Redox regulation of tumor cell toxicity by flavones from Lethedon tannaensis.

The purpose of the present work has been to seek a correlation of potential predictive value, demonstrating redox control of cytotoxicity toward human nasopharynx carcinoma (KB) cells by seven 5-hydroxy-7-methoxyflavones, together with two glycoside derivatives, all extracted from Lethedon tannaensis. In this approach, redox control is characterized by a physicochemical parameter expressing quantitatively the relative electron-donating power of the flavones, this parameter being the second order rate constant, kdelta, for quenching of singlet oxygen O2 (1deltag). This rate constant kdelta is usually related to the ability of a given molecule D to donate an electron and, thus, with the reduction potential E of the couple (D*+/D). Our results show that the flavone toxicity is linearly correlated with ease of oxidation: the higher the rate constant of reaction with singlet oxygen, the easier the oxidation, the less positive or more negative the reduction potential ((D*+/D), the higher the cytotoxicity. The results suggest new screening strategies to identify and improve potential antitumor drugs.     

Structural and electronic properties of tyrosine kinases inhibitors.

Protein tyrosine kinases (TKs) regulate cell proliferation, cell differentiation, and play a fundamental role in signal transduction pathway. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases was related to diseases such as cancer, atherosclerosis and psoriasis. For the present study, we selected a number of structurally related ATP-binding site inhibitors of EGF-receptors of diverse classes. Molecular properties of competitive inhibitors are key features for the action mechanism of these compounds. We performed a theoretical study at the RHF/6-311G* level of theory, in order to correlate the molecular parameters with the biological inhibitory activities. Species stability as evaluated by ionization potentials as well as the E(HOMO)-E(LUMO) energy gap, is in very good correlation with higher inhibitory potency (IP). The most active species, 1, 5, 6,10,11 and 12 exhibited strongly negative charged atoms over the C6 and C7 positions, the higher IP, higher mu and higher energy gap. In summary, a good correlation was observed between the molecular parameters, such as ionization potential, dipolar moment and E(HOMO)-E(LUMO) energy gap and inhibitory potency, suggesting that these properties play an important role for the interaction at the ATP-binding site of EGF-receptors.     

Increasing hydrophobicity of residues in an anti-HIV-1 Env peptide synergistically improves potency

T-20/Fuzeon/Enfuvirtide (ENF), a peptide inhibitor of HIV-1 infection, targets the grooves created by heptad repeat 2 (HR2) of Env's coiled-coil, but mutants resistant to ENF emerge. In this study, ENF-resistant mutants—V38A, N43D, N43D/S138A, Q40H/L45M—were combined with modified inhibitory peptides to identify what we believe to be novel ways to improve peptide efficacy. V38A did not substantially reduce infectivity, but was relatively resistant to inhibitory peptides. N43D was more resistant to inhibitory peptides than wild-type, but infectivity was reduced. The additional mutation S138A (N43D/S138A) increased infectivity and further reduced peptide inhibitory potency. It is concluded that S138A increased binding of HR2/ENF into grooves and that S138A compensated for electrostatic repulsion between N43D and HR2. The six-helix bundle structure indicated that E148A should increase hydrophobic interactions between the coiled-coil and peptide. Importantly, the modifications S138A and E148A in the same peptide retained potency against ENF-escape mutants. The double mutant's increase in potency was greater than the increases from the sum of S138A and E148A individually, showing that these two altered residues synergistically contributed to peptide binding. Isothermal titration calorimetry established that hydrophobic substitutions at positions S138 and E148 improved potency of inhibitory peptides against escape mutants by increasing enthalpic release of energy upon peptide binding.     

Evidence for NQO2-mediated reduction of the carcinogenic estrogen ortho-quinones

The physiological function of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase) is to detoxify potentially reactive quinones by direct transfer of two electrons. A similar detoxification role has not been established for its homologue NRH:quinone oxidoreductase 2 (NQO2). Estrogen quinones, including estradiol(E(2))-3,4-Q, generated by estrogen metabolism, are thought to be responsible for estrogen-initiated carcinogenesis. In this investigation, we have shown for the first time that NQO2 catalyzes the reduction of electrophilic estrogen quinones and thereby may act as a detoxification enzyme. ESI and MALDI mass spectrometric binding studies involving E(2)-3,4-Q with NQO2 clearly support the formation of an enzyme-substrate physical complex. The problem of spontaneous reduction of substrate by cofactor, benzyldihydronicotinamide riboside (BNAH), was successfully overcome by taking advantage of the ping-pong mechanism of NQO2 catalysis. The involvement of the enzyme in the reduction of E(2)-3,4-Q was further supported by addition of the inhibitor quercetin to the assay mixture. NQO2 is a newly discovered binding site (MT3) of melatonin. However, addition of melatonin to the assay mixture did not affect the catalytic activity of NQO2. Preliminary kinetic studies show that NQO2 is faster in reducing estrogen quinones than its homologue NQO1. Both UV and liquid chromatography-tandem mass spectrometry assays unequivocally corroborate the reduction of estrogen ortho-quinones by NQO2, indicating that it could be a novel target for prevention of breast cancer initiation.     

Effects of dibromothymoquinone on oxidation-reduction reactions and the midpoint potential of the Rieske iron-sulfur center in photosynthetic electron transport of Synechococcus Sp.

The effects of 2,5-dibromo-3-methyl-p-benzoquinone (DBMIB) on the reduction kinetics of flash-oxidized P-700 and cytochrome c-553 were studied in the thermophilic cyanobacterium Synechococcus sp. (1) The reduction kinetics of P-700 showed two exponential phases with half-times of 0.2 and 2 ms at the recording time used (Nanba, M. and Katoh, S. (1983) Biochim. Biophys. Acta 725, 272–279). DBMIB strongly slowed down the 2 ms reduction phase but not the 0.2 ms phase. (2) The content of an electron donor which transfers its electrons to P-700 with the half time of 0.2 ms was estimated to be comparable to that of cytochrome f. (3) The magnitudes of the 0.2 ms reduction phase and cytochrome c-553 oxidation decreased as the flash interval was shortened below 2 s in the poisoned cells. Assuming a rapid equilibrium of electrons in the electron donor pool of Photosystem I, the midpoint potential of the 0.2 ms donor was estimated as 280 mV by comparing its percent reduction with that of cytochrome c-553 at three different flash intervals. (4) A similar value was obtained for the midpoint potential of the 0.2 ms donor in the cells in which the plastoquinone pool had been oxidized by dark starvation. It is concluded that the 0.2 ms reduction phase of P-700 is due to the electron donation from the Rieske iron-sulfur center and that DBMIB inhibits strongly but incompletely the reduction of the iron-sulfur center with electrons from the plastoquinone pool, whereas the inhibitor has no effect on the midpoint potential and Photosystem-I-dependent oxidation of the iron-sulfur center.     

Radiosensitization of mammalian cells in vitro by nitroacridines

The nitroacridine nitracrine (1-NC) is a DNA intercalator and a hypoxia-selective, electron-affinic radiosensitizer. Sensitization of Chinese hamster fibroblast cultures at 0 degrees C by the nitro positional isomers of 1-NC has now been compared to help establish structure-activity relationships. The des-nitro analog (E(1) at pH 7 = -899 mV) did not sensitize, suggesting that an electron-affinic chromophore is required. All the nitroacridines (E(1) range -376 to -257 mV) sensitized hypoxic cells with a maximum sensitizer enhancement ratio of about 1.7, but with a 200-fold range in potency. When mean intracellular drug concentrations were compared, 2-, 3-, and 4-NC had potencies which were similar, independent of E(1), and no greater than predicted for non-DNA binding nitroheterocycles. Sensitization by these three isomers occurred at intracellular concentrations likely to saturate the potential intercalation sites on DNA. A large fraction of the radical sites sensitized by O2 are apparently inaccessible to these drugs. It is suggested that sensitization results from electron transfer from migrating transient charge carriers of low reduction potential to immobile bound intercalators. An additional sensitizing mechanism may be available to 1-NC, which was 20 times more potent, a potency not accounted for by E(1), cell uptake, or DNA binding affinity. The dissociation kinetics of the DNA-drug complex was faster for 1-NC than for the other isomers. The higher potency of 1-NC may reflect a short mean residence time (less than 1 ms) in its intercalation site, allowing significant mobility on the DNA within the lifetime of relatively stable radiation-induced target radicals.     

Structure–activity relationships and colorimetric properties of specific probes for the putative cancer biomarker human arylamine N-acetyltransferase 1

A naphthoquinone inhibitor of human arylamine N-acetyltransferase 1 (hNAT1), a potential cancer biomarker and therapeutic target, has been reported which undergoes a distinctive concomitant color change from red to blue upon binding to the enzyme. Here we describe the use of in silico modeling alongside structure–activity relationship studies to advance the hit compound towards a potential probe to quantify hNAT1 levels in tissues. Derivatives with both a fifty-fold higher potency against hNAT1 and a two-fold greater absorption coefficient compared to the initial hit have been synthesized; these compounds retain specificity for hNAT1 and its murine homologue mNat2 over the isoenzyme hNAT2. A relationship between pK_a, inhibitor potency and colorimetric properties has also been uncovered. The high potency of representative examples against hNAT1 in ZR-75-1 cell extracts also paves the way for the development of inhibitors with improved intrinsic sensitivity which could enable detection of hNAT1 in tissue samples and potentially act as tools for elucidating the unknown role hNAT1 plays in ER+ breast cancer; this could in turn lead to a therapeutic use for such inhibitors.     

Estradiol enhances the resistance of LDL to oxidation by stabilizing apoB-100 conformation

Among different proposed mechanisms to account for the protection exerted by estrogens against cardiovascular diseases, the antioxidant effect has attracted considerable attention. We confirmed that 17-beta-estradiol (E2), when added to human LDL at a 6:1 ratio to apoB-100, markedly delays the phase of massive LDL lipid peroxidation induced by Cu(2+). We also observed an increased oxidative resistance of E2-treated LDL by monitoring the early phase of oxidative degradation on the basis of increased LDL surface polarity by the generalized polarization of the lipophilic fluorescent probe 2-(dimethylamino)-6-lauroylnaphthalene (Laurdan). A scavenging of free radicals by E2 is ruled out since, consistent with its structure, its rate constant for the reduction of peroxy radicals is extremely low, i.e., 0.02% of that of vitamin E. Tryptophan fluorescence lifetime and circular dichroism measurements revealed that (i) apoB-100 undergoes a conformational modification and a progressive loss of secondary structure during lipid peroxidation; (ii) E2 increases apoB-100 secondary structure and modifies its conformation; and (iii) the apoB-100 conformational change induced by E2 makes this protein resistant to modifications brought about by lipid peroxidation. We propose that E2, by affecting apoB-100 secondary structure and conformation, modifies the interaction of this protein with the outer layer of the LDL particle thus increasing its overall oxidative resistance.     

Protection against hydrogen peroxide-induced cytotoxicity in PC12 cells by scutellarin

The present study investigated the protective actions of the antioxidant scutellarin against the cytotoxicity produced by exposure to H2O2 in PC12 cells. This was done by assaying for MTT (3,(4,5-dimethylthiazole-2-yl)2,5-diphenyl-tetrazolium bromide) reduction and lactate dehydrogenase (LDH) release. Reactive oxygen species (ROS) and Ca2+ in cells were evaluated by fluorescent microplate reader using DCFH and Fura 2-AM, respectively, as probes. Lipid peroxidation was quantified using thiobarbituric acid-reactive substances (TBARS). Mitochondrial membrane potential (MMP) was assessed by the retention of rhodamine123 (Rh123), a specific fluorescent cationic dye that is readily sequestered by active mitochondria, depending on their transmembrane potential. The DNA content and percentage of apoptosis were monitored with flow cytometry. Vitamin E, a potent antioxidant, was employed as a comparative agent. Preincubation of PC12 cells with scutellarin prevented cytotoxicity induced by H2O2. Intracellular accumulation of ROS, Ca2+ and products of lipid peroxidation, resulting from H2O2 were significantly reduced by scutellarin. Incubation of cells with H2O2 caused a marked decrease in MMP, which was significantly inhibited by scutellarin. PC12 cells treated with H2O2 underwent apoptotic death as determined by flow cytometric assay. The percentage of this H2O2-induced apoptosis in the cells was decreased in the presence of different concentrations of scutellarin. Scutellarin exhibited significantly higher potency compared to the antioxidant vitamin E. The present findings showed that scutellarin attenuated H2O2-induced cytotoxicity, intracellular accumulation of ROS and Ca2+, lipid peroxidation, and loss of MMP and DNA, which may represent the cellular mechanisms for its neuroprotective action.     

The synthesis and evaluation of thymoquinone analogues as anti-ovarian cancer and antimalarial agents

Thymoquinone (TQ), 2-isopropyl-5-methyl-1,4-benzoquinone, a natural product isolated from Nigella sativa L., has previously been demonstrated to exhibit antiproliferative activity in vitro against a range of cancers as well as the human malarial parasite Plasmodium falciparum. We describe here the synthesis of a series of analogues of TQ that explore the potential for nitrogen-substitution to this scaffold, or reduction to a hydroquinone scaffold, in increasing the potency of this antiproliferative activity against ovarian cancer cell lines and P. falciparum. In addition, alkyl or halogen-substituted analogues were commercially sourced and tested in parallel. Several TQ analogues with improved potency against ovarian cancer cells and P. falciparum were found, although this increase is suggested to be moderate. Key aspects of the structure activity relationship that could be further explored are highlighted.     

Calculated proton uptake on anaerobic reduction of cytochrome C oxidase: is the reaction electroneutral?

Cytochrome c oxidase is a transmembrane proton pump that builds an electrochemical gradient using chemical energy from the reduction of O(2). Ionization states of all residues were calculated with Multi-Conformation Continuum Electrostatics (MCCE) in seven anaerobic oxidase redox states ranging from fully oxidized to fully reduced. One long-standing problem is how proton uptake is coupled to the reduction of the active site binuclear center (BNC). The BNC has two cofactors: heme a(3) and Cu(B). If the protein needs to maintain electroneutrality, then 2 protons will be bound when the BNC is reduced by 2 electrons in the reductive half of the reaction cycle. The effective pK(a)s of ionizable residues around the BNC are evaluated in Rhodobacter sphaeroides cytochrome c oxidase. At pH 7, only a hydroxide coordinated to Cu(B) shifts its pK(a) from below 7 to above 7 and so picks up a proton when heme a(3) and Cu(B) are reduced. Glu I-286, Tyr I-288, His I-334, and a second hydroxide on heme a(3) all have pK(a)s above 7 in all redox states, although they have only 1.6-3.5 DeltapK units energy cost for deprotonation. Thus, at equilibrium, they are protonated and cannot serve as proton acceptors. The propionic acids near the BNC are deprotonated with pK(a)s well below 7. They are well stabilized in their anionic state and do not bind a proton upon BNC reduction. This suggests that electroneutrality in the BNC is not maintained during the anaerobic reduction. Proton uptake on reduction of Cu(A), heme a, heme a(3), and Cu(B) shows approximately 2.5 protons bound per 4 electrons, in agreement with prior experiments. One proton is bound by a hydroxyl group in the BNC and the rest to groups far from the BNC. The electrochemical midpoint potential (E(m)) of heme a is calculated in the fully oxidized protein and with 1 or 2 electrons in the BNC. The E(m) of heme a shifts down when the BNC is reduced, which agrees with prior experiments. If the BNC reduction is electroneutral, then the heme a E(m) is independent of the BNC redox state.     

Mechanism of the malabaricone C-induced toxicity to the MCF-7 cell line

Abstract

In this study, we studied the mechanism of the cytotoxicity of malabaricone C (mal C) against human breast cancer MCF-7 cell line. Mal C dose-dependently increased the sub G1 cell population, associated with cytoplasmic oligonucleosome formation and chromatin condensation. The mal C-induced apoptosis led to mitochondrial damage as revealed by fluorescence microscopy and flow cytometry of the JC-1-stained cells as well as from the release of mitochondrion-specific nuclease proteins AIF and endo G. Mal C also released intracellular Ca²⁺ from the MCF-7 cells, but the Ca²⁺-modulators BAPTA-AM and Ru360 only partially abrogated the apoptosis. The calpain activation by mal C did not have any effect on its cytotoxicity. On the other hand, after mal C treatment significant lysosomal membrane permeabilization (LMP), along with release of cathepsin B, as well as Bid-cleavage and its translocation to mitochondria were observed much earlier than the mitochondrial damage. This suggested that cytotoxicity of mal C against human MCF-7 human breast cancer cell line may proceed through LMP as the initial event that triggered a caspase-independent, but cathepsin B and t-Bid-dependent intrinsic mitochondrial apoptotic pathway. A significant accumulation of cells in the S or G2-M phases along with upregulation of the cyclins E and A due to mal C exposure promises it to be a potential anti-cancer agent.     

Enhanced antioxidant effect of prenylated polyphenols as Fyn inhibitor

Polyphenols have antioxidant effects. In view of the diverse biological activities of prenylated natural products, this study investigated whether polyphenols with prenyl residues have improved antioxidant and cytoprotective activity against oxidative stress, and explored the underlying basis for this effect. A set of structurally related polyphenols exhibited varying degrees of antioxidant effect in HepG2 cells, as evidenced by increases in cell viability against oxidative injury; kazinol E and C with three prenyls had greater potency than other kazinols having fewer prenyl chains. Polyphenols without prenyl (tupichinol C and resveratrol) showed weaker potency. Treatment with kazinol E diminished H(2)O(2) production and enabled cells to protect the mitochondria, as indicated by the inhibition of mitochondrial fragmentation, mitochondrial permeability transition, and cytochrome c release. Moreover, kazinol E activated LKB1 by its phosphorylation and cytoplasmic translocation, contributing to the protection of mitochondria via AMPK. In vitro or in a cell-based assay, tyrosine phosphorylation of Fyn was prohibited by kazinol E, which led to LKB1 activation, as shown by the experiments using Fyn over-expression construct or siRNA. SU6656, a known Fyn inhibitor, had a similar effect. Moreover, oxidative stress facilitated Fyn phosphorylation with repression of AMPKα and GSK3β phosphorylation, which was abolished by kazinol E treatment. The role of Fyn inhibition by kazinol E in AMPK-mediated protection of the cell viability and mitochondrial function was strengthened by ectopically expressed Fyn's reversal of these effects. In conclusion, kazinols as multi-prenylated polyphenols possess increased antioxidant and cytoprotective activity, which depends on the activation of LKB1-AMPK pathway downstream of Fyn inhibition.     

Protection of PC12 cells from hydrogen peroxide-induced cytotoxicity by salvianolic acid B, a new compound isolated from Radix Salviae miltiorrhizae

A number of studies indicate that reactive oxygen species (ROS) are involved in neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). The neuroprotective effects of salvianolic acid B (SalB) from Radix Salviae miltiorrhizae (RSM) against hydrogen peroxide (H2O2)-induced rat pheochromocytoma line PC12 injury were evaluated in the present study. Vitamin E, a potent antioxidant, was employed as a positive control agent. Following exposure of cells to H2O2 (150 microM), a marked decrease in cell survival and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), as well as increased levels of malondialdehyde (MDA) production and lactate dehydrogenase (LDH) release were observed. In parallel, H2O2 caused significant elevation in intracellular Ca2+ level and caspase-3 activity, and induced apoptotic death as determined by flow cytometric assay. However, pretreatment of the cells with SalB (0.1-10 microM) prior to H2O2 exposure blocked these H2O2-induced cellular events noticeably. Moreover, SalB exhibited significantly higher potency as compared to Vitamin E. The present findings indicated that SalB exerts neuroprotective effects against H2O2 toxicity, which might be of importance and contribute to its clinical efficacy for the treatment of neurodegenerative diseases.     

Mechanism of action in a 4,5-diarylpyrrole series of selective cyclo-oxygenase-2 inhibitors

Using semi-empirical AM1 calculation and 6.31G* basis sets, we have calculated the energy of the highest-occupied molecular orbital (E(HOMO)) for anti-inflammatory 4,5-diarylpyrroles which have been shown to have inhibitory activity on cyclooxygenase COX-2, an inducible enzyme expressed during inflammation. We have found a correlation between the E(HOMO) of a molecule and its COX-2 inhibition. However, no correlation was observed between E(HOMO) and the inhibition efficiency of cyclooxygenase-1 (COX-1), the constitutively expressed enzyme, protective to the organism. This result suggests that the inhibitions of the two isoforms follow different molecular mechanisms.     

Dimeric unnatural polyproline-rich peptides with enhanced antibacterial activity

We report a dimerization strategy to enhance the antibacterial potency of an otherwise weak cationic amphiphilic polyproline helical (CAPH) peptide. Overall, the dimeric CAPHs were more active against Escherichia coli and Staphylococcus aureus than the monomeric counterpart, reaching up to a 60-fold increase in potency. At their minimum inhibitory concentration (MIC), the dimeric peptides demonstrated no hemolytic activity or bacterial membrane disruption as monitored by β-galactosidase release in E. coli. At higher concentrations the dimeric agents were found to induce β-galactosidase release, but maintained negligible hemolytic activity, pointing to a potential shift in the mechanism of action at higher concentrations. Thus, discontinuous dimerization of an unnatural proline-rich peptide was a successful strategy to create potent de novo antibacterial peptides without membrane lysis.     

4-Deoxyraputindole C induces cell death and cell cycle arrest in tumor cell lines

Several molecules extracted from natural products exhibit different biological activities, such as ion channel modulation, activation of signaling pathways, and anti-inflammatory or antitumor activity. In this study, we tested the antitumor ability of natural compounds extracted from the Raputia praetermissa plant. Among the compounds tested, an alkaloid, here called compound S4 (4-Deoxyraputindole C), showed antitumor effects against human tumor lineages. Compound S4 was the most active against Raji, a lymphoma lineage, promoting cell death with characteristics that including membrane permeabilization, dissipation of the mitochondrial potential, increased superoxide production, and lysosomal membrane permeabilization. The use of cell death inhibitors such as Z-VAD-FMK (caspase inhibitor), necrostatin-1 (receptor-interacting serine/threonine-protein kinase 1 inhibitor), E-64 (cysteine peptidases inhibitor), and N-acetyl- L -cysteine (antioxidant) did not decrease compound S4-dependent cell death. Additionally, we tested the effect of cellular activity on adherent human tumor cells. The highest reduction of cellular activity was observed in A549 cells, a lung carcinoma lineage. In this lineage, the effect on the reduction of the cellular activity was due to cell cycle arrest, without plasma membrane permeabilization, loss of the mitochondrial potential or lysosomal membrane permeabilization. Compound S4 was able to inhibit cathepsin B and L by a nonlinear competitive (negative co-operativity) and simple-linear competitive inhibitions, respectively. The potency of inhibition was higher against cathepsin L. Compound S4 promoted cell cycle arrest at G ₀ and G ₂ phase, and increase the expression of p16 and p21 proteins. In conclusion, compound S4 is an interesting molecule against cancer, promoting cell death in the human lymphoma lineage Raji and cell cycle arrest in the human lung carcinoma lineage A549.     

Synthesis and biological study of 4-aminopyrimidine-5-carboxaldehyde oximes as antiproliferative VEGFR-2 inhibitors

A novel 4-aminopyrimidine-5-carboxaldehyde oxime scaffold with inhibitory activity against VEGFR-2 kinase has been identified. With a 4-fluoro-2-methylindol-5-yloxy group at the 6-position and alkyl groups as the oxime side chains, many analogues showed good potency for VEGFR-2. This series also exhibited antiproliferative activity against cancer cells, causing cell accumulation at the G2/M phase of the cell cycle and preventing cells from entering mitosis. Described here are the chemistry, structure-activity relationships (SAR), and biological testing for this series.     

The effects of cucurbitacin E on GADD45β‐trigger G2/M arrest and JNK‐independent pathway in brain cancer cells

Cucurbitacin E (CuE), an active compound of the cucurbitacin family, possesses a variety of pharmacological functions and chemotherapy potential. Cucurbitacin E exhibits inhibitory effects in several types of cancer; however, its anticancer effects on brain cancer remain obscure and require further interpretation. In this study, efforts were initiated to inspect whether CuE can contribute to anti‐proliferation in human brain malignant glioma GBM 8401 cells and glioblastoma‐astrocytoma U‐87‐MG cells. An MTT assay measured CuE's inhibitory effect on the growth of glioblastomas (GBMs). A flow cytometry approach was used for the assessment of DNA content and cell cycle analysis. DNA damage 45β (GADD45β) gene expression and CDC2/cyclin‐B1 disassociation were investigated by quantitative real‐time PCR and Western blot analysis. Based on our results, CuE showed growth‐inhibiting effects on GBM 8401 and U‐87‐MG cells. Moreover, GADD45β caused the accumulation of CuE‐treated G2/M‐phase cells. The disassociation of the CDC2/cyclin‐B1 complex demonstrated the known effects of CuE against GBM 8401 and U‐87‐MG cancer cells. Additionally, CuE may also exert antitumour activities in established brain cancer cells. In conclusion, CuE inhibited cell proliferation and induced mitosis delay in cancer cells, suggesting its potential applicability as an antitumour agent.