mycophenolic-acid and Neuroblastoma

Topics: Amino Acid Sequence; Animals; Cell Line; Drug Resistance; Humans; IMP Dehydrogenase; Kinetics; Mice; Molecular Sequence Data; Mycophenolic Acid; Neuroblastoma; Sequence Homology, Amino Acid; Tumor Cells, Cultured

Tacrolimus (FK506)/mycophenolate mofetil (MMF) has been demonstrated to be an effective salvage therapy for steroid-resistant chronic graft-versus-host disease (GVHD), but its effectiveness as prophylaxis for acute GVHD (aGVHD) is unknown. We investigated the safety and efficacy of FK506/MMF in preventing aGVHD and sparing the use of methotrexate and methylprednisolone in childhood and adolescent allogeneic stem cell transplant (AlloSCT) recipients. Thirty-four childhood and adolescent patients (median age, 7 years; range, 0.5-21 years; 24 males and 10 females) undergoing 37 AlloSCTs for malignant (n = 22) and nonmalignant (n = 12) disorders received FK506 (0.03 mg/kg/d by continuous intravenous infusion) and MMF (15 mg/kg per dose orally or intravenously twice daily). Stem cell sources included 22 umbilical cord blood donors (21 unrelated and 1 related), 6 related bone marrow donors, and 9 related peripheral blood donors. Malignant diagnoses included 7 acute lymphoblastic leukemias, 3 acute myeloid leukemias, 1 acute promyelocytic leukemia, 2 non-Hodgkin lymphomas, 4 Hodgkin diseases, 3 chronic myeloid leukemias, and 2 neuroblastomas; nonmalignant diagnoses included 2 beta-thalassemias, 1 sickle cell disease, 4 aplastic anemias, 1 Wiskott-Aldrich syndrome, 1 Hurler syndrome, 2 hemophagocytic lymphohistiocytoses, and 1 myelodysplastic syndrome. The probability of developing grade > or =II aGVHD was 45.4% +/- 9.7% (7 related bone marrow/related peripheral blood; 5 umbilical cord blood), and for chronic GVHD it was 38.1% +/- 19.7%. FK506/MMF was well tolerated. Three patients had grade III to IV neurotoxicity (disorientation and leukoencephalopathy); 4 patients developed grade III to IV nephrotoxicity (all received concomitant nephrotoxins). Patients who achieved target mycophenolic acid levels (1.0-3.5 microg/mL) before day +30 had a significantly reduced incidence of developing grade >/=II aGVHD (16.7% +/- 15.2% versus 100%; P <.02). These results suggest that FK506/MMF is well tolerated and may be a safe and effective methotrexate- and methylprednisolone-sparing alternative GVHD prophylaxis regimen after AlloSCT. Further pharmacokinetic and pharmacodynamic studies are ongoing in pediatric and adolescent AlloSCT recipients to define optimal MMF dosing.

Topics: Adolescent; Adult; Child; Child, Preschool; Drug Resistance; Drug Therapy, Combination; Female; Graft vs Host Disease; Hematologic Diseases; Humans; Immunosuppressive Agents; Infant; Male; Mucopolysaccharidosis I; Mycophenolic Acid; Neuroblastoma; Pilot Projects; Prognosis; Stem Cell Transplantation; Tacrolimus; Transplantation Conditioning; Transplantation, Homologous; Treatment Outcome

Twenty six propargylamine mycophenolate analogues were designed and synthesized from mycophenolic acid 1 employing a key step A

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Molecular Docking Simulation; Molecular Structure; Mycophenolic Acid; Neuroblastoma; Pargyline; Propylamines; Structure-Activity Relationship

Lesch-Nyhan disease (LND), caused by complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT), is characterized by a neurological deficit, the etiology of which is unknown. Evidence has accumulated indicating that it might be related to dysfunction of the basal ganglia with a prominent loss of striatal dopamine fibers. Guanine nucleotide depletion has been shown to occur in cells from Lesch-Nyhan patients. In this study we demonstrate that chronic guanine nucleotide depletion induced by inhibition of inosine monophosphate dehydrogenase with low levels (50 nM) of mycophenolic acid (MPA) lead human neuroblastoma cell lines to differentiate toward the neuronal phenotype. The MPA-induced morphological changes were more evident in the dopaminergic line LAN5, than in the cholinergic line IMR32. MPA-induced differentiation, unlike that induced by retinoic acid, caused a less extensive neurite outgrowth and branching (similar to that observed in cultured HPRT-deficient dopaminergic neurons) and involved up-regulation of p53, p21 and bax, and bcl-2 down-regulation without p27 protein accumulation. These results suggest that guanine nucleotide depletion following HPRT deficiency, might lead to earlier and abnormal brain development mainly affecting the basal ganglia, displaying the highest HPRT activity, and could be responsible for the specific neurobehavioral features of LND.

Topics: Basal Ganglia; Basal Ganglia Diseases; Cell Cycle Proteins; Cell Differentiation; Cell Enlargement; Cell Line; Enzyme Inhibitors; Guanine Nucleotides; Humans; Hypoxanthine Phosphoribosyltransferase; IMP Dehydrogenase; Lesch-Nyhan Syndrome; Models, Neurological; Mycophenolic Acid; Neurites; Neuroblastoma; Neurofilament Proteins; Tretinoin; Up-Regulation

Mycophenolic acid (MPA) specifically inhibits inosine-5'-monophosphate dehydrogenase, the first committed step toward GMP biosynthesis. In its morpholinoethyl ester pro-drug form it is one of the most promising immunosuppressive drugs recently developed. The aim of the present study was to investigate the in vitro effects of MPA, at concentrations readily attainable during immunosuppressive therapy, on 3 human neuroblastoma cell lines (LAN5, SHEP and IMR32). Mycophenolic acid (0.1-10 microM) caused a decrease of intracellular levels of guanine nucleotides, a G(1) arrest and a time- and dose-dependent death by apoptosis. These effects, associated with an up-regulation of p53, p21 and bax, a shuttling of p53 protein into the nucleus and a down-regulation of bcl-2, survivin and p27 protein, were reversed by the simultaneous addition of guanine or guanosine and were more evident using nondialysed serum containing hypoxanthine. These results suggest that in neuroblastoma cell lines clinically attainable concentrations of mycophenolic acid deplete guanine nucleotide pools triggering G(1) arrest and apoptosis through p53-mediated pathways, indicating a potential role of its morpholinoethyl ester pro-drug in the management of patients with neuroectodermal tumors.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Nucleus; Dose-Response Relationship, Drug; Down-Regulation; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Hypoxanthine; Immunohistochemistry; Immunosuppressive Agents; Inhibitor of Apoptosis Proteins; Microfilament Proteins; Microtubule-Associated Proteins; Muscle Proteins; Mycophenolic Acid; Neoplasm Proteins; Neuroblastoma; Prodrugs; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins p21(ras); Reverse Transcriptase Polymerase Chain Reaction; Survivin; Time Factors; Tumor Suppressor Protein p53

Topics: Antineoplastic Agents; Cell Cycle; Cell Differentiation; Dose-Response Relationship, Drug; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Mycophenolic Acid; Neuroblastoma; Tumor Cells, Cultured

In human neuroblastoma cell lines (LAN5, SHEP and IMR32), mycophenolic acid (MPA) at concentrations (10(-7)-10(-6) M) readily attainable during immunosuppressive therapy with mycophenolate mofetil (Cellcept), induces guanine nucleotide depletion leading to cell cycle arrest and apoptosis through a p53 mediated pathway (up-regulation of p53, p21 and bax and down-regulation of bcl-2 and survivin). MPA-induced apoptosis is also associated to a marked decrease of p27 protein. In the same cell lines MPA, at lower concentrations (50 nM), corresponding to the plasma levels of the active free drug during Cellcept therapy, induces differentiation toward the neuronal phenotype by causing a partial chronic guanine nucleotide depletion. MPA-induced differentiation is not associated to p27 accumulation as occurs using retinoic acid. At a fixed concentration of MPA a higher percentage of apoptotic or differentiated cells is obtained when non dialysed serum substitutes for the dialysed one, due to the higher hypoxanthine concentration in the former (about 10 microM) leading to competition on HPRT-mediated salvage of guanine. At hypoxanthine or oxypurinol concentrations higher than 1 microM (up to 100 microM) no further enhancement of MPA effects was obtained, in agreement with the recently described safety of the allopurinol-mycophenolate mofetil combination in the treatment of hyperuricemia of kidney transplant recipients. The apoptotic effects of MPA do not appear to be significantly increased by the UDP-glucuronosyltransferase inhibitor niflumic acid.

Topics: Allopurinol; Antineoplastic Agents; Apoptosis; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Guanine; Humans; Hypoxanthine; Hypoxanthine Phosphoribosyltransferase; Immunosuppressive Agents; Mycophenolic Acid; Neuroblastoma; Niflumic Acid; Phenotype

Topics: Cell Differentiation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Hypoxanthine Phosphoribosyltransferase; IMP Dehydrogenase; Kinetics; Mycophenolic Acid; Neuroblastoma; Nucleotides; Phosphoric Diester Hydrolases; Tumor Cells, Cultured

Topics: Biopterins; Cell Differentiation; Guanosine Triphosphate; Humans; Mycophenolic Acid; Neuroblastoma; Tumor Cells, Cultured

Topics: Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Cell Division; Gene Expression Regulation, Neoplastic; Guanosine; Humans; Mycophenolic Acid; Neuroblastoma; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Incubation of SH-SY5Y neural cells with mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (the key enzyme in purine nucleotide biosynthesis), reduced the cellular content of GTP by 94% relative to its concentration in control cells (43 nmol/mg protein) without altering the level of GDP. Although in GTP-depleted intact cells the receptor binding parameters (Kd and Bmax) of the opioid antagonist [3H]naltrexone were unchanged from those in untreated cells, the binding affinity of the mu-selective opioid agonist [3H]Tyr-D-Ala-Gly-(Me)- Phe-Gly-ol ([3H]DAMGO) was enhanced 2-fold. Furthermore, the kinetics of ligand/receptor interaction revealed that in the nucleotide-depleted cells, the dissociation rate constant for [3H]DAMGO was reduced by 44%. Initial exposure of SH-SY5Y cells to pertussis toxin reduced high-affinity ligand binding by 95% and abolished the effect of MPA treatment. Renewed incubation of the GTP-depleted cells with guanosine restored the original GTP levels and agonist binding. Neither MPA nor guanosine treatment changed the Bmax of [3H]DAMGO binding. Forskolin- and prostaglandin E1-stimulated adenylyl cyclase activities were decreased significantly in GTP-depleted cells. DAMGO and [D-Pen2,D-Pen5]enkephalin inhibitions of adenylyl cyclase were also affected with MPA treatment. Maximal inhibition of forskolin-stimulated adenylyl cyclase activity by both of the agonists was reduced, whereas MPA caused a 2-fold reduction in potency for DAMGO. The results show that reduction in endogenous GTP levels leads to noticeable changes in agonist, receptor, and G protein interactions, as measured by agonist binding, and to subsequent diminution of the signal transduction, as reflected by the cAMP levels.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Analgesics; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Kinetics; Molecular Sequence Data; Mycophenolic Acid; Naltrexone; Narcotic Antagonists; Neuroblastoma; Neurons; Receptors, Opioid, mu; Tritium; Tumor Cells, Cultured

Topics: Alcohol Oxidoreductases; Animals; Biopterins; Cell Line; Dihydropteridine Reductase; GTP Cyclohydrolase; Guanosine Triphosphate; Kinetics; Mice; Mycophenolic Acid; Neuroblastoma; Phosphorus-Oxygen Lyases; Tumor Cells, Cultured

GTP cyclohydrolase I exhibits a positive homotropic cooperative binding to GTP, which raises the possibility of a role for GTP in regulating the enzyme reaction (Hatakeyama, K., Harada, T., Suzuki, S., Watanabe, Y., and Kagamiyama, H. (1989) J. Biol. Chem. 264, 21660-21664). We examined whether or not the intracellular GTP level is within the range of affecting GTP cyclohydrolase I activity, using PC-12 rat pheochromocytoma and IMR-32 human neuroblastoma cells. Since GTP cyclohydrolase I was the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in these cell lines, the intracellular activities of this enzyme were reflected in the tetrahydrobiopterin contents. We found that the addition of guanine or guanosine increased GTP but not tetrahydrobiopterin in these cells. On the other hand, three IMP dehydrogenase inhibitors, tiazofurin, 2-amino-1,3,4-thiadiazole, and mycophenolic acid, decreased both GTP and tetrahydrobiopterin in a parallel and dose-dependent manner, and these effects were reversed by the simultaneous addition of guanine or guanosine. There was no evidence suggesting that these inhibitors inhibited other enzymes involved in the biosynthesis and regeneration of tetrahydrobiopterin. Comparing intracellular activities of GTP cyclohydrolase I in the inhibitor-treated cells with its substrate-velocity curve, we estimated that the intracellular concentration of free GTP is 150 microM at which point the activity of GTP cyclohydrolase I is elicited at its maximum velocity. Below this GTP concentration, GTP cyclohydrolase I activity is rapidly decreased. Therefore GTP can be a regulator for tetrahydrobiopterin biosynthesis.

Topics: Animals; Antineoplastic Agents; Biopterins; GTP Cyclohydrolase; Guanine; Guanosine; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Kinetics; Mycophenolic Acid; Neuroblastoma; PC12 Cells; Ribavirin; Thiadiazoles; Tumor Cells, Cultured

Mouse wild-type neuroblastoma cells (NB cells) were stepwise selected for 10,000-fold increased resistance to mycophenolic acid (NB-Myco cells), an inhibitor of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205). IMP dehydrogenase activity was increased 25-fold, from 3.1 to 75 nmol/min.mg of protein; and a 56.7-kDa peptide was increased in abundance 200-500-fold in NB-Myco as compared to NB cells. Purification and sequence analysis confirmed that the abundant protein was IMP dehydrogenase. The stepwise selection, increased activity and protein abundance, and unstable phenotype are indirect evidence for a process of gene amplification. Kinetic findings consistent with an Ordered Bi Bi mechanism were indicative of IMP dehydrogenase having undergone mutation. The Michaelis constants were unchanged for IMP (14 and 13 microM) and increased 4-fold for NAD from 25 to 94 microM for NB and NB-Myco cells, respectively. The Ki for mycophenolic acid was increased 2400-fold from 1.4 nM to 3.4 microM for the enzyme from NB versus NB-Myco cells, and the Ki for XMP was increased 4-fold from 78 to 336 microM. Mycophenolic acid exhibited uncompetitive inhibition with IMP, consistent with the formation of a dead end E-XMP-inhibitor complex. The cellular GTP concentration was increased 2-fold in resistant cells and, upon removal of mycophenolic acid, further increased to 4.5-fold that of NB cells.

Topics: Amino Acid Sequence; Animals; Cell Line; Drug Resistance; Humans; IMP Dehydrogenase; Ketone Oxidoreductases; Kinetics; Mice; Molecular Sequence Data; Mycophenolic Acid; Neuroblastoma; Peptide Fragments; Sequence Homology, Nucleic Acid; Tumor Cells, Cultured

Topics: Animals; Cell Line; Drug Resistance; IMP Dehydrogenase; Ketone Oxidoreductases; Mice; Mycophenolic Acid; Neuroblastoma; Ribonucleotides; Time Factors; Xanthine

Mycophenolic acid, an inhibitor of inosinate dehydrogenase, had cytostatic and cytotoxic effects on cultured neuroblastoma cells. Proliferation was inhibited by 50% when cells were incubated with 0.07 micrometerM mycophenolic acid, and cell viability was reduced by 83% when cells were treated with 10 micrometerM mycophenolic acid for 24 hr. Treatment of monolayer cultures with mycophenolic acid reduced intracellular concentrations of guanosine triphosphate by 70% within 3 hr, whereas cytidine triphosphate and uridine triphosphate concentrations were significantly elevated, and adenosine triphosphate concentrations were increased only slightly. Reduction of cellular guanine nucleotides had differential effects on rates of macromolecular synthesis: incorporation of radioactive thymidine into acid-insoluble material was inhibited by mycophenolic acid to a much greater extent than was that of adenosine and leucine. Although proliferation of neuroblastoma cells was inhibited, differentiation, as judged by formation of neuronlike processes in serum-free medium, was unaffected by decreased intracellular concentrations of guanosine triphosphate.

Topics: Adenosine Triphosphate; Cell Differentiation; Cell Division; Cell Survival; Cells, Cultured; Cytosine Nucleotides; DNA, Neoplasm; Guanine Nucleotides; Guanosine Monophosphate; Guanosine Triphosphate; IMP Dehydrogenase; Mycophenolic Acid; Neoplasm Proteins; Neoplasms, Experimental; Neuroblastoma; RNA, Neoplasm; Uracil Nucleotides