Insulin-like growth factors modulate the growth inhibitory effects of retinoic acid on MCF-7 breast cancer cells

Retinoids are currently being tested for the treatment and prevention of several human cancers, including breast cancer. However, the anti-cancer and growth inhibitory mechanisms of retinoids are not well understood. All-trans retinoic acid (RA) inhibits the growth of the estrogen receptor-positive (ER+) breast cancer cell line, MCF-7, in a reversible and dose-dependent manner. In contrast, insulin-like growth factors (IGF-I,IGF-II) and insulin are potent stimulators of the proliferation of MCF-7 and several other breast cancer cell lines. Pharmacologic doses of RA (≤10^?6M) completely inhibit IGF-I-stimulated MCF-7 cell growth. Published data suggest that the growth inhibitory action of RA on IGF-stimulated cell growth is linear and dose-dependent, similar to RA inhibition of unstimulated or estradiol-stimulated MCF-7 cell growth. Surprisingly, we have found that IGF-I or insulin-stimulated cell growth is increased to a maximum of 132% and 127%, respectively, by cotreatment with 10^?7 M RA, and that 10^?9–10^?7 M RA increase cell proliferation compared to IGF-I or insulin alone. MCF-7 cells that stably overexpress IGF-II are also resistant to the growth inhibitory effects of 10^?9–10^?7 M RA. Treatment with the IGF-I receptor blocking antibody, αIR-3, restores RA-induced growth inhibition of IGF-I-treated or IGF-II-overexpressing MCF-7 cells, indicating that the IGF-I receptor is mediating these effects. IGFs cannot reverse all RA effects since the altered cell culture morphology of RA-treated cells is similar in growth-inhibited cultures and in IGF-II expressing clones that are resistant to RA-induced growth inhibition. These results indicate that RA action on MCF-7 cells is biphasic in the presence of IGF-I or insulin with 10^?9–10^?7 M RA enhancing cell proliferation and ≥ 10^?6M RA causing growth inhibition. As IGF-I and IGF-II ligands are frequently detectable in breast tumor tissues, their potential for modulation of RA effects should be considered when evaluating retinoids for use in in vivo experimental studies and for clinical purposes. Additionally, the therapeutic use of inhibitors of IGF action in combination with RA is suggested by these studies. © 1995 Wiley-Liss Inc.     

Differential binding to soluble nuclear receptors and effects on cell viability of retinol and retinoic acid in cultured retinoblastoma cells

Human retinoblastoma cells in culture contain soluble receptors for both ³H-retinol and ³H-retinoic acid which are separate and distinct as assessed by specificity, enzyme susceptibility and binding affinity. Total cellular binding of retinol correlates well with its rapid effect on cell mortality. A limited number of soluble nuclear receptor sites for ³H-retinoic acid but not ³H-retinol are observed after incubation of the ³H-retinoid with intact cells indicating a possible preferential effect of retinoic acid at the gene level.     

Structure-based design of a novel synthetic spiroketal pyran as a pharmacophore for the marine natural product spongistatin 1

SPIKET-P, a novel synthetic spiroketal pyran, was rationally designed as a pharmocophore for the tubulin depolymerizing marine natural product Spongistatin 1. SPIKET-P was prepared from the commercially available benzyl (R)-(-)-glycidyl ether using a versatile 11-step synthetic scheme in a stereocontrolled fashion. At nanomolar concentrations, SPIKET-P caused tubulin depolymerization in cell-free turbidity assays and exhibited potent cytotoxic activity against cancer cells as evidenced by destruction of microtubule organization, and prevention of mitotic spindle formation in human breast cancer cells.     

Structure basis of bigelovin as a selective RXR agonist with a distinct binding mode

The nuclear receptor retinoid X receptor (RXR) functions potently in the regulation of homeostasis and cell development, while rexinoids as RXR agonists have proved their therapeutic potential in the treatment of metabolic diseases and cancer. Here, the natural product bigelovin was identified as a selective RXRα agonist. Interestingly, this compound could not transactivate RXRα:RXRα homodimer but could enhance the transactivation of RXRα:peroxisome proliferator-activated receptor γ heterodimer and repress that of RXRα:liver X receptor (LXR) α heterodimer, while it had no effects on RXRα:farnesoid X receptor heterodimer. Considering that the effective role of LXR response element involved transactivation of sterol regulatory element-binding protein-1c mediated by RXRα:LXRα in triglyceride elevation, such LXR response element repressing by bigelovin has obviously addressed its potency for further research. Moreover, our determined crystal structure of the bigelovin-activated RXRα ligand-binding domain with the coactivator human steroid receptor coactivator-1 peptide revealed that bigelovin adopted a distinct binding mode. Compared with the known RXR ligands, bigelovin lacks the acidic moiety in structure, which indicated that the acidic moiety rendered little effects on RXR activation. Our results have thereby provided new insights into the structure-based selective rexinoids design with bigelovin as a potential lead compound.     

Purification and partial characterization of a novel binding protein for retinoic acid from neonatal rat

This report describes the purification and partial characterization of a novel retinoic acid-binding protein (CRABP-II) from neonatal rat pups. The isolation procedure included gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-cellulose, and high performance liquid chromatography (HPLC) on a DEAE 5PW column. Two retinoic acid-binding peaks were resolved at the DEAE-cellulose step, with CRABP-I in the major peak and CRABP-II in the minor peak. Apparent homogeneity was achieved for both binding proteins after the HPLC step. CRABP-II consists of a single polypeptide, migrating with an apparent Mr of 15,000 in sodium dodecyl sulfate-polyacrylamide gel. It has an isoelectric point of 5.0. The dissociation constant for CRABP-II of retinoic acid was estimated to be 65 nM by fluorescence titration. Amino-terminal sequence analysis showed that CRABP-II has a distinct sequence, while the CRABP-I sequence is exactly identical to that of the rat testis CRABP. Despite the extensive sequence homology between CRABP-I and CRABP-II, antibodies directed against CRABP-I did not cross-react with CRABP-II.     

Anibamine,a natural product CCR5 antagonist,as a novel lead for the development of anti-prostate cancer agents

Accumulating evidence indicates that the chemokine receptor CCR5 and the chemokine CCL5 may be involved in the proliferation and metastasis of prostate cancer. Consequently, chemokine receptor CCR5 antagonists could potentially act as anti-prostate cancer agents. As the first natural product CCR5 antagonist, anibamine provides a novel chemical structural skeleton compared with other known antagonists identified through high-throughput screening. Our studies demonstrate that anibamine produces significant inhibition of prostate cancer cell proliferation at micromolar to submicromolar concentrations as well as suppressing adhesion and invasion of the highly metastatic M12 prostate cancer cell line. Preliminary in vivo studies indicate that anibamine also inhibits prostate tumor growth in mice. These findings indicate that anibamine may prove to be a novel lead compound for the development of prostate cancer therapeutic agents.     

1-Methylisoguanosine: An orally active marine natural product with skeletal muscle and cardiovascular effects

1-Methylisoguanosine is a marine natural product with skeletal muscle relaxant, hypothermic and cardiovascular effects following oral administration in mice and rats. The cardiovascular effects (hypotension associated with bradycardia) are similar to those of adenosine. Structure-activity studies with a series of 1-methylisoguanosine analogues suggest a reasonable correlation between ability of the compounds to stimulate adenosine-sensitive adenylate cyclase and effects on skeletal muscular and cardiovascular responses. Thus, it appears that 1-methylisoguanosine may function as a long-acting adenosine analogue.     

The crystal structure of human geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement and inhibitory product binding

Modification of GTPases with isoprenoid molecules derived from geranylgeranyl pyrophosphate or farnesyl pyrophosphate is an essential requisite for cellular signaling pathways. The synthesis of these isoprenoids proceeds in mammals through the mevalonate pathway, and the final steps in the synthesis are catalyzed by the related enzymes farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase. Both enzymes play crucial roles in cell survival, and inhibition of farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates is an established concept in the treatment of bone disorders such as osteoporosis or certain forms of cancer in bone. Here we report the crystal structure of human geranylgeranyl pyrophosphate synthase, the first mammalian ortholog to have its x-ray structure determined. It reveals that three dimers join together to form a propeller-bladed hexameric molecule with a mass of approximately 200 kDa. Structure-based sequence alignments predict this quaternary structure to be restricted to mammalian and insect orthologs, whereas fungal, bacterial, archaeal, and plant forms exhibit the dimeric organization also observed in farnesyl pyrophosphate synthase. Geranylgeranyl pyrophosphate derived from heterologous bacterial expression is tightly bound in a cavity distinct from the chain elongation site described for farnesyl pyrophosphate synthase. The structure most likely represents an inhibitory complex, which is further corroborated by steady-state kinetics, suggesting a possible feedback mechanism for regulating enzyme activity. Structural comparisons between members of this enzyme class give deeper insights into conserved features important for catalysis.     

A novel binding site for a synthetic progestagen in breast cancer cells

A novel, high-affinity saturable binding site for the synthetic 19-nor testosterone progestagen, 17 alpha-ethinyl-13 beta-ethyl 17 beta-hydroxy-4,15-oestradiene-3-one (gestodene) has been detected using a sensitive affinity chromatography technique. This binding site is present in a range of malignant breast-derived cells lines, regardless of the presence of oestrogen and progesterone receptors, but is absent from endometrial carcinoma cells that contain both oestrogen and progesterone receptors. Competition studies show that this binding is not attributable to the receptors for the progestagens, androgens, glucocorticoids or mineralocorticoids. Cytosolic gestodene binding is refractory to competition with oestradiol but nuclear gestodene binding is completely abolished by oestradiol. The binding of oestradiol to the oestrogen receptor is reduced 40-50% by competition with gestodene. Non-dissociating polyacrylamide gel electrophoresis and size-exclusion high performance liquid chromatography reveal that this binding activity is associated with a protein of mean molecular mass 47 +/- 9 kDa. Ligand binding studies with a range of other cell lines indicates that this binding site appears to be specific to breast cancer cells. These data show the presence of a partly oestrogen competable novel binding protein in breast cancer cells which does not appear to be due to any of the conventional steroid receptors.     

Dynamics of cellular retinoic acid binding protein I on multiple time scales with implications for ligand binding

Cellular retinoic acid binding protein I (CRABPI) belongs to the family of intracellular lipid binding proteins (iLBPs), all of which bind a hydrophobic ligand within an internal cavity. The structures of several iLBPs reveal minimal structural differences between the apo (ligand-free) and holo (ligand-bound) forms, suggesting that dynamics must play an important role in the ligand recognition and binding processes. Here, a variety of nuclear magnetic resonance (NMR) spectroscopy methods were used to systematically study the dynamics of both apo and holo CRABPI at various time scales. Translational and rotational diffusion constant measurements were used to study the overall motions of the proteins. Both apo and holo forms of CRABPI tend to self-associate at high (1.2 mM) concentrations, while at low concentrations (0.2 mM), they are predominantly monomeric. Rapid amide exchange rate and laboratory frame relaxation rate measurements at two spectrometer field strengths (500 and 600 MHz) were used to probe the internal motions of the individual residues. Several residues in the apo form, notably within the ligand recognition region, exhibit millisecond time scale motions that are significantly arrested in the holo form. In contrast, no significant differences in the high-frequency motions were observed between the two forms. These results provide direct experimental evidence for dynamics-induced ligand recognition and binding at a specifically defined time scale. They also exemplify the importance of dynamics in providing a more comprehensive understanding of how a protein functions.     

Inhibition of mesothelin as a novel strategy for targeting cancer cells

Mesothelin, a differentiation antigen present in a series of malignancies such as mesothelioma, ovarian, lung and pancreatic cancer, has been studied as a marker for diagnosis and a target for immunotherapy. We, however, were interested in evaluating the effects of direct targeting of Mesothelin on the viability of cancer cells as the first step towards developing a novel therapeutic strategy. We report here that gene specific silencing for Mesothelin by distinct methods (siRNA and microRNA) decreased viability of cancer cells from different origins such as mesothelioma (H2373), ovarian cancer (Skov3 and Ovcar-5) and pancreatic cancer (Miapaca2 and Panc-1). Additionally, the invasiveness of cancer cells was also significantly decreased upon such treatment. We then investigated pro-oncogenic signaling characteristics of cells upon mesothelin-silencing which revealed a significant decrease in phospho-ERK1 and PI3K/AKT activity. The molecular mechanism of reduced invasiveness was connected to the reduced expression of β-Catenin, an important marker of EMT (epithelial-mesenchymal transition). Ero1, a protein involved in clearing unfolded proteins and a member of the ER-Stress (endoplasmic reticulum-stress) pathway was also markedly reduced. Furthermore, Mesothelin silencing caused a significant increase in fraction of cancer cells in S-phase. In next step, treatment of ovarian cancer cells (OVca429) with a lentivirus expressing anti-mesothelin microRNA resulted in significant loss of viability, invasiveness, and morphological alterations. Therefore, we propose the inhibition of Mesothelin as a potential novel strategy for targeting human malignancies.     

Differential capacity of wild type promoter elements for binding and trans-activation by retinoic acid and thyroid hormone receptors.

Retinoic acid receptor (RAR) and thyroid hormone receptor (T3R) are structurally similar and can bind as homodimers or T3R-RAR heterodimers to a single synthetic DNA response element. The interaction of these two types of receptors with wild type elements, however, has not been systematically investigated. Promoter elements from genes regulated by retinoic acid (RA) or thyroid hormone (T3) were tested for response to T3 and RA in transient transfections in both JEG and COS cells. The elements were classified as primarily responsive to RA or to T3 or responsive to both ligands. Binding of highly purified RAR alpha and T3R alpha to the various elements was assessed using the gel shift assay. Those elements predominantly responsive to one ligand showed preferential binding to the appropriate receptor. A series of point mutations were introduced into the rat GH T3 response element to further define sequence requirements for response to both RA and T3. Down-mutations in any of the three hexamers (previously demonstrated to be required for full response to T3 and full binding of T3R) also decreased RA induction and RAR binding. However, only one of two sets of up-mutations for T3 response also increased RA induction, demonstrating differences in hexamer preference between RAR and T3R. Variation in spacing of the three hexamers did not influence RA vs. T3 induction or RAR vs. T3R binding according to the predictions of a simple hexamer spacing model. There was a strong correlation between the extent of T3R dimer binding and strength of T3 induction for a subset of elements studied in JEG cells (r = 0.97, P < 0.01) and a weaker but significant correlation in COS cells (r = 0.65, P < 0.05)). In contrast, RAR dimer binding by the wild type elements did not quantitatively correlate with RA induction in either JEG (r = 0.13, P > 0.05) or COS cells (r = 0.21, P > 0.05). These results suggests that RAR interacts with a heterodimer partner(s) which influences binding site specificity, whereas T3R heterodimer partner(s) is less likely to alter binding site recognition. The observed difference in COS and JEG cells as well as the weak T3R binding-function relationship of the malic enzyme element, however, suggest that the influence of T3R heterodimer partner(s) on binding site specificity is likely to vary with cell type and the specific element tested.     

A novel binding assay for metabotropic glutamate receptors using [3H] L-quisqualic acid and recombinant receptors

We established a methodology to analyze radioligand binding to the recombinant type la metabotropic glutamate receptor (mGluRla). A full-length cDNA encoding mGluR1a, which was isolated from a lambda gt 11 cDNA library of human cerebellar origin, was expressed in a baculovirus/Sf9 insect cell system. Membrane fractions with recombinant receptor expression were analyzed for the binding of [3H]L-quisqualic acid (L-QA), which is known to be a potent agonist of mGluRla. Efficient binding of the radioligand to the human receptor was observed in a saturable manner, giving an apparent Kd= 0.091 microM. [3H]L-QA bound to the human mGluR1a was displaced by known ligands such as L-QA, L-Glu, t-ACPD ((+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid) with IC50s = 0.056, 0.97 and 4.0 microM, respectively. MCPG (alpha-methyl-4-carboxyphenylglycine) displaced the radioligand binding with lower potency. Using this binding protocol, we then evaluated the ligand ability of synthetic dipeptides. Among peptides tested, only Glu-containing dipeptides inhibited the radioligand binding, e.g. IC50 of L-Met-L-Glu was 4.3 microM. When phosphatidyl inositol turnover was assayed in mGluR1a-expressing CHO cells, L-Met-L-Glu was partially agonistic. We further expanded this [3H]L-QA binding protocol to type 5a mGluR, another member of group I mGluRs, as well as to AMPA receptor, a member of ionotropic glutamate receptors, since L-QA is also known to be a potent ligand for these receptors. Data shown here will provide a novel system not only to search for ligands for the glutamate receptors, but also to biochemically analyze the interaction modes between glutamate receptors and their ligands.     

Stereospecific effects of ascorbic acid and analogues on D1 and D2 agonist binding.

Ascorbic acid inhibited the specific binding of both the D1 agonist, [3H] SKF 38393, and the D2 agonist, [3H] N-0437 at physiologically relevant concentrations. This inhibition was both stereospecific and receptor selective. Using ligand concentrations approximating their KD's, the IC50's for ascorbate and two structural analogues, isoascorbate and D-glucoascorbate, were determined. The rank order of IC50's at both D1 and D2 were D-glucoascorbate greater than isoascorbate greater than ascorbate. However, the IC50 for each compound was greater at D1 than D2. Evaluation of the relationship between the IC50 for ascorbate and the ligand concentration using both the D1 and the D2 ligand yielded data inconsistent with competitive inhibition models. Preliminary experiments were conducted to evaluate the site and type of inhibition with results consistent with an allostearic effect at the level of the receptor.