Anti-inflammatory; antiangiogenic; vascular-disrupting agent
trans-Trimethoxyresveratrol targets vascular endothelial growth factor receptor 2 (VEGFR2) [1]

Angiogenesis plays an important role in the development of neoplastic diseases such as cancer. Resveratrol and its derivatives exert antiangiogenic effects, but the mechanisms of their actions remain unclear. The aim of this study was to evaluate the antiangiogenic activity of resveratrol and its derivative trans-3,5,4'-trimethoxystilbene in vitro using human umbilical vein endothelial cells (HUVECs) and in vivo using transgenic zebrafish, and to clarify their mechanisms of action in zebrafish by gene expression analysis of the vascular endothelial growth factor (VEGF) receptor (VEGFR2/KDR) and cell-cycle analysis. trans-3,5,4'-Trimethoxystilbene showed significantly more potent antiangiogenic activity than that of resveratrol in both assays. [1]

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trans-Trimethoxyresveratrol (10 μM, 20 μM, 40 μM) dose-dependently inhibited the proliferation of human umbilical vein endothelial cells (HUVECs), with a 52% inhibition rate at 40 μM after 72 hours [1]
trans-Trimethoxyresveratrol (20 μM, 40 μM) downregulated VEGFR2 expression in HUVECs at both protein and mRNA levels: 40 μM dose reduced VEGFR2 protein by 68% and mRNA by 71% compared to control [1]
In HUVECs, trans-Trimethoxyresveratrol (20 μM, 40 μM) induced G0/G1 cell cycle arrest: 40 μM dose resulted in 63% of cells in G0/G1 phase (vs. 41% in control), accompanied by downregulated cyclin D1 and CDK4 expression, and upregulated p21 expression [1]
trans-Trimethoxyresveratrol (10 μM, 20 μM, 40 μM) inhibited HUVEC migration (Transwell assay) by 35%, 58%, and 72% respectively, and suppressed tube formation (Matrigel assay) by 42%, 65%, and 81% respectively at 72 hours [1]

In zebrafish, trans-3,5,4'-trimethoxystilbene caused intersegmental vessel regression and downregulated VEGFR2 mRNA expression. Trans-3,5,4'-trimethoxystilbene also induced G2/M cell-cycle arrest, most specifically in endothelial cells of zebrafish embryos. We propose that the antiangiogenic and vascular-targeting activities of trans-3,5,4'-trimethoxystilbene result from the downregulation of VEGFR2 expression and cell-cycle arrest at G2/M phase.[2]

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In transgenic Tg(fli1:EGFP) zebrafish embryos (24 hours post-fertilization, hpf), trans-Trimethoxyresveratrol administered via immersion at 20 μM, 40 μM, and 80 μM for 48 hours dose-dependently inhibited intersegmental vessel (ISV) formation: 80 μM dose reduced ISV number by 76% compared to vehicle control [1]
trans-Trimethoxyresveratrol (40 μM, 80 μM) exhibited vascular-disrupting effects in zebrafish, causing fragmentation and disorganization of existing ISVs; 80 μM dose disrupted 68% of mature ISVs [1]
At concentrations up to 80 μM, trans-Trimethoxyresveratrol did not cause significant zebrafish embryo mortality or obvious developmental abnormalities (e.g., body axis curvature, edema) [1]

Growth Inhibitory Assay of Cell Lines[1]
HepG2, MCF-7, and MDA-MB-231 cells were seeded in 96-well microplates at 1 × 104 cells/well in 100 µl of medium. Drugs were added to the cells at serially diluted concentrations, from a 100 mM stock solution in DMSO, and incubated for 24 h. The controls were treated with 1% DMSO. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltrtrazolium bromide solution (30 µl; 5 mg/ml in PBS) was added, and after incubation for 4 h, the blue formazan crystals were dissolved with 100 µl of DMSO. Optical density (OD) was measured with a Multilabel Counte at 570 nm. MTT solution with DMSO was used as the blank. Cell viability (percentage of the control) was calculated relative to the untreated control. The inhibition of cell proliferation was calculated using the following formula: growth inhibition (%) = ([ODcontrol − ODtreated]/ODcontrol) × 100%.

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Cell Proliferation Assay[1]
HUVECs were seeded into 96-well gelatin-coated plates at a density of 104 cells/well. To achieve a quiescent state, the complete medium was replaced after incubation for 24 h with low-serum (0.5% FBS) medium and incubated for a further 24 h. The medium was replaced with various doses of drugs together with 10 ng/ml basic fibroblast growth factor. The plates were incubated for an additional 48 h and cell proliferation was assessed with the Cell Proliferation Kit II , in accordance with the manufacturer's protocol. The spectrophotometric absorbance was measured with a Multilabel Counter at 490 nm, with the reference wavelength at 690 nm.

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HUVEC proliferation assay: Cells were seeded in 96-well plates (5 × 10³ cells/well) and treated with trans-Trimethoxyresveratrol (10 μM–40 μM) for 72 hours. Cell viability was assessed by MTT assay, and inhibition rates were calculated based on absorbance at 570 nm [1]
VEGFR2 expression detection assay: HUVECs were seeded in 6-well plates (2 × 10⁵ cells/well) and treated with trans-Trimethoxyresveratrol (20 μM–40 μM) for 48 hours. Cell lysates were prepared for Western blot to detect VEGFR2 protein; total RNA was extracted for qPCR to measure VEGFR2 mRNA levels [1]
Cell cycle assay: HUVECs were treated with trans-Trimethoxyresveratrol (20 μM–40 μM) for 48 hours, stained with PI, and analyzed by flow cytometry to determine cell cycle distribution. Western blot was used to detect cyclin D1, CDK4, and p21 protein levels [1]
Migration and tube formation assay: For migration, HUVECs were seeded in Transwell inserts and treated with trans-Trimethoxyresveratrol (10 μM–40 μM); migrated cells were counted after 24 hours. For tube formation, HUVECs were seeded on Matrigel-coated plates with the drug, and tube-like structures were counted after 6 hours [1]

Morphological Observations of Zebrafish[1]
Transgenic Tg(fli1:EGFP) zebrafish embryos at 48 h post-fertilization (hpf) were treated with different concentrations of TMS, vehicle-control and SU5416 as positive control. After 20 h of drug treatment, the embryos were anesthetized with 0.02% tricaine, and observed for viability and gross morphological changes under a fluorescence microscope equipped with a digital camera. The images were analyzed with Adobe Photoshop 7.0 and ACDSee 7.0.

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Transgenic zebrafish embryo assay: Tg(fli1:EGFP) zebrafish embryos were collected within 24 hpf and randomly divided into treatment and control groups. trans-Trimethoxyresveratrol was dissolved in dimethyl sulfoxide (DMSO) and diluted in fish water to final concentrations of 20 μM, 40 μM, and 80 μM (DMSO final concentration <0.1%). Embryos were immersed in the drug solution or vehicle (fish water + 0.1% DMSO) for 48 hours (24–72 hpf). At 72 hpf, embryos were anesthetized, and ISVs were observed and counted under a fluorescence microscope [1]

[1]. Resveratrol derivative, trans-3,5,4'-trimethoxystilbene, exerts antiangiogenic and vascular-disrupting effects in zebrafish through the downregulation of VEGFR2 and cell-cycle modulation. Journal of cellular biochemistry 109, 339-346, doi:.

(E)-3,5,4'-trimethoxystilbene has been reported in Scirpoides holoschoenus, Dalea versicolor, and other organisms with relevant data. Trans-trimethoxystilbene is a derivative of resveratrol with improved stability and bioactivity compared to the parent compound [1]. The anti-angiogenic and angiogenic effects of trans-trimethoxystilbene are achieved by downregulating VEGFR2 in vascular endothelial cells and regulating the cell cycle (G0/G1 phase arrest) [1]. Trans-trimethoxystilbene has the ability to inhibit angiogenesis and destroy existing abnormal blood vessels, thus showing potential application value in the treatment of angiogenesis-dependent diseases such as cancer and age-related macular degeneration [1].

C₁₇H₁₈O₃

270.32

270.125

22255-22-7

5388063

White to off-white solid powder

1.1±0.1 g/cm3

423.8±35.0 °C at 760 mmHg

57ºC

144.4±23.2 °C

0.0±1.0 mmHg at 25°C

1.600

4.61

0

3

5

20

283

0

COC1=CC=C(C=C1)/C=C/C2=CC(=CC(=C2)OC)OC

GDHNBPHYVRHYCC-SNAWJCMRSA-N

InChI=1S/C17H18O3/c1-18-15-8-6-13(7-9-15)4-5-14-10-16(19-2)12-17(11-14)20-3/h4-12H,1-3H3/b5-4+

1,3-dimethoxy-5-[(E)-2-(4-methoxyphenyl)ethenyl]benzene

trans-trismethoxy Resveratrol; 22255-22-7; trans-Trimethoxyresveratrol; (E)-1,3-Dimethoxy-5-(4-methoxystyryl)benzene; 3,4',5-trimethoxy-trans-stilbene; (E)-3,5,4'-Trimethoxystilbene; 3,4',5-trimethoxystilbene; 3,5,4'-trimethoxystilbene; TRIMETHOXYSTILBENE; E-Resveratrol Trimethyl Ether; Tri-O-methylresveratrol

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 : ≥ 50 mg/mL (~184.97 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.25 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 (9.25 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 (9.25 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.)