Brucine, a weak alkaline indole alkaloid, is one of the main bioactive and toxic constituents of Nux-vomica. Modern pharmacology studies and clinical practice demonstrate that brucine possesses wide pharmacological activities, such as anti-tumor, anti-inflammatory, analgesic, and the effects on cardiovascular system and nervous system, etc. However, its central nervous system toxicity severely limits its clinical application. Herein, the physicochemical properties, pharmacological activities, and toxicity of brucine were reviewed, and the novel strategies to address the toxicity issues were discussed, aiming to bring new insights into further research and application of this active component.

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Brucine: A Review of Phytochemistry, Pharmacology, and Toxicology.

Ferroptotic cell death is characterized by iron-dependent lipid peroxidation that is initiated by ferrous iron and H2O2 via Fenton reaction, in which the role of activating transcription factor 3 (ATF3) remains elusive. Brucine is a weak alkaline indole alkaloid extracted from the seeds of Strychnos nux-vomica, which has shown potent antitumor activity against various tumors, including glioma. In this study, we showed that brucine inhibited glioma cell growth in vitro and in vivo, which was paralleled by nuclear translocation of ATF3, lipid peroxidation, and increases of iron and H2O2. Furthermore, brucine-induced lipid peroxidation was inhibited or exacerbated when intracellular iron was chelated by deferoxamine (500 μM) or improved by ferric ammonium citrate (500 μM). Suppression of lipid peroxidation with lipophilic antioxidants ferrostatin-1 (50 μM) or liproxstatin-1 (30 μM) rescued brucine-induced glioma cell death. Moreover, knockdown of ATF3 prevented brucine-induced accumulation of iron and H2O2 and glioma cell death. We revealed that brucine induced ATF3 upregulation and translocation into nuclei via activation of ER stress. ATF3 promoted brucine-induced H2O2 accumulation via upregulating NOX4 and SOD1 to generate H2O2 on one hand, and downregulating catalase and xCT to prevent H2O2 degradation on the other hand. H2O2 then contributed to brucine-triggered iron increase and transferrin receptor upregulation, as well as lipid peroxidation. This was further verified by treating glioma cells with exogenous H2O2 alone. Moreover, H2O2 reversely exacerbated brucine-induced ER stress. Taken together, ATF3 contributes to brucine-induced glioma cell ferroptosis via increasing H2O2 and iron.

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ATF3 contributes to brucine-triggered glioma cell ferroptosis via promotion of hydrogen peroxide and iron

Despite a dogma that apoptosis does not induce inflammation, Fas ligand (FasL), a well‐known death factor, possesses pro‐inflammatory activity. For example, FasL induces nuclear factor κB (NF‐κB) activity and interleukin 8 (IL‐8) production by engagement of Fas in human cells. Here, we found that a dominant negative mutant of c‐Jun, a component of the activator protein‐1 (AP‐1) transcription factor, inhibits FasL‐induced AP‐1 activity and IL‐8 production in HEK293 cells. Selective inhibition of AP‐1 did not affect NF‐κB activation and vice versa, indicating that their activations were not sequential events. The FasL‐induced AP‐1 activation could be inhibited by deleting or introducing the lymphoproliferation (lpr)‐type point mutation into the Fas death domain (DD), knocking down the Fas‐associated DD protein (FADD), abrogating caspase‐8 expression with small interfering RNAs, or using inhibitors for pan‐caspase and caspase‐8 but not caspase‐1 or caspase‐3. Furthermore, wildtype, but not a catalytically inactive mutant, of caspase‐8 reconstituted the FasL‐induced AP‐1 activation in caspase‐8‐deficient cells. Fas ligand induced the phosphorylation of two of the three major mitogen‐activated protein kinases (MAPKs): extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) but not p38 MAPK. Unexpectedly, an inhibitor for JNK but not for MAPK/ERK kinase inhibited the FasL‐induced AP‐1 activation and IL‐8 production. These results demonstrate that FasL‐induced AP‐1 activation is required for optimal IL‐8 production, and this process is mediated by FADD, caspase‐8, and JNK.

Caspase-8-and JNK-dependent AP-1 activation is required Fas Ligand-induced IL-8 production

To examine the effect of brucine on the migration, invasion, adhesion and expressions of epithelial-to-mesenchymal transition (EMT) markers and matrix metalloproteinases (MMPs) in the highly metastatic breast cancer cell lines MDA-MB-231 and Hs578-T. MDA-MB-231 and Hs578-T cells were divided to 4 groups: the control group (0.1% DMSO), and 25, 50 and 100 μmol/L brucine groups. The cell viability was determined using a CellTiter-Glo® luminescent cell viability. The scratch wound healing assay and tanswell migration assay were used to determine the migration ability of these cells treated by different concentrations of brucine. The proliferation rate, invasive potential and adhesive ability were respectively performed by colony formation assay, transwell invasion assay and adhension assay. The protein and mRNA expressions of EMT biomarkers, MMP-2 and MMP-9 were investigated by real-time reverse transcription polymerase chain reaction and Western blot. Compared with the control group, brucine had little effect on cell viability or proliferation (P>0.05), but led to a dose-dependent decrease on migration, invasion, adhension of MDA-MB-231 and Hs578-T cells (P<0.01). Furthermore, brucine increased the protein and mRNA levels of EMT markers such as E-cadherin and β-catenin in MDA-MB-231 and Hs578-T cells, and decreased the protein and mRNA levels of mesenychmal markers such as vimentin and fibronectin, as well as the expressions of MMP-2 and MMP-9 (all P<0.01). Brucine inhibited triple negative breast cancer cells metastasis potentially through EMT reversion and MMP-2 and MMP-9 inhibition.

Brucine suppresses breast cancer metastasis via inhibiting epithelial mesenchymal transition and matrix metalloproteinases expressions.

Brucine PEGylated nanoemulsion: In vitro and in vivo evaluation

The aim of this study was to investigate the mechanism of the apoptotic effect of brucine on human multiple myeloma (MM) cells. U266 cells (5x104) were plated in the presence or absence of brucine (0, 0.05, 0.1, 0.2 and 0.4 mg/ml) in 96‑well culture plates for 24‑72 h. The anti-proliferative response to brucine was assessed by MTT assay. Analysis of the cell cycle of U266 cells treated with or without brucine was performed using flow cytometry. The expression change of c-Jun following treatment with brucine or brucine plus the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 was detected using RT-PCR. Brucine appeared to have an effect on apoptosis in a dose- and time‑dependent manner. Cell cycle analysis using flow cytometry revealed the accumulation of cells at the sub-G0/G1 phase. The apoptotic rates were 4.137, 10.55, 12.31, 27.67 and 29.67% (0, 0.05, 0.1, 0.2, 0.4 mg/ml brucine, respectively; P<0.01). The gray scale values were 0.7961 ± 0.007 and 0.4683 ± 0.003 (mRNA expression of c-Jun of U266 cells with or without SP600125, respectively). Concentrations of ≤ 0.4 mg/ml brucine induced apoptosis in U266 cells. Thus, brucine‑induced apoptosis in U266 cells occurs via the JNK signaling pathway and phosphorylation of c-Jun.

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Brucine induces the apoptosis of U266 multiple myeloma cells by phosphorylation of c-Jun.

The aim of this study was to explore the effects of brucine on bone metabolism in multiple myeloma (MM) and to compare brucine and bortezomib regarding the effects on MM in vitro. The half maximal inhibitory concentration (IC50) values of brucine and bortezomib in the MM cell line U266 were detected by MTT assay. In addition, the expression of alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG) and osteoprotegerin ligand (also termed receptor activator of nuclear factor κB ligand) (RANKL) at mRNA levels were measured by RT-PCR. IC50 of bortezomib in the U266 cell line at 48 h was 22.4 nmol/l, and that of brucine was 0.16 nmol/l. Compared with osteoblasts incubated with MM cell supernatant alone, the mRNA levels of ALP, OC and OPG in osteoblasts co-treated with brucine and MM cell supernatant were higher (p<0.05), while the mRNA expression of RANKL was lower, and the ranges of the changes were all larger than those of the group treated with bortezomib (P<0.05). Brucine exerts effects on bone metabolism in multiple myeloma through the regulation of osteoclasts by osteoblasts.

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Zhihua Li

Papers

Brucine induces the apoptosis of U266 multiple myeloma cells by phosphorylation of c-Jun.

Effects of brucine on bone metabolism in multiple myeloma