OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: Brusatol improves anti-PD-1 antibody efficacy by targeting PD-L1, suggesting its potential as an adjuvant in anti-PD-1 immunotherapy.

BACKGROUND: 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.
METHODS: 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 Krastm4TyjTrp53tm1BrnTg (Pdx1-cre/Esr1*) #Dam/J (KPC) mouse model.
RESULTS: 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.
CONCLUSIONS: 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.

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.

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.

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.

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.

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.
RESULTS: 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.
CONCLUSIONS: 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.

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.

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.

BACKGROUND: Poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) can be developed from differentiated thyroid cancer, and this dedifferentiated transformation leads to poor prognosis and high mortality. The role of Nrf2 in the dedifferentiation of differentiated thyroid cancer (DTC) induced by KRAS remains unclear.
METHODS: In this study, two DTC cell lines, BCPAP and WRO, were used to evaluate the function of Nrf2 in the dedifferentiation caused by wild-type KRAS (KRAS-WT) and G12V point mutation KRAS (KRAS-G12V).
RESULTS: The overexpression of KRAS-WT and KRAS-G12V increased the proliferative and invasive ability of BCPAP and WRO cells. Aggressive morphology was observed in KRAS-WT and KRAS-G12V overexpressed WRO cells. These results suggested that overexpression of KRAS-WT or KRAS-G12V may induce dedifferentiation in DTC cells. The expression of Nrf2 was increased by KRAS-WT and KRAS-G12V in DTC cells. In addition, compared with normal thyroid tissues, the expression of Nrf2 protein was considerably higher in thyroid cancer tissues on immunohistochemistry (IHC) staining, and the increased expression of Nrf2 indicated a poor prognosis of thyroid cancer. These results indicated that Nrf2 is the KRAS downstream molecule in thyroid cancer. Functional studies showed that the Nrf2 inhibitor Brusatol counteracted the proliferative and invasive abilities induced by KRAS-WT and KRAS-G12V in BCPAP and WRO cells. In addition, the xenograft assay further confirmed that Brusatol inhibits tumor growth induced by KRAS-WT and KRAS-G12V.
CONCLUSIONS: Collectively, this study suggests that Nrf2 could be a promising therapeutic target in KRAS-mediated dedifferentiation of thyroid cancer.