guanosine-5--o-(3-thiotriphosphate) and Breast-Neoplasms

Tamoxifen (Tam) is a selective estrogen receptor (ER) modulator (SERM) that is an essential drug to treat ER-positive breast cancer. Aside from known actions at ERs, recent studies have suggested that some SERMs like Tam also exhibit novel activity at cannabinoid subtype 1 and 2 receptors (CB1R and CB2Rs). Interestingly, cis- (E-Tam) and trans- (Z-Tam) isomers of Tam exhibit over a 100-fold difference in affinity for ERs. Therefore, the current study assessed individual isomers of Tam and subsequent cytochrome P450 metabolic products, 4-hydroxytamoxifen (4OHT) and 4-hydroxy-N-desmethyl tamoxifen (End) for affinity and activity at CBRs. Results showed that Z-4OHT, but not Z-Tam or Z-End, exhibits higher affinity for both CB1 and CB2Rs relative to the E-isomer. Furthermore, Z- and E-isomers of Tam and 4OHT show slightly higher affinity for CB2Rs, while both End isomers are relatively CB1R-selective. When functional activity was assessed by G-protein activation and regulation of the downstream effector adenylyl cyclase, all isomers examined act as full CB1 and CB2R inverse agonists. Interestingly, Z-Tam appears to be more efficacious than the full inverse agonist AM630 at CB2Rs, while both Z-Tam and Z-End exhibit characteristics of insurmountable antagonism at CB1 and CB2Rs, respectively. Collectively, these results suggest that the SERMs Tam, 4OHT and End elicit ER-independent actions via CBRs in an isomer-specific manner. As such, this novel structural scaffold might be used to develop therapeutically useful drugs for treatment of a variety of diseases mediated via CBRs.

Topics: Adenylyl Cyclases; Animals; Binding, Competitive; Breast Neoplasms; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; CHO Cells; Colforsin; Cricetinae; Cricetulus; Cyclic AMP; Cyclohexanols; Female; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Indoles; Isomerism; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Selective Estrogen Receptor Modulators; Tamoxifen

The functional characteristics of membrane progesterone receptors (mPRs) have been investigated using recombinant mPR proteins over-expressed in MDA-MB-231 breast cancer cells. Although these cells do not express the full-length progesterone receptor (PR), it is not known whether they express N-terminally truncated PR isoforms which could possibly account for some progesterone receptor functions attributed to mPRs. In the present study, the presence of N-terminally truncated PR isoforms was investigated in untransfected and mPR-transfected MDA-MB-231 cells, and in MDA-MB-468 breast cancer cells. PCR products were detected in PR-positive T47D Yb breast cancer cells using two sets of C-terminus PR primers, but not in untransfected and mPR-transfected MDA-MB-231 cells, nor in MDA-MB-468 cells. Western blot analysis using a C-terminal PR antibody, 2C11F1, showed the same distribution pattern for PR in these cell lines. Another C-terminal PR antibody, C-19, detected immunoreactive bands in all the cell lines, but also recognized α-actinin, indicating that the antibody is not specific for PR. High affinity progesterone receptor binding was identified on plasma membranes of MDA-MB-468 cells which was significantly decreased after treatment with siRNAs for mPRα and mPRβ. Plasma membranes of MDA-MB-468 cells showed very low binding affinity for the PR agonist, R5020, ≤1% that of progesterone, which is characteristic of mPRs. Progesterone treatment caused G protein activation and decreased production of cAMP in MDA-MB-468 cells, which is also characteristic of mPRs. The results indicate that the progestin receptor functions in these cell lines are mediated through mPRs and do not involve any N-terminally truncated PR isoforms.

Topics: Adenylyl Cyclases; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cyclic AMP; Enzyme Assays; Female; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Peptide Fragments; Progesterone; Protein Isoforms; Protein Structure, Tertiary; Receptors, Progesterone; Recombinant Proteins; RNA Interference; Transcription, Genetic

Increased circulating levels of L-alpha-lysophosphatidylinositol (LPI) are associated with cancer and LPI is a potent, ligand for the G-protein-coupled receptor GPR55. Here we have assessed the modulation of breast cancer cell migration, orientation and polarization by LPI and GPR55.. Quantitative RT-PCR was used to measure GPR55 expression in breast cancer cell lines. Cell migration and invasion were measured using a Boyden chamber chemotaxis assay and Cultrex invasion assay, respectively. Cell polarization and orientation in response to the microenvironment were measured using slides containing nanometric grooves.. GPR55 expression was detected in the highly metastatic MDA-MB-231 breast cancer cell line. In these cells, LPI stimulated binding of [(35)S]GTPgammaS to cell membranes (pEC(50) 6.47 +/- 0.45) and significantly enhanced cell chemotaxis towards serum. MCF-7 cells expressed low levels of GPR55 and did not migrate or invade towards serum factors. When GPR55 was over-expressed in MCF-7 cells, serum induced a robust migratory and invasive response, which was further enhanced by LPI and prevented by siRNA to GPR55. The physical microenvironment has been identified as a key factor in determining breast tumour cell metastatic fate. LPI endowed MDA-MB-231 cells with the capacity to detect shallow (40 nm deep) grooved slides and induced marked cancer cell polarization on both flat and grooved surfaces.. LPI and GPR55 play a role in the modulation of migration, orientation and polarization of breast cancer cells in response to the tumour microenvironment.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Polarity; Chemotaxis; Female; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Lysophospholipids; Neoplasm Invasiveness; Neoplasm Metastasis; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; RNA, Small Interfering

Inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in the de novo synthesis of guanine nucleotides, is a major therapeutic target. A prototypic uncompetitive inhibitor of IMPDH, mycophenolic acid (MPA), is the active form of mycophenolate mofeteil (CellCept), a widely used immunosuppressive drug. We have found that MPA interacts with intracellular IMPDH in vivo to alter its mobility on SDS-polyacrylamide gels. MPA also induces a striking conformational change in IMPDH protein in intact cells, resulting in the formation of annular aggregates of protein with concomitant inhibition of IMPDH activity. These aggregates are not associated with any known intracellular organelles and are reversible by incubating cells with guanosine, which repletes intracellular GTP, or with GTPgammaS. GTP also restores IMPDH activity. Treatment of highly purified IMPDH with MPA also results in the formation of large aggregates of protein, a process that is both prevented and reversed by the addition of GTP. Finally, GTP binds to IMPDH at physiologic concentrations, induces the formation of linear arrays of tetrameric protein, and prevents the aggregation of protein induced by MPA. We conclude that intracellular GTP acts as an antagonist to MPA by directly binding to IMPDH and reversing the conformational changes in the protein.

Topics: Breast Neoplasms; Cell Line, Tumor; Drug Interactions; Enzyme Activation; Enzyme Inhibitors; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; IMP Dehydrogenase; Mycophenolic Acid; Protein Conformation

The WTH3 gene's biological characteristics and relationship to multidrug resistance (MDR) were investigated further. Results showed that WTH3 was mainly located in the cytosol and capable of binding to GTP. In addition, WTH3's promoter function was significantly attenuated in MDR (MFC7/AdrR) relative to non-MDR (MCF7/WT) cells. Advanced analyses indicated that two mechanisms could be involved in WTH3's down-regulation: DNA methylation and trans-element modulations. It was found that the 5' end portion of a CpG island in WTH3's promoter was hypermethylated in MCF7/AdrR but not MCF7/WT cells, which could have a negative effect on the WTH3 promoter. This idea was supported by the observation that a 45-bp sequence (DMR45) in this differentially methylated region positively influenced promoter activity. We also discovered that different nuclear proteins in MCF7/AdrR and MCF7/WT cells bound to methylated or nonmethylated DMR45. Moreover, a sequence containing a unique repeat that was also a positive cis-element for the promoter was attached by different transcription factors depending on whether they were prepared from MCF7/AdrR or MCF7/WT cells. These molecular changes, apparently induced by drug treatment, resulted in WTH3's down regulation in MDR cells. Therefore, present studies support previous observations that WTH3, as a negative regulator, participates in MDR development in MCF7/AdrR cells.

Topics: Base Sequence; Breast Neoplasms; Cell Line, Tumor; DNA Methylation; Drug Resistance, Multiple; Gene Expression Regulation, Neoplastic; Glutathione Transferase; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); HeLa Cells; Humans; Molecular Sequence Data; Promoter Regions, Genetic; rab GTP-Binding Proteins; Recombinant Fusion Proteins; Transfection; Untranslated Regions

In this study we have examined CD44 (a hyaluronan (HA) receptor) interaction with a RhoA-specific guanine nucleotide exchange factor (p115RhoGEF) in human metastatic breast tumor cells (MDA-MB-231 cell line). Immunoprecipitation and immunoblot analyses indicate that both CD44 and p115RhoGEF are expressed in MDA-MB-231 cells and that these two proteins are physically associated as a complex in vivo. The binding of HA to MDA-MB-231 cells stimulates p115RhoGEF-mediated RhoA signaling and Rho kinase (ROK) activity, which, in turn, increases serine/threonine phosphorylation of the adaptor protein, Gab-1 (Grb2-associated binder-1). Phosphorylated Gab-1 promotes PI 3-kinase recruitment to CD44v3. Subsequently, PI 3-kinase is activated (in particular, alpha, beta, gamma forms but not the delta form of the p110 catalytic subunit), AKT signaling occurs, the cytokine (macrophage-colony stimulating factor (M-CSF)) is produced, and tumor cell-specific phenotypes (e.g. tumor cell growth, survival and invasion) are up-regulated. Our results also demonstrate that HA/CD44-mediated oncogenic events (e.g. AKT activation, M-CSF production and breast tumor cell-specific phenotypes) can be effectively blocked by a PI 3-kinase inhibitor (LY294002). Finally, we have found that overexpression of a dominant-negative form of ROK (by transfection of MBA-MD-231 cells with the Rho-binding domain cDNA of ROK) not only inhibits HA/CD44-mediated RhoA-ROK activation and Gab-1 phosphorylation but also down-regulates oncogenic signaling events (e.g. Gab-1.PI 3-kinase-CD44v3 association, PI 3-kinase-mediated AKT activation, and M-CSF production) and tumor cell behaviors (e.g. cell growth, survival, and invasion). Taken together, these findings strongly suggest that CD44 interaction with p115RhoGEF and ROK plays a pivotal role in promoting Gab-1 phosphorylation leading to Gab-1.PI 3-kinase membrane localization, AKT signaling, and cytokine (M-CSF) production during HA-mediated breast cancer progression.

Topics: Adaptor Proteins, Signal Transducing; Adenosine Triphosphate; Adjuvants, Immunologic; Animals; Breast Neoplasms; Cell Division; Cell Survival; Cloning, Molecular; COS Cells; Cytoplasm; Disease Progression; DNA, Complementary; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Escherichia coli; Genetic Vectors; GRB2 Adaptor Protein; Green Fluorescent Proteins; Guanine Nucleotide Exchange Factors; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hyaluronan Receptors; Hyaluronic Acid; Immunoblotting; Intracellular Signaling Peptides and Proteins; Kinetics; Luminescent Proteins; Macrophage Colony-Stimulating Factor; Models, Biological; Neoplasm Invasiveness; Phenotype; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Precipitin Tests; Protein Binding; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Proteins; Rho Guanine Nucleotide Exchange Factors; rho-Associated Kinases; Signal Transduction; Time Factors; Transfection; Tumor Cells, Cultured; Up-Regulation

The anti-proliferative activity of the somatostatin analog RC-160 is limited by its short serum half life. To circumvent this limitation, fatty acids of chain lengths ranging from 4 to 18 were individually conjugated to the N-terminal residue of RC-160. The lipophilized derivatives of RC-160 were synthesized, purified and characterized. The anti-proliferative activity of lipophilized-RC-160 on the human breast carcinoma cell line MCF-7, was evaluated in vitro. The long chain lipopeptides like pamitoyl-RC-160 exhibited significantly higher anti-proliferative activity on MCF-7 cells (p<0.001), relative to RC-160. The affinity of RC-160 towards somatostatin receptors remained unaltered by pamitoylation. However, the observed increase in bioactivity was manifested within an optimum range of chain length of the lipoppetide. Increasing the peptide hydrophobicity beyond this range reduced the bioactivity of lipophilized-RC-160. Accordingly, stearoyl-RC-160, manifested lower anti-neoplastic activity and receptor affinity relative to pamitoyl-RC-160 and RC-160 itself. The signaling pathways underlying the antineoplastic activity of these lipopeptides were found to be similar to RC-160. Pamitoyl-RC-160 displayed enhanced inhibition of protein tyrosine kinase activity and intracellular cAMP levels in MCF-7 cells, relative to butanoyl-RC-160 or RC-160 itself. Pamitoyl-RC-160 also displayed greater resistance towards trypsin and serum degradation than RC-160. Lipophilization of RC-160 with long chain fatty acids like pamitic acid improves its stability and anti-proliferative activity, thereby improving the scope of enhancing its therapeutic index. However, the optimization of peptide hydrophobicity seems to be a crucial factor governing the efficacy of bioactive lipopeptides.

Topics: Acylation; Adenocarcinoma; Antineoplastic Agents, Hormonal; Binding, Competitive; Breast Neoplasms; Cell Division; Cyclic AMP; Endopeptidases; Enzyme Inhibitors; Fatty Acids; Female; Freeze Drying; Guanosine 5'-O-(3-Thiotriphosphate); Half-Life; Hormone Antagonists; Humans; Protein-Tyrosine Kinases; Signal Transduction; Somatostatin; Structure-Activity Relationship; Tumor Cells, Cultured

Topics: Breast Neoplasms; Female; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Intracellular Fluid; Neoplasm Invasiveness; Phosphatidic Acids; Phospholipase D; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Several G-proteins (GTP-binding proteins) were identified by SDS/PAGE in the cytosol (105,000 g supernatant) and membrane fractions of the oestrogen-dependent human mammary-tumour cell line ZR-75-1. These proteins, with molecular masses in the range 18-29 kDa, specifically bind [alpha-32P]GTP, which can be displaced by unlabelled GTP, GDP and their non-hydrolysable analogues guanosine 5'-[delta-thio]triphosphate (GTP[S]) and guanosine 5'-[beta-thio]diphosphate (GDP[S]), but not by GMP, ATP, ADP, AMP and other unrelated nucleotides. The apparent dissociation constant for GTP was approx. 2 x 10(-8)M. Homogenization of ZR-75-1 cells in high-salt buffer (1 M-KCl), and successive washing of the membrane fraction, suggested that, among the major G-proteins found, the 18 kDa protein is predominantly soluble, whereas the 27-29 kDa complex is primarily bound to the membrane fraction under the experimental conditions employed. Possible translocation of these G-proteins between membrane and cytosol was analysed. No redistribution of the 27-29 kDa complex was observed, whereas GTP[S] in the presence of Mg2+ caused apparent translocation of the 18 kDa protein to the membrane fraction. This effect was specific for GTP and stable GTP analogues, whereas GDP, GMP, ATP, ADP, AMP and other unrelated nucleotides were ineffective. GTP[S] and guanosine 5'-[beta gamma-imido]-triphosphate (p[NH]ppG) were equally potent (apparent Kd approximately 5 x 10(-6)M), whereas GTP was rather weak. The nucleotide effect is temperature-, time- and concentration-dependent. The translocation process was reversible, slow, and reached its maximum between 30 and 60 min at 37 degrees C. The apparent translocation of this small G-protein from the cytosol to the membrane fraction, and the specific effect of GTP analogues, suggest that this process may have functional significance in mammary-tumour cells.

Topics: Biological Transport; Breast Neoplasms; Cell Membrane; Cytosol; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Kinetics; Magnesium; Molecular Weight; Temperature; Thionucleotides; Tumor Cells, Cultured