Integrated multi-omics analysis of Periplocymarin to identify the mechanism of p38α MAPK phosphorylation inhibition in Non-small cell lung cancer

Integrated multi-omics analysis of Periplocymarin to identify the mechanism of p38α MAPK phosphorylation inhibition in Non-small cell lung cancer | 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 Article Integrated multi-omics analysis of Periplocymarin to identify the mechanism of p38α MAPK phosphorylation inhibition in Non-small cell lung cancer Xinye Wang, Xiao Liang, Zhibin Song, Mingwei Wang, Hui Sun, Xiaoting Ma, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9282067/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background: Periplocymarin (PPM) is a plant-derived natural product, which is isolated and purified from the dried root bark of Periploca sepium. Previous studies have shown that Periplocymarin exhibits significantly and broad-spectrum anti-tumor pharmacological activity. To investigate the anti-tumor activity and mechanism of the natural product Periplocymarin against Non-small cell lung cancer (NSCLC). Methods: This study screened a library of 1459 anti-tumor natural products using CCK-8 assays in three NSCLC cell lines and two normal lung epithelial cell lines, identifying Periplocymarin as a selective candidate against NSCLC. The effects of Periplocymarin on NSCLC proliferation, apoptosis, and migration were assessed in cell lines and patient-derived organoid (PDO) models. Transcriptome sequencing, network pharmacology, proteome thermal stability profiling (TPP), Microscale Thermophoresis (MST) and Molecular Simulation were integrated to identify the target of Periplocymarin. Western blot and CCK-8 assays were performed to verify Periplocymarin’s effect on p38α mitogen-activated protein kinase (p38α MAPK, MAPK14) phosphorylation. In vivo anti-tumor efficacy and safety of Periplocymarin were evaluated in nude mouse xenograft and patient-derived xenograft (PDX) models. Results: Periplocymarin exhibited concentration-dependent inhibition of proliferation, promoted apoptosis, and reduced cell migration, while also suppressing growth in organoids. Integrated analyses indicated MAPK14 as the target, and Western blot confirmed markedly inhibition of MAPK14 phosphorylation by Periplocymarin. MAPK14 knockdown attenuated Periplocymarin’s anti-tumor effects. In vivo , Periplocymarin significantly inhibited tumor growth, with decreased Ki-67 and phospho-MAPK14 (p-MAPK14) expression in tumor tissues and no evident organ toxicity or hematological abnormalities. Conclusions: Periplocymarin, by predominantly inhibiting MAPK14 phosphorylation, presents a robust anti-tumor effect in vitro and in vivo . These findings provide a theoretical and experimental basis for the application of Periplocymarin and for the development of novel anticancer drugs targeting MAPK14 in NSCLC. NSCLC natural product library screen Periplocymarin multi-omics analysis MAPK14 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Lung cancer is characterized by lack of specific early symptoms, and approximately 70% of patients are diagnosed at advanced stage, making it one of the malignancies with high incidence and mortality worldwide( 1 ). Non-small cell lung cancer (NSCLC), the predominant histological subtype of lung cancer, is marked by high invasiveness, a propensity for metastasis, and a high rate of recurrence, resulting in poor prognosis( 2 ). Currently, NSCLC treatments primarily include surgical resection, radiotherapy, chemotherapy, targeted therapies, and immunotherapies( 3 ). Tyrosine kinase inhibitors against driver alterations such as EGFR and ALK have significantly improved the prognosis( 4 , 5 ). Anti-angiogenic therapies can synergize by modulating the tumor microenvironment to exert better therapeutic effect: Ivonescimab improved the progression free survival (PFS) of untreated patients with advanced PD-L1 positive NSCLC( 6 ). Moreover, PD-1/PD-L1–based immunotherapy has prolonged the survival of lung cancer patients( 7 ). Despite these advances, challenges including acquired resistance and treatment-related toxicities, illustrated the need for novel strategies. Natural products are characterized by chemical and structural diversity, as well as intrinsic multi-target mechanisms, which enable them and their derivatives with unique advantages in antitumor activity and pharmacodynamic synergy( 8 ). Compared with single-target drugs, natural products often modulate multiple pathways involved in tumor initiation and progression( 9 ). Substantial studies showed that natural products can induce tumor cell apoptosis, inhibit proliferation, and block angiogenesis( 10 ). Moreover, they can exert anti-tumor effects by modulating tumor metabolism (e.g., Polyphyllin VII reversing PARP inhibitor resistance( 11 )), remodeling the tumor microenvironment (e.g., Chelidonine-induced disrupts M2 macrophage polarization in breast cancer( 12 )), and regulating immune responses (e.g., Yi-Fei-San-Jie-Fang modulating immune evasion( 13 )). Importantly, many chemotherapeutic agents widely used in lung cancer are derived from natural products or their derivatives. Paclitaxel, extracted from Taxus species, stabilizes microtubule dynamics to prevent mitosis and suppress tumor growth( 14 ). Vincristine and Vinblastine, derived from Catharanthus roseus (Madagascar periwinkle), inhibit microtubule polymerization to block cell division, widely used in chemotherapy for various solid tumors( 15 ). In addition, Etoposide is a semi-synthetic derivative of podophyllotoxin from Podophyllum species, which induces DNA breaks by inhibiting topoisomerase II, thereby promoting tumor cell apoptosis( 16 ). Nevertheless, clinical translation of natural products remains challenged by compound stability, targeted delivery, and the complexity of their mechanisms. The diversity and unclear mechanisms of natural products limit their clinical applications as well as pharmaceutical innovation and development. Currently, there is a lack of systematic and standardized screening processes, and the specific targets and regulatory mechanisms of active constituents remain unclear, limiting the clinical utility and translational potential of natural products. Systematic screening of natural products and utilization of multi-omics, can help to elucidate their targets and molecular mechanisms( 17 ). This study identified Periplocymarin as a potent inhibitor of lung cancer cell proliferation through screening of a natural product library. Periplocymarin exerts selective cytotoxicity against NSCLC cells and showing tumor growth inhibition in cell-based assays, patient-derived organoids (PDOs), nude mouse xenograft, and patient-derived xenograft (PDX) models. Characterizing the target-binding affinities of natural products is essential for the discovery of novel anticancer agents and their biological activities( 18 ). Therefore, this study employs thermal proteome profiling (TPP) as a pivotal method for target discovery, combined with transcriptome sequencing, network pharmacology, and molecular computational analyses to elucidate Periplocymarin’s target as p38α mitogen-activated protein kinase (p38α MAPK, MAPK14). MAPK14 exhibits dual roles in cancer: its activation can induce cell cycle arrest and apoptosis and promote tumor cell proliferation( 19 , 20 ). The study demonstrates that Periplocymarin targets MAPK14 and inhibits its phosphorylation, thereby exerting anti-tumor effects. In summary, lung cancer, a highly heterogeneous and aggressive malignant tumor, urgently requires the development of definite-target, low-toxicity therapeutic pharmaceuticals. These present findings show that Periplocymarin can effectively inhibit proliferation and metastasis, promote apoptosis by suppressing MAPK14 phosphorylation in NSCLC. It can also suppress tumor growth in both nude mouse models and PDO/PDX xenografts, demonstrating favorable anti-tumor effects. These findings, together with natural product library screening and target-discovery technologies, will help elucidate the direct mechanisms of natural products and promote their clinical application in the treatment of lung cancer. Materials and Methods Reagents and Antibodies Natural product library (Cat. #HY-L107) and Periplocymarin (PPM, Cat. #HY-N4252) were purchased from MedChemExpress (MCE, Shanghai, China). Phospho-MAPK14 (p-MAPK14) (Cat. #28796-1-AP), MAPK14 (Cat. #14064-1-AP), Ki-67 antibody (Cat. #27309-1-AP) and α-tubulin (Cat. #66031-1-Ig) were obtained from Proteintech (Wuhan, China). Cell Culture and siRNA Transfection Normal human bronchial epithelial cell lines (Beas-2B and 16HBE cells) and NSCLC cell lines (A549, H1299, H292, HCC827, H1975, and H3122 cells) were obtained from the Cell Bank of the Chinese Academy of Sciences. Cells were maintained in RPMI-1640 or DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Servicebio, China). Cells were transfected with either non-targeting control siRNA (siRNA-NC) or siRNA targeting MAPK14 (si-MAPK14; interference sequence: 5’-3’: UUAAGUAACCGCAGUUCUC (dT) (dT)) using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. Forty-eight hours post-transfection, cells were collected for subsequent analyses. Natural Products Screen A library of 1459 anti-tumor natural products were screened for cytotoxic effects against NSCLC cells. In the initial screen, Beas-2B, 16HBE, A549, H1299, and H3122 cells were treated with 10 µM of each natural product for 48 hours, followed by CCK-8 assay. Candidate compounds producing cell viability less than 10% across the three cell lines were involved. In the secondary screen, these candidates were retested at a 5 µM concentration, and those showing markedly higher toxicity in lung cancer cells than in lung epithelial cells were confirmed. CCK-8 Assay Cells were treated with Periplocymarin at the concentrations of 0, 0.01, 0.05, 0.1, 1, or 10 µM for 24 or 48 hours. After treatment, 10% CCK-8 reagent (MeilunBio, China) in complete medium was added and incubated for 2 hours. IC 50 values were calculated by nonlinear regression using GraphPad Prism 9.0. To assess effects on proliferation capacity, cells were treated with Periplocymarin at the concentrations of 0, 50, and 100 nM. OD450 nm values were recorded at 0, 24, 48, and 72 hours with CCK-8 assay, and calculated using GraphPad Prism 9.0 for proliferation rates. Colony Formation Assay Cells were treated with Periplocymarin at 0, 20, 50, or 100 nM for 10 ~ 14 days. Following incubation, colonies were fixed with methanol for 15 minutes and stained with crystal violet for 45 minutes. After washing, colonies were quantified using ImageJ, and results were plotted in GraphPad Prism 9.0. EdU Assay Cells were treated with Periplocymarin at 0, 50, or 100 nM for 24 hours. EdU labeling was performed for 2 hours, after which cells were fixed with methanol for 10 minutes and permeabilized with 0.1% Triton X-100. EdU-positive cells were detected using the Azide 555 reagent (Beyotime, China), and nuclei were counterstained with Hoechst. Fluorescent images were captured, and EdU-positive and total-cell counts were quantified with ImageJ to calculate the proliferation rate. Wound-Healing Experiment Cells were seeded in 6-well plates and grown to 100% confluence. A vertical scratch was created using a 100 µL pipette tip. After washing with PBS to remove the debris, cells were incubated in serum-free RPMI-1640 containing Periplocymarin at 0, 20, or 50 nM. Images of the wound area were captured at 0, 24, and 48 hours. The scratch area was quantified with ImageJ, and migration was analyzed by plotting the closure over time. Transwell Assay Cells were suspended in 200 µL serum-free medium containing 0, 20, or 50 nM Periplocymarin and seeded into the upper chamber of Transwell inserts. The lower chamber contained 700 µL complete medium with 20% FBS and the corresponding Periplocymarin concentration. After 24 hours at 37°C with 5% CO 2 , non-migrated cells on the upper surface were removed, and migrated cells were fixed in 4% paraformaldehyde and stained with crystal violet. Images were captured and migrated cells were counted using ImageJ. Data were analyzed and presented using GraphPad Prism 9.0. Flow Cytometric Analysis Cells were seeded in 12-well plates and treated with final concentrations of Periplocymarin (0, 100, 200 nM) for 48 hours. Cells were digested with trypsin without EDTA, collected by centrifugation, and the supernatant was discarded. Cells were resuspended in 50 µL binding buffer, and 5 µL of PI and FITC-labeled Annexin V were added. Cells were then incubated at room temperature, followed by the addition of 300 µL binding buffer for flow cytometry analysis. FlowJo software was used to quantify the proportions of early apoptotic cells (Annexin V-positive, PI-negative) and late apoptotic cells (Annexin V-positive, PI-positive). Meanwhile, flow cytometry was performed to evaluate the effect of Periplocymarin on cell cycle progression. A549 and H1299 cells were treated with Periplocymarin at concentrations of 0, 100 and 200 nM for 24 hours. Subsequently, cells were collected and fixed overnight at 4°C with pre‑cooled 70% ethanol. After removal of ethanol, an appropriate amount of staining solution was added to the cell pellets, followed by incubation at 37°C in the dark for 30 min. Finally, samples were analyzed by flow cytometry and data were measured by FlowJo software. Measurement of intracellular ROS Cells were seeded in 35 mm confocal dishes and treated with different concentrations of Periplocymarin (0, 50, 100 nM) for 24 hours. Subsequently, cells were washed with pre-cooled PBS and incubated with 10 µM DCFH-DA (Beyotime, China) for 30 minutes. Cells were washed with PBS to remove unbound probes. Fluorescence images were captured using a confocal microscope (Nikon, Japan) to identify the ROS content. MDA Assay Cells were seeded in 6-well plates and treated with different concentrations of Periplocymarin (0, 50, 100 nM) for 24 hours, collected and homogenized on ice using pre-cooled lysis buffer. The lysate was centrifuged, and the supernatant was collected for MDA content determination, in accordance with the instructions of the MDA assay kit (Beyotime, China). Western Blot Analysis The lysate of treated cells were centrifuged, and the supernatant was collected. An appropriate amount of 5 × loading buffer was added, and samples were boiled at 100°C for 10 minutes. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 5% non-fat milk in TBST at room temperature for 2 hours. Primary antibodies against p-MAPK14 (1:1000), MAPK14 (1:1000), and α-Tubulin (1:5000) were incubated with the membranes in TBST at 4°C overnight. The next day, membranes were washed with TBST. Corresponding secondary antibodies were incubated at room temperature for 2 hours, followed by washes with TBST. Target protein bands were detected and quantified using the Tanon imaging system. Immunohistochemistry (IHC) Paraffin-embedded tumor tissue sections were prepared at a thickness of 4 ~ 5 µm and deparaffinized at 65°C. Sections were subsequently rehydrated sequentially in xylene, gradient ethanol (100%, 85%, 50%, 25%, subsequently), and deionized water. Antigen retrieval was performed using citrate buffer using high-temperature and high-pressure methods. Endogenous peroxidase activity was inhibited using 3% hydrogen peroxide solution, followed by blocking of non-specific binding sites with 5% goat serum at room temperature. Primary antibodies diluted in PBS were applied to tissue sections and incubated at 4°C overnight. The next day, sections were washed with PBS, followed by incubation with secondary antibodies from Maixin Biotechnology (China) at room temperature for 30 minutes. The expression of the indicated protein was visualized by DAB (Dako, Denmark). Sections were counterstained with hematoxylin, and immunostaining results were observed under microscope. Network Pharmacological Analysis To identify potential targets of Periplocymarin in lung cancer, the compound’s two-dimensional structure was first retrieved from PubChem. Using this structure, potential human targets were predicted with SwissTargetPrediction. Simultaneously, a lung cancer target set was assembled by integrating targets from DisGeNET, Online Mendelian Inheritance in Man (OMIM), and GeneCards, followed by deduplication. Overlaps between predicted drug targets and lung cancer targets were identified via Venn diagram analysis, and the intersecting targets were designated as candidate Periplocymarin targets for lung cancer. The overlapping target list was submitted to the STRING database (Homo sapiens; confidence score ≥ 0.9), and isolated nodes were removed to construct a protein–protein interaction (PPI) network. Subsequently, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on the candidate targets with a significance threshold of P < 0.05, clarifying the potential molecular mechanisms and critical pathways of Periplocymarin in NSCLC. Thermal Proteome Profiling (TPP) A549 cells were cultured and collected, then allocated into two groups and treated with either DMSO (vehicle) or Periplocymarin. The treatment temperatures were set at 37°C, 42°C, 46°C, 50°C, 54°C, 58°C, 62°C, and 67°C. Quantitative mass spectrometry (MS) was employed to comprehensively assess protein states. Dynamic changes in protein thermal stability parameters, notably the melting temperature (Tm), were quantitatively measured, and proteins displaying significantly enhanced stability following compound treatment were identified. Microscale thermophoresis (MST) Cells were transfected with the GFP-MAPK14 plasmid and harvested 48 h later. MST measurements were performed using a Monolith NT.115 Pico (NanoTemper Technologies). Periplocymarin was prepared as a 15-point serial dilution with identical final DMSO concentrations and mixed at equal volumes with GFP-MAPK14–containing cell lysates, ensuring constant protein concentrations and uniform DMSO content across all samples. The mixtures were loaded into capillaries and subjected to MST analysis to calculate the dissociation constant (K d ). RNA Sequencing A549 and H1299 cells were cultured and treated with DMSO or 200 nM Periplocymarin for 48 hours, and then collected for RNA sequencing analysis. Differentially expressed genes (DEGs) were identified for each cell line and subjected to downstream enrichment analyses. KEGG pathway enrichment analyses and Gene Set Enrichment Analysis (GSEA) trend analyses were conducted to identify signaling pathways potentially involved in Periplocymarin’s mechanism. PDOs Culture and Calcein AM staining Fresh tumor specimens were collected from NSCLC patients undergoing surgical resection at the Affiliated Jiangyin Hospital of Nantong University, with all patients providing informed consent. The study protocol was approved by the Institutional Ethics Committee (Approval No.: L028). Tumor tissues were enzymatically dissociated into single cells or small cell clusters using digestion buffer containing collagenase IV and DNase I. The resulting cell suspension was filtered through 70 µm cell strainer and centrifuged at 300 × g for 5 min. Cells were resuspended in low-growth-factor Matrigel (Corning, USA), and droplets were plated into 24-well plates to solidify at 37°C for 15 minutes. PDOs passaged three times were treated with Periplocymarin at 0, 50, and 100 nM for 7 days. Organoids were incubated with Calcein AM solution for 15 minutes, and the organoid morphology was captured by fluorescence microscopy to evaluate effects on growth and structural integrity. In Vivo Assay BALB/c nude mice (aged 4ཞ6 weeks, male) were purchased from GemPharmatech (Jiangsu, China) for xenograft experiments. A549 cells were prepared and mixed with Matrigel at a ratio of 3 × 10 6 cells in 40 µL of cell suspension to 40 µL Matrigel, then inoculated into the left axilla of each mouse. When tumor diameter reached approximately 5 mm, mice were randomly allocated into two groups (n = 5 per group): the control group received intraperitoneal injections of 100 µL PBS, while the treatment group received intraperitoneal injections of Periplocymarin at 5 mg/kg, every two days for 14 consecutive days. During treatment, body weight and tumor volume were measured every two days. After two weeks, mice were euthanized, tumors were excised and weighed, and tumor tissues were fixed and embedded for subsequent IHC analyses. All experimental protocols were approved by the Animal Ethics Committee of Nantong University (Approval No.: P20250310-012). Patient-Derived Xenograft (PDX) Models This study evaluated in vivo therapeutic effects in PDXs using male NCG mice aged 4ཞ6 weeks, purchased from GemPharmatech. Fresh tissues from NSCLC patients undergoing surgical resection at the Affiliated Hospital of Nantong University were collected with informed consent, and approved by the Animal Ethics Committee of Nantong University (Approval No.: 2025-L111). Tumor tissues were cut into sterile 1ཞ2 mm 3 fragments and subcutaneously transplanted into the left axilla of recipient mice. The tumor was passed into a new cohort of mice when reached approximately 15 mm in diameter. When the third-generation PDX tumors reached about 5 mm in diameter, mice were randomly assigned to two groups. Each group received intraperitoneal injections of PBS or Periplocymarin at 5 mg/kg every two days for 14 days. Body weight and tumor volume were recorded every two days. Then, mice were euthanized and tumors were excised and weighed, and the PDX tumor tissues were fixed and embedded for subsequent IHC analyses. All animal experiments were approved by the Animal Ethics Committee of Nantong University (Approval No.: P20250310-012). Statistical Analysis Statistical analyses were performed using GraphPad Prism 9.0. Data are presented as mean ± SD or SEM. Two-tailed unpaired Student’s t -test were used for comparisons between two groups. Two-way ANOVA was conducted for comparisons among multiple groups. P value < 0.05 was considered statistically significant. Results Screening of active compounds against NSCLC cells from a natural product library To identify natural products with cytotoxic effects against NSCLC cells, 1459 anti-tumor natural products were obtained for two-stage screening. A549, H1299, and H3122 cells were exposed to each compound at 10 µM for 48 hours, and cell viability was assessed using the CCK-8 assay (Fig. 1 A, B). Cytotoxicity was calculated for each compound. Analysis revealed 28 compounds that exhibited ≥ 90% cytotoxicity in three cancer cell lines (Fig. 1 C). To identify tumor cell-selective agents, the cytotoxic effects of these candidates were further evaluated in Beas-2B and 16HBE cells and three NSCLC cancer cells at a concerntration of 5 µM (Fig. 1 D). Periplocymarin displayed minimal toxicity to normal lung epithelial cells while demonstrating substantial cytotoxicity toward NSCLC cell lines, consistent with potential anti-tumor activity. Periplocymarin inhibits the proliferation and migration, promote apoptosis of NSCLC cells This study assessed the anti-tumor activity of Periplocymarin in NSCLC using six cell lines (A549, H1299, H292, HCC827, H1975, and H3122). Cells were treated with Periplocymarin at 0, 0.01, 0.05, 0.1, 1, and 10 µM for 24 or 48 hours, and viability was measured by the CCK-8 assay. Periplocymarin inhibited cell growth in a dose-dependent manner. The 24-hour IC 50 values were: A549, 0.43 µM; H1299, 0.37 µM; H292, 0.15 µM; HCC827, 3.78 µM; H1975, 0.50 µM; H3122, 0.32 µM, separately. The 48-hour IC 50 values were: A549, 0.13 µM; H1299, 0.39 µM; H292, 0.09 µM; HCC827, 0.44 µM; H1975, 0.19 µM; H3122, 0.08 µM, separately (Fig. 2 A). To further characterize dose-dependent effects, cells were treated with 0, 50, 100, and 200 nM Periplocymarin and viability was assessed at 0, 24, 48, and 72 hours by CCK-8 assay (Fig. 2 B), demonstrating continued inhibition of proliferation over time. Colony formation assays revealed a dose-dependent reduction in colony number following Periplocymarin exposure (Fig. 2 C, D), indicating substantial suppression of long-term proliferative capacity. Furthermore, the EdU assay showed that the EdU-positive signal decreased as the concentration of Periplocymarin increased (Fig. 3 A). These results suggest that Periplocymarin has a significant inhibitory effect on proliferation. To analyze whether Periplocymarin affects apoptosis, we treated A549, H1299, H1975 and H3122 cells with 0, 100, and 200 nM Periplocymarin for 48 hours, after which the cells were collected for flow cytometry analysis of apoptosis. The results showed that the apoptosis rate in the Periplocymarin-treated groups was higher than in the control group (Fig. 3 B, C), indicating that Periplocymarin promoted apoptosis in NSCLC cells. To thoroughly validate the inhibitory effect of Periplocymarin on cell proliferation, we established a PDO model and compared the effects of Periplocymarin (0, 100, and 200 nM) on PDO morphology and viability. Bright-field images showed that Periplocymarin treatment caused the organoids’ three-dimensional structures to become increasingly disrupted. Calcein-AM fluorescence decreased in a concentration-dependent manner, the signal diminished sequentially at 100 nM and 200 nM (Fig. 3 D, E), suggesting that higher concentrations of Periplocymarin could suppress organoid viability. Currently, the function of Periplocymarin on NSCLC remains unclear. To further investigate its effects, this study employed wound-healing and transwell assays to analyze the impact of Periplocymarin on NSCLC cell migration. In the wound-healing assay, A549, H1299, H1975, and H3122 cells were treated with 0, 20, and 50 nmol/L Periplocymarin, and scratch closure images were collected at 0, 24, and 48 hours, with the migrated area quantified. The results (Fig. 4 A, B) showed that increasing Periplocymarin concentration and longer exposure time markedly reduced the migrated area. In the transwell assay, A549, H1299, and H1975 cells were treated with 0, 20, and 50 nM Periplocymarin for 48 hours. The results (Fig. 4 C, D) demonstrated a significant decline in the number of migrated cells with increasing drug concentration. Notably, H3122 cells exhibited relatively weak migratory capacity and were therefore not included in the Transwell assay. In summary, Periplocymarin possesses the ability to inhibit the migration of NSCLC cells. RNA Sequencing indicates the possible pathways of Periplocymarin To elucidate the specific mechanism this study performed RNA sequencing on A549 and H1299 cells treated with Periplocymarin, followed by KEGG pathway enrichment and GSEA trend analyses based on the differentially expressed genes for each cell line. The results showed a high degree of concordance in pathway-level changes between the two cell lines, with MAPK pathway alterations ranking among the top enriched pathways (Fig. 5 A–C). Additionally, several classical tumor biology processes were enriched in both cell lines, including cell cycle regulation and p53-related regulatory networks. Further GSEA analyses of the transcriptomes from A549 and H1299 individually suggested that after Periplocymarin treatment, processes related to extracellular matrix organization, integrin-mediated adhesion, calcium signaling pathway, and drug metabolism cytochrome P450 were generally downregulated; conversely, pathways related to G2/M DNA damage checkpoint, G1/S DNA damage checkpoints, DNA damage response, the p53 signaling axis, and programmed cell death were significantly upregulated (Fig. 5 D–E). Collectively, the results support a consistent regulatory response induced by Periplocymarin, with MAPK signaling probably the principal regulatory pathway. Network pharmacology analysis suggests possible mechanisms of Periplocymarin in NSCLC By integrating data from GeneCards, OMIM, and DisGeNET, 3151 NSCLC-related targets were identified. Concurrently, Swiss Target Prediction predicted 100 potential targets of Periplocymarin. Intersection analysis revealed 54 overlapping targets between Periplocymarin and NSCLC (Fig. 6 A). These 54 targets were then input into STRING to construct a protein–protein interaction (PPI) network (Fig. 6 B). After removing isolated nodes, the network comprised 53 nodes and 380 edges. Core targets were identified through topological analysis, including BCL2L1, MTOR, CASP3, EGFR, STAT3, MAPK1, MAPK14, PIK3CA, ABL1, PRKCA, HDAC6, HDAC1, and PTGS2 (Fig. 6 C). Based on these core targets, GO functional enrichment and KEGG pathway analyses were performed. In the GO enrichment analysis ( P < 0.05), the top ten terms across molecular function (MF), biological process (BP), and cellular component (CC) are shown in Fig. 6 D. Notably, the BP term “regulation of reactive oxygen species metabolic process” suggesting that Periplocymarin might exert effects by modulating ROS levels, indicating a focus for subsequent investigations. KEGG pathway analysis (Fig. 6 E) highlighted the apoptosis pathway as significantly enriched, indicating that Periplocymarin might exert anti-tumor effects through regulation of apoptosis-related signaling cascades. Simultaneously, by integrating RNA sequencing analyses, MAPK14 was identified among the key genes, which strongly suggested that Periplocymarin might exert its inhibitory effect on NSCLC by targeting MAPK14. TPP and MST assays identify MAPK14 as a poteintial target A549 cells were used to elucidate the potential mechanisms by which Periplocymarin exerted cytotoxicity in NSCLC cells. Total cellular proteins were extracted and incubated with Periplocymarin. The samples were subjected to a temperature gradient (37°C, 42°C, 46°C, 50°C, 54°C, 58°C, 62°C, and 67°C), followed by centrifugation to separate soluble and insoluble fractions. Soluble proteins were quantified by Western blot and mass spectrometry using a label-free approach combined with data-independent acquisition (DIA) (Fig. 7 A). Protein thermal stability curves were generated. Through network pharmacology analysis, MAPK14 was found among the common interacting proteins. TPP experiments showed that with increasing temperature, MAPK14 maintained relatively high stability in the presence of Periplocymarin (Fig. 7 B, C). To validate this conclusion, we treated A549 cells at different temperatures and likewise found that Periplocymarin could enhance the thermal stability of MAPK14 (Fig. 7 D), suggesting that Periplocymarin is likely to interact with MAPK14 to exert cytotoxic effects against NSCLC. To evaluate the direct interaction between Periplocymarin and MAPK14, MST was employed under defined aqueous conditions. The equilibrium dissociation constant (K d ) was determined to be 0.11 mM (Fig. 7 E). The binding profile exhibited a characteristic sigmoidal trajectory, indicative of specific and saturable interaction between Periplocymarin and MAPK14, which mean K d was 0.11 ± 0.06 mM. Collectively, these MST-derived findings provided direct in vitro biochemical evidence of physical association between Periplocymarin and MAPK14, thereby offering a mechanistic foundation for subsequent investigations into phosphorylation-dependent regulation and downstream signaling events. Dynamic Stability and Interaction Mechanism of MAPK14–Periplocymarin complex The dynamic behavior of the MAPK14–Periplocymarin complex was characterized over a 100 ns trajectory. The Root Mean Square Deviation (RMSD) profile (Fig. 7 F) reveals a synchronized upward trend for both the complex and the protein, while the ligand Periplocymarin remains remarkably stable throughout the simulation. This pattern indicates that the global structural changes are driven by the receptor’s internal dynamics rather than ligand instability. As evidenced by the structural snapshots (Fig. 7 L), these fluctuations originate from the inherent flexibility of the N-terminal disordered region and a significant expansion of the binding pocket. This expansion is further quantified by the Radius of Gyration (Rg) (Fig. 7 G). The total Rg, along with its components in the X and Z directions, shows a clear increasing trend, directly reflecting the protein’s transition into a more extended state. This phenomenon is consistent with the visual evidence in Fig. 7 L, where the binding cleft expands to accommodate a conformational rearrangement of the surrounding motifs. The binding interface properties provide a nuanced view of this expansion. The interaction SASA (Fig. 7 H) exhibits a progressive decrease, stabilizing at an average of 3.15 nm 2 . While pocket expansion often increases total protein surface area, the reduction in interaction surface area indicates a relocation of the drug. As the pocket opens, Periplocymarin shifts from an initially encapsulated state toward the deeper ATP-binding region, effectively increasing its distance from the phosphorylation sites Thr180 and Tyr182. Despite this relocation and pocket expansion, the number of hydrogen bonds (Fig. 7 I) remains consistently maintained (Average: 1.18). This persistent hydrogen bonding network, even within a dynamic and expanding pocket, underscores the high binding stability of Periplocymarin, which is essential for its long-term inhibitory effect on MAPK14. Binding Free Energy and Phosphorylation Inhibition The binding affinity was quantified at − 34.41 kcal/mol (Fig. 7 J), primarily stabilized by van der Waals interactions (–31.61 kcal/mol). The temporal energy heatmap (Fig. 7 K) and per-residue decomposition ( Fig. 7 M) reveal the precise molecular mechanism behind the structural changes observed: ATP-Binding Core Preservation: Residues 31–36 and 38, located within the ATP-binding region, maintain consistent and strong interactions with the drug throughout the 100 ns simulation. Expansion-Induced Recruitment: Residues 63, 64, and 67 show negligible interaction initially but are recruited into the binding network as the pocket expands (observed in the latter half of Fig. 7 K). Decoupling from Phosphorylation Sites: In contrast, residues 150, 174, and the critical phosphorylation site Tyr182 exhibit favorable interactions during the early phase (pre-expansion) but eventually lose contact as the pocket enlarges and the drug relocates. As visualized in the detailed View 1 and View 2 of Fig. 7 N, the expansion and subsequent ligand shift result in Thr180 and Tyr182 moving significantly further away from the ATP-binding region. This movement causes the activation loop to become increasingly buried and distorted (as labeled “up” in Fig. 7 N). By sequestering the drug in the ATP pocket and inducing a conformational state where the 180/182 sites are buried, Periplocymarin effectively blocks the access of upstream kinases, providing a structural basis for the inhibition of MAPK14 phosphorylation. Periplocymarin inhibits tumor growth by suppressing MAPK14 phosphorylation Molecular docking and TPP experiments strongly suggested an interaction between Periplocymarin and MAPK14. To further elucidate the effect of Periplocymarin on MAPK14, A549, H1299, H1975, and H3122 cells were treated with 0, 50, 100, and 200 nM Periplocymarin for 24 hours, and the cells were collected for Western blot analysis. The results showed that Periplocymarin could inhibit MAPK14 phosphorylation in a dose-dependent manner (Fig. 8 A). Subsequently, in A549, H1299, H1975, and H3122 cells, MAPK14 was knocked down by transfecting siRNA-MAPK14. Western blot analysis confirmed downregulation of MAPK14 expression in NSCLC cells (Fig. 8 B). After MAPK14 knockdown, cells were treated with varying concentrations of Periplocymarin in siRNA-NC and siRNA-MAPK14 groups, and cell viability was assessed by CCK-8 assay and to determine the IC 50 of Periplocymarin (Fig. 8 C). The IC 50 increased following MAPK14 silencing, suggesting that Periplocymarin might exert its cytotoxic effects on NSCLC cells via MAPK14. Transcriptomic results indicated that Periplocymarin engages the ROS pathway. To validate these results, we treated A549 and H1299 cells with Periplocymarin and measured lipid peroxidation levels using an MDA assay. The results showed MDA content rising with increasing Periplocymarin concentration (Fig. 8 D). ROS probes further revealed enhanced ROS signals in A549 and H1299 cells after Periplocymarin treatment (Fig. 8 E), indicating increased cellular oxidative stress. Additionally, flow cytometry showed that Periplocymarin induced G2/M-phase cell cycle prolonged in A549 and H1299 cells, thereby inhibiting cell cycle progression (Fig. 8 F, G). Taken together, these findings suggested that Periplocymarin could promote redox activity and cause cell cycle arrest, contributing to its anti-tumor effect. Periplocymarin exerts an inhibitory effect on NSCLC progression in vivo To further validate the in vivo anti-tumor efficacy of Periplocymarin, subcutaneous tumor models in immunodeficient mice and PDXs of lung adenocarcinoma were established. In the subcutaneous model, A549 cells were inoculated into the left axilla of BALB/c nude mice. When the tumor length reached 5 mm, mice were randomly assigned to control group or Periplocymarin-treated group (Fig. 9 A). Periplocymarin or vehicle was administered intraperitoneally every two days for two weeks. Periplocymarin-treated tumors showed reduced tumor mass and weight (Fig. 9 B, C). IHC revealed decreased Ki-67 and p-MAPK14 expression in the Periplocymarin-treated group (Fig. 9 D), suggesting that in vivo Periplocymarin could inhibit MAPK14 phosphorylation and thereby suppress tumor proliferation. Given the relatively limited in vivo studies of Periplocymarin in tumors, we observed no difference in body weight between the Periplocymarin-treated mice and controls (Fig. 9 E). To assess potential toxicity, hearts, livers, spleens, lungs, and kidneys were collected, fixed, and subjected to HE staining, which showed no obvious organ toxicity in the Periplocymarin group (Fig. 9 F). Moreover, peripheral blood was obtained for complete blood count, hepatic and renal function analyses, with no significant differences between treated and control groups (Fig. 9 G), supporting the conclusion that Periplocymarin provided effectiveness in vivo tumor suppression with acceptable safety. Furthermore, to more rigorously confirm Periplocymarin’s effect on patient tumor tissue, we applied NCG mice to establish lung adenocarcinoma xenograft model and randomly assigned the third-generation PDX to control and Periplocymarin groups (Fig. 9 H). The dosing regimen was the same as above, and Periplocymarin similarly inhibited PDX tumor growth (Fig. 9 I, J), without affecting body weight (Fig. 9 K). Relative to controls, Ki-67 and p-MAPK14 expression were markedly reduced in the Periplocymarin-treated tumors (Fig. 9 L), indicating diminished proliferative activity and inhibited MAPK14 signaling within the tumor xenografts. H&E staining showed no obvious organ toxicity in the hearts, livers, spleens, lungs, or kidneys of the NCG mice treated with Periplocymarin (Fig. 9 M). Collectively, these in vivo findings demonstrate substantial anti-tumor activity of Periplocymarin, likely mediated through suppression of MAPK14 signaling. Discussion Lung cancer is one of the malignancies with the highest incidence and mortality worldwide( 21 ). Therapeutic strategies for NSCLC remain constrained by drug resistance, substantial treatment-related toxicities, and limited efficacy( 2 ). Natural products represent substantial bioactive compounds that demonstrated anti-tumor activity, offering advantages such as multi-target regulation and comparatively lower toxicity( 22 ). However, due to their complex composition and variability in active constituents, the precise mechanisms of action remain unclear, which constrains clinical translation and the development of novel therapeutics. In this study, through screening 1459 anti-tumor natural products, Periplocymarin was found to have significant selective cytotoxicity against NSCLC cells while showing low toxicity to normal lung epithelial cells, indicating favorable tumor specificity and safety. Periplocymarin, a plant-derived cardiac glycoside natural product isolated from Periploca sepium, has been studied in cardiac diseases( 23 ). It mainly inhibits Na+/K+-ATPase activity, regulating intracellular Na+/K+ balance and thereby affecting the cellular ionic environment and signaling( 24 ). In cardiovascular disease research, Periplocymarin has shown protective effects against myocardial fibrosis induced by adrenergic activation in mice, exerts a positive inotropic effect by promoting Ca2 + influx, mitigates doxorubicin-induced heart failure and excessive ceramide accumulation, and attenuates pathological myocardial hypertrophy by inhibiting the JAK2/STAT3 signaling pathway( 25 ). More recently, Periplocymarin has demonstrated potential in oncology, such as inducing apoptosis in colon cancer cells by disrupting the PI3K/AKT pathway and triggering ferroptosis in gastric cancer via the ATP1A1-Src-YAP/TAZ-TFRC axis, indicating notable anti-tumor potential( 26 ). However, its role in lung cancer remains unclear. This study found that Periplocymarin inhibited proliferation and migratory capacity across multiple lung cancer cell lines and induced apoptosis. In addition, Periplocymarin can suppress the proliferation of organoids derived from lung cancer tissues. In vivo , it significantly suppressed tumor growth in both nude mouse xenograft and PDX models. Collectively, these results illustrate the potential of Periplocymarin for lung cancer therapy, though its mechanism remains unclear. To elucidate the mechanism, this study utilized multi-omics, integrative approach, consistent with recent reports that combining transcriptomics, network pharmacology, proteomics, and molecular docking can reveal how natural products regulate cancer signaling pathways( 27 ). For example, Li et al. utilized network pharmacology to identify the mechanism of quercetin in ovarian syndrome and endometrial cancer by AKT inhibition( 28 ). Multi-omics technologies were used to explore that triptolide overcame paclitaxel resistance in NSCLC by targeting the HNF1A/SHH axis( 29 ). Erianin, isolated from Dendrobium chrysotoxum, suppresses tumor growth in BRAFV600E or KRAS-mutant melanoma and colorectal carcinoma mouse models by targeting CRAF and MEK1/2( 30 ). In this study, network pharmacology and TPP technology both identified an interaction between Periplocymarin and MAPK14. Subsequently, molecular docking revealed a relatively high binding affinity between Periplocymarin and MAPK14. Therefore, we conclude that Periplocymarin exerts antitumor effects by modulating the MAPK14 signaling pathway. MAPK14 is a core member of the MAPK family and a key signaling molecule in cellular stress responses, widely participating in biological processes such as cell proliferation, differentiation, apoptosis, and inflammatory responses( 19 , 31 ). Inflammatory cytokines, oxidative stress, and various cellular stimuli commonly trigger its activation, which regulates cell fate by phosphorylating downstream transcription factors such as ATF2 and p53( 32 , 33 ). In tumor initiation and progression, MAPK14 exhibits a complex dual role. p38α MAPK signaling suppresses tumor initiation in epithelial cells, while contributes to the proliferation and survival of tumor cells, thus to potentiate colon tumor formation( 34 ). Additionally, MAPK14 modulates inflammatory responses within tumor microenvironment, influencing the function and thereby participating in tumor immune evasion mechanisms. p38α MAPK can downregulate cyclins, upregulate cyclin-dependent kinase (CDK) inhibitors and modulate p53, to negatively regulate G1/S and the G2/M transitions by several mechanisms( 35 ), which corresponds to the results about Periplocymarin prolonged G2/M phase in NSCLC cells. Given its multifaceted regulatory roles in inflammation and tumor initiation and progression, MAPK14 has emerged as an important potential therapeutic target for inflammatory diseases( 31 ) and various cancers, including breast and colon cancer, and inhibitors targeting MAPK14 can effectively suppress tumor progression( 36 ). First-in-class p38α inhibitor designated ULTR-p38i, performs as a mitosis-targeted therapy for colorectal cancer( 37 ). In this study, Western blot analysis revealed that Periplocymarin markedly inhibited MAPK14 phosphorylation in lung cancer cells, thereby blocking signal transduction, promoting tumor cell apoptosis, and suppressing migratory capacity. MAPK14 knockdown attenuated the anti-tumor effects of Periplocymarin, indicating that Periplocymarin exerted its anticancer action, at least in part, by inhibiting MAPK14 phosphorylation. The results also showed that Periplocymarin promoted ROS generation and inhibited cell cycle progression, suggesting that its antitumor activity might rely on MAPK14, promoting tumor cell apoptosis while inhibiting proliferation and migration. In addition, Periplocymarin significantly reduced the expression level of p-MAPK14 in nude mice and PDX models. In vivo , no significant organ toxicity or hematologic abnormalities were observed in the Periplocymarin-treated group, indicating a favorable safety. These findings collectively supported its safety in the treatment of NSCLC. Our study provided a mechanistic basis for MAPK14-targeted therapeutic development. Conclusion To summarize, Periplocymarin, a natural product, exhibits potent activity against NSCLC, with its mechanism primarily through inhibiting MAPK14 phosphorylation, thereby regulating tumor cell proliferation, migration, and apoptosis (Fig. 10 ). Considering the pivotal role and therapeutic potential of MAPK14 in cancer, this study not only broadens the mechanistic understanding of natural products against NSCLC but also provides a theoretical basis for developing innovative MAPK14-targeted anticancer therapies. Abbreviations ALK Anaplastic Lymphoma Kinase AM Acetoxymethyl ATP Adenosine Triphosphate AKT Protein kinase B ANOVA Analysis of Variance ABL1 ABL proto-oncogene 1, non-receptor tyrosine kinase BCA Bicinchoninic Acid BP Biological Process BCL2L1 BCL2-like 1 CO 2 Carbon Dioxide CCK-8 Cell Counting Kit-8 CC Cellular Component CRAF C-Raf Proto-Oncogene, Serine/Threonine Kinase CDK Cyclin-dependent Kinase CASP3 Caspase 3 DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid DEGs Differentially Expressed Genes EdU 5-Ethynyl-2'-deoxyuridine EDTA Ethylenediaminetetraacetic Acid EGFR Epidermal Growth Factor Receptor FBS Fetal Bovine Serum FITC Fluorescein Isothiocyanate GSEA Gene Set Enrichment Analysis HDAC1 Histone deacetylase 1 HDAC6 Histone deacetylase 6 IHC Immunohistochemistry KEGG Kyoto Encyclopedia of Genes and Genomes KRAS Kirsten Rat Sarcoma Viral Oncogene Homolog MDA Malondialdehyde MST Microscale thermophoresis MF Molecular Function MTOR Mechanistic target of rapamycin MAPK1 Mitogen-activated protein kinase 1 MAPK14 Mitogen-activated protein kinase 14（p38α） NSCLC Non-Small Cell Lung Cancer NC Negative Control NCG NOD CRISPR Prkdc Il2r Gamma OMIM Online Mendelian Inheritance in Man PPM Periplocymarin PD-L1 Programmed Death-Ligand 1 PI Propidium Iodide PPI Protein-Protein Interaction PBS Phosphate-Buffered Saline PFS Progression-Free Survival PI3K Phosphatidylinositol 3-Kinase PIK3CA Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α PRKCA Protein kinase C α PTGS2 Prostaglandin-endoperoxide synthase 2 PDO Patient-Derived Organoid PDX Patient-Derived Xenograft RNA Ribonucleic Acid ROS Reactive Oxygen Species RMSD Root‑Mean‑Square Deviation SDS Sodium Dodecyl Sulfate SD Standard Deviation SEM Standard Error of the Mean SASA Solvent-Accessible Surface Area STAT3 Signal transducer and activator of transcription 3 TBST Tris-buffered saline with Tween 20 TPP Thermal Proteome Profiling Declarations Supplementary materials Supplementary material associated with this article can be found in the online version. Acknowledgements Thanks to all authors for their contributions to the manuscript. Author contributions Xinye Wang: Writing (original draft), Data curation; Xiao Liang: Writing (review & editing), Software; Zhibin Song: Methodology; Mingwei Wang: Formal analysis; Hui Sun: Project administration; Xiaoting Ma: Methodology; Yiyan Miao: Methodology; Xingqin Zhou: Visualization, Validation; Yifei Liu: Investigation; Jiahai Shi: Supervision, Funding acquisition; Liting Lv: Resources, Conceptualization. Funding This research was supported by Project of the Nantong Municipal Health Commission (No.: MS2024010, MSZ2024057), Nantong Social Livelihood Science and Technology Program (No.: MSZ2025007), Scientific Research Grant Project under the Senior Aging Pilot Program (No.: 323) and Jiangsu Province Graduate Practice and Innovation Program (No.: SJCX25 2067). Availability of date and materials The data used to support the finding of this study are available on request. 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Additional Declarations No competing interests reported. Supplementary Files MolecularSimulation.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 02 May, 2026 Reviews received at journal 29 Apr, 2026 Reviewers agreed at journal 21 Apr, 2026 Reviews received at journal 20 Apr, 2026 Reviews received at journal 17 Apr, 2026 Reviewers agreed at journal 13 Apr, 2026 Reviewers agreed at journal 10 Apr, 2026 Reviewers invited by journal 09 Apr, 2026 Editor assigned by journal 01 Apr, 2026 Submission checks completed at journal 01 Apr, 2026 First submitted to journal 31 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9282067","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":622596845,"identity":"89964813-41c1-4b9f-bd05-e286abd2fdd9","order_by":0,"name":"Xinye Wang","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Xinye","middleName":"","lastName":"Wang","suffix":""},{"id":622596846,"identity":"224dc2fd-5b9b-4492-9ebe-a92f2240a4d6","order_by":1,"name":"Xiao Liang","email":"","orcid":"","institution":"Jiangyin People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Liang","suffix":""},{"id":622596847,"identity":"8628c2f3-15c6-489a-90cf-9125e52dae4f","order_by":2,"name":"Zhibin Song","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Zhibin","middleName":"","lastName":"Song","suffix":""},{"id":622596848,"identity":"56904798-d407-4cf6-88be-d8c8913b676f","order_by":3,"name":"Mingwei Wang","email":"","orcid":"","institution":"Nantong Third People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mingwei","middleName":"","lastName":"Wang","suffix":""},{"id":622596849,"identity":"c3760a6a-b036-4928-bac1-d987ff8148dc","order_by":4,"name":"Hui Sun","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Sun","suffix":""},{"id":622596850,"identity":"b226db6c-7efe-4df9-ba36-483020316b75","order_by":5,"name":"Xiaoting Ma","email":"","orcid":"","institution":"Jiangyin People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoting","middleName":"","lastName":"Ma","suffix":""},{"id":622596851,"identity":"e8b69bae-a8be-45d5-950a-aeb27f8f035c","order_by":6,"name":"Yiyan Miao","email":"","orcid":"","institution":"The Jiangyin Clinical College of Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yiyan","middleName":"","lastName":"Miao","suffix":""},{"id":622596853,"identity":"3678f151-6ce2-4d15-96cb-5b16a052c70e","order_by":7,"name":"Xingqin Zhou","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Xingqin","middleName":"","lastName":"Zhou","suffix":""},{"id":622596855,"identity":"9cf0bd78-da75-4a28-baa6-f9e7e1a5ea43","order_by":8,"name":"Yifei Liu","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Yifei","middleName":"","lastName":"Liu","suffix":""},{"id":622596856,"identity":"60031582-fb6c-43aa-8cd1-c2ee533f7780","order_by":9,"name":"Jiahai Shi","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Jiahai","middleName":"","lastName":"Shi","suffix":""},{"id":622596858,"identity":"6d049761-24ee-4dc6-8c62-ec4173aba960","order_by":10,"name":"Liting Lv","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACPnYYi70BTDE2ENLCxswMJBOAmOcAWDUpWiQSiNbCf0ya94dNnnzk2+OPeRhsZDccYH72gIAtbNI8CWnFhrfzEpt5GNKMNxxgMzcgQsvhxI2zcwyBWg4nbjjAwyZBhJb/iRtnngFp+U+0lgOJ8yV4QFoOEKXF2HJOWnLiBp4cw5lzDJKNZx5mM8OrhZ+98eGNNzZ2ifPbzxh8eFNhJ9t3vPkZXi1AwAJWYHAATAIxMwH1ICUfQKR8A2GVo2AUjIJRMEIBAB6NP2pFgNoYAAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":true,"prefix":"","firstName":"Liting","middleName":"","lastName":"Lv","suffix":""}],"badges":[],"createdAt":"2026-03-31 15:38:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9282067/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9282067/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107485987,"identity":"3cccc527-799e-4453-a6ec-eb35b7f60a84","added_by":"auto","created_at":"2026-04-22 02:37:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":15369506,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening for NSCLC cell selective compounds.\u003c/strong\u003e(A) Screening of a library of 1459 compounds. (B) Scatter plots of cell viability for A549, H3122 and H1299 cells with exposure to the compound library. (C) Venn diagram showing 28 effective compounds among three NSCLC cell lines. (D) Heatmap of anti-tumor efficacy for five cell lines, including normal lung epithelia (Beas-2B and 16HBE) cells and lung cancer lines (A549, H3122 and H1299 cells).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9282067/v1/9165ddc4fe09ec6b2e68e124.png"},{"id":107485906,"identity":"ac1589d6-19b5-42ef-b5e0-bd1f5e7798b8","added_by":"auto","created_at":"2026-04-22 02:36:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":53721040,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of PPM on viability and proliferation in NSCLC cell lines. \u003c/strong\u003e(A) Relative cell viability curves for six NSCLC cell lines treated with PPM at the concerntration of 0, 0.01, 0.05, 0.1, 1, and 10 μM for 24 h and 48 h. (B) Cell viability for each cell line treated with 0, 50, 100, and 200 nM PPM for 0, 24, 48 and 72 h. (C) Representative colony formation images and quantification of colony numbers (D) for cells treated with 0, 20, 50, and 100 nM PPM. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 and ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 were considered of great significance.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9282067/v1/8da3f288756b2e0182af4374.png"},{"id":107485986,"identity":"04c9b4f8-60c3-4956-8623-3c1171daae02","added_by":"auto","created_at":"2026-04-22 02:37:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73145546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEdU assay, apoptosis assay, and PDOs response in NSCLC.\u003c/strong\u003e(A) Representative EdU images for A549, H1299, H1975, and H3122 cells treated with 0, 50, and 100 nM PPM for 24 h. Quantification shows a dose-dependent decrease in EdU-positive cells. (B) Apoptosis analysis by Annexin V-FITC/PI flow cytometry in cells after 48 h treatment. (C) Apoptosis rate for cells in response to PPM. (D) Representative images of PDOs viability with exposure to 0, 100 and 200 nM over 7 days. (E) Fluorescence intensity in PDOs treated with different concentrations of PPM. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, and ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 were considered of great significance.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9282067/v1/da3b5406de41f18674d14f2f.png"},{"id":107485763,"identity":"2c6992e6-a39d-42d1-983e-6391d94f2648","added_by":"auto","created_at":"2026-04-22 02:36:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":59824099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWound-healing and transwell migration assays evaluating the effect of PPM on NSCLC cell lines. \u003c/strong\u003e(A) Representative wound-healing images for A549, H1299, H1975, and H3122 cells treated with 0, 20, and 50 nM PPM, captured at 0, 24, and 48 hours. (B) Quantification of wound closure as migration area. (C) Representative Transwell migration images after 0, 20, and 50 nM PPM treatment. (D) Quantification of migrated cell numbers from Transwell assays. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 and ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 were considered of great significance. Ns, no significance.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9282067/v1/b634d4b969b66101d9f7b055.png"},{"id":107485985,"identity":"0795505b-7f8d-40eb-a5f3-018c72c62266","added_by":"auto","created_at":"2026-04-22 02:37:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":26104403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA-sequence analysis of treated A549 and H1299 cells.\u003c/strong\u003e(A) Volcano plots of differential expression. Red indicates upregulated genes, purple indicates downregulated genes, gray indicates non-significant genes. (B-C) KEGG pathway enrichment results for A549 and H1299 cells. (D) GSEA enrichment plots for A549 cells. (E) GSEA enrichment plots for H1299 cells.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-9282067/v1/a3566479ab17a4cdf7794a02.png"},{"id":107485867,"identity":"3c4bcad2-4f2b-4453-9daa-a0bc571404be","added_by":"auto","created_at":"2026-04-22 02:36:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":26889219,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNetwork pharmacology analysis of PPM against NSCLC.\u003c/strong\u003e(A) Venn diagram showing the overlap between PPM targets and NSCLC-related genes. A bar chart beneath illustrates the genes of each group. (B) PPM-NSCLC targets interaction network. (C) Flowchart and core-network extraction. Left: initial network; middle: core network after applying thresholds; right: final subnetworks prepared for visualization. (D) GO functional enrichment analysis for 54 genes. Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). (E) KEGG pathway analysis for 54 genes.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-9282067/v1/5e00c30b91dae8c4de6523be.png"},{"id":107485868,"identity":"d8eb0989-d592-4248-8011-5333a67333e8","added_by":"auto","created_at":"2026-04-22 02:36:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":38666690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTPP, molecular dynamics and inhibitory mechanism analysis of the MAPK14–Periplocymarin complex.\u003c/strong\u003e (A) A549 cells were treated with vehicle (DMSO) or PPM, then subjected to a temperature gradient (37-67 ℃). After lysis, the soluble fractions were analyzed by mass spectrometry. (B) MAPK14 melting curve showing the Tm shift between DMSO- and PPM-treated samples. (C) Relative MAPK14 expression illustrated altered thermal stability with PPM treatment. (D) Temperature-dependent expression of MAPK14 in DMSO and PPM groups. (E) MST confirmed the binding of Periplocymarin to MAPK14 with an affinity of 0.11 mM. (F) RMSD trajectories of the complex, protein backbone, and ligand over 100 ns. (G) Rg including total value and components in X, Y, and Z directions. (H) Interaction SASA between the protein and the ligand over time. (I) Number of hydrogen bonds formed between the protein and the ligand. (J) Binding free energy components calculated via the MM/GBSA method. (K) Temporal energy heatmap showing the contribution of individual residues to binding over time. (L) Structural snapshots comparing the complex conformation at 0 ns and 100 ns. (M) Per-residue energy decomposition of key amino acid residues. (N) 3D binding mode visualization (View 1 and View 2) of the ligand and key residues within the expanded pocket.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-9282067/v1/a565604a9f1f5dd907d7ba7d.png"},{"id":107486009,"identity":"a8cba6b5-4e6e-4193-a7f2-5dbb64369f0b","added_by":"auto","created_at":"2026-04-22 02:37:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":27711173,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePeriplocymarin inhibits tumor growth by suppressing MAPK14 phosphorylation. \u003c/strong\u003e(A) Western blots for p-MAPK14, MAPK14, and α-tubulin in A549, H1299, H1975, and H3122 cells treated with 0, 50, 100, and 200 nM PPM. (B) Western blots showing p-MAPK14, MAPK14 and α-tubulin in cells transfected with si-NC or si-MAPK14. (C) Relative cell viability with PPM treatment for each cell line comparing si-NC and si-MAPK14. (D) MDA content in A549 and H1299 cells under 0, 100, and 200 nM PPM. (E) ROS fluorescence in A549 and H1299 cells treated with 0, 100, and 200 nM PPM. (F) The cell cycle distribution of A549 and H1299 cells treated with 0, 100, and 200 nM PPM for 24 h, as assessed by FL2-A-based flow cytometry, revealed an accumulation in the G2/M phase. The quantitative data and statistical analysis are shown as (G). *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, and ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 were considered of great significance. Ns, no significance.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-9282067/v1/194d656feb67e73fb0939ecd.png"},{"id":107487017,"identity":"480405ba-a0bb-42bc-81e7-bc2e7f82b88e","added_by":"auto","created_at":"2026-04-22 02:39:36","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1002540,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo anti-tumor efficacy and safety of PPM. \u003c/strong\u003e(A) Representative images of A549 cells xenograft tumors growth in BALB/c nude mice receiving Vehicle or PPM (5 mg/kg). (B) Tumor growth curves for control and PPM groups in BALB/c nude mice. (C) Tumor weight changes for two groups of BALB/c nude mice. (D) Representative images of H\u0026amp;E staining, Ki-67, and p-MAPK14 in two groups of BALB/c nude mice. (E) Mouse weight changes for BALB/c nude mice. (F) H\u0026amp;E staining of heart, liver, spleen, lung, and kidney from control and PPM group of BALB/c nude mice. (G) Measurement of complete blood count, hepatic and renal function in tumor bearing mice receiving indicated treatment. WBC, White blood cell count; HGB, Hemoglobin; PLT, Platelet count; ALT, alanine transaminase; AST, aspartate transaminase; CREA, Creatinine; BUN, blood urea nitrogen. (H) Representative images of PDX tumor growth receiving Vehicle or PPM (5 mg/kg). (I) Tumor growth curves for control and PPM groups in PDX model. (J) Tumor weight changes for two groups of PDX model. (K) Mouse weight changes for NCG mice. (L) Representative images of H\u0026amp;E staining, Ki-67, and p-MAPK14 in two groups of NCG mice tumors. (M) H\u0026amp;E staining of heart, liver, spleen, lung, and kidney from control and PPM group of NCG mice. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 and ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 were considered of great significance.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9282067/v1/ca91a36535ea8581f2b3634b.png"},{"id":107485869,"identity":"6a2d7a41-defb-495f-9fc4-61b0037f1635","added_by":"auto","created_at":"2026-04-22 02:36:41","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":21890440,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA graphical abstract summarizing the molecular mechanisms and experimental methods of Periplocymarin in inhibiting Non-small cell lung cancer.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-9282067/v1/aa4ab06c009c5e99c084fe9f.png"},{"id":107254929,"identity":"f659fddc-edbe-4022-9175-c6fb8df10354","added_by":"auto","created_at":"2026-04-19 12:06:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":376320,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9282067/v1/e8cf550b-b8fb-48de-ad0b-f1d853886d18.pdf"},{"id":107486929,"identity":"2bac463d-f326-431b-a790-206848148fed","added_by":"auto","created_at":"2026-04-22 02:39:18","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":27340,"visible":true,"origin":"","legend":"","description":"","filename":"MolecularSimulation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9282067/v1/0468e8513c99f82212722383.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrated multi-omics analysis of Periplocymarin to identify the mechanism of p38α MAPK phosphorylation inhibition in Non-small cell lung cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer is characterized by lack of specific early symptoms, and approximately 70% of patients are diagnosed at advanced stage, making it one of the malignancies with high incidence and mortality worldwide(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Non-small cell lung cancer (NSCLC), the predominant histological subtype of lung cancer, is marked by high invasiveness, a propensity for metastasis, and a high rate of recurrence, resulting in poor prognosis(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Currently, NSCLC treatments primarily include surgical resection, radiotherapy, chemotherapy, targeted therapies, and immunotherapies(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Tyrosine kinase inhibitors against driver alterations such as EGFR and ALK have significantly improved the prognosis(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Anti-angiogenic therapies can synergize by modulating the tumor microenvironment to exert better therapeutic effect: Ivonescimab improved the progression free survival (PFS) of untreated patients with advanced PD-L1 positive NSCLC(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Moreover, PD-1/PD-L1\u0026ndash;based immunotherapy has prolonged the survival of lung cancer patients(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Despite these advances, challenges including acquired resistance and treatment-related toxicities, illustrated the need for novel strategies.\u003c/p\u003e \u003cp\u003eNatural products are characterized by chemical and structural diversity, as well as intrinsic multi-target mechanisms, which enable them and their derivatives with unique advantages in antitumor activity and pharmacodynamic synergy(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Compared with single-target drugs, natural products often modulate multiple pathways involved in tumor initiation and progression(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Substantial studies showed that natural products can induce tumor cell apoptosis, inhibit proliferation, and block angiogenesis(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Moreover, they can exert anti-tumor effects by modulating tumor metabolism (e.g., Polyphyllin VII reversing PARP inhibitor resistance(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)), remodeling the tumor microenvironment (e.g., Chelidonine-induced disrupts M2 macrophage polarization in breast cancer(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)), and regulating immune responses (e.g., Yi-Fei-San-Jie-Fang modulating immune evasion(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)). Importantly, many chemotherapeutic agents widely used in lung cancer are derived from natural products or their derivatives. Paclitaxel, extracted from Taxus species, stabilizes microtubule dynamics to prevent mitosis and suppress tumor growth(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Vincristine and Vinblastine, derived from Catharanthus roseus (Madagascar periwinkle), inhibit microtubule polymerization to block cell division, widely used in chemotherapy for various solid tumors(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In addition, Etoposide is a semi-synthetic derivative of podophyllotoxin from Podophyllum species, which induces DNA breaks by inhibiting topoisomerase II, thereby promoting tumor cell apoptosis(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Nevertheless, clinical translation of natural products remains challenged by compound stability, targeted delivery, and the complexity of their mechanisms.\u003c/p\u003e \u003cp\u003eThe diversity and unclear mechanisms of natural products limit their clinical applications as well as pharmaceutical innovation and development. Currently, there is a lack of systematic and standardized screening processes, and the specific targets and regulatory mechanisms of active constituents remain unclear, limiting the clinical utility and translational potential of natural products. Systematic screening of natural products and utilization of multi-omics, can help to elucidate their targets and molecular mechanisms(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). This study identified Periplocymarin as a potent inhibitor of lung cancer cell proliferation through screening of a natural product library. Periplocymarin exerts selective cytotoxicity against NSCLC cells and showing tumor growth inhibition in cell-based assays, patient-derived organoids (PDOs), nude mouse xenograft, and patient-derived xenograft (PDX) models. Characterizing the target-binding affinities of natural products is essential for the discovery of novel anticancer agents and their biological activities(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Therefore, this study employs thermal proteome profiling (TPP) as a pivotal method for target discovery, combined with transcriptome sequencing, network pharmacology, and molecular computational analyses to elucidate Periplocymarin\u0026rsquo;s target as p38α mitogen-activated protein kinase (p38α MAPK, MAPK14). MAPK14 exhibits dual roles in cancer: its activation can induce cell cycle arrest and apoptosis and promote tumor cell proliferation(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The study demonstrates that Periplocymarin targets MAPK14 and inhibits its phosphorylation, thereby exerting anti-tumor effects.\u003c/p\u003e \u003cp\u003eIn summary, lung cancer, a highly heterogeneous and aggressive malignant tumor, urgently requires the development of definite-target, low-toxicity therapeutic pharmaceuticals. These present findings show that Periplocymarin can effectively inhibit proliferation and metastasis, promote apoptosis by suppressing MAPK14 phosphorylation in NSCLC. It can also suppress tumor growth in both nude mouse models and PDO/PDX xenografts, demonstrating favorable anti-tumor effects. These findings, together with natural product library screening and target-discovery technologies, will help elucidate the direct mechanisms of natural products and promote their clinical application in the treatment of lung cancer.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents and Antibodies\u003c/h2\u003e \u003cp\u003eNatural product library (Cat. #HY-L107) and Periplocymarin (PPM, Cat. #HY-N4252) were purchased from MedChemExpress (MCE, Shanghai, China). Phospho-MAPK14 (p-MAPK14) (Cat. #28796-1-AP), MAPK14 (Cat. #14064-1-AP), Ki-67 antibody (Cat. #27309-1-AP) and α-tubulin (Cat. #66031-1-Ig) were obtained from Proteintech (Wuhan, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Culture and siRNA Transfection\u003c/h3\u003e\n\u003cp\u003eNormal human bronchial epithelial cell lines (Beas-2B and 16HBE cells) and NSCLC cell lines (A549, H1299, H292, HCC827, H1975, and H3122 cells) were obtained from the Cell Bank of the Chinese Academy of Sciences. Cells were maintained in RPMI-1640 or DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Servicebio, China). Cells were transfected with either non-targeting control siRNA (siRNA-NC) or siRNA targeting MAPK14 (si-MAPK14; interference sequence: 5\u0026rsquo;-3\u0026rsquo;: UUAAGUAACCGCAGUUCUC (dT) (dT)) using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer\u0026rsquo;s instructions. Forty-eight hours post-transfection, cells were collected for subsequent analyses.\u003c/p\u003e\n\u003ch3\u003eNatural Products Screen\u003c/h3\u003e\n\u003cp\u003eA library of 1459 anti-tumor natural products were screened for cytotoxic effects against NSCLC cells. In the initial screen, Beas-2B, 16HBE, A549, H1299, and H3122 cells were treated with 10 \u0026micro;M of each natural product for 48 hours, followed by CCK-8 assay. Candidate compounds producing cell viability less than 10% across the three cell lines were involved. In the secondary screen, these candidates were retested at a 5 \u0026micro;M concentration, and those showing markedly higher toxicity in lung cancer cells than in lung epithelial cells were confirmed.\u003c/p\u003e\n\u003ch3\u003eCCK-8 Assay\u003c/h3\u003e\n\u003cp\u003eCells were treated with Periplocymarin at the concentrations of 0, 0.01, 0.05, 0.1, 1, or 10 \u0026micro;M for 24 or 48 hours. After treatment, 10% CCK-8 reagent (MeilunBio, China) in complete medium was added and incubated for 2 hours. IC\u003csub\u003e50\u003c/sub\u003e values were calculated by nonlinear regression using GraphPad Prism 9.0. To assess effects on proliferation capacity, cells were treated with Periplocymarin at the concentrations of 0, 50, and 100 nM. OD450 nm values were recorded at 0, 24, 48, and 72 hours with CCK-8 assay, and calculated using GraphPad Prism 9.0 for proliferation rates.\u003c/p\u003e\n\u003ch3\u003eColony Formation Assay\u003c/h3\u003e\n\u003cp\u003eCells were treated with Periplocymarin at 0, 20, 50, or 100 nM for 10\u0026thinsp;~\u0026thinsp;14 days. Following incubation, colonies were fixed with methanol for 15 minutes and stained with crystal violet for 45 minutes. After washing, colonies were quantified using ImageJ, and results were plotted in GraphPad Prism 9.0.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEdU Assay\u003c/h2\u003e \u003cp\u003eCells were treated with Periplocymarin at 0, 50, or 100 nM for 24 hours. EdU labeling was performed for 2 hours, after which cells were fixed with methanol for 10 minutes and permeabilized with 0.1% Triton X-100. EdU-positive cells were detected using the Azide 555 reagent (Beyotime, China), and nuclei were counterstained with Hoechst. Fluorescent images were captured, and EdU-positive and total-cell counts were quantified with ImageJ to calculate the proliferation rate.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWound-Healing Experiment\u003c/h3\u003e\n\u003cp\u003eCells were seeded in 6-well plates and grown to 100% confluence. A vertical scratch was created using a 100 \u0026micro;L pipette tip. After washing with PBS to remove the debris, cells were incubated in serum-free RPMI-1640 containing Periplocymarin at 0, 20, or 50 nM. Images of the wound area were captured at 0, 24, and 48 hours. The scratch area was quantified with ImageJ, and migration was analyzed by plotting the closure over time.\u003c/p\u003e\n\u003ch3\u003eTranswell Assay\u003c/h3\u003e\n\u003cp\u003eCells were suspended in 200 \u0026micro;L serum-free medium containing 0, 20, or 50 nM Periplocymarin and seeded into the upper chamber of Transwell inserts. The lower chamber contained 700 \u0026micro;L complete medium with 20% FBS and the corresponding Periplocymarin concentration. After 24 hours at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, non-migrated cells on the upper surface were removed, and migrated cells were fixed in 4% paraformaldehyde and stained with crystal violet. Images were captured and migrated cells were counted using ImageJ. Data were analyzed and presented using GraphPad Prism 9.0.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometric Analysis\u003c/h2\u003e \u003cp\u003eCells were seeded in 12-well plates and treated with final concentrations of Periplocymarin (0, 100, 200 nM) for 48 hours. Cells were digested with trypsin without EDTA, collected by centrifugation, and the supernatant was discarded. Cells were resuspended in 50 \u0026micro;L binding buffer, and 5 \u0026micro;L of PI and FITC-labeled Annexin V were added. Cells were then incubated at room temperature, followed by the addition of 300 \u0026micro;L binding buffer for flow cytometry analysis. FlowJo software was used to quantify the proportions of early apoptotic cells (Annexin V-positive, PI-negative) and late apoptotic cells (Annexin V-positive, PI-positive). Meanwhile, flow cytometry was performed to evaluate the effect of Periplocymarin on cell cycle progression. A549 and H1299 cells were treated with Periplocymarin at concentrations of 0, 100 and 200 nM for 24 hours. Subsequently, cells were collected and fixed overnight at 4\u0026deg;C with pre‑cooled 70% ethanol. After removal of ethanol, an appropriate amount of staining solution was added to the cell pellets, followed by incubation at 37\u0026deg;C in the dark for 30 min. Finally, samples were analyzed by flow cytometry and data were measured by FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of intracellular ROS\u003c/h2\u003e \u003cp\u003eCells were seeded in 35 mm confocal dishes and treated with different concentrations of Periplocymarin (0, 50, 100 nM) for 24 hours. Subsequently, cells were washed with pre-cooled PBS and incubated with 10 \u0026micro;M DCFH-DA (Beyotime, China) for 30 minutes. Cells were washed with PBS to remove unbound probes. Fluorescence images were captured using a confocal microscope (Nikon, Japan) to identify the ROS content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMDA Assay\u003c/h2\u003e \u003cp\u003eCells were seeded in 6-well plates and treated with different concentrations of Periplocymarin (0, 50, 100 nM) for 24 hours, collected and homogenized on ice using pre-cooled lysis buffer. The lysate was centrifuged, and the supernatant was collected for MDA content determination, in accordance with the instructions of the MDA assay kit (Beyotime, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e \u003cp\u003eThe lysate of treated cells were centrifuged, and the supernatant was collected. An appropriate amount of 5 \u0026times; loading buffer was added, and samples were boiled at 100\u0026deg;C for 10 minutes. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 5% non-fat milk in TBST at room temperature for 2 hours. Primary antibodies against p-MAPK14 (1:1000), MAPK14 (1:1000), and α-Tubulin (1:5000) were incubated with the membranes in TBST at 4\u0026deg;C overnight. The next day, membranes were washed with TBST. Corresponding secondary antibodies were incubated at room temperature for 2 hours, followed by washes with TBST. Target protein bands were detected and quantified using the Tanon imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eParaffin-embedded tumor tissue sections were prepared at a thickness of 4\u0026thinsp;~\u0026thinsp;5 \u0026micro;m and deparaffinized at 65\u0026deg;C. Sections were subsequently rehydrated sequentially in xylene, gradient ethanol (100%, 85%, 50%, 25%, subsequently), and deionized water. Antigen retrieval was performed using citrate buffer using high-temperature and high-pressure methods. Endogenous peroxidase activity was inhibited using 3% hydrogen peroxide solution, followed by blocking of non-specific binding sites with 5% goat serum at room temperature. Primary antibodies diluted in PBS were applied to tissue sections and incubated at 4\u0026deg;C overnight. The next day, sections were washed with PBS, followed by incubation with secondary antibodies from Maixin Biotechnology (China) at room temperature for 30 minutes. The expression of the indicated protein was visualized by DAB (Dako, Denmark). Sections were counterstained with hematoxylin, and immunostaining results were observed under microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eNetwork Pharmacological Analysis\u003c/h2\u003e \u003cp\u003eTo identify potential targets of Periplocymarin in lung cancer, the compound\u0026rsquo;s two-dimensional structure was first retrieved from PubChem. Using this structure, potential human targets were predicted with SwissTargetPrediction. Simultaneously, a lung cancer target set was assembled by integrating targets from DisGeNET, Online Mendelian Inheritance in Man (OMIM), and GeneCards, followed by deduplication. Overlaps between predicted drug targets and lung cancer targets were identified via Venn diagram analysis, and the intersecting targets were designated as candidate Periplocymarin targets for lung cancer. The overlapping target list was submitted to the STRING database (Homo sapiens; confidence score\u0026thinsp;\u0026ge;\u0026thinsp;0.9), and isolated nodes were removed to construct a protein\u0026ndash;protein interaction (PPI) network. Subsequently, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on the candidate targets with a significance threshold of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, clarifying the potential molecular mechanisms and critical pathways of Periplocymarin in NSCLC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThermal Proteome Profiling (TPP)\u003c/h2\u003e \u003cp\u003eA549 cells were cultured and collected, then allocated into two groups and treated with either DMSO (vehicle) or Periplocymarin. The treatment temperatures were set at 37\u0026deg;C, 42\u0026deg;C, 46\u0026deg;C, 50\u0026deg;C, 54\u0026deg;C, 58\u0026deg;C, 62\u0026deg;C, and 67\u0026deg;C. Quantitative mass spectrometry (MS) was employed to comprehensively assess protein states. Dynamic changes in protein thermal stability parameters, notably the melting temperature (Tm), were quantitatively measured, and proteins displaying significantly enhanced stability following compound treatment were identified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMicroscale thermophoresis (MST)\u003c/h2\u003e \u003cp\u003eCells were transfected with the GFP-MAPK14 plasmid and harvested 48 h later. MST measurements were performed using a Monolith NT.115 Pico (NanoTemper Technologies). Periplocymarin was prepared as a 15-point serial dilution with identical final DMSO concentrations and mixed at equal volumes with GFP-MAPK14\u0026ndash;containing cell lysates, ensuring constant protein concentrations and uniform DMSO content across all samples. The mixtures were loaded into capillaries and subjected to MST analysis to calculate the dissociation constant (K\u003csub\u003ed\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRNA Sequencing\u003c/h2\u003e \u003cp\u003eA549 and H1299 cells were cultured and treated with DMSO or 200 nM Periplocymarin for 48 hours, and then collected for RNA sequencing analysis. Differentially expressed genes (DEGs) were identified for each cell line and subjected to downstream enrichment analyses. KEGG pathway enrichment analyses and Gene Set Enrichment Analysis (GSEA) trend analyses were conducted to identify signaling pathways potentially involved in Periplocymarin\u0026rsquo;s mechanism.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePDOs Culture and Calcein AM staining\u003c/h2\u003e \u003cp\u003eFresh tumor specimens were collected from NSCLC patients undergoing surgical resection at the Affiliated Jiangyin Hospital of Nantong University, with all patients providing informed consent. The study protocol was approved by the Institutional Ethics Committee (Approval No.: L028). Tumor tissues were enzymatically dissociated into single cells or small cell clusters using digestion buffer containing collagenase IV and DNase I. The resulting cell suspension was filtered through 70 \u0026micro;m cell strainer and centrifuged at 300 \u0026times; g for 5 min. Cells were resuspended in low-growth-factor Matrigel (Corning, USA), and droplets were plated into 24-well plates to solidify at 37\u0026deg;C for 15 minutes. PDOs passaged three times were treated with Periplocymarin at 0, 50, and 100 nM for 7 days. Organoids were incubated with Calcein AM solution for 15 minutes, and the organoid morphology was captured by fluorescence microscopy to evaluate effects on growth and structural integrity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eIn Vivo Assay\u003c/h2\u003e \u003cp\u003eBALB/c nude mice (aged 4ཞ6 weeks, male) were purchased from GemPharmatech (Jiangsu, China) for xenograft experiments. A549 cells were prepared and mixed with Matrigel at a ratio of 3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells in 40 \u0026micro;L of cell suspension to 40 \u0026micro;L Matrigel, then inoculated into the left axilla of each mouse. When tumor diameter reached approximately 5 mm, mice were randomly allocated into two groups (n\u0026thinsp;=\u0026thinsp;5 per group): the control group received intraperitoneal injections of 100 \u0026micro;L PBS, while the treatment group received intraperitoneal injections of Periplocymarin at 5 mg/kg, every two days for 14 consecutive days. During treatment, body weight and tumor volume were measured every two days. After two weeks, mice were euthanized, tumors were excised and weighed, and tumor tissues were fixed and embedded for subsequent IHC analyses. All experimental protocols were approved by the Animal Ethics Committee of Nantong University (Approval No.: P20250310-012).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ePatient-Derived Xenograft (PDX) Models\u003c/h2\u003e \u003cp\u003eThis study evaluated \u003cem\u003ein vivo\u003c/em\u003e therapeutic effects in PDXs using male NCG mice aged 4ཞ6 weeks, purchased from GemPharmatech. Fresh tissues from NSCLC patients undergoing surgical resection at the Affiliated Hospital of Nantong University were collected with informed consent, and approved by the Animal Ethics Committee of Nantong University (Approval No.: 2025-L111). Tumor tissues were cut into sterile 1ཞ2 mm\u003csup\u003e3\u003c/sup\u003e fragments and subcutaneously transplanted into the left axilla of recipient mice. The tumor was passed into a new cohort of mice when reached approximately 15 mm in diameter. When the third-generation PDX tumors reached about 5 mm in diameter, mice were randomly assigned to two groups. Each group received intraperitoneal injections of PBS or Periplocymarin at 5 mg/kg every two days for 14 days. Body weight and tumor volume were recorded every two days. Then, mice were euthanized and tumors were excised and weighed, and the PDX tumor tissues were fixed and embedded for subsequent IHC analyses. All animal experiments were approved by the Animal Ethics Committee of Nantong University (Approval No.: P20250310-012).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism 9.0. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or SEM. Two-tailed unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test were used for comparisons between two groups. Two-way ANOVA was conducted for comparisons among multiple groups. \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eScreening of active compounds against NSCLC cells from a natural product library\u003c/h2\u003e \u003cp\u003eTo identify natural products with cytotoxic effects against NSCLC cells, 1459 anti-tumor natural products were obtained for two-stage screening. A549, H1299, and H3122 cells were exposed to each compound at 10 \u0026micro;M for 48 hours, and cell viability was assessed using the CCK-8 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Cytotoxicity was calculated for each compound. Analysis revealed 28 compounds that exhibited\u0026thinsp;\u0026ge;\u0026thinsp;90% cytotoxicity in three cancer cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To identify tumor cell-selective agents, the cytotoxic effects of these candidates were further evaluated in Beas-2B and 16HBE cells and three NSCLC cancer cells at a concerntration of 5 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Periplocymarin displayed minimal toxicity to normal lung epithelial cells while demonstrating substantial cytotoxicity toward NSCLC cell lines, consistent with potential anti-tumor activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003ePeriplocymarin inhibits the proliferation and migration, promote apoptosis of NSCLC cells\u003c/h2\u003e \u003cp\u003eThis study assessed the anti-tumor activity of Periplocymarin in NSCLC using six cell lines (A549, H1299, H292, HCC827, H1975, and H3122). Cells were treated with Periplocymarin at 0, 0.01, 0.05, 0.1, 1, and 10 \u0026micro;M for 24 or 48 hours, and viability was measured by the CCK-8 assay. Periplocymarin inhibited cell growth in a dose-dependent manner. The 24-hour IC\u003csub\u003e50\u003c/sub\u003e values were: A549, 0.43 \u0026micro;M; H1299, 0.37 \u0026micro;M; H292, 0.15 \u0026micro;M; HCC827, 3.78 \u0026micro;M; H1975, 0.50 \u0026micro;M; H3122, 0.32 \u0026micro;M, separately. The 48-hour IC\u003csub\u003e50\u003c/sub\u003e values were: A549, 0.13 \u0026micro;M; H1299, 0.39 \u0026micro;M; H292, 0.09 \u0026micro;M; HCC827, 0.44 \u0026micro;M; H1975, 0.19 \u0026micro;M; H3122, 0.08 \u0026micro;M, separately (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To further characterize dose-dependent effects, cells were treated with 0, 50, 100, and 200 nM Periplocymarin and viability was assessed at 0, 24, 48, and 72 hours by CCK-8 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), demonstrating continued inhibition of proliferation over time. Colony formation assays revealed a dose-dependent reduction in colony number following Periplocymarin exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D), indicating substantial suppression of long-term proliferative capacity. Furthermore, the EdU assay showed that the EdU-positive signal decreased as the concentration of Periplocymarin increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). These results suggest that Periplocymarin has a significant inhibitory effect on proliferation. To analyze whether Periplocymarin affects apoptosis, we treated A549, H1299, H1975 and H3122 cells with 0, 100, and 200 nM Periplocymarin for 48 hours, after which the cells were collected for flow cytometry analysis of apoptosis. The results showed that the apoptosis rate in the Periplocymarin-treated groups was higher than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C), indicating that Periplocymarin promoted apoptosis in NSCLC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo thoroughly validate the inhibitory effect of Periplocymarin on cell proliferation, we established a PDO model and compared the effects of Periplocymarin (0, 100, and 200 nM) on PDO morphology and viability. Bright-field images showed that Periplocymarin treatment caused the organoids\u0026rsquo; three-dimensional structures to become increasingly disrupted. Calcein-AM fluorescence decreased in a concentration-dependent manner, the signal diminished sequentially at 100 nM and 200 nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E), suggesting that higher concentrations of Periplocymarin could suppress organoid viability.\u003c/p\u003e \u003cp\u003eCurrently, the function of Periplocymarin on NSCLC remains unclear. To further investigate its effects, this study employed wound-healing and transwell assays to analyze the impact of Periplocymarin on NSCLC cell migration. In the wound-healing assay, A549, H1299, H1975, and H3122 cells were treated with 0, 20, and 50 nmol/L Periplocymarin, and scratch closure images were collected at 0, 24, and 48 hours, with the migrated area quantified. The results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B) showed that increasing Periplocymarin concentration and longer exposure time markedly reduced the migrated area. In the transwell assay, A549, H1299, and H1975 cells were treated with 0, 20, and 50 nM Periplocymarin for 48 hours. The results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D) demonstrated a significant decline in the number of migrated cells with increasing drug concentration. Notably, H3122 cells exhibited relatively weak migratory capacity and were therefore not included in the Transwell assay. In summary, Periplocymarin possesses the ability to inhibit the migration of NSCLC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eRNA Sequencing indicates the possible pathways of Periplocymarin\u003c/h2\u003e \u003cp\u003eTo elucidate the specific mechanism this study performed RNA sequencing on A549 and H1299 cells treated with Periplocymarin, followed by KEGG pathway enrichment and GSEA trend analyses based on the differentially expressed genes for each cell line. The results showed a high degree of concordance in pathway-level changes between the two cell lines, with MAPK pathway alterations ranking among the top enriched pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;C). Additionally, several classical tumor biology processes were enriched in both cell lines, including cell cycle regulation and p53-related regulatory networks. Further GSEA analyses of the transcriptomes from A549 and H1299 individually suggested that after Periplocymarin treatment, processes related to extracellular matrix organization, integrin-mediated adhesion, calcium signaling pathway, and drug metabolism cytochrome P450 were generally downregulated; conversely, pathways related to G2/M DNA damage checkpoint, G1/S DNA damage checkpoints, DNA damage response, the p53 signaling axis, and programmed cell death were significantly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026ndash;E). Collectively, the results support a consistent regulatory response induced by Periplocymarin, with MAPK signaling probably the principal regulatory pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eNetwork pharmacology analysis suggests possible mechanisms of Periplocymarin in NSCLC\u003c/h2\u003e \u003cp\u003eBy integrating data from GeneCards, OMIM, and DisGeNET, 3151 NSCLC-related targets were identified. Concurrently, Swiss Target Prediction predicted 100 potential targets of Periplocymarin. Intersection analysis revealed 54 overlapping targets between Periplocymarin and NSCLC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). These 54 targets were then input into STRING to construct a protein\u0026ndash;protein interaction (PPI) network (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). After removing isolated nodes, the network comprised 53 nodes and 380 edges. Core targets were identified through topological analysis, including BCL2L1, MTOR, CASP3, EGFR, STAT3, MAPK1, MAPK14, PIK3CA, ABL1, PRKCA, HDAC6, HDAC1, and PTGS2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Based on these core targets, GO functional enrichment and KEGG pathway analyses were performed. In the GO enrichment analysis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the top ten terms across molecular function (MF), biological process (BP), and cellular component (CC) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD. Notably, the BP term \u0026ldquo;regulation of reactive oxygen species metabolic process\u0026rdquo; suggesting that Periplocymarin might exert effects by modulating ROS levels, indicating a focus for subsequent investigations. KEGG pathway analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) highlighted the apoptosis pathway as significantly enriched, indicating that Periplocymarin might exert anti-tumor effects through regulation of apoptosis-related signaling cascades. Simultaneously, by integrating RNA sequencing analyses, MAPK14 was identified among the key genes, which strongly suggested that Periplocymarin might exert its inhibitory effect on NSCLC by targeting MAPK14.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eTPP and MST assays identify MAPK14 as a poteintial target\u003c/h2\u003e \u003cp\u003eA549 cells were used to elucidate the potential mechanisms by which Periplocymarin exerted cytotoxicity in NSCLC cells. Total cellular proteins were extracted and incubated with Periplocymarin. The samples were subjected to a temperature gradient (37\u0026deg;C, 42\u0026deg;C, 46\u0026deg;C, 50\u0026deg;C, 54\u0026deg;C, 58\u0026deg;C, 62\u0026deg;C, and 67\u0026deg;C), followed by centrifugation to separate soluble and insoluble fractions. Soluble proteins were quantified by Western blot and mass spectrometry using a label-free approach combined with data-independent acquisition (DIA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Protein thermal stability curves were generated. Through network pharmacology analysis, MAPK14 was found among the common interacting proteins. TPP experiments showed that with increasing temperature, MAPK14 maintained relatively high stability in the presence of Periplocymarin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C). To validate this conclusion, we treated A549 cells at different temperatures and likewise found that Periplocymarin could enhance the thermal stability of MAPK14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), suggesting that Periplocymarin is likely to interact with MAPK14 to exert cytotoxic effects against NSCLC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the direct interaction between Periplocymarin and MAPK14, MST was employed under defined aqueous conditions. The equilibrium dissociation constant (K\u003csub\u003ed\u003c/sub\u003e) was determined to be 0.11 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). The binding profile exhibited a characteristic sigmoidal trajectory, indicative of specific and saturable interaction between Periplocymarin and MAPK14, which mean K\u003csub\u003ed\u003c/sub\u003e was 0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 mM. Collectively, these MST-derived findings provided direct \u003cem\u003ein vitro\u003c/em\u003e biochemical evidence of physical association between Periplocymarin and MAPK14, thereby offering a mechanistic foundation for subsequent investigations into phosphorylation-dependent regulation and downstream signaling events.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDynamic Stability and Interaction Mechanism of MAPK14–Periplocymarin complex\u003c/h3\u003e\n\u003cp\u003eThe dynamic behavior of the MAPK14\u0026ndash;Periplocymarin complex was characterized over a 100 ns trajectory. The Root Mean Square Deviation (RMSD) profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF) reveals a synchronized upward trend for both the complex and the protein, while the ligand Periplocymarin remains remarkably stable throughout the simulation. This pattern indicates that the global structural changes are driven by the receptor\u0026rsquo;s internal dynamics rather than ligand instability. As evidenced by the structural snapshots (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL), these fluctuations originate from the inherent flexibility of the N-terminal disordered region and a significant expansion of the binding pocket. This expansion is further quantified by the Radius of Gyration (Rg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). The total Rg, along with its components in the X and Z directions, shows a clear increasing trend, directly reflecting the protein\u0026rsquo;s transition into a more extended state. This phenomenon is consistent with the visual evidence in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL, where the binding cleft expands to accommodate a conformational rearrangement of the surrounding motifs. The binding interface properties provide a nuanced view of this expansion. The interaction SASA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH) exhibits a progressive decrease, stabilizing at an average of 3.15 nm\u003csup\u003e2\u003c/sup\u003e. While pocket expansion often increases total protein surface area, the reduction in interaction surface area indicates a relocation of the drug. As the pocket opens, Periplocymarin shifts from an initially encapsulated state toward the deeper ATP-binding region, effectively increasing its distance from the phosphorylation sites Thr180 and Tyr182. Despite this relocation and pocket expansion, the number of hydrogen bonds (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI) remains consistently maintained (Average: 1.18). This persistent hydrogen bonding network, even within a dynamic and expanding pocket, underscores the high binding stability of Periplocymarin, which is essential for its long-term inhibitory effect on MAPK14.\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eBinding Free Energy and Phosphorylation Inhibition\u003c/h2\u003e \u003cp\u003eThe binding affinity was quantified at \u0026minus;\u0026thinsp;34.41 kcal/mol (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ), primarily stabilized by van der Waals interactions (\u0026ndash;31.61 kcal/mol). The temporal energy heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK) and per-residue decomposition \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eM) reveal the precise molecular mechanism behind the structural changes observed: ATP-Binding Core Preservation: Residues 31\u0026ndash;36 and 38, located within the ATP-binding region, maintain consistent and strong interactions with the drug throughout the 100 ns simulation. Expansion-Induced Recruitment: Residues 63, 64, and 67 show negligible interaction initially but are recruited into the binding network as the pocket expands (observed in the latter half of Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK). Decoupling from Phosphorylation Sites: In contrast, residues 150, 174, and the critical phosphorylation site Tyr182 exhibit favorable interactions during the early phase (pre-expansion) but eventually lose contact as the pocket enlarges and the drug relocates. As visualized in the detailed View 1 and View 2 of Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eN, the expansion and subsequent ligand shift result in Thr180 and Tyr182 moving significantly further away from the ATP-binding region. This movement causes the activation loop to become increasingly buried and distorted (as labeled \u0026ldquo;up\u0026rdquo; in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eN). By sequestering the drug in the ATP pocket and inducing a conformational state where the 180/182 sites are buried, Periplocymarin effectively blocks the access of upstream kinases, providing a structural basis for the inhibition of MAPK14 phosphorylation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003ePeriplocymarin inhibits tumor growth by suppressing MAPK14 phosphorylation\u003c/h2\u003e \u003cp\u003eMolecular docking and TPP experiments strongly suggested an interaction between Periplocymarin and MAPK14. To further elucidate the effect of Periplocymarin on MAPK14, A549, H1299, H1975, and H3122 cells were treated with 0, 50, 100, and 200 nM Periplocymarin for 24 hours, and the cells were collected for Western blot analysis. The results showed that Periplocymarin could inhibit MAPK14 phosphorylation in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Subsequently, in A549, H1299, H1975, and H3122 cells, MAPK14 was knocked down by transfecting siRNA-MAPK14. Western blot analysis confirmed downregulation of MAPK14 expression in NSCLC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). After MAPK14 knockdown, cells were treated with varying concentrations of Periplocymarin in siRNA-NC and siRNA-MAPK14 groups, and cell viability was assessed by CCK-8 assay and to determine the IC\u003csub\u003e50\u003c/sub\u003e of Periplocymarin (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). The IC\u003csub\u003e50\u003c/sub\u003e increased following MAPK14 silencing, suggesting that Periplocymarin might exert its cytotoxic effects on NSCLC cells via MAPK14.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTranscriptomic results indicated that Periplocymarin engages the ROS pathway. To validate these results, we treated A549 and H1299 cells with Periplocymarin and measured lipid peroxidation levels using an MDA assay. The results showed MDA content rising with increasing Periplocymarin concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). ROS probes further revealed enhanced ROS signals in A549 and H1299 cells after Periplocymarin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE), indicating increased cellular oxidative stress. Additionally, flow cytometry showed that Periplocymarin induced G2/M-phase cell cycle prolonged in A549 and H1299 cells, thereby inhibiting cell cycle progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF, G). Taken together, these findings suggested that Periplocymarin could promote redox activity and cause cell cycle arrest, contributing to its anti-tumor effect.\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003ePeriplocymarin exerts an inhibitory effect on NSCLC progression in vivo\u003c/h2\u003e \u003cp\u003eTo further validate the \u003cem\u003ein vivo\u003c/em\u003e anti-tumor efficacy of Periplocymarin, subcutaneous tumor models in immunodeficient mice and PDXs of lung adenocarcinoma were established. In the subcutaneous model, A549 cells were inoculated into the left axilla of BALB/c nude mice. When the tumor length reached 5 mm, mice were randomly assigned to control group or Periplocymarin-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Periplocymarin or vehicle was administered intraperitoneally every two days for two weeks. Periplocymarin-treated tumors showed reduced tumor mass and weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB, C). IHC revealed decreased Ki-67 and p-MAPK14 expression in the Periplocymarin-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD), suggesting that \u003cem\u003ein vivo\u003c/em\u003e Periplocymarin could inhibit MAPK14 phosphorylation and thereby suppress tumor proliferation. Given the relatively limited \u003cem\u003ein vivo\u003c/em\u003e studies of Periplocymarin in tumors, we observed no difference in body weight between the Periplocymarin-treated mice and controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE). To assess potential toxicity, hearts, livers, spleens, lungs, and kidneys were collected, fixed, and subjected to HE staining, which showed no obvious organ toxicity in the Periplocymarin group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF). Moreover, peripheral blood was obtained for complete blood count, hepatic and renal function analyses, with no significant differences between treated and control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG), supporting the conclusion that Periplocymarin provided effectiveness \u003cem\u003ein vivo\u003c/em\u003e tumor suppression with acceptable safety.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, to more rigorously confirm Periplocymarin\u0026rsquo;s effect on patient tumor tissue, we applied NCG mice to establish lung adenocarcinoma xenograft model and randomly assigned the third-generation PDX to control and Periplocymarin groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eH). The dosing regimen was the same as above, and Periplocymarin similarly inhibited PDX tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eI, J), without affecting body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eK). Relative to controls, Ki-67 and p-MAPK14 expression were markedly reduced in the Periplocymarin-treated tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eL), indicating diminished proliferative activity and inhibited MAPK14 signaling within the tumor xenografts. H\u0026amp;E staining showed no obvious organ toxicity in the hearts, livers, spleens, lungs, or kidneys of the NCG mice treated with Periplocymarin (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eM). Collectively, these \u003cem\u003ein vivo\u003c/em\u003e findings demonstrate substantial anti-tumor activity of Periplocymarin, likely mediated through suppression of MAPK14 signaling.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLung cancer is one of the malignancies with the highest incidence and mortality worldwide(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Therapeutic strategies for NSCLC remain constrained by drug resistance, substantial treatment-related toxicities, and limited efficacy(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Natural products represent substantial bioactive compounds that demonstrated anti-tumor activity, offering advantages such as multi-target regulation and comparatively lower toxicity(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). However, due to their complex composition and variability in active constituents, the precise mechanisms of action remain unclear, which constrains clinical translation and the development of novel therapeutics. In this study, through screening 1459 anti-tumor natural products, Periplocymarin was found to have significant selective cytotoxicity against NSCLC cells while showing low toxicity to normal lung epithelial cells, indicating favorable tumor specificity and safety. Periplocymarin, a plant-derived cardiac glycoside natural product isolated from Periploca sepium, has been studied in cardiac diseases(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). It mainly inhibits Na+/K+-ATPase activity, regulating intracellular Na+/K+ balance and thereby affecting the cellular ionic environment and signaling(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In cardiovascular disease research, Periplocymarin has shown protective effects against myocardial fibrosis induced by adrenergic activation in mice, exerts a positive inotropic effect by promoting Ca2\u0026thinsp;+\u0026thinsp;influx, mitigates doxorubicin-induced heart failure and excessive ceramide accumulation, and attenuates pathological myocardial hypertrophy by inhibiting the JAK2/STAT3 signaling pathway(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). More recently, Periplocymarin has demonstrated potential in oncology, such as inducing apoptosis in colon cancer cells by disrupting the PI3K/AKT pathway and triggering ferroptosis in gastric cancer via the ATP1A1-Src-YAP/TAZ-TFRC axis, indicating notable anti-tumor potential(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). However, its role in lung cancer remains unclear.\u003c/p\u003e \u003cp\u003eThis study found that Periplocymarin inhibited proliferation and migratory capacity across multiple lung cancer cell lines and induced apoptosis. In addition, Periplocymarin can suppress the proliferation of organoids derived from lung cancer tissues. \u003cem\u003eIn vivo\u003c/em\u003e, it significantly suppressed tumor growth in both nude mouse xenograft and PDX models. Collectively, these results illustrate the potential of Periplocymarin for lung cancer therapy, though its mechanism remains unclear. To elucidate the mechanism, this study utilized multi-omics, integrative approach, consistent with recent reports that combining transcriptomics, network pharmacology, proteomics, and molecular docking can reveal how natural products regulate cancer signaling pathways(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). For example, Li et al. utilized network pharmacology to identify the mechanism of quercetin in ovarian syndrome and endometrial cancer by AKT inhibition(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Multi-omics technologies were used to explore that triptolide overcame paclitaxel resistance in NSCLC by targeting the HNF1A/SHH axis(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Erianin, isolated from Dendrobium chrysotoxum, suppresses tumor growth in BRAFV600E or KRAS-mutant melanoma and colorectal carcinoma mouse models by targeting CRAF and MEK1/2(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In this study, network pharmacology and TPP technology both identified an interaction between Periplocymarin and MAPK14. Subsequently, molecular docking revealed a relatively high binding affinity between Periplocymarin and MAPK14. Therefore, we conclude that Periplocymarin exerts antitumor effects by modulating the MAPK14 signaling pathway.\u003c/p\u003e \u003cp\u003eMAPK14 is a core member of the MAPK family and a key signaling molecule in cellular stress responses, widely participating in biological processes such as cell proliferation, differentiation, apoptosis, and inflammatory responses(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Inflammatory cytokines, oxidative stress, and various cellular stimuli commonly trigger its activation, which regulates cell fate by phosphorylating downstream transcription factors such as ATF2 and p53(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In tumor initiation and progression, MAPK14 exhibits a complex dual role. p38α MAPK signaling suppresses tumor initiation in epithelial cells, while contributes to the proliferation and survival of tumor cells, thus to potentiate colon tumor formation(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Additionally, MAPK14 modulates inflammatory responses within tumor microenvironment, influencing the function and thereby participating in tumor immune evasion mechanisms. p38α MAPK can downregulate cyclins, upregulate cyclin-dependent kinase (CDK) inhibitors and modulate p53, to negatively regulate G1/S and the G2/M transitions by several mechanisms(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), which corresponds to the results about Periplocymarin prolonged G2/M phase in NSCLC cells. Given its multifaceted regulatory roles in inflammation and tumor initiation and progression, MAPK14 has emerged as an important potential therapeutic target for inflammatory diseases(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) and various cancers, including breast and colon cancer, and inhibitors targeting MAPK14 can effectively suppress tumor progression(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). First-in-class p38α inhibitor designated ULTR-p38i, performs as a mitosis-targeted therapy for colorectal cancer(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, Western blot analysis revealed that Periplocymarin markedly inhibited MAPK14 phosphorylation in lung cancer cells, thereby blocking signal transduction, promoting tumor cell apoptosis, and suppressing migratory capacity. MAPK14 knockdown attenuated the anti-tumor effects of Periplocymarin, indicating that Periplocymarin exerted its anticancer action, at least in part, by inhibiting MAPK14 phosphorylation. The results also showed that Periplocymarin promoted ROS generation and inhibited cell cycle progression, suggesting that its antitumor activity might rely on MAPK14, promoting tumor cell apoptosis while inhibiting proliferation and migration. In addition, Periplocymarin significantly reduced the expression level of p-MAPK14 in nude mice and PDX models. \u003cem\u003eIn vivo\u003c/em\u003e, no significant organ toxicity or hematologic abnormalities were observed in the Periplocymarin-treated group, indicating a favorable safety. These findings collectively supported its safety in the treatment of NSCLC. Our study provided a mechanistic basis for MAPK14-targeted therapeutic development.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo summarize, Periplocymarin, a natural product, exhibits potent activity against NSCLC, with its mechanism primarily through inhibiting MAPK14 phosphorylation, thereby regulating tumor cell proliferation, migration, and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Considering the pivotal role and therapeutic potential of MAPK14 in cancer, this study not only broadens the mechanistic understanding of natural products against NSCLC but also provides a theoretical basis for developing innovative MAPK14-targeted anticancer therapies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALK\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Anaplastic Lymphoma Kinase\u003cbr\u003eAM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Acetoxymethyl\u003c/p\u003e\n\u003cp\u003eATP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Adenosine Triphosphate\u003cbr\u003eAKT\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Protein kinase B\u003cbr\u003eANOVA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Analysis of Variance\u003cbr\u003eABL1\u0026nbsp; \u0026nbsp;\u0026nbsp;ABL proto-oncogene 1, non-receptor tyrosine kinase\u003c/p\u003e\n\u003cp\u003eBCA\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Bicinchoninic Acid\u003cbr\u003eBP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Biological Process\u003cbr\u003eBCL2L1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;BCL2-like 1\u003c/p\u003e\n\u003cp\u003eCO\u003csub\u003e2\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Carbon Dioxide\u003cbr\u003eCCK-8\u0026nbsp;\u0026nbsp;Cell Counting Kit-8\u003cbr\u003eCC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cellular Component\u003cbr\u003eCRAF\u0026nbsp; \u0026nbsp;C-Raf Proto-Oncogene, Serine/Threonine Kinase\u003cbr\u003eCDK\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cyclin-dependent Kinase\u003cbr\u003eCASP3\u0026nbsp;\u0026nbsp;Caspase 3\u003c/p\u003e\n\u003cp\u003eDMSO\u0026nbsp;\u0026nbsp;Dimethyl Sulfoxide\u003c/p\u003e\n\u003cp\u003eDNA\u0026nbsp; \u0026nbsp; \u0026nbsp;Deoxyribonucleic Acid\u003cbr\u003eDEGs\u0026nbsp; \u0026nbsp;\u0026nbsp;Differentially Expressed Genes\u003cbr\u003eEdU\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;5-Ethynyl-2'-deoxyuridine\u003c/p\u003e\n\u003cp\u003eEDTA\u0026nbsp; \u0026nbsp;\u0026nbsp;Ethylenediaminetetraacetic Acid\u003c/p\u003e\n\u003cp\u003eEGFR\u0026nbsp; \u0026nbsp;\u0026nbsp;Epidermal Growth Factor Receptor\u003c/p\u003e\n\u003cp\u003eFBS Fetal Bovine Serum\u003cbr\u003eFITC Fluorescein Isothiocyanate\u003cbr\u003eGSEA Gene Set Enrichment Analysis\u003cbr\u003eHDAC1 Histone deacetylase 1\u003cbr\u003eHDAC6 Histone deacetylase 6\u003cbr\u003eIHC Immunohistochemistry\u003cbr\u003eKEGG Kyoto Encyclopedia of Genes and Genomes\u003cbr\u003eKRAS Kirsten Rat Sarcoma Viral Oncogene Homolog\u003cbr\u003eMDA Malondialdehyde\u003cbr\u003eMST Microscale thermophoresis\u003cbr\u003eMF Molecular Function\u003cbr\u003eMTOR Mechanistic target of rapamycin\u003cbr\u003eMAPK1 Mitogen-activated protein kinase 1\u003cbr\u003eMAPK14 Mitogen-activated protein kinase 14（p38α）\u003c/p\u003e\n\u003cp\u003eNSCLC\u0026nbsp;Non-Small Cell Lung Cancer\u003cbr\u003eNC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Negative Control\u003cbr\u003eNCG\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;NOD CRISPR Prkdc Il2r Gamma\u003cbr\u003eOMIM\u0026nbsp;\u0026nbsp;Online Mendelian Inheritance in Man\u003cbr\u003ePPM\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Periplocymarin\u003c/p\u003e\n\u003cp\u003ePD-L1\u0026nbsp; \u0026nbsp;Programmed Death-Ligand 1\u003cbr\u003ePI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Propidium Iodide\u003cbr\u003ePPI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Protein-Protein Interaction\u003cbr\u003ePBS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Phosphate-Buffered Saline\u003c/p\u003e\n\u003cp\u003ePFS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Progression-Free Survival\u003c/p\u003e\n\u003cp\u003ePI3K\u0026nbsp; \u0026nbsp; \u0026nbsp;Phosphatidylinositol 3-Kinase\u003cbr\u003ePIK3CA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α\u003cbr\u003ePRKCA\u0026nbsp;Protein kinase C α\u003cbr\u003ePTGS2\u0026nbsp;\u0026nbsp;Prostaglandin-endoperoxide synthase 2\u003cbr\u003ePDO\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Patient-Derived Organoid\u003c/p\u003e\n\u003cp\u003ePDX\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Patient-Derived Xenograft\u003c/p\u003e\n\u003cp\u003eRNA\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Ribonucleic Acid\u003cbr\u003eROS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Reactive Oxygen Species\u003cbr\u003eRMSD\u0026nbsp;\u0026nbsp;Root‑Mean‑Square Deviation\u003c/p\u003e\n\u003cp\u003eSDS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Sodium Dodecyl Sulfate\u003cbr\u003eSD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Standard Deviation\u003cbr\u003eSEM\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Standard Error of the Mean\u003cbr\u003eSASA\u0026nbsp; \u0026nbsp;\u0026nbsp;Solvent-Accessible Surface Area\u003cbr\u003eSTAT3\u0026nbsp;\u0026nbsp;Signal transducer and activator of transcription 3\u003cbr\u003eTBST\u0026nbsp; \u0026nbsp;\u0026nbsp;Tris-buffered saline with Tween 20\u003c/p\u003e\n\u003cp\u003eTPP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Thermal Proteome Profiling\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary material associated with this article can be found in the online version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks to all authors for their contributions to the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXinye Wang: Writing (original draft), Data curation; Xiao Liang: Writing (review \u0026amp; editing), Software; Zhibin Song: Methodology; Mingwei Wang: Formal analysis; Hui Sun: Project administration; Xiaoting Ma: Methodology; Yiyan Miao: Methodology; Xingqin Zhou: Visualization, Validation; Yifei Liu: Investigation; Jiahai Shi: Supervision, Funding acquisition; Liting Lv: Resources, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Project of the Nantong Municipal Health Commission (No.: MS2024010, MSZ2024057), Nantong Social Livelihood Science and Technology Program (No.: MSZ2025007), Scientific Research Grant Project under the Senior Aging Pilot Program (No.: 323) and Jiangsu Province Graduate Practice and Innovation Program (No.: SJCX25 2067).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of date and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used to support the finding of this study are available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur animal experiment was approved by the Animal Ethics Committee of Nantong University (Approval No.: P20250310-012).And in this study all fresh tissues from NSCLC patients undergoing surgical resection at the Affiliated Hospital of Nantong University were collected with informed consent, and approved by the Animal Ethics Committee of Nantong University (Approval No.: 2025-L111).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHendriks LEL, Remon J, Faivre-Finn C, Garassino MC, Heymach JV, Kerr KM, et al. 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Thermal proteome profiling (TPP) reveals NAMPT as the anti-glioma target of phenanthroindolizidine alkaloid PF403. Acta Pharm Sin B. 2025;15(4):2008-23.\u003c/li\u003e\n \u003cli\u003eJoseph S, Zhang X, Droby GN, Wu D, Bae-Jump V, Lyons S, et al. MAPK14/p38alpha shapes the molecular landscape of endometrial cancer and promotes tumorigenic characteristics. Cell Rep. 2025;44(1):115104.\u003c/li\u003e\n \u003cli\u003eHui L, Bakiri L, Stepniak E, Wagner EF. p38alpha: a suppressor of cell proliferation and tumorigenesis. Cell Cycle. 2007;6(20):2429-33.\u003c/li\u003e\n \u003cli\u003eHuang Q, Li Y, Huang Y, Wu J, Bao W, Xue C, et al. Advances in molecular pathology and therapy of non-small cell lung cancer. Signal Transduct Target Ther. 2025;10(1):186.\u003c/li\u003e\n \u003cli\u003eChen B, Chen Q, Lu M, Zou E, Lin G, Yao J, et al. Hypocrellin A against intrahepatic Cholangiocarcinoma via multi-target inhibition of the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways. Phytomedicine. 2024;135:156022.\u003c/li\u003e\n \u003cli\u003eYun W, Qian L, Yuan R, Xu H. Periplocymarin protects against myocardial fibrosis induced by beta-adrenergic activation in mice. Biomed Pharmacother. 2021;139:111562.\u003c/li\u003e\n \u003cli\u003ePacker M. Qiliqiangxin: A multifaceted holistic treatment for heart failure or a pharmacological probe for the identification of cardioprotective mechanisms? Eur J Heart Fail. 2023;25(12):2130-43.\u003c/li\u003e\n \u003cli\u003eFan CL, Liang S, Ye MN, Cai WJ, Chen M, Hou YL, et al. Periplocymarin alleviates pathological cardiac hypertrophy via inhibiting the JAK2/STAT3 signalling pathway. J Cell Mol Med. 2022;26(9):2607-19.\u003c/li\u003e\n \u003cli\u003eKe A, Yang W, Zhang W, Chen Y, Meng X, Liu J, et al. The cardiac glycoside periplocymarin sensitizes gastric cancer to ferroptosis via the ATP1A1-Src-YAP/TAZ-TFRC axis. Phytomedicine. 2025;142:156804.\u003c/li\u003e\n \u003cli\u003eWu Z, Xiang H, Wang X, Zhang R, Guo Y, Qu L, et al. Integrating network pharmacology, molecular docking and experimental verification to explore the therapeutic effect and potential mechanism of nomilin against triple-negative breast cancer. Mol Med. 2024;30(1):166.\u003c/li\u003e\n \u003cli\u003eLi M, Cui Y, Wu X, Yang X, Huang C, Yu L, et al. Integrating network pharmacology to investigate the mechanism of quercetin\u0026apos;s action through AKT inhibition in co-expressed genes associated with polycystic ovary syndrome and endometrial cancer. Int J Biol Macromol. 2025;297:139468.\u003c/li\u003e\n \u003cli\u003eLi LB, Yang LX, Liu L, Liu FR, Li AH, Zhu YL, et al. Targeted inhibition of the HNF1A/SHH axis by triptolide overcomes paclitaxel resistance in non-small cell lung cancer. Acta Pharmacol Sin. 2024;45(5):1060-76.\u003c/li\u003e\n \u003cli\u003eWang P, Jia X, Lu B, Huang H, Liu J, Liu X, et al. Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2. Signal Transduct Target Ther. 2023;8(1):96.\u003c/li\u003e\n \u003cli\u003eKumar S, Boehm J, Lee JC. p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases. Nat Rev Drug Discov. 2003;2(9):717-26.\u003c/li\u003e\n \u003cli\u003eKirsch K, Zeke A, Toke O, Sok P, Sethi A, Sebo A, et al. Co-regulation of the transcription controlling ATF2 phosphoswitch by JNK and p38. Nat Commun. 2020;11(1):5769.\u003c/li\u003e\n \u003cli\u003eOno K, Han J. The p38 signal transduction pathway: activation and function. Cell Signal. 2000;12(1):1-13.\u003c/li\u003e\n \u003cli\u003eGupta J, del Barco Barrantes I, Igea A, Sakellariou S, Pateras IS, Gorgoulis VG, et al. Dual function of p38alpha MAPK in colon cancer: suppression of colitis-associated tumor initiation but requirement for cancer cell survival. Cancer Cell. 2014;25(4):484-500.\u003c/li\u003e\n \u003cli\u003eWagner EF, Nebreda AR. Signal integration by JNK and p38 MAPK pathways in cancer development. Nat Rev Cancer. 2009;9(8):537-49.\u003c/li\u003e\n \u003cli\u003eIgea A, Nebreda AR. The Stress Kinase p38alpha as a Target for Cancer Therapy. Cancer Res. 2015;75(19):3997-4002.\u003c/li\u003e\n \u003cli\u003eRudalska R, Harbig J, Forster M, Woelffing P, Esposito A, Kudolo M, et al. First-in-class ultralong-target-residence-time p38alpha inhibitors as a mitosis-targeted therapy for colorectal cancer. Nat Cancer. 2025;6(2):259-77.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"NSCLC, natural product library screen, Periplocymarin, multi-omics analysis, MAPK14","lastPublishedDoi":"10.21203/rs.3.rs-9282067/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9282067/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003ePeriplocymarin (PPM) is a plant-derived natural product, which is isolated and purified from the dried root bark of Periploca sepium. Previous studies have shown that Periplocymarin exhibits significantly and broad-spectrum anti-tumor pharmacological activity. To investigate the anti-tumor activity and mechanism of the natural product Periplocymarin against Non-small cell lung cancer (NSCLC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThis study screened a library of 1459 anti-tumor natural products using CCK-8 assays in three NSCLC cell lines and two normal lung epithelial cell lines, identifying Periplocymarin as a selective candidate against NSCLC. The effects of Periplocymarin on NSCLC proliferation, apoptosis, and migration were assessed in cell lines and patient-derived organoid (PDO) models. Transcriptome sequencing, network pharmacology, proteome thermal stability profiling (TPP), Microscale Thermophoresis (MST) and Molecular Simulation were integrated to identify the target of Periplocymarin. Western blot and CCK-8 assays were performed to verify Periplocymarin’s effect on p38α mitogen-activated protein kinase (p38α MAPK, MAPK14) phosphorylation. \u003cem\u003eIn vivo\u003c/em\u003e anti-tumor efficacy and safety of Periplocymarin were evaluated in nude mouse xenograft and patient-derived xenograft (PDX) models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003ePeriplocymarin exhibited concentration-dependent inhibition of proliferation, promoted apoptosis, and reduced cell migration, while also suppressing growth in organoids. Integrated analyses indicated MAPK14 as the target, and Western blot confirmed markedly inhibition of MAPK14 phosphorylation by Periplocymarin. MAPK14 knockdown attenuated Periplocymarin’s anti-tumor effects. \u003cem\u003eIn vivo\u003c/em\u003e, Periplocymarin significantly inhibited tumor growth, with decreased Ki-67 and phospho-MAPK14 (p-MAPK14) expression in tumor tissues and no evident organ toxicity or hematological abnormalities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003ePeriplocymarin, by predominantly inhibiting MAPK14 phosphorylation, presents a robust anti-tumor effect \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. These findings provide a theoretical and experimental basis for the application of Periplocymarin and for the development of novel anticancer drugs targeting MAPK14 in NSCLC.\u003c/p\u003e","manuscriptTitle":"Integrated multi-omics analysis of Periplocymarin to identify the mechanism of p38α MAPK phosphorylation inhibition in Non-small cell lung cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-19 12:06:28","doi":"10.21203/rs.3.rs-9282067/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-03T03:04:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T14:56:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199516012174662390053831619727764309454","date":"2026-04-21T04:34:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-20T09:23:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-17T13:11:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"253770819881862422913269946137674477113","date":"2026-04-13T23:23:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139633044244361110353886711125631771718","date":"2026-04-10T13:58:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-09T12:14:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-01T12:35:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-01T12:35:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chinese Medicine","date":"2026-03-31T15:30:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9a067ac5-59f9-465e-8d40-db1c8eda0279","owner":[],"postedDate":"April 19th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-03T03:04:56+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T05:53:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-19 12:06:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9282067","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9282067","identity":"rs-9282067","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}