Cucurbitacin E induces G2/M phase cell cycle arrest via the integration of GADD45γ with CDC2, involving the downregulation of CDC2 and cyclin B1. [1]

Cucurbitacin E (CuE) was exposed to increasing concentrations of colorectal cancer (CRC) cell lines (0, 2.5, 5, and 7.5 μM) for a duration of 24 hours in an in vitro study to investigate the anticancer effect of CuE on CRC cells. We next used the MTT method to measure the proliferation of cancer cells treated with cucurbitacin E. For primary colon cancer cells, curcumin E promotes morphological alterations. Microscopically, cucurbitacin E (5 μM) treatment caused a considerable alteration in the morphology of primary colon cancer cells between 6 to 24 hours. By preventing the production of the GADD45γ gene and the cyclin B1/CDC2 complex in primary CRC cells, curcumin E inhibits the cell cycle in the G2/M phase of the cell cycle, which in turn stops its proliferation [1].

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Cucurbitacin E (CuE) inhibited the survival and proliferation of five primary human colorectal cancer (CRC) cell lines (CP1-CP5) in a dose-dependent manner after 24 hours of treatment with concentrations ranging from 0 to 7.5 μM, as measured by MTT assay. The inhibitory effect was partially irreversible upon removal of CuE.
CuE induced G2/M phase cell cycle arrest in a dose-dependent manner in these CRC cell lines, as determined by propidium iodide staining and flow cytometry. The mitotic index, assessed by MPM-2 antibody staining, was significantly increased, indicating arrest in metaphase.
CuE treatment did not induce significant apoptosis, necrosis, or caspase-3 activation in CRC cells at concentrations up to 7.5 μM for 4-6 hours, as shown by Annexin V/PI staining and flow cytometry. It also did not significantly reduce mitochondrial membrane potential.
Intracellular reactive oxygen species (ROS) levels were not significantly increased by CuE treatment.
Western blot and co-immunoprecipitation analyses showed that CuE treatment led to the downregulation of CDC2 protein expression and dissociation of the cyclin B1/CDC2 complex.
Gene expression profiling (microarray), RT-PCR, and qRT-PCR analyses confirmed that CuE downregulated cyclin B1 and CDC2 mRNA levels and upregulated the mRNA levels of the growth arrest and DNA-damage-inducible protein 45 (GADD45) family genes (GADD45α, β, γ) in CRC cells.
Co-immunoprecipitation further demonstrated that CuE treatment enhanced the binding of GADD45γ protein to CDC2, leading to the inhibition of the cyclin B1/CDC2 complex activity. [1]

The effects of cucurbitacin E (CuE) on body weight and adipose tissue biology were assessed using the high-fat diet mouse model of metabolic syndrome (HFD-MetS). When compared to HFD-MetS mice treated with vehicle alone, cucurbitacin E (0.5 mg/kg)-treated mice showed a significant reduction in body weight. In HFD-MetS mice, cucurbitacin E treatment decreased all fat pad weights. Following treatment with cucurbitacin E, mice's total fat decreased by 55% when compared to HFD-MetS mice. Metabolic syndrome is closely linked to abdominal obesity. Following cucurbitacin E treatment, central adiposity in HFD MetS mice was reduced to 50%, demonstrating the efficacy of cucurbitacin E in targeting MetS [2].

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In a high-fat diet (HFD)-induced metabolic syndrome (MetS) mouse model, oral administration of Cucurbitacin E (0.5 mg/kg/day for 10 weeks) significantly reduced body weight gain and decreased the weights of various fat pads (perigonadal, perirenal, mesenteric, subcutaneous). Total body fat content and visceral fat index were also reduced.
CuE treatment attenuated adipocyte hypertrophy in visceral fat and reduced the mRNA expression of lipogenic genes (SREBP, FASN, ACACA) in visceral fat tissue.
Serum adiponectin levels were increased, while serum leptin and TNF-α levels were decreased in CuE-treated HFD-MetS mice.
CuE treatment reduced the mRNA expression of macrophage infiltration markers (CD11b, MCP-1, CCR2) in visceral fat.
CuE improved glucose intolerance, as shown by a glucose tolerance test (GTT), and ameliorated insulin resistance, as evidenced by reduced basal insulin levels and maintained lower insulin secretion during a glucose-stimulated insulin secretion (GSIS) test.
Western blot analysis of skeletal muscle from CuE-treated mice showed diminished IRS-1 serine phosphorylation (Ser307) and enhanced phosphorylation (activation) of AKT (Ser473) and AMPK (Thr172) following insulin injection.
CuE treatment significantly reduced serum levels of free fatty acids (FFA), triglycerides (TG), low-density lipoprotein (LDL), and total cholesterol in HFD-MetS mice. [2]

For the cell proliferation assay (MTT), cells were seeded in 96-well plates and treated with 0, 2.5, 5, or 7.5 μM Cucurbitacin E for 1 to 3 days. MTT dye was added, incubated for at least 4 hours, the reaction was stopped with DMSO, and absorbance was measured at 540 nm.
For cell cycle analysis, cells were treated with CuE (0, 2.5, 5, 7.5 μM) for 24 hours, harvested, fixed in cold ethanol, stained with propidium iodide/RNaseA solution, and analyzed by flow cytometry.
For apoptosis evaluation, cells were treated with CuE (0, 2.5, 5, 7.5 μM) for 6 hours, and apoptotic cells were detected using an Annexin V-FITC/PI apoptosis detection kit followed by flow cytometry analysis.
For mitochondrial membrane potential (ΔΨm) evaluation, cells treated with CuE for 6 hours were stained with JC-1 dye, fixed, and analyzed by flow cytometry.
For intracellular ROS measurement, cells were treated with CuE, incubated with DCFH-DA fluorescent dye, and ROS/superoxide levels were assessed using a detection kit followed by flow cytometry.
For mitotic index analysis, cells treated with CuE for 24 hours were fixed, stained with MPM-2 primary antibody and a FITC-conjugated secondary antibody, counterstained with propidium iodide, and analyzed by flow cytometry.
For Western blotting, proteins from CuE-treated cells were separated by SDS-PAGE, transferred to PVDF membranes, blocked, and incubated with primary antibodies against β-actin and CDC2, followed by incubation with a fluorescent secondary antibody and detection using an infrared imaging system.
For co-immunoprecipitation (Co-IP), cellular proteins were incubated with antibodies against cyclin B1 or GADD45γ overnight. Protein-antibody complexes were precipitated using protein A/G agarose beads. The precipitates were then analyzed by Western blotting for CDC2 protein.
For gene expression profiling, total RNA was isolated from untreated or CuE-treated cells (4 hours) and analyzed using whole human genome microarrays.
For RT-PCR, cDNA was synthesized from total RNA. PCR was performed using gene-specific primers for cyclin B1, CDC2, and GADD45γ under defined thermal cycling conditions. Products were analyzed by agarose gel electrophoresis.
For quantitative real-time PCR (qRT-PCR), SYBR Green PCR MasterMix and gene-specific primers were used. Reactions were run on a real-time PCR system under standard cycling conditions, and data were normalized using 18S rRNA as an internal control. [1]

C57BL/6 male mice were fed a high-fat diet (HFD; 60% fat) for 8 weeks to induce metabolic syndrome (MetS). Mice with significant obesity and fasting blood glucose ≥126 mg/dl were selected as MetS mice.
MetS mice were then orally administered Cucurbitacin E dissolved in 0.5% carboxymethylcellulose (CMC) by gavage daily for 10 weeks. Two dose groups were used: low dose (0.25 mg/kg/day) and high dose (0.5 mg/kg/day).
A positive control group received Orlistat (50 mg/kg/day) orally.
Control groups included mice on a standard diet (SD) administered 0.5% CMC vehicle.
Body weight was monitored weekly. After the treatment period, mice were fasted for specific tests (GTT, GSIS), and tissues (fat, muscle) and blood were collected for biochemical and molecular analyses. [2]

[1]. Therapeutic ROS targeting of GADD45γ in the induction of G2/M arrest in primary human colorectal cancer cell lines by cucurbitacin E. Cell Death Dis. 2014 Apr 24;5:e1198.

[2]. Cucurbitacin E reduces obesity and related metabolic dysfunction in mice by targeting JAK-STAT5 signaling pathway. PLoS One. 2017 Jun 9;12(6):e0178910.

Cucurbitacin E is a cucurbitacin compound with a lanosterane skeleton that is multiplely substituted with hydroxyl, methyl, and oxo groups, and has unsaturated bonds at positions 1, 5, and 23. It is both a cucurbitacin and a tertiary α-hydroxy ketone. Cucurbitacin E has been reported to exist in begonia nantoensis, purslane (Bacopa monnieri), and other organisms with relevant data. Cucurbitacin E is a natural tetracyclic triterpenoid compound, also known as α-elaterin, derived from plants such as melon (Cucumis melo L.). It has been used in traditional medicine and possesses anti-inflammatory and anticancer properties. In this study, at the tested concentrations, cucurbitacin E exhibited potent anticancer activity against primary human colorectal cancer cells by inducing irreversible G2/M phase cell cycle arrest rather than apoptosis. The proposed mechanism involves the upregulation of the GADD45γ gene, which then binds to and inhibits the activity of CDC2 kinase, leading to the dissociation of the cyclin B1/CDC2 complex, which is crucial for the G2/M phase transition. This study suggests that CuE may be a promising antitumor drug for colorectal cancer. [1]

C32H44O8

556.6870

556.303

18444-66-1

Cucurbitacin B;6199-67-3;Cucurbitacin I;2222-07-3

5281319

White to off-white solid powder

1.2±0.1 g/cm3

712.6±60.0 °C at 760 mmHg

228-234ºC

224.4±26.4 °C

0.0±5.2 mmHg at 25°C

1.579

3.15

3

8

6

40

1270

8

CC(=O)OC(C)(C)/C=C/C(=O)[C@@](C)([C@H]1[C@@H](C[C@@]2([C@@]1(CC(=O)[C@@]3([C@H]2CC=C4[C@H]3C=C(C(=O)C4(C)C)O)C)C)C)O)O

NDYMQXYDSVBNLL-MUYMLXPFSA-N

InChI=1S/C32H44O8/c1-17(33)40-27(2,3)13-12-23(36)32(9,39)25-21(35)15-29(6)22-11-10-18-19(14-20(34)26(38)28(18,4)5)31(22,8)24(37)16-30(25,29)7/h10,12-14,19,21-22,25,34-35,39H,11,15-16H2,1-9H3/b13-12+/t19-,21-,22+,25+,29+,30-,31+,32+/m1/s1

[(E,6R)-6-[(8S,9R,10R,13R,14S,16R,17R)-2,16-dihydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-8,10,12,15,16,17-hexahydro-7H-cyclopenta[a]phenanthren-17-yl]-6-hydroxy-2-methyl-5-oxohept-3-en-2-yl] acetate

Cucurbitacin E; α-Elaterin; α-Elaterine

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 (~89.82 mM)

Solubility in Formulation 1: 2.5 mg/mL (4.49 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 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 (4.49 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.)