In breast cancer models, Tryptanthrin modulates the inflammatory tumor microenvironment (TME), affecting the expression of E-cadherin, Snail, MMP-2, COX-2, NOS1, and NF-κB p65. It does not directly inhibit 5-lipoxygenase (5-LO) enzymatic activity in cell-free assays. In human neutrophils, it potently reduces 5-LO product formation (IC50 = 0.6 ± 0.2 μmol L⁻¹ for LTB4 and 5-H(P)ETE synthesis). [1]
Tryptanthrin inhibits 5-LO product formation in intact human neutrophils stimulated with LPS/fMLP with an IC50 of 0.6 μM. It does not directly inhibit 5-LO in cell-free assays (e.g., neutrophil homogenates or recombinant human 5-LO), nor does it inhibit cPLA₂, FLAP, MAPKs, or Ca²⁺ mobilization. It also shows inhibitory effects on COX-2 (IC50 = 0.83 μM for isolated enzyme) and PGE₂ synthesis. [2]

Tryptamine (0–60 μM; 24, 48, and 72 h) inhibits MCF-7 cell colony growth and proliferation [1]. EMT-associated E-calcium is restored by tryptophan (6.25–25 μM; 48 h; MCF-7 cells). With an IC50 value of 0.6 μM [Mucin, MMP-2, and Snail], tryptamine (0-30 μM; 15 min; MCF-7 cells) decreases leukotriene (LT)-formation in human neutrophils.

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Tryptanthrin inhibited the proliferation of human breast adenocarcinoma MCF-7 cells in a time- and concentration-dependent manner (1.56-50.0 μmol L⁻¹). At 50.0 μmol L⁻¹ for 72 h, the proliferation rate was most significantly decreased (p < 0.001). It also inhibited colony formation at concentrations of 1.56, 3.13, and 6.25 μmol L⁻¹. H&E staining revealed morphologic changes including poor cell adherence, darkened nuclei, disappeared cell membrane, and diffused cytoplasm. [1]
Tryptanthrin blocked migration and invasion of MCF-7 cells. In wound healing assays, it inhibited TGF-β1-induced migration at 3.13 and 6.25 μmol L⁻¹ (p < 0.05, p < 0.01). In Transwell chamber assays, it reduced TGF-β1-induced invasion, with the highest dose (6.25 μmol L⁻¹) maximally reducing invasion (p < 0.001). [1]
Tryptanthrin reversed EMT-associated protein expression in TGF-β1-stimulated MCF-7 cells. It upregulated E-cadherin protein levels at 6.25, 12.5, and 25.0 μmol L⁻¹ (p < 0.05) and downregulated MMP-2 and Snail protein levels (p < 0.05, p < 0.01). [1]
Tryptanthrin potently reduced 5-LO product (LTB₄ and 5-H(P)ETE) formation in human neutrophils stimulated with LPS (1 μg/mL) and fMLP (1 μM), with an IC50 of 0.6 ± 0.2 μM, comparable to zileuton (IC50 = 0.7 ± 0.1 μM). It did not inhibit arachidonic acid (AA) release in neutrophils at up to 30 μM. [2]
Tryptanthrin (30 μM) did not directly inhibit 5-LO activity in neutrophil homogenates or partially purified recombinant human 5-LO. It showed no significant radical scavenging activity in the DPPH assay. It did not inhibit fMLP-induced phosphorylation of ERK1/2 or p38 MAPK, nor did it suppress fMLP-stimulated increases in intracellular Ca²⁺ or ROS formation. It did not prevent LPS-induced degradation of IκBα or nuclear accumulation of p65 (NF-κB). [2]
Tryptanthrin caused a redistribution of 5-LO in human neutrophils. Immunofluorescence microscopy showed it induced 5-LO accumulation within the perinuclear region, preventing its full translocation to the nuclear membrane upon stimulation with LPS/fMLP. [2]

Tryptamine (25-100 mg/kg; intraperitoneal; daily for 13 days; female Bal b/c mice with 4T1 xenografts) suppresses the formation of tumors and modifies IL-2, IL-6, IL-10, and IL-Tryptophan (10 mg/kg; powder; Vehicle Wistar Han Stent) to produce LTB4 in vivo [2].

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In a 4T1 murine breast cancer model, oral administration of Tryptanthrin (25.0, 50.0, 100.0 mg kg⁻¹) for 13 consecutive days inhibited tumor growth. Tumor volumes in treated groups were smaller than in model groups, with 100.0 mg kg⁻¹ showing remarkable efficacy. It did not significantly affect body mass or organ coefficients (liver, spleen, lung, kidney) (p > 0.05), unlike cyclophosphamide (40 mg kg⁻¹) which reduced body mass and organ coefficients (p < 0.05 for lung and kidney, p < 0.01 for spleen). [1]
Tryptanthrin regulated serum cytokine levels in 4T1 tumor-bearing mice. It increased IL-2 and TNF-α levels (e.g., 1.93 and 1.37 times higher than model group for IL-2 at 100.0 mg kg⁻¹ and TNF-α at 100.0 mg kg⁻¹, respectively) (p < 0.05, p < 0.01). It also reduced the elevated serum IL-10 levels observed in the model group (p < 0.05). [1]
Tryptanthrin inhibited the expression of inflammatory proteins in tumor tissues of 4T1-bearing mice. IHC analysis showed downregulation of COX-2 (100.0 mg kg⁻¹) and NOS1 (25.0, 50.0, 100.0 mg kg⁻¹) (p < 0.05). Western blotting showed downregulation of NF-κB p65 protein expression in the tryptanthrin-treated groups (p < 0.05, p < 0.01). [1]
Tryptanthrin reduced LTB₄ levels in vivo. In a rat model of carrageenan-induced pleurisy, a single oral dose of 10 mg kg⁻¹ given 1 hour before carrageenan significantly reduced LTB₄ pleural levels by 46% (p < 0.01), PGE₂ levels by 42% (p < 0.01), exudate volume by 80% (p < 0.001), and the number of infiltrating inflammatory cells by 41% (p < 0.001). [2]

For determining 5-LO product formation in cell-free systems, neutrophil homogenates or partially purified human recombinant 5-LO were used. The homogenates or enzyme were incubated with Tryptanthrin or vehicle for 10 min at 4°C, then pre-warmed for 30 s at 37°C. The reaction was initiated by adding 2 mmol L⁻¹ CaCl₂ and arachidonic acid (AA, 20 μmol L⁻¹). After 10 min at 37°C, the reaction was stopped with ice-cold methanol and HCl. Formed 5-LO metabolites (LTB₄, 5-H(P)ETE) were extracted using C-18 solid-phase columns and analyzed by reversed-phase HPLC. [2]
The redox potential and radical scavenging property of Tryptanthrin were evaluated using the DPPH (diphenylpicrylhydrazyl) assay. A solution of DPPH radical in ethanol (buffered to pH 5.5) was added to increasing concentrations of tryptanthrin (25-200 μmol L⁻¹) in ethanol. The absorbance was recorded at 520 nm after 30 min incubation in the dark. Ascorbic acid and L-cysteine were used as reference compounds. [2]
The inhibition of isolated COX-2 by Tryptanthrin was mentioned, with an IC50 of 0.83 μmol L⁻¹. [2]

Cell viability assay [1]
Cell Types: MCF-7 cells
Tested Concentrations: 0-60 μM
Incubation Duration: 24, 48 and 72 hrs (hours)
Experimental Results: The inhibition rate of MCF-7 cells increased in a dose- and time-dependent manner. 2]. Way.

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Western Blot Analysis[1]
Cell Types: MCF-7 Cell
Tested Concentrations: 6.25, 12.5 and 25 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: E-cadherin protein levels increased, MMP-2 and Snail protein expression levels were Dramatically up-regulated TGF-β1 induction of MCF-7 cells.

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MCF-7 human breast adenocarcinoma cells were used to assess the antiproliferative effect of Tryptanthrin. Cells were seeded into 96-well plates (1.0 x 10⁵ cells mL⁻¹) and treated with various concentrations (1.56-50.0 μmol L⁻¹) for 24, 48, and 72 h. Then, 15 μL of 5% MTT solution was added. After 4 h of culture, the supernatant was discarded, and 150 μL DMSO was added to dissolve the crystals. Absorbance (OD) was measured at 490 nm using a microplate reader. [1]
Cell colony-forming ability of MCF-7 cells was assessed. Cells were plated into 6-well plates (250 cells mL⁻¹) and treated with Tryptanthrin (1.56, 3.13, 6.25 μmol L⁻¹) or left untreated for 2 weeks. Colonies containing at least 50 cells were stained with Giemsa, photographed, and counted. [1]
For H&E staining of MCF-7 cells, cells were cultured on glass slides and treated with Tryptanthrin (6.25, 12.5, 25.0 μmol L⁻¹) for 24 h. Cells were then fixed with 95% ethanol, stained with hematoxylin and eosin, and photographed under an optical microscope. [1]
Wound healing assay was used to detect migration of MCF-7 cells. Cells were treated with TGF-β1 (5 μg L⁻¹) and/or Tryptanthrin (1.56, 3.13, 6.25 μmol L⁻¹). A scratch was made using a pipette tip, and images were taken at 0 and 24 h. Scratch width was measured using ImageJ software, and the scratch healing rate was calculated. [1]
Transwell chamber assay was used to assess MCF-7 cell invasion. Cells were pre-treated with TGF-β1 (5 μg L⁻¹) and/or Tryptanthrin (1.56, 3.13, 6.25 μmol L⁻¹) for 24 h. Matrigel-coated inserts were used. A cell suspension (5 x 10⁵ cells mL⁻¹, 200 μL) was added to the upper chamber, and medium with 10% FBS was added to the lower chamber. After 24 h, non-invading cells were removed, and invading cells were fixed, stained with crystal violet, and counted. [1]
Western blotting was performed on MCF-7 cells treated with TGF-β1 (5 μg L⁻¹) and/or Tryptanthrin (6.25, 12.5, 25.0 μmol L⁻¹) for 24 h. Cellular proteins were extracted, separated by SDS-PAGE, and transferred to PVDF membranes. Membranes were incubated with primary antibodies against E-cadherin, Snail, MMP-2, and GAPDH, followed by HRP-labeled secondary antibodies. Bands were visualized by chemiluminescence and analyzed. [1]
For assessment of 5-LO product formation in intact neutrophils, freshly isolated human neutrophils were primed with LPS (1 μg mL⁻¹) and adenosine deaminase (0.3 U mL⁻¹) for 30 min at 37°C. Tryptanthrin or vehicle was added 15 min before stimulation with fMLP (1 μmol L⁻¹). After 5 min, the reaction was stopped with methanol and HCl. 5-LO metabolites were extracted and analyzed by HPLC. [2]
To measure arachidonic acid (AA) release, neutrophils were pre-labelled with [³H]-AA. Labelled neutrophils were primed with LPS and stimulated with fMLP. Tryptanthrin was added 15 min before fMLP. The amount of [³H]-AA released into the medium was measured by liquid scintillation counting. [2]
For analysis of 5-LO subcellular localization by immunofluorescence, neutrophils were primed with LPS/Ada, treated with Tryptanthrin (30 μmol L⁻¹) or vehicle, and then activated with fMLP. Cells were centrifuged onto poly-L-lysine-coated coverslips, fixed in methanol, and permeabilized. They were then incubated with anti-5-LO antibody, followed by Alexa Fluor 488-conjugated secondary antibody. Fluorescence was visualized using a fluorescence microscope. [2]

Animal/Disease Models: α levels in female Bal b/c mice 12 and TNF-tumor-bearing mice [1]. 4T1 Xenograft[1]
Doses: 25, 50 and 100 mg/kg
Route of Administration: po (oral gavage); one time/day for 13 days
Experimental Results: Inhibition of tumor growth in a dose-dependent manner.

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Animal/Disease Models: Male Wistar Han rat (220-230 g) [2]
Doses: 10 mg/kg
Route of Administration: Oral; primary
Experimental Results: LTB4 pleural levels were diminished by 46%. diminished PGE2 levels (42% reduction), exudate volume (80% reduction), and infiltrating cell number (41% reduction).

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For the 4T1 murine breast cancer model, female Balb/c mice were injected with 0.1 mL of 1 x 10⁶ mL⁻¹ 4T1 cell suspension into the right forelimb armpit. When tumor size reached ~100 mm³, mice were randomly divided into groups (n=9). Tryptanthrin (25.0, 50.0, 100.0 mg kg⁻¹) was administered through oral gavage once daily for 13 consecutive days. The positive control group received cyclophosphamide (40 mg kg⁻¹). Model groups received normal saline (NS) or 0.5% sodium carboxymethyl cellulose (CMCNa). Tumor volume was measured daily with a vernier caliper and calculated as V = 1/2 x a x b² (a = longest diameter, b = shortest diameter). On day 13, mice were anesthetized, blood was collected, and serum was obtained by centrifugation. Mice were sacrificed, and tumors, livers, spleens, and lungs were harvested. Organ coefficients were calculated as (organ mass / body mass) x 100%. [1]
For the carrageenan-induced pleurisy model in rats, male Wistar Han rats received an oral dose of Tryptanthrin (10 mg kg⁻¹) or vehicle (0.5 mL of 0.5% carboxymethylcellulose and 10% Tween 20) 1 hour before carrageenan injection. Rats were anesthetized, and 0.2 mL of 1% λ-carrageenan was injected into the pleural cavity. At 4 hours after carrageenan injection, animals were sacrificed, and the pleural cavity was rinsed with 2 mL saline containing heparin. The exudate volume was measured. Leukocytes in the exudate were counted. LTB₄ levels in the exudate supernatant were assayed by enzyme immunoassay, and PGE₂ levels by radioimmunoassay. [2]

In the 4T1 mouse breast cancer model, Tryptanthrin (25.0, 50.0, 100.0 mg kg⁻¹, p.o. for 13 days) showed favorable safety. It did not induce significant fluctuations in body mass or organ coefficients (liver, spleen, lung, kidney) of tumor-bearing mice compared to model groups (p > 0.05). In contrast, the positive drug cyclophosphamide (40 mg kg⁻¹) significantly reduced body mass and organ coefficients (p < 0.05 for lung and kidney, p < 0.01 for spleen). H&E staining of organs (liver, spleen, lung) from tryptanthrin-treated mice showed no obvious toxic effects. [1]
In human neutrophils, incubation with Tryptanthrin up to 30 μmol L⁻¹ for 30 min at 37°C caused no significant change in cell viability, as analyzed by light microscopy and trypan blue exclusion. [2]

[1]. Tryptanthrin exerts anti-breast cancer effects both in vitro and in vivo through modulating the inflammatory tumor microenvironment. Acta Pharm. 2021 Jun 1;71(2):245-266.

[2]. On the inhibition of 5-lipoxygenase product formation by tryptanthrin: mechanistic studies and efficacy in vivo. Br J Pharmacol. 2012 Feb;165(3):765-76.

Tryptanthrine is an organic nitrogen heterocyclic compound, an organic heterotetracyclic compound, and an alkaloid antibiotic. Indole[2,1-b]quinazolin-6,12-dione has been reported to exist in Strobilanthes, Strobilanthes, and other organisms with relevant data.

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The inflammatory tumor microenvironment (TME) plays a crucial role in oncogenesis. Tryptanthrin exerts its anti-breast cancer activities by modulating the inflammatory TME both in vitro and in vivo, inhibiting proliferation, migration, and invasion of cancer cells, partly through upregulating E-cadherin and downregulating MMP-2 and Snail. It also suppresses tumor growth in mice by downregulating NOS1, COX-2, and NF-κB in tumor tissues, and upregulating IL-2 and TNF-α while normalizing IL-10 levels in serum. [1]
Tryptanthrin is a potent natural inhibitor of cellular leukotriene (LT) biosynthesis with proven efficacy in human whole blood (IC50 ~10 μmol L⁻¹ for both A23187 and LPS/fMLP stimulations) and in vivo after oral administration. Its unique pharmacological profile, including causing a subcellular redistribution of 5-LO and not acting as a direct 5-LO inhibitor or redox-active compound, suggests a novel molecular mechanism. It exhibits comparable potencies for inhibition of both LT and PG biosynthesis, suggesting a possible dual or multiple-target inhibition of arachidonic acid metabolism pathways. It has also shown antitumoural effects in leukemia cells and breast cancer cells, indicating potential as an anticancer agent. It is noted for its stability and ease of synthesis. [2]

C15H8N2O2

248.2362

248.059

C, 72.58; H, 3.25; N, 11.29; O, 12.89

13220-57-0

73549

Light yellow to yellow solid powder

1.45g/cm3

469.3ºC at 760 mmHg

237.7ºC

5.55E-09mmHg at 25°C

1.762

1.93

0

3

0

19

471

0

O=C1C2=C([H])C([H])=C([H])C([H])=C2N2C(C3=C([H])C([H])=C([H])C([H])=C3N=C21)=O

VQQVWGVXDIPORV-UHFFFAOYSA-N

InChI=1S/C15H8N2O2/c18-13-10-6-2-4-8-12(10)17-14(13)16-11-7-3-1-5-9(11)15(17)19/h1-8H

indolo[2,1-b]quinazoline-6,12-dione

Tryptanthrin

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 : ~7.14 mg/mL (~28.76 mM)

Solubility in Formulation 1: 0.71 mg/mL (2.86 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 7.1 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: 0.67 mg/mL (2.70 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 6.7 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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 (Please use freshly prepared in vivo formulations for optimal results.)