Alantolactone inhibits T-cell lymphoma progression by suppressing CD47 expression via the PI3K/AKT and ERK signaling pathways

Alantolactone inhibits T-cell lymphoma progression by suppressing CD47 expression via the PI3K/AKT and ERK signaling pathways | 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 Alantolactone inhibits T-cell lymphoma progression by suppressing CD47 expression via the PI3K/AKT and ERK signaling pathways Xiaodong Li, Ningbo Pang, Yingcong Chen, Tingting Pan, Wenwen Sun, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8529840/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 16 You are reading this latest preprint version Abstract T-cell lymphoma (TCL) is a malignant tumor caused by abnormal proliferation of T cells, and its specific pathogenesis remains unclear. Currently, there is still a lack of highly effective therapeutic drugs in clinic. As a semi-terpene lactone compound, alantolactone (ATL) is mainly used to treat diseases such as asthma, and its role in TCL has not been revealed. This study demonstrated that ATL not only significantly inhibits the proliferation and migration of TCL cells and induces apoptosis in vitro, but also exhibits significant anti-tumor effects in tumor-bearing mice. Meanwhile, ATL can markedly enhance the sensitivity of TCL cells to chemotherapeutic drugs such as decitabine. Mechanistically, multi-omics analysis confirmed that ATL restricts TCL progression by negatively regulating the PI3K/AKT and ERK signaling pathway. Furthermore, our study also found that ATL-mediated suppression of these pathways leads to significant downregulation of CD47 expression. In summary, this study is the first to elucidate that ATL exhibits significant anti-TCL effects both in vitro and in vivo, suggesting its potential as a novel clinical strategy for the treatment of TCL. T-cell lymphoma Alantolactone Decitabine CD47 PI3K/AKT ERK Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction T-cell lymphoma (TCL) is a subtype of non-Hodgkin lymphoma caused by malignant proliferation of T cells[ 1 , 2 ]. The disease progresses rapidly and is highly aggressive, with generally poor patient prognosis and a five-year survival rate of only about 20%[ 3 , 4 ]. The pathogenesis of TCL remains incompletely elucidated, and is generally attributed to various factors such as genetic mutations, chemicals, and infection [ 5 – 7 ]. Due to the atypical early symptoms and lack of specific markers, some patients are easily misdiagnosed, thereby missing the optimal treatment period. Currently, multi-drug combination regimens are the primary clinical approach for treating TCL[ 8 , 9 ]. For instance, Makoto Yoshimitsu et al. reported that Mogamulizumab combined with the CHOP regimen can significantly improve progression-free survival (PFS) in patients with adult T-cell leukemia/lymphoma, potentially emerging as a new first-line treatment option[ 10 ]. However, for patients with relapsed or advanced TCL, multi-drug resistance is common, and existing therapeutic drugs face efficacy limitations. Therefore, further elucidating the pathogenesis of TCL and developing novel therapeutic strategies are urgently needed. Currently, the search for effective and safe therapeutic agents represents a prominent focus in cancer research. However, the development of new drugs not only involves high time and cost, but also has great uncertainty. Drug retargeting aims to explore the novel pharmacological functions of existing drugs[ 11 , 12 ]. Certain agents originally intended for non-cancer therapies have shown promising antitumor activity in clinical studies. Given that these agents have been used in clinical practice with a well-established safety profile in both preclinical and clinical settings, they represent potential candidates for antitumor therapy. Elucidating novel targets and mechanisms of existing drugs can unlock their untapped therapeutic potential, thereby providing patients with additional treatment options. Alantolactone (ALT) is a sesquiterpene lactone extracted from elecampane, which exhibits broad-spectrum inhibitory effects against pathogens such as viruses, bacteria, and fungi[ 13 – 15 ]. In clinical practice, ATL has been widely used in the treatment of inflammatory, infectious, and asthmatic diseases, significantly improving clinical management strategies for related conditions[ 16 ]. For instance, studies by Jiong Wang et al. demonstrated that ATL exerts a protective effect against alcoholic fatty liver disease by blocking high-fat diet-induced inflammatory responses and oxidative stress[ 17 ]. Moreover, from the perspective of drug repurposing, researchers continue to uncover new pharmacological potentials of ATL. For example, Zhiwen Fu et al. reported that ATL effectively suppresses the malignant proliferation and migration of non-small-cell lung cancer cells by inhibiting the activity of aldo-keto reductase family 1 member C1 (AKR1C1)[ 18 ]. However, the role of ATL in TCL progression has yet to be reported, and the underlying mechanisms remain a mystery. CD47 is an immunoglobulin widely expressed on the surface of various tumor cells[ 19 , 20 ]. It binds to signal regulatory protein α (SIRPα), thereby helping tumor cells evade immune clearance by phagocytes, which is one of the reasons for the impaired immune response in cancer patients[ 21 , 22 ]. Recent studies have shown that high expression of CD47 is closely associated with poor prognosis in cancer patients[ 23 ]. In this context, several monoclonal antibodies targeting CD47 have entered clinical trials for the treatment of various lymphomas and other diseases, demonstrating the potential to significantly improve patient outcomes. In the field of basic research, Hiroaki Kamijo et al. discovered that CD47 is highly expressed in cutaneous T-cell lymphoma (CTCL), and the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) was found to promote the proliferation of CTCL cells, while CD47 monoclonal antibodies significantly reversed the proliferative effect of TSP-1 on CTCL[ 24 ]. These findings suggest that CD47 mediates TSP-1-induced malignant progression of CTCL. However, the role of CD47 in the regulation of TCL progression by ATL remains unclear. The PI3K/AKT and ERK signaling pathways are critical regulatory networks in tumor cells, extensively involved in key biological processes such as cell proliferation, migration, and apoptosis[ 25 – 28 ]. In-depth exploration of the regulatory mechanisms of these signaling pathways not only helps elucidate the pathogenesis of diseases, but also provides potential directions for developing new clinical treatment strategies. Our previous studies have confirmed that the AKT/mTOR pathway is involved in the growth and migration of TCL cells and serves as a target for antitumor drugs such as maprotiline[ 29 ]. Given the current challenges in TCL, including unclear pathogenic mechanisms and a lack of highly effective therapeutic drugs, this research focuses on investigating the specific role of ATL in TCL progression and its molecular mechanisms, aiming to provide novel targeted drug options for the clinical treatment. In this study, it was found for the first time that ATL downregulates the expression of CD47 by modulating the PI3K/AKT and ERK signaling axes, thereby inhibiting the malignant progression of TCL. In a xenograft mouse model, ATL also demonstrated highly efficient therapeutic effects on TCL, with good safety and no obvious drug side effects. Furthermore, the combination of ATL with chemotherapeutic drugs such as decitabine demonstrates a synergistic inhibitory effect on TCL growth both in vitro and in vivo. These findings not only expand the understanding of ATL's pharmacological functions but also offer new insights and strategies for the clinical treatment of TCL. 2. Materials and Methods 2.1. Cell lines and culture The human TCL cell lines (Jurkat, H9, Hut-78, and HH) used in this study were purchased from the American Type Culture Collection (ATCC). All cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum in a cell incubator at 37°C, 95% air and 5% CO2. 2.2. Reagents and Materials Alantolactone, Decitabine, Belinostat, Z-VAD-FMK, FR180204, Chidamide, MK2206, SC79 were purchased from Selleck (Shanghai, China). C16-PAF were purchased from MCE (Monmouth Junction, NJ, USA). Protease and phosphatase inhibitors (78442) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The primary antibodies including AKT (4691), phospho-AKT (p-AKT) (4060), ERK (4695), phospho-ERK (4370), caspase-3 (9662), phospho-H2A.X (γ-H2A.X) (60566), cleaved caspase-3 (9664), cleaved PARP (5625), CD47(63000), and GAPDH (5174) were purchased from Cell Signaling Technology (Massachusetts, USA). The CD47 monoclonal antibodies for the cell viability assay (sc-12730) were purchased from Santa Cruz (Dallas, Texas, USA), while those used for immunofluorescence staining (ab284132) were obtained from abcam (Cambridge, UK). The secondary antibodies were purchased from Huabio (Hangzhou, China). 2.3. Cell viability Assay For cell viability assay, TCL cells were seeded into 96-well plates at a density of 8×10³ cells per well and treated with varying concentrations of ATL or other reagents. Following incubation for the indicated time, cell viability was assessed using a CCK-8 kit (Yeasen Biotechnology, Shanghai, China). The absorbance of each well was read at 450 nm using a microplate spectrophotometer (BioTek Instruments Winooski, VT, USA). 2.4. Cell migration Assay Cell migration ability was detected using Transwell chambers (Corning, NY, USA). Briefly, serum-free medium containing 2 × 10⁵ TCL cells was added to the upper chamber, while the lower chamber was filled with RPMI 1640 medium supplemented with different reagents. After cultured at 37°C for 24 h, the upper chamber was carefully removed, and the culture medium in the plate was gently mixed. The migrated cells in the lower chamber were then collected and quantified. 2.5. Flow cytometric Analysis TCL cells were seeded in plates and exposed to varying concentrations of different agents for 48 h. For cell apoptosis analysis, TCL cells were stained with Annexin V-APC and 7-AAD (Liankebio, Hangzhou, China) in the dark at room temperature for 10 min. Then, TCL cells were quantified by flow cytometry using a FACSCalibur system (BD Biosciences, San Jose, CA, USA). 2.6. RNA sequencing and analysis Total RNA was extracted from TCL cells using Trizol reagentt (Thermo Fisher Scientific, Waltham, MA, USA). RNA sequencing (RNA-seq) was performed by Novogene Bioinformatics Technology Co, Ltd (Beijing, China). The sequencing data were analyzed with the DEseq2 software, and genes with a fold change in transcript expression ≥ 1.5 and a P-value < 0.05 were identified as differentially expressed genes (DEGs). 2.7. Western blot Analysis Total proteins were extracted from TCL cells using M-PER buffer (Thermo Fisher, Waltham, MA, USA) supplemented with protease and phosphatase inhibitors (100×) (Thermo Scientific, Waltham, MA, USA) for 10 min on ice. The lysates were centrifuged at 14000 g at 4°C to harvest protein extracts and quantified using a BCA protein assay kit (Thermo Fisher, Waltham, USA). Protein samples from each group were then separated by 10% SDS-PAGE and subsequently transferred onto nitrocellulose (NC) membranes. 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (1×TBST) was used to block the membranes for 1h at room temperature, then incubated with primary antibodies at 4°C overnight. After washed with 1× TBST for three times, the HRP-conjugated secondary antibodies were used to incubate the membranes for 1 h at room temperature. Following another three times washed with 1× TBST, protein bands were visualized using a chemiluminescence imaging system (Azure Biosystems, Dublin, OH, USA) with a chemiluminescence Western blot kit (Thermo Fisher Scientific, Waltham, MA, USA). 2.8. Immunofluorescence (IF) experiment TCL cells were harvested, washed twice with PBS buffer, and resuspended in PBS buffer at a concentration of 1×10⁶ cells/mL. Centrifuge cell suspension onto a slide. After fixed with 4% paraformaldehyde for 20 min at room temperature, the cells were washed three times with PBS buffer. Permeabilization was performed by treating the slides with 0.1% Triton X-100 for 5 min, and then washed again with PBS buffer. Next, the samples were blocked with 5% BSA for 30 min at room temperature, followed by washing with PBS buffer. The samples were incubated with primary antibody at 4°C overnight. After washed with PBS buffer, the samples were incubated with secondary antibody in the dark for 1 h at room temperature. Washed with PBS buffer, the samples were then treated with DAPI-containing mounting medium and incubated for 5 min at room temperature. Finally, the samples were covered with a coverslip and observed under a fluorescence microscope. 2.9. Animal experiments Five-week-old female NSG mice were obtained from GemPharmatech (Nanjing, China). All animals were raised under specific pathogen-free (SPF) conditions in a light/dark cycle-and temperature-controlled environment, and provided with sufficient food and water. To establish a TCL xenograft model, 4×10 6 Jurkat cells suspended in 150 µL PBS were injected subcutaneously into the flanks of NSG mice. When the tumor volume reached approximately 100 mm³, the mice were intraperitoneally injected with different agents every two days. The tumor volume (mm 3 ) was measured with a vernier caliper and calculated as length × width 2 /2, while changes in body weight were recorded simultaneously. At the experimental endpoint, peripheral blood was collected from mice and analyzed with a Sysmex XN-1000 hematology analyzer for blood cell counts. Serum was separated and subjected to biochemical parameter analysis using a HITACHI automated biochemistry analyzer. The tumor-bearing mice were then euthanized, tumor tissues and organs (such as liver, kidneys and lungs) were harvested for immunohistochemical (IHC) analysis and hematoxylin and eosin (H&E) staining according to the manufacturer’s instructions (Haokebio, Hangzhou, China). Additionally, the expression of signaling pathway proteins was analyzed in tumor tissues. All animal procedures were approved by the Institution’s Ethics Committee. 2.10. Statistical analysis Statistical analysis was performed using GraphPad Prism software (San Diego, CA, USA). All data are presented as mean ± standard error of the mean (SEM), and group comparisons were conducted using two-tailed t-tests. p-value of less than 0.05 (p < 0.05) was considered statistically significant. *represents p < 0.05, **represents p < 0.01, and ***represents p < 0.001. ns indicates no significance. 3. Results 3.1. Alantolactone exerts anti-TCL effects both in vitro and in vivo Alantolactone (ATL), a sesquiterpene lactone derived from Agarwood, exhibits multiple pharmacological properties including deworming, antibacterial, anti-inflammatory, and hepatoprotective effects. In this study, we aim to investigate the regulatory role of ATL in T-cell lymphoma (TCL). As illustrated in Fig 1A–B, ATL treatment markedly suppresses the proliferation and migration capacities of TCL cells. To further evaluate the in vivo efficacy of ATL, a xenograft tumor model was established by subcutaneously injecting Jurkat cells into NSG mice (Fig. 1C). As shown in Fig 1D, administration of ATL significantly inhibits the growth of transplanted tumors in mice. Additionally, it was found that both tumor size and weight in the ATL-treated group were markedly reduced compared to the control group (Fig. 1E–F). IHC analysis revealed that ATL significantly reduces the levels of Ki67, indicating a marked inhibitory effect on the malignant progression of transplanted tumors in mice (Fig 1G). During the experimental period, no significant differences in body weight was observed between the control and ATL-treated group (Fig. 1H). Serum biochemical analysis further indicated that ATL has no significant effect on the levels of alanine transaminase (ALT), aspartate transaminase (AST), creatinine (CREA), and uric acid (UA), suggesting no obvious adverse effects on the metabolic functions of the liver and kidney (Fig. 1I-L). Meanwhile, the results of H&E staining showed that ATL treatment did not cause significant morphological changes in the liver, kidney and lung tissues of the mice (Fig. 1M). The results of animal experiments demonstrated that while effectively suppressing TCL, ATL does not exhibit significant toxicity in vivo and possesses a favorable safety profile. Collectively, these findings provide preliminary evidence that ATL exerts novel anti-TCL pharmacological activities both in vitro and in vivo. 2. The Caspase-3-dependent pathway mediates alantolactone-induced apoptosis of TCL cells To further investigate whether ATL induces apoptosis in TCL cells, we evaluated the apoptosis rate and the levels of relevant molecular markers. As key executors in the regulation of apoptosis, the cleavage of PARP and caspase-3 represents a critical step in initiating the apoptotic process in tumor cells. It was demonstrated that ATL treatment markedly enhances the cleavage of both caspase-3 and PARP in a dose-dependent manner (Fig. 2A). Additionally, the level of phosphorylated H2A.X (γ-H2A.X), a marker for DNA damage and apoptosis was also observed to increase in a dose-dependent manner following ATL treatment (Fig. 2B). Subsequently, we conducted RNA-seq on the TCL cells to analyze the differentially expressed genes (DEGs) after ATL treatment. The results of GO (Gene Ontology) analysis further showed that the differential genes were enriched in biological processes such as cell growth, migration and apoptosis (Fig. 2C). Furthermore, the pan-caspase inhibitor Z-VAD-FMK significantly reverses the anti-proliferative effect of ATL on TCL cells (Fig. 2D) and attenuates ATL-induced cell apoptosis (Fig. 2E-F). Taken together, these findings indicate that ATL triggers apoptosis and DNA damage in TCL cells. 3. The PI3K/AKT and ERK signaling axis mediates the anti-TCL effect of Alantolactone To further elucidate the molecular mechanism by which ATL regulates the TCL progression, we performed KEGG enrichment analysis on differentially expressed genes (DEGs). As illustrated in the Fig. 3A, DEGs were enriched in PI3K/AKT and MAPK pathways, both of which are closely associated with tumorigenesis. Western blot analysis further confirmed that ATL treatment markedly reduced the levels of p-AKT and p-ERK without affecting total AKT and ERK levels in TCL cells (Fig. 3B). In contrast, the phosphorylation and total protein levels of p38 and JNK1/2, two other pathways downstream of MAPK, were not significantly changed (Fig. 3C). These findings were consistently corroborated in tumor tissues in mice (Fig. 3D). Similarly, IHC analysis revealed substantially lower expression of p-AKT and p-ERK in ATL-treated groups compared with controls (Fig. 3E). Moreover, the AKT activator SC79 significantly attenuated the suppressive effects of ATL on both cell growth (Fig. 3F-G) and PI3K/AKT signaling pathway (Fig. 3H) in TCL. Likewise, the ERK activator C16-PAF also largely reversed the inhibitory effect of ATL in TCL cells (Fig. 3I-K). Collectively, these results indicated that the PI3K/AKT and ERK signaling pathways mediate the inhibitory effects of ATL on TCL both in vitro and in vivo. 4. Alantolactone reduces the expression level of CD47 via PI3K/AKT and ERK axis in TCL cells CD47 is highly expressed in malignant tumors such as TCL and is closely associated with disease progression. As shown in Fig. 4A, we found that CD47 monoclonal antibody significantly inhibits the proliferation of TCL cells. Further analysis revealed that ALT markedly downregulates CD47 expression in TCL cells, suggesting that CD47 serves as one of the targets through which ALT exerts its antitumor effects (Fig. 4B-C). Given that the PI3K/AKT and ERK signaling axis mediates ATL-induced inhibition of TCL, we further investigated the molecular mechanism by which ALT regulates CD47 expression. Subsequently, we observed that specific inhibition of either PI3K/AKT (Fig. 4D-E) or ERK (Fig. 4F-G) signaling pathway significantly suppresses both TCL cell proliferation and CD47 expression. This suppressive effect was reversed by corresponding activators of PI3K/AKT and ERK signaling pathways, which restored CD47 levels in the presence of ALT (Fig. 4H-I). These results confirmed that ALT downregulates CD47 via the PI3K/AKT and ERK signaling pathways in TCL cells. 5. Alantolactone exerts synergistic effects combined with clinical chemotherapy agents in TCL Drug resistance poses a major challenge in first-line cancer therapy, often leading to disease relapse or malignant progression. Currently, combination therapies employing agents with different mechanisms of action have shown considerable promise in clinical practice. Decitabine, a first-line treatment for TCL, has garnered significant interest for its potential synergistic effects when used in combination with other agents. To explore whether ATL and decitabine exert synergistic antitumor activity, TCL cells were treated with ATL, decitabine, or the combination of both. As shown in Fig. 5A, the combination of ATL and decitabine markedly inhibits TCL cell proliferation compared to monotherapy with either agent alone. Moreover, ATL also demonstrates significant synergistic inhibition of TCL cell proliferation when used in combination with the chemotherapeutic agents Belinostat and Chidamide (Fig. 5B-C). Meanwhile, the combination of ATL and decitabine significantly increases the apoptotic rates and markedly upregulates the expression of apoptosis-related proteins in TCL cells compared to monotherapy with either agent alone (Fig. 5D-E). Mechanistically, it was found that these combinations notably suppress both the PI3K/AKT and ERK signaling pathways in TCL cells (Fig. 5F). These in vitro experiments indicate that ATL exerts synergistic effects combined with clinical chemotherapy agents such as decitabine in TCL. Additionally, to further investigate the antitumor effects and underlying mechanisms of the combination of ATL (10 mg/kg) and decitabine (0.4 mg/kg), we also conducted in vivo experiments in tumor‑bearing mice (Fig. 6A). The results showed that the combination of the two agents significantly reduced tumor weight in mice compared to the single-agent group (Fig. 6B). Consistent with the in vitro experimental results, Western blot analysis revealed that the combined treatment significantly inhibited both the PI3K/AKT and ERK signaling pathways. (Fig. 6C). In terms of safety, no significant decrease in body weight of mice was observed throughout the administration period (Fig. 6D). It was also revealed that the combination therapy did not significantly affect the levels of ALT, AST, CREA, and UA in mice, and no notable morphological changes were detected in the liver, kidney, or lung tissues, suggesting that this combined regimen exhibits high antitumor efficacy without significant toxicity (Fig. 6E-I). Together, these findings indicate that the combination of ATL and decitabine holds potential as a candidate first-line treatment option for TCL. 4. Discussion Although T-cell lymphoma (TCL) accounts for a relatively low proportion among all tumor types, its incidence rate has been increasing year by year, imposing a heavy burden on society and patients' family. The pathogenesis of TCL is complex, and no specific biomarkers have been identified, which severely limits early diagnosis and development of targeted drugs[ 30 ]. Although first-line chemotherapy agents demonstrate a relatively high rate of cure and remission, their effectiveness remains limited overall due to several challenges, including the adverse reactions and side effects associated with the drugs themselves, as well as the strong heterogeneity of TCL and its tendency to develop drug resistance. Consequently, current treatment strategies still fall short of fully meeting clinical needs. Therefore, elucidating the pathogenesis of TCL and developing novel effective drugs have become urgent tasks to address the current challenges in clinical treatment. Benefiting from its multi-target effects and low toxicity, the traditional Chinese medicine alantolactone (ALT) is now widely used in clinical practice for the treatment of diseases such as cancer[ 31 , 32 ]. With the application of new technologies such as metabolomics, researchers are attempting to reveal novel pharmacological functions of ATL from multiple dimensions. Currently, the regulation of TCL by ATL remains uninvestigated. Here, we reported for the first time that ATL markedly inhibits TCL cell growth in vitro and in vivo, thereby revealing its anti-tumor potential in this study. Notably, during drug administration in tumor-bearing mice, no toxic reactions such as weight loss, liver, kidney or lung damage, or hematopoietic system injury were observed, suggesting that ATL possesses a favorable safety profile while exerting its anti-TCL effects. Additionally, no metastasis of tumor lesions to vital organs such as the lungs or liver was observed, which may be related to the construction method of constructing the transplanted tumor model by subcutaneous inoculation rather than tail vein injection, and the relevant molecular mechanisms requires further investigation. TCL is a malignant tumor originating from the lymphatic system, and the occurrence of distant metastasis markedly elevates disease malignancy, often resulting in a dismal prognosis. Therefore, understanding the metastatic characteristics of TCL is crucial for developing individualized treatment strategies. In cell model experiments, we observed that ATL significantly inhibits the migratory ability of TCL cells. Given the complexity and interindividual variability of TCL metastasis, further studies using diverse cellular and animal models are warranted to elucidate the role of ATL in this process. Inducing apoptosis is one of the key mechanisms by which many chemotherapy drugs exert anti-tumor effects[ 33 , 34 ]. In this study, we found that ATL significantly promotes apoptosis in TCL cells. Specifically, ATL-induced apoptosis is closely related to the activation of caspase and PARP, and the broad-spectrum caspase inhibitor Z-VAD-FMK can significantly reverse the pro-apoptotic effect of ATL on TCL cells. In summary, this study confirmed that ATL has significant anti-TCL activity through in vivo and in vitro experiments, providing a strong basis for its further development as a clinical candidate drug. On this basis, we plan to conduct a series of experiments to deeply analyze the specific molecular mechanisms of ATL against TCL. In terms of molecular mechanism exploration, we performed RNA-seq sequencing to systematically screen differentially expressed genes (DEGs) in TCL cells after ATL treatment. KEGG pathway enrichment analysis of DEGs revealed that the PI3K/AKT and MAPK signaling pathways may be involved in the biological activity of ATL. Multi-omics analysis in cells and mice models showed that ATL inhibits the progression of TCL by negatively regulating PI3K/AKT and ERK signaling axes. It was known that abnormal activation of the PI3K/AKT and ERK pathways is closely associated with the development and progression of various malignant tumors, involving processes such as cell proliferation, drug resistance, and migration. Therefore, these pathways are considered important potential targets for cancer therapy. Several small-molecule drugs targeting these pathways have entered clinical trials and showed promising prospects. For example, Carlos Fernandez Teruel et al. reported that the AKT inhibitor capivasertib combined with fulvestrant demonstrates good efficacy in breast cancer patients[ 35 ]. Romain Sigaud et al. confirmed that the ERK inhibitor ulixertinib exhibits significant activity in low-grade gliomas[ 35 ]. Further, rescue experiments indicated that reactivating PI3K/AKT or ERK signaling significantly reverse the regulatory effects of ATL on TCL cell proliferation and apoptosis. These results collectively suggest that the PI3K/AKT and ERK signaling axes play a critical role in ATL-mediated regulation of TCL development, supporting their value as potential therapeutic targets for TCL. Multiple studies have confirmed the close relationship between the immune system and tumor development, and anti-tumor immunotherapy has shown promising therapeutic prospects[ 36 ]. In this study, we found that anti-CD47 monoclonal antibodies significantly inhibits TCL cell proliferation. CD47, as an important "don't eat me" signal molecule, is highly expressed on the surface of various tumor cells, including TCL[ 37 – 40 ]. By binding to its ligand SIRPα on immune cells such as macrophages, CD47 inhibits phagocytosis, thereby mediating immune escape. Our results further indicated that ATL downregulates CD47 expression levels by negatively regulating the PI3K/AKT and ERK signaling pathways, while activators of PI3K/AKT and ERK signaling axes can effectively reverse the inhibitory effect of ATL on CD47, respectively. These findings underscore the pivotal role of ATL in TCL immunotherapy by reducing CD47 expression, highlighting the need for further experimental research to assess its clinical application value. Although single-drug therapy can temporarily delay the malignant progression of tumors, long-term use often leads to the development of drug resistance, which has become one of the major reasons for the failure of tumor treatment. The mechanisms of drug resistance are complex and diverse, primarily involving factors such as alterations in drug targets, enhanced DNA damage repair capacity, and epigenetic changes[ 41 – 43 ]. Therefore, combination therapy has emerged as a promising strategy in current cancer research and plays a crucial role in enhancing clinical outcomes. Taking the classic CHOP regimen for treating TCL as an example, its success demonstrates the significant value of combination therapy in achieving long-term disease control[ 44 ]. As a DNA methyltransferase inhibitor, decitabine can effectively induce apoptosis and plays a crucial role in the first-line treatment of TCL[ 45 ]. This study showed that the combination of ATL and decitabine synergistically inhibits the activation of the PI3K/AKT and ERK signaling pathways in both in vitro and in vivo, demonstrating significant inhibitory effects on TCL growth without observable notable drug toxicity in mouse models. It indicates that this combination regimen can intervene in TCL progression and possesses favorable safety, providing novel targeted therapy strategies for clinical first-line treatment. In summary, this study is the first to demonstrate that ATL effectively inhibits the malignant progression of TCL in vitro and in vivo, and significantly enhances the sensitivity of TCL to first-line chemotherapeutic drugs such as decitabine. Mechanically, we found that ATL suppresses TCL by negatively regulating the PI3K/AKT and ERK signaling pathways and downregulating CD47 protein expression. These findings provide a solid experimental basis for ATL as a candidate agent for TCL treatment, offering a potential new strategy to improve the current therapeutic landscape. Declarations Acknowledgments We gratefully acknowledge the technical and experimental support from all team members. Author Contributions Validation and investigation: Xiaodong Li, Yinyu Mu, and Ni Li. Analyzed the data: Xiaodong Li and Yinyu Mu. Writing: Xiaodong Li and Ni Li. Resources: Bingrong Chen, Wenwen Sun and Fuyi Xie. Methodology: Ningbo Pang, Yingcong Chen, and Tingting Pan. Supervision: Fuyi Xie and Ni Li. Funding: Xiaodong Li, Tingting Pan and Ni Li. All authors have reviewed and agreed to the publication of this manuscript. Funding This work was supported by grants from the Zhejiang Province Health industry Science and Technology Plan (2025HY0868), Ningbo Municipal Program for Innovative and Leading Talents (2024QL025), and the Natural Science Foundation of Ningbo (2024J337). Data Availability Statement: No datasets were generated or analysed during the current study. Conflicts of Interest: The authors declare no conflict of interest. Fig 1C, Fig 6A, and Fig 7 were drawn using BioGDP. 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Meng, M., et al., Efficacy and mechanism of the XPO1 inhibitor selinexor combined with decitabine in T-cell lymphoblastic lymphoma. Ann Hematol, 2025. 104 (3): p. 1747-1756. Additional Declarations No competing interests reported. Supplementary Files ATLSupplementarymaterials.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 13 Jan, 2026 Reviews received at journal 13 Jan, 2026 Reviews received at journal 12 Jan, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviews received at journal 11 Jan, 2026 Reviewers agreed at journal 10 Jan, 2026 Reviewers agreed at journal 09 Jan, 2026 Reviewers agreed at journal 07 Jan, 2026 Reviewers agreed at journal 07 Jan, 2026 Reviewers agreed at journal 07 Jan, 2026 Reviewers invited by journal 07 Jan, 2026 Editor assigned by journal 07 Jan, 2026 Submission checks completed at journal 07 Jan, 2026 First submitted to journal 06 Jan, 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. 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11:12:56","extension":"png","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":42724,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8529840/v1/683efef326de86bd11748d93.png"},{"id":99880680,"identity":"71e7065f-2737-449a-a88d-d6dba8a2126b","added_by":"auto","created_at":"2026-01-09 11:12:55","extension":"xml","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":98551,"visible":true,"origin":"","legend":"","description":"","filename":"0671bfe4dfb74a1da699cfc95b5633201structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8529840/v1/4c03e4c696bf9089b3ef7c5d.xml"},{"id":99880686,"identity":"3bc073c4-58be-4066-9c8a-f49a750337f7","added_by":"auto","created_at":"2026-01-09 11:12:55","extension":"html","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109966,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8529840/v1/99103564426f9d5946cf3ed3.html"},{"id":99880645,"identity":"8d845968-8e54-4818-9743-8b2ba0a73ada","added_by":"auto","created_at":"2026-01-09 11:12:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3265131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATL exerts anti-TCL effects both in vitro and in vivo. \u0026nbsp;A. \u003c/strong\u003eViability of TCL cells (Jurkat, H9, Hut-78, and HH) following treatment with varying concentrations of ATL for 96 hwas detected using the CCK-8 assay. \u003cstrong\u003eB.\u003c/strong\u003eTCL cells were treated with ATL for 24 h, and the migration capacity was determined by chamber migration assays. \u003cstrong\u003eC. \u003c/strong\u003eThe schematic diagram of ATL treatment for TCL xenograft mice. \u003cstrong\u003eD. \u003c/strong\u003eThe growth curve showed that ATL significantly suppresses the tumor growth in tumor-bearing mice. \u003cstrong\u003eE.\u003c/strong\u003eRepresentative images of xenograft tumors from the control and ATL-treated groups. \u003cstrong\u003eF. \u003c/strong\u003eComparison of tumor weights between the two groups. \u003cstrong\u003eG. \u003c/strong\u003eThe results of IHC staining showed that ATL significantly reduces the levels of Ki 67 in tumor tissues. Scale bars, 100 μm. \u003cstrong\u003eH. \u003c/strong\u003eThe body weight change curve of mice during ATL treatment. \u003cstrong\u003eI-L.\u003c/strong\u003e The ALT (I), AST (J), CREA (K), and UA (L) levels in mice of control and ATL-treated groups. \u003cstrong\u003eM.\u003c/strong\u003e H\u0026amp;E analysis results of the liver, kidney and lung in mice. Scale bars, 100 μm. **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8529840/v1/e0f59f6c1af770cdfb2c0307.jpg"},{"id":99880644,"identity":"9cee7923-8365-443a-ad8a-98bed82bca7e","added_by":"auto","created_at":"2026-01-09 11:12:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3070008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATL induces apoptosis in TCL cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-B. TCL cells were treated with ATL for 48 h, the levels of cleaved caspase-3, caspase-3, cleaved PARP (A) and γ-H2A.X (B) were analyzed by Western blot. C. The data of RNA-seq were analyzed by GO analysis. D-E. TCL cells were pre-incubated with or without Z-VAD-FMK (50 μM) for 1 h, and then treated with ATL (4 µM). Cell viability was measured using the CCK-8 assay after 72 h (D), while the expression of cleaved caspase-3, caspase-3, and cleaved PARP was evaluated by Western blot at 48 h (E).\u003c/p\u003e\n\u003cp\u003eF. The results illustrated that Z-VAD-FMK (50 μM) effectively reverses ATL (2 µM)-induced apoptosis in TCL cells after 48 h. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8529840/v1/7d993cd2a3ef13e76108dc2e.jpg"},{"id":100358386,"identity":"36a78428-966f-4c46-9681-52e33c340281","added_by":"auto","created_at":"2026-01-16 07:21:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3035770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATL\u003c/strong\u003e \u003cstrong\u003eexerts an antitumor effect in TCL by inhibiting the PI3K/AKT and ERK signaling axis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eKEGG enrichment was performed on DEGs. \u003cstrong\u003eB. \u003c/strong\u003eWestern blot analysis of p-AKT, AKT, p-ERK, and ERK protein levels in TCL cells treated with ATL for 48h. \u003cstrong\u003eC.\u003c/strong\u003e It was showed that ATL did not significantly affect the levels of either phosphorylated or total p38 and JNK. \u003cstrong\u003eD. \u003c/strong\u003eThe expression levels of p-AKT, AKT, p-ERK, and ERK in tumor tissue from mice treated with vehicle or ATL was analyzed by Western blot. \u003cstrong\u003eE.\u003c/strong\u003e Representative images of IHC staining for p-ERK and p-AKT in tumor tissues from control and ATL-treated mice. Scale bars, 100 μm. \u003cstrong\u003eF-H. \u003c/strong\u003eTCL cells were pretreated with 8 μM AKT activator (SC79) for 1 h prior to ATL treatment (4 μM). Cell viability was determined by CCK-8 assay at 96 h \u003cstrong\u003e(F-G)\u003c/strong\u003e, and protein levels of p-AKT and AKT were assessed by Western blot at 48 h \u003cstrong\u003e(H)\u003c/strong\u003e. \u003cstrong\u003eI-K. \u003c/strong\u003eERK activator C16-PAF (2 µM) significantly reversed the inhibitory effect of ATL (4 μM) on cell viability at 96 h\u003cstrong\u003e (I-J) \u003c/strong\u003eand ERK signaling pathway\u003cstrong\u003e \u003c/strong\u003eat 48 h\u003cstrong\u003e (K)\u003c/strong\u003e. **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8529840/v1/c39fb36bb74ff6efdc8abc12.jpg"},{"id":99880646,"identity":"b839f4fc-4a19-46d4-ac4f-9de5d2140e5d","added_by":"auto","created_at":"2026-01-09 11:12:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2413250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATL reduces the expression level of CD47 via PI3K/AKT and ERK signaling axis in TCL cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Anti-CD47 antibody (5 μg/ml) significantly inhibits the proliferation of TCL cells at 72 h. \u003cstrong\u003eB-C.\u003c/strong\u003e TCL cells were treated with ATL for 48 h, the CD47 level was detected by Western blot \u003cstrong\u003e(B)\u003c/strong\u003e and immunofluorescence assay \u003cstrong\u003e(C)\u003c/strong\u003e. Scale bars, 50 μm.\u003cstrong\u003e D-E.\u003c/strong\u003eTCL cells were treated with MK2206 (an inhibitor of AKT), the cell viability was detected by CCK-8 at 72 h\u003cstrong\u003e (D)\u003c/strong\u003e, the levels of CD47, p-AKT, and AKT were assessed by Western blot at 48 h \u003cstrong\u003e(E)\u003c/strong\u003e. \u003cstrong\u003eF-G.\u003c/strong\u003e TCL cells were treated with FR180204 (an inhibitor of ERK), the cell viability was detected by CCK-8 at 72 h \u003cstrong\u003e(F)\u003c/strong\u003e, the levels of CD47, p-ERK, and ERK were assessed by Western blot at 48 h \u003cstrong\u003e(G)\u003c/strong\u003e. \u003cstrong\u003eH. \u003c/strong\u003ePretreatment of TCL cells with SC79 (8 µM), and then incubated with ATL (4 µM), the levels of CD47, p-AKT, and AKT were assessed by Western blot at 48 h. \u003cstrong\u003eI. \u003c/strong\u003eSimilarly, TCL cells were pretreated with C16-PAF (2 µM) followed by incubation with ATL (4 µM), while the expression levels of CD47, p-ERK, and ERK were evaluated by Western blot at 48 h. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8529840/v1/7c4430ba9296eb588863f36b.jpg"},{"id":100357807,"identity":"df2c7c5a-a374-4e64-be98-be0729cf15b5","added_by":"auto","created_at":"2026-01-16 07:20:22","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3449440,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATL exerts synergistic effects combined with clinical chemotherapy agents in TCL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eTCL cells were treated with 2 μM ATL, 0.5 μM decitabine, or the combination of both for 96 h, and cell viability was determined by CCK-8 assay. \u003cstrong\u003eB-C.\u003c/strong\u003eThe combination of 2 μM ATL with 0.2 µM Belinostat \u003cstrong\u003e(B)\u003c/strong\u003e or 0.5 µM Chidamide \u003cstrong\u003e(C)\u003c/strong\u003ealso synergistically inhibits the proliferation of TCL cells at 96 h. \u003cstrong\u003eD-F.\u003c/strong\u003e TCL cells were treated with 2 µM ATL, 0.5 µM decitabine, or the combination of both for 48 h, cell apoptosis was assessed by flow cytometry \u003cstrong\u003e(D)\u003c/strong\u003e, the levels of caspase-3, cleaved caspase-3, cleaved PARP and γ-H2A.X were analyzed by Western blot assay \u003cstrong\u003e(E)\u003c/strong\u003e, and the levels of p-AKT, AKT, p-ERK, and ERK were assessed by Western blot \u003cstrong\u003e(F)\u003c/strong\u003e. **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8529840/v1/7344ce99a671c956d8d37430.jpg"},{"id":99880655,"identity":"91596303-279b-4528-af8a-d067ebf5b86f","added_by":"auto","created_at":"2026-01-09 11:12:55","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5121140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATL exerts synergistic effects combined with clinical chemotherapy agents in TCL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eThe schematic diagram of the TCL xenograft mice experiments. \u003cstrong\u003eB.\u003c/strong\u003eThe tumor weight of mice in each group was analyzed. \u003cstrong\u003eC. \u003c/strong\u003eCombination of ATL and decitabine markedly suppresses the PI3K/AKT and ERK signaling pathways in tumors from mice. \u003cstrong\u003eD.\u003c/strong\u003e The body weight of mice was recorded. \u003cstrong\u003eE-H. \u003c/strong\u003eThe ALT \u003cstrong\u003e(E)\u003c/strong\u003e, AST\u003cstrong\u003e (F)\u003c/strong\u003e, CREA \u003cstrong\u003e(G)\u003c/strong\u003e, and UA \u003cstrong\u003e(H) \u003c/strong\u003elevels in mice of each group. \u003cstrong\u003eI.\u003c/strong\u003e Representative H\u0026amp;E staining of liver, kidney and lung of mice was shown. Scale bars, 100 μm. **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8529840/v1/154eeefaa1005390bec89a60.jpg"},{"id":99880651,"identity":"70acf76c-96dd-4999-94b6-f5700f690a08","added_by":"auto","created_at":"2026-01-09 11:12:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":174036,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram illustrating the anti-tumor mechanism of alantolactone in TCL\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8529840/v1/dd1846aa9c821189b4506e1e.png"},{"id":100546048,"identity":"0597d177-edb6-4470-b036-4ae754f1e931","added_by":"auto","created_at":"2026-01-19 07:37:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20974704,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8529840/v1/10691aed-d53e-4247-9537-0af7547fa144.pdf"},{"id":99880649,"identity":"a5e4c7f1-8ca3-458e-a844-705a022902b7","added_by":"auto","created_at":"2026-01-09 11:12:55","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":366037,"visible":true,"origin":"","legend":"","description":"","filename":"ATLSupplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8529840/v1/24b783f8f64e21c71003ea12.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Alantolactone inhibits T-cell lymphoma progression by suppressing CD47 expression via the PI3K/AKT and ERK signaling pathways","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eT-cell lymphoma (TCL) is a subtype of non-Hodgkin lymphoma caused by malignant proliferation of T cells[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The disease progresses rapidly and is highly aggressive, with generally poor patient prognosis and a five-year survival rate of only about 20%[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The pathogenesis of TCL remains incompletely elucidated, and is generally attributed to various factors such as genetic mutations, chemicals, and infection [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Due to the atypical early symptoms and lack of specific markers, some patients are easily misdiagnosed, thereby missing the optimal treatment period. Currently, multi-drug combination regimens are the primary clinical approach for treating TCL[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. For instance, Makoto Yoshimitsu et al. reported that Mogamulizumab combined with the CHOP regimen can significantly improve progression-free survival (PFS) in patients with adult T-cell leukemia/lymphoma, potentially emerging as a new first-line treatment option[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, for patients with relapsed or advanced TCL, multi-drug resistance is common, and existing therapeutic drugs face efficacy limitations. Therefore, further elucidating the pathogenesis of TCL and developing novel therapeutic strategies are urgently needed.\u003c/p\u003e \u003cp\u003eCurrently, the search for effective and safe therapeutic agents represents a prominent focus in cancer research. However, the development of new drugs not only involves high time and cost, but also has great uncertainty. Drug retargeting aims to explore the novel pharmacological functions of existing drugs[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Certain agents originally intended for non-cancer therapies have shown promising antitumor activity in clinical studies. Given that these agents have been used in clinical practice with a well-established safety profile in both preclinical and clinical settings, they represent potential candidates for antitumor therapy. Elucidating novel targets and mechanisms of existing drugs can unlock their untapped therapeutic potential, thereby providing patients with additional treatment options. Alantolactone (ALT) is a sesquiterpene lactone extracted from elecampane, which exhibits broad-spectrum inhibitory effects against pathogens such as viruses, bacteria, and fungi[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In clinical practice, ATL has been widely used in the treatment of inflammatory, infectious, and asthmatic diseases, significantly improving clinical management strategies for related conditions[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For instance, studies by Jiong Wang et al. demonstrated that ATL exerts a protective effect against alcoholic fatty liver disease by blocking high-fat diet-induced inflammatory responses and oxidative stress[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Moreover, from the perspective of drug repurposing, researchers continue to uncover new pharmacological potentials of ATL. For example, Zhiwen Fu et al. reported that ATL effectively suppresses the malignant proliferation and migration of non-small-cell lung cancer cells by inhibiting the activity of aldo-keto reductase family 1 member C1 (AKR1C1)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the role of ATL in TCL progression has yet to be reported, and the underlying mechanisms remain a mystery.\u003c/p\u003e \u003cp\u003eCD47 is an immunoglobulin widely expressed on the surface of various tumor cells[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. It binds to signal regulatory protein α (SIRPα), thereby helping tumor cells evade immune clearance by phagocytes, which is one of the reasons for the impaired immune response in cancer patients[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Recent studies have shown that high expression of CD47 is closely associated with poor prognosis in cancer patients[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this context, several monoclonal antibodies targeting CD47 have entered clinical trials for the treatment of various lymphomas and other diseases, demonstrating the potential to significantly improve patient outcomes. In the field of basic research, Hiroaki Kamijo et al. discovered that CD47 is highly expressed in cutaneous T-cell lymphoma (CTCL), and the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) was found to promote the proliferation of CTCL cells, while CD47 monoclonal antibodies significantly reversed the proliferative effect of TSP-1 on CTCL[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These findings suggest that CD47 mediates TSP-1-induced malignant progression of CTCL. However, the role of CD47 in the regulation of TCL progression by ATL remains unclear.\u003c/p\u003e \u003cp\u003eThe PI3K/AKT and ERK signaling pathways are critical regulatory networks in tumor cells, extensively involved in key biological processes such as cell proliferation, migration, and apoptosis[\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In-depth exploration of the regulatory mechanisms of these signaling pathways not only helps elucidate the pathogenesis of diseases, but also provides potential directions for developing new clinical treatment strategies. Our previous studies have confirmed that the AKT/mTOR pathway is involved in the growth and migration of TCL cells and serves as a target for antitumor drugs such as maprotiline[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Given the current challenges in TCL, including unclear pathogenic mechanisms and a lack of highly effective therapeutic drugs, this research focuses on investigating the specific role of ATL in TCL progression and its molecular mechanisms, aiming to provide novel targeted drug options for the clinical treatment. In this study, it was found for the first time that ATL downregulates the expression of CD47 by modulating the PI3K/AKT and ERK signaling axes, thereby inhibiting the malignant progression of TCL. In a xenograft mouse model, ATL also demonstrated highly efficient therapeutic effects on TCL, with good safety and no obvious drug side effects. Furthermore, the combination of ATL with chemotherapeutic drugs such as decitabine demonstrates a synergistic inhibitory effect on TCL growth both in vitro and in vivo. These findings not only expand the understanding of ATL's pharmacological functions but also offer new insights and strategies for the clinical treatment of TCL.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cell lines and culture\u003c/h2\u003e \u003cp\u003eThe human TCL cell lines (Jurkat, H9, Hut-78, and HH) used in this study were purchased from the American Type Culture Collection (ATCC). All cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum in a cell incubator at 37\u0026deg;C, 95% air and 5% CO2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Reagents and Materials\u003c/h2\u003e \u003cp\u003eAlantolactone, Decitabine, Belinostat, Z-VAD-FMK, FR180204, Chidamide, MK2206, SC79 were purchased from Selleck (Shanghai, China). C16-PAF were purchased from MCE (Monmouth Junction, NJ, USA). Protease and phosphatase inhibitors (78442) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The primary antibodies including AKT (4691), phospho-AKT (p-AKT) (4060), ERK (4695), phospho-ERK (4370), caspase-3 (9662), phospho-H2A.X (γ-H2A.X) (60566), cleaved caspase-3 (9664), cleaved PARP (5625), CD47(63000), and GAPDH (5174) were purchased from Cell Signaling Technology (Massachusetts, USA). The CD47 monoclonal antibodies for the cell viability assay (sc-12730) were purchased from Santa Cruz (Dallas, Texas, USA), while those used for immunofluorescence staining (ab284132) were obtained from abcam (Cambridge, UK). The secondary antibodies were purchased from Huabio (Hangzhou, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Cell viability Assay\u003c/h2\u003e \u003cp\u003eFor cell viability assay, TCL cells were seeded into 96-well plates at a density of 8\u0026times;10\u0026sup3; cells per well and treated with varying concentrations of ATL or other reagents. Following incubation for the indicated time, cell viability was assessed using a CCK-8 kit (Yeasen Biotechnology, Shanghai, China). The absorbance of each well was read at 450 nm using a microplate spectrophotometer (BioTek Instruments Winooski, VT, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cell migration Assay\u003c/h2\u003e \u003cp\u003eCell migration ability was detected using Transwell chambers (Corning, NY, USA). Briefly, serum-free medium containing 2 \u0026times; 10⁵ TCL cells was added to the upper chamber, while the lower chamber was filled with RPMI 1640 medium supplemented with different reagents. After cultured at 37\u0026deg;C for 24 h, the upper chamber was carefully removed, and the culture medium in the plate was gently mixed. The migrated cells in the lower chamber were then collected and quantified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Flow cytometric Analysis\u003c/h2\u003e \u003cp\u003eTCL cells were seeded in plates and exposed to varying concentrations of different agents for 48 h. For cell apoptosis analysis, TCL cells were stained with Annexin V-APC and 7-AAD (Liankebio, Hangzhou, China) in the dark at room temperature for 10 min. Then, TCL cells were quantified by flow cytometry using a FACSCalibur system (BD Biosciences, San Jose, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. RNA sequencing and analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from TCL cells using Trizol reagentt (Thermo Fisher Scientific, Waltham, MA, USA). RNA sequencing (RNA-seq) was performed by Novogene Bioinformatics Technology Co, Ltd (Beijing, China). The sequencing data were analyzed with the DEseq2 software, and genes with a fold change in transcript expression\u0026thinsp;\u0026ge;\u0026thinsp;1.5 and a P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were identified as differentially expressed genes (DEGs).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Western blot Analysis\u003c/h2\u003e \u003cp\u003eTotal proteins were extracted from TCL cells using M-PER buffer (Thermo Fisher, Waltham, MA, USA) supplemented with protease and phosphatase inhibitors (100\u0026times;) (Thermo Scientific, Waltham, MA, USA) for 10 min on ice. The lysates were centrifuged at 14000 g at 4\u0026deg;C to harvest protein extracts and quantified using a BCA protein assay kit (Thermo Fisher, Waltham, USA). Protein samples from each group were then separated by 10% SDS-PAGE and subsequently transferred onto nitrocellulose (NC) membranes. 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (1\u0026times;TBST) was used to block the membranes for 1h at room temperature, then incubated with primary antibodies at 4\u0026deg;C overnight. After washed with 1\u0026times; TBST for three times, the HRP-conjugated secondary antibodies were used to incubate the membranes for 1 h at room temperature. Following another three times washed with 1\u0026times; TBST, protein bands were visualized using a chemiluminescence imaging system (Azure Biosystems, Dublin, OH, USA) with a chemiluminescence Western blot kit (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Immunofluorescence (IF) experiment\u003c/h2\u003e \u003cp\u003eTCL cells were harvested, washed twice with PBS buffer, and resuspended in PBS buffer at a concentration of 1\u0026times;10⁶ cells/mL. Centrifuge cell suspension onto a slide. After fixed with 4% paraformaldehyde for 20 min at room temperature, the cells were washed three times with PBS buffer. Permeabilization was performed by treating the slides with 0.1% Triton X-100 for 5 min, and then washed again with PBS buffer. Next, the samples were blocked with 5% BSA for 30 min at room temperature, followed by washing with PBS buffer. The samples were incubated with primary antibody at 4\u0026deg;C overnight. After washed with PBS buffer, the samples were incubated with secondary antibody in the dark for 1 h at room temperature. Washed with PBS buffer, the samples were then treated with DAPI-containing mounting medium and incubated for 5 min at room temperature. Finally, the samples were covered with a coverslip and observed under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Animal experiments\u003c/h2\u003e \u003cp\u003eFive-week-old female NSG mice were obtained from GemPharmatech (Nanjing, China). All animals were raised under specific pathogen-free (SPF) conditions in a light/dark cycle-and temperature-controlled environment, and provided with sufficient food and water. To establish a TCL xenograft model, 4\u0026times;10\u003csup\u003e6\u003c/sup\u003e Jurkat cells suspended in 150 \u0026micro;L PBS were injected subcutaneously into the flanks of NSG mice. When the tumor volume reached approximately 100 mm\u0026sup3;, the mice were intraperitoneally injected with different agents every two days. The tumor volume (mm\u003csup\u003e3\u003c/sup\u003e) was measured with a vernier caliper and calculated as length \u0026times; width\u003csup\u003e2\u003c/sup\u003e/2, while changes in body weight were recorded simultaneously. At the experimental endpoint, peripheral blood was collected from mice and analyzed with a Sysmex XN-1000 hematology analyzer for blood cell counts. Serum was separated and subjected to biochemical parameter analysis using a HITACHI automated biochemistry analyzer. The tumor-bearing mice were then euthanized, tumor tissues and organs (such as liver, kidneys and lungs) were harvested for immunohistochemical (IHC) analysis and hematoxylin and eosin (H\u0026amp;E) staining according to the manufacturer\u0026rsquo;s instructions (Haokebio, Hangzhou, China). Additionally, the expression of signaling pathway proteins was analyzed in tumor tissues. All animal procedures were approved by the Institution\u0026rsquo;s Ethics Committee.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism software (San Diego, CA, USA). All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM), and group comparisons were conducted using two-tailed t-tests. p-value of less than 0.05 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was considered statistically significant. *represents p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **represents p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***represents p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. ns indicates no significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Alantolactone exerts anti-TCL effects both in vitro and in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlantolactone (ATL), a sesquiterpene lactone derived from Agarwood, exhibits multiple pharmacological properties including deworming, antibacterial, anti-inflammatory, and hepatoprotective effects. In this study, we aim to investigate the regulatory role of ATL in T-cell lymphoma (TCL). As illustrated in Fig 1A\u0026ndash;B, ATL treatment markedly suppresses the proliferation and migration capacities of TCL cells. To further evaluate the in vivo efficacy of ATL, a xenograft tumor model\u0026nbsp;was established by subcutaneously injecting Jurkat cells into NSG mice (Fig. 1C). As shown in Fig 1D, administration of ATL significantly inhibits the growth of transplanted tumors in mice. Additionally, it was found that both tumor size and weight in the ATL-treated group were markedly reduced compared to the control group (Fig. 1E\u0026ndash;F). IHC analysis revealed that ATL significantly reduces the levels of Ki67, indicating a marked inhibitory effect on the malignant progression of transplanted tumors in mice (Fig 1G). During the experimental period, no significant differences in body weight was observed between the control and ATL-treated group (Fig. 1H). Serum biochemical analysis further indicated that ATL has no significant effect on the levels of alanine transaminase (ALT), aspartate transaminase (AST), creatinine (CREA), and uric acid (UA), suggesting no obvious adverse effects on the metabolic functions of the liver and kidney (Fig. 1I-L). Meanwhile, the results of H\u0026amp;E staining showed that ATL treatment did not cause\u0026nbsp;significant morphological changes\u0026nbsp;in the liver, kidney and lung tissues of the mice (Fig. 1M).\u0026nbsp;The results of animal experiments demonstrated that while effectively suppressing TCL, ATL does not exhibit significant toxicity in vivo and possesses a favorable safety profile.\u0026nbsp;Collectively, these findings provide preliminary evidence that ATL exerts novel anti-TCL pharmacological activities both in vitro and in vivo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. The Caspase-3-dependent pathway mediates alantolactone-induced apoptosis of TCL cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate whether ATL induces apoptosis in TCL cells, we evaluated the apoptosis rate and the levels of relevant molecular markers. As key executors in the regulation of apoptosis, the cleavage of PARP and caspase-3 represents a critical step in initiating the apoptotic process in tumor cells. It was demonstrated that ATL treatment markedly enhances the cleavage of both caspase-3 and PARP in a dose-dependent manner (Fig. 2A). Additionally, the level of phosphorylated H2A.X (\u0026gamma;-H2A.X), a marker for DNA damage and apoptosis was also observed to increase in a dose-dependent manner following ATL treatment (Fig. 2B). Subsequently, we conducted RNA-seq on the TCL cells to analyze the differentially expressed genes (DEGs) after ATL treatment. The results of GO (Gene Ontology) analysis further showed that the differential genes were enriched in biological processes such as cell growth, migration and apoptosis (Fig. 2C). Furthermore, the pan-caspase inhibitor Z-VAD-FMK significantly reverses the anti-proliferative effect of ATL on TCL cells (Fig. 2D) and attenuates ATL-induced cell apoptosis (Fig. 2E-F). Taken together, these findings indicate that ATL triggers apoptosis and DNA damage in TCL cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. The PI3K/AKT and ERK signaling axis mediates the anti-TCL effect of Alantolactone\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further elucidate the molecular mechanism by which ATL regulates the TCL progression, we performed KEGG enrichment analysis on differentially expressed genes (DEGs). As illustrated in the Fig. 3A, DEGs were enriched in PI3K/AKT and MAPK pathways, both of which are closely associated with tumorigenesis. Western blot analysis further confirmed that ATL treatment markedly reduced the levels of p-AKT and p-ERK without affecting total AKT and ERK levels in TCL cells (Fig. 3B). In contrast, the phosphorylation and total protein levels of p38 and JNK1/2, two other pathways downstream of MAPK, were not significantly changed (Fig. 3C). These findings were consistently corroborated in tumor tissues in mice (Fig. 3D). Similarly, IHC analysis revealed substantially lower expression of p-AKT and p-ERK in ATL-treated groups compared with controls (Fig. 3E). Moreover, the AKT activator SC79 significantly attenuated the suppressive effects of ATL on both cell growth (Fig. 3F-G) and PI3K/AKT signaling pathway (Fig. 3H) in TCL. Likewise, the ERK activator C16-PAF also largely reversed the inhibitory effect of ATL in TCL cells (Fig. 3I-K). Collectively, these results indicated that the PI3K/AKT and ERK signaling pathways mediate the inhibitory effects of ATL on TCL both in vitro and in vivo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Alantolactone reduces the expression level of CD47 via\u003c/strong\u003e \u003cstrong\u003ePI3K/AKT and ERK\u003c/strong\u003e \u003cstrong\u003eaxis in TCL cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCD47 is highly expressed in malignant tumors such as TCL and is closely associated with disease progression. As shown in Fig. 4A, we found that CD47 monoclonal antibody significantly inhibits the proliferation of TCL cells. Further analysis revealed that ALT markedly downregulates CD47 expression in TCL cells, suggesting that CD47 serves as one of the targets through which ALT exerts its antitumor effects (Fig. 4B-C). Given that the PI3K/AKT and ERK signaling axis mediates ATL-induced inhibition of TCL, we further investigated the molecular mechanism by which ALT regulates CD47 expression. Subsequently, we observed that specific inhibition of either PI3K/AKT (Fig. 4D-E) or ERK (Fig. 4F-G) signaling pathway significantly suppresses both TCL cell proliferation and CD47 expression. This suppressive effect was reversed by corresponding activators of PI3K/AKT and ERK signaling pathways, which restored CD47 levels in the presence of ALT (Fig. 4H-I). These results confirmed that ALT downregulates CD47 via the PI3K/AKT and ERK signaling pathways in TCL cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Alantolactone exerts synergistic effects combined with clinical chemotherapy agents in TCL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDrug resistance poses a major challenge in first-line cancer therapy, often leading to disease relapse or malignant progression. Currently, combination therapies employing agents with different mechanisms of action have shown considerable promise in clinical practice. Decitabine, a first-line treatment for TCL, has garnered significant interest for its potential synergistic effects when used in combination with other agents. To explore whether ATL and decitabine exert synergistic antitumor activity, TCL cells were treated with ATL, decitabine, or the combination of both. As shown in Fig. 5A, the combination of ATL and decitabine markedly inhibits TCL cell proliferation compared to monotherapy with either agent alone. Moreover, ATL also demonstrates significant synergistic inhibition of TCL cell proliferation\u0026nbsp;when used in combination with the chemotherapeutic agents Belinostat and Chidamide\u0026nbsp;(Fig. 5B-C). Meanwhile, the combination of ATL and decitabine significantly increases the apoptotic rates and markedly upregulates the expression of apoptosis-related proteins in TCL cells compared to monotherapy with either agent alone (Fig. 5D-E). Mechanistically, it was found that these combinations notably suppress both the PI3K/AKT and ERK signaling pathways in TCL cells (Fig. 5F).\u0026nbsp;These in vitro experiments indicate that ATL exerts synergistic effects combined with clinical chemotherapy agents such as decitabine in TCL.\u003c/p\u003e\n\u003cp\u003eAdditionally, to further investigate the antitumor effects and underlying mechanisms of the combination of ATL (10 mg/kg) and decitabine (0.4 mg/kg), we also conducted in vivo experiments in tumor‑bearing mice (Fig. 6A). The results showed that the combination of the two agents significantly reduced tumor weight in mice compared to the single-agent group (Fig. 6B). Consistent with the in vitro experimental results, Western blot analysis revealed that the combined treatment significantly inhibited both the PI3K/AKT and ERK signaling pathways. (Fig. 6C). In terms of safety, no significant decrease in body weight of mice was observed throughout the administration period (Fig. 6D). It was also revealed that the combination therapy did not significantly affect the levels of ALT, AST, CREA, and UA in mice, and no notable morphological changes were detected in the liver, kidney, or lung tissues, suggesting that this combined regimen exhibits high antitumor efficacy without significant toxicity (Fig. 6E-I). Together, these findings indicate that the combination of ATL and decitabine holds potential as a candidate first-line treatment option for TCL.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAlthough T-cell lymphoma (TCL) accounts for a relatively low proportion among all tumor types, its incidence rate has been increasing year by year, imposing a heavy burden on society and patients' family. The pathogenesis of TCL is complex, and no specific biomarkers have been identified, which severely limits early diagnosis and development of targeted drugs[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Although first-line chemotherapy agents demonstrate a relatively high rate of cure and remission, their effectiveness remains limited overall due to several challenges, including the adverse reactions and side effects associated with the drugs themselves, as well as the strong heterogeneity of TCL and its tendency to develop drug resistance. Consequently, current treatment strategies still fall short of fully meeting clinical needs. Therefore, elucidating the pathogenesis of TCL and developing novel effective drugs have become urgent tasks to address the current challenges in clinical treatment.\u003c/p\u003e \u003cp\u003eBenefiting from its multi-target effects and low toxicity, the traditional Chinese medicine alantolactone (ALT) is now widely used in clinical practice for the treatment of diseases such as cancer[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. With the application of new technologies such as metabolomics, researchers are attempting to reveal novel pharmacological functions of ATL from multiple dimensions. Currently, the regulation of TCL by ATL remains uninvestigated. Here, we reported for the first time that ATL markedly inhibits TCL cell growth in vitro and in vivo, thereby revealing its anti-tumor potential in this study. Notably, during drug administration in tumor-bearing mice, no toxic reactions such as weight loss, liver, kidney or lung damage, or hematopoietic system injury were observed, suggesting that ATL possesses a favorable safety profile while exerting its anti-TCL effects. Additionally, no metastasis of tumor lesions to vital organs such as the lungs or liver was observed, which may be related to the construction method of constructing the transplanted tumor model by subcutaneous inoculation rather than tail vein injection, and the relevant molecular mechanisms requires further investigation. TCL is a malignant tumor originating from the lymphatic system, and the occurrence of distant metastasis markedly elevates disease malignancy, often resulting in a dismal prognosis. Therefore, understanding the metastatic characteristics of TCL is crucial for developing individualized treatment strategies. In cell model experiments, we observed that ATL significantly inhibits the migratory ability of TCL cells. Given the complexity and interindividual variability of TCL metastasis, further studies using diverse cellular and animal models are warranted to elucidate the role of ATL in this process. Inducing apoptosis is one of the key mechanisms by which many chemotherapy drugs exert anti-tumor effects[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this study, we found that ATL significantly promotes apoptosis in TCL cells. Specifically, ATL-induced apoptosis is closely related to the activation of caspase and PARP, and the broad-spectrum caspase inhibitor Z-VAD-FMK can significantly reverse the pro-apoptotic effect of ATL on TCL cells. In summary, this study confirmed that ATL has significant anti-TCL activity through in vivo and in vitro experiments, providing a strong basis for its further development as a clinical candidate drug. On this basis, we plan to conduct a series of experiments to deeply analyze the specific molecular mechanisms of ATL against TCL.\u003c/p\u003e \u003cp\u003eIn terms of molecular mechanism exploration, we performed RNA-seq sequencing to systematically screen differentially expressed genes (DEGs) in TCL cells after ATL treatment. KEGG pathway enrichment analysis of DEGs revealed that the PI3K/AKT and MAPK signaling pathways may be involved in the biological activity of ATL. Multi-omics analysis in cells and mice models showed that ATL inhibits the progression of TCL by negatively regulating PI3K/AKT and ERK signaling axes. It was known that abnormal activation of the PI3K/AKT and ERK pathways is closely associated with the development and progression of various malignant tumors, involving processes such as cell proliferation, drug resistance, and migration. Therefore, these pathways are considered important potential targets for cancer therapy. Several small-molecule drugs targeting these pathways have entered clinical trials and showed promising prospects. For example, Carlos Fernandez Teruel et al. reported that the AKT inhibitor capivasertib combined with fulvestrant demonstrates good efficacy in breast cancer patients[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Romain Sigaud et al. confirmed that the ERK inhibitor ulixertinib exhibits significant activity in low-grade gliomas[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Further, rescue experiments indicated that reactivating PI3K/AKT or ERK signaling significantly reverse the regulatory effects of ATL on TCL cell proliferation and apoptosis. These results collectively suggest that the PI3K/AKT and ERK signaling axes play a critical role in ATL-mediated regulation of TCL development, supporting their value as potential therapeutic targets for TCL. Multiple studies have confirmed the close relationship between the immune system and tumor development, and anti-tumor immunotherapy has shown promising therapeutic prospects[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, we found that anti-CD47 monoclonal antibodies significantly inhibits TCL cell proliferation. CD47, as an important \"don't eat me\" signal molecule, is highly expressed on the surface of various tumor cells, including TCL[\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. By binding to its ligand SIRPα on immune cells such as macrophages, CD47 inhibits phagocytosis, thereby mediating immune escape. Our results further indicated that ATL downregulates CD47 expression levels by negatively regulating the PI3K/AKT and ERK signaling pathways, while activators of PI3K/AKT and ERK signaling axes can effectively reverse the inhibitory effect of ATL on CD47, respectively. These findings underscore the pivotal role of ATL in TCL immunotherapy by reducing CD47 expression, highlighting the need for further experimental research to assess its clinical application value.\u003c/p\u003e \u003cp\u003eAlthough single-drug therapy can temporarily delay the malignant progression of tumors, long-term use often leads to the development of drug resistance, which has become one of the major reasons for the failure of tumor treatment. The mechanisms of drug resistance are complex and diverse, primarily involving factors such as alterations in drug targets, enhanced DNA damage repair capacity, and epigenetic changes[\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Therefore, combination therapy has emerged as a promising strategy in current cancer research and plays a crucial role in enhancing clinical outcomes. Taking the classic CHOP regimen for treating TCL as an example, its success demonstrates the significant value of combination therapy in achieving long-term disease control[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. As a DNA methyltransferase inhibitor, decitabine can effectively induce apoptosis and plays a crucial role in the first-line treatment of TCL[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. This study showed that the combination of ATL and decitabine synergistically inhibits the activation of the PI3K/AKT and ERK signaling pathways in both in vitro and in vivo, demonstrating significant inhibitory effects on TCL growth without observable notable drug toxicity in mouse models. It indicates that this combination regimen can intervene in TCL progression and possesses favorable safety, providing novel targeted therapy strategies for clinical first-line treatment.\u003c/p\u003e \u003cp\u003eIn summary, this study is the first to demonstrate that ATL effectively inhibits the malignant progression of TCL in vitro and in vivo, and significantly enhances the sensitivity of TCL to first-line chemotherapeutic drugs such as decitabine. Mechanically, we found that ATL suppresses TCL by negatively regulating the PI3K/AKT and ERK signaling pathways and downregulating CD47 protein expression. These findings provide a solid experimental basis for ATL as a candidate agent for TCL treatment, offering a potential new strategy to improve the current therapeutic landscape.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge the technical and experimental support from all team members.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValidation and investigation: Xiaodong Li, Yinyu Mu, and Ni Li. Analyzed the data: Xiaodong Li and Yinyu Mu. Writing: Xiaodong Li and Ni Li. Resources: Bingrong Chen, Wenwen Sun and Fuyi Xie. Methodology: Ningbo Pang, Yingcong Chen, and Tingting Pan. Supervision: Fuyi Xie and Ni Li. Funding: Xiaodong Li, Tingting Pan and Ni Li. All authors have reviewed and agreed to the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Zhejiang Province Health industry Science and Technology Plan (2025HY0868), Ningbo Municipal Program for Innovative and Leading Talents (2024QL025), and the Natural Science Foundation of Ningbo (2024J337).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003eThe authors declare no conflict of interest. Fig 1C, Fig 6A, and Fig 7 were drawn using BioGDP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with protocols approved by the Animal Experimentation Ethics Committee of Zhejiang University of Chinese Medicine (Approval code: IACUC-202501-17).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMohammed Saleh, M.F., et al., Recent Advances in Diagnosis and Therapy of Angioimmunoblastic T Cell Lymphoma. Curr Oncol, 2021. \u003cstrong\u003e28\u003c/strong\u003e(6): p. 5480-5498.\u003c/li\u003e\n\u003cli\u003eWang, C., et al., TRIM24 promotes T-cell lymphoma development and glucocorticoid resistance via FUS-mediated phase separation of the glucocorticoid receptor. 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Ann Hematol, 2025. \u003cstrong\u003e104\u003c/strong\u003e(3): p. 1747-1756.\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":"apoptosis","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"appt","sideBox":"Learn more about [Apoptosis](http://link.springer.com/journal/10495)","snPcode":"10495","submissionUrl":"https://submission.nature.com/new-submission/10495/3","title":"Apoptosis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"T-cell lymphoma, Alantolactone, Decitabine, CD47, PI3K/AKT, ERK","lastPublishedDoi":"10.21203/rs.3.rs-8529840/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8529840/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eT-cell lymphoma (TCL) is a malignant tumor caused by abnormal proliferation of T cells, and its specific pathogenesis remains unclear. Currently, there is still a lack of highly effective therapeutic drugs in clinic. As a semi-terpene lactone compound, alantolactone (ATL) is mainly used to treat diseases such as asthma, and its role in TCL has not been revealed. This study demonstrated that ATL not only significantly inhibits the proliferation and migration of TCL cells and induces apoptosis in vitro, but also exhibits significant anti-tumor effects in tumor-bearing mice. Meanwhile, ATL can markedly enhance the sensitivity of TCL cells to chemotherapeutic drugs such as decitabine. Mechanistically, multi-omics analysis confirmed that ATL restricts TCL progression by negatively regulating the PI3K/AKT and ERK signaling pathway. Furthermore, our study also found that ATL-mediated suppression of these pathways leads to significant downregulation of CD47 expression. In summary, this study is the first to elucidate that ATL exhibits significant anti-TCL effects both in vitro and in vivo, suggesting its potential as a novel clinical strategy for the treatment of TCL.\u003c/p\u003e","manuscriptTitle":"Alantolactone inhibits T-cell lymphoma progression by suppressing CD47 expression via the PI3K/AKT and ERK signaling pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-09 11:12:48","doi":"10.21203/rs.3.rs-8529840/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-13T09:22:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-13T06:52:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-13T02:51:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32365110833314311090851582116396690282","date":"2026-01-13T00:07:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81628351808025875228296343736194430461","date":"2026-01-12T15:58:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161772893399683341082147588871791896913","date":"2026-01-12T14:23:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-11T16:25:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"23010290682262274732559028889189971848","date":"2026-01-11T02:56:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290491857266912668515979564542073931073","date":"2026-01-09T12:19:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4525140450599896424435683417298503857","date":"2026-01-08T00:25:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13044604658314123488978862132543763218","date":"2026-01-07T12:56:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132530605768022965361577168074548335534","date":"2026-01-07T10:29:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-07T10:21:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-07T10:16:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-07T10:12:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Apoptosis","date":"2026-01-06T09:39:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"apoptosis","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"appt","sideBox":"Learn more about [Apoptosis](http://link.springer.com/journal/10495)","snPcode":"10495","submissionUrl":"https://submission.nature.com/new-submission/10495/3","title":"Apoptosis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5443c029-83be-473b-9656-f347f234edb8","owner":[],"postedDate":"January 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-17T03:55:02+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-09 11:12:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8529840","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8529840","identity":"rs-8529840","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}