The quassinoids bruceines A-M: pharmacology, mechanism of action, synthetic advance, and pharmacokinetics-a review.

Bruceines A-L are among the quassinoid representatives found in the medicinal plant Brucea javanica (L.). An overview of their pharmacological activities is still unknown. The given research deals with highlights in their pharmacological result, molecular mechanism of action, synthetic progress, and pharmacokinetics. From previous evidence, bruceine derivatives are potential agents for anticancer treatments, as well as they are appropriate to treat inflammation, diabetes, and parasitic infections, and protect the neurons, kidneys, and lungs. Cytokine inhibitions, oxidative stress responses, and various signaling pathways, such as MAPK (mitogen-activated protein kinase) and NF-κB (nuclear factor-kappa B), have been proposed as the underlying mechanisms of action. Synthetic approaches to synthesize new derivatives with enhancement activities are based on free hydroxyl group modifications. Bruceines seem to be promptly absorbed by both oral and intravenous administrations, but their bioavailability is not high (less than 6%). Pre-clinical and clinical studies to prove their anticancer potential and other activities are urgent. Structural modifications, nano-combinations, and synergistic effects are necessary.

Bruceine A alleviates alcoholic liver disease by inhibiting AIM2 inflammasome activation via activating FXR.

BACKGROUND: Alcoholic liver disease (ALD), a public health challenge worldwide caused by long-term persistent drinking, is life-threatening with minimal approved therapies. Hepatic steatosis accompanied by inflammation is an initial and inevitable stage in the complex progression of simple alcoholic liver injury to more severe liver diseases such as hepatitis, liver fibrosis, cirrhosis and liver cancer. PURPOSE: We aimed to identify the therapeutic role of Bruceine A (BA) in ALD whilst attempting to explore whether its protective effects depend specifically on the farnesoid X receptor (FXR). METHODS: Autodock was applied to detect the affinity between BA and FXR. Lieber-DeCarli liquid diet with 5 % ethanol (v/v) was adopted to establish the mouse ALD model. The lentivirus mediating FXR (LV-FXR) was injected into mice via the tail vein to establish FXR-overexpressed mice. FXR silencing or overexpression plasmids were transfected into AML-12 cells prior to ethanol stimulation. Quantitative real-time PCR, Western blotting and immunofluorescence assays were employed to determine the expression of related genes. We subjected liver sections to H&E and Oil Red O staining to evaluate the liver histological injury and the deposition of lipid droplets. RESULTS: BA significantly reduced body weight and liver-to-body weight ratios as well as biochemical indexes in mice. Ethanol-induced liver damage and lipid accumulation could be alleviated by BA treatment. BA bound to FXR by two hydrogen bonds. There was a positive correlation between BA administration and FXR expression. BA inhibited the expression of lipid synthesis genes and enhanced the expression of lipid metabolism genes by activating FXR, thus alleviating steatosis in ALD. Moreover, BA exerted an ameliorative effect against inflammation by inhibiting the activation of absent in melanoma 2 (AIM2) inflammasome by activating FXR. FXR overexpression possessed the ability to counter the accumulation of lipid and the activation of AIM2 inflammasome caused by ethanol. FXR deficiency exacerbated ethanol-induced liver steatosis and inflammation. The hepatoprotective effect of BA could be disrupted by FXR antagonist guggulsterone (GS) in vivo and FXR siRNA in vitro. CONCLUSION: BA alleviated alcoholic liver disease by inhibiting AIM2 inflammasome activation through an FXR-dependent mechanism. This study may potentially represent a new therapeutic approach for ALD.

Discovery of Ten Anti-HIV Hit Compounds and Preliminary Pharmacological Mechanisms Studies.

BACKGROUND: The research and development of HIV drugs is very important, but at the same time it is a long cycle and expensive system project. High-throughput drug screening systems and molecular libraries of potential hit compounds remain the main ways for the discovery of hit compounds with anti-HIV activity. OBJECTIVE: The aim of this study was to screen out the hit compounds against HIV-1 in the natural product molecule library and the antiviral molecule library, and elucidate the molecular mechanism of their inhibition of HIV-1, so as to provide a new choice for AIDS drug research. METHODS: In this study, a drug screening system using HIV Rev-dependent indicator cell line (Rev-A3R5-GFP reporter cells) with pseudoviruses (pNL4-3) was used. The natural drug molecule library and antiviral molecule library were screened, and preliminary drug mechanism studies were performed. RESULTS: Ten promising hit compounds were screened. These ten molecules and their drug inhibitory IC50 were as follows: Cephaeline (0.50 µM), Yadanziolide A (8.82 µM), Bruceine D (2.48 µM), Astragaloside IV (4.30 µM), RX-3117 (1.32 µM), Harringtonine (0.63 µM), Tubercidin (0.41 µM), Theaflavine-3, 3'-digallate (0.41 µM), Ginkgetin (10.76 µM), ZK756326 (5.97 µM). The results of the Time of additions showed that except for Astragaloside IV and Theaflavine-3, 3'-digallate had a weak entry inhibition effect, and it was speculated that all ten compounds had an intracellular inhibition effect. Cephaeline, Harringtonine, Astragaloside IV, Bruceine D, and Tubercidin may have pre-reverse transcriptional inhibition. Yadanziolide A, Theaflavine-3, 3'-digallate, Ginkgetin and RX-3117 may be in the post-reverse transcriptional inhibition. The inhibitory effect of ZK 75632 may be in the reverse transcriptional process. CONCLUSION: A drug screening system using Rev-A3R5-GFP reporter cells with pseudoviruses (pNL4-3) is highly efficient. This study provided potential hit compounds for new HIV drug research.

Bruceine D inhibits the growth of Spodoptera litura by inducing cell apoptosis in the midgut via an oxidative burst.

BACKGROUND: Spodoptera litura is one of the most harmful lepidoptera pests in China, and is difficult to control due to its strong resistance to the current frequently used insecticide species. The requirement to develop pesticides with novel toxicology mechanisms to control S. litura is urgent. The quassinoid of bruceine D display outstanding systemic properties and strong insecticidal activity against S. litura, which possess notable application potential for integrative management of S. litura, but the mechanism of toxicity remains unclear. RESULTS: In this study, we found that bruceine D exerts potent growth inhibitory activity against S. litura, disrupting the ecdysone and juvenile hormone titers, and causing long-term adverse effects. Association analysis between transcriptomics and metabolomics suggested that bruceine D affected the digestion and absorption capacity of S. litura larvae by inducing a strong oxidative stress response and cell apoptosis in the intestine. Further analysis demonstrated that bruceine D can inhibit the activities of digestive and antioxidant enzymes and induce malondialdehyde (MDA) and reactive oxygen species (ROS) overaccumulation in the midgut. Moreover, the protein level of Bax, cleavage caspase 3, and cytochrome c expressed in cytoplasm (cyto) were up-regulated by bruceine D, while Bcl-2 and cytochrome c expressed in mitochondria (mito) were down-regulated. In addition, there was a noticeable increase in caspase-3 protease activity. Histopathological observations revealed that bruceine D damages the structure of midgut epithelial cells and activates lysosomes, which subsequently disrupts the midgut tissue. CONCLUSION: Overall, our findings suggested that bruceine D induced excessive ROS accumulation in midgut epithelial cells. The resulting cell apoptosis disrupted midgut tissue, leading ultimately to reduced nutrient digestion and absorption in the midgut and the inhibition of larval growth. © 2024 Society of Chemical Industry.

Bruceine A inhibited breast cancer proliferation and metastasis by inducing autophagy via targeting PI3K-AKT signaling pathway.

Although there have been significant advances in cancer treatment, the urgent need to inhibit breast cancer metastasis remained unmet. Bruceine A (BA) is a natural compound extracted from Bruceae Fructus and has long been recognized to have antitumor effects with high safety and biocompatibility. However, the mechanisms and/or targets of BA for metastatic breast cancer treatment are still not fully elucidated. In this study, we systematically investigated the effects of BA on inhibition of breast cancer metastasis and its underlying mechanisms. We found that, in addition to its cytotoxic effects, BA significantly inhibited the invasion and migration capabilities of two types of breast cancer cell lines (MDA-MB-231 and MCF-7) while concurrently promoting apoptosis in these cells. Further mechanistic studies revealed that, by targeting the canonical PI3K-AKT signaling pathway, BA initiated autophagy of both types of breast cancer cell lines in vitro. In vivo results further confirmed the in vitro findings, manifested by shrinkage of size and weight of breast tumor as well as initiation of autophagy (indicated by upregulation of LC3I/II) through targeting PI3K-AKT pathway on mice model. These data collectively demonstrated the potential of BA in antimetastasis of breast cancer cells, suggesting its future clinical transformation in metastatic breast cancer therapy.

Bruceine D suppresses CAF-promoted TNBC metastasis under TNF-α stimulation by inhibiting Notch1-Jagged1/NF-κB(p65) signaling.

BACKGROUND: Triple-negative breast cancer (TNBC) has a poor prognosis because of its high degree of malignancy and the lack of effective treatment options. Cancer-associated fibroblasts (CAFs) comprise the most abundant stromal cells in the tumor microenvironment (TME), leading to functional impairments and facilitating tumor metastasis. Excessive TNF-α further promotes cross-talk between different cells in TME. Therefore, there is an urgent need to develop more effective therapies and potential drugs that target the key factors that promote TNBC metastasis. PURPOSE: The study aimed to evaluate the efficacy of Bruceine D, an active compound derived from the Chinese herb Brucea javanica, in inhibiting metastasis and elucidate the underlying mechanism of action in TNBC. METHODS: In vitro, the clonogenic and the Transwell assays were used to assess the effects of Bruceine D on the proliferation, migration and invasion abilities of co-cultured CAFs and MDA-MB-231 (4T1) cells under TNF-α stimulation. TNF-α, IL-6, CXCL12, TGF-ß1, and MMP9 levels in the supernatant of co-cultured cells were determined using ELISA. Western blotting was utilized to detect the expression levels of proteins related to the Notch1-Jagged1/NF-κB(p65) pathway. In vivo, the anti-tumor growth and anti-metastatic effectiveness of Bruceine D was evaluated by determining tumor weight, number of metastatic lesions, and pathological changes in the tumor and lung/liver tissues. The inhibitory effect of Bruceine D on α-SMA+ CAFs activation and CAF-medicated extracellular matrix remodeling was accessed using immunohistochemistry, immunofluorescence, and Masson and Sirius Red staining. The expression levels of Notch1, Jagged1 and p-NF-κB(p65) proteins in the primary tumors were measured by immunohistochemistry and western blotting. RESULTS: In vitro, Bruceine D significantly inhibited the migration and invasion of co-cultured CAFs and MDA-MB-231 (4T1) cells under TNF-α stimulation, reduced the expression of tumor-promoting and matrix-remodeling cytokines secreted by CAFs, and hindered the mutual activation of Notch1-Jagged1 and NF-κB(p65). In vivo, Bruceine D significantly suppressed tumor growth and the formation of lung and liver metastases by decreasing TNF-α stimulated α-SMA+ CAFs activation, collagen fibers, MMPs production, and inhibited Notch1-Jagged1/NF-κB(p65) signaling in TNBC-bearing mice. CONCLUSION: Bruceine D effectively weakened the "tumor-CAF-inflammation" network by inhibiting the mutual activation of Notch1-Jagged1 and NF-κB(p65) and thereby suppressed TNBC metastasis. This study first explored that Bruceine D disrupted the cross-talk between CAFs and tumor cells under TNF-α stimulation to inhibit the metastasis of TNBC, and highlighted the potential of Bruceine D as therapeutic agent for suppressing tumor metastasis.

Brucine D restrains colorectal cancer tumorigenesis and autophagy by downregulating circ_0068464.

Bruceine D (BD) from Brucea javanica (L) exerts an antitumor effect in several human cancers. At present, it has not been reported whether BD inhibits the malignancy of colorectal cancer (CRC) cells. Therefore, investigating the role and regulatory mechanisms of BD in CRC is the main thrust of this study. Effect of BD on CRC cell viability, proliferation, apoptosis, invasion, and autophagy was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, 5-ethynyl-2'-deoxyuridine, flow cytometry, transwell invasion, and western blotting assays. Expression changes of has_circ_0068464 (circ_0068464) were detected using real time quantitative polymerase chain reaction. The molecular mechanisms related to circ_0068464 were predicted through online prediction websites Starbase 2.0, circinteractome, and CircBank and validated using dual-luciferase reporter and RNA pull-down assays. The tumorigenic ability of BD and circ_0068464 on CRC was confirmed by xenograft experiments. The results showed that BD lessened CRC cell proliferation, invasion, autophagy, and prompted cell apoptosis. Circ_0068464 was overexpressed in CRC samples and cells. BD led to a significant reduction in circ_0068464 levels in cells of this carcinoma, but circ_0068464 overexpression partially rescued these effects urged by BD. Also, the combination of BD and circ_0068464 silencing decreased xenograft tumor growth compared to BD alone. Importantly, circ_0068464 could regulate ATG5 expression by functioning as a miR-520h molecular sponge. In conclusion, BD might suppress CRC growth by inhibiting the circ_0068464/miR-520h/ATG5 axis, providing a new perspective for the molecular pathogenesis of CRC and preliminarily indicating that BD may be a promising drug for CRC treatment.

Bruceine A: Suppressing metastasis via MEK/ERK pathway and invoking mitochondrial apoptosis in triple-negative breast cancer.

Triple-negative breast cancer (TNBC), as the most aggressive subtype of breast cancer, presents a scarcity of miraculous drugs in suppressing its proliferation and metastasis. Bruceine A (BA) is a functional group-rich quassin compound with extensive and distinctive pharmacological activities. Within the present study, we investigated the capabilities of BA in suppressing TNBC proliferation and metastasis as well as its potential mechanisms. The results displayed that BA dramatically repressed the proliferation of MDA-MB-231 and 4T1 cells with corresponding IC50 values of 78.4 nM and 524.6 nM, respectively. Concurrently, BA arrested cells in G1 phase by downregulating cycle-related proteins Cyclin D1 and CDK4. Furthermore, BA distinctly induced mitochondrial dysfunction as manifested by diminished mitochondrial membrane potential, elevated reactive oxygen species generation, minimized ATP production, and Caspase-dependent activation of the mitochondrial apoptosis pathway. Additionally, BA restrained the invasion and metastasis of TNBC cells by repressing MMP9 and MMP2 expression. Intriguingly, after pretreatment with MEK activator C16-PAF, the inhibitory effect of BA on MEK/ERK pathway was notably diminished, while the proliferation suppression and metastasis repression exerted by BA were all strikingly curtailed. Molecular docking illustrated that BA potently combined with residues on the MEK1 protein with the presence of diverse intermolecular interactions. Ultimately, BA effectively suppressed tumor growth in the 4T1 xenograft tumor model with no detectable visceral toxicity in the high-dose group and, astonishingly, repressed tumor metastasis in the 4T1-luc lung metastasis model. Collectively, our study demonstrates that BA is a promising chemotherapeutic agent for treating TNBC and suppressing lung metastasis.

Inhibition of Hepatitis B Virus (HBV) replication and antigen expression by Brucea javanica (L.) Merr. oil emulsion.

Introduction: The seeds of Brucea javanica (L.) Merr. (BJ) have been traditionally used to treat various types of cancers for many years in China. In this study, we systematically investigated a BJ oil emulsion (BJOE) produced from BJ seeds with the purpose of evaluating its antiviral effect against hepatitis B virus (HBV). Methods: HepG2.215 (a wild-type HBV cell line), HepG2, and Huh7, transfected with wildtype (WT) or lamivudine-resistance mutant (LMV-MT) HBV replicon plasmids, were treated with different doses of BJOE and then used for pharmacodynamic evaluation. Cell viability was determined using CCK8 assay. The levels of HBsAg/HBeAg in cell cultured supernatant, HBcAg in cell lysis solution, and HBV DNA in both were evaluated. Results: BJOE at ≤5 mg/ml was nontoxic to carcinoma cell lines, but could significantly inhibit WT/LMV-MT HBV replication and HBs/e/c antigen expression in a dose-dependent manner by upregulating interleukin-6 (IL-6), demonstrating that it possesses moderate anti-HBV activity. As one of the major components of BJOE, bruceine B was found to play a dominant role in IL-6 induction and HBV inhibition. Discussion: Our results demonstrated that BJOE suppressed HBV replication by stimulating IL-6, indicating that it has promising clinical therapeutic potential for both WT and LMV-MT HBV.

Bruceine D Acts as a Potential Insecticide by Antagonizing 20E-EcR/USP Signal Transduction.

Bruceine D (BD) is an effective insecticidal compound found in the Chinese herb Brucea javanica (L.) Merr. BD inhibits the growth and metamorphosis of Plutella xylostella and Drosophila melanogaster; however, its target protein and the molecular mechanism of insecticidal activity remain unclear. In this study, proteins with high affinity for BD were screened using surface plasmon resonance and high-performance liquid chromatography coupled with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, revealing the ecdysone receptor (EcR) is the main target of BD. In vivo results showed that BD inhibited insect growth and metamorphosis through inhibition of the expression of 20E response genes. In vitro dual luciferase and enhanced green fluorescent protein (EGFP) fluorescence experiments indicated that BD suppressed the transcriptional activation activity of EcR by blocking the ecdysone response element (EcRE)-triggered transcriptional cascade, suggesting that BD inhibits the formation of the 20E-EcR-USP-EcRE complex. Moreover, molecular docking demonstrated that BD bound well to EcR. Elucidating the insecticidal mechanism of BD will be helpful in the development of green pesticides to control pests.

Bruceine a exerts antitumor effect against colon cancer by accumulating ROS and suppressing PI3K/Akt pathway.

Bruceine A (BA), a quassic ester from bruceine javanica, regulates diverse intracellular signal transduction pathways and manifests a variety of biological activities, however, its pharmacological mechanism in treating colon cancer (CC) is unclear. In this study, we investigated the anticancer effects of BA on CC cells and the underlying mechanisms. The network pharmacology research indicated that Akt1 and Jun and PI3K/Akt pathways are the predominant targets and critical signaling pathways, respectively, for BA treatment of CC. Meanwhile, molecular docking results implied that BA could conjugate to pivotal proteins in the PI3K/Akt pathway. BA remarkably suppressed the proliferation of CC cells HCT116 and CT26 with 48-h IC50 of 26.12 and 229.26 nM, respectively, and the expression of p-PI3K/p-Akt was restrained by BA at the molecular level as verified by Western blot assay. Further mechanistic studies revealed BA impacted cell cycle-related proteins by regulating the expression of P27 (a protein bridging the PI3K/Akt signaling pathway with cycle-related proteins), arresting the cell cycle in the G2 phase, inhibiting the proliferation of HCT116 and CT26, and facilitated the apoptosis in CC cells by activating the mitochondria-associated apoptosis protein Bax and accumulating reactive oxygen species, in addition to BA apparently inhibited the migration of CC cells. Taken together, our results demonstrated that BA might be a promising chemotherapy drug in the treatment of CC.

Anti-tumor Mechanisms of Bruceine D： A Review / 中国实验方剂学杂志

Malignant tumors seriously threaten human life and health. Radiotherapy and chemotherapy are the conventional methods for the clinical treatment of advanced tumors. The prognosis and efficacy are still far from satisfactory due to the radiotherapy has serious adverse effects on the body and the chemotherapy often causes problems such as tumor resistance and cell proliferationinhibition. Therefore， the search for new， safe， and effective anti-tumor drugs and the elucidation of their molecular mechanisms are effective measures for clinical treatment of tumors and improvement of patients' quality of life. Active ingredients derived from Chinese herbal medicines and natural products have gradually become a hot spot in the research and development of anti-tumor drugs due to their multi-target and multi-channel anti-tumor pharmacological activity characteristics and their advantages such as less adverse reaction on the body. Bruceine D is a class of tetracyclic triterpenoids extracted from the fruit of the Chinese herbal medicine Bruceae Fructus， with anti-inflammatory， anti-malarial， anti-parasitic， and other pharmacological activities， and its anti-tumor activity is particularly significant. Pharmacological studies have found that bruceine D can regulate various cellular physiological activities such as proliferation， apoptosis， invasion， and migration of lung cancer， liver cancer， pancreatic cancer， intestinal cancer， and other cancer cells by targeting different signaling pathways. Bruceine D can be used in combination with other chemotherapeutic drugs to improve the sensitivity of tumor cells to chemotherapeutic drugs， thereby reducing the adverse effect of chemotherapy. Clinical application practice has shown that Bruceae Fructus oil emulsion injection containing bruceine D has significant advantages in the efficacy and safety of tumor treatment. Although there are many studies on the antitumor pharmacological activity of bruceine D and its clinical efficacy is significant， the specific antitumor molecular mechanism of bruceine D is still unclear， and there is a lack of systematic review on the existing antitumor mechanism of bruceine D. Therefore， based on the research on bruceine D in China and abroad in recent years， this paper reviewed the anti-tumor effect and related molecular mechanisms of bruceine D from six aspects， namely， tumor cell proliferation， apoptosis， metastasis and invasion， glucose metabolism process， autophagy， and chemotherapy sensitivity. This paper is expected to provide a pharmacological basis and scientific reference for the antitumor drug development and clinical application of bruceine D.

Effects of KCNQ1OT1 Gene Knockout Combined with Bruceine D on Proliferation, Migration, and Invasion of Breast Cancer MDA-MB-231 Cells / 肿瘤防治研究

Objective To explore the effect of KCNQ1OT1 gene knockout combined with bruceine D on the proliferation, migration, and invasion of breast cancer MDA-MB-231 cells. Methods Cell Counting Kit-8, wound healing, and Transwell invasion assay were used to detect the effects of bruceine D and siKCNQ1OT1 on the viability, migration, and invasion of MDA-MB-231 cells. Effect of bruceine D and siKCNQ1OT1 on the expression of KCNQ1OT1 in MDA-MB-231 cells was detected by qRT-PCR. Western blot was used to detect the effect of bruceine D and siKCNQ1OT1 on the expression of EMT-related proteins and CDC42, p-MKK7, MKK7 proteins in MDA-MB-231 cells. Results Bruceine D and siKCNQ1OT1 could significantly inhibit the viability, migration, and invasion of MDA-MB-231 cells, and the inhibitory effect was enhanced when they were combined (all P < 0.05); bruceine D downregulated the expression of KCNQ1OT1 in MDA-MB-231 cells (all P < 0.05); bruceine D combined with siKCNQ1OT1 significantly decreased CDC42, p-MKK7, N-cadherin, and Vimentin expression in MDA-MB-231 cells and increased the expression of E-cadherin (all P < 0.05). Conclusion Bruceine D combined with siKCNQ1OT1 significantly inhibit the proliferation, migration, invasion, and EMT of human breast cancer MDA-MB-231 cells, and its molecular mechanism may be related to the blocking of CDC42/MKK7 signaling pathway.

Bruceine D may affect the phenylpropanoid biosynthesis by acting on ADTs thus inhibiting Bidens pilosa L. seed germination.

Bruceine D is a natural quassinoid, which was successfully isolated in our research group from the residue of Brucea javanica (L.) seeds. Our previous research showed that Bruceine D prevented Bidens pilosa L. seed germination by suppressing the activity of key enzymes and the expression levels of key genes involved in the phenylpropanoid biosynthesis pathway. In this study, integrated analyses of non-targeted metabolomic and transcriptomic were performed. A total of 356 different accumulated metabolites (DAMs) were identified, and KEGG pathway analyses revealed that most of these DAMs were involved in phenylpropanoid biosynthesis. The decreased expression of ADTs and content of L-phenylalanine implicates that Bruceine D may suppress the downstream phenylpropanoid biosynthesis pathway by disrupting primary metabolism, that is, the phenylalanine biosynthesis pathway, thus inhibiting the final products, resulting in the interruption of B. pilosa seed germination. These results suggest that Bruceine D may inhibit the B. pilosa seed germination by suppressing phenylpropanoid biosynthesis through acting on ADTs.

Bruceine A protects against diabetic kidney disease via inhibiting galectin-1.

Bruceine A is a natural quassinoid compound extracted from the fruit of the Traditional Chinese Medicine Brucea javanica (L.) Merr. that has various types of various biological activities. However, whether the compound has a protective effect on diabetic kidney disease remains unknown. Galectin-1 is actively involved in a variety of chronic inflammation-relevant human diseases including diabetic kidney disease. Here, we identified Bruceine A as a kidney protective molecule against a model of diabetic kidney disease in db/db mice with potent anti-inflammatory activity both in vitro and in vivo. Mechanistically, by selectively binding to the conserved carbohydrate-recognition domain of galectin-1 and disrupting the interaction between galectin-1 and the receptor for activated protein C kinase 1, Bruceine A was found to inhibit galectin-1-mediated inflammatory signal transduction under high glucose stress in rat mesangial HBZY-1 cells. Thus, our findings reveal Bruceine A as an unidentified galectin-1 inhibitor affording significant protection against diabetic kidney disease and may provide novel pharmacological therapeutics for the disease.

Bruceine H Mediates EGFR-TKI Drug Persistence in NSCLC by Notch3-Dependent ß-Catenin Activating FOXO3a Signaling.

Tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) protein serve as a critical pillar in the treatment of non-small cell lung cancer (NSCLC), but resistance is universal. Identifying the potential key factors of drug resistance to EGFR-TKIs is essential to treat patients with EGFR mutant lung cancer. Our research here shows that bruceine H suppressed the proliferation, migration, and invasion of lung cancer cells; inhibited the growth of human NSCLC cell xenografts; and enhanced the therapeutic effects of gefitinib in the PC-9/GR xenograft models, possibly by inhibiting Notch3. In order to analyze the potential targets of the combination of Notch3 and EGFR-TKIs on resistance to EGFR, we analyzed the differences of gene expression between NSCLC tissues and EGFR-driven gefitinib-resistant tumoral groups and then identify through the WGCNA key genes that may provide therapeutic targets for TKI-resistant lung cancer xenograft models. We confirmed that EGFR-TKI in combination with Notch3 inhibitor can inhibit the expression of ß-catenin and enhance the level of FOXO3a, leading to improved recurrence-free survival and overall survival of the xenotransplantation model. These results support that the combination of gefitinib and bruceine H may provide a promising alternative strategy for treating acquired EGFR-TKI resistance in patients with NSCLC.

Bruceine D Identified as a Drug Candidate against Breast Cancer by a Novel Drug Selection Pipeline and Cell Viability Assay.

The multi-target effects of natural products allow us to fight complex diseases like cancer on multiple fronts. Unlike docking techniques, network-based approaches such as genome-scale metabolic modelling can capture multi-target effects. However, the incompleteness of natural product target information reduces the prediction accuracy of in silico gene knockout strategies. Here, we present a drug selection workflow based on context-specific genome-scale metabolic models, built from the expression data of cancer cells treated with natural products, to predict cell viability. The workflow comprises four steps: first, in silico single-drug and drug combination predictions; second, the assessment of the effects of natural products on cancer metabolism via the computation of a dissimilarity score between the treated and control models; third, the identification of natural products with similar effects to the approved drugs; and fourth, the identification of drugs with the predicted effects in pathways of interest, such as the androgen and estrogen pathway. Out of the initial 101 natural products, nine candidates were tested in a 2D cell viability assay. Bruceine D, emodin, and scutellarein showed a dose-dependent inhibition of MCF-7 and Hs 578T cell proliferation with IC50 values between 0.7 to 65 µM, depending on the drug and cell line. Bruceine D, extracted from Brucea javanica seeds, showed the highest potency.

Bruceine D inhibits HIF-1α-mediated glucose metabolism in hepatocellular carcinoma by blocking ICAT/ß-catenin interaction.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths, characterized by highly hypoxic tumor microenvironment. Hypoxia-inducible factor-1α (HIF-1α) is a major regulator involved in cellular response to changes of oxygen levels, supporting the adaptation of tumor cells to hypoxia. Bruceine D (BD) is an isolated natural quassinoid with multiple anti-cancer effects. Here, we identified BD could significantly inhibit the HIF-1α expression and its subsequently mediated HCC cell metabolism. Using biophysical proteomics approaches, we identified inhibitor of ß-catenin and T-cell factor (ICAT) as the functional target of BD. By targeting ICAT, BD disrupted the interaction of ß-catenin and ICAT, and promoted ß-catenin degradation, which in turn induced the decrease of HIF-1α expression. Furthermore, BD could inhibit HCC cells proliferation and tumor growth in vivo, and knockdown of ICAT substantially increased resistance to BD treatment in vitro. Our data highlight the potential of BD as a modulator of ß-catenin/HIF-1α axis mediated HCC metabolism.

The Anthelmintic Quassinoids Ailanthone and Bruceine a Induce Infertility in the Model Organism Caenorhabditis elegans by an Apoptosis-like Mechanism Induced in Gonadal and Spermathecal Tissues.

In continuation of the search for new anthelmintic natural products, the study at hand investigated the nematicidal effects of the two naturally occurring quassinoids ailanthone and bruceine A against the reproductive system of the model nematode Caenorhabditis elegans to pinpoint their anthelmintic mode of action by the application of various microscopic techniques. Differential Interference Contrast (DIC) and the epifluorescence microscopy experiments used in the presented study indicated the genotoxic effects of the tested quassinoids (c ailanthone = 50 µM, c bruceine A = 100 µM) against the nuclei of the investigated gonadal and spermathecal tissues, leaving other morphological key features such as enterocytes or body wall muscle cells unimpaired. In order to gain nanoscopic insight into the morphology of the gonads as well as the considerably smaller spermathecae of C. elegans, an innovative protocol of polyethylene glycol embedding, ultra-sectioning, acridine orange staining, tissue identification by epifluorescence, and subsequent AFM-based ultrastructural data acquisition was applied. This sequence allowed the facile and fast assessment of the impact of quassinoid treatment not only on the gonadal but also on the considerably smaller spermathecal tissues of C. elegans. These first-time ultrastructural investigations on C. elegans gonads and spermathecae by AFM led to the identification of specific quassinoid-induced alterations to the nuclei of the reproductive tissues (e.g., highly condensed chromatin, impaired nuclear membrane morphology, as well as altered nucleolus morphology), altogether implying an apoptosis-like effect of ailanthone and bruceine A on the reproductive tissues of C. elegans.

Bruceine D ameliorates the balance of Th1/Th2 in a mouse model of ovalbumin-induced allergic asthma via inhibiting the NOTCH pathway.

Allergic asthma is a heterogeneous inflammatory disorder triggered by inhaled allergens, leading to airflow obstruction, bronchial inflammation, and airway hyperresponsiveness (AHR). T helper (Th) 2 cell-mediated immune response and airway inflammation are the key features of allergic asthma. Bruceine D (BD) is a bioactive compound extracted from the seeds of Brucea javanica. The present study aimed to investigate the effects of increased doses of BD on AHR, secretion of Th1-/Th2-associated cytokines, and inflammatory cell infiltration in ovalbumin (OVA)-induced allergic asthma mice. The results showed that BD reduced OVA-induced inflammatory cell infiltration and bronchial hyperresponsiveness into the peribronchial tissues and perivascular areas. Mice treated with BD also showed significantly decreased expressions of Th2-associated cytokines (i.e., interleukin (IL)-4, IL-5, and IL-13) and elevated production of Th1-associated cytokines (i.e., interferon gamma and IL-2) following OVA stimulation. BD treatment dose-dependently inhibited OVA-induced accumulation of inflammatory cells in asthmatic mice. Further analysis revealed that OVA exposure upregulated pulmonary expressions of NOTCH signaling receptors, a group of transmembrane proteins that communicate signals upon binding to transmembrane ligands expressed on adjacent cells, while BD treatment significantly abolished OVA-induced activation of the NOTCH pathway. In conclusion, BD protected mice against OVA-induced allergic asthma by reducing AHR and restoring the Th1/Th2 balance through the NOTCH signaling pathway. Our findings highlighted the potential of BD as a therapeutic agent for allergic asthma.