ACT001 inhibits primary central nervous system lymphoma tumor growth by enhancing the anti-tumor effect of T cells.

Primary central nervous system lymphoma (PCNSL) is a group of malignant brain tumors with a poor prognosis, and new therapeutic approaches for this tumor urgently need to be investigated. Formulated from a long-standing anti-inflammatory drugs, ACT001 has demonstrated in clinical research to be able to pass through the blood-brain barrier (BBB) and affect the central nervous system. The effects of ACT001 on PCNSL cell apoptosis, proliferation and immune-related indexes were detected by flow cytometry, and the efficacy of ACT001 was verified in vivo by constructing a mouse PCNSL tumor model. ACT001 significantly inhibited PCNSL cell proliferation and induced apoptosis in vitro. In addition, ACT001 can significantly inhibit the PD-1/PD-L1 expression and restore the function of T cells, so that the immune system cannot allow tumor cells to escape. In vivo experiments show that co-infusion of ACT001 and T cells effectively inhibits PCNSL tumor growth in NSG mice. Our work describes the inhibitory effect of ACT001 on the PCNSL cell line and demonstrated the inhibitory effect of ACT001 on immune checkpoints.

Alleviative effects of the parthenolide derivative ACT001 on insulin resistance induced by sodium propionate combined with a high-fat diet and its potential mechanisms.

The increasing side effects of traditional medications used to treat type II diabetes have made research into the development of safer and more effective natural medications necessary. ACT001, a derivative of parthenolide, has been shown to have good anti-inflammatory and antitumor effects; however, its role in diabetes is unclear. The short-chain fatty acid propionate is a common food preservative that has been found to cause disturbances in glucose metabolism in mice and humans. This study aimed to investigate whether sodium propionate could aggravate insulin resistance in obese mice and cause diabetes and to study the alleviative effects and potential mechanisms of action of ACT001 on insulin resistance in diabetic mice. Type II diabetic mice were adminietered sodium propionate combined with a high-fat diet (HFD + propionate) by gavage daily for four weeks. Biochemical analysis showed that ACT001 significantly affected blood glucose concentration in diabetic mice, mainly by downregulating the expression of phosphoenolpyruvate carboxykinase 2 and glucose-6-phosphatase. Meanwhile, the level of fatty acid-binding protein 4 in the liver was significantly decreased. ACT001 has a protective effect on the liver and adipose tissue of mice. In addition, the results of the running wheel experiment indicated that ACT001 alleviated the circadian rhythm disorder caused by insulin resistance to a certain extent. This study revealed the potential mechanism by which ACT001 alleviates insulin resistance and provides ideas for developing natural antidiabetic drugs.

ACT001 alleviates inflammation and pyroptosis through the PPAR-γ/NF-κB signaling pathway in LPS-induced alveolar macrophages.

BACKGROUND: ACT001 is an anti-inflammatory agent that has been widely investigated for its role in tumors, intracranial diseases, and fibrotic diseases, but its effect on acute lung injury is less known. OBJECTIVE: The purpose of this study was to investigate the effect and mechanism of ACT001 on regulating inflammation and pyroptosis in lipopolysaccharide (LPS)-induced alveolar macrophages. METHODS: NR8383 alveolar macrophages treated with LPS were used to replicate the proinflammatory macrophage phenotype observed during acute lung injury. After ACT001 treatment, we measured the secretion and expression levels of critical inflammatory cytokines, the rate of pyroptosis, and the expression of NLRP3 inflammasome-associated proteins and pyroptosis-associated proteins. In addition, we assessed the role of the PPAR-γ/NF-κB signaling pathways and further validated the results with a PPAR-γ inhibitor. RESULTS: Our findings confirmed that ACT001 reduced the expression and release of inflammatory factors, attenuated cell pyroptosis, and downregulated the expression of NLRP3, ASC, caspase-1 p20, and GSDMD-N. These effects may be achieved by activating PPAR-γ expression and then inhibiting the NF-κB signaling pathway. When macrophages were treated with the PPAR-γ inhibitor, the protective effects of ACT001 were reversed. CONCLUSION: ACT001 significantly ameliorated inflammation and pyroptosis via the PPAR-γ/NF-κB signaling pathways in LPS-induced NR8383 alveolar macrophages.

ACT001 improved cardiovascular function in septic mice by inhibiting the production of proinflammatory cytokines and the expression of JAK-STAT signaling pathway.

Sepsis is a life-threatening multiple organ dysfunction syndrome (MODS) caused by a microbial infection that leads to high morbidity and mortality worldwide. Sepsis-induced cardiomyopathy (SIC) and coagulopathy promote the progression of adverse outcomes in sepsis. Here, we reported that ACT001, a modified compound of parthenolide, improved the survival of sepsis mice. In this work, we used cecal ligation and puncture (CLP) model to induce SIC. Transthoracic echocardiography and HE staining assays were adopted to evaluate the influence of ACT001 on sepsis-induced cardiac dysfunction. Our results showed that ACT001 significantly improved heart function and reduced SIC. Coagulation accelerates organ damage in sepsis. We found that ACT001 decreased blood clotting in the FeCl3-induced carotid artery thrombosis experiment. ACT001 also reduced the production of neutrophil extracellular traps (NETs). RNA-sequencing of heart tissues revealed that ACT001 significantly downregulated the expression of pro-inflammatory cytokines and the JAK-STAT signaling pathway. These results were confirmed with real-time PCR and ELISA. In summary, we found ACT001 rescued mice from septic shock by protecting the cardiovascular system. This was partially mediated by inhibiting pro-inflammatory cytokine production and down-regulating the JAK-STAT signaling.

ACT001 Alleviates chronic kidney injury induced by a high-fat diet in mice through the GPR43/AMPK pathway.

BACKGROUND: Roughly 10 -15% of global populace suffer from Chronic Kidney Disease(CKD). A major secondary disease that can progress to end-stage renal disease (ESRD) is obesity-associated kidney disease (ORG). Although clinical management strategies are currently available, morbidity and mortality rates are increasing. Thus, new solutions are needed. Intestinal permeability, systemic inflammation, and aberrant intestinal metabolites have all been linked to ORG. PURPOSE: ACT001 has anti-inflammatory, redox-regulatory and antitumour activities. The current study was designed to examine how ACT001 affects ORG and analyze the fundamental processes. METHODS: A high-fat diet (HFD) was used to generate ORG in female C57BL/6 J mice. ORG mice were divided into three groups at random: HFD, HFD + ACT001, HFD + polyphosphocholine (PPC). To assess renal and colonic damage, periodic acid-Schiff (PAS) and hematoxylin-eosin (HE) staining were used. Following that, renal inflammation, oxidative stress, lipid deposition, colonic inflammation, and intestinal permeability were evaluated by protein blotting, polymerase chain reaction (PCR), immunohistochemistry, and immunofluorescence staining. Lastly, the SCFAs content was assessed by gas chromatographymass spectrometry. RESULTS: Mice in the HFD group displayed more severe albuminuria, glomerular hypertrophy, renal oxidative damage, inflammation, and lipid accumulation than mice with the normal diet (ND) group, as well as lower levels of intestinal SCFA valproic acid, colonic inflammation, and tight junction protein downregulation. ACT001 treatment restores the content of valproic acid in intestinal SCFAs, promotes the binding of SCFAs to renal GPR43, activates the AMPK signalling pathway. Therefore, it promotes the Nrf2-Keap1 signalling pathway and inhibits the NF-κB signalling pathway. SCFAs, additionally, augment colonic GPR43 concentrations, diminishing NLRP3 inflammasome expression and restoring ZO-1 and occludin protein levels. CONCLUSION: This study is the first to look at ACT001's potential as a treatment for obesity-related kidney disease. Regulating GPR43 and AMPK signalling pathways, By controlling the GPR43 and AMPK signalling pathways, ACT001 improves colitis and the intestinal mucosal barrier, decreases renal lipid deposition, and suppresses inflammation and oxidative stress in the kidneys. According to this study, ACT001 could be a viable ORG therapy option.

ACT001 Ameliorates ionizing radiation-induced lung injury by inhibiting NLRP3 inflammasome pathway.

Radiotherapy is a prevalent treatment modality for thoracic tumors; however, it can lead to radiation-induced lung injury (RILI), which currently lacks effective interventions. ACT001, a prodrug of micheliolide, has demonstrated promising clinical application potential, yet its impact on RILI requires further validation. This study aims to investigate the radioprotective effects of ACT001 on RILI and elucidate its underlying mechanism. Sprague-Dawley rats were utilized to induce RILI following 20 Gy X-ray chest irradiation, and lung tissue inflammation and fibrosis were assessed using hematoxylin and eosin (H&E) and Masson staining. Lung injury, inflammation, and oxidative stress markers were evaluated employing commercial kits. Pyroptosis-related differentially expressed genes (DEGs) were analyzed using a microarray dataset from the Gene Expression Omnibus (GEO) database, and their functions and hub genes were identified through protein-protein interaction networks. Pyroptosis-related genes were detected via RT-qPCR, western blotting, immunofluorescence, and immunohistochemistry. The results demonstrated that ACT001 ameliorated RILI, diminished pro-inflammatory cytokine release and fibrosis, and mitigated the activation of the NLRP3 inflammasome while inhibiting pyroptosis in lung tissue. In conclusion, our study reveals that ACT001 can suppress NLRP3 inflammasome-mediated pyroptosis and improve RILI, suggesting its potential as a novel protective agent for RILI.

ACT001 Relieves NMOSD Symptoms by Reducing Astrocyte Damage with an Autoimmune Antibody.

Neuromyelitis optica spectrum disorder (NMOSD) is a central nervous system inflammatory demyelinating disease, the pathogenesis of which involves autoantibodies targeting the extracellular epitopes of aquaporin-4 on astrocytes. We neutralized the AQP4-IgG from NMOSD patient sera using synthesized AQP4 extracellular epitope peptides and found that the severe cytotoxicity produced by aquaporin-4 immunoglobin (AQP4-IgG) could be blocked by AQP4 extracellular mimotope peptides of Loop A and Loop C in astrocyte protection and animal models. ACT001, a natural compound derivative, has shown anti-tumor activity in various cancers. In our study, the central nervous system anti-inflammatory effect of ACT001 was investigated. The results demonstrated the superior astrocyte protection activity of ACT001 at 10 µM. Furthermore, ACT001 decreases the behavioral score in the mouse NMOSD model, which was not inferior to Methylprednisolone Sodium Succinate, the first-line therapy of NMOSD in clinical practice. In summary, our study showed that astrocytes are protected by specific peptides, or small molecular drugs, which is a new strategy for the treatment of NMOSD. It is possible for ACT001 to be a promising therapy for NMOSD.

The antitumor effect of the novel agent MCL/ACT001 in pancreatic ductal adenocarcinoma.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a major challenge in cancer therapy, there are more than four hundred thousand deaths per year, and the 5-year survival rate is less than 10%. The incidence continues to rise. Treatment with classic drugs offers limited therapeutic benefits. The aim of this study was to investigate the mechanism and effect of the new agent ACT001, the active metabolite of Micheliolide (MCL), in vitro and in vivo against PDAC. METHODS: MTT assay, wound healing assay, and flow cytometry were used to assess the effects of MCL/ACT001 in vitro. DCFH-DA assay was used to assess ROS accumulation. Western blotting, immunohistochemical staining and TUNEL assay were also conducted to determine the mechanisms. PANC-1-Luc cells and bioluminescent reporter imaging were used to assess antitumor effect of ACT001 using a orthotopic xenograft model in vivo. RESULTS: MCL/ACT001 significantly inhibited cell growth in PDAC in a dose-dependent manner, induced cell apoptosis, cell migration and reactive oxygen species (ROS) accumulation in vitro. In vivo, ACT001 (400 mg/kg/day) inhibited PDAC tumor growth in orthotopic xenograft mice. We verified that EGFR and Akt were markedly overexpressed in PDAC cells and patient tumors. Mechanistic investigations revealed that MCL exerted its antitumor activity via regulation of the EGFR-Akt-Bim signaling pathway, thus inducing Bim expression both in vitro and in vivo. CONCLUSION: MCL/ACT001 is a highly promising agent in the treatment of PDAC patients.

ACT001 exerts anti-inflammatory and antioxidative activity to alleviate sepsis-induced acute lung injury through STAT1/CIITA/MHC- II pathway / 中国药理学通报

Aim This study aimed to assess the therapeutic potential of ACT001, a micheliolide derivative, in the treatment of acute lung injury ( ALI) induced by sepsis and investigate its pharmacological mechanisms. Methods At the animal level, an ALI model was established in mice through intraperitoneal injection of li-popolysaccharide (LPS). Subsequently, ACT001 was administered to the ALI-afflicted mice. The therapeutic effects of ACT001 were assessed by evaluating factors such as individual survival rate, lung inflammation, and pulmonary edema. At the cellular level, RAW264. 7 cells were stimulated with LPS to explore the pharmacological mechanism of ACT001. The study examined inflammatory response and oxidative stress levels, and proteomics analysis was conducted to investigate the underlying molecular mechanisms. Results At the animal level, ACT001 can improve the survival of mice with ALI, reduce lung inflammation, and reduce the levels of inflammatory cytokines in serum. At the cellular level, ACT001 promotes the polarization of RAW264. 7 cells toward an anti-inflammatory pheno-type by inhibiting MHC-II related pathways, inhibiting the production of NO and related inflammatory cytokines while increasing SOD content and scavenging ROS. Conclusions ACT001 exhibited the potential to alleviate ALI via its anti-inflammatory and antioxidative activity, mainly by inhibiting the STAT1/ CIITA/ MHC-II pathway. ACT001 holds promise as a novel therapeutic candidate for the treatment of ALI induced by sepsis.

ACT001 suppressing M1 polarization against inflammation via NF-κB and STAT1 signaling pathways alleviates acute lung injury in mice.

ACT001 has been shown to exhibit excellent antitumor and anti-fibrosis activities. However, the role of ACT001 in acute lung injury (ALI) and the underlying mechanism remains largely unclear. The present study aimed to investigate the protective effects of ACT001 on ALI and explore the potential mechanisms. Herein, we firstly established the ALI mouse model induced by intratracheal instillation of lipopolysaccharide (LPS). ACT001 treatment significantly alleviated histopathological changes of lung tissues with lower infiltration of pulmonary M1 macrophages in ALI mice. Then, we performed in vitro experiment and found that ACT001 treatment effectively inhibited the M1 phenotype of RAW264.7 and THP-1.. Next, we performed pull-down and mass spectrometry analysis to screen the interacting proteins of ACT001, identifying IKKß and STAT1 as the critical target proteins of ACT001. And ACT001 treatment significantly suppressed the NF-κB and STAT1 pathways, thereby inhibiting the M1 polarization against inflammation in vivo and in vitro. Finally, we used IMD 0354 (IMD) and Fludarabine (Flud) to specifically block the activity of IKKß and STAT1, and stimulated macrophages through IKKß and STAT1 overexpression. Our data clearly showed that ACT001-induced decrease of the M1 polarization was blocked by IMD and Flud treatment, and reversed by IKKß and STAT1 overexpression in RAW264.7 cells. In conclusion, we discovered that ACT001 significantly alleviates inflammation and limits M1 phenotype of pulmonary macrophages via suppressing NF-κB and STAT1 signaling pathways, providing new insights for the development of drugs to treat ALI/ARDS.

ACT001 Inhibits TLR4 Signaling by Targeting Co-Receptor MD2 and Attenuates Neuropathic Pain.

Neuropathic pain is a common and challenging neurological disease, which renders an unmet need for safe and effective new therapies. Toll-like receptor 4 (TLR4) expressed on immune cells in the central nervous system arises as a novel target for treating neuropathic pain. In this study, ACT001, an orphan drug currently in clinical trials for the treatment of glioblastoma, was identified as a TLR4 antagonist. In vitro quenching titrations of intrinsic protein fluorescence and saturation transfer difference (STD)-NMR showed the direct binding of ACT001 to TLR4 co-receptor MD2. Cellular thermal shift assay (CETSA) showed that ACT001 binding affected the MD2 stability, which implies that MD2 is the endogenous target of ACT001. In silico simulations showed that ACT001 binding decreased the percentage of hydrophobic area in the buried solvent-accessible surface areas (SASA) of MD2 and rendered most regions of MD2 to be more flexible, which is consistent with experimental data that ACT001 binding decreased MD2 stability. In keeping with targeting MD2, ACT001 was found to restrain the formation of TLR4/MD2/MyD88 complex and the activation of TLR4 signaling axes of NF-κB and MAPKs, therefore blocking LPS-induced TLR4 signaling downstream pro-inflammatory factors NO, IL-6, TNF-α, and IL-1ß. Furthermore, systemic administration of ACT001 attenuated allodynia induced by peripheral nerve injury and activation of microglia and astrocyte in vivo. Given the well-established role of neuroinflammation in neuropathic pain, these data imply that ACT001 could be a potential drug candidate for the treatment of chronic neuropathic pain.

ACT001 inhibits the proliferation of non-small cell lung cancer cells by upregulating NKTR expression.

BACKGROUND: Lung cancer, the primary cause of cancer-related deaths worldwide, is diagnosed at an advanced stage and has a poor prognosis. Non-small cell lung cancer (NSCLC) is a major histological type of lung malignancy. This study investigated the effect of ACT001, a novel sesquiterpene lactone derivative, on the proliferation of NSCLC cells and explored the underlying mechanism. METHODS: The effect of ACT001 on cell proliferation was examined by clone formation and MTT assay. Differentially expressed genes and enrichment pathways were analyzed by RNA-seq. Flow cytometry and cell cycle-related protein expression analysis were performed to study the cell cycle. Phosphorylated AKT was detected to explore the mechanism in natural killer cell triggering receptor (NKTR) KD cells with AKT activator and/or inhibitor. The therapeutic effect of ACT001 in vivo was studied in the xenograft tumor model. RESULTS: ACT001 inhibited the proliferation and G1/S transition in NSCLC cell lines. By RNA-seq analysis, NKTR may be the target of ACT001. Moreover, knockdown NKTR promoted cell proliferation and reversed the effects of ACT001. In addition, ACT001 inhibited AKT phosphorylation, but NKTR knockdown promoted AKT phosphorylation. CONCLUSION: Our results suggested NKTR may be the target of ACT001 in NSCLC. ACT001 holds promise as a novel method for the treatment of NSCLC.

ACT001 attenuates microglia-mediated neuroinflammation after traumatic brain injury via inhibiting AKT/NFκB/NLRP3 pathway.

BACKGROUND: Microglia-mediated neuroinflammatory response following traumatic brain injury (TBI) is considered as a vital secondary injury factor, which drives trauma-induced neurodegeneration and is lack of efficient treatment. ACT001, a sesquiterpene lactone derivative, is reportedly involved in alleviation of inflammatory response. However, little is known regarding its function in regulating innate immune response of central nervous system (CNS) after TBI. This study aimed to investigate the role and underlying mechanism of ACT001 in TBI. METHODS: Controlled cortical impact (CCI) models were used to establish model of TBI. Cresyl violet staining, evans blue extravasation, neurobehavioral function assessments, immunofluorescence and transmission electron microscopy were used to evaluate therapeutic effects of ACT001 in vivo. Microglial depletion was induced by administering mice with colony stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622. Cell-cell interaction models were established as co-culture system to simulate TBI conditions in vitro. Cytotoxic effect of ACT001 on cell viability was assessed by cell counting kit-8 and activation of microglia cells were induced by Lipopolysaccharides (LPS). Pro-inflammatory cytokines expression was determined by Real-time PCR and nitric oxide production. Apoptotic cells were detected by TUNEL and flow cytometry assays. Tube formation was performed to evaluate cellular angiogenic ability. ELISA and western blot experiments were used to determine proteins expression. Pull-down assay was used to analyze proteins that bound ACT001. RESULTS: ACT001 relieved the extent of blood-brain barrier integrity damage and alleviated motor function deficits after TBI via reducing trauma-induced activation of microglia cells. Delayed depletion of microglia with PLX5622 hindered therapeutic effect of ACT001. Furthermore, ACT001 alleviated LPS-induced activation in mouse and rat primary microglia cells. Besides, ACT001 was effective in suppressing LPS-induced pro-inflammatory cytokines production in BV2 cells, resulting in reduction of neuronal apoptosis in HT22 cells and improvement of tube formation in bEnd.3 cells. Mechanism by which ACT001 functioned was related to AKT/NFκB/NLRP3 pathway. ACT001 restrained NFκB nuclear translocation in microglia cells through inhibiting AKT phosphorylation, resulting in decrease of NLRP3 inflammasome activation, and finally down-regulated microglial neuroinflammatory response. CONCLUSIONS: Our study indicated that ACT001 played critical role in microglia-mediated neuroinflammatory response and might be a novel potential chemotherapeutic drug for TBI. Video Abstract.

ACT001 inhibits pituitary tumor growth by inducing autophagic cell death via MEK4/MAPK pathway.

ACT001, derived from traditional herbal medicine, is a novel compound with effective anticancer activity in clinical trials. However, little is known regarding its role in pituitary adenomas. Here, we demonstrated that ACT001 suppressed cell proliferation and induced cell death of pituitary tumor cells in vitro and in vivo. ACT001 was also effective in suppressing the growth of different subtypes of human pituitary adenomas. The cytotoxic mechanism ACT001 employed was mainly related to autophagic cell death (ACD), indicated by autophagosome formation and LC3-II accumulation. In addition, ACT001-mediated inhibitory effect decreased when either ATG7 was downregulated or cells were cotreated with autophagy inhibitor 3-methyladenine (3-MA). RNA-seq analysis showed that mitogen-activated protein kinase (MAPK) pathway was a putative target of ACT001. Specifically, ACT001 treatment promoted the phosphorylation of JNK and P38 by binding to mitogen-activated protein kinase kinase 4 (MEK4). Our study indicated that ACT001-induced ACD of pituitary tumor cells via activating JNK and P38 phosphorylation by binding with MEK4, and it might be a novel and effective anticancer drug for pituitary adenomas.

ACT001 reverses resistance of prolactinomas via AMPK-mediated EGR1 and mTOR pathways.

Dopamine agonist (DA) is the first choice for the treatment of prolactinomas, and drug resistance is unavoidable during treatment due to the heterogeneity of tumors. The two prolactinoma cell lines (GH3 cells and MMQ cells) were found to have different sensitivity and responding modes to the cabergoline (CAB) and bromocriptine (BRC). In this research, we disclosed the capability of ACT001, a derivative of parthenolide analogs, to activate AMPK by increasing the intracellular reactive oxygen species (ROS) level and AMP/ATP ratio to reverse DA resistance through dual pathways in prolactinoma cells. The results indicated that ACT001 could reverse the CAB resistance in GH3 cells by inhibiting the mTOR signaling pathway, inducing cell death through autophagy, and reverse the BRC resistance in MMQ cells by activating the EGR1 signaling pathway, inducing cell death through apoptosis. Our results suggested that ACT001 is a promising therapeutic compound for treating DA-resistant prolactinomas.

Targeting of glioma stem-like cells with a parthenolide derivative ACT001 through inhibition of AEBP1/PI3K/AKT signaling.

Glioblastoma (GBM) is the most lethal primary brain tumor in adults with a median survival of around 15 months. A potential treatment strategy involves targeting glioma stem-like cells (GSCs) that are able to initiate, maintain, and repopulate the tumor mass. Here, we identify ACT001, a parthenolide derivative, targeting GSCs through regulation of adipocyte enhancer binding protein 1 (AEBP1) signaling. Methods: The effects of ACT001 on cell survival of normal human astrocytes (NHA) and patient-derived glioma stem-like cells (GSCs) were evaluated. RNA-Seq were performed to detect differentially expressed genes. ACT001 efficacy as a single agent or in combination with SHP-2 inhibitor SHP099 was assessed using a GSC orthotopic xenograft model. Results: GSCs exhibit high response to ACT001 in compared with normal human astrocytes. AEBP1 is a putative target of ACT001 by RNA-Seq analysis, which expression associates with prognosis of GBM patients. Knockdown of AEBP1 inhibits GSC proliferation and glioma sphere formation. Treatment with ACT001 or PI3K inhibitor or AEBP1 depletion would impair AKT phosphorylation and GSC proliferation, whereas constitutive AKT activation rescues ACT001 treatment or AEBP1 depletion-inhibited cell proliferation. Moreover, ACT001 blocks TGF-ß-activated AEBP1/AKT signaling in GSCs. ACT001 exhibits antitumor activity either as a single agent or in combination with SHP099, which provides significant survival benefits for GSC tumor xenograft-bearing animals. Conclusions: Our data demonstrate AEBP1 as a new druggable target in GBM and ACT001 as a potential therapeutic option for improving the clinical treatment of GBM in combination with SHP099.

Anticancer Effects of ACT001 via NF-κB Suppression in Murine Triple-Negative Breast Cancer Cell Line 4T1.

PURPOSE: ACT001 is a novel sesquiterpene lactone derivative with anticancer effects, including the reversal of tamoxifen resistance in estrogen receptor-positive breast cancer cells. However, few studies have investigated the anticancer effects of ACT001 in triple-negative breast cancer (TNBC), a highly aggressive cancer with a poor prognosis. This study aimed to investigate the effects of ACT001 on TNBC and the potential mechanism underlying these effects. MATERIALS AND METHODS: The anticancer effects of ACT001 on the murine TNBC cell line 4T1 were evaluated by Cell Counting Kit-8 assay, animal experiments, TUNEL staining, flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay, and Western blotting analysis. RESULTS: ACT001 induced apoptosis in 4T1 cells by upregulating B cell lymphoma 2-associated X protein expression. Moreover, ACT001 markedly decreased levels of secretory granulocyte-macrophage colony stimulating factor (GM-CSF) in 4T1 tumors, decreased the number of myeloid-derived suppressor cells (MDSCs), and reduced angiogenesis. Furthermore, GM-CSF promoted angiogenesis and the proliferation of MDSCs in a dose-dependent manner. Finally, ACT001 suppressed phospho-NF-κB and IκB-α levels in 4T1 cells, thereby further decreasing GM-CSF levels. CONCLUSION: Our results suggest that ACT001 exerts its anticancer effects by inducing apoptosis in murine TNBC cell line 4T1 and regulates the tumor microenvironment by attenuating angiogenesis and accumulation of MDSCs in 4T1 tumors. The underlying mechanism may involve the suppression of NF-κB activity.

ACT001 reduces the expression of PD-L1 by inhibiting the phosphorylation of STAT3 in glioblastoma.

ACT001, which is derived from an ancient anti-inflammatory drug, has been shown to cross the blood-brain barrier in preclinical studies and has demonstrated anti-glioblastoma (GBM) activity in clinical trials. However, its pharmacological potential for anti-GBM immune response modulation remains unclear. The chemical structure of ACT001 indicates that it may bind to STAT3 and thus modulate antitumor immune response. Methods: Bioinformatics and immunohistochemistry (IHC) were used to assess STAT3 and PD-L1 expression in gliomas. Western blotting, RT-PCR and immunofluorescence were used to detect PD-L1 and p-STAT3 expression in glioma cells exposed to ACT001. Chromatin immunoprecipitation, an ACT001-Biotin probe, and a dual-luciferase reporter assay were used to detect direct modulation. The in vivo efficacy of ACT001 was evaluated in GL261 murine glioma model. Survival analyses were conducted using the log-rank (Mantel-Cox) test. Results: Bioinformatic analysis of 1,837 samples from 4 public glioma datasets showed that STAT3 mRNA expression was correlated with the degree of malignancy and therapeutic resistance and that STAT3 mRNA expression was related to immunosuppression, leukocyte infiltration, and PD-L1 expression. IHC staining of 53 tissue samples confirmed that relatively high phosphorylated STAT3 and PD-L1 protein expression was associated with a relatively advanced World Health Organization (WHO) glioma grade. Next, we confirmed that ACT001 treatment reduced PD-L1 expression and STAT3 phosphorylation. An ACT001-biotin probe was used to verify that ACT001 bound to STAT3. We also demonstrated that STAT3 bound to the PD-L1 promoter. The inhibition of PD-L1 expression and STAT3 phosphorylation by ACT001 could be rescued by STAT3 overexpression. Additionally, ACT001 inhibited GBM growth and decreased PD-L1 expression in vivo. The expression of the M2 markers CD206 and CD163 was decreased, while that of the antitumor immune markers iNOS and IFNγ was increased by ACT001 in vivo. Conclusion: Our results demonstrate that STAT3 plays a key role in immunosuppression of glioma and is inhibited by ACT001. ACT001 inhibits PD-L1 transcription and modulates anti-tumor immune response in glioma bearing mice. These findings will help us to understand the mechanism of ACT001 in GBM therapy.

Anti-neuroinflammatory effects of dimethylaminomylide (DMAMCL, i.e., ACT001) are associated with attenuating the NLRP3 inflammasome in MPTP-induced Parkinson disease in mice.

Parthenolide (PTL) is a natural compound with anti-inflammatory and antioxidant properties and is an active ingredient extracted from the medicinal plant Tanacetum parthenium. ACT001 is derived from parthenolide and is a fumarate form of dimethylaminomylide (DMAMCL). Its effect is equivalent to that of PTL, but it is more stable in plasma and has lower acquisition costs. Related reports indicate that NLRP3-mediated neuroinflammation is involved in the progression of Parkinson's disease (PD). In our research, we explored whether ACT001 alleviates NLRP3-mediated neuroinflammation in PD mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our results revealed that ACT001 reduces movement impairment and cognitive deficit in PD mice. In addition, it alleviates dopaminergic neurodegeneration in the nigrostriatal pathway and inhibits oxidative stress, the inflammatory response and activation of the NLRP3 inflammasome in the midbrain of MPTP-induced PD mice. Moreover, it attenuates microglial activation in the nigrostriatal pathway. Overall, our study showed that ACT001 alleviates NLRP3-mediated neuroinflammation in PD mice induced by MPTP.

The parthenolide derivative ACT001 synergizes with low doses of L-DOPA to improve MPTP-induced Parkinson's disease in mice.

L-3,4-dihydroxyphenylalanine (L-DOPA) is currently the main drug used to treat Parkinson's disease (PD). However, long-term use of l-DOPA causes substantial side effects, and we hope to find a biological active ingredient that synergizes with a low-dose of l-DOPA to achieve the same therapeutic effect as that of a high-dose of l-DOPA. The natural product parthenolide (PTL) is the active ingredient in the medicinal plant feverfew (Tanacetum parthenium) and has antioxidant and anti-inflammatory properties. ACT001, a fumarate salt form of dimethylaminomicheliolide (DMAMCL), is a derivative of parthenolide and has comparable effects to those of PTL but exhibits higher stability in the plasma and is available at a lower cost. In our study, we used ACT001 in combination with l-DOPA to treat 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced Parkinson's disease in mice. Specifically, ACT001 significantly reduced motor dysfunction and dopaminergic neurodegeneration in MPTP-treated mice. Furthermore, ACT001 abolished MPTP-induced α-synuclein overexpression, astrocyte activation and interleukin-1ß (IL-1ß) production in the substantia nigra and striatum of the mouse brain. In addition, ACT001 increased the levels of the anti-apoptotic signalling molecule Bcl-2 and the pAkt/Akt ratio and reduced the levels of the pro-apoptotic signalling molecule Bax and the activation of Caspase3 in the substantia nigra and striatum. We found that the effects of the co-administration of ACT001 and l-DOPA (5 mg/kg) were equivalent to those of the administration of 8 mg/kg l-DOPA in MPTP-induced Parkinson's disease in mice. Then, this evidence suggests that l-DOPA + ACT001 may be used for the treatment of PD.