Sodium new houttuyfonate inhibits pancreatic cancer by suppressing the MAPK pathway and tumor angiogenesis

Sodium new houttuyfonate inhibits pancreatic cancer by suppressing the MAPK pathway and tumor angiogenesis | 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 Sodium new houttuyfonate inhibits pancreatic cancer by suppressing the MAPK pathway and tumor angiogenesis Xin Yu, Lihong Jiang, Xiaoyu Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5756684/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Sodium new houttuyfonate (SNH) is derived from Houttuynia cordata Thunb. (HCT), which is a famous edible and medicinal plant in China. Although research findings suggest that SNH possesses anticancer properties, its role and mechanisms in pancreatic cancer remain unclear. Methods We investigated the effect of SNH on the malignant pancreatic cancer cell line PANC-1 using cell counting kit-8 (CCK-8), colony formation, apoptosis assays, transwell migration and electron microscopy. Additionally, we generated a mouse xenograft model to verify the potential anticancer effect of SNH in vivo. Moreover, RNA sequencing, immunohistochemical assay, and western blotting were performed on tumor cells and tissues to elucidate the potential regulatory pathways of SNH in pancreatic cancer. Results SNH treatment suppressed the proliferation and invasive abilities of pancreatic cancer cells (PANC-1 and SW1990). Additionally, SNH treatment significantly downregulated Fibroblast Growth Factor 1 (FGF1) expression in tumor tissues and cell lines. In vivo and in vitro experiments confirmed that SNH inhibited pancreatic cancer cell proliferation and promoted apoptosis, thereby inhibiting tumor progression. Mechanistically, SNH downregulated FGF1 expression and inactivated the p38/MAPK (mitogen-activated protein kinase) pathway, thereby reducing tumor angiogenesis. Conclusion Our study revealed the anticancer effect of SNH in pancreatic cancer and provides a potentially effective strategy for the treatment of pancreatic cancer. pancreatic cancer sodium new houttuyfonate FGF1 angiogenesis p38/MAPK Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Pancreatic cancer is one of the most common tumors of the digestive system and is the seventh leading cause of cancer-related deaths in both male and female populations( 1 ). In the past few decades, the global burden of pancreatic cancer has increased dramatically and is expected to become the third leading cause of cancer death in European countries( 1 ). Surgical resection, radiotherapy, and chemotherapy are currently the primary treatments for pancreatic cancer( 2 ). However, the optimal time for radical resection is often missed because most patients are diagnosed at an advanced stage. Moreover, prognosis may remain poor even after surgery, and postoperative recurrence and metastasis are common. Therefore, surgery is only suitable in 15–20% of patients( 3 ). Systemic chemotherapy is the most commonly used treatment for advanced pancreatic cancer. However, pancreatic cancer is resistant to several chemotherapeutic drugs( 4 , 5 ), and the effects of chemotherapy are relatively poor( 6 ). Additionally, chemotherapy drugs often have serious side effects and reduce the quality of life of patients( 7 ). Therefore, reducing the toxic side effects of drugs, improving the quality of life of patients, and prolonging survival are important research areas. Phytochemicals are considered potential alternative or complementary anticancer drugs because of their safety, low-cost, oral bioavailability, and potential anticancer effects( 8 – 11 ). Houttuynia cordata Thunb. (HCT), a member of the Saururaceae family, is a well-known edible and medicinal plant that is widely distributed in China and has complex medicinal ingredients. Although the volatile oil is the active component in HCT, it is unstable and easily degraded. SNH is a compound of sodium bisulfite and dodecanoyl acetaldehyde in the volatile oil of HCT and is relatively stable. Research findings indicate that SNH has anti-inflammatory( 12 , 13 ), anti-fungal( 14 , 15 ), antiviral( 16 , 17 ), and anticancer properties( 18 – 21 ). For example, SNH promoted breast cancer cell apoptosis by inhibiting the activation of the PDK1-AKT-GSK3β pathway( 18 ). Additionally, SNH suppressed non-small cell lung cancer (NSCLC) by activating pyroptosis( 19 ) and attenuated NSCLC metastasis via the Linc00668/miR-147a/slug axis( 22 ). Collectively, these findings indicate that SNH has potential anticancer effects. However, the therapeutic effect and potential molecular mechanisms of SNH in pancreatic cancer remain unclear. Therefore, we investigated the therapeutic effect and mechanism of action of SNH in pancreatic cancer using in vitro and in vivo models. Materials and methods Cell culture Human pancreatic cancer cell lines (PANC-1 and SW1990) and human umbilical vein cells (HUVECs) were purchased from the BeiNa Culture Collection. PANC-1 and SW1990 cells were cultured in Dulbecco’s modified eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Biological Industries). HUVECs were cultured in endothelial cell medium (ECM; Gibco) supplemented with 10% FBS. All cells were incubated at 37°C in a humidified atmosphere containing 95% air and 5% CO 2 . Cell viability assay Briefly, PANC-1 and SW1990 cells were seeded in 96-well plates at a density of 3,000 cells/well and cultured in the presence of 10% FBS at 37°C. The next day, the culture medium in each well (final volume, 100 µL) was supplemented with various concentrations of SNH (MCE). Thereafter, 10 µL of CCK-8 (MCE) was added to each well at 24, 48, and 72 h after drug treatment. After 2–4 h of incubation, absorbance was measured with a microplate reader (Allsheng) at a wavelength of 450 nm. Colony formation assay PANC-1 and SW1990 cells were seeded in 6-well plates (4,000 cells/well), incubated overnight, and treated with SNH (10, 20, 30, 40, and 50 µΜ) for 14 days. Thereafter, the cells were fixed with 4% paraformaldehyde (Biosharp) and stained with 0.04% crystal violet (Biosharp) for 30 min at 25°C. Finally, the stained plates were scanned using a printer. Cell invasion assay Briefly, 200 µL of PANC-1 cell suspension (5 * 10 5 cells/mL) was added into 8 µm transwell chambers, followed by the addition of DMEM containing 10% FBS and 45 µΜ of SNH to the lower compartment of the chamber (24-well plate). After culturing in an incubator at 37°C for 24 h, upper chamber cells were first fixed with 4% formaldehyde for 20 min before staining with crystal violet and finally photographed by light microscopy (Olympus). Apoptosis assay Apoptosis was assessed via flow cytometry using annexin V (AV) and propidium iodide (PI). Briefly, PANC-1 cells were seeded in 6-well plates at a density of 2 * 10 5 cells/well, followed by treatment with SNH (45 µΜ) for 72 h starting from the next day. Thereafter, the cells were trypsinized and stained with AV and PI using the FITC Annexin V/Dead Cell Apoptosis Kit (Beyotime), according to the manufacturer’s instructions. Data acquisition and analysis were performed using a flow cytometer (Agilent). Transmission electron microscopy PANC-1 cells were seeded in 6-well plates at a density of 2 * 10 5 cells/well, followed by treatment with SNH (45 µΜ) for 72 h starting from the next day. Thereafter, the cells were fixed, dehydrated, permeated, and sectioned. Finally, the sections were stained with uranyl acetate and photographed by a Scanning Electron Microscope (Zeiss) at 100 kV. Tube formation assay For the endothelial tube formation assay, 50 µL of Matrigel was added to each well of a 96-well plate and incubated at 37°C for 1 h. Thereafter, HUVECs (2 * 10 4 ) were added to each well, followed by further incubation for 4 h. The degree of tube formation was evaluated using an inverted microscope (Olympus). Western blot analysis PANC-1 cells were seeded in 6-well plates at a density of 2 * 10 5 cells/well, followed by treatment with SNH (45 µΜ) for 72 h starting from the next day. Thereafter, the cells were lysed with RIPA buffer, and the protein concentration was measured using Bradford method with BSA as the standard. Protein samples (30 µg) were separated using 12% gel electrophoresis and transferred to nitrocellulose membranes. After blocking, the cells were incubated overnight at 4°C with antibodies targeting GAPDH (AF7021, Affinity), MMP2 (ab92536, Abcam), Bax (ab32503, Abcam), Bcl-2 (ab196495, Abcam), p-p38/MAPK (AF5887, Beyotime), p38/MAPK (AF7668, Beyotime), p-JNK (AF1762, Beyotime), JNK (AF1048, Beyotime), p-ERK (ab201015, Abcam), ERK (ab17942, Abcam), and FGF1 (ab207321, Abcam), followed by incubation with specific secondary antibodies (ab6721, Abcam) at 20°C for 1 h. Finally, protein bands were quantified using Image J software. In vivo experiment Female BALB/c mice (6–8 weeks old) were purchased from the Animal Research and Resource Center of Yunnan University. The mice were housed and maintained under specific pathogen-free conditions and heedfully reared throughout the study. All mouse experiments were performed following protocols approved by the Ethics Review Committee of Animal Experimentation of Yunnan University. Briefly, the mice were subcutaneously injected with PANC-1 cells (1 * 10 7 ) to establish a xenograft model. When the tumor volume reached 100–150 mm 3 , the mice were randomly assigned to three treatment groups (n = 4 mice/group): saline (control), low-dose SNH (15 mg/kg SNH), and high-dose SNH (30 mg/kg SNH). All treatments were administered once every two days via intraperitoneal injection. Tumor size was measured every two days using the same caliper. Tumor volumes were calculated using the following formula: 0.5 * (length * width 2 ). Mice were euthanized on the 12th day of drug administration or when the tumor volume reached 1000 mm 3 . Tumor, kidney, heart, liver, lung, and spleen were harvested and fixed with 4% paraformaldehyde. Mouse tissue staining Briefly, tumor and organs were dehydrated after fixing in 4% paraformaldehyde for 48 h and embedded in paraffin. Thereafter, the tissues were cut into 4 µm thick sections, stained with hematoxylin and eosin (H&E), and observed using an optical microscope. Additionally, tumor sections were used for immumohistochemical (IHC) and immunofluorescence (IF) assays. For IHC assay, the tumor sections were dewaxed with xylene and ethanol, soaked in citric acid antigen repair buffer (pH 6.0), and heated in a microwave oven for antigen repair. After blocking with serum at room temperature for 30 min, the sections were incubated overnight at 4°C with primary antibody, washed, and incubated with secondary antibody at room temperature for 50 min. Thereafter, the sections were stained DAB for color development, and the nuclei were counterstained with hematoxylin. For IF assay, the nuclei were stained with DAPI after washing and incubated in the dark at room temperature for 10 min. Finally, the stained sections were observed using a microscope. Positive results were quantified using Image J software. Statistical analysis All data are expressed as mean ± standard error of mean (SEM). Significant differences between groups were determined using a one-way ANOVA or nonlinear regression in GraphPad Prism 9 software. Statistical significance was set at p 0.05 considered non-significant (ns). Results SNH exerts anticancer activities in pancreatic cancer cells in vitro To examine the anticancer effects SNH, the pancreatic cancer cell lines PANC-1 and SW1990 were treated with different concentrations of SNH at various time points, followed by cell viability assay. SNH treatment for 72 h significantly inhibited the growth and proliferation of PANC-1 and SW1990 cells in a concentration-dependent manner (Fig. 1 a), with a stronger inhibitory effect observed in PANC-1 cells (50% inhibitory concentration [IC 50 ] = 44.53 µM) than in SW1990 cells line (IC 50 = 54.27 µM). Additionally, colony formation assay showed that SNH treatment markedly reduced the colony formation efficiency of the cells (Fig. 1 b). Based on the results of the cell viability assay, we focused on PANC-1 cells in this study. Transwell invasion assay was performed to assess the effects of SNH on the invasive ability of PANC-1 cell. SNH treatment significantly inhibited the invasive ability of PANC-1 cells (Fig. 3 c and d). Additionally, SNH treatment downregulated the expression of Matrix Metallopeptidase 2 (MMP2), a critical factor in tumor metastasis (Fig. 3 e). Moreover, we examined the effect of SNH on cell apoptosis using AV–FITC dual staining assay and found that SNH treatment induced apoptosis in PANC-1 cells (Fig. 1 f and g). SNH treatment upregulated the expression of Bax (BCL-2-associated X protein, pro-apoptotic protein) and downregulated the expression of Bcl-2 (B-cell lymphoma-2, anti-apoptotic protein) in PANC-1 cells (Fig. 1 h). Bax and Bcl-2 belong to the Bcl-2 family and initiate the apoptotic process by regulating mitochondrial membrane permeability( 23 ). Therefore, we examined mitochondrial morphology in PANC-1 cells using transmission electron microscopy. Compared with that in the control group, abnormalities were observed in the mitochondrial morphology of SNH-treated cells, indicating mitochondrial damage (Fig. 1 i). Collectively, these results indicate that SNH induces mitochondrial damage in cells, resulting in apoptosis. SNH suppresses pancreatic tumor growth in vivo, without causing significant host toxicity To investigate the anticancer effect of SNH in vivo, we established a PANC-1 subcutaneous tumor model. Notably, the xenograft mice were intraperitoneally injected with saline (control), low-dose SNH (15 mg/kg), or high-dose SNH (30 mg/kg) once every two days. Tumor growth curves were obtained by measuring tumor volumes every two days (Fig. 2 a). Tumor growth was rapid in the control group, but suppressed in the SNH groups (Fig. 2 b). SNH treatments, especially the high-dose treatment, significantly decreased tumor cell proliferation, as indicated by the final tumor morphology and weight (Fig. 2 c and d). Additionally, IHC assay showed that SNH treatments significantly inhibited Bcl-2 expression (Fig. 2 e and f) and upregulated Bax expression (Fig. 2 g and h) in tumor tissues, with the high-dose group showing a stronger effect. Overall, these findings were consistent with the in vitro results. Importantly, no distinct abnormalities were observed in the heart, liver, spleen, lung, and kidneys in each treatment group (Fig. 2 i). The MAPK pathway is involved in the anticancer activity of SNH To investigate the molecular mechanisms by which SNH inhibits pancreatic cancer, we performed RNA sequencing of mouse tumor tissues and PANC-1 cells after SNH and DMSO (control) treatments. Principal component analysis (PCA) showed a shift in principal component 1 (PC1) and PC2 in the SNH vs. control groups (Fig. 3 a). Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed on downregulated genes in the control vs. SNH groups of PANC-1 cells (Fig. 3 b) and the control vs. 30mg/kg SNH (Fig. 3 c), 15mg/kg SNH vs. 30mg/kg SNH (Fig. 3 d) of tumor tissues. Notably, the MAPK signaling pathway was downregulated in both tissues and cells in the SNH group. Multiple signaling pathways, including the MAPK signaling pathway, are involved in the development and progression of pancreatic cancer ( 24 ). Overall, these results suggest that SNH exerts its anticancer effects by downregulating the MAPK signaling pathway. Research has established that there are three main components of the MAPK pathway in mammals, including extracellular signal-regulated kinases (ERK), Jun N-terminal kinases (JNK), and p38/MAPK. JNK and p38/MAPK have similar functions and are involved in inflammation, apoptosis, and growth, whereas ERK is mainly involved in cell growth and differentiation. Western blotting showed that SNH treatment downregulated the phosphorylation of JNK (p-JNK) and p38/MAPK (p-p38/MAPK) in PANC-1 cells, but did not affect ERK phosphorylation (p-ERK) compared with those in the control group (Fig. 3 e). Additionally, SNH had a stronger effect on p-p38/MAPK protein than on other proteins examined (Fig. 3 e). Moreover, SNH treatment downregulated p-p38/MAPK protein expression in the tumor tissues in a concentration-dependent manner compared with that in the control group (Fig. 3 f and g). Collectively, these results suggest that SNH suppresses pancreatic cancer development by inhibiting the p38/MAPK pathway. SNH inhibits FGF1 expression and tumor angiogenesis To investigate the genes involved in SNH-mediated regulation of the MAPK pathway, we analyzed transcriptomic data related to the MAPK pathway in SNH-treated cells and tumors. Additionally, differential expression analysis was performed to identify differentially expressed genes (DEGs) related to the MAPK pathway in the control vs. SNH groups. Compared with those in the control group, we identified 51 and 12 downregulated DEGs in SNH-treated cells and tumor tissues, respectively. Additionally, we identified eight downregulated DEGs in tumor tissues in the high-dose group compared with those in the low-dose group. A combined analysis of the three datasets revealed that FGF1 and DUSP4 (Dual Specificity Phosphatase 4) may be involved in SNH-mediated regulation of the MAPK pathway in in vitro and in vivo tumor models (Fig. 4 a). FGF1 showed the highest variation in cellular expression and a better trend in animal tumors (Fig. 4 b). Therefore, we focused on the role of FGF1 in the anticancer effect of SNH. Further experiments showed that SNH inhibited FGF1 protein expression in the cells regardless of the presence or absence of exogenous FGF1 protein (Fig. 4 c). Similarly, SNH treatment downregulated FGF1 protein expression in the xenograft tumors (Fig. 4 d and e). Collectively, these results suggest that FGF1 may be the key gene responsible for the anti-tumor effects of SNH. FGF1 has been shown to exert pro-angiogenic effects on endothelial cells( 25 ). A previous study showed that ILT4 (Leukocyte Immunoglobulin Like Receptor B2) activated MAPK phosphorylation and upregulated FGF1 expression, leading to angiogenesis in colorectal cancer( 26 ). Therefore, we hypothesized that SNH exerts its anti-tumor effects by disrupting tumor angiogenesis via downregulation of FGF1 expression. Expectedly, SNH treatment impaired endothelial tube formation by HUVECs in vitro, which was alleviated following treatment with exogenous FGF1 (Fig. 4 f and g). Additionally, IF and IHC assay were performed to examine CD31 expression in tumor blood vessels (Fig. 4 h). SNH treatment significantly decreased CD31 + area and microvascular density (MVD) in tumors compared with those in the control group (Fig. 4 i and j). Discussion Pancreatic cancer is a refractory cancer with one of the lowest 5-year survival rates. Chemoresistance and side effects associated with most chemotherapy drugs are critical challenges affecting pancreatic cancer treatment. Recently, phytochemicals have attracted attention owing to their therapeutic potential and high safety profiles. SNH, derived from HCT, has been clinically used to treat inflammatory diseases. Although SNH has shown anticancer activity, studies on its effect and mechanism in pancreatic cancer are limited. In this study, SNH treatment inhibited the proliferation and invasive abilities of pancreatic cancer cells in vitro and upregulated apoptosis in PANC-1 cells. Additionally, transmission electron microscopy showed that SNH treatment induced mitochondrial damage in pancreatic cells, indicating that that the pro-apoptotic effects of SNH may be related to mitochondrial function. Importantly, our results were consistent with previous findings in various disease models( 18 , 21 , 22 ). To investigate the anticancer effect of SNH in vivo, we generated a xenograft model of pancreatic cancer. Consistent with the in vitro results, SNH treatment suppressed pancreatic cancer development in vivo. Research findings indicate that KRAS (KRAS Proto-Oncogene, GTPase)-activating mutations are present in most pancreatic cancers( 27 ). Mutant KRAS proteins play oncogenic roles by participating in multiple downstream signaling cascades, including the MAPK and PI3K-AKT-mTOR pathways( 28 ). The MAPK pathway regulates multiple transcription factors, cell cycle progression, and cell proliferation. Additionally, the PI3K-AKT-mTOR pathway regulates protein synthesis, cell growth, and survival( 29 ). Based on previous findings, the MAPK pathway is considered as one of the most critical therapeutic targets( 30 ). Generally, the MAPK pathway includes ERK, JNK, and p38/MAPK in mammals. Our study showed that SNH inhibited the p38/MAPK pathway in pancreatic cancer. Notably, the p38/MAPK pathway is activated by most growth factors, cytokines, and immune receptors as well as by several integrin and chemokine receptors. In the present study, transcriptome sequencing revealed that FGF1 and DUSP4 were potential drivers of SNH-mediated regulation of p38/MAPK pathway in pancreatic cancer. FGF1 is the most important cytokine involved in pancreatic cancer formation and has been shown to interact with the p38/MAPK pathway( 31 ). FGF1 is a member of the FGF family and is involved in cell proliferation and chemotherapy resistance in human pancreatic cancer( 32 , 33 ). Importantly, our experiments confirmed that SNH downregulated FGF1 expression in pancreatic cancer cells In addition to playing an important role in tumor cell motility and survival, the FGF family is involved in tumor angiogenesis( 34 , 35 ). Tumor angiogenesis is a key determining step in tumor growth and metastasis( 36 ). In the last few decades, anti-angiogenic therapy has become a rational strategy for the treatment of various cancers, including pancreatic cancer. For example, inhibition of VEGF (Vascular Endothelial Growth Factor) activity through the p38/MAPK pathway may attenuate drug resistance in breast cancer( 37 ). Additionally, several p38/MAPK pathway inhibitors have been shown to inhibit the progression of prostate cancer( 38 ) and head and neck squamous cell carcinoma( 39 ). Our study confirmed that SNH not only inactivated the p38/MAPK pathway but also inhibited tumor angiogenesis. Collectively, these results suggest that SNH inhibits tumor angiogenesis by downregulating FGF1 expression via the p38/MAPK pathway, thereby suppressing pancreatic cancer progression. Conclusions Our work reveals SNH inhibits the proliferation and invasive abilities of PANC-1 cells, induces mitochondrial damage and apoptosis, and suppresses the growth of pancreatic tumors in vivo. Mechanistically, SNH inhibits the expression of FGF1 and inactivates the p38/MAPK pathway, thereby inhibiting tumor angiogenesis. Based on our findings, it can be concluded that SNH possesses potential application as a therapeutic agent against pancreatic cancer. Declarations Competing interest The 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. Ethics approval and consent to participate This study was approved by the Ethics Review Committee of Animal Experimentation of Yunnan University (No. YNU20241109). Author Contributions Xin Yu performed the experiments, analyzed and interpreted the data, and wrote the paper. Lihong Jiang supervised the study, provided important advice, and received financial support. Xiaoyu Yang participated in the study design, performed bioinformatics analysis and received financial support. Funding This work was supported by Science and Technology plan project of the First People’s Hospital of Yunnan Province (Project number: KHBS-2022-024), the Yunnan Science and Technology Commission of the Yunnan Provincial Science and Technology Department and Kunming Medical University (Project number: 202401AY070001-123), and Yunnan Provincial Key Laboratory for Innovative Application of Traditional Chinese Medicine (Project number: 202205AG070005). Availability of data The RNA-Seq data generated in this study have been deposited in the National Center for Biotechnology Information (NCBI) resource under accession number PRJNA1201330 (PANC-1 cells) and PRJNA1202402 (mouse tumor tissues). All data are also available from the corresponding author (Xiaoyu Yang) upon reasonable request. References H. Sung, J. Ferlay, R.L. Siegel, M. Laversanne, I. Soerjomataram, A. 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Lijing et al., Dipalmitoylphosphatidic acid inhibits breast cancer growth by suppressing angiogenesis via inhibition of the CUX1/FGF1/HGF signalling pathway. J. Cell. Mol. Med. 2018; 22 (10) X. Li, Z. Jiang, J. Li, K. Yang, J. He, Q. Deng et al., PRELP inhibits colorectal cancer progression by suppressing epithelial-mesenchymal transition and angiogenesis via the inactivation of the FGF1/PI3K/AKT pathway. Apoptosis: Int. J. Program. cell. death 2024 J F. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971;285(21) A. Reidun, S. Betzabe Chavez, N. Jens Henrik, L. Rolf, V. Kristina, L. Barbro, An autocrine VEGF/VEGFR2 and p38 signaling loop confers resistance to 4-hydroxytamoxifen in MCF-7 breast cancer cells. Mol. Cancer Res. 2008; 6 (10) R. Gangaraju, K. Malgorzata, M. Abby, A.S. Mark, R.W. William, B. Sunil et al., Pro-inflammatory angiogenesis is mediated by p38 MAP kinase. J. Cell. Physiol. 2010; 226 (3) L. Kantima, A. Panomwat, A.M. Alfredo, R.B. John, K. Sittichai, J Silvio G. A role for p38 MAPK in head and neck cancer cell growth and tumor-induced angiogenesis and lymphangiogenesis. Mol. Oncol. 2013; 8 (1) Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5756684","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":398267282,"identity":"72a858f8-89f1-4479-8e96-5944e8f76100","order_by":0,"name":"Xin Yu","email":"","orcid":"","institution":"Faculty of Life science and Technology, Kunming University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Yu","suffix":""},{"id":398267284,"identity":"d8eaf314-2939-4898-aabd-8c257cb9cc7f","order_by":1,"name":"Lihong Jiang","email":"","orcid":"","institution":"Regenerative Medicine Research Center, The First People’s Hospital of Yunnan Province","correspondingAuthor":false,"prefix":"","firstName":"Lihong","middleName":"","lastName":"Jiang","suffix":""},{"id":398267285,"identity":"4df325a3-bfbc-4273-b6a3-aeea7b1d96f2","order_by":2,"name":"Xiaoyu Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYDADfmbGxgcfGCRI0CLZ3nzYcAZJWgzOHEsT5iFK5fEes4dfc+zyGG7kmDHb/LHI429gfvjoBl7Dz5gby25LLmackWP2OLdNoljiAJuxcQ4+LUDDpSW3MSc2S+SYG+c2SCQ2HOBhkyZCS31imwSQYfFHInE+MVokP247nNjDcyxNmoFNInEDIS2SZ46VSTNuO544gx0YyL1tEokbDxPwC9/x5m2SP7dVJ+4/DIzKH3/qEucdb374GJ8WhQMMDMyo0cGMRzkIyDcwMDD+IKBoFIyCUTAKRjgAAOUPUAbsD2HKAAAAAElFTkSuQmCC","orcid":"","institution":"Regenerative Medicine Research Center, The First People’s Hospital of Yunnan Province","correspondingAuthor":true,"prefix":"","firstName":"Xiaoyu","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-01-03 08:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5756684/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5756684/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73305812,"identity":"2fe7557d-0650-4f83-b6f8-325d087d23d8","added_by":"auto","created_at":"2025-01-08 16:56:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1423254,"visible":true,"origin":"","legend":"\u003cp\u003eSNH inhibited the invasive and proliferative abilities of PANC-1 and promoted apoptosis.\u003cstrong\u003e \u003c/strong\u003e(a) Cell Counting Kit-8 kits were used to detect the activity of PANC-1 and SW1990 cells after treatment with different concentrations of SNH at 24, 48, and 72 h. N=4-6. (b) Clone formation assay of PANC-1 and SW1990 cells treated with different concentrations of SNH. (c, d) Transwell chambers were used to examine the cell invasion ability of PANC-1 cells after SNH treatment. Scale bar: 100 μm. N=3. (e) Western blot was used to detect the expression levels of MMP2 in PANC-1 cells treated with SNH for 72 h. (f, g) Flow cytometry was used to examine the effect of SNH on apoptosis. N=3. (h) Western blot was used to detect the expression levels of apoptosis-related proteins Bax, and Bcl-2 in PANC-1cells treated with SNH for 72 h. (i) TEM was used to observe the mitochondria of PANC-1 cells after treatment with SNH. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001, ns = not significant. SNH: Sodium new houttuyfonate, MMP2: Matrix metallopeptidase 2, Bax: BCL-2-associated X protein, Bci-2: B-cell lymphoma-2, TEM: Transmission electron microscopy.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5756684/v1/c16833efb8b41dd62ffe00d1.png"},{"id":73305816,"identity":"7ded617c-4132-47b8-9f3c-7ff7d17d70f9","added_by":"auto","created_at":"2025-01-08 16:56:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1992162,"visible":true,"origin":"","legend":"\u003cp\u003eSNH suppresses pancreatic tumor growth in vivo. (a) Schematic representation of treatment protocols for animal experiments. (b) Changes in tumor volume during treatment in each group of mice. N=4. (c, d) The tumors of the mice were collected and weighed after the mice were euthanized. N=4. (e, f) The expression of Bax in tumors was detected by immunohistochemical staining. Scale bar: 50 μm (up), 20 μm (down). N=4. (g, h) The expression of Bcl-2 in tumors was detected by immunohistochemicalstaining. Scale bar: 50 μm (up), 20 μm (down). N=4. (i) H\u0026amp;E staining of heart, liver, spleen, lung and kidney in each group of mice. Scale bar: 100 μm. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001, ns = not significant. SNH: Sodium new houttuyfonate, Bax: BCL-2-associated X protein, Bci-2: B-cell lymphoma-2, H\u0026amp;E: Hematoxylin and eosin.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5756684/v1/ab31965e0332bdcdb2d8aa1d.png"},{"id":73305814,"identity":"70d65b40-c6b4-4452-8cbb-9cb971186ffc","added_by":"auto","created_at":"2025-01-08 16:56:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1038443,"visible":true,"origin":"","legend":"\u003cp\u003eThe MAPK pathway is involved in the anticancer activity of SNH.\u003cstrong\u003e \u003c/strong\u003e(a) PCA cluster analysis showed statistically significant differences among the groups. PANC-1 cells (up, n=3), tumor tissues (down, n=4). (b) KEGG down-regulated pathway enrichment analysis in the control vs. SNH groups of PANC-1 cells (top 6). (c) KEGG down-regulated pathway enrichment analysis in the control vs. 30 mg/kg SNH groups of tumor tissues (top 6). (d) KEGG down-regulated pathway enrichment analysis in the 15 mg/kg SNH vs. 30 mg/kg SNH groups of tumor tissues (top 6). (e) Western blot was used to detect the expression levels of p-ERK, ERK, p-JNK, JNK, p-p38/MAPK, and p38/MAPK in PANC-1 cells treated with SNH for 72 h. (f, g) The expression of p-p38/MAPK in tumors was detected by immunofluorescence staining. Scale bar: 100 μm (up), 20 μm (down). N=3. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001, ns = not significant. SNH: Sodium new houttuyfonate, PCA: Principal component analysis, KEGG: Kyoto Encyclopedia of Genes and Genomes, ERK: Extracellular signal-regulated kinases, JNK: Jun N-terminal kinases, MAPK: Mitogen-activated protein kinase.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5756684/v1/7e42ec6e19331789a5dcf34f.png"},{"id":73305818,"identity":"71cb2f46-9229-43e0-88d9-87dd6f9c7c11","added_by":"auto","created_at":"2025-01-08 16:56:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1770075,"visible":true,"origin":"","legend":"\u003cp\u003eSNH inhibits FGF1 expression and tumor angiogenesis. (a) Venn graph showed the intersection downregulated genes of MAPK pathways in each group. (b) FPKM of FGF1 and DUSP4 in PANC-1cells (left) and tumor tissues (right). (c) Western blot was used to detect the expression levels of FGF1 in PANC-1 cells treated with different drugs. (d, e) The expression of FGF1 in tumors was detected by immunohistochemical staining. Scale bar: 100 μm (up), 20 μm (down). N=4. (f, g) HUVECs tube formation treated by different drugs. Scale bar: 100 μm. N=3. FGF1 (100 ng/ml) was used in the detection of HUVECs tube formation. (h) Upper: immunofluorescence analysis with CD31 (red), and DAPI (blue) of tumor tissues. Scale bar: 100 μm (up), 20 μm (down). Lower: immunohistochemical analysis with CD31 in tumor tissues. Scale bar: 100 μm (up), 20 μm (down). (i, j) Bar graphs indicated the quantitative data for (I) MVD (n=3) or (J) CD31\u003csup\u003e+ \u003c/sup\u003earea (n=4). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001, ns = not significant. SNH: Sodium new houttuyfonate, FGF1: Fibroblast growth factor 1, MAPK: Mitogen-activated protein kinase, FPKM: Fragments per kilobase of exon model per million mapped fragments, DUSP4: Dual specificity phosphatase 4, HUVECs: Human umbilical vein cells, MVD: Microvascular density.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5756684/v1/9724053bcfafac77101b3efb.png"},{"id":73332854,"identity":"af21c684-0926-41c4-bb15-2ea617a4fbed","added_by":"auto","created_at":"2025-01-09 03:31:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7370806,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5756684/v1/681adcde-8c76-4155-a830-cb74565f77dc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sodium new houttuyfonate inhibits pancreatic cancer by suppressing the MAPK pathway and tumor angiogenesis","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic cancer is one of the most common tumors of the digestive system and is the seventh leading cause of cancer-related deaths in both male and female populations(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In the past few decades, the global burden of pancreatic cancer has increased dramatically and is expected to become the third leading cause of cancer death in European countries(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSurgical resection, radiotherapy, and chemotherapy are currently the primary treatments for pancreatic cancer(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). However, the optimal time for radical resection is often missed because most patients are diagnosed at an advanced stage. Moreover, prognosis may remain poor even after surgery, and postoperative recurrence and metastasis are common. Therefore, surgery is only suitable in 15\u0026ndash;20% of patients(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Systemic chemotherapy is the most commonly used treatment for advanced pancreatic cancer. However, pancreatic cancer is resistant to several chemotherapeutic drugs(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), and the effects of chemotherapy are relatively poor(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Additionally, chemotherapy drugs often have serious side effects and reduce the quality of life of patients(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Therefore, reducing the toxic side effects of drugs, improving the quality of life of patients, and prolonging survival are important research areas.\u003c/p\u003e \u003cp\u003ePhytochemicals are considered potential alternative or complementary anticancer drugs because of their safety, low-cost, oral bioavailability, and potential anticancer effects(\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). \u003cem\u003eHouttuynia cordata\u003c/em\u003e Thunb. (HCT), a member of the Saururaceae family, is a well-known edible and medicinal plant that is widely distributed in China and has complex medicinal ingredients. Although the volatile oil is the active component in HCT, it is unstable and easily degraded. SNH is a compound of sodium bisulfite and dodecanoyl acetaldehyde in the volatile oil of HCT and is relatively stable. Research findings indicate that SNH has anti-inflammatory(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), anti-fungal(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), antiviral(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), and anticancer properties(\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). For example, SNH promoted breast cancer cell apoptosis by inhibiting the activation of the PDK1-AKT-GSK3β pathway(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Additionally, SNH suppressed non-small cell lung cancer (NSCLC) by activating pyroptosis(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) and attenuated NSCLC metastasis via the Linc00668/miR-147a/slug axis(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Collectively, these findings indicate that SNH has potential anticancer effects. However, the therapeutic effect and potential molecular mechanisms of SNH in pancreatic cancer remain unclear.\u003c/p\u003e \u003cp\u003eTherefore, we investigated the therapeutic effect and mechanism of action of SNH in pancreatic cancer using in vitro and in vivo models.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHuman pancreatic cancer cell lines (PANC-1 and SW1990) and human umbilical vein cells (HUVECs) were purchased from the BeiNa Culture Collection. PANC-1 and SW1990 cells were cultured in Dulbecco\u0026rsquo;s modified eagle\u0026rsquo;s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Biological Industries). HUVECs were cultured in endothelial cell medium (ECM; Gibco) supplemented with 10% FBS. All cells were incubated at 37\u0026deg;C in a humidified atmosphere containing 95% air and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eBriefly, PANC-1 and SW1990 cells were seeded in 96-well plates at a density of 3,000 cells/well and cultured in the presence of 10% FBS at 37\u0026deg;C. The next day, the culture medium in each well (final volume, 100 \u0026micro;L) was supplemented with various concentrations of SNH (MCE). Thereafter, 10 \u0026micro;L of CCK-8 (MCE) was added to each well at 24, 48, and 72 h after drug treatment. After 2\u0026ndash;4 h of incubation, absorbance was measured with a microplate reader (Allsheng) at a wavelength of 450 nm.\u003c/p\u003e\n\u003ch3\u003eColony formation assay\u003c/h3\u003e\n\u003cp\u003ePANC-1 and SW1990 cells were seeded in 6-well plates (4,000 cells/well), incubated overnight, and treated with SNH (10, 20, 30, 40, and 50 \u0026micro;Μ) for 14 days. Thereafter, the cells were fixed with 4% paraformaldehyde (Biosharp) and stained with 0.04% crystal violet (Biosharp) for 30 min at 25\u0026deg;C. Finally, the stained plates were scanned using a printer.\u003c/p\u003e\n\u003ch3\u003eCell invasion assay\u003c/h3\u003e\n\u003cp\u003eBriefly, 200 \u0026micro;L of PANC-1 cell suspension (5 * 10\u003csup\u003e5\u003c/sup\u003e cells/mL) was added into 8 \u0026micro;m transwell chambers, followed by the addition of DMEM containing 10% FBS and 45 \u0026micro;Μ of SNH to the lower compartment of the chamber (24-well plate). After culturing in an incubator at 37\u0026deg;C for 24 h, upper chamber cells were first fixed with 4% formaldehyde for 20 min before staining with crystal violet and finally photographed by light microscopy (Olympus).\u003c/p\u003e\n\u003ch3\u003eApoptosis assay\u003c/h3\u003e\n\u003cp\u003eApoptosis was assessed via flow cytometry using annexin V (AV) and propidium iodide (PI). Briefly, PANC-1 cells were seeded in 6-well plates at a density of 2 * 10\u003csup\u003e5\u003c/sup\u003e cells/well, followed by treatment with SNH (45 \u0026micro;Μ) for 72 h starting from the next day. Thereafter, the cells were trypsinized and stained with AV and PI using the FITC Annexin V/Dead Cell Apoptosis Kit (Beyotime), according to the manufacturer\u0026rsquo;s instructions. Data acquisition and analysis were performed using a flow cytometer (Agilent).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy\u003c/h2\u003e \u003cp\u003ePANC-1 cells were seeded in 6-well plates at a density of 2 * 10\u003csup\u003e5\u003c/sup\u003e cells/well, followed by treatment with SNH (45 \u0026micro;Μ) for 72 h starting from the next day. Thereafter, the cells were fixed, dehydrated, permeated, and sectioned. Finally, the sections were stained with uranyl acetate and photographed by a Scanning Electron Microscope (Zeiss) at 100 kV.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTube formation assay\u003c/h3\u003e\n\u003cp\u003eFor the endothelial tube formation assay, 50 \u0026micro;L of Matrigel was added to each well of a 96-well plate and incubated at 37\u0026deg;C for 1 h. Thereafter, HUVECs (2 * 10\u003csup\u003e4\u003c/sup\u003e) were added to each well, followed by further incubation for 4 h. The degree of tube formation was evaluated using an inverted microscope (Olympus).\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003ePANC-1 cells were seeded in 6-well plates at a density of 2 * 10\u003csup\u003e5\u003c/sup\u003e cells/well, followed by treatment with SNH (45 \u0026micro;Μ) for 72 h starting from the next day. Thereafter, the cells were lysed with RIPA buffer, and the protein concentration was measured using Bradford method with BSA as the standard. Protein samples (30 \u0026micro;g) were separated using 12% gel electrophoresis and transferred to nitrocellulose membranes. After blocking, the cells were incubated overnight at 4\u0026deg;C with antibodies targeting GAPDH (AF7021, Affinity), MMP2 (ab92536, Abcam), Bax (ab32503, Abcam), Bcl-2 (ab196495, Abcam), p-p38/MAPK (AF5887, Beyotime), p38/MAPK (AF7668, Beyotime), p-JNK (AF1762, Beyotime), JNK (AF1048, Beyotime), p-ERK (ab201015, Abcam), ERK (ab17942, Abcam), and FGF1 (ab207321, Abcam), followed by incubation with specific secondary antibodies (ab6721, Abcam) at 20\u0026deg;C for 1 h. Finally, protein bands were quantified using Image J software.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo experiment\u003c/h2\u003e \u003cp\u003eFemale BALB/c mice (6\u0026ndash;8 weeks old) were purchased from the Animal Research and Resource Center of Yunnan University. The mice were housed and maintained under specific pathogen-free conditions and heedfully reared throughout the study. All mouse experiments were performed following protocols approved by the Ethics Review Committee of Animal Experimentation of Yunnan University. Briefly, the mice were subcutaneously injected with PANC-1 cells (1 * 10\u003csup\u003e7\u003c/sup\u003e) to establish a xenograft model. When the tumor volume reached 100\u0026ndash;150 mm\u003csup\u003e3\u003c/sup\u003e, the mice were randomly assigned to three treatment groups (n\u0026thinsp;=\u0026thinsp;4 mice/group): saline (control), low-dose SNH (15 mg/kg SNH), and high-dose SNH (30 mg/kg SNH). All treatments were administered once every two days via intraperitoneal injection. Tumor size was measured every two days using the same caliper. Tumor volumes were calculated using the following formula: 0.5 * (length * width\u003csup\u003e2\u003c/sup\u003e). Mice were euthanized on the 12th day of drug administration or when the tumor volume reached 1000 mm\u003csup\u003e3\u003c/sup\u003e. Tumor, kidney, heart, liver, lung, and spleen were harvested and fixed with 4% paraformaldehyde.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMouse tissue staining\u003c/h2\u003e \u003cp\u003eBriefly, tumor and organs were dehydrated after fixing in 4% paraformaldehyde for 48 h and embedded in paraffin. Thereafter, the tissues were cut into 4 \u0026micro;m thick sections, stained with hematoxylin and eosin (H\u0026amp;E), and observed using an optical microscope. Additionally, tumor sections were used for immumohistochemical (IHC) and immunofluorescence (IF) assays.\u003c/p\u003e \u003cp\u003eFor IHC assay, the tumor sections were dewaxed with xylene and ethanol, soaked in citric acid antigen repair buffer (pH 6.0), and heated in a microwave oven for antigen repair. After blocking with serum at room temperature for 30 min, the sections were incubated overnight at 4\u0026deg;C with primary antibody, washed, and incubated with secondary antibody at room temperature for 50 min. Thereafter, the sections were stained DAB for color development, and the nuclei were counterstained with hematoxylin. For IF assay, the nuclei were stained with DAPI after washing and incubated in the dark at room temperature for 10 min. Finally, the stained sections were observed using a microscope. Positive results were quantified using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM). Significant differences between groups were determined using a one-way ANOVA or nonlinear regression in GraphPad Prism 9 software. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 considered non-significant (ns).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSNH exerts anticancer activities in pancreatic cancer cells in vitro\u003c/h2\u003e \u003cp\u003eTo examine the anticancer effects SNH, the pancreatic cancer cell lines PANC-1 and SW1990 were treated with different concentrations of SNH at various time points, followed by cell viability assay. SNH treatment for 72 h significantly inhibited the growth and proliferation of PANC-1 and SW1990 cells in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), with a stronger inhibitory effect observed in PANC-1 cells (50% inhibitory concentration [IC\u003csub\u003e50\u003c/sub\u003e]\u0026thinsp;=\u0026thinsp;44.53 \u0026micro;M) than in SW1990 cells line (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;54.27 \u0026micro;M). Additionally, colony formation assay showed that SNH treatment markedly reduced the colony formation efficiency of the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Based on the results of the cell viability assay, we focused on PANC-1 cells in this study.\u003c/p\u003e \u003cp\u003eTranswell invasion assay was performed to assess the effects of SNH on the invasive ability of PANC-1 cell. SNH treatment significantly inhibited the invasive ability of PANC-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and d). Additionally, SNH treatment downregulated the expression of Matrix Metallopeptidase 2 (MMP2), a critical factor in tumor metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Moreover, we examined the effect of SNH on cell apoptosis using AV\u0026ndash;FITC dual staining assay and found that SNH treatment induced apoptosis in PANC-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef and g). SNH treatment upregulated the expression of Bax (BCL-2-associated X protein, pro-apoptotic protein) and downregulated the expression of Bcl-2 (B-cell lymphoma-2, anti-apoptotic protein) in PANC-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Bax and Bcl-2 belong to the Bcl-2 family and initiate the apoptotic process by regulating mitochondrial membrane permeability(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Therefore, we examined mitochondrial morphology in PANC-1 cells using transmission electron microscopy. Compared with that in the control group, abnormalities were observed in the mitochondrial morphology of SNH-treated cells, indicating mitochondrial damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). Collectively, these results indicate that SNH induces mitochondrial damage in cells, resulting in apoptosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSNH suppresses pancreatic tumor growth in vivo, without causing significant host toxicity\u003c/h2\u003e \u003cp\u003eTo investigate the anticancer effect of SNH in vivo, we established a PANC-1 subcutaneous tumor model. Notably, the xenograft mice were intraperitoneally injected with saline (control), low-dose SNH (15 mg/kg), or high-dose SNH (30 mg/kg) once every two days. Tumor growth curves were obtained by measuring tumor volumes every two days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Tumor growth was rapid in the control group, but suppressed in the SNH groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). SNH treatments, especially the high-dose treatment, significantly decreased tumor cell proliferation, as indicated by the final tumor morphology and weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and d).\u003c/p\u003e \u003cp\u003eAdditionally, IHC assay showed that SNH treatments significantly inhibited Bcl-2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and f) and upregulated Bax expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg and h) in tumor tissues, with the high-dose group showing a stronger effect. Overall, these findings were consistent with the in vitro results. Importantly, no distinct abnormalities were observed in the heart, liver, spleen, lung, and kidneys in each treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThe MAPK pathway is involved in the anticancer activity of SNH\u003c/h2\u003e \u003cp\u003eTo investigate the molecular mechanisms by which SNH inhibits pancreatic cancer, we performed RNA sequencing of mouse tumor tissues and PANC-1 cells after SNH and DMSO (control) treatments. Principal component analysis (PCA) showed a shift in principal component 1 (PC1) and PC2 in the SNH vs. control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed on downregulated genes in the control vs. SNH groups of PANC-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and the control vs. 30mg/kg SNH (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), 15mg/kg SNH vs. 30mg/kg SNH (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) of tumor tissues. Notably, the MAPK signaling pathway was downregulated in both tissues and cells in the SNH group. Multiple signaling pathways, including the MAPK signaling pathway, are involved in the development and progression of pancreatic cancer (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Overall, these results suggest that SNH exerts its anticancer effects by downregulating the MAPK signaling pathway. Research has established that there are three main components of the MAPK pathway in mammals, including extracellular signal-regulated kinases (ERK), Jun N-terminal kinases (JNK), and p38/MAPK. JNK and p38/MAPK have similar functions and are involved in inflammation, apoptosis, and growth, whereas ERK is mainly involved in cell growth and differentiation. Western blotting showed that SNH treatment downregulated the phosphorylation of JNK (p-JNK) and p38/MAPK (p-p38/MAPK) in PANC-1 cells, but did not affect ERK phosphorylation (p-ERK) compared with those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Additionally, SNH had a stronger effect on p-p38/MAPK protein than on other proteins examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Moreover, SNH treatment downregulated p-p38/MAPK protein expression in the tumor tissues in a concentration-dependent manner compared with that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef and g). Collectively, these results suggest that SNH suppresses pancreatic cancer development by inhibiting the p38/MAPK pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSNH inhibits FGF1 expression and tumor angiogenesis\u003c/h2\u003e \u003cp\u003eTo investigate the genes involved in SNH-mediated regulation of the MAPK pathway, we analyzed transcriptomic data related to the MAPK pathway in SNH-treated cells and tumors. Additionally, differential expression analysis was performed to identify differentially expressed genes (DEGs) related to the MAPK pathway in the control vs. SNH groups. Compared with those in the control group, we identified 51 and 12 downregulated DEGs in SNH-treated cells and tumor tissues, respectively. Additionally, we identified eight downregulated DEGs in tumor tissues in the high-dose group compared with those in the low-dose group. A combined analysis of the three datasets revealed that FGF1 and DUSP4 (Dual Specificity Phosphatase 4) may be involved in SNH-mediated regulation of the MAPK pathway in in vitro and in vivo tumor models (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). FGF1 showed the highest variation in cellular expression and a better trend in animal tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Therefore, we focused on the role of FGF1 in the anticancer effect of SNH. Further experiments showed that SNH inhibited FGF1 protein expression in the cells regardless of the presence or absence of exogenous FGF1 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Similarly, SNH treatment downregulated FGF1 protein expression in the xenograft tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed and e). Collectively, these results suggest that FGF1 may be the key gene responsible for the anti-tumor effects of SNH.\u003c/p\u003e \u003cp\u003eFGF1 has been shown to exert pro-angiogenic effects on endothelial cells(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). A previous study showed that ILT4 (Leukocyte Immunoglobulin Like Receptor B2) activated MAPK phosphorylation and upregulated FGF1 expression, leading to angiogenesis in colorectal cancer(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Therefore, we hypothesized that SNH exerts its anti-tumor effects by disrupting tumor angiogenesis via downregulation of FGF1 expression. Expectedly, SNH treatment impaired endothelial tube formation by HUVECs in vitro, which was alleviated following treatment with exogenous FGF1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef and g). Additionally, IF and IHC assay were performed to examine CD31 expression in tumor blood vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). SNH treatment significantly decreased CD31\u003csup\u003e+\u003c/sup\u003e area and microvascular density (MVD) in tumors compared with those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei and j).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePancreatic cancer is a refractory cancer with one of the lowest 5-year survival rates. Chemoresistance and side effects associated with most chemotherapy drugs are critical challenges affecting pancreatic cancer treatment. Recently, phytochemicals have attracted attention owing to their therapeutic potential and high safety profiles. SNH, derived from HCT, has been clinically used to treat inflammatory diseases. Although SNH has shown anticancer activity, studies on its effect and mechanism in pancreatic cancer are limited. In this study, SNH treatment inhibited the proliferation and invasive abilities of pancreatic cancer cells in vitro and upregulated apoptosis in PANC-1 cells. Additionally, transmission electron microscopy showed that SNH treatment induced mitochondrial damage in pancreatic cells, indicating that that the pro-apoptotic effects of SNH may be related to mitochondrial function. Importantly, our results were consistent with previous findings in various disease models(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). To investigate the anticancer effect of SNH in vivo, we generated a xenograft model of pancreatic cancer. Consistent with the in vitro results, SNH treatment suppressed pancreatic cancer development in vivo.\u003c/p\u003e \u003cp\u003eResearch findings indicate that KRAS (KRAS Proto-Oncogene, GTPase)-activating mutations are present in most pancreatic cancers(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Mutant KRAS proteins play oncogenic roles by participating in multiple downstream signaling cascades, including the MAPK and PI3K-AKT-mTOR pathways(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). The MAPK pathway regulates multiple transcription factors, cell cycle progression, and cell proliferation. Additionally, the PI3K-AKT-mTOR pathway regulates protein synthesis, cell growth, and survival(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Based on previous findings, the MAPK pathway is considered as one of the most critical therapeutic targets(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Generally, the MAPK pathway includes ERK, JNK, and p38/MAPK in mammals. Our study showed that SNH inhibited the p38/MAPK pathway in pancreatic cancer. Notably, the p38/MAPK pathway is activated by most growth factors, cytokines, and immune receptors as well as by several integrin and chemokine receptors. In the present study, transcriptome sequencing revealed that FGF1 and DUSP4 were potential drivers of SNH-mediated regulation of p38/MAPK pathway in pancreatic cancer. FGF1 is the most important cytokine involved in pancreatic cancer formation and has been shown to interact with the p38/MAPK pathway(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). FGF1 is a member of the FGF family and is involved in cell proliferation and chemotherapy resistance in human pancreatic cancer(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Importantly, our experiments confirmed that SNH downregulated FGF1 expression in pancreatic cancer cells\u003c/p\u003e \u003cp\u003eIn addition to playing an important role in tumor cell motility and survival, the FGF family is involved in tumor angiogenesis(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Tumor angiogenesis is a key determining step in tumor growth and metastasis(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In the last few decades, anti-angiogenic therapy has become a rational strategy for the treatment of various cancers, including pancreatic cancer. For example, inhibition of VEGF (Vascular Endothelial Growth Factor) activity through the p38/MAPK pathway may attenuate drug resistance in breast cancer(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Additionally, several p38/MAPK pathway inhibitors have been shown to inhibit the progression of prostate cancer(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) and head and neck squamous cell carcinoma(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Our study confirmed that SNH not only inactivated the p38/MAPK pathway but also inhibited tumor angiogenesis. Collectively, these results suggest that SNH inhibits tumor angiogenesis by downregulating FGF1 expression via the p38/MAPK pathway, thereby suppressing pancreatic cancer progression.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur work reveals SNH inhibits the proliferation and invasive abilities of PANC-1 cells, induces mitochondrial damage and apoptosis, and suppresses the growth of pancreatic tumors in vivo. Mechanistically, SNH inhibits the expression of FGF1 and inactivates the p38/MAPK pathway, thereby inhibiting tumor angiogenesis. Based on our findings, it can be concluded that SNH possesses potential application as a therapeutic agent against pancreatic cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eCompeting interest\u0026nbsp;\u003c/em\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\n\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Review Committee of Animal Experimentation of Yunnan University (No. YNU20241109).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eXin Yu performed the experiments, analyzed and interpreted the data, and wrote the paper. Lihong Jiang supervised the study, provided important advice, and received financial support. Xiaoyu Yang participated in the study design, performed bioinformatics analysis and received financial support.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Science and Technology plan project of the First People’s Hospital of Yunnan Province (Project number: KHBS-2022-024), the Yunnan Science and Technology Commission of the Yunnan Provincial Science and Technology Department and Kunming Medical University (Project number: 202401AY070001-123), and Yunnan Provincial Key Laboratory for Innovative Application of Traditional Chinese Medicine (Project number: 202205AG070005).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-Seq data generated in this study have been deposited in the National Center for Biotechnology Information (NCBI) resource under accession number PRJNA1201330 (PANC-1 cells) and PRJNA1202402 (mouse tumor tissues). 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Oncol. 2013;\u003cb\u003e8\u003c/b\u003e(1)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"pancreatic cancer, sodium new houttuyfonate, FGF1, angiogenesis, p38/MAPK","lastPublishedDoi":"10.21203/rs.3.rs-5756684/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5756684/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eSodium new houttuyfonate (SNH) is derived from \u003cem\u003eHouttuynia cordata\u003c/em\u003e Thunb. (HCT), which is a famous edible and medicinal plant in China. Although research findings suggest that SNH possesses anticancer properties, its role and mechanisms in pancreatic cancer remain unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe investigated the effect of SNH on the malignant pancreatic cancer cell line PANC-1 using cell counting kit-8 (CCK-8), colony formation, apoptosis assays, transwell migration and electron microscopy. Additionally, we generated a mouse xenograft model to verify the potential anticancer effect of SNH in vivo. Moreover, RNA sequencing, immunohistochemical assay, and western blotting were performed on tumor cells and tissues to elucidate the potential regulatory pathways of SNH in pancreatic cancer.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSNH treatment suppressed the proliferation and invasive abilities of pancreatic cancer cells (PANC-1 and SW1990). Additionally, SNH treatment significantly downregulated Fibroblast Growth Factor 1 (FGF1) expression in tumor tissues and cell lines. In vivo and in vitro experiments confirmed that SNH inhibited pancreatic cancer cell proliferation and promoted apoptosis, thereby inhibiting tumor progression. Mechanistically, SNH downregulated FGF1 expression and inactivated the p38/MAPK (mitogen-activated protein kinase) pathway, thereby reducing tumor angiogenesis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur study revealed the anticancer effect of SNH in pancreatic cancer and provides a potentially effective strategy for the treatment of pancreatic cancer.\u003c/p\u003e","manuscriptTitle":"Sodium new houttuyfonate inhibits pancreatic cancer by suppressing the MAPK pathway and tumor angiogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-08 16:56:49","doi":"10.21203/rs.3.rs-5756684/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d867ec1-dd75-4730-ae14-c0396e3cd157","owner":[],"postedDate":"January 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-09T03:23:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-08 16:56:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5756684","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5756684","identity":"rs-5756684","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}