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Biochemical Pharmacology (v.82, #9)

Editorial Board (pp. iii).

Understanding SOS (Son of Sevenless) by Stéphane Pierre; Anne-Sophie bats; Xavier Coumoul (pp. 1049-1056).

Son of Sevenless (SOS) was discovered in Drosophila melanogaster. Essential for normal eye development in Drosophila, SOS has two human homologues, SOS1 and SOS2. The SOS1 gene encodes the Son of Sevenless 1 protein, a Ras and Rac guanine nucleotide exchange factor. This protein is composed of several important domains. The CDC25 and REM domains provide the catalytic activity of SOS1 towards Ras and the histone fold DH/PH (Dbl homology and Pleckstrin homology) domains function, in tandem, to stimulate GTP/GDP exchange for Rac. In contrast to Ras, there have been few studies that implicate SOS1 in human disease and, initially, less attention was given to this gene. However, mutations in SOS1 have been reported recently in Noonan syndrome and in type 1 hereditary gingival fibromatosis. Although, there have been very few studies that focus on the regulation of this important gene by physiological or exogenous factors, we recently found that the SOS1 gene was induced by the environmental toxin, dioxin, and that this effect was mediated by the aryl hydrocarbon receptor (AhR). These recent observations raise the possibility that alterations in the expression of the SOS1 gene and, consequently, in the activity of the SOS1 protein may affect toxicological endpoints and lead to clinical disease. These possibilities, thus, have stimulated much interest in SOS1 recently. In this article, we review the functions of SOS1 and the evidence for its roles in physiology and pathology across species.

Keywords: Abbreviations; AhR; aryl hydrocarbon receptor; Cdc 25; cell division cycle 25; CFC; cardio-facio-cutaneous syndrome; DH; Dbl homology; E3b1; Eps8 SH3 domain binding protein 1; Eps8; EGFR pathway substrate 8; ERK; extracellular signal-regulated protein kinase; Grb2; growth factor receptor bound 2; FAK; focal adhesion kinase; MAPK; mitogen activated protein kinase; MEK; MAP ERK kinase; NS; Noonan syndrome; PH; Pleckstrin homology; PTB; phosphotyrosine binding; PTPN; tyrosine-protein phosphatase non-receptor type 1; PxxP; proline rich domain; REM; Ras exchange motif; SOS; Son of Sevenless; TCDD; 2,3,7,8-tetrachlorodibenzodioxin; TGF; tumor growth factorSon of Sevenless; Ras guanine nucleotide exchange factors; Rac guanine nucleotide exchange factors; Noonan syndrome; Dioxin; Carcinogenesis

Understanding SOS (Son of Sevenless) by Stéphane Pierre; Anne-Sophie bats; Xavier Coumoul (pp. 1049-1056).

Linear ubiquitination in NF-κB signaling and inflammation: What we do understand and what we do not by Kelly Verhelst; Lynn Verstrepen; Isabelle Carpentier; Rudi Beyaert (pp. 1057-1065).

Despite its small size, ubiquitin is one of the most versatile signaling molecules in the cell and affects distinct cellular processes. It forms the building block of a repertoire of posttranslational modifications of cellular proteins, ranging from the attachment of a single ubiquitin to ubiquitin chains of different linkage. Proteins that contain ubiquitin chain-specific ubiquitin-binding domains recognize different types of ubiquitination and determine the mode of signaling of modified proteins. Polyubiquitin chains were thought to be formed only by the conjugation of the ubiquitin C-terminal Gly to one of the seven internal Lys residues of another ubiquitin. However, the C-terminal Gly was recently shown to also link to the N-terminus of another ubiquitin to form head-to-tail polyubiquitin chains, which is referred to as linear ubiquitination. These linear linkages can be assembled and conjugated to another protein by an E3 ligase complex known as LUBAC, and are recognized by a particular ubiquitin-binding domain known as UBAN. Both have been implicated in the regulation of TNF-induced NF-κB signaling, which induces the expression of a wide range of proteins that mediate many biological processes including inflammation and cell survival. We discuss the molecular players and mechanisms that determine the specificity and outcome of linear ubiquitination in NF-κB signaling, as well as future directions and challenges ahead.

Keywords: Immunity; NF-κB; Signaling; TNF; Ubiquitin

Linear ubiquitination in NF-κB signaling and inflammation: What we do understand and what we do not by Kelly Verhelst; Lynn Verstrepen; Isabelle Carpentier; Rudi Beyaert (pp. 1057-1065).

Keywords: Immunity; NF-κB; Signaling; TNF; Ubiquitin

Mcl-1 downregulation by YM155 contributes to its synergistic anti-tumor activities with ABT-263 by Haikuo Tang; Huanjie Shao; Chunrong Yu; Jinsong Hou (pp. 1066-1072).

YM155, a small-molecule survivin suppressant, exhibits anti-tumor activities in vitro, in vivo and in clinical trials. However, the mechanism of YM155 action remains unclear. In this study, YM155 was administered to a panel of cell lines and the effects of YM155 on Bcl-2 family members were analyzed. Our results show that YM155 strikingly downregulates Mcl-1 in a broad spectrum of cancer cell lines and that the Mcl-1 modulation occurs at the transcriptional level, independently of survivin modulation or caspase activity. Furthermore, analysis of the contribution of Mcl-1 or survivin downregulation to YM155-induced cell death in vitro showed that knockdown of Mcl-1 sensitizes cells to YM155-induced cytotoxicity. Finally, our data demonstrate that downregulation of Mcl-1 by YM155 synergistically lowers the threshold of Bcl-2 family member inhibitor ABT-263-induced cell death. Our findings reveal a novel mechanism by which survivin-independent Mcl-1 suppression plays a critical role in YM155-mediated anti-tumor activities. YM155 treatment in combination with ABT-263 thus affords a new strategy for cancer treatment.

Keywords: Abbreviations; IAP; inhibitors of apoptosis; NSCLC; non-small cell lung cancer; XIAP; X-linked inhibitor of apoptosis protein; DMSO; dimethyl sulfoxide; PARP; poly (ADP-ribose) polymerase; STAT3; signal transducer and activator of transcription 3YM155; Mcl-1; Survivin; ABT-263; Cell death

Mcl-1 downregulation by YM155 contributes to its synergistic anti-tumor activities with ABT-263 by Haikuo Tang; Huanjie Shao; Chunrong Yu; Jinsong Hou (pp. 1066-1072).

Pharmacodynamics of curcumin as DNA hypomethylation agent in restoring the expression of Nrf2 via promoter CpGs demethylation by Tin Oo Khor; Ying Huang; Tien-Yuan Wu; Limin Shu; Jonghun Lee; Ah-Ng Tony Kong (pp. 1073-1078).

Prostate cancer (PCa) is one of the most deadly malignancies among men in the United States. Although localized prostate cancer can be effectively treated via surgery or radiation, metastatic disease is usually lethal. Recent evidence suggests that the development and progression of human prostate cancer involves complex interplay between epigenetic alterations and genetic defects. We have recently demonstrated that Nrf2, a master regulator of cellular antioxidant defense systems, was epigenetically silenced during the progression of prostate tumorigenesis in TRAMP mice. The aim of this study is to investigate the potential of curcumin (CUR), a dietary compound that we have reported to be able to prevent the development of prostate cancer in TRAMP mice, as a DNA hypomethylation agent. Using bisulfite genomic sequencing (BGS), treatment of TRAMP C1 cells we showed that CUR reversed the methylation status of the first 5 CpGs in the promoter region of the Nrf2 gene. Methylation DNA immunoprecipitation (MeDIP) analysis revealed that CUR significantly reduced the anti-mecyt antibody binding to the first 5 CpGs of the Nrf2 promoter, corroborated the BGS results. Demethylation of Nrf2 was found to be associated with the re-expression of Nrf2 and one of its downstream target gene, NQO-1, one of the major anti-oxidative stress enzymes, both at the mRNA and protein levels. Taken together, our current study suggests that CUR can elicit its prostate cancer chemopreventive effect, potentially at least in part, through epigenetic modification of the Nrf2 gene with its subsequent induction of the Nrf2-mediated anti-oxidative stress cellular defense pathway.

Keywords: Abbreviations; PCa; prostate cancer; Nrf2; nuclear factor erythroid-2 (NF-E2) related factor-2; CUR; curcumin; BGS; bisulfite genomic sequencing; MeDIP; methylation DNA immunoprecipitation; anti-mecyt; anti-methycytosine; HDAC; histone deacetylases; DNMTs; DNA methyltransferases; GST; glutathione-S-transferases; NQO1; NAD(P)H:quinone oxidoreductase-1; HO-1; heme oxygenase-1; Gpx; glutathione peroxidases; TIS; translation initiation site; 5-Aza; 5-azadeoxycytidine; TSA; Trichostatin ACurcumin; DNA hypomethylation; Nrf2; Prostate cancer; TRAMP C1; Epigenetics

Atorvastatin modulates anti-proliferative and pro-proliferative signals in Her2/neu-positive mammary cancer by Chiara Riganti; Hedwige Pinto; Elisabetta Bolli; Dimas C. Belisario; Raffaele A. Calogero; Amalia Bosia; Federica Cavallo (pp. 1079-1089).

The widely used anticholesterolemic drugs statins decrease the synthesis of cholesterol and the isoprenylation and activity of small G-proteins such as Ras and Rho, the effectors of which are often critical in cell proliferation. Thanks to this property, it has been hypothesized that statins may have anti-tumor activities. We investigated this issue in BALB-neuT mice, which developed Her2/neu-positive mammary cancers with 100% penetrance, and in TUBO cells, a cell line established from these tumors.Contrary to the mammary glands of BALB/c mice, the tumor tissue from BALB-neuT animals had constitutively activated Ras and ERK1/2. These were reduced by the oral administration of atorvastatin, but the statin did not prevent tumor growth in mice nor reduce the proliferation of TUBO cells, although it lowered the activity of mevalonate pathway and Ras/ERK1/2 signaling. By decreasing the mevalonate pathway-derived metabolite geranylgeranyl pyrophosphate and the RhoA/RhoA kinase signaling, atorvastatin activated NF-κB, that sustained cell proliferation. Unexpectedly Her2-positive cells were much more sensitive to the inhibition of RhoA-dependent pathways than to the suppression of Ras-dependent pathways elicited by atorvastatin. Only the simultaneous inhibition of RhoA/RhoA-kinase/NF-κB and Ras/ERK1/2 signaling allowed the statin to decrease tumor cell proliferation.Our study demonstrates that Her2-positive mammary cancers have redundant signals to sustain their proliferation and shows that statins simultaneously reduce the pro-proliferative Ras/ERK1/2 axis and activate the pro-proliferative RhoA/RhoA-kinase/NF-κB axis. The latter event dissipates the antitumor efficacy that may arise from the former one. Only the association of statins and NF-κB-targeted therapies efficiently decreased proliferation of tumor cells.

Keywords: Atorvastatin; Ras; RhoA; NF-κB; Her2-positive mammary cancer

Growth differentiation factor 15 (GDF15)-mediated HER2 phosphorylation reduces trastuzumab sensitivity of HER2-overexpressing breast cancer cells by Jayashree P. Joshi; Nicole E. Brown; Samantha E. Griner; Rita Nahta (pp. 1090-1099).

Resistance to the anti-HER2 monoclonal antibody trastuzumab is a major problem in the treatment of HER2-overexpressing metastatic breast cancer. Growth differentiation factor 15 (GDF15), which is structurally similar to TGF beta, has been reported to stimulate phosphorylation of HER2. We tested the hypothesis that GDF15-mediated phosphorylation of HER2 reduces the sensitivity of HER2-overexpressing breast cancer cell lines to trastuzumab. Gene microarray analysis, real-time PCR, and ELISA were used to assess GDF15 expression. Growth inhibition and proliferation assays in response to pharmacologic inhibitors of HER2, TGF beta receptor, or Src were performed on cells stimulated with recombinant human GDF15 or stable GDF15 transfectants. Western blotting was performed to determine effects of GDF15 on HER2 signaling. Cells were infected with lentiviral GDF15 shRNA plasmid to determine effects of GDF15 knockdown on cell survival in response to trastuzumab. Cells with acquired or primary trastuzumab resistance showed increased GDF15 expression. Exposure of trastuzumab-sensitive cells to recombinant human GDF15 or stable transfection of a GDF15 expression plasmid inhibited trastuzumab-mediated growth inhibition. HER2 tyrosine kinase inhibition abrogated GDF15-mediated Akt and Erk1/2 phosphorylation and blocked GDF15-mediated trastuzumab resistance. Pharmacologic inhibition of TGF beta receptor blocked GDF15-mediated phosphorylation of Src. Further, TGF beta receptor inhibition or Src inhibition blocked GDF15-mediated trastuzumab resistance. Finally, lentiviral GDF15 shRNA increased trastuzumab sensitivity in cells with acquired or primary trastuzumab resistance. These results support GDF15-mediated activation of TGF beta receptor-Src-HER2 signaling crosstalk as a novel mechanism of trastuzumab resistance.

Keywords: Breast cancer; erbB2; Herceptin; Resistance; Trastuzumab; Lapatinib

Anti-cancer effects of novel flavonoid vicenin-2 as a single agent and in synergistic combination with docetaxel in prostate cancer by Lokesh Dalasanur Nagaprashantha; Rit Vatsyayan; Jyotsana Singhal; Spence Fast; Rhonda Roby; Sanjay Awasthi; Sharad S. Singhal (pp. 1100-1109).

The present study was conducted to determine the efficacy of novel flavonoid vicenin-2 (VCN-2), an active constituent of the medicinal herb Ocimum Sanctum Linn or Tulsi, as a single agent and in combination with docetaxel (DTL) in carcinoma of prostate (CaP). VCN-2 effectively induced anti-proliferative, anti-angiogenic and pro-apoptotic effect in CaP cells (PC-3, DU-145 and LNCaP) irrespective of their androgen responsiveness or p53 status. VCN-2 inhibited EGFR/Akt/mTOR/p70S6K pathway along with decreasing c-Myc, cyclin D1, cyclin B1, CDK4, PCNA and hTERT in vitro. VCN-2 reached a level of 2.6±0.3μmol/l in serum after oral administration in mice which reflected that VCN-2 is orally absorbed. The i.v. administration of docetaxel (DTL), current drug of choice in androgen-independent CaP, is associated with dose-limiting toxicities like febrile neutropenia which has lead to characterization of alternate routes of administration and potential combinatorial regimens. In this regard, VCN-2 in combination with DTL synergistically inhibited the growth of prostate tumors in vivo with a greater decrease in the levels of AR, pIGF1R, pAkt, PCNA, cyclin D1, Ki67, CD31, and increase in E-cadherin. VCN-2 has been investigated for radioprotection and anti-inflammatory properties. This is the first study on the anti-cancer effects of VCN-2. In conclusion, our investigations collectively provide strong evidence that VCN-2 is effective against CaP progression along with indicating that VCN-2 and DTL co-administration is more effective than either of the single agents in androgen-independent prostate cancer.

Keywords: Abbreviations; CaP; carcinoma of prostate; VCN-2; vicenin-2; DTL; docetaxel; PCNA; proliferating cell nuclear antigen; AR; androgen receptorProstate cancer; Vicenin-2; Docetaxel; Tumor xenografts

Proteasome inhibitor MG132-induced apoptosis via ER stress-mediated apoptotic pathway and its potentiation by protein tyrosine kinase p56^lck in human Jurkat T cells by Hae Sun Park; Do Youn Jun; Cho Rong Han; Hyun Ju Woo; Young Ho Kim (pp. 1110-1125).

Exposure of human Jurkat T cells to MG132 caused apoptosis along with upregulation of Grp78/BiP and CHOP/GADD153, activation of JNK and p38MAPK, activation of Bak, mitochondrial membrane potential (Δψm) loss, cytochrome c release, activation of caspase-12, -9, -3, -7, and -8, cleavage of Bid and PARP, and DNA fragmentation. However, these MG132-induced apoptotic events, with the exceptions of upregulation of Grp78/BiP and CHOP/GADD153 and activation of JNK and p38MAPK, were abrogated by overexpression of Bcl-xL. Pretreatment with the pan-caspase inhibitor z-VAD-fmk prevented MG132-induced apoptotic caspase cascade, but allowed upregulation of Grp78/BiP and CHOP/GADD153 levels, activation of JNK and p38MAPK, Δψm loss, and cleavage of procaspase-9 (47kDa) to active form (35kDa). Further analysis using selective caspase inhibitors revealed that caspase-12 activation was required for activation of caspase-9 and -3 to the sufficient level for subsequent activation of caspase-7 and -8. MG132-induced cytotoxicity, apoptotic sub-G₁ peak, Bak activation, and Δψm loss were markedly reduced by p38MAPK inhibitor, but not by JNK inhibitor. MG132-induced apoptotic changes, including upregulation of Grp78/BiP and CHOP/GADD153 levels, activation of caspase-12, p38MAPK and Bak, and mitochondria-dependent activation of caspase cascade were more significant in p56^lck-stable transfectant JCaM1.6/lck than in p56^lck-deficient JCaM1.6/vector. The cytotoxicity of MG132 toward p56^lck-positive Jurkat T cell clone was not affected by the Src-like kinase inhibitor PP2. These results demonstrated that MG132-induced apoptosis was caused by ER stress and subsequent activation of mitochondria-dependent caspase cascade, and that the presence of p56^lck enhances MG132-induced apoptosis by augmenting ER stress-mediated apoptotic events in Jurkat T cells.

Keywords: Proteasome inhibitor; Apoptosis; ER stress; Mitochondria-dependent caspase cascade; p56; ^lck

UTP reduces infarct size and improves mice heart function after myocardial infarct via P2Y₂ receptor by R. Cohen; A. Shainberg; E. Hochhauser; Y. Cheporko; A. Tobar; E. Birk; L. Pinhas; J. Leipziger; J. Don; E. Porat (pp. 1126-1133).

Ischemia causes cardiac damage. Treatment with UTP protects the heart from ischemic damage by activating P2Y₂ receptors, resulting in smaller infarct size. No protection obtained in P2Y₂ receptor knockout mice.Pyrimidine nucleotides are signaling molecules, which activate G protein-coupled membrane receptors of the P2Y family. P2Y₂ and P2Y₄ receptors are part of the P2Y family, which is composed of 8 subtypes that have been cloned and functionally defined. We have previously found that uridine-5′-triphosphate (UTP) reduces infarct size and improves cardiac function following myocardial infarct (MI). The aim of the present study was to determine the role of P2Y₂ receptor in cardiac protection following MI using knockout (KO) mice, in vivo and wild type (WT) for controls. In both experimental groups used (WT and P2Y₂^−/− receptor KO mice) there were 3 subgroups: sham, MI, and MI+UTP. 24h post MI we performed echocardiography and measured infarct size using triphenyl tetrazolium chloride (TTC) staining on all mice. Fractional shortening (FS) was higher in WT UTP-treated mice than the MI group (44.7±4.08% vs. 33.5±2.7% respectively, p<0.001). However, the FS of P2Y₂^−/− receptor KO mice were not affected by UTP treatment (34.7±5.3% vs. 35.9±2.9%). Similar results were obtained with TTC and hematoxylin and eosin stainings. Moreover, troponin T measurements demonstrated reduced myocardial damage in WT mice pretreated with UTP vs. untreated mice (8.8±4.6 vs. 12±3.1 p<0.05). In contrast, P2Y₂^−/− receptor KO mice pretreated with UTP did not demonstrate reduced myocardial damage. These results indicate that the P2Y₂ receptor mediates UTP cardioprotection, in vivo.

Keywords: Cardioprotection; Ischemia; Knockout mice; P2Y; ₂; receptor; UTP

Triptolide, histone acetyltransferase inhibitor, suppresses growth and chemosensitizes leukemic cells through inhibition of gene expression regulated by TNF-TNFR1-TRADD-TRAF2-NIK-TAK1-IKK pathway by Byoungduck Park; Bokyung Sung; Vivek R. Yadav; Madan M. Chaturvedi; Bharat B. Aggarwal (pp. 1134-1144).

Triptolide, a diterpene triepoxide, from the Chinese herb Tripterygium wilfordii Hook.f, exerts its anti-inflammatory and immunosuppressive activities by inhibiting the transcription factor nuclear factor-κB (NF-κB) pathway, through a mechanism not yet fully understood. We found that triptolide, in nanomolar concentrations, suppressed both constitutive and inducible NF-κB activation, but did not directly inhibit binding of p65 to the DNA. The diterpene did block TNF-induced ubiquitination, phosphorylation, and degradation of IκBα, the inhibitor of NF-κB and inhibited acetylation of p65 through suppression of binding of p65 to CBP/p300. Triptolide also inhibited the IκBα kinase (IKK) that activates NF-κB and phosphorylation of p65 at serine 276, 536. Furthermore, the NF-κB reporter activity induced by TNF-TNFR1-TRADD-TRAF2-NIK-TAK1-IKKβ was abolished by the triepoxide. Triptolide also abrogated TNF-induced expression of cell survival proteins (XIAP, Bcl-x_L, Bcl-2, survivin, cIAP-1 and cIAP-2), cell proliferative proteins (cyclin D1, c-myc and cyclooxygenase-2), and metastasis proteins (ICAM-1 and MMP-9). This led to enhancement of apoptosis induced by TNF, taxol, and thalidomide by the diterpene and to suppression of tumor invasion. Overall, our results demonstrate that triptolide can block the inflammatory pathway activated by TNF-TNFR1-TRADD-TRAF2-NIK-TAK1-IKK, sensitizes cells to apoptosis, and inhibits invasion of tumor cells.

Keywords: Triptolide; TNF; NF-κB; CBP/p300

The NADPH oxidase cytosolic component p67phox is constitutively phosphorylated in human neutrophils: Regulation by a protein tyrosine kinase, MEK1/2 and phosphatases 1/2A by Pham My-Chan Dang; Houssam Raad; Riad Arabi Derkawi; Tarek Boussetta; Marie-Hélène Paclet; Sahra Amel Belambri; Karama Makni-Maalej; Yolande Kroviarski; Françoise Morel; Marie-Anne Gougerot-Pocidalo; Jamel El-Benna (pp. 1145-1152).

Neutrophils play a key role in host defense and inflammation through the production of superoxide anion and other reactive oxygen species (ROS) by the enzyme complex NADPH oxidase. The cytosolic NADPH oxidase component, p67phox, has been shown to be phosphorylated in human neutrophils but the pathways involved in this process are largely unknown. In this study, we show that p67phox is constitutively phosphorylated in resting human neutrophils and that neutrophil stimulation with PMA further enhanced this phosphorylation. Inhibition of the constitutively active serine/threonine phosphatases type 1 and type 2A (PP1/2A) by calyculin A resulted in the enhancement of p67phox phosphorylation. Constitutive and calyculin A-induced phosphorylation of p67phox was completely inhibited by the protein tyrosine kinase inhibitor genistein and partially inhibited by the MEK1/2 inhibitor PD98059, but was unaffected by GF109203X, wortmannin and SB203580, inhibitors of PKC, PI3K and p38MAP kinase, respectively. Two-dimensional phosphopeptide mapping revealed that constitutive and calyculin A-induced p67phox phosphorylation occurred on the same major sites. Interestingly, calyculin A enhanced formyl-Met-Leu-Phe (fMLP)-induced superoxide production, while genistein inhibited this process. Taken together, these results suggest that (i) p67phox undergoes a continual cycle of phosphorylation/dephosphorylation in resting cells; (ii) p67phox phosphorylation is controlled by MEK1/2 and an upstream tyrosine kinase; (iii) PP1/2A directly or indirectly antagonize this process. Thus, these pathways could play a role in regulating ROS production by human neutrophils at inflammatory sites.

Keywords: Neutrophils; NADPH oxidase; p67phox; ROS; Inflammation; Phosphorylation

EGCG downregulates IL-1RI expression and suppresses IL-1-induced tumorigenic factors in human pancreatic adenocarcinoma cells by Julia Hoffmann; Heike Junker; Annett Schmieder; Simone Venz; Rowena Brandt; Gabriele Multhoff; Werner Falk; Jürgen Radons (pp. 1153-1162).

Human pancreatic cancer is currently one of the fifth-leading causes of cancer-related mortality with a 5-year survival rate of less than 5%. Since pancreatic carcinoma is largely refractory to conventional therapies, there is a strong medical need for the development of novel and innovative therapeutic strategies. Increasing evidence suggests an association of carcinogenesis and chronic inflammation. Because IL-1 plays a crucial role in inflammation-associated carcinogenesis, we analyzed the biological effects of IL-1 and its modulation by the chemopreventive green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) in the human pancreatic adenocarcinoma cell line Colo357. Proinflammatory IL-6 and PGHS-2 as well as proangiogenic IL-8 and VEGF were induced by IL-1, whereas the secretion of invasion-promoting MMP-2 remained unaffected. IL-1 responsiveness and constitutive MMP-2 release in Colo357 were downregulated by EGCG in a dose- and time-dependent manner. Moreover, EGCG reduced cell viability via induction of apoptosis in Colo357. Since EGCG effects on cytokine production precede reduction in cell viability, we hypothesize that these findings are not only a result of cell death but also depend on alterations in the IL-1 signaling cascade. In this context, we found for the first time an EGCG-induced downregulation of the IL-1RI expression possibly being caused by NF-κB inhibition and causative for its inhibitory action on the production of tumorigenic factors. Thus, our data might have future clinical implications with respect to the development of novel approaches as an adjuvant therapy in high-risk patients with human pancreatic carcinoma.

Keywords: Pancreatic carcinoma; IL-1; EGCG; IL-1RAcP; IL-1RI; NF-κB

Direct inhibition of NF-κB activation by peptide targeting the NOA ubiquitin binding domain of NEMO by Jeanne Chiaravalli; Elisabeth Fontan; Hafida Fsihi; Yves-Marie Coic; Françoise Baleux; Michel Véron; Fabrice Agou (pp. 1163-1174).

Aberrant and constitutive NF-κB activation are frequently reported in numerous tumor types, making its inhibition an attractive target for the treatment of certain cancers. NEMO (NF-κB essential modulator) is the crucial component of the canonical NF-κB pathway that mediates IκB kinase (IKK) complex activation. IKK activation resides in the ability of the C-terminal domain of NEMO to properly dimerize and interact with linear and K63-linked polyubiquitin chains. Here, we have identified a new NEMO peptide inhibitor, termed UBI (ubiquitin binding inhibitor) that derives from the NOA/NUB/UBAN ubiquitin binding site located in the CC2-LZ domain of NEMO. UBI specifically inhibits the NF-κB pathway at the IKK level in different cell types stimulated by a variety of NF-κB signals. Circular dichroïsm and fluorescence studies showed that UBI exhibits an increased α-helix character and direct, good-affinity binding to the NOA-LZ region of NEMO. We also showed that UBI targets NEMO in cells but its mode of inhibition is completely different from the previously reported LZ peptide (herein denoted NOA-LZ). UBI does not promote dissociation of NEMO subunits in cells but impairs the interaction between the NOA UBD of NEMO and polyubiquitin chains. Importantly, we showed that UBI efficiently competes with the in vitro binding of K63-linked chains, but not with linear chains. The identification of this new NEMO inhibitor emphasizes the important contribution of K63-linked chains for IKK activation in NF-κB signaling and would provide a new tool for studying the complex role of NF-κB in inflammation and cancer.

Keywords: Abbreviations; NF-κB; nuclear factor-κB; NEMO; NF-κB essential modulator; LZ; leucine zipper; CC2; coiled-coil 2; UBI; ubiquitin binding inhibitor derived from NEMO; NOA; NEMO Optineurin ABIN; CPP; cell permeable peptide; BA-peptide; peptide fused at its N-terminus with the cell permeable sequence antennapedia and conjugated with the bodipy fluorophore; A-peptide; Antennapedia tagged peptide; R; ₇; - or R; ₉; -peptide; peptide fused at its N-terminus with a hepta- or nona-arginine sequence; DDM; dodecyl maltoside; PBS; phosphate-buffered saline; LPS; lipopolysaccharide; PMA; phorbol 12-myristate 13-acetate; IL-1β; interleukine 1β; TNF-α; tumor necrosis factor α; UBD; ubiquitin binding domainIKK complex; NF-κB; Ubiquitin binding domain; Protein–protein interaction inhibitor; Ubiquitin linkage

Activation of mitogen-activated protein kinases by 5,6-dimethylxanthenone-4-acetic acid (DMXAA) plays an important role in macrophage stimulation by Jing Sun; Liang-Chuan S. Wang; Zvi G. Fridlender; Veena Kapoor; Guanjun Cheng; Lai-Ming Ching; Steven M. Albelda (pp. 1175-1185).

This study confirms that DMXAA activates both p38 and ERK1/2 MAPKs through unknown mechanisms. By using selective inhibitors of MAPKs, BIRB796 and FR180204, we confirmed that both p38 and ERK1/2 MAPK play a significant role in regulation of TNF-α and IL-6 protein production induced by DMXAA at the post-transcriptional level.The small molecule anti-tumor agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA, now called Vadimezan) is a potent macrophage and dendritic cell activating agent that, in the murine system, results in the release of large amounts of cytokines and chemokines. The mechanisms by which this release is mediated have not been fully elucidated. The mitogen-activated protein kinase (MAPK) pathways play an important role in the regulation of proinflammatory cytokines, such as TNF-α, IL-1β, as well as the responses to extracellular stimuli, such as lipopolysaccharide (LPS). The results of this study demonstrate that DMXAA activates three members of mitogen-activated protein kinase (MAPK) superfamily, namely p38 MAPK, extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), and c-Jun N-terminal kinases (JNKs) via a RIP2-independent mechanism in murine macrophages. By using selective inhibitors of MAPKs, this study confirms that both activated p38/MK2 pathways and ERK1/2 MAPK play a significant role in regulation of both TNF-α and IL-6 protein production induced by DMXAA at the post-transcriptional level. Our findings also show that interferon-γ priming can dramatically augment TNF-α protein secretion induced by DMXAA through enhancing activation of multiple MAPK pathways at the post-transcriptional level. This study expands current knowledge on mechanisms of how DMXAA acts as a potent anti-tumor agent in murine system and also provides useful information for further study on the mechanism of action of this potential anti-tumor compound in human macrophages.

Keywords: Abbreviations; ARE; adenylate/uridylate-rich elements; DMXAA; 5,6-dimethylxanthenone-4-acetic acid; ELISA; enzyme-linked immunosorbent assay; ERK; extracellular signal-regulated kinase; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; IFN; interferon; IFNAR; interferon alpha receptor; IL-6; interleukin 6; IP-10; interferon gamma-induced protein 10; kDa; JNK; c-Jun N-terminal kinase; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; MCP-1; monocyte chemotactic protein-1; MK-2; MAP kinase activated kinase-2; NF-κB; nuclear factor kappa-light-chain-enhancer of activated B cells; NO; nitric oxide; NOD; nucleotide oligomerization domain; PCR; polymerase chain reaction; RANTES; regulated upon Activation, Normal T-cell Expressed, and Secreted or CCL5; RIP2; receptor-interacting protein 2 or RICK; TBK–IRF3; TANK-binding kinase-interferon regulatory factor 3; TBP; TATA-binding protein; TLR; toll-like receptor; TNF-α; tumor necrosis factor-alpha; TTP; tristetraprolin; 3′-UTR; 3′-untranslated regionMAPK; Post-transcriptional regulation; TNF-α; DMXAA; Proinflammatory cytokines

Carlinoside reduces hepatic bilirubin accumulation by stimulating bilirubin-UGT activity through Nrf2 gene expression by Rakesh Kundu; Suman Dasgupta; Anindita Biswas; Sushmita Bhattacharya; Bikas C. Pal; Shelley Bhattacharya; P.G. Rao; N.C. Barua; Manobjyoti Bordoloi; Samir Bhattacharya (pp. 1186-1197).

Accumulation of bilirubin, primarily because of its insolubility, has been found to be associated with liver diseases including jaundice. Free bilirubin is insoluble; its glucuronidation by bilirubin-UGT enzyme (UGT1A1) makes it soluble and eliminates it through urine and faeces. Taking CCl₄ induced rat liver dysfunction model, we demonstrated that suppression of UGT1A1 activity in rat liver increased serum bilirubin level which could be reversed by carlinoside (Cln), a flavone glycoside. Although Cln is a flavone compound, it escaped self-glucuronidation in the intestine and readily absorbed. Kinetic study of microsomal UGT1A1 from HepG2 cells suggested that Cln enhanced enzyme activity by increasing V_max without altering K_m. This altered V_max was found to be due to UGT1A1 overexpression by Cln which was observed in both HepG2 and rat primary hepatocytes. Since Nrf2 is the transcription factor of UGT1A1, we examined whether Cln effect on UGT1A1 overexpression is mediated through Nrf2. In Nrf2 knock-out cells, Cln could not elevate UGT1A1 activity indicating Nrf2 to be its target. Cln significantly increased Nrf2 gene expression in HepG2 cells which was subsequently localized in nuclear region. Results from ChIP assay showed that Cln markedly augmented Nrf2 binding to UGT1A1 promoter that consequently enhanced reporter activity. Our findings therefore show that Cln upregulated Nrf2 gene expression, increased its nuclear translocation and stimulated UGT1A1 promoter activity. Total outcome of these events brought about a significant increase of bilirubin glucuronidation. Cln therefore could be a worthy choice to intervene hyperbilirubinemia due to liver dysfunction.

Keywords: Bilirubin; Carlinoside; Liver damage; Nrf2; UGT1A1

Des-aspartate-angiotensin-I and angiotensin IV improve glucose tolerance and insulin signalling in diet-induced hyperglycaemic mice by Yong-Chiat Wong; Meng-Kwoon Sim; Kok-Onn Lee (pp. 1198-1208).

Des-aspartate-angiotensin I and angiotensin IV attenuate hyperglycemia in C57BL/6J mice fed on a high fat and high glucose diet for 52 weeks.Although clinical studies suggested that blockade of the renin-angiotensin system may prevent diabetes, the mechanism is uncertain. As a follow-up to an earlier study, we investigated how des-aspartate-angiotensin-1 (DAA-1) and its metabolite, angiotensin IV (Ang-IV) improved glucose tolerance in diet-induced hyperglycaemic mice. Male C57BL/6J mice were fed a high-fat-high-sucrose (HFD) or normal (ND) diet for 52 weeks. HFD animals were orally administered either DAA-I (600nmol/kg/day), Ang-IV (400nmol/kg/day) or distilled water. Body weight, blood glucose and insulin were measured fortnightly. Inflammatory and insulin signalling transducers that are implicated in hyperglycaemia were analyzed in skeletal muscles at 52 weeks. HFD animals developed hyperglycemia, hyperinsulinemia and obesity. DAA-I and Ang-IV improved glucose tolerance but had no effect on hyperinsulinemia and obesity. Skeletal muscles of HFD animals showed increased level of ROS, gp91 of NADPH oxidase, pJNK and AT₁R-JAK-2-IRS-1 complex. Both DAA-I and Ang-IV attenuated these increases. Insulin-induced activation of IR, IRS-1, IRS-1-PI3K coupling, phosphorylation of Akt, and GLUT4 translocation were attenuated in skeletal muscles of HFD animals. The attenuation was significantly ameliorated in DAA-I-treated HFD animals. In corresponding Ang-IV treated animals, insulin induced IRAP and PI3K interaction, activation of pAkt and GLUT4 translocation, but no corresponding activation of IR, IRS-1 and IRS-1-PI3K coupling were observed. DAA-I and Ang-IV improved glucose tolerance, insulin signalling, and para-inflammatory processes linked to hyperglycaemia. DAA-I acts via the angiotensin AT₁ receptor and activates the insulin pathway. Ang-IV acts via IRAP, which couples PI3K and activates the later part of the insulin pathway.

Keywords: Abbreviations; Akt; protein kinase B; Ang-II; angiotensin II; Ang-IV; angiotensin IV; DAA-I; des-aspartate-angiotensin I; HFD; high-fat-high-sucrose diet; IR; insulin receptor; IRAP; insulin-related aminopeptidase; JAK; Janus kinase; JNK; c-Jun N-terminal kinases; ND; normal diet; PI3K; phosphoinositide 3-kinase; ROS; reactive oxygen speciesDes-aspartate-angiotensin I; Angiotensin IV; Diet-induced hyperglycaemia; Insulin resistance; Angiotensin receptor type I; Insulin regulated aminopeptidase

Des-aspartate-angiotensin-I and angiotensin IV improve glucose tolerance and insulin signalling in diet-induced hyperglycaemic mice by Yong-Chiat Wong; Meng-Kwoon Sim; Kok-Onn Lee (pp. 1198-1208).

Lycium barbarum (Goji Berry) extracts and its taurine component inhibit PPAR-γ-dependent gene transcription in human retinal pigment epithelial cells: Possible implications for diabetic retinopathy treatment by M.K. Song; N.K. Salam; Basil D. Roufogalis; T.H.W. Huang (pp. 1209-1218).

The peroxisome proliferator activated receptor-γ (PPAR-γ) is involved in the pathogenesis of diabetic retinopathy. Diabetic retinopathy is a preventable microvascular diabetic complication that damages human retinal pigment epithelial cells. Taurine is abundant in the fruit of Lycium barbarum (Goji Berry), and is reportedly beneficial for diabetic retinopathy. However, the mechanism of its action is unknown. Hence, we have investigated the mechanism of action of an extract from L. barbarum on a model of diabetic retinopathy, the retinal ARPE-19 cell line, and identified the receptor function of taurine, an active component of L. barbarum (Goji Berry) extract, which is potentially responsible for the protective effect on diabetic retinopathy. We demonstrate for the first time that L. barbarum extract and its taurine component dose-dependently enhance PPAR-γ luciferase activity in HEK293 cell line transfected with PPAR-γ reporter gene. This activity was significantly decreased by a selective PPAR-γ antagonist GW9662. Moreover, L. barbarum extract and taurine dose-dependently enhanced the expression of PPAR-γ mRNA and protein. In an inflammation model where ARPE-19 cells were exposed to high glucose L. barbarum extract and taurine down-regulated the mRNA of pro-inflammatory mediators encoding MMP-9, fibronectin and the protein expression of COX-2 and iNOS proteins. The predicted binding mode of taurine in the PPAR-γ ligand binding site mimics key electrostatic interactions seen with known PPAR-γ agonists. We conclude that PPAR-γ activation by L. barbarum extract is associated with its taurine content and may explain at least in part its use in diabetic retinopathy progression.

Keywords: Abbreviations; PPAR-γ; peroxisome proliferator activated receptor-γ; LB; fruit from; Lycium barbarum; RT-PCR; reverse transcription-polymerase chain reaction; HEK; human embryonic kidney; ARPE-19; human retinal epithelial cell line; DR; diabetic retinopathy; RG; rosiglitazone; PG; 15-deoxy-Δ; ^12,14; -prostaglandin J; ₂ Lycium barbarum; Taurine; Peroxisome proliferator activated receptor-γ; Diabetic retinopathy; Human retinal pigment epithelial cell

OATP1B1/1B3 activity in plated primary human hepatocytes over time in culture by Maria Ulvestad; Petter Björquist; Espen Molden; Anders Åsberg; Tommy B. Andersson (pp. 1219-1226).

Primary human hepatocytes are widely used as an in vitro model for evaluation of drug metabolism and transport. However, it has been shown that the gene expression of many drug-metabolizing enzymes and transporters change in culture. The aim of the present study was to evaluate the activity of organic anion-transporting polypeptide 1B1 (OATP1B1) and 1B3 (OATP1B3) in plated primary human hepatocytes over time in culture. The uptake kinetics of the OATP1B1/1B3 substrate [³H]-estradiol-17β-d-glucuronide was determined in cells from five donors. An extensive and variable decrease in OATP1B1/1B3 activity and/or increase in passive diffusion was observed over time. Already after 6h in culture, the OATP1B1/1B3 activity was not possible to determine in liver cells from one donor, while after 24h, the uptake activity was not measurable in one additional donor. In the other three, the decrease in CL_int ( V_max/ K_m) values ranged from 15% to 86% after 24h in culture compared to the values measured at 2h. Visual examination of OATP1B1 protein expression by confocal microscopy showed localization to the plasma membrane as expected, and an extensive decrease in OATP1B1 expression over time in culture supported the decline in activity. The significant reduction in SLCO1B1 and SLCO1B3 gene expression over time determined by RT-PCR also supported the loss of OATP1B1/1B3 activity. In conclusion, plated primary human hepatocytes are useful as an in vitro model for OATP1B1/1B3-mediated uptake studies, but the culture time may substantially change the uptake kinetics.

Keywords: Abbreviations; OATP; organic anion-transporting polypeptide; SNP; single nucleotide polymorphism; E17βG; estradiol-17β-; d; -glucuronide; FBS; fetal bovine serum; HBSS; Hanks’ balanced salt solution; PBS; phosphate-buffered saline; PCR; polymerase chain reaction; Ct; cycle threshold; OCT1; organic cation transporter 1; OAT2; organic anion transporter 2; NTCP; sodium-taurocholate cotransporting polypeptideHepatocytes; Transporters; OATP1B1; OATP1B3; Pharmacokinetics

OATP1B1/1B3 activity in plated primary human hepatocytes over time in culture by Maria Ulvestad; Petter Björquist; Espen Molden; Anders Åsberg; Tommy B. Andersson (pp. 1219-1226).

Efavirenz is a substrate and in turn modulates the expression of the efflux transporter ABCG2/BCRP in the gastrointestinal tract of the rat by Roxana N. Peroni; Stefania S. Di Gennaro; Christian Hocht; Diego A. Chiappetta; Modesto C. Rubio; Alejandro Sosnik; Guillermo F. Bramuglia (pp. 1227-1233).

The oral bioavailability of the antiretroviral efavirenz (EFV) undergoes high inter and intra-individual variability, this fact supporting its therapeutic drug monitoring. Previously, it was demonstrated that the encapsulation of EFV within polymeric micelles increases the oral bioavailability of the drug. The breast cancer resistant protein (BCRP, ABCG2) is known to be inhibited by EFV in vitro. Since ABCG2 is profusely expressed in the gastrointestinal tract, the aim of the present work was to thoroughly investigate whether the intestinal permeability of EFV is modulated by ABCG2. The functional role of ABCG2 in mediating the transport of EFV at the intestinal level was consistent with the following findings: (a) an ABCG2 inhibitor, fumitremorgin C (5–10μM), significantly potentiated the mucosal-to-serosal permeation of the drug in everted gut sacs; (b) a five-day oral treatment with 20mg/kg EFV promotes the over-expression of ABCG2 in about 100%, this phenomenon being accompanied by a clear decline in the intestinal permeability of the antiretroviral and (c) the normalization of the ABCG2 expression within 24h after the last administration of EFV was coincident with the recovery of the ability of the drug to permeate through the small intestine wall. Interestingly, no interactions between EFV and P-glycoprotein (ABCB1) were apparent. Since the intestinal permeability of a drug could be associated with its in vivo absorbability, we suggest that the oral absorption of EFV is affected by modifications in the ABCG2 intestinal expression contributing to the intra-individual bioavailability variations.

Keywords: Abcg2; Efavirenz; Hiv; Oral bioavailability; Intestine

Amiodarone impairs trafficking through late endosomes inducing a Niemann-Pick C-like phenotype by Elena Piccoli; Matteo Nadai; Carla Mucignat Caretta; Valeria Bergonzini; Claudia Del Vecchio; Huy Riem Ha; Laurent Bigler; Daniele Dal Zoppo; Elisabetta Faggin; Andrea Pettenazzo; Rocco Orlando; Cristiano Salata; Arianna Calistri; Giorgio Palù; Aldo Baritussio (pp. 1234-1249).

Amiodarone, having basic pKa and high water solubility at acidic pH, accumulates within late endocytic compartments, blocking fluid-phase endocytosis, proteolysis and lipid trafficking and inducing a Niemann-Pick C-like phenotype.Patients treated with amiodarone accumulate lysobisphosphatidic acid (LBPA), also known as bis(monoacylglycero)phosphate, in airway secretions and develop in different tissues vacuoles and inclusion bodies thought to originate from endosomes. To clarify the origin of these changes, we studied in vitro the effects of amiodarone on endosomal activities like transferrin recycling, Shiga toxin processing, ESCRT-dependent lentivirus budding, fluid phase endocytosis, proteolysis and exosome secretion. Furthermore, since the accumulation of LBPA might point to a broader disturbance in lipid homeostasis, we studied the effect of amiodarone on the distribution of LBPA, unesterified cholesterol, sphingomyelin and glycosphyngolipids. Amiodarone analogues were also studied, including the recently developed derivative dronedarone. We found that amiodarone does not affect early endosomal activities, like transferrin recycling, Shiga toxin processing and lentivirus budding. Amiodarone, instead, interferes with late compartments of the endocytic pathway, blocking the progression of fluid phase endocytosis and causing fusion of organelles, collapse of lumenal structures, accumulation of undegraded substrates and amassing of different types of lipids. Not all late endocytic compartments are affected, since exosome secretion is spared. These changes recall the Niemann-Pick type-C phenotype (NPC), but originate by a different mechanism, since, differently from NPC, they are not alleviated by cholesterol removal. Studies with analogues indicate that basic pKa and high water-solubility at acidic pH are crucial requirements for the interference with late endosomes/lysosomes and that, in this respect, dronedarone is at least as potent as amiodarone. These findings may have relevance in fields unrelated to rhythm control.

Keywords: Abbreviations; LBPA; lysobisphosphatidic acid; ESCRT; endosomal sorting complex required for transport; VLP; virus like particlesAmiodarone; Dronedarone; Late endosomes; Niemann-Pick C; Phospholipidosis

Corrigendum to “Jingzhaotoxin-II, a novel tarantula toxin preferentially targets rat cardiac sodium channel” [Biochem. Pharmacol. 76 (2008) 1716–1727] by Meichi Wang; Qingping Liu; Haiyong Luo; Jiang Li; Jianzhou Tang; Yucheng Xiao; Songping Liang (pp. 1250-1251).

Corrigendum to “Effects of AZD1152, a selective Aurora B kinase inhibitor, on Burkitt's and Hodgkin's lymphomas” [Biochem. Pharmacol. 81 (2011) 1106–1115] by Naoki Mori; Chie Ishikawa; Masachika Senba; Masashi Kimura; Yukio Okano (pp. 1252-1252).

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Biochemical Pharmacology (v.82, #9)