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Currently, primaquine is the only malaria transmission-blocking drug recommended by the WHO. Recent efforts have highlighted the importance of discovering new agents that regulate malarial transmission, with particular interest in agents that can be administered in a single low dose, ideally with a discrete and Plasmodium-selective mechanism of action. Here, our team demonstrates an approach to identify malaria transmission-blocking agents through a combination of in vitro screening and in vivo analyses. Using a panel of natural products, our approach identified potent transmission blockers, as illustrated by the discovery of the transmission-blocking efficacy of brusatol. As a member of a large family of biologically active natural products, this discovery provides a critical next step toward developing methods to rapidly identify quassinoids and related agents with valuable pharmacological therapeutic properties.

Chemoresistance to gemcitabine (Gem) remains a substantial obstacle in the treatment of pancreatic ductal adenocarcinoma carcinoma (PDAC). Nrf2, a transcription factor responsive to oxidative stress, has been implicated as a key contributor to chemoresistance. Previous studies have demonstrated anti-tumor effects of brusatol (BRT) in PDAC. Herein, we aimed to investigate the efficacy of BRT in enhancing chemosensitivity to Gem and to elucidate the underlying mechanisms involving Nrf2. Gain- and-loss-of-function experiments revealed that Nrf2 exacerbated Gem chemoresistance in PDAC cells. Additionally, BRT effectively inhibited PDAC cell proliferation and enhanced Gem chemosensitivity. Mechanistic investigations demonstrated that BRT sensitized PDAC cells to Gem by suppressing Nrf2 at the transcriptional level. Activation of Nrf2 conteracted BRT’s effects on chemosensitivity. In contrast, combination treatment with Nrf2 silencing and BRT demonstrated a more potent inhibitory effect on Gem in vitro and in vivo, thereby indicating the Nrf2 dependence of BRT action. These findings highlight BRT’s ability to enhance Gem chemosensitivity by inhibiting Nrf2 signaling in PDAC; therefore, BRT may serve as a potential adjuvant therapy for PDAC.

Objective: Brusatol (BT) is a quassinoid compound extracted from Brucea javanica that is a traditional Chinese herbal medicine. Brusatol possesses biological and medical activity, including antitumor, antileukemia, anti-inflammatory, antitrypanosomal, antimalarial, and antitobacco mosaic virus activity. To summarize and discuss the antitumor effects of BT and its mechanisms of actions, we compiled this review by combining the extensive relevant literature and our previous studies. Methods: We searched and retrieved the papers that reported the pharmacological effects of BT and the mechanism of BT antitumor activity from PubMed until July 2023. Key findings: Numerous studies have shown that BT is a unique nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor that acts on various signaling pathways and has good antitumor properties. Brusatol shows great potential in cancer therapy by inhibiting cell proliferation, blocking the cell cycle, promoting tumor cell differentiation, accelerating tumor cell apoptosis, inducing autophagy, suppressing angiogenesis, inhibiting tumor invasion and metastasis, and reversing multidrug resistance. Conclusion: This review summarizes recent updates on the antitumor activity and molecular mechanisms of BT and provides references for future development and clinical translation of BT and its derivatives as antitumor drugs. [ABSTRACT FROM AUTHOR]

BackgroundSilicosis is a diffuse fibrosis of the lungs caused by long-term inhalation of free silicon dioxide (SiO2). It has a complex pathogenesis and lacks effective treatment. Brusatol (Bru) has a variety of biological activities, and its role in silicosis fibrosis is unclear yet. ObjectiveTo investigate the effects of different concentrations of Bru on SiO2-induced silicosis fibrosis in mice. MethodsThirty male C57BL/6J mice were randomly divided into five groups: a control group, a silica group, and three Bru intervention groups with low, medium, and high doses (1, 2, and 4 mg·kg−1), with 6 mice in each group. Except the control group, the remaining groups were established as SiO2-induced silicosis mouse models by using a single tracheal infusion of 50 μL 60 mg·mL−1 SiO2 suspension. The control group was dosed with equal amount of saline. The Bru intervention groups were injected intraperitoneally with Bru for 5 consecutive days and then injected every other day. After 28 d of exposure, the mice were executed and lung tissues were collected. The lung coefficient of the mice was measured, and the pathological changes of the lung tissues were observed after hematoxylin-eosin (HE) and Masson staining. The levels of apoptotic protein Cleaved-caspase 3, fibrosis-related protein α-smooth muscle actin (α-SMA), type I collagen (Col-I), autophagy-associated protein Beclin1, microtubule-associated protein 1 light chain 3 (LC3), Sequestosome 1 (p62/SQSTM1), Kelch like ECH-associated protein-1 (Keap1), and nuclear factor erythroid 2 related factor 2 (Nrf2) were detected by Western blot. The mRNA levels of Caspase 3, α-SMA, and Col-I were measured by realtime fluorescence-based quantitative PCR. ResultsCompared with the control group, the lung coefficient of mice in the silica group was significantly increased (P < 0.01); the lung tissues of the silicosis mice showed damaged alveolar walls, along with infiltration of inflammatory cells, fibrous nodules, and collagen deposition; furthermore, the protein and mRNA levels of Cleaved-caspase 3, α-SMA, and Col-I were significantly increased (P < 0.01); the expression levels of Beclin1, LC3-II/I, p62, and Nrf2 were increased, while that of Keap1 was decreased (P < 0.05). The interventions with low and medium doses of Bru reduced lung coefficient (P < 0.05) and protected against pathological damage and collagen deposition in the lung tissues of the silicosis mice; the protein and mRNA expression levels of Cleaved-caspase 3, α-SMA, and Col-I were significantly decreased in the low and medium dose groups (P < 0.05, P < 0.01), the expression levels of Beclin1, LC3-II/I, p62, and Nrf2 were also decreased (P < 0.05, P < 0.01), and the expression level of Keap1 was increased in the medium dose group (P < 0.05). However, compared with the silica group, the differences in lung coefficient, pathological damage, and protein and mRNA expression levels of Cleaved-caspase 3, α-SMA, and Col-I in the Bru high dose group were not statistically significant (P > 0.05). In addition, the high dose of Bru decreased Beclin1, LC3-II/I, and Nrf2 expression levels (P < 0.01), did not change p62 protein expression level (P > 0.05), while increased Keap1 protein level (P < 0.01). ConclusionLow and medium doses of Bru might regulate autophagy through the Keap1-Nrf2 pathway, ameliorate autophagic degradation impairment, reduce pulmonary coefficient, attenuate apoptosis, and delay the progression of fibrosis in SiO2-induced silicosis mice.

Ferroptosis is a novel form of programmed cell death that is triggered by iron‐dependent lipid peroxidation. Brusatol (BRU), a natural nuclear factor erythroid 2‐related factor 2 inhibitor, exhibits potent anticancer effects in various types of cancer. However, the exact mechanism of BRU in the treatment of hepatocellular carcinoma (HCC) remains unknown. The anticancer effects of BRU in HCC were detected using cell counting kit‐8 and colony formation assays and a xenograft model. RNA sequencing (RNA‐seq) and bioinformatics analyses of HCC cells were utilized to elucidate the mechanism underlying the effects of BRU in HCC. The levels of reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and Fe2+ were measured using assay kits. The expression of activating transcription factor 3 (ATF3) was tested using RT‐qPCR, western blotting, and immunofluorescence staining. The role of ATF3 in BRU‐induced ferroptosis was examined using siATF3. BRU significantly inhibited HCC cell proliferation, both in vitro and in vivo. BRU activated the ferroptosis signaling pathway and increased ATF3 expression. Furthermore, ATF3 knockdown impeded BRU‐induced ferroptosis. BRU suppressed HCC growth through ATF3‐mediated ferroptosis, supporting BRU as a promising therapeutic agent for HCC. [ABSTRACT FROM AUTHOR]

Changes during the production cycle of dairy cattle can leave these animals susceptible to oxidative stress and reduced antioxidant health. In particular, the periparturient period, when dairy cows must rapidly adapt to the sudden metabolic demands of lactation, is a period when the production of damaging free radicals can overwhelm the natural antioxidant systems, potentially leading to tissue damage and reduced milk production. Central to the protection against free radical damage and antioxidant defense is the transcription factor NRF2, which activates an array of genes associated with antioxidant functions and cell survival. The objective of this study was to evaluate the effect that two natural NRF2 modulators, the NRF2 agonist sulforaphane (SFN) and the antagonist brusatol (BRU), have on the transcriptome of immortalized bovine mammary alveolar cells (MACT) using both the RT-qPCR of putative NRF2 target genes, as well as RNA sequencing approaches. The treatment of cells with SFN resulted in the activation of many putative NRF2 target genes and the upregulation of genes associated with pathways involved in cell survival, metabolism, and antioxidant function while suppressing the expression of genes related to cellular senescence and DNA repair. In contrast, the treatment of cells with BRU resulted in the upregulation of genes associated with inflammation, cellular stress, and apoptosis while suppressing the transcription of genes involved in various metabolic processes. The analysis also revealed several novel putative NRF2 target genes in bovine. In conclusion, these data indicate that the treatment of cells with SFN and BRU may be effective at modulating the NRF2 transcriptional network, but additional effects associated with cellular stress and metabolism may complicate the effectiveness of these compounds to improve antioxidant health in dairy cattle via nutrigenomic approaches. [ABSTRACT FROM AUTHOR]

The activity of known modulators of the Nrf2 signaling pathway (bardoxolone and brusatol) was studied on cultures of tumor organoids of metastatic colorectal cancer previously obtained from three patients. The effect of modulators was studied both as monotherapy and in combination with standard chemotherapy drugs used to treat colorectal cancer. The Nrf2 inhibitor brusatol and the Nrf2 activator bardoxolone have antitumor activity. Moreover, bardoxolone and brusatol also significantly enhance the effect of the chemotherapy drugs 5-fluorouracil, oxaliplatin, and irinotecan metabolite SN-38. Thus, bardoxolone and brusatol can be considered promising candidates for further preclinical and clinical studies in the treatment of colorectal cancer. [ABSTRACT FROM AUTHOR]

Challenges to docetaxel use in prostate cancer treatment include several resistance mechanisms as well as toxicity. To overcome these challenges and to improve the therapeutic efficacy in heterogeneous prostate cancer, the use of multiple agents that can destroy different subpopulations of the tumor is required. Brusatol, a multitarget inhibitor, has been shown to exhibit potent anticancer activity and play an important role in drug response and chemoresistance. Thus, the combination of brusatol and docetaxel in a nanoparticle platform for the treatment of prostate cancer is expected to produce synergistic effects. In this study, we reported the development of polymeric nanoparticles for the delivery of brusatol and docetaxel in the treatment of prostate cancer. The one-factor-at-a-time method was used to screen for formulation and process variables that impacted particle size. Subsequently, factors that had modifiable effects on particle size were evaluated using a 24 full factorial statistical experimental design followed by the optimization of drug loading. The optimization of blank nanoparticles gave a formulation with a mean size of 169.1 nm ± 4.8 nm, in agreement with the predicted size of 168.333 nm. Transmission electron microscopy showed smooth spherical nanoparticles. The drug release profile showed that the encapsulated drugs were released over 24 h. Combination index data showed a synergistic interaction between the drugs. Cell cycle analysis and the evaluation of caspase activity showed differences in PC-3 and LNCaP prostate cancer cell responses to the agents. Additionally, immunoblots showed differences in survivin expression in LNCaP cells after treatment with the different agents and formulations for 24 h and 72 h. Therefore, the nanoparticles are potentially suitable for the treatment of advanced prostate cancer. [ABSTRACT FROM AUTHOR]

Renal cell carcinoma (RCC) is the most aggressive subtype of kidney tumour with a poor prognosis and an increasing incidence rate worldwide. Brusatol, an essential active ingredient derived from Brucea javanica, exhibits potent antitumour properties. Our study aims to explore a novel treatment strategy for RCC patients. We predicted 37 molecular targets of brusatol based on the structure of brusatol, and MEF2A (Myocyte Enhancer Factor 2A) was selected as our object through bioinformatic analyses. We employed various experimental techniques, including RT‐PCR, western blot, CCK8, colony formation, immunofluorescence, wound healing, flow cytometry, Transwell assays and xenograft mouse models, to investigate the impact of MEF2A on RCC. MEF2A expression was found to be reduced in patients with RCC, indicating a close correlation with MEF2A deubiquitylation. Additionally, the protective effects of brusatol on MEF2A were observed. The overexpression of MEF2A inhibits RCC cell proliferation, invasion and migration. In xenograft mice, MEF2A overexpression in RCC cells led to reduced tumour size compared to the control group. The underlying mechanism involves the inhibition of RCC cell proliferation, invasion, migration and epithelial‐mesenchymal transition (EMT) through the modulation of Wnt/β‐catenin signalling. Altogether, we found that MEF2A overexpression inhibits RCC progression by Wnt/β‐catenin signalling, providing novel insight into diagnosis, treatment and prognosis for RCC patients. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this study was to investigate the inhibitory effect of brusatol, a nigakilactone extracted from Brucea javanica, on lung cancer for development of therapeutic drugs. We explored the effects of brusatol on the proliferation, migration, and invasion of lung cancer PC-9 cells in vitro and analyzed the mechanisms involved. Materials and Methods: MTT assay was used to determine the effect of brusatol on the proliferative capacity of PC-9 and H1975 cells in vitro. The half-maximal inhibitory concentrations (IC50) were calculated and used as a reference for subsequent experiments. Variations in the number and size of tumor cell clusters were monitored by the colony formation assay as evidence for the effect of brusatol on cell proliferation. The effect of brusatol on the migration and invasion of PC-9 cells was evaluated using wound healing and transwell assays, respectively. Apoptosis in lung cancer cells was detected using the Annexin V-FITC/propidium iodide assay. The correlated anticancer mechanism was detected using Western blotting. Results: The IC50 values of brusatol acting on PC-9 and H1975 cells were 1.58 ± 0.30 μM and 31.34 ± 2.72 μM, respectively, according to the MTT experiment. In addition, brusatol suppressed PC-9 cell proliferation, migration, and invasion, as well as induced apoptosis, which may be related to the downregulation of epidermal growth factor receptor (EGFR), β-catenin, Akt, and STAT3 expression. Conclusions: Brusatol showed potent anticancer activity against lung cancer PC-9 cells, inhibiting the proliferative capacity and metastatic potential of PC-9 cells. Its anticancer effect may be related to the downregulation of EGFR, β-catenin, Akt, and STAT3.

Triple-negative breast cancer (TNBC) has been clearly recognized as a heterogeneous tumor with the worst prognosis among the subtypes of breast cancer (BC). The advent and application of current small-molecule drugs for treating TNBC, as well as other novel inhibitors, among others, have made treatment options for TNBC more selective. However, there are still problems, such as poor patient tolerance, large administration doses, high dosing frequency, and toxic side effects, necessitating the development of more efficient and less toxic treatment strategies. High expression of Nrf2, a vital antioxidant transcription factor, often promotes tumor progression, and it is also one of the most effective targets in BC therapy. We found that in MDA-MB-231 cells and SUM159 cells, brusatol (BRU) combined with polydatin (PD) could significantly inhibit cell proliferation in vitro, significantly downregulate the expression of Nrf2 protein as well as the expression of downstream related target genes Heme Oxygenase-1 (HO-1) and NAD(P)H dehydrogenase, quinone 1 (NQO1), and promote reactive oxygen species (ROS) levels to further strengthen the anti-tumor effect. Furthermore, we discovered in our in vivo experiments that by reducing the drug dosage three times, we could significantly reduce tumor cell growth while avoiding toxic side effects, providing a treatment method with greater clinical application value for TNBC treatment. [ABSTRACT FROM AUTHOR]

Challenges to docetaxel use in prostate cancer treatment include several resistance mechanisms as well as toxicity. To overcome these challenges and to improve the therapeutic efficacy in heterogeneous prostate cancer, the use of multiple agents that can destroy different subpopulations of the tumor is required. Brusatol, a multitarget inhibitor, has been shown to exhibit potent anticancer activity and play an important role in drug response and chemoresistance. Thus, the combination of brusatol and docetaxel in a nanoparticle platform for the treatment of prostate cancer is expected to produce synergistic effects. In this study, we reported the development of polymeric nanoparticles for the delivery of brusatol and docetaxel in the treatment of prostate cancer. The one-factor-at-a-time method was used to screen for formulation and process variables that impacted particle size. Subsequently, factors that had modifiable effects on particle size were evaluated using a 24 full factorial statistical experimental design followed by the optimization of drug loading. The optimization of blank nanoparticles gave a formulation with a mean size of 169.1 nm ± 4.8 nm, in agreement with the predicted size of 168.333 nm. Transmission electron microscopy showed smooth spherical nanoparticles. The drug release profile showed that the encapsulated drugs were released over 24 h. Combination index data showed a synergistic interaction between the drugs. Cell cycle analysis and the evaluation of caspase activity showed differences in PC-3 and LNCaP prostate cancer cell responses to the agents. Additionally, immunoblots showed differences in survivin expression in LNCaP cells after treatment with the different agents and formulations for 24 h and 72 h. Therefore, the nanoparticles are potentially suitable for the treatment of advanced prostate cancer.

Cancer is a consequence of uncontrolled cell proliferation that is associated with cell-cycle disruption. It is a multifactorial disease that depends on the modulation of numerous oncogenic signaling pathways and targets. Although a battle against cancer has been waged for centuries, this disease remains a major cause of death worldwide. Because of the development of resistance to current anticancer drugs, substantial effort has been focused on discovering more effective agents for tumor therapy. Natural products have powerful prospects as anticancer drugs. Brusatol, a component isolated from the plant Brucea javanica, has been demonstrated to efficiently combat a wide variety of tumors. Extensive studies have indicated that brusatol exhibits anticancer effects by arresting the cell cycle; promoting apoptosis; inducing autophagy; attenuating epithelial-mesenchymal transition; inhibiting migration, invasion and angiogenesis; and increasing chemosensitivity and radiosensitivity. These effects involve various oncogenic signaling pathways, including the MAPK, NF-κB, PI3K/AKT/mTOR, JAK/STAT and Keap1/Nrf2/ARE signaling pathways. This review describes the evidence suggesting that brusatol is a promising drug candidate for cancer therapeutics.

Hepatocellular carcinoma (HCC) is a common malignancy with a poor prognosis. The recommended treatment of unresectable HCC involves targeted therapy, for example sorafenib, combined with immunotherapy. A recent article reported that sorafenib could induce ferroptosis escape in HCC. Brusatol is a novel Nrf2 inhibitor that takes effects in various diseases. In our study, we aimed to identify whether the addition of Brusatol to sorafenib could reverse ferroptosis escape in Huh7 cells. The cultured Huh7 cells treated by sorafenib with or without Brusatol addition were harvested for ferroptotic phenotype experiments and ferroptosis-related markers such as GPX4 and SLC7A11 were detected. In vivo experiments were conducted to discover the effect of Brusatol in combination with sorafenib in liver tumor bearing mice. Mechanism signaling pathways were detected by RNA-sequencing. Brusatol alone could induce Huh7 cell death and sorafenib could moderately mediate Huh7 cell ferroptosis by paradoxically inhibiting GPX4. However, sorafenib simultaneously upregulates Nrf2 signaling in Huh7 cells fighting against ferroptosis to result in sorafenib resistance. The addition of Brusatol could potentiate ferroptosis in Huh7 cells through downregulating Nrf2 and the downstream HO-1 and NQO1, thus enhancing the efficacy of sorafenib, which could be reversed by ferrostatin-1 treatment. In conclusion, Brusatol improves the efficacy of sorafenib by inducing ferroptosis via hindering Nrf2 signaling activation in HCC. [Display omitted] • Brusatol triggers cell death in Huh7 cells in a dose-dependent manner. • Brusatol potentiates sorafenib-induced ferroptosis in Huh7 cells and tumor-bearing mice. • Brusatol improves the efficacy of sorafenib by hindering Nrf2 signaling activation. [ABSTRACT FROM AUTHOR]

Combing PD-1/PD-L1 immune checkpoint inhibitors with natural products has exhibited better efficacy than monotherapy. Hence, the purpose of this research was to examine the anti-cancer effects of brusatol, a natural quassinoid-terpenoid derived from Brucea javanica , when used in conjunction with an anti-mouse-PD-1 antibody in a murine head and neck squamous cell carcinoma (HNSCC) model and elucidate underlying mechanisms. A murine HNSCC model and an SCC-15 cell xenograft nude mouse model were established to investigate the anti-cancer effects of brusatol and anti-PD-1 antibody. Mechanistic studies were performed using immunohistochemistry. Cell proliferation, migration, colony formation, and invasion were evaluated by MTT, migration, colony formation, and transwell invasion assays. PD-L1 levels in oral squamous cell carcinoma (OSCC) cells were assessed through qRT-PCR, flow cytometry, and western blotting assays. The impact of brusatol on Jurkat T cell function was assessed by an OSCC/Jurkat co-culture assay. Brusatol improved tumor suppression by anti-PD-1 antibody in HNSCC mouse models. Mechanistic studies revealed brusatol inhibited tumor cell growth and angiogenesis, induced apoptosis, increased T lymphocyte infiltration, and reduced PD-L1 expression in tumors. Furthermore, in vitro assays confirmed brusatol inhibited PD-L1 expression in OSCC cells and suppressed cell migration, colony formation, and invasion. Co-culture assays indicated that brusatol's PD-L1 inhibition enhanced Jurkat T cell-mediated OSCC cell death and reversed the inhibitory effect induced by OSCC cells. Brusatol improves anti-PD-1 antibody efficacy by targeting PD-L1, suggesting its potential as an adjuvant in anti-PD-1 immunotherapy. • Brusatol improves anti-PD-1 therapy efficacy in a murine head and neck squamous cell carcinoma model. • Brusatol promotes T lymphocyte infiltration into tumor tissues. • Brusatol inhibits programmed cell death 1 ligand 1 expression in oral squamous cell carcinoma cells. [ABSTRACT FROM AUTHOR]

Three fluorinated quassinoid analogues—4α-fluorobruceosin, 1α-fluorobrusatol, and 1-fluorodehydrobrusatol—were synthesized from brusatol, bruceosin, and dehydrobrusatol, respectively, by treatment with Selectfluor. These analogues were less cytotoxic to HL-60 cells than their non-fluorinated counterparts.

Esophageal adenocarcinoma (EAC), the predominant type of esophageal cancer in the United States, develops through Barrett's esophagus (BE)-dysplasia-carcinoma cascade. Gastroesophageal reflux disease, where acidic bile salts refluxate into the esophagus, is the main risk factor for the development of BE and its progression to EAC. The NFE2-related factor 2 (NRF2) is the master cellular antioxidant regulator. We detected high NRF2 protein levels in the EAC cell lines and primary tissues. Knockdown of NRF2 significantly enhanced acidic bile salt-induced oxidative stress, DNA damage, and inhibited EAC cell growth. Brusatol, an NRF2 inhibitor, significantly inhibited NRF2 transcriptional activity and downregulated the NRF2 target genes. We discovered that in addition to inducing apoptosis, Brusatol alone or in combination with cisplatin (CDDP) induced significant lipid peroxidation and ferroptosis, as evidenced by reduced xCT and GPX4 expression, two known ferroptosis markers. The combination of Brusatol and CDDP significantly inhibited EAC tumor xenograft growth in vivo and confirmed the in vitro data showing ferroptosis as an important mechanism in the tumors treated with Brusatol or Brusatol and CDDP combination. Our data support the role of NRF2 in protecting against stress-induced apoptosis and ferroptosis in EACs. Targeting NRF2 in combination with platinum therapy can be an effective strategy for eliminating cancer cells in EAC. [ABSTRACT FROM AUTHOR]

Overexpression of programed death-ligand 1 (PD-L1) is associated with poor prognosis in leukemia. Moreover, antitumor pharmaceuticals have been shown to induce immunoresistance, leading to reduced efficacy. Previous studies have indicated that arsenic trioxide (ATO) promotes immune evasion by inducing PD-L1 expression in solid tumors; however, little is known about its role in leukemia. A proportion of patients with acute promyelocytic leukemia were resistant to ATO therapy. Thus, this study aimed to investigate the effect of ATO on the expression of PD-L1 in leukemia cells and the underlying mechanism mediated through the nuclear factor erythroid 2 related factor (NRF2) protein. Brusatol, extracted from Brucea javanica, was selected as a unique NRF2 inhibitor, and we evaluated the possibility of using a regimen combining ATO/Brusatol in leukemia therapy. Promyelocytic NB4 and lymphocytic Jurkat cells were treated with ATO and brusatol either alone or in combination. We found that ATO significantly upregulated the expression of PD-L1 in NB4 and Jurkat cells at both the protein and mRNA levels compared with its expression in the untreated cell group. Mechanistically, ATO increased nuclear NRF2 expression and the extent of NRF2 binding to the PD-L1 promoter. Pharmacological inhibition of NRF2 by brusatol significantly blocked this effect, thereby reducing ATO-induced PD-L1 expression. In addition, the combination of brusatol and ATO showed stronger cytotoxicity than ATO alone indicated by cell counting kit-8 assay. Therefore, brusatol may further enhance the antileukemia effect of ATO not only by inhibiting ATO-induced PD-L1 expression but also by enhancing ATO-induced cytotoxicity. Our study provides a rationale for the clinical application of ATO/brusatol combination therapy. [ABSTRACT FROM AUTHOR]

Brusatol, a major quassinoid extract of Bruceae fructus, is an important bioactive component with antineoplastic capacity. Several beneficial pharmacological and biological properties of brusatol have been uncovered to date, including anti-inflammatory, anticolitis, antimalarial, and anticancer activities. To confer anticancer benefits, brusatol is reported to effectively inhibit the Nrf2-mediated antioxidant response and trigger apoptotic signaling. In this study, we investigated the regulatory mechanisms underlying apoptotic processes in brusatol-treated A549 cells in detail. Our experiments showed that brusatol induces cell death through intracellular ROS-triggered mitochondria-dependent apoptotic events and does not involve necrosis. Mechanistically, p21-activated protein kinase 2 (PAK2) was cleaved by caspase-3 to generate an activated p34 fragment involved in brusatol-induced apoptosis of A549 cells. Notably, PAK2 knockdown led to downregulation of caspase-3-mediated PAK2 activity, in turn, effectively attenuating brusatol-induced apoptosis, highlighting a crucial role of caspase-3-activated PAK2 in this process. Moreover, knockdown of PAK2 resulted in significant inhibition of c-Jun N-terminal kinase (JNK) activity in brusatol-treated A549 cells, clearly suggesting that JNK serves as a downstream substrate of caspase-3-cleaved/activated PAK2 in the apoptotic cascade. SP600125, a specific JNK inhibitor, significantly suppressed brusatol-induced JNK activity but only partially prevented apoptosis, implying that JNK serves as only one of a number of substrates for PAK2 in the brusatol-triggered apoptotic cascade. Based on the collective results, we propose a signaling cascade model for brusatol-induced apoptosis in human A549 cells involving ROS, caspases, PAK2, and JNK. [ABSTRACT FROM AUTHOR]

Brusatol is a natural quassinoid terpenoid that shows a potential therapeutic use in treating various ailments. Recently, brusatol was shown to have anti-tumor activity and ameliorating chemoresistance or enhancing chemotherapeutic drug efficacy in vitro and in vivo cancer models, but its molecular mechanisms remained to be clarified. Here, we aim to investigate the roles of brusatol in oral squamous cell carcinoma (OSCC) cells and its possible mechanism, especially on tumor cell glycolytic metabolism. In this study, we found that brusatol exerted significant anti-tumor effects in OSCC cells by repressing tumor cells proliferation, cycles, migration, invasion, and colony formation and inducing cell apoptosis in vitro through MTT, cell cycle, cell migration and invasion, colony formation and cell apoptosis assays. The result of metabolic assay and Seahorse XF glycolysis stress test assay showed that brusatol directly targeted glycolysis by inhibiting the extracellular acidification rate represented by a decreased glycolysis, glycolytic capacity and reserve capacity, and repressing activity of the key glycolytic enzymes pyruvate kinase and hexokinase. We also found that the expression of glucose transporter 1 (GLUT1) and several important glycolysis related genes, including HIF-1α and c-Myc was downregulated by brusatol through real-time PCR. Furthermore, in vivo studies supported anti-tumor role for brusatol, demonstrating that brusatol inhibited tumor growth, angiogenesis, glycolysis metabolism and induction of apoptosis in OSCC cells nude mice xenografts. Our data clearly demonstrated the anti-tumor effects of brusatol in OSCC cells by targeting glycolytic metabolism, and it could become a promising tumor chemotherapeutic agent in oral cancer.

Colorectal cancer is among the most frequently diagnosed cancers in patients today. In the treatment of this disease, combination or multicomponent therapy has been identified as a potential method to improve patient response and delay side effects. The aim of this study was to determine the effects on cell viability of commercial Bru and Erio used together with the anticancer drug 5-FU in the human colorectal cancer (CRC) cell line (HT-29 cell line) for the first time, as far as can be determined from available literature at this time. Additionally, the research seeks to study any potential effects on apoptosis. For this purpose, the effects of independent and combined treatments of the aforementioned agents on cell viability were investigated through the MTT experiment. Apoptotic effects were determined by Annexin V/PI and real-time PCR methods. In addition, a cell cycle analysis was used to determine the distribution of cells in the cycle. Data from experiments for 48 h showed that Bru, alone or in combination with 5-FU, is capable of causing an increase in the percentage of apoptotic cells in HT-29 cells compared to those of Erio alone or in combination with 5-FU. A significant increase in the level of bax and caspase-3 apoptotic genes was also detected in combinations of IC50 concentrations of Bru and 5-FU. These findings suggest that unlike Erio, Bru alone or in combination with 5-FU may be useful for increasing the effects of 5-FU used in the treatment of CRC and to provide data on alternative treatment approaches. [ABSTRACT FROM AUTHOR]

BACKGROUND: Prevention or reduction of kidney ischemia-reperfusion injury can improve the livability of kidney transplant. Therefore, it is necessary to develop safer, cheaper and more effective drugs to prevent or treat kidney ischemia-reperfusion injury. OBJECTIVE: To investigate the protective effect of asiaticoside on kideny ischemia-reperfusion injury in a rat model and its relevant mechanism. METHODS: A total of 40 Sprague-Dawley rats were randomly divided into four groups: sham surgery group, model group, asiaticoside group, and asiaticoside+brusatol group, with 10 rats in each group. The kidney pedicles were clamped for 45 minutes in the latter three groups to establish the rat kidney ischemia-reperfusion injury model. The rats in the asiaticoside and asiaticoside+brusatol groups were given asiaticoside suspension by gavage for 4 weeks in advance. Before modeling, the asiaticoside+brusatol group was given brusatol, a nuclear factor E2-related factor 2-specific inhibitor, by intraperitoneal injection for 5 continuous days. The levels of serum creatinine and urea nitrogen were detected. The expression of kidney injury molecule-1 in urine was detected by enzyme-linked immunosorbent assay. The expression levels of superoxide dismutase and malondialdehyde in kidney tissue were measured. Hematoxylineosin staining was used to observe the pathological changes of liver tissue. The protein expression of nuclear factor E2-related factor 2 protein in kidney tissue was detected by immunohistochemistry. The protein expression of nuclear factor E2-related factor 2 and hemeoxygenase-1 protein in kidney tissue cells was detected by western blot assay. RESULTS AND CONCLUSION: Compared with the model group, the levels of serum creatinine and urea nitrogen and urinary kidney injury molecule 1 were significantly reduced in the asiaticoside group (all P < 0.05). Asiaticoside could also significantly increase the expression of superoxide dismutase (P < 0.05), decrease the expression of malondialdehyde in kidney tissue (P < 0.05), and improve the pathological changes of kidney tissue. Compared with the asiaticoside group, the combined use of asiaticoside and bruceol could increase the levels of serum creatinine and urea nitrogen, urinary kidney injury molecule 1, and malondialdehyde in the kidney tissue, while decrease the level of superoxide dismutase in the kidney tissue (all P < 0.05). Meanwhile, asiaticoside could significantly induce the translocation of nuclear factor E2-related factor 2, and increased the expression level of hemeoxygenase-1. Overall, these findings indicate that asiaticoside can protect against kidney ischemia-reperfusion injury by activating nuclear factor E2-related factor 2/hemeoxygenase-1 pathway. [ABSTRACT FROM AUTHOR]

The dual tyrosine kinase (EGFR/HER2) inhibitor lapatinib is currently used to clinically treat HER2-positive breast cancer. However, a majority of patients do not respond to lapatinib therapy within 6 months. Therefore, potentiating the anti-tumor effect of lapatinib by combination treatment has a great potential to overcome the obstacle. Herein, we aim to investigate the anti-tumor activity of lapatinib in combination with brusatol and explore the potential mechanism involved in the combinatorial treatment. Our findings revealed that the Nrf2 inhibitor brusatol potently enhanced the anti-tumor effect of lapatinib against SK-BR-3, SK-OV-3 and AU565 cancer cells in a synergistic manner. Furthermore, we found that lapatinib plus brusatol more effectively decreased Nrf2 level and induced ROS generation in both SK-BR-3 and SK-OV-3 cells. Moreover, we also observed a significant reduction on the phosphorylation of HER2, EGFR, AKT and ERK1/2 in SK-BR-3 and SK-OV-3 cells when treated with lapatinib plus brusatol compared to either agent alone. More importantly, brusatol significantly augmented the anti-tumor effects of lapatinib in the SK-OV-3 xenograft model. In summary, these data provide a potential rationale for the combination of brusatol and lapatinib on the treatment of HER2-positive cancers.

Pancreatic cancer (PanCa), ranked as the 4th leading cause of cancer-related death worldwide, exhibits an dismal 5-year survival rate of less than 5 %. Chronic pancreatitis (CP) is a known major risk factor for PanCa. Brusatol (BRT) possesses a wide range of biological functions, including the inhibition of PanCa proliferation. However, its efficacy in halting the progression from CP to pancreatic carcinogenesis remains unexplored. We assess the effects of BRT against pancreatic carcinogenesis from CP using an experimentally induced CP model with cerulein, and further evaluate the therapeutic efficacy of BRT on PanCa by employing Kras tm4Tyj Trp53 tm1Brn Tg (Pdx1-cre/Esr1*) #Dam/J (KPC) mouse model. Our finding demonstrated that BRT mitigated the severity of cerulein-induced pancreatitis, reduced pancreatic fibrosis and decreased the expression of α-smooth muscle actin (α-SMA), which is a biomarker for pancreatic fibrosis. In addition, BRT exerted effects against cerulein-induced pancreatitis via inactivation of NLRP3 inflammasome. Moreover, BRT significantly inhibited tumor growth and impeded cancer progression. The observed effect of BRT on impeding pancreatic carcinogenesis through targeting NLRP3 inflammasome suggests its good potential as a potential agent for treatment of PanCa. [Display omitted] • Chronic pancreatitis poses a significant risk for Pancreatic cancer. • Brusatol lessened the severity of cerulein-induced chronic pancreatitis and reduced pancreatic fibrosis. • Brusatol administration impeded cancer progression in transgenic Krastm4Tyj Trp53tm1Brn Tg (Pdx1-cre/Esr1*) #Dam/J (KPC) mouse model. • Brusatol protects against pancreatitis and suppresses Pancreatic carcinogenesis by modulating NLRP3 inflammasome. [ABSTRACT FROM AUTHOR]

Introduction: Epithelial-mesenchymal transition (EMT) is a process of transdifferentiation where epithelial cells attain mesenchymal phenotype to gain invasive properties and thus, can contribute to metastasis of tumor cells. Objectives: The antimetastatic and antitumor efficacy of brusatol (BT) was investigated in a hepatocellular carcinoma (HCC) model. Methods: We evaluated the action of BT on EMT process using various biological assays in HCC cell lines and its effect on tumorigenesis in an orthotopic mouse model. Results: We found that BT treatment restored the expression of Occludin, E-cadherin (epithelial markers) while suppressing the levels of different mesenchymal markers in HCC cells and tumor tissues. Moreover, we observed a decline in the expression of transcription factors (Snail, Twist). Since the expression of these two factors can be regulated by STAT3 signaling, we deciphered the influence of BT on modulation of this pathway. BT suppressed the phosphorylation of STAT3Y705 and STAT3 depletion using siRNA resulted in the restoration of epithelial markers. Importantly, BT (1mg/kg) reduced the tumor burden in orthotopic mouse model with a concurrent decline in lung metastasis. Conclusions: Overall, our results demonstrate that BT interferes with STAT3 induced metastasis by altering the expression of EMT-related proteins in HCC model.

Abstract Background Tumor progression and tumor response to anticancer therapies may be affected by activation of oncogenic pathways such as the antioxidant one induced by NRF2 (nuclear factor erythroid 2-related factor 2) transcription factor and the pathways modified by deregulation of oncosuppressor p53. Often, oncogenic pathways may crosstalk between them increasing tumor progression and resistance to anticancer therapies. Therefore, understanding that interplay is critical to improve cancer cell response to therapies. In this study we aimed at evaluating NRF2 and p53 in several cancer cell lines carrying different endogenous p53 status, using a novel curcumin compound since curcumin has been shown to target both NRF2 and p53 and have anti-tumor activity. Methods We performed biochemical and molecular studies by using pharmacologic of genetic inhibition of NRF2 to evaluate the effect of curcumin compound in cancer cell lines of different tumor types bearing wild-type (wt) p53, mutant (mut) p53 or p53 null status. Results We found that the curcumin compound induced a certain degree of cell death in all tested cancer cell lines, independently of the p53 status. At molecular level, the curcumin compound induced NRF2 activation, mutp53 degradation and/or wtp53 activation. Pharmacologic or genetic NRF2 inhibition further increased the curcumin-induced cell death in both mutp53- and wtp53-carrying cancer cell lines while it did not increase cell death in p53 null cells, suggesting a cytoprotective role for NRF2 and a critical role for functional p53 to achieve an efficient cancer cell response to therapy. Conclusions These findings underline the prosurvival role of curcumin-induced NRF2 expression in cancer cells even when cells underwent mutp53 downregulation and/or wtp53 activation. Thus, NRF2 inhibition increased cell demise particularly in cancer cells carrying p53 either wild-type or mutant suggesting that p53 is crucial for efficient cancer cell death. These results may represent a paradigm for better understanding the cancer cell response to therapies in order to design more efficient combined anticancer therapies targeting both NRF2 and p53.

Background: Kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor erythroid-2-related factor 2 (NRF2) pathway is a major regulator protecting cells from oxidative and metabolic stress. Studies have revealed that this pathway is involved in mediating resistance to cytotoxic chemotherapy and immunotherapy, however, its implications in oncogene-addicted tumors are largely unknown. This study aimed to elucidate whether this pathway could be a potential therapeutic target for epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer., Methods: We measured the baseline expression of NRF2 using EGFR-mutant parental cells and acquired gefitinib resistant cells. We investigated whether NRF2 inhibition affected cell death in vitro and tumor growth in vivo using a xenograft mouse model, and compared the transcriptional changes before and after NRF2 inhibition., Results: Baseline NRF2 expression was enhanced in PC9 and PC9 with gefitinib resistance (PC9/GR) cells than in other cell lines, with a more prominent expression in PC9/GR. The NRF2 inhibitor induced NRF2 downregulation and cell death in a dose-dependent manner. Co-treatment with an NRF2 inhibitor enhanced osimertinib-induced cell death in vitro, and potentiated tumor growth inhibition in a PC9/GR xenograft model. Finally, RNA sequencing revealed that NRF2 inhibition resulted in the altered expression of multiple genes involved in various signaling pathways., Conclusion: We identified that NRF2 inhibition enhanced cell death and inhibited tumor growth in TKI-resistant lung cancer with EGFR-mutation. Thus, NRF2 modulation may be a novel therapeutic strategy to overcome the resistance to EGFR-tyrosine kinase inhibitors.

Brusatol (Bru), a Chinese herbal extract, has a variety of anti-tumor effects. However, little is known regarding its role and underlying mechanism in glioblastoma cells. Here, we found that Bru could inhibit the proliferation of glioblastoma cells in vivo and in vitro. Besides, it also had an inhibitory effect on human primary glioblastoma cells. RNA-seq analysis indicated that Bru possibly achieved these effects through inhibiting the expression of extracellular matrix protein 1 (ECM1). Down-regulating the expression of ECM1 via transfecting siRNA could weaken the proliferation and invasion of glioblastoma cells and promote the inhibitory effect of Bru treatment. Lentivirus-mediated overexpression of ECM1 could effectively reverse this weakening effect. Our findings indicated that Bru could inhibit the proliferation and invasion of glioblastoma cells by suppressing the expression of ECM1, and Bru might be a novel effective anticancer drug for glioblastoma cells. [ABSTRACT FROM AUTHOR]

Brusatol (Bru), a Chinese herbal extract, has a variety of anti-tumor effects. However, little is known regarding its role and underlying mechanism in glioblastoma cells. Here, we found that Bru could inhibit the proliferation of glioblastoma cells in vivo and in vitro. Besides, it also had an inhibitory effect on human primary glioblastoma cells. RNA-seq analysis indicated that Bru possibly achieved these effects through inhibiting the expression of extracellular matrix protein 1 (ECM1). Down-regulating the expression of ECM1 via transfecting siRNA could weaken the proliferation and invasion of glioblastoma cells and promote the inhibitory effect of Bru treatment. Lentivirus-mediated overexpression of ECM1 could effectively reverse this weakening effect. Our findings indicated that Bru could inhibit the proliferation and invasion of glioblastoma cells by suppressing the expression of ECM1, and Bru might be a novel effective anticancer drug for glioblastoma cells.

Diabetes mellitus induces testicular damage, increases sperm abnormalities, and impairs reproductive dysfunction due to induction of endocrine disturbance and testicular oxidative stress. This study evaluated the reproductive protective effect of ellagic acid (EA) against testicular damage and abnormalities in sperm parameters in Streptozotocin (STZ)-induced diabetic rats (T1DM) and examined some possible mechanisms of protection. Adult male rats were segregated into 5 groups (n = 12 rat/each) as control, control + EA (50 mg/kg/day), T1DM, T1DM + EA, and T1DM + EA + brusatol (an Nrf-2 inhibitor) (2 mg/twice/week). All treatments were conducted for 12 weeks, daily. EA preserved the structure of the seminiferous tubules, prevented the reduction in sperm count, motility, and viability, reduced sperm abnormalities, and downregulated testicular levels of cleaved caspase-3 and Bax in diabetic rats. In the control and diabetic rats, EA significantly increased the circulatory levels of testosterone, reduced serum levels of FSH and LH, and upregulated Bcl-2 and all steroidogenic genes (StAr, 3β-HSD1, and 11β-HSD1). Besides, it reduced levels of ROS and MDA but increased levels of GSH and MnSOD and the transactivation of Nrf2. All these biochemical alterations induced by EA were associated with increased activity and nuclear accumulation of Nrf2. However, all these effects afforded by EA were weakened in the presence of brusatol. In conclusion, EA could be an effective therapy to alleviated DM-induced reproductive toxicity and dysfunction in rats by a potent antioxidant potential mediated by the upregulation of Nrf2. [ABSTRACT FROM AUTHOR]

Bladder cancer is a common tumor that impacts the urinary system and marked by a significant fatality rate and an unfavorable prognosis. Promising antineoplastic properties are exhibited by brusatol, which is obtained from the dried ripe fruit of Brucea javanica. The present study aimed to evaluate the influence of brusatol on the progression of bladder cancer and uncover the molecular mechanism involved. We used Cell Counting Kit-8, colony formation and EdU assays to detect cell numbers, viability and proliferation. We used transwell migration assay to detect cell migration ability. The mechanism of brusatol inhibition of bladder cancer proliferation was studied by flow cytometry and western blotting. It was revealed that brusatol could reduce the viability and proliferation of T24 and 5637 cells. The transwell migration assay revealed that brusatol was able to attenuate the migration of T24 and 5637 cells. We found that treatment with brusatol increased the levels of reactive oxygen species, malondialdehyde and Fe2+, thereby further promoting ferroptosis in T24 and 5637 cells. In addition, treatment with RSL3 (an agonistor of ferroptosis) ferrostatin-1 (a selective inhibitor of ferroptosis) enhanced or reversed the brusatol-induced inhibition. In vivo, treatment with brusatol significantly suppressed the tumor growth in nude mice. Mechanistically, brusatol induced ferroptosis by upregulating the expression of ChaC glutathione-specific gamma-glutamylcyclotransferase (Chac1) and decreasing the expression of SLC7A11 and Nrf2 in T24 and 5637 cells. To summarize, the findings of this research demonstrated that brusatol hindered the growth of bladder cancer and triggered ferroptosis via the Chac1/Nrf2/SLC7A11 pathway. [ABSTRACT FROM AUTHOR]

Esophageal adenocarcinoma (EAC), the predominant type of esophageal cancer in the United States, develops through Barrett’s esophagus (BE)-dysplasia-carcinoma cascade. Gastroesophageal reflux disease, where acidic bile salts refluxate into the esophagus, is the main risk factor for the development of BE and its progression to EAC. The NFE2-related factor 2 (NRF2) is the master cellular antioxidant regulator. We detected high NRF2 protein levels in the EAC cell lines and primary tissues. Knockdown of NRF2 significantly enhanced acidic bile salt-induced oxidative stress, DNA damage, and inhibited EAC cell growth. Brusatol, an NRF2 inhibitor, significantly inhibited NRF2 transcriptional activity and downregulated the NRF2 target genes. We discovered that in addition to inducing apoptosis, Brusatol alone or in combination with cisplatin (CDDP) induced significant lipid peroxidation and ferroptosis, as evidenced by reduced xCT and GPX4 expression, two known ferroptosis markers. The combination of Brusatol and CDDP significantly inhibited EAC tumor xenograft growth in vivo and confirmed the in vitro data showing ferroptosis as an important mechanism in the tumors treated with Brusatol or Brusatol and CDDP combination. Our data support the role of NRF2 in protecting against stress-induced apoptosis and ferroptosis in EACs. Targeting NRF2 in combination with platinum therapy can be an effective strategy for eliminating cancer cells in EAC.

Background Taxane based conventional chemotherapy serves as the standard treatment regimen for triple-negative breast cancer (TNBC). However, the efficacy is plateaued due to toxicities, chemoresistance and metastasis. Hence, the development of new therapies that provide long-term cover is needed. Brusatol, a natural quassinoid, has been implicated to inhibit the migration and proliferation of metastatic cells in lung and liver carcinoma, but its efficacy in TNBC has not been explored. Methods The growth inhibitory activity on TNBC cells was measured using MTT assay and flow cytometry. Epithelial to mesenchymal transition (EMT) and apoptotic markers were quantified using western blotting. The caspases using Calorimetric assay. Results Brusatol along with paclitaxel showed an enhanced growth inhibitory activity and a combined synergistic effect. In addition, brusatol was also observed to inhibit the invasion, migratory potential of TNBC cells. Mechanistically, brusatol and its combination were observed to decrease the matrix metalloproteinase (MMP) and a modest increase in the reactive oxygen species (ROS) production. Furthermore, brusatol treatment activated both intrinsic and extrinsic pathways with morphological changes of apoptosis in TNBC cells. Conclusion This is the first in vitro report demonstrating antineoplastic, anti-EMT and synergistic activity of brusatol and in combination with paclitaxel in TNBC cell. Further in-vivo studies are needed to substantiate the above findings. [ABSTRACT FROM AUTHOR]

Abstract Background We previously showed that knockdown of nuclear factor E2-related factor 2 (Nrf2) resulted in suppression of hepatitis C virus (HCV) infection. In this study, whether brusatol, an Nrf2 inhibitor, has dual anti-HCV and anticancer effects was explored. Methods The anti-HCV effect of brusatol was investigated by analyzing HCV RNA and proteins in a hepatic cell line persistently-infected with HCV, HPI cells, and by analyzing HCV replication in a replicon-replicating hepatic cell line, OR6 cells. Then, dual anti-HCV and anticancer effects of brusatol and enhancement of the effects by the combination of brusatol with anticancer drugs including sorafenib, which has been reported to have the dual effects, were then investigated. Results Brusatol suppressed the persistent HCV infection at both the RNA and protein levels in association with a reduction in Nrf2 protein in the HPI cells. Analysis of the OR6 cells treated with brusatol indicated that brusatol inhibited HCV persistence by inhibiting HCV replication. Combination of brusatol with an anticancer drug not only enhanced the anticancer effect but also, in the case of the combination with sorafenib, strongly suppressed HCV infection. Conclusions Brusatol has dual anti-HCV and anticancer effects and can enhance the comparable effects of sorafenib. There is therefore the potential for combination therapy of brusatol and sorafenib for HCV-related hepatocellular carcinoma.

Jinming Zhang,1,* Xiaobin Fang,1,* Zeyong Li,2 Hon Fai Chan,3 Zhixiu Lin,4 Yitao Wang,1 Meiwan Chen1 1State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China; 2Department of Laboratory Medicine, Guangdong No 2 Provincial People’s Hospital, Guangzhou, China; 3Department of Biomedical Engineering, Columbia University, New York, NY, USA; 4Faculty of Science, School of Chinese Medicine, Chinese University of Hong Kong, Shatin, Hong Kong, China *These authors contributed equally to this work Abstract: Brusatol (Bru) exhibits promising anticancer effects, with both proliferation inhibition and chemoresistance amelioration activity. However, the poor solubility and insufficient intracellular delivery of Bru greatly restrict its application. Herein, to simultaneously utilize the advantages of Pluronics as drug carriers and tumor microenvironment-responsive drug release profiles, a flexible amphiphilic copolymer with a polymer skeleton, that is, Pluronic® F68 grafting with linoleic acid moieties by redox-reducible disulfide bonds (F68-SS-LA), was synthesized. After characterization by 1H-nuclear magnetic resonance and Fourier transform infrared spectroscopy, the redox-sensitive F68-SS-LA micelles were self-assembled in a much lower critical micelle concentration than that of the unmodified F68 copolymer. Bru was loaded in micelles (Bru/SS-M) with high loading efficiency, narrow size distribution, and excellent storage stability. The redox-sensitive Bru/SS-M exhibited rapid particle dissociation and drug release in response to a redox environment. Based on the enhanced cellular internalization, Bru/SS-M achieved higher cytotoxicity in both Bel-7402 and MCF-7 cells compared with free Bru and nonreducible micelles. The improved anticancer effect was attributed to the remarkably decreased mitochondrial membrane potential and increased reactive oxygen species level as well as ­apoptotic rate. These results demonstrated that F68-SS-LA micelles possess great potential as an efficient delivery vehicle for Bru to promote its anticancer efficiency via an oxidation pathway. Keywords: poloxamer188, tumor micro-environment, glutathione, Brucea javanica, cancer treatment

Abstract Background Natural products from herbal medicines have long been investigated for their potentials as cancer therapeutics. Besides the development of several herbal medicine-derived anti-cancer agents, such as paclitaxel, vincristine and podophyllotoxin, many recent laboratory findings demonstrated that brusatol, a quassinoid from the seeds of Brucea sumatrana, exhibits potent tumor suppressing effect with improved disease outcome. Our recent finding further demonstrated that brusatol synergizes with the intrinsic metabolic burden in cancer cells. Main body Here, we summarized the recent investigations of brusatol as an experimental therapeutic for human malignancies, such as leukemia, lung cancer, pancreatic cancer and brain tumor. We also discussed the molecular target brusatol, with a focus on the Nuclear factor erythroid 2-related factor 2 (NRF2)-guided gene transcription, as well as glutathione de novo synthesis. Further, we discussed the challenges and future applications of brusatol for cancer therapy. Conclusion In conclusion, we believe increasing evidences have shown the value of brusatol as a novel strategy for cancer treatment, which may indicate future drug development and clinical translation.

Objective: The aim of this study is to investigate the possible beneficial effects of brusatol on lung injury caused by cecal ligation and puncture (CLP) model in rats. Methods: In this study, 32 Sprague Dawley male rats were divided into 4 random groups. The groups were programmed as group 1 (sham), group 2 (CLP), group 3 (DMSO), and group 4 (0.5 mg/ml brusatol). In group 1, a midline vertical incision was applied to the abdominal region and closed again without forming a CLP model. Group 2 performed CLP for 18 hours. In group 3, 1% DMSO was administered intraperitoneally 0.3 ml once every two days for 10 days. The last application was made 30 minutes before CLP. In group 4, brusatol was administered intraperitoneally at 0.5 mg/ml once every two days for 10 days. The last application was made 30 minutes before CLP. After CLP was over, the rats were sacrificed and lung tissues were removed. Results: MDA, MPO, OSI, TOS, TNF-α and IL-1β values increased significantly in groups 2 and 3 compared to group 1, while SOD and TAS values decreased. Unlike the increase in antioxidant enzyme activity; MDA level, MPO activity, TOS, OSI, TNF-a and IL-1β values decreased significantly in group 4 compared to groups 2 and 3. Conclusion: As a result, brusatol may play an effective role against CLD-induced lung injury in rats. [ABSTRACT FROM AUTHOR]

• Brusatol affects migration and invasion ability of HCC cells. • Brusatol affects EMT process through modulation of STAT3 activation pathway. • Brusatol mitigates tumorigenesis and metastasis in HCC preclinical model. Epithelial-mesenchymal transition (EMT) is a process of transdifferentiation where epithelial cells attain mesenchymal phenotype to gain invasive properties and thus, can contribute to metastasis of tumor cells. The antimetastatic and antitumor efficacy of brusatol (BT) was investigated in a hepatocellular carcinoma (HCC) model. We evaluated the action of BT on EMT process using various biological assays in HCC cell lines and its effect on tumorigenesis in an orthotopic mouse model. We found that BT treatment restored the expression of Occludin, E-cadherin (epithelial markers) while suppressing the levels of different mesenchymal markers in HCC cells and tumor tissues. Moreover, we observed a decline in the expression of transcription factors (Snail, Twist). Since the expression of these two factors can be regulated by STAT3 signaling, we deciphered the influence of BT on modulation of this pathway. BT suppressed the phosphorylation of STAT3Y705 and STAT3 depletion using siRNA resulted in the restoration of epithelial markers. Importantly, BT (1mg/kg) reduced the tumor burden in orthotopic mouse model with a concurrent decline in lung metastasis. Overall, our results demonstrate that BT interferes with STAT3 induced metastasis by altering the expression of EMT-related proteins in HCC model. [ABSTRACT FROM AUTHOR]

Background: It has been reported that brusatol (BRU) reduces cellular reactive oxygen species (ROS) level under hypoxia; here the protective effect of BRU against oxygen-glucose deprivation/reoxygenation (OGD-R)-induced injury in HepG2 cells and against anoxia/reoxygenation (A/R)-induced injury in rat liver mitochondria was investigated. Materials and Methods: OGD-R-induced HepG2 cell viability loss was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and trypan blue staining. Mitochondrial ROS level in HepG2 cells was measured by MitoSOX staining. The cellular malondialdehyde and adenosine triphosphate level was measured by commercial kits. The mitochondrial membrane potential in HepG2 cells was measured by JC-1 staining. The protein level was detected by Western blotting. Rat liver mitochondria were separated by differential centrifugation. A/R-induced injury in isolated rat liver mitochondria was established by using a Clark oxygen electrode. The ROS generation in isolated mitochondria was evaluated using Amplex red/horseradish peroxidase. Results: BRU reduced mitochondrial ROS level and alleviated oxidative injury in HepG2 cells, thereby significantly inhibited OGD-R-induced cell death. During OGD-R, BRU improved mitochondrial function and inhibited the release of cytochrome c. Furthermore, BRU showed a clear protective effect against A/R-induced injury in isolated rat liver mitochondria. When isolated rat liver mitochondria were pretreated with BRU, A/R-induced ROS generation was significantly decreased, and mitochondrial respiratory dysfunction was ameliorated. Conclusions: BRU pretreatment attenuated OGD-R-induced injury in HepG2 cells and A/R-induced injury in isolated rat liver mitochondria by inhibiting mitochondrial ROS-induced oxidative stress. [ABSTRACT FROM AUTHOR]

Background: Tumor progression and tumor response to anticancer therapies may be affected by activation of oncogenic pathways such as the antioxidant one induced by NRF2 (nuclear factor erythroid 2-related factor 2) transcription factor and the pathways modified by deregulation of oncosuppressor p53. Often, oncogenic pathways may crosstalk between them increasing tumor progression and resistance to anticancer therapies. Therefore, understanding that interplay is critical to improve cancer cell response to therapies. In this study we aimed at evaluating NRF2 and p53 in several cancer cell lines carrying different endogenous p53 status, using a novel curcumin compound since curcumin has been shown to target both NRF2 and p53 and have anti-tumor activity. Methods: We performed biochemical and molecular studies by using pharmacologic of genetic inhibition of NRF2 to evaluate the effect of curcumin compound in cancer cell lines of different tumor types bearing wild-type (wt) p53, mutant (mut) p53 or p53 null status. Results: We found that the curcumin compound induced a certain degree of cell death in all tested cancer cell lines, independently of the p53 status. At molecular level, the curcumin compound induced NRF2 activation, mutp53 degradation and/or wtp53 activation. Pharmacologic or genetic NRF2 inhibition further increased the curcumin-induced cell death in both mutp53- and wtp53-carrying cancer cell lines while it did not increase cell death in p53 null cells, suggesting a cytoprotective role for NRF2 and a critical role for functional p53 to achieve an efficient cancer cell response to therapy. Conclusions: These findings underline the prosurvival role of curcumin-induced NRF2 expression in cancer cells even when cells underwent mutp53 downregulation and/or wtp53 activation. Thus, NRF2 inhibition increased cell demise particularly in cancer cells carrying p53 either wild-type or mutant suggesting that p53 is crucial for efficient cancer cell death. These results may represent a paradigm for better understanding the cancer cell response to therapies in order to design more efficient combined anticancer therapies targeting both NRF2 and p53. [ABSTRACT FROM AUTHOR]

Purpose: Brusatol, a natural quassinoid that is isolated from a traditional Chinese herbal medicine known as Bruceae Fructus, possesses biological activity in various types of human cancers, but its effects in nasopharyngeal carcinoma (NPC) have not been reported. This study aimed to explore the effect and molecular mechanism of brusatol in NPC in vivo and in vitro.Methods: The antiproliferative effect of brusatol was assessed by MTT and colony formation assays. Apoptosis was determined by flow cytometry. The expression of mitochondrial apoptosis, cell cycle arrest, and Akt/mTOR pathway proteins were determined by western blot analysis. Further in vivo confirmation was performed in a nude mouse model.Results: Brusatol showed antiproliferative activity against four human NPC cell lines (CNE-1, CNE-2, 5-8F, and 6-10B) in a dose-dependent manner. This antiproliferative effect was accompanied by mitochondrial apoptosis and cell cycle arrest through the modulation of several key molecular targets, such as Bcl-xl, Bcl-2, Bad, Bax, PARP, Caspase-9, Caspase-7, Caspase-3, Cdc25c, Cyclin B1, Cdc2 p34, and Cyclin D1. In addition, we found that brusatol inhibited the activation of Akt, mTOR, 4EBP1, and S6K, suggesting that the Akt/mTOR pathway is a key underlying mechanism by which brusatol inhibits growth and promotes apoptosis. Further in vivo nude mouse models proved that brusatol significantly inhibited the growth of CNE-1 xenografts with no significant toxicity.Conclusions: These observations indicate that brusatol is a promising antitumor drug candidate or a supplement to current chemotherapeutic therapies to treat NPC. [ABSTRACT FROM AUTHOR]

Nuclear factor erythroid-2 related factor-2 (Nrf2) is an oxidative stress-response transcriptional activator that promotes carcinogenesis through metabolic reprogramming, tumor promoting inflammation, and therapeutic resistance. However, the extension of Nrf2 expression and its involvement in regulation of breast cancer (BC) responses to chemotherapy remain largely unclear. This study determined the expression of Nrf2 in BC tissues (n = 46) and cell lines (MDA-MB-453, MCF-7, MDA-MB-231, MDA-MB-468) with diverse phenotypes. Immunohistochemical (IHC)analysis indicated lower Nrf2 expression in normal breast tissues, compared to BC samples, although the difference was not found to be significant. However, pharmacological inhibition and siRNA-induced downregulation of Nrf2 were marked by decreased activity of NADPH quinone oxidoreductase 1 (NQO1), a direct target of Nrf2. Silenced or inhibited Nrf2 signaling resulted in reduced BC proliferation and migration, cell cycle arrest, activation of apoptosis, and sensitization of BC cells to cisplatin in vitro. Ehrlich Ascites Carcinoma (EAC) cells demonstrated elevated levels of Nrf2 and were further tested in experimental mouse models in vivo. Intraperitoneal administration of pharmacological Nrf2 inhibitor brusatol slowed tumor cell growth. Brusatol increased lymphocyte trafficking towards engrafted tumor tissue in vivo, suggesting activation of anti-cancer effects in tumor microenvironment. Further large-scale BC testing is needed to confirm Nrf2 marker and therapeutic capacities for chemo sensitization in drug resistant and advanced tumors.

Brusatol (BR) is one of the main bioactive components derived from Brucea javanica, a medicinal herb historically used in the treatment of dysenteric disorders (also known as ulcerative colitis(UC)). Due to its poor aqueous solubility, a novel brusatol self-microemulsifying drug delivery system (BR-SMEDDS) nanoformulation with smaller size, higher negative zeta potential and drug content, and excellent stability was developed. The appearance of BR-SMEDDS remained clear and transparent, and transmission electron microscopy showed microemulsion droplets to be spherical with homogeneous distribution. Pharmacokinetic parameters indicated that oral bioavailability was greatly improved by BR-SMEDDS as compared with aqueous suspension. Meanwhile, the anti-colitis activity of BR-SMEDDS was evaluated on dextran sodium sulfate (DSS)-induced colitis mice model. The result illustrated that the nano-formation significantly reduced the body weight loss, recovered colon length, decreased disease activity index and microscopic score, regulated immune-inflammatory cytokines, diminished oxidative stress and repressed the colonic expression of myeloid differentiation factor 88 (MyD88), toll-like receptor 4 (TLR4) and nuclear factor kappa B p65 (NF-κB p65) proteins. Our findings demonstrated for the first time that BR could effectively attenuate colonic inflammation in mice, at least partially, via favorable regulation of anti-oxidative and anti-inflammatory status and inhibition of the TLR4-linked NF-κB signaling pathway. The BR nano-formulation was superior to BR suspension and sulphasalazine, in treating experimental UC, and exhibited similar effect with azathioprine, with much smaller dosage. The enhanced anti-UC effect of BR might be intimately associated with the improved pharmacokinetic property by SMEDDS. The developed nano-delivery system might thus be a promising candidate for colitis treatment.

Background: Vector control is still emphasized on the using of chemical insecticide which can cause death of non-target organisms, pollution and vector resistance. Therefore, natural insecticides/larvicides are an alternative to using chemical insecticides to control the mosquito vector. Methods: Brusatol was isolated from the seeds of Makassar Fruit (Brucea javanica L. Merr). Culex quinquefasciatus larvae were divided into 3 groups, i.e. 6 testing groups and one negative and positive control group. In the negative control group, the larvae were treated with 100 ml aquadest and positive control was treated with temephos 1 ppm. After 24 hours, dead larvae were calculated and the percentage of death was determined. The dead larvae were then examined for morphological changes using a light microscope. Results: The higher of the concentration level of brusatol, the higher number of the death of Cx. quinquefasciatus larvae (P

Gastroenteropancreatic neuroendocrine neoplasms (GEP‑NEN) are a group of rare tumors whose specific pathogenetic mechanisms of resistance to therapies have not been completely revealed yet. Chemotherapy is the main therapeutic approach in patients with GEP‑NEN, however, novel combination regimens and targeted therapy are continuously explored. In the present study, the anticancer effect of a novel Ruthenium (Ru)(II)‑Bisdemethoxycurcumin (Ru‑bdcurc) compound was evaluated in BON‑1 cell line, one of the few cell lines derived from GEP‑NEN, largely used in experimental research of this type of tumors. The experimental data revealed that the Ru‑bdcurc compound induced cell death in a dose‑dependent manner, in vitro. Biochemical studies demonstrated that, in response to the lower dose of treatment, BON‑1 cells activated the nuclear factor erythroid 2‑related factor 2 (NRF2) pathway with induction of some of its targets including catalase and p62 as well as of the antiapoptotic marker Bcl2, all acting as chemoresistance mechanisms. NRF2 induction associated also with increased expression of endogenous p53 which is reported to be dysfunctional in BON‑1 cells and to inhibit apoptosis. Genetic or pharmacologic targeting of NRF2 inhibited the activation of the NRF2 pathway, as well as of endogenous dysfunctional p53, in response to the lower dose of Ru‑bdcurc, increasing the cell death. To assess the interplay between NRF2 and dysfunctional p53, genetic targeting of p53 showed reduced activation of the NRF2 pathway in response to the lower dose of Ru‑bdcurc, increasing the cell death. These findings identified for the first time a possible dysfunctional p53/NRF2 interplay in BON‑1 cell line that can be a novel key determinant in cell resistance to cytotoxic agents to be evaluated also in GEP‑NEN.

Graphical abstract Highlights • Pharmacokinetic study of quassinoid glycoside was performed for the first time. • Bruceoside A could serve as the precursor of the potent anticancer brusatol in vivo , rather than its direct deglycosylated metabolite bruceosin. • Quassinoid glycosides were proposed to be potential contributors to the anticancer properties of Fructus Bruceae. Abstract Bruceoside A, an abundant quassinoid glycoside in Fructus Bruceae, was chosen for the pharmacokinetic study. It is the first case report on the pharmacokinetic study of quassinoid glycosides so far. A sensitive, accurate, and repeatable UHPLC–MS/MS method was developed for the determination of bruceoside A and its major metabolite. The results showed bruceoside A could be transformed into the potent anticancer component brusatol in vivo , rather than its direct deglycosylated metabolite bruceosin. And the intestinal bacteria were proposed to take a potential role during such transformation. Based on the present study, it could be concluded that the quassinoid glycosides possessing weak activities in vitro could do contribution to the anticancer properties of Fructus Bruceae in vivo via transforming into more active metabolites. [ABSTRACT FROM AUTHOR]

Brusatol (Bru), a bioactive compound found in Brucea sumatrana, exerts antitumour effects on several malignancies. However, the role and molecular mechanism of Bru in squamous cell carcinoma of the oesophagus (ESCC) remain unclear. Here, we found that Bru decreased the survival of ESCC cells. Subsequently, the ferroptosis inhibitors, deferoxamine and liproxstatin-1, rescued Bru-induced cell death, indicating that ferroptosis plays a major role in Bru-induced cell death. Furthermore, Bru promoted lipid peroxidation, glutathione (GSH) depletion, and ferrous iron overload in vitro. Consistent with these in vitro results, Bru significantly inhibited tumour growth in KYSE150 xenograft nude mice by triggering ferroptosis. Mechanistically, nuclear factor E2-related factor 2 (NRF2) inactivation via increased ubiquitin-proteasome degradation was found to be a vital determinant of ferroptosis induced by Bru. Notably, Bru significantly decreases GSH synthesis, iron storage, and efflux by downregulating the expression of NRF2 target genes (glutamate-cysteine ligase catalytic subunit (GCLC), solute carrier family 7 member 11 (SLC7A11), ferritin heavy chain 1 (FTH1), and solute carrier family 40 member 1 (SLC40A1)), resulting in the accumulation of lethal lipid-based reactive oxygen species (ROS) and intracellular enrichment of chelated iron. Taken together, our findings indicate that ferroptosis is a novel mechanism underlying Bru-induced antitumour activity and will hopefully provide a valuable compound for ESCC treatment. [Display omitted] • Brusatol induces ferroptosis in oesophageal squamous cell carcinoma in vitro and in vivo. • Brusatol inhibits the level of GCLC and SLC7A11 to promote intracellular GSH depletion. • Brusatol down-regulates the expression of FTH1 and SLC40A1 to induce Fe2+ overload. • Brusatol reduces the expression of GCLC, SLC7A11, FTH1 and SLC40A1 by inducing NRF2 inactivation. [ABSTRACT FROM AUTHOR]

Abstract Brusatol is a main bioactive component derived from the Chinese medicinal plant Brucea javanica , which is traditionally used for the treatment of dysentery (also known as ulcerative colitis, UC). Previously, we have designed a novel brusatol self-microemulsifying drug delivery system (BR-SMEDDS) to increase its solubility and bioavailability, and enhance its bioactivities. In the present study, we established 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model in vivo and lipopolysaccharide (LPS)-induced RAW 264.7 macrophages in vitro, to investigate the potential anti-inflammatory effect and underlying mechanism of BR-SMEDDS. Disease activity index (DAI) including body weight, stool consistency and gross bleeding was measured. Macroscopic and histological evaluations of colons were conducted. Relevant molecular events were determined by ELISA, qRT-PCR, immunohistochemistry or Western blotting. The results showed that BR notably inhibited the productions of TNF-α, pro-IL-1β, PGE 2 and NO, and suppressed the NF-κB signaling pathway in LPS-stimulated macrophages. In parallel with the vitro experimental results, BR significantly attenuated diarrhea, colonic shortening, macroscopic damage and histological injury. BR treatment also increased the levels of TGF-β and IL-4, decreased the contents of IL-1β and IL-18, and elevated the levels of CAT, GSH and SOD in the colons. Furthermore, BR also markedly activated the Nrf2 expression and suppressed the NLRP3 inflammasome activation. Taken together, the anti-UC effect of BR might be intimately associated with the suppression of NF-κB and NLRP3-mediated inflammatory responses, and regulation of Nrf2-mediated oxidative stress. BR might have the potential to be further developed into a promising therapeutic agent for colitis treatment. Highlights • BR significantly attenuated the inflammatory response and suppressed the NF-κB signaling pathway in vitro. • BR effectively decreased DAI scores of rats and ameliorated macroscopic and histological colon pathological deterioration. • BR favorably regulated inflammatory status and diminished oxidative stress in TNBS-induced colitis. • BR markedly activated the Nrf2 expression and suppressed the NLRP3 inflammasome activation in the colons. • BR might have the potential to be further developed into a promising therapeutic agent for colitis treatment. [ABSTRACT FROM AUTHOR]