Eng-Hui Chew

Targeting thioredoxin reductase is a basis for cancer therapy by arsenic trioxide

Arsenic trioxide (ATO) is an effective cancer therapeutic drug for acute promyelocytic leukemia and has potential anticancer activity against a wide range of solid tumors. ATO exerts its effect mainly through elevated oxidative stress, but the exact molecular mechanism remains elusive. The thioredoxin (Trx) system comprising NADPH, thioredoxin reductase (TrxR), and Trx and the glutathione (GSH) system composed of NADPH, glutathione reductase, and GSH supported by glutaredoxin are the two electron donor systems that control cellular proliferation, viability, and apoptosis. Recently, the selenocysteine-dependent TrxR enzyme has emerged as an important molecular target for anticancer drug development. Here, we have discovered that ATO irreversibly inhibits mammalian TrxR with an IC50 of 0.25 μM. Both the N-terminal redox-active dithiol and the C-terminal selenothiol-active site of reduced TrxR may participate in the reaction with ATO. The inhibition of MCF-7 cell growth by ATO was correlated with irreversible inactivation of TrxR, which subsequently led to Trx oxidation. Furthermore, the inhibition of TrxR by ATO was attenuated by GSH, and GSH depletion by buthionine sulfoximine enhanced ATO-induced cell death. These results strongly suggest that the ATO anticancer activity is by means of a Trx system-mediated apoptosis. Blocking cancer cell DNA replication and repair and induction of oxidative stress by the inhibition of both Trx and GSH systems are suggested as cancer chemotherapeutic strategies.

Inhibition of the Human Thioredoxin System A MOLECULAR MECHANISM OF MERCURY TOXICITY

Mercury toxicity mediated by different forms of mercury is a major health problem; however, the molecular mechanisms underlying toxicity remain elusive. We analyzed the effects of mercuric chloride (HgCl2) and monomethylmercury (MeHg) on the proteins of the mammalian thioredoxin system, thioredoxin reductase (TrxR) and thioredoxin (Trx), and of the glutaredoxin system, glutathione reductase (GR) and glutaredoxin (Grx). HgCl2 and MeHg inhibited recombinant rat TrxR with IC50 values of 7.2 and 19.7 nm, respectively. Fully reduced human Trx1 bound mercury and lost all five free thiols and activity after incubation with HgCl2 or MeHg, but only HgCl2 generated dimers. Mass spectra analysis demonstrated binding of 2.5 mol of Hg2+ and 5 mol of MeHg+/mol of Trx1 with the very strong Hg2+ complexes involving active site and structural disulfides. Inhibition of both TrxR and Trx activity was observed in HeLa and HEK 293 cells treated with HgCl2 or MeHg. GR was inhibited by HgCl2 and MeHg in vitro, but no decrease in GR activity was detected in cell extracts treated with mercurials. Human Grx1 showed similar reactivity as Trx1 with both mercurial compounds, with the loss of all free thiols and Grx dimerization in the presence of HgCl2, but no inhibition of Grx activity was observed in lysates of HeLa cells exposed to mercury. Overall, mercury inhibition was selective toward the thioredoxin system. In particular, the remarkable potency of the mercury compounds to bind to the selenol-thiol in the active site of TrxR should be a major molecular mechanism of mercury toxicity.

6‐Shogaol, an active constituent of ginger, inhibits breast cancer cell invasion by reducing matrix metalloproteinase‐9 expression via blockade of nuclear factor‐κB activation

BACKGROUND AND PURPOSE Shogaols are reported to possess anti‐inflammatory and anticancer activities. However, the antimetastatic potential of shogaols remains unexplored. This study was performed to assess the effects of shogaols against breast cancer cell invasion and to investigate the underlying mechanisms.

Substrate-mediated Regulation of Cullin Neddylation

Cullin-based E3 ligases are a large family of multi-subunit ubiquitin ligases with diverse cellular functions, including the regulation of the cell cycle, of the DNA damage response, and of various transcription factors. These ligases are composed of one of six mammalian cullin homologs (Cul1, Cul2, Cul3, Cul4a, Cul4b, and Cul5), the Ring finger containing protein Roc1/Rbx1, and cullin homolog-specific adaptor and substrate recognition subunits. To be active, cullin-based ligases require the covalent modification of a conserved lysine residue in the cullin protein with the ubiquitin-like protein Nedd8. We show in this study that in intact cells Cul1 neddylation is dependent on binding to adaptor proteins and substrate recognition subunits. Mutant Cul1 that is unable to recruit adaptor and substrate recognition subunits exhibits markedly reduced neddylation, and inhibiting binding of adaptor and substrate recognition subunits to wild type Cul1 reduces Nedd8 modification. This regulatory mechanism also extends to other cullin-based E3 ligases, including Cul2, Cul3, and Cul4a. The regulation of cullin neddylation by adaptor proteins and substrate recognition subunits in cells was found to be independent of both CAND1 and the COP9 signalosome, two negative regulators of cullin Nedd8 modification. Using hypoxia-inducible factor-1α (HIF-1α), a substrate of the Elongin B/C-Cul2-VHL ligase, we demonstrate the critical role of substrate binding to promote Cul2 neddylation in a manner that does not require substrate ubiquitination but may involve a conformational change. These findings suggest a mechanism through which availability of substrate recognition subunits and substrates can regulate the ubiquitin ligase activity.

Elucidation of thioredoxin as a molecular target for antitumor quinols

Heteroaromatic quinols 4-(benzothiazol-2-yl)-4-hydroxycyclohexa-2,5-dienone (1) and 4-(1-benzenesulfonyl-1H-indol-2-yl)-4-hydroxycyclohexa-2,5-dienone (2) exhibit potent and selective antitumor activity against colon, renal, and breast carcinoma cell lines in vitro (GI50 < 500 nmol/L). In vivo growth inhibition of renal, colon, and breast xenografts has been observed. Profound G2-M cell cycle block accompanied down-regulation of cdk1 gene transcription was corroborated by decreased CDK1 protein expression following treatment of HCT 116 cells with growth inhibitory concentrations of 1 or 2. The chemical structure of the quinol pharmacophore 4-(hydroxycyclohexa-2,5-dienone) suggested that these novel agents would readily react with nucleophiles in a double Michael (beta-carbon) addition. Indeed, COMPARE analysis within the National Cancer Institute database revealed a number of chemically related quinone derivatives that could potentially react with sulfur nucleophiles in a similar manner and suggested that thioredoxin/thioredoxin reductase signal transduction could be a putative target. Molecular modeling predicted covalent irreversible binding between quinol analogues and cysteine residues 32 and 35 of thioredoxin, thereby inhibiting enzyme activity. Binding has been confirmed, via mass spectrometry, between reduced human thioredoxin and 1. Microarray analyses of untreated HCT 116 cells and those exposed to either 1 (1 micromol/L) or 2 (500 nmol/L and 1 micromol/L) determined that of > or =10,000 cancer-related genes, expression of thioredoxin reductase was up-regulated >3-fold. Furthermore, quinols 1 and 2 inhibited insulin reduction, catalyzed by thioredoxin/thioredoxin reductase signaling in a dose-dependent manner (IC50 < 6 micromol/L). Results are consistent with a mechanism of action of novel antitumor quinols involving inhibition of the small redox protein thioredoxin.

Antioxidant and Nrf2 inducing activities of luteolin, a flavonoid constituent in Ixeris sonchifolia Hance, provide neuroprotective effects against ischemia-induced cellular injury

Ixeris sonchifolia Hance is an herb distributed in northeastern part of China and has been used by natives to invigorate circulation. In the present study, bioactivity-guided fractionation of I. sonchifolia Hance extract was performed with the aim to isolate and identify the compounds underlying the potential protective effects against ischemia brain injury. Among the four fractions isolated from the herb extract, the ethyl acetate fraction was found to scavenge DPPH radicals, induce ARE-dependent transcriptional activity and upregulate Nrf2 protein levels. The isolation work focused on this fraction revealed the presence of two categories of compounds: flavonoids and sesquiterpene lactones. Among the five isolated flavonoids, luteolin was evaluated to possess direct and indirect antioxidant activities by scavenging free radicals and inducing the upregulation of ARE-dependent phase II enzymes. Concomitant with the findings from the cell-based assays, in the middle cerebral artery occlusion-induced ischemia rat model, administration of luteolin at 4 mg/kg displayed neuroprotective effects by reducing infarct area and inhibiting neuronal cell death. In summary, the obtained results suggest that flavonoids in I. sonchifolia Hance, in particular luteolin, contribute at least partly to the neuroprotective effects against ischemia-induced cellular injury and can be potentially developed for treatment of ischemia-reperfusion induced diseases.

Functionalized aurones as inducers of NAD(P)H:quinone oxidoreductase 1 that activate AhR/XRE and Nrf2/ARE signaling pathways: synthesis, evaluation and SAR.

The chemopreventive potential of functionalized aurones and related compounds as inducers of NAD(P)H:quinone oxidoreductase 1 (NQO1, EC 1.6.99.2) are described. Several 4,6-dimethoxy and 5-hydroxyaurones induced NQO1 activity of Hepa1c1c7 cells by 2-fold at submicromolar concentrations, making these the most potent inducers to be identified from this class. Mechanistically, induction of NQO1 was mediated by the activation of AhR/XRE and Nrf2/ARE pathways, indicating that aurones may be mixed activators of NQO1 induction or agents capable of exploiting the proposed cross-talk between the AhR and Nrf2 gene batteries. QSAR analysis by partial least squares projection to latent structures (PLS) identified size parameters, in particular those associated with non-polar surface areas, as an important determinant of induction activity. These were largely determined by the substitution on rings A and B. A stereoelectronic role for the exocyclic double bond as reflected in the E(LUMO) term was also identified. The electrophilicity of the double bond or its effect on the conformation of the target compound are possible key features for induction activity.

Thioredoxin reductase inhibition by antitumor quinols: a quinol pharmacophore effect correlating to antiproliferative activity

Novel heteroaromatic‐substituted 4‐hy‐ droxycyclohexa‐2,5‐dienones (quinols) demonstrate po tent in vitro antiproliferative activity and in vivo antitu mor activity in tumor xenografts. The mechanism of action of these promising novel anticancer agents, however, remains to be fully elucidated. The thiore‐ doxin (Trx) system comprising Trx, thioredoxin reduc tase (TrxR), and NADPH participates in a broad range of cellular functions involved in cell survival and pro liferation. Accumulating evidence has indicated that the selenocysteine‐containing mammalian TrxR is a valid molecular target for development of novel cancer therapeutics. In this study, we demonstrate that struc tural analogs containing a quinol pharmacophore inhib ited TrxR with potencies correlated with their antipro liferative and cytotoxic efficacies. Benzenesulfonyl‐6F‐ indole‐substituted quinol (compound 6) irreversibly inhibited TrxR most strongly with a half‐maximal inhib itory concentration of 2.7 μ,M after 1 h of incubation with recombinant rat TrxR. The inhibition was shown to be concentration‐, time‐, and NADPH‐dependent and mediated through a direct quinol attack on the penultimate C‐terminal selenocysteine residue. More over, TrxR activity in lysates of HCT 116 cells treated with apoptosis‐inducing doses of quinols was signifi cantly reduced. From the results obtained, we propose that TrxR inhibition is a critical cellular event that contributes to the proapoptotic effects of quinols.— Chew, E. H., Lu, J., Bradshaw, T. D., Holmgren, A. Thioredoxin reductase inhibition by antitumor quinols: a quinol pharmacophore effect correlating to antipro liferative activity. FASEB J. 22, 2072–2083 (2008)

Shogaols at proapoptotic concentrations induce G2/M arrest and aberrant mitotic cell death associated with tubulin aggregation

Shogaols have been previously reported to induce cancer cell death via multiple mechanisms, among which one analog 6-shogaol has been reported to cause microtubule damage through specific reaction with sulfhydryl groups in tubulin. In this study, a series of shogaols with different side chain lengths (4-, 6-, 8- and 10-shogaol) was synthesized and evaluated for antiproliferative activity in HCT 116 colon carcinoma and SH-SY5Y neuroblastoma cells. 4- and 6-shogaol were identified as lead compounds possessing the strongest antiproliferative activity. In the soft agar assay, the lead shogaols displayed dose-dependent inhibition on cancer cell colony formation under anchorage-independent conditions. Using HCT 116 as the selected cancer cell line, the molecular events linking shogaols-induced G2/M cell cycle arrest to apoptosis characterized by caspase 3 and PARP cleavage were investigated. At sublethal concentrations, the halt at G2/M phase was alleviated along time and cells survived. Conversely, proapoptotic concentrations of 4- and 6-shogaol induced irreversible G2/M arrest that was at least in part associated with down-regulation of cell cycle checkpoint proteins cdk1, cyclin B and cdc25C, as well as spindle assembly checkpoint proteins mad2, cdc20 and survivin. A dose- and time-dependent accumulation of insoluble tubulin in the insoluble fractions of cell lysates provided evidence that G2 checkpoint failure led to disruption of microtubule turnover. In summary, our results conclude that shogaols cause apoptosis by inducing aberrant mitosis at least through the attenuation of cell cycle and spindle assembly checkpoint proteins.

Sulforaphane and its methylcarbonyl analogs inhibit the LPS-stimulated inflammatory response in human monocytes through modulating cytokine production, suppressing chemotactic migration and phagocytosis in a NF-κB- and MAPK-dependent manner.

Sulforaphane [SF; 1-isothiocyanato-4-(methylsulfinyl)-butane], an aliphatic isothiocyanate (ITC) naturally derived from cruciferous vegetables and largely known for its chemopreventive potential also appears to possess anti-inflammatory potential. In this study, structural analogs of SF {compound 1 [1-isothiocyanato-4-(methylcarbonyl)-butane] and 2 [1-isothiocyanato-3-(methylcarbonyl)-propane]} containing a carbonyl group in place of the sulfinyl group in SF, were evaluated for their anti-inflammatory activities. In RAW 264.7 cells, the ITCs at non-toxic concentrations caused an inhibition of NO and prostaglandin E2 (PGE2) release through suppressing expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as a reduction in matrix metalloproteinase-9 (MMP-9) expression, secretion and gelatinolytic activity. Further work performed on human monocytes isolated from blood of healthy donors revealed that the ITCs not only suppressed the expression and release of pro-inflammatory mediators IL-1β, IL-6, TNF-α and MMP-9, but also suppressed their antibody-independent phagocytic and chemotactic migratory abilities. These anti-inflammatory activities were mediated through suppression of the NF-κB and MAPK signaling pathways. In addition, the ITCs were revealed to interact with the cysteines in inhibitor of nuclear factor-κB kinase β subunit (IKKβ), which could contribute at least partly to the suppression of NF-κB signaling. In conclusion, results obtained in this study provide deeper insights into the anti-inflammatory properties of SF and its methylcarbonyl analogs and the underlying mechanisms. These compounds thus serve as promising candidates for clinical applications in controlling inflammatory conditions.