Because TMS is a strong CYP1B1 inhibitor, it is regarded as a possible cancer preventative drug and an equivalent of resveratrol. Cells were cultured for up to 72 hours without changing the media in order to assess the survival of MCF-7 cells exposed to 1 μM benzo[a]pyrene (BP), 1 μM BP + 1 μM TMS, and 1 μM BP + 4 μM TMS. cells exposed to light expressed as a percentage of cells treated with solvent (DMSO) at the same time interval in terms of luminescence units. For the first 24 hours, cell viability in all exposure groups was >90%; however, by the 72nd hour, it had decreased to 60–70% [1].

Because TMS is a strong CYP1B1 inhibitor, it is regarded as a possible cancer preventative drug and an equivalent of resveratrol. Cells were cultured for up to 72 hours without changing the media in order to assess the survival of MCF-7 cells exposed to 1 μM benzo[a]pyrene (BP), 1 μM BP + 1 μM TMS, and 1 μM BP + 4 μM TMS. cells exposed to light expressed as a percentage of cells treated with solvent (DMSO) at the same time interval in terms of luminescence units. For the first 24 hours, cell viability in all exposure groups was >90%; however, by the 72nd hour, it had decreased to 60–70% [1].

---

In MCF-7 human breast cancer cells co-exposed to 1 μM Benzo[a]pyrene (BP) and TMS (1 or 4 μM) for 96 hours, TMS did not reduce the overall formation of BP-DNA adducts (BPdG). The maximum level of BPdG adducts reached was similar across all groups (BP alone: ~15.7 adducts/10⁶ nucleotides at 16h; BP + 1μM TMS: ~15.9 at 24h; BP + 4μM TMS: ~16.6 at 48h). However, TMS delayed the time to reach this maximum.[2]
TMS, in combination with BP, significantly increased the overall gene expression of CYP1A1 and CYP1B1 over the 96-hour exposure period (calculated as Area Under the Curve, AUC₄₋₉₆), in a dose-dependent manner, compared to BP exposure alone. For example, the CYP1A1 expression AUC₄₋₉₆ was about 10-fold higher with 4 μM TMS than with BP alone.[2]
TMS also significantly increased the combined CYP1A1/1B1 enzyme activity (measured by EROD assay) over the 96-hour period in a dose-dependent manner. The EROD activity AUC₄₋₉₆ was about 2.8-fold higher with 4 μM TMS than with BP alone.[2]
Exposure to TMS alone (1.0 or 4.0 μM) for 24 hours significantly induced CYP gene expression in MCF-7 cells (CYP1A1: 780-fold and 360-fold induction; CYP1B1: 3-fold and 2.5-fold induction, for 1 and 4 μM TMS respectively), but did not significantly affect cell viability or basal EROD activity.[2]

The effect of TMS was investigated in SHR and WKY rats in order to ascertain the function of CYP1B1 in the development of hypertension in spontaneously hypertensive rats (SHR). Starting at 4 weeks of age, the systolic blood pressure in SHR grew gradually. Daily TMS injections started to lower the SHR's systolic blood pressure at 8 weeks of age, returning it to the initial values (207±7 vs. 129±2 mmHg). In WKY treated with TMS or its carrier, systolic blood pressure did not alter (129±7 vs. 127±4 mmHg) [1].

---

Daily intraperitoneal administration of TMS (600 µg/kg), starting from 8 weeks of age, reversed the development of hypertension in SHR, reducing systolic blood pressure from 207 ± 7 mmHg to 129 ± 2 mmHg (levels comparable to normotensive WKY rats), without altering blood pressure in WKY rats.[3]
TMS treatment reduced the increased CYP1B1 enzymatic activity observed in the aorta, heart, and kidney of SHR, but did not alter CYP1B1 protein expression in these tissues.[3]
TMS minimized increased vascular reactivity to phenylephrine and endothelin-1 in the aorta, mesenteric, and renal arteries of SHR, and reduced the media-to-lumen ratio (an indicator of vascular smooth muscle hypertrophy) in these vessels.[3]
TMS improved endothelial dysfunction in SHR, as evidenced by enhanced acetylcholine-induced relaxation in the aorta, mesenteric, and renal arteries. Endothelium-independent relaxation to sodium nitroprusside was unaffected.[3]
TMS improved renal dysfunction in SHR: it increased glomerular filtration rate (creatinine clearance), urinary sodium excretion, and urine osmolality, while reducing serum creatinine and proteinuria.[3]
TMS reduced cardiac hypertrophy (heart-to-body weight ratio) and minimized cardiac and renal fibrosis (evidenced by reduced α-smooth muscle actin-positive myofibroblasts and collagen deposition) in SHR.[3]
TMS reduced superoxide anion production (measured by dihydroethidium fluorescence) and NADPH oxidase activity in the aorta, heart, and kidney of SHR.[3]
TMS reduced increased plasma and urinary levels of oxidative stress markers in SHR, including hydrogen peroxide (H₂O₂), thiobarbituric acid reactive substances (TBARS, a marker of lipid peroxidation), and nitrate/nitrite (NOx).[3]
TMS decreased elevated plasma levels of pro-inflammatory cytokines in SHR, specifically interleukin-1β (IL-1β), IL-2, IL-6, and IL-12.[3]
TMS lowered increased plasma levels of catecholamines (norepinephrine and epinephrine) in SHR.[3]
TMS reduced the increased cardiac activity (phosphorylation) of signaling molecules ERK1/2, p38 mitogen-activated protein kinase (MAPK), c-Src tyrosine kinase, and protein kinase B (Akt) in SHR.[3]

An ethoxyresorufin-O-deethylase (EROD) assay was used to measure the combined specific activity of CYP1A1 and CYP1B1 enzymes. Briefly, MCF-7 cells cultured in multi-well plates were exposed to treatments. At designated time points, the culture medium was removed, cells were washed, and an EROD solution containing ethoxyresorufin and salicylamide in buffer was added. After incubation, an aliquot of the reaction solution was transferred to a plate, and the conversion of ethoxyresorufin to resorufin was measured fluorometrically. Protein content from the lysed cells was quantified to normalize the activity, which was expressed as picomoles of resorufin produced per minute per microgram of protein.[2]

Cell viability was assessed using a luminescent assay. MCF-7 cells were seeded in multi-well plates, exposed to treatments, and lysed at various time points. A portion of the cell lysate was mixed with a luminescent reagent, and the luminescent signal, proportional to ATP content/viable cells, was measured. Viability was expressed as a percentage of the signal from solvent-treated control cells.[2]
For DNA adduct analysis, MCF-7 cells were cultured in flasks and exposed to treatments. Cells were harvested at multiple time points over 96 hours. DNA was isolated using a non-organic procedure involving RNase A and Proteinase K digestion, followed by purification. DNA quantity and quality were assessed spectrophotometrically and fluorometrically.[2]
BP-DNA adducts (BPdG) were quantified using a BPDE-DNA chemiluminescence immunoassay (CIA). Opaque high-binding plates were coated with DNA. Sample DNA or standard BPDE-modified DNA was sonicated and mixed with a primary antibody specific for BPDE-DNA adducts. After incubation and washing, sequential incubations with a biotinylated secondary antibody and streptavidin-alkaline phosphatase were performed. Chemiluminescent substrate was added, and the signal was measured. Adduct levels were determined by comparison to a standard curve.[2]
For gene expression analysis, RNA was isolated from treated cells at various time points, treated with DNase, and reverse transcribed into cDNA. Quantitative real-time PCR was performed using gene-specific primers for CYP1A1 and CYP1B1, with GAPDH as a housekeeping gene. The fold change in gene expression was calculated using the ΔΔCt method.[2]

Male Spontaneously Hypertensive Rats (SHR) and normotensive Wistar-Kyoto (WKY) rats (3 weeks old) were used. Starting at 8 weeks of age, rats received daily intraperitoneal injections of TMS (600 µg/kg) or its vehicle (dimethyl sulfoxide, DMSO, 100 µl) for a period of 6 weeks (until 14 weeks of age). Blood pressure was measured twice weekly using a non-invasive tail-cuff method. At the end of the treatment period (14 weeks), animals were anesthetized, and blood, aorta, heart, kidneys, and other tissues were collected for various biochemical, histological, and functional analyses.[3]

In MCF-7 cells, after exposure to 1 μM BP alone or in combination with 1 or 4 μM TMS for up to 72 hours, cell viability remained above 90% for the first 24 hours, but decreased to 60-70% after 72 hours. This cytotoxicity was mainly attributed to BP, as the addition of TMS did not alter the cell survival pattern. Exposure to TMS alone (1.0 or 4.0 μM) for 24 hours did not result in significant cytotoxicity. [2]

[1]. Design, synthesis, and discovery of novel trans-stilbene analogues as potent and selective human cytochrome P450 1B1 inhibitors. J Med Chem. 2002 Jan 3;45(1):160-4.

[2]. The resveratrol analogue, 2,3',4,5'-tetramethoxystilbene, does not inhibit CYP gene expression, enzyme activity and benzo[a]pyrene-DNA adduct formation in MCF-7 cells exposed to benzo[a]pyrene. Mutagenesis. 2011 Sep;26(5):629-35.

[3]. Cytochrome P450 1B1 contributes to increased blood pressure and cardiovascular and renal dysfunction in spontaneously hypertensive rats. Cardiovasc Drugs Ther. 2014 Apr;28(2):145-61.

1-[2-(2,4-dimethoxyphenyl)vinyl]-3,5-dimethoxybenzene is a stilbene compound. 2,3',4,5'-tetramethoxystilbene has been reported in apple wood (Maclura pomifera), and data are available. TMS is an analog of resveratrol and is considered a potential cancer preventative agent due to its potent inhibition of CYP1B1. [2] Contrary to the initial hypothesis, in a chronic model of 96 hours of co-exposure to BP in MCF-7 cells, TMS did not inhibit the formation of BP-DNA adducts (BPdG). Instead, it slowed the rate of BP biotransformation, prolonged the time required to reach maximum adduct levels, and unexpectedly increased the overall expression and activity of BP-induced CYP1A1 and CYP1B1 enzymes. [2]
This study suggests that the potential application of TMS in human chemoprevention should be approached with caution, as it failed to inhibit the formation of carcinogen-DNA adducts under the tested chronic exposure conditions. [2]

C18H20O4

300.35

300.136

24144-92-1

5354004

Light yellow to yellow solid powder

1.1±0.1 g/cm3

459.9±40.0 °C at 760 mmHg

152.3±34.2 °C

0.0±1.1 mmHg at 25°C

1.588

4.49

0

4

6

22

332

0

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

JDBCWSHYEQUBLW-AATRIKPKSA-N

InChI=1S/C18H20O4/c1-19-15-8-7-14(18(12-15)22-4)6-5-13-9-16(20-2)11-17(10-13)21-3/h5-12H,1-4H3/b6-5+

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.32 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (8.32 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.

---

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