Glutathione (GSH) – Diethylmaleate depletes cellular GSH by conjugation [1].
MAPK pathway components (ERK, p38, JNK) – activation via phosphorylation (Thr202/Tyr204 for ERK; Thr180/Tyr182 for p38; Thr183/Tyr185 for JNK) [1].

DEM induces upregulation of GSH(L-c-glutamyl-L-cysteinyl-glycine) metabolism, and the downregulation of pathways of cancer, chemokine signaling, cytokine-cytokine receptor, and focal adhesion in transformed cells. DEM appears to modify microenvironment of transformed cells thereby restraining tumor cell growth. DEM is cytotoxic to the transformed cells in a concentration dependent manner. DEM at 0.25 mM decreases cell viability to 75%. The co-exposure of cells to DEM+GSHe inhibits DEM induced cytotoxicity. DEM exposure increases the ROS generation by multiple orders of magnitude in transformed cells. This is evident from the dose and time dependent increase in fluorescence intensity of CMH2DCFDA. Moreover, DEM activates MAPK pathway and DEM induced activation of ERK is found to be due to phosphorylation at Thr 202/204.

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Cell Assay: A stock solution of DEM is prepared in 100% DMSO and diluted in DMEM to achieve the desired concentration with less than 0.1% DMSO in the culture wells. Briefly, cells are seeded (1 ×104 cells/well) in 96-well plates and allowed to adhere overnight. Subsequently, these cells are exposed to DEM at various test concentrations (0.05-1 mM) or co-exposed to equimolar (0.25 mM) DEM+GSHe for 24 h. Cell viability is determined by recording the OD at 570 nm in an Elisa microplate reader.

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Diethylmaleate (0.25 mM) decreased viability of transformed C3H10T1/2 cells to approximately 75% after 24 h in a concentration‑dependent manner (0.05–1 mM); co‑exposure with equimolar GShE (0.25 mM) inhibited this cytotoxicity [1].
Diethylmaleate (0.1–0.5 mM, 18 h) reduced cellular GSH content in a concentration‑ and time‑dependent manner; at 0.25 mM for 24 h, GSH decreased by nearly 50%; 0.5 mM for 18 h substantially decreased GSH [1].
Diethylmaleate increased ROS generation in transformed cells in a dose‑ and time‑dependent manner (measured by H2DCFDA fluorescence) [1].
Diethylmaleate (0.25 mM, 18 h) induced cell cycle arrest at G0/G1 phase in transformed cells; 0.25 mM caused a 6–8‑fold increase in TUNEL‑positive nuclei, and 0.5 mM caused a 15–18‑fold increase [1].
Diethylmaleate (0.25 mM) activated MAPK pathway: ERK phosphorylation at Thr202/Tyr204 (detectable after 15 min), p38 phosphorylation at Thr180/Tyr182 (after 30 min), and JNK phosphorylation at Thr183/Tyr185 (after 12–18 h) in tC3H10T1/2 cells. Pre‑incubation with ERK inhibitor PD98059 (50 μM) or p38 inhibitor SB203580 (30 μM) prevented DEM‑induced cytotoxicity up to 95%; JNK inhibitor SP600125 (20 μM) was partially effective (increased viability up to 85%). ERK1/2 and p38 activations were independent of each other [1].
Diethylmaleate (0.25 mM) reduced the frequency of colony formation in soft agar by 27% in tC3H10T1/2 cells and decreased DNA content in colonies [1].
Diethylmaleate (0.25 mM, 18 h) upregulated genes involved in glutathione metabolism: GSTA1 (9.19‑fold), GSTA2 (10.03‑fold), GSTA3 (7.17‑fold), GCLC (3.61‑fold), GCLM (3.56‑fold), GSS (2.66‑fold), ODC1 (2.05‑fold). Downregulated pathways included cancer pathway, cytokine‑cytokine receptor interaction (18 genes, p=0.0008), focal adhesion (18 genes, p=0.0176), chemokine signaling (12 genes, p=0.0015), and others. Downregulated genes included FGF18, CXCL12, PDGFB, TGFB2, CAMK2A, etc. [1].

Sperm motility and epididymal sperm count are significantly reduced in the DEM treated animals. Fertility status is also affected by DEM exposure as is evident from the percent fertility and the litter size. consequences of the oxidative stress produced by the DEM induces glutathione depletion, on the reproductive ability of male mice and modulation of the various components of the antioxidant defense system at the transcriptional level.

Cell viability assay (MTT): transformed C3H10T1/2 or BALB/c 3T3 cells were seeded at 1×10⁴ cells/well in 96‑well plates, allowed to adhere overnight, then exposed to DEM at 0.05–1 mM or co‑exposed to equimolar (0.25 mM) DEM+GShE for 24 h. Viability was determined by OD at 570 nm using a microplate reader [1].
GSH determination (DTNB method): cells seeded at 2×10⁶ per flask, exposed to DEM (0.1, 0.25, 0.5 mM) or DEM+GShE for 6,12,18,24 h. Cells lysed in extraction buffer (0.1% Triton‑X, 0.6% sulfo‑salicylic acid in 0.1 M potassium phosphate buffer pH 7.5), sonicated, centrifuged. Supernatant mixed with DTNB and glutathione reductase, NADPH added, absorbance at 412 nm recorded [1].
GSH measurement using CMFDA dye: cells seeded at 2×10⁴/well in black‑bottom 96‑well plate, exposed to DEM or DEM+GShE for 18 h, stained with 1 μM CMFDA for 30 min, fluorescence measured at Ex 488 nm/Em 510–540 nm; also imaged with Hoechst nuclear stain [1].
ROS measurement (H2DCFDA): cells seeded at 2×10⁴/well in black‑bottom 96‑well plate, exposed to DEM or DEM+GShE for 0,1,2,4,6 h, incubated with 5 μM H2DCFDA for 30 min, fluorescence measured at Ex 485 nm/Em 530 nm [1].
Cell cycle analysis: cells seeded at 1×10⁶/well in 6‑well plate, exposed to DEM or DEM+GShE for 18 h, harvested, ethanol‑fixed, incubated with RNase (250 μg, 1 h, 37 °C), stained with propidium iodide (50 μg), analyzed by flow cytometry (BD FACS Canto II) [1].
TUNEL assay: cells seeded on coverslips (0.5×10⁶), exposed to DEM or DEM+GShE for 18 h, stained with Hoechst, TUNEL‑positive nuclei counted under fluorescence microscope (five random areas, % of 100 nuclei) [1].
Western blot for MAPK: cells seeded at 2×10⁶/well in 6‑well plate, pre‑incubated with kinase inhibitors (PD98059 50 μM, SB203580 30 μM, SP600125 20 μM) for 2 h, then exposed to DEM (0.25 mM) ± GShE for 1,6,12,18 h. Cells lysed in Cell Lytic‑M with protease inhibitor cocktail, protein quantified (Bradford), 50 μg protein run on 10% SDS‑PAGE, transferred to PVDF, probed with primary antibodies (p‑ERK, ERK; p‑p38, p38; p‑JNK, JNK; 1:1000), then HRP‑conjugated secondary antibody (1:10,000), detected by chemiluminescence [1].
Soft agar colony formation assay: cells (5000/well) suspended in 0.33% agarose in complete medium over a base layer of 0.5% agarose in 24‑well plates, incubated for 15 days with medium refresh every 5th day. Colonies (>50 cells) stained with 5% Giemsa and counted. DNA content in colonies measured using CyQuant GR dye (Ex 485 nm/Em 520 nm) after lysing with matrix solubilization solution [1].
Microarray: tC3H10T1/2 cells exposed to DEM (0.25 mM) for 18 h, total RNA isolated, cRNA labeled with Cy3 using Agilent Low input RNA linear amplification kit, hybridized to Whole Genome Mouse 8×60K slide (16 h at 65 °C), scanned with Agilent Microarray Scanner, analyzed with Gene Spring GX v11.5 [1].
qRT‑PCR validation: RNA from tC3H10T1/2 and tBALB/c cells exposed to DEM (0.25 mM) or DEM+GShE for 18 h. cDNA synthesis, SYBR Green qPCR with primers for GSTA1, GSTA3, GCLM, GSS, FGF18, CXCL12, PDGFB, TGFB2, CAMK2A, normalized to GAPDH. Fold change calculated by 2^‑ΔΔCT [1].

Dissolved in corn oil; 6mmol/kg; i.p. Sprague-Dawley rats

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Diethylmaleate at 0.25 mM was non‑cytotoxic to non‑transformed cells but reduced viability of transformed cells to 75% [1].
Co‑exposure with equimolar GShE (0.25 mM) blocked DEM‑induced cytotoxicity, GSH depletion, ROS increase, cell cycle arrest, TUNEL positivity, MAPK activation, and gene expression changes [1].
No in vivo toxicity data are presented in this study; the authors cite other reports (not extracted) that DEM delays tumor induction and reduces tumor incidence in vivo [1].

Chem Biol Interact.2014 Aug 5;219:37-47;Biochem Pharmacol.1992 Feb 4;43(3):451-6.

Diethyl maleate is a maleic ester formed by the condensation of the two carboxyl groups of maleic acid with ethanol. It is a colorless liquid at room temperature (melting point -10°C) and has a boiling point of 220°C at 1 atmosphere. It is commonly used as a diephile in Diels-Alder cycloaddition reactions in organic synthesis. It also has the effect of consuming glutathione. It is both a maleic ester and an ethyl ester. Its functional group is related to ethanol.
See also: 2-Butenic acid (2Z)-, di-C8-15-alkyl ester (note moved to).

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Diethylmaleate is a maleate derivative that depletes cellular glutathione (GSH) by conjugation via glutathione S‑transferases. It is not an inhibitor of GSH biosynthetic enzymes. In this study, DEM was used at concentrations of 0.05–1 mM, with 0.25 mM selected for most experiments as a moderate GSH‑depleting dose. The effects of DEM were reversible by co‑treatment with glutathione monoethyl ester (GShE), confirming the specificity of GSH depletion [1].
The transformed cells (tC3H10T1/2 and tBALB/c) had 1.6‑ to 1.8‑fold higher basal GSH levels than non‑transformed cells [1].
DEM exposure dysregulated 2260 up‑regulated and 2920 down‑regulated genes (p<0.05), with 52.91% changed >2‑fold, 27.9% >3‑fold, 12.39% >4‑fold, 6.8% >5‑fold. Microarray data deposited in GEO with accession GSE44650 [1].
DEM downregulated pathways including cytokine‑cytokine receptor interaction, focal adhesion, cancer pathway, chemokine signaling, axon guidance, gap junction, TGF‑beta signaling, etc. (detailed in Table 4 and Supplementary Table S1) [1].

C8H12O4

172.18

172.073

141-05-9

5271566

Colorless to light yellow liquid

1.1±0.1 g/cm3

214.0±0.0 °C at 760 mmHg

−10 °C(lit.)

93.3±0.0 °C

0.2±0.4 mmHg at 25°C

1.443

1.68

0

4

6

12

164

0

CCOC(=O)/C=C\C(=O)OCC

IEPRKVQEAMIZSS-WAYWQWQTSA-N

InChI=1S/C8H12O4/c1-3-11-7(9)5-6-8(10)12-4-2/h5-6H,3-4H2,1-2H3/b6-5-

2-Butenedioic acid (2Z)-, diethyl ester

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: ≥ 9.09 mg/mL (52.79 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 90.9 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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 (Please use freshly prepared in vivo formulations for optimal results.)