Anticancer

Bruceantin was first isolated from Brucea antidysenterica, a tree used in Ethiopia for the treatment of cancer, and activity was observed against B16 melanoma, colon 38, and L1210 and P388 leukemia in mice. Phase I and II clinical trials were then initiated, but no objective tumor regressions were observed and clinical development was terminated. Recently, the activity of bruceantin has been studied with a number of leukemia, lymphoma, and myeloma cell lines. Cell differentiation was induced and c-MYC was down-regulated, suggesting a mechanistic correlation between c-MYC down-regulation and induction of cell differentiation or cell death. Treatment of HL-60 and RPMI 8226 cell lines induced apoptosis, and this involved the caspase and mitochondrial pathways. Moreover, an in vivo study using RPMI 8226 human-SCID xenografts demonstrated that bruceantin induced regression in early as well as advanced tumors, and these significant antitumor responses were facilitated in the absence of overt toxicity. Apoptosis was significantly elevated in tumors derived from animals treated with bruceantin. In sum, bruceantin interferes with the growth of leukemia, lymphoma, and myeloma cells in culture and xenograft models. Responses of this type suggest bruceantin should be reinvestigated for clinical efficacy against hematological malignancies.[1]

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c-MYC was strongly down-regulated in cultured RPMI 8226 cells by treatment with bruceantin for 24 h. With U266 and H929 cells, bruceantin did not regulate c-MYC in this manner. Apoptosis was induced in the three cell lines. In RPMI 8226 cells, apoptosis occurred through proteolytic processing of procaspases and degradation of poly(ADP-ribose) polymerase. The mitochondrial pathway was also involved[2].

Because RPMI 8226 cells were the most sensitive, they were used in a xenograft model. Bruceantin treatment (2.5-5 mg/kg) resulted in a significant regression of tumors without overt toxicity. Apoptosis was significantly elevated in tumors derived from animals treated with bruceantin (37%) as compared with the control tumors (14%)[2].

Analysis of Mitochondrial Membrane Potential[2]
3,3′-Dihexyloxacarbocyanine iodide (DiOC6) is a dye used to measure mitochondrial membrane potential (ΔΨm). In brief, cells (5 × 106) were treated with bruceantin (2.5, 5, 10, 20, or 40 ng/ml) for 6, 12, 18, or 24 h. Fifteen min before collection of cells after drug treatment, 40 nm DiOC6 was added to the cells. Cells were washed once with PBS before resuspending in 300 μl PBS containing 40 nm DiOC6 and 30 μg/ml propidium iodide. Fluorescence intensities of DiOC6 were analyzed by flow cytometry with excitation and emission settings of 484 and 500 nm, respectively. Propidium iodide was added to gate out dead cells. Histograms show only propidium iodide-negative cells.

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Analysis of Caspase-3/7-like Activity[2]
The Apo-ONE Homogeneous Caspase-3/7 Assay kit was used to measure the activities of caspase-3/7. Cells (1.5 × 104) were treated with bruceantin (10 ng/ml) for 6, 12, 18, or 24 h in a black 96-well plate. At the end of the treatment, lysis buffer and the substrate (Z-DEVD-rhodamine 110) were mixed and added to the cells. Upon sequential cleavage and removal of the DEVD peptides by caspase-3/7 activity and excitation at 499 nm, the rhodamine 110 leaving group becomes intensely fluorescent. The emission maximum is 521 nm. The amount of fluorescent product generated is proportional to the amount of caspase-3/7 cleavage activity present in the sample. The samples were measured in triplicate. Results were expressed as fold of induction relative to the control (DMSO treated cells).

c-MYC. The expression of c-MYC protein was assessed by immunoblots as described previously. In brief, cells (10Million) were treated with brusatol (25 ng/ml) or bruceantin (10 ng/ml) and harvested after 4 or 24 h. Whole-cell pellets were lysed with detergent lysis buffer [1 ml/107 cells, 50 mm Tris-HCl buffer (pH 8.0), 150 mm NaCl, 1 mm DTT, 0.5 mm EDTA, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 2 μg/ml leupeptin, and 100 μm sodium vanadate] to obtain protein lysates, and protein concentrations were quantified using a bicinchoninic acid kit. Because c-MYC is labile, cell lysates were not frozen, but stored at 4°C until all of the lysates were ready for a particular cell line, then a Western blot was performed. Total protein (30 μg) was separated by 10% SDS-PAGE, electroblotted to polyvinylidene difluoride membranes, and blocked overnight with 5% nonfat dry milk. The membrane was incubated with a 2.5 μg/ml solution of the primary antibody, prepared in 1% blocking solution, for 2 h at room temperature, washed three-times for 15 min with PBS-T (PBS with 0.1%, v/v, and Tween 20), and incubated with a 1:2500 dilution of horseradish peroxidase-conjugated secondary antibody for 30 min at 37°C. Blots were again washed three-times for 10 min each in PBS-T and developed by enhanced chemiluminescence. Membranes were exposed to Kodak Biomax film and the resulting film analyzed using Kodak 1D Image Analysis Software. Membranes were then stripped and reprobed for β-actin.[2]

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Caspase-3, -8, -9, BID, and Poly(ADP-ribose) Polymerase (PARP).The expression of caspase-3, -8, -9, BID, and PARP protein was assessed by immunoblots. In brief, cells (10 million) were treated with bruceantin (2.5, 5, 10, 20, or 40 ng/ml) and harvested after 24 h. Whole-cell pellets were lysed with detergent lysis buffer [1 ml/107 cells, 62.5 mm Tris-HCl buffer (pH 6.8), 6 m urea, 10% glycerol, 2% SDS, 0.00125% bromphenol blue and 5% β-mercaptoethanol], then sonicated for 15 s and incubated at 65°C for 15 min to obtain protein lysates, and protein concentrations were quantified using a bicinchoninic acid kit. Total protein (30 μg) was separated by 7.5–15% SDS-PAGE, electroblotted to polyvinylidene difluoride membranes, and blocked overnight with 5% nonfat dry milk. The membrane was incubated with anticaspase-3 monoclonal antibody (1:100), anticaspase-8 polyclonal antibody (1:200), anticaspase-9 polyclonal antibody (1:200), anti-BID polyclonal antibody (1:1000), or anti-PARP monoclonal antibody (1:100), prepared, washed, and developed as described above for c-MYC.[2]

RPMI 8226 cells (1 × 107) were injected s.c. into the right rear flank of 6- or 13-week-old male and/or female SCID mice. Cells were injected in a final volume of 0.1 ml. The area of inoculation was shaved before inoculation. Approximately 10–14 days after inoculation, or when the tumor size was 5 mm, bruceantin (0–12 mg/kg, dissolved in 100% ethanol, sonicated, and diluted to a 5% ethanol solution with saline) was injected i.p. every 3 days. About 40 days after the inoculation of RPMI 8226, treatment with bruceantin was terminated. The vehicle-treated control tumor-bearing group was divided into two groups. Some of these mice continued to receive the vehicle and others were treated with bruceantin (2.5 or 5.0 mg/kg) every 3 days. Animals were weighed twice weekly and observed daily. Tumors were measured twice a week. The volume at the site of the tumor was calculated in mm3 according to the formula (D × d × 0.2/6) x π, where D is the longer diameter and d is the shorter diameter.[2]

[1]. Antitumor activity of bruceantin: an old drug with new promise. J Nat Prod. 2004 Feb;67(2):269-72.
[2]. Multiple myeloma regression mediated by bruceantin. Clin Cancer Res. 2004 Feb 1;10(3):1170-9.

Bruceantin is a triterpenoid.
Bruceantin has been reported in Brucea antidysenterica and Brucea javanica with data available.
Bruceantin is a triterpene quassinoid antineoplastic antibiotic isolated from the plant Brucea antidysenterica. Bruceantin inhibits the peptidyl transferase elongation reaction, resulting in decreased protein and DNA synthesis. Bruceantin also has antiamoebic and antimalarial activity. (NCI04)

C28H36O11

548.5788

548.225

C, 61.30; H, 6.61; O, 32.08

41451-75-6

41451-75-6 （Bruceantin); 79439-84-2 (Bruceantinoside B); 95258-15-4 (Yadanzioside A); 95258-18-7 (Yadanzioside B); 95258-16-5 (Yadanzioside C)

5281304

White to off-white solid powder

1.4±0.1 g/cm3

731.0±60.0 °C at 760 mmHg

239.4±26.4 °C

0.0±5.4 mmHg at 25°C

1.598

2.96

3

11

6

39

1200

10

CC1=C(C(=O)C[C@]2([C@H]1C[C@@H]3[C@]45[C@@H]2[C@H]([C@@H]([C@]([C@@H]4[C@H](C(=O)O3)OC(=O)/C=C(\C)/C(C)C)(OC5)C(=O)OC)O)O)C)O

IRQXZTBHNKVIRL-GOTQHHPNSA-N

InChI=1S/C28H36O11/c1-11(2)12(3)7-17(30)39-20-22-27-10-37-28(22,25(35)36-6)23(33)19(32)21(27)26(5)9-15(29)18(31)13(4)14(26)8-16(27)38-24(20)34/h7,11,14,16,19-23,31-33H,8-10H2,1-6H3/b12-7+/t14-,16+,19+,20+,21+,22+,23-,26-,27+,28-/m0/s1

Picras-3-en-21-oic acid, 15-((3,4-dimethyl-1-oxo-2-pentenyl)oxy)-13,20-epoxy-3,11,12-trihydroxy-2,16-dioxo-, methyl ester, (11beta,12alpha,15beta(E))- (9CI)

(-)-Bruceantin; NCI165563; NCI-165563; NCI 165563; NSC165563; NSC-165563; BRUCEANTIN; 41451-75-6; Bruceantine; NSC 165563; NSC-165563; NSC165563; Bruceantin(NSC165563); S3NW88DI4T;NSC 165563;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~182.29 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.56 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.56 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.56 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)