Modulation of Brahma expression by the mitogen-activated protein kinase/extracellular signal regulated kinase pathway is associated with changes in melanoma proliferation.

Brahma (BRM) and Brahma-related gene 1(BRG1) are catalytic subunits of SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes. BRM is epigenetically silenced in a wide-range of tumors. Mutations in the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene occur frequently in melanoma and lead to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK1/2) pathway. We tested the hypothesis that BRM expression is modulated by oncogenic BRAF and phosphorylation of ERK1/2 in melanocytes and melanoma cells. Expression of oncogenic BRAF in melanocytes and melanoma cells that are wild-type for BRAF decreased BRM expression and increased BRG1 expression. Inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) or selective inhibition of BRAF in melanoma cells that harbor oncogenic BRAF increased BRM expression and decreased BRG1 expression. Increased BRM expression was associated with increased histone acetylation on the BRM promoter. Over-expression of BRM in melanoma cells that harbor oncogenic BRAF promoted changes in cell cycle progression and apoptosis consistent with a tumor suppressive role. Upon inhibition of BRAF(V600E) with PLX4032, BRM promoted survival. PLX4032 induced changes in BRM function were correlated with increased acetylation of the BRM protein. This study provides insights into the epigenetic consequences of inhibiting oncogenic BRAF in melanoma through modulation of SWI/SNF subunit expression and function.

Syntheses and structures of copper(I) complexes based on Cu(n)X(n) (X = Br and I; n = 1, 2 and 4) units and bis(pyridyl) ligands with longer flexible spacer.

Self-assembly of four bis(pyridyl) ligands with longer flexible spacer: 1,4-bis(3-pyridylaminomethyl)benzene (L1), 1,4-bis(2-pyridylaminomethyl)benzene (L2), 1,3-bis(3-pyridylaminomethyl)benzene (L3) and 1,3-bis(2-pyridylaminomethyl)benzene (L4), and CuX (X = Br and I) leads to the formation of eight [Cu(n)X(n)]-based (X = Br and I; n = 1, 2, and 4) complexes, [Cu(2)I(2)L1(PPh(3))(4)] (1), [Cu(4)Cl(2)Br(2)(L4)(2)(PPh(3))(6)]·(CH(3)CN)(2) (2), [Cu(2)I(2)(L3)(2)] (3), {[Cu(2)Br(2)L2(PPh(3))(2)]·(CH(2)Cl(2))(2)}(n) (4), [CuIL1](n)·nCH(2)Cl(2) (5), [CuIL1](n) (6), [CuIL4](n) (7) and [Cu(2)I(2)L4](n) (8), which have been synthesized and characterized by elemental analysis, IR, TG, powder and single-crystal X-ray diffraction. Structural analyses show that the eight complexes possess an increasing dimensionality from 0D (1-3) to 1D (4) to 2D (5-8), in which 1 and 2 contain a CuX unit, 2-7 contain a Cu(2)X(2) unit and 8 contains a Cu(4)X(4) unit. Such evolvement indicates that the conformation of flexible bis(pyridyl) ligands and the participation of triphenylphosphine (PPh(3)) as a second ligand take an essential role in the framework formation of the Cu(i) complexes. Moreover, a pair of symmetry-related L3 ligands in complex 3 coordinate to the rhomboid Cu(2)I(2) dimer to form "handcuff-shaped" dinuclear structures, which are further joined together through intermolecular N-HI hydrogen bonds to furnish a 2D (4,4) layer. Although complexes 5 and 6 exhibit a similar 2D (4,4) layer constructed from L1 ligand bridging [Cu(2)I(2)](n) units, the different packing fashion of the layers leads to the formation of 3D porous frameworks of 5 and dense 3D frameworks of 6. The "twisted-boat" conformation of the Cu(4)I(4) tetramer unit in complex 8 has not been reported so far.

Modulation of extracellular matrix/adhesion molecule expression by BRG1 is associated with increased melanoma invasiveness.

BACKGROUND: Metastatic melanoma is an aggressive malignancy that is resistant to therapy and has a poor prognosis. The progression of primary melanoma to metastatic disease is a multi-step process that requires dynamic regulation of gene expression through currently uncharacterized epigenetic mechanisms. Epigenetic regulation of gene expression often involves changes in chromatin structure that are catalyzed by chromatin remodeling enzymes. Understanding the mechanisms involved in the regulation of gene expression during metastasis is important for developing an effective strategy to treat metastatic melanoma. SWI/SNF enzymes are multisubunit complexes that contain either BRG1 or BRM as the catalytic subunit. We previously demonstrated that heterogeneous SWI/SNF complexes containing either BRG1 or BRM are epigenetic modulators that regulate important aspects of the melanoma phenotype and are required for melanoma tumorigenicity in vitro. RESULTS: To characterize BRG1 expression during melanoma progression, we assayed expression of BRG1 in patient derived normal skin and in melanoma specimen. BRG1 mRNA levels were significantly higher in stage IV melanomas compared to stage III tumors and to normal skin. To determine the role of BRG1 in regulating the expression of genes involved in melanoma metastasis, we expressed BRG1 in a melanoma cell line that lacks BRG1 expression and examined changes in extracellular matrix and adhesion molecule expression. We found that BRG1 modulated the expression of a subset of extracellular matrix remodeling enzymes and adhesion proteins. Furthermore, BRG1 altered melanoma adhesion to different extracellular matrix components. Expression of BRG1 in melanoma cells that lack BRG1 increased invasive ability while down-regulation of BRG1 inhibited invasive ability in vitro. Activation of metalloproteinase (MMP) 2 expression greatly contributed to the BRG1 induced increase in melanoma invasiveness. We found that BRG1 is recruited to the MMP2 promoter and directly activates expression of this metastasis associated gene. CONCLUSIONS: We provide evidence that BRG1 expression increases during melanoma progression. Our study has identified BRG1 target genes that play an important role in melanoma metastasis and we show that BRG1 promotes melanoma invasive ability in vitro. These results suggest that increased BRG1 levels promote the epigenetic changes in gene expression required for melanoma metastasis to proceed.

Folate receptor beta as a potential delivery route for novel folate antagonists to macrophages in the synovial tissue of rheumatoid arthritis patients.

OBJECTIVE: To determine the expression of folate receptor beta (FRbeta) in synovial biopsy tissues and peripheral blood lymphocytes from rheumatoid arthritis (RA) patients and to identify novel folate antagonists that are more selective in the targeting and internalization of FRbeta than methotrexate (MTX). METHODS: Immunohistochemistry and computer-assisted digital imaging analyses were used for the detection of FRbeta protein expression on immunocompetent cells in synovial biopsy samples from RA patients with active disease and in noninflammatory control synovial tissues. FRbeta messenger RNA (mRNA) levels were determined by reverse transcription-polymerase chain reaction analysis. Binding affinities of FRbeta for folate antagonists were assessed by competition experiments for 3H-folic acid binding on FRbeta-transfected cells. Efficacy of FRbeta-mediated internalization of folate antagonists was evaluated by assessment of antiproliferative effects against FRbeta-transfected cells. RESULTS: Immunohistochemical staining of RA synovial tissue showed high expression of FRbeta on macrophages in the intimal lining layer and synovial sublining, whereas no staining was observed in T cell areas or in control synovial tissue. Consistently, FRbeta mRNA levels were highest in synovial tissue extracts and RA monocyte-derived macrophages, but low in peripheral blood T cells and monocytes. Screening of 10 new-generation folate antagonists revealed 4 compounds for which FRbeta had a high binding affinity (20-77-fold higher than for MTX). One of these, the thymidylate synthase inhibitor BCG 945, displayed selective targeting against FRbeta-transfected cells. CONCLUSION: Abundant FRbeta expression on activated macrophages in synovial tissue from RA patients deserves further exploration for selective therapeutic interventions with high-affinity-binding folate antagonists, of which BCG 945 may be a prototypical representative.

Synergistic induction of folate receptor beta by all-trans retinoic acid and histone deacetylase inhibitors in acute myelogenous leukemia cells: mechanism and utility in enhancing selective growth inhibition by antifolates.

The folate receptor (FR) type beta is a promising target for therapeutic intervention in acute myelogenous leukemia (AML), owing particularly to its selective up-regulation in the leukemic cells by all-trans retinoic acid (ATRA). Here we show, using KG-1 and MV4-11 AML cells and recombinant 293 cells, that the histone deacetylase (HDAC) inhibitors trichostatin A (TSA), valproic acid (VPA), and FK228 potentiated ATRA induction of FR-beta gene transcription and FR-beta mRNA/protein expression. ATRA and/or TSA did not induce de novo FR synthesis in any of a variety of FR-negative cell lines tested. TSA did not alter the effect of ATRA on the expression of retinoic acid receptor (RAR) alpha, beta, or gamma. Chromatin immunoprecipitation assays indicate that HDAC inhibitors act on the FR-beta gene by enhancing RAR-associated histone acetylation to increase the association of Sp1 with the basal FR-beta promoter. Under these conditions, the expression level of Sp1 is unaltered. A decreased availability of putative repressor AP-1 proteins may also indirectly contribute to the effect of HDAC inhibitors. Finally, FR-beta selectively mediated growth inhibition by (6S) dideazatetrahydrofolate in a manner that was greatly potentiated in AML cells by ATRA and HDAC inhibition. Therefore, the combination of ATRA and innocuous HDAC inhibitors may be expected to facilitate selective FR-beta-targeted therapies in AML.

Forskolin up-regulates metastasis-related phenotypes and molecules via protein kinase B, but not PI-3K, in H7721 human hepato-carcinoma cell line.

Forskolin (FSK) is known as an up-regulator of intracellular cAMP and inhibitor of cancer growth and metastasis. The effects of FSK on the metastasis potential and its mechanisms were studied using a human hepatocarcinoma cell line, H7721. It was found that FSK stimulated cell growth, increased cAMP in the cells, and enhanced the metastasis-related phenotypes, including adhesion to laminin (Ln) and human umbilical vein epithelial cells (HUVEC), chemotactic migration and invasion. These effects were supposed to result from the increase of the SLex expression induced by FSK, since only the monoclonal antibody of SLex showed a significant attenuation of the enhanced metastasis-associated phenotypes. Using H7721 cells transfected with the sense or antisense cDNA of protein kinase B (PKB) and some inhibitors of signal transduction, it was discovered that FSK up-regulated the expression of SLex via PKB, but it was independent of phosphotidylinositide-3-kinase (PI-3K). A subtype of atypical protein kinase C (a-PKC) might also participate in the up-regulation of SLex expression by FSK, and cAMP/PKA pathway is a negative regulator of SLex expression on H7721 cells. It can be concluded that FSK shows a metastasis-promoting effect ex vivo.

Insulin/protein kinase B signalling pathway upregulates metastasis-related phenotypes and molecules in H7721 human hepatocarcinoma cell line.

The effect of insulin on cancer metastatic potential was studied in a human hepatocarcinoma cell line, H7721. Cell adhesion to human umbilical vein endothelial cells (HUVECs) and laminin as well as chemotactic cell migration and invasion were selected as the indices of metastasis-related phenotypes for assessment of metastatic potential ex vivo. The results indicated that insulin enhanced all of these metastasis-related phenotypes. After the cells were treated with specific inhibitor of PI3K (LY294002) or transfected with antisense cDNA of PKB (AS-PKB), all of the above phenotypes were attenuated, and they could not be significantly stimulated by insulin, indicating that the insulin effect on metastatic potential was mediated by PI3K and PKB. Only the monoclonal antibody to the sialyl Lewis X (SLe(x)), but not antibodies to other Lewis antigens, significantly blocked the cell adhesion to HUVECs, cell migration and invasion, suggesting that SLe(x) played a crucial role in the metastatic potential of H7721 cells. The upregulation of cell surface SLe(x) and alpha-1,3-fucosyltransferase-VII (alpha-1,3 Fuc T-VII, enzyme for SLe(x) synthesis) was also mediated by PI3K and PKB, since LY294002 and AS-PKB also reduced the expressions of SLe(x) and alpha-1,3 FucT-VII, and attenuated the response to insulin. Furthermore, the alterations in the expressions of PKB protein and activity were correlated to the changes of metastatic phenotypes and SLe(x) expression. Taken together, the insulin/PKB signalling pathway participated in the enhancement of metastatic potential of H7721 cells, which was mediated by the upregulation of the expression of SLe(x) and alpha-1,3 FucT-VII.

Receptor induction and targeted drug delivery: a new antileukaemia strategy.

Strategic modalities of drug delivery have the potential to greatly improve the therapeutic efficacy of available drugs in acute myelogenous leukaemia (AML). Folate receptor (FR) type beta is selectively expressed on the surface of approximately 70% of AMLs. Increased FR-beta expression in these cells can be induced by all-trans retinoic acid (ATRA) and other retinoid compounds in the absence of terminal differentiation or cell growth inhibition. An apparent post-transcriptional modification prevents FR-beta in normal haematopoietic cells from binding folate, in contrast to AML cells. FR-beta may, therefore, be used as a target for the selective delivery of chemotherapeutic drugs to AML cells; this treatment modality appears to be particularly efficacious when administered in conjunction with retinoid-induction of FR-beta. FR-targeted liposomal drug delivery can also bypass the P-glycoprotein (P-gp)-mediated drug efflux pump commonly associated with multiple drug resistance in AML. The rationale and merits of this novel experimental treatment for AML and the current status of this research are provided.

Modulation of the folate receptor type beta gene by coordinate actions of retinoic acid receptors at activator Sp1/ets and repressor AP-1 sites.

Folate receptor (FR) type beta is a promising target for therapeutic intervention in acute myelogenous leukemia (AML) owing particularly to its specific up-regulation in AML cells by all-trans retinoic acid (ATRA). Here we identify functional elements in the FR-beta gene and examine the molecular mechanism of transcriptional induction of FR-beta by ATRA. The basal promoter activity of FR-beta resulted from synergistic interaction between Sp1 and ets binding sites (EBSs) and repression by upstream AP-1-like elements, whose action required EBSs. A minimal promoter containing the Sp1 and ets elements was ATRA-responsive. The repressor elements bound Fos family proteins; association of the proteins with the repressor elements correlated negatively with FR-beta expression in peripheral blood neutrophils and monocytes and also in KG-1 (AML) cells grown in the absence or in the presence of ATRA. Furthermore, down-regulation of FR-beta in KG-1 cells treated with O-tetradecanoylphorbol 13-acetate (TPA) was accompanied by increased AP-1 binding to the repressor elements. From chromatin immunoprecipitation (ChIP) assays, the nuclear retinoic acid receptor alpha (RARalpha) associated with the Sp1 region, and RARs beta and gamma associated with the AP-1 and Sp1 regions; treatment of KG-1 cells with ATRA did not alter Sp1 binding but increased the association of RARalpha and decreased the association of RARs beta and gamma. ATRA also decreased RAR expression levels. The results suggest that the FR-beta gene is a target for multiple coordinate actions of nuclear receptors for ATRA directly and indirectly acting on a transcriptional complex containing activating Sp1/ets and inhibitory AP-1 proteins. The multiple mechanisms favor the prediction that ATRA will induce FR-beta expression in a broad spectrum of AML cells. Further, optimal FR-beta induction may be expected when all 3 RAR subtypes bind agonist.