (#2880), p-Foxo1 (#84192), p-Rb
   (Ser807/811,#8516), CyclinB1 (#4138), P21 (#2947), Cleaved-PARP
   (Asp214, #9541), JNK (#9252), and p-JNK (#4668), the above antibodies
   were purchased from Cell Signaling Technology (Beverly, California,
   USA).

RNA-seq and pathway enrichment analysis

   Total RNA was isolated from SW1990 cells treated with either 0 or 100
   nM Erianin for 48 h using TRIzol Reagent (#15596026, Invitrogen,
   Waltham, MA, USA). Subsequent RNA sequencing was performed using an
   Illumina NovaseqTM 6000 (Illumina, Beijing, China). Raw reads were
   filtered and cleaned using FASTq. Sequences were aligned to the GRCh38
   reference genome using STAR. The gene expression levels were quantified
   using FPKM. Differentially expressed genes (DEGs) were identified using
   the Deseq2 package with a p-value < 0.05. Enrichment analysis was
   performed using the ClusterProfiler software package. R software was
   used for visualization.

Animal experiments

   Female BALB/c nude mice (4–6 week) were purchased from Tianjin Jinke
   Bona Biotechnology Co., LTD (Tianjin, China). The Laboratory Animal
   Ethics Committee approved the animal experiments, which were conducted
   in strict accordance with the NIH Guide for the Care and Use of
   Laboratory Animals.

Statistical analysis

   Significant differences between two groups were analyzed using an
   unpaired t-test, and differences between more than two groups were
   analyzed using a one-way ANOVA. Statistical analyses were performed
   using GraphPad Prism 9.5 software. Statistical significance was set at
   P < 0.05.

Results

Identification of Erianin and pancreatic cancer-related targets and
construction of ER-PC intersection network

   The structure of Erianin is shown in Fig. [68]1A. After overlapping,
   105 Erianin targets were identified from the Swiss Target Prediction
   database. A total of 4,131 pancreatic cancer targets were obtained from
   the above four databases after removing overlapping targets. A total of
   75 common targets (Fig. [69]1B) were screened based on the Venn
   diagram, and an ER-PC-target network diagram (Fig. [70]1C) was
   generated using Cytoscape software. In total, 77 nodes and 150 edges
   were identified (Fig. [71]1D). These targets were filtered according to
   the cytoHubba analysis, and 16 key target genes were obtained for
   Erianin treatment of pancreatic cancer (Fig. [72]1E). KEGG enrichment
   analysis showed that the key targets were mainly enriched in cellular
   senescence, focal adhesion, cell cycle, PI3K-AKT signaling pathway,
   MAPK signaling pathway, and FOXO signaling pathway (Fig. [73]1F,
   Supplementary Table [74]S1). These results revealed the signaling
   pathways and biological processes of Erianin against pancreatic cancer,
   showing that its tumor suppression is multi-targeted and multi-pathway.

Fig. 1.

   [75]Fig. 1
   [76]Open in a new tab

   Intersection targets and bioinformatic analysis between ER and PC. (A)
   Chemical structure of Erianin. (B, C) 75 intersection targets of ER and
   PC. (D) The PPI network of ER and PC intersection targets. (E) Key
   targets of ER for treating PC based on cytoHubba analysis. (F) KEGG
   pathway analysis

Erianin suppresses pancreatic cancer cell proliferation

   To clarify whether Erianin affects the proliferation of pancreatic
   cancer cells, two pancreatic cancer cell lines (SW1990 and L3.7) were
   treated with different concentrations of Erianin for 48 h. CCK8 results
   showed that Erianin significantly inhibited the proliferative viability
   of pancreatic cancer cells in a dose-dependent manner (Fig. [77]2A-B).
   The 48-h inhibitory concentration 50 (IC50) values of SW1990 and L3.7
   cells were 472.8 and 101.0 nM, respectively (Fig. [78]2C-E). Based on
   these results, Erianin concentrations approaching the IC50 value were
   used for subsequent experiments. Morphological observations showed that
   in both the SW1990 and L3.7 cell lines, the number of shrunken,
   rounded, and detached cells significantly increased with Erianin
   treatment (Fig. [79]2F). To further demonstrate the inhibitory effects
   of Erianin on pancreatic cancer cells, we performed colony formation
   and EdU proliferation assays. Compared to the control group, there was
   a significant reduction in the number of clones and actively dividing
   cells after 48 h of Erianin treatment, indicating the potent inhibitory
   capacity of Erianin (Fig. [80]2G-J). These experiments demonstrated
   that Erianin significantly inhibited the proliferative ability of
   pancreatic cancer cells in vitro.

Fig. 2.

   [81]Fig. 2
   [82]Open in a new tab

   Erianin effectively suppresses the proliferation of PC cells. (A, B)
   Cell viability determined by CCK8 assay for SW1990 and L3.7 cells
   treated with Erianin (0, 25, 50nM) for 24,48,72 and 96 h. (C) IC50
   values of the SW1990 and L3.7 cells after Erianin exposure for 48 h.
   (D, E) Cell viability determined by CCK8 assay for SW1990 and L3.7
   cells treated with Erianin. (F) Cell morphology of SW1990 and L3.7
   cells treated with Erianin for 48 h. (G) Cell colony formation assays
   of SW1990 and L3.7 cells exposed to Erianin for 14 day. (H) The number
   of colonies of those in Fig. [83]1G. The data are presented as
   means ± SD, n = 3, *P < 0.05 vs. control group. (I, J) EdU
   proliferation assays of SW1990 and L3.7 cells exposed to Erianin for
   48 h

Erianin inhibits cell migration and induces G2/M phase arrest in pancreatic
cancer cells

   To further investigate the inhibitory effect of Erianin on pancreatic
   cancer cells, cell wound-healing assays were performed to analyze the
   effect of Erianin on the migratory ability of pancreatic cancer cells.
   After 48 h, the migrated cells in the control group had almost
   completely filled the scratch area. However, in the Erianin-treated
   group, minimal migration distance was observed for SW1990 and L3.7
   cells, indicating that Erianin significantly decreased the migratory
   ability of pancreatic cancer cells (Fig. [84]3A-B). Transwell assays
   also showed that Erianin significantly inhibited the migration of
   SW1990 and L3.7 cells (Fig. [85]3C-D). Western blotting showed that
   Erianin significantly down-regulated the expression of N-cadherin,
   vimentin, and β-catenin, while E-cadherin was up-regulated
   (Fig. [86]3E).

Fig. 3.

   [87]Fig. 3
   [88]Open in a new tab

   Erianin suppresses migration and cell cycle of PC cells. (A, B)
   Wound-healing ability of SW1990 and L3.7 cells with Erianin for 48 h.
   (C, D) The effect of Erianin on the migration in SW1990 and L3.7 cells.
   (E) Expression of N-cadherin, E-cadherin, β-catenin, and vimentin in
   SW1990 and L3.7 cells by western blotting. β-actin served as a loading
   control. (F, G) Cell cycle distribution as analyzed by flow cytometry.
   (H) Western blotting analyses of the expression of the cell cycle
   regulators phosphorylated Rb (p-Rb), Cyclin B1, and P21 in SW1990 and
   L3.7 cells. β-actin served as a loading control

   In addition, we investigated whether Erianin affected the cell cycle of
   pancreatic cancer cells. Flow cytometric analysis revealed that Erianin
   induced G2/M phase arrest (Fig. [89]3F-G). Western blotting showed that
   the expression of P21 was elevated, whereas the expression of p-Rb and
   the CDK regulator CyclinB1 was decreased after treatment with Erianin
   for 48 h (Fig. [90]3H). These findings further confirmed that Erianin
   suppressed epithelial-mesenchymal transition(EMT), inhibited pancreatic
   cancer cell migration, and arrested the cell cycle.

Erianin promotes apoptosis in pancreatic cancer cells

   To elucidate the mechanisms underlying Erianin-induced cytotoxicity in
   pancreatic cancer cells, we first investigated apoptosis. Flow
   cytometric analysis revealed that Erianin significantly increased the
   apoptotic rate of SW1990 and L3.7 cells (Fig. [91]4A). Correspondingly,
   the production of reactive oxygen species (ROS) increased after Erianin
   treatment and the mitochondrial membrane potential of the cells was
   significantly reduced (Fig. [92]4B-C). After Erianin treatment, the
   protein expression of BCL-2 and apoptosis-related proteins
   Cleaved-PARP, BAX, and Cleaved-Caspase3 increased (Fig. [93]4D). These
   results suggested that Erianin promoted ROS production and induced
   apoptosis via the mitochondrial pathway.

Fig. 4.

   [94]Fig. 4
   [95]Open in a new tab

   Erianin promotes apoptosis of PC cells. (A) Apoptosis of PC cells after
   Erianin for 48 h measured by flow cytometry. (B) ROS of PC cells after
   Erianin for 48 h. (C) JC-1 staining of PC cells after Erianin for 48 h.
   (D) Expression of Cleaved-PARP, BCL2, BAX and Cleave-Caspase3 in SW1990
   and L3.7 cells by western blotting. β-ACTIN served as internal standard

Erianin inhibits the AKT/FOXO1 pathway and activates the ASK1/JNK/p38 MAPK
pathway in pancreatic cancer cells

   To further explore the potential molecular mechanisms of Erianin in
   pancreatic cancer, we performed RNA Sequencing analysis to identify
   DEGs under Erianin stimulation in SW1990 cells (Supplementary Table
   [96]S2). GO enrichment analysis showed that Erianin-associated DEGs
   were enriched in important biological processes, such as focal
   adhesion, cadherin binding, cell-substrate junction, and ATP hydrolysis
   activity (Fig. [97]5A, Supplementary Table [98]S3). Consistent with the
   ER-PC-target network, KEGG pathway enrichment analysis also showed that
   Erianin-associated DEGs were enriched in the PI3K-AKT, FOXO, and MAPK
   signaling pathways, suggesting that these pathways may play key roles
   in the anti-pancreatic cancer effects of Erianin (Fig. [99]5B,
   Supplementary Table [100]S4). Western blotting showed that Erianin
   significantly downregulated the phosphorylation level of AKT and the
   phosphorylation and total expression level of FOXO1 (downstream protein
   of AKT). In addition, Erianin significantly upregulated the
   phosphorylation of the MAPK pathway-related proteins ASK1, p38, and JNK
   in pancreatic cancer cells (Fig. [101]5C-E). Together, these results
   confirm that Erianin inhibited AKT/FOXO1 and activated the ASK1/JNK/p38
   MAPK signaling pathway in pancreatic cancer cells.

Fig. 5.

   [102]Fig. 5
   [103]Open in a new tab

   Enrichment analysis and mechanism of Erianin inhibition in pancreatic
   cancer cells. (A, B) GO and KEGG pathway analysis (C-E) Expression of
   AKT and JNK/p38 MAPK pathway. β-ACTIN served as internal standard

Erianin binds to target proteins with better thermal stability

   We analyzed whether Erianin could bind to AKT or ASK1 proteins.
   Molecular docking predictions showed that Erianin has potential binding
   affinity with ASK1 and AKT, with binding energies of -6.6 kcal/mol and
   − 4.7 kcal/mol, respectively (Fig. [104]6A-B). Cellular thermal shift
   assay(CETSA) experiments showed that Erianin shifted the melting curve
   of ASK1 to the right (Fig. [105]6C), implying that Erianin binds
   directly to ASK1. The same trend was observed for AKT. The results
   showed that Erianin binds to ASK1 and AKT with better protein thermal
   stability.

Fig. 6.

   [106]Fig. 6
   [107]Open in a new tab

   Molecular docking and CETSA results of Hub protein and ER. (A, B)
   Molecular docking between ER complexed with AKT and ASK1. (C) Cellular
   thermal shift assay between Erianin AKT and ASK1. The curve was fitted
   using GraphPad Prism 9.5

Erianin inhibits tumorigenicity of pancreatic cancer in vivo

   To further determine whether Erianin inhibited the tumorigenicity of
   pancreatic cancer, we constructed a subcutaneous xenograft tumor model
   using SW1990 cells and BALB/c nude mice. The results showed that
   Erianin inhibited tumor growth in tumor-bearing nude mice, and the
   tumor size and weight were significantly lower than those in the
   control group (Fig. [108]7A-D). In addition, HE and TUNEL staining
   showed that Erianin significantly induced apoptosis in tumor tissues
   (Fig. [109]7E-F). IHC showed increased phosphorylation levels of JNK
   and P38 and cleaved-caspase3, and decreased phosphorylation levels of
   AKT and ki-67 in the Erianin group (Fig. [110]7G). In conclusion, these
   findings demonstrate that Erianin inhibited tumor growth in pancreatic
   cancer in vivo.

Fig. 7.

   [111]Fig. 7
   [112]Open in a new tab

   Erianin exhibits anti-cancer effects in vivo. (A)Xenograft model in
   nude mice. (B, D) Tumor volume and weight of SW1990 xenograft tumors
   after Erianin treatment for 14 days. (C) Growth curve of the tumor
   volumes measured on the indicated days. Error bars represent the
   mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t-test. (E)
   TUNEL staining was used to detect apoptosis in tumour tissues. (F) HE
   staining was used to observe the changes in the morphology and
   structure of the tumour tissue. (G) IHC was used to detect the
   expression levels of Ki-67, Cleaved-Caspase3 and the phosphorylation
   levels of AKT, JNK, and P38 in tissue cells

Discussion

   Changes in cell signaling are the main mechanisms employed by cells in
   the development and progression of multiple cancers [[113]15]. AKT and
   MAPK pathways are important signal transduction pathways in a variety
   of malignancies because they are critically involved in tumor cell
   growth, metabolism, proliferation, invasion, and apoptosis [[114]16,
   [115]17]. For instance, the AKT/FOXO1 and MAPK pathways act as tumor
   factors in lung, colorectal, breast, bladder, and prostate cancer
   [[116]18–[117]23]. Both pathways are expected to be useful in cancer
   treatment, especially for pancreatic cancer.

   Natural products and their derivatives, which are characterized by
   structural diversity and high biological activity, are important
   sources for drug discovery and development. A total of 75% of antitumor
   compounds currently used to treat human cancers are natural products
   [[118]24]. With advancements in natural compound extraction and
   separation technologies, a growing number of compounds with
   anti-pancreatic cancer activities have been identified in plants
   [[119]25–[120]29]. For instance, CK21 is a novel pro-drug of triptolide
   that exerts potent anti-proliferative effects on human pancreatic
   tumors by inhibiting the NF-κB pathway, ultimately leading to
   mitochondrial-mediated tumor cell apoptosis [[121]25]. DT-13 inhibits
   proliferation and induces apoptosis of pancreatic cancer cells by
   activating the AMPK/mTOR signaling pathway and suppressing p70 S6K
   [[122]29].

   In recent years, the tumor-suppressive effects of Erianin have been
   confirmed in studies on various diseases such as liver [[123]30,
   [124]31], lung [[125]13], breast [[126]32, [127]33], and colorectal
   cancer [[128]34]. These findings illustrate that Erianin exhibits
   different targets and phenotypes in diverse tumor cell types.
   Consistent with previous reports, we found that Erianin suppressed the
   proliferation, migration, and EMT of pancreatic cancer cells and
   promoted apoptosis at nanomolar concentrations. Further experiments
   revealed that Erianin inhibited AKT/FOXO1, while activating the
   ASK1/JNK/p38 MAPK pathway. Our results are consistent with those of
   previous reports showing that Erianin promotes apoptosis in tumor cells
   by inhibiting AKT and activating the MAPK pathway [[129]35, [130]36].
   However, another study found that Erianin inhibited p38 phosphorylation
   to prevent human oral cancer cell proliferation, while JNK
   phosphorylation was also activated [[131]37]. This discrepancy may be
   because JNK and p38 signaling outcomes depend on cell types and
   contexts. In our study, ASK1 activated the MAPK pathway to exert
   antitumor effects. Our findings further refine the mechanism of action
   of Erianin in various tumors. Therefore, Erianin may have a
   bidirectional regulatory role; it is necessary to explore in depth the
   role of Erianin in tumors in future studies.

   Molecular docking techniques use receptors and ligands of known
   structures to identify intermolecular interactions, which can identify
   the binding of traditional Chinese medicine active ingredients to
   potential targets [[132]38]. Molecular docking results indicated that
   Erianin could bind directly to AKT and ASK1 proteins. This was verified
   by CETSA experiments. The results indicated that the thermodynamic
   stability of the target protein was significantly improved and that
   Erianin bound tightly to the target proteins. These results emphasize
   the potential mechanism of Erianin for treating pancreatic cancer via
   AKT and ASK1 pathway (Fig. [133]8).

Fig. 8.

   [134]Fig. 8
   [135]Open in a new tab

   The potential mechanism for the treatment of pancreatic cancer with
   Erianin

Conclusions

   In conclusion, Erianin inhibited the proliferation and migration of
   pancreatic cancer cells and induced apoptosis by targeting the
   AKT/FOXO1 and ASK1/JNK/p38 MAPK pathways. Our study provides a
   rationale for the use of Erianin as a potential agent for pancreatic
   cancer treatment.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [136]Supplementary Material 1^ (339.5KB, xlsx)
   [137]Supplementary Material 2^ (2.7MB, xlsx)
   [138]Supplementary Material 3^ (108.7KB, xlsx)
   [139]Supplementary Material 4^ (47.3KB, xlsx)

Acknowledgements