Structure-toxicity relationships of the tetramethylated tetralin and indane analogs of retinoic acid

The teratogenicity of retinoids containing either tetramethylated tetralin (Ro 13-6307 or Ro 13-2389) or tetramethylated indane (Ro 13-4306) ring system substitutions was compared to the teratogenic potency of all-trans-retinoic acid. Single oral doses, administered to Syrian Golden hamsters at 10:00 A.M. on day 8 of gestation, induced a syndrome of malformations identical to that induced by treatment with all-trans-retinoic acid. These retinoids failed to induce signs of maternal hypervitaminosis A at doses associated with a significant teratogenic response. The tetramethylated tetralin retinoids and indane retinoid were 18 and 2.4 times as embryotoxic on a molar basis, respectively, as all-trans-retinoic acid. Introduction of a supplementary ring in the side-chain restricted polyene chain flexibility and maintained the hydrophobic plane of the chain. The present results are consistent with previous studies showing that the presence of or biotransformation to a free acid congener was necessary for retinoid teratogenic activity in hamsters and that increasing conformational restriction of acidic retinoids increased teratogenic potency.     

N4-aminocytidine, a nucleoside analog that has an exceptionally high mutagenic activity.

The reaction of cytidine with hydrazine to give N4-aminocytidine was greatly promoted by addition of a less-than-stoichiometric amount of bisulfite, and the product was isolated in a good yield. N4-Aminocytidine was strongly mutagenic to bacteria (Salmonella typhimurium TA100 and TA1535, and E. coli WP2 uvrA) and to phage (phi X174 am3). The activity did not require the presence of mammalian microsomal fraction in the system. The mutagenic potency of N4-aminocytidine in these systems was two orders of magnitude greater than that of N4-amino-2'-deoxycytidine, and more than two orders of magnitude greater than that of N4-hydroxycytidine. The greater activity of the riboside than the deoxyriboside was ascribed to the lack of deoxycytidine kinase in these cells. This compound may be useful as a powerful mutagen to induce a transition mutation in microorganisms.     

Anti-tumour effect of an aromatic retinoic acid analog in a mouse syngeneic transplantable sarcoma

Anti-tumour activity of an aromatic retinoic acid analog has been tested on Sarcoma J in syngeneic system. Tumour complete regression rate and median survival time of progressors mice are significantly improved. These results are at variance with previous observations suggesting the lack of activity of this compound on transplanted murine tumours. Arguments in favour of an immunological stimulation responsible for the observed anti-tumour effect are presented and discussed.     

Antifungal activity in vitro of Ro 14-4767/002, a phenylpropyl-morpholine

Minimal inhibitory concentrations (MICs) of Ro 14-4767/002 for pathogenic yeasts, Aspergillus spp., dermatophytes and other filamentous fungi were determined in dilution tests under a variety of experimental conditions and, for the most of the species and a number of different isolates. Ro 14-4767/002 showed the highest effect against dermatophytes and Cryptococcus neoformans, followed by Candida spp., whereas its activity against Aspergillus spp. was weak. Its activity against most pathogens compared favourably with antifungals of the imidazole class. The activity of Ro 14-4767/002 not only differed between the species but there was also a significant intra-species variation. The MICs were influenced by the inoculum size, the incubation time, and by the composition of the medium. The activity of the compound was significantly higher on Casitone agar than on a chemically defined medium (Yeast Nitrogen Base + glucose). Ro 14-4767/002 was also found to exert fungicidal activity which was time- and concentration-dependent.     

Regionalized metabolic activity establishes boundaries of retinoic acid signalling.

The competence of a cell to respond to the signalling molecule retinoic acid (RA) is thought to depend largely on its repertoire of cognate zinc finger nuclear receptors. XCYP26 is an RA hydroxylase that is expressed differentially during early Xenopus development. In Xenopus embryos, XCYP26 can rescue developmental defects induced by application of exogenous RA, suggesting that the enzymatic modifications introduced inhibit RA signalling activities in vivo. Alterations in the expression pattern of a number of different molecular markers for neural development induced upon ectopic expression of XCYP26 reflect a primary function of RA signalling in hindbrain development. Progressive inactivation of RA signalling results in a stepwise anteriorization of the molecular identity of individual rhombomeres. The expression pattern of XCYP26 during gastrulation appears to define areas within the prospective neural plate that develop in response to different concentrations of RA. Taken together, these observations appear to reflect an important regulatory function of XCYP26 for RA signalling; XCYP26-mediated modification of RA modulates its signalling activity and helps to establish boundaries of differentially responsive and non-responsive territories.     

Modulation of glutathione transferase P1-1 activity by retinoic acid in neuroblastoma cells.

The ability of retinoic acid to modulate glutathione S-transferase P1-1 (GSTP1-1) activity has important implications both for cancer prevention and for anticancer therapy. We investigated GSTP1-1 expression and activity in the human neuroblastoma cell line SK-N-BE(2) (genotype A*/B*) under basal conditions and during 48-h incubation with 0.1 microM all-trans-retinoic acid. The steady-state levels of glutathione transferase P1-1 mRNA and protein during 48-h incubation with all-trans-retinoic acid did not increase substantially, but we detected a significant reduction of GSTP1-1 specific activity. This reduction in enzymatic activity could not be ascribed to a differential action of retinoic acid on the gene variants A* and B*; indeed, the two GSTP1-1 isoforms have different affinities toward 1-chloro-2,4-dinitrobenzene (CDNB), while we found a substantial invariance of the K(m) (CDNB) in the cytosol during retinoid treatment. A modulatory effect of retinoic acid on other enzymes involved in glutathione transferase P1-1 metabolism, such as the retinoic acid-induced tissue trans-glutaminase, might be hypothesized, as well as a direct inactivation of GSTP1-1 by the oxidative stress that characterizes the early phases of apoptosis.     

Synthesis of apo-13- and apo-15-lycopenoids,cleavage products of lycopene that are retinoic acid antagonists

Consumption of the tomato carotenoid, lycopene, has been associated with favorable health benefits. Some of lycopene's biological activity may be due to metabolites resulting from cleavage of the lycopene molecule. Because of their structural similarity to the retinoic acid receptor (RAR) antagonist, β-apo-13-carotenone, the “first half” putative oxidative cleavage products of the symmetrical lycopene have been synthesized. All transformations proceed in moderate to good yield and some with high stereochemical integrity allowing ready access to these otherwise difficult to obtain terpenoids. In particular, the methods described allow ready access to the trans isomers of citral (geranial) and pseudoionone, important flavor and fragrance compounds that are not readily available isomerically pure and are building blocks for many of the longer apolycopenoids. In addition, all of the apo-11, apo-13, and apo-15 lycopenals/lycopenones/lycopenoic acids have been prepared. These compounds have been evaluated for their effect on RAR-induced genes in cultured hepatoma cells and, much like β-apo-13-carotenone, the comparable apo-13-lycopenone and the apo-15-lycopenal behave as RAR antagonists. Furthermore, molecular modeling studies demonstrate that the apo-13-lycopenone efficiently docked into the ligand binding site of RARα. Finally, isothermal titration calorimetry studies reveal that apo-13-lycopenone acts as an antagonist of RAR by inhibiting coactivator recruitment to the receptor.     

A retinoic acid responsive gene, MK, produces a secreted protein with heparin binding activity

MK is a gene whose expression increases transiently during retinoic acid-induced differentiation of embryonal carcinoma cells. MK polypeptide was secreted by differentiating HM-1 embryonal carcinoma cells and by L-cells transfected with an MK cDNA under the control of the beta-actin promoter and Rous sarcoma virus enhancer. MK polypeptide was found to have heparin binding activity. Conditioned medium of the transfected L-cells promoted growth of PC-12 pheochromocytoma cells. These findings support the view that MK polypeptide is a secreted factor involved in regulation of growth and differentiation.     

Hemolytic activity of a cyclic peptide Ro09-0198 isolated from Streptoverticillium

Ro09-0198, a cyclic peptide isolated from culture filtrates of Streptoverticillium griseoverticillatum, induced lysis of erythrocytes. Preincubation of the peptide with phosphatidylethanolamine reduced the hemolytic activity, whereas other phospholipids present in erythrocytes in nature had no effect. A study of the structural requirements on phosphatidylethanolamine necessary for interaction with the peptide indicates that Ro09-0198 recognizes strictly a particular chemical structure of phosphatidylethanolamine: dialkylphosphoethanolamine as well as 1-acylglycerophosphoethanolamine showed the same inhibitory effect on hemolysis induced by Ro09-0198 as diacylphosphatidylethanolamine, whereas phosphoethanolamine gave no inhibitory effect. Neither phosphatidyl-N-monomethylethanolamine nor alkylphosphopropanolamine had an inhibitory effect. Consequently, the hydrophobic chain is necessary for the interaction and the phosphoethanolamine moiety is exactly recognized by the peptide. Ro-09-0198-induced hemolysis was temperature-dependent and the sensitivity of hemolysis differed greatly among animal species.     

Catalytic inhibition of gamma-aminobutyric acid - alpha-ketoglutarate transaminase of bacterial origin by 4-aminohex-5-ynoic acid, a substrate analog.

γ-Aminobutyric acid-α-ketoglutarate transaminase from $\text{Pseudomonas fluorescens}$ is irreversibly inhibited by 4-aminohex-5-yhoic acid, a new structural analog of GABA. The fact that this inhibition requires the pyridoxal form of the holoenzyme, and the formation of a Michaelis complex is in support of a catalytic mechanism. The compound is also active $\text{in vitro}$ and $\text{in vivo}$ on the same enzyme from mammalian brain.     

A new family of proteins with high activity for heparin and regulated by retinoic acid]

An heparin binding protein (RIHB) was purified from chick embryos. Essentially expressed during early embryogenesis it is mainly localized within basement membranes. Its synthesis and that of the RIHB mRNA are induced by retinoic acid in chicken myoblasts cell culture. This protein belongs to the same family that HBGAM or Pleiotropin and MK protein two other heparin binding proteins exhibiting growth and/or neurotrophic activities.     

13-cis retinoic acid and all-trans retinoic acid in the regulation of the proliferation and survival of human breast cancer cell line MCF-7.

Retinoids are a group of compounds which inhibit cell proliferation and induce cellular differentiation. The aim of this study was to compare the antiproliferative activity of various concentrations of 13-cis retinoic acid (isotretinoin) and all-trans retinoic acid (tretinoin) in a culture of the estrogen-sensitive human breast cancer cell line MCF-7. Evaluation was based on [3H]thymidine incorporation into the cancer cells and through immunocytochemical analysis of cell cycle-associated PCNA and Ki-67 protein expression. Both retinoids inhibited [3H]thymidine incorporation into the cancer cells most effectively at a concentration of 3x10(-3) M. Two basic substances used for line MCF-7 culture experiments, one stimulating - estradiol - and the other inhibiting - tamoxifen - were applied. Estradiol added to a culture containing decreasing concentrations of isotretinoin (from 3x10(-3) to 3x10(-8) M) caused a statistically significant reduction in the percentage of [3H]thymidine incorporation into the cancer cell line MCF-7, compared to the 17 beta estradiol group (189.25%+/-62.64, control=100%, p<0.05). In the group of decreasing tretinoin concentrations, statistically significant differences were found only at 3x10(-3), 3x10(-4) and 3x10(-8) M. Following culture supplementation with tamoxifen (1 microM), statistically significant differences were observed only at the highest concentrations of both retinoids (3x10(-3) and 3x10(-4) M). The evaluation of breast carcinoma cells with a positive immunocytochemical reaction to PCNA and Ki-67 has revealed that isotretinoin reduces their percentage in the most determined and statistically significant way (38.00%+/-2.58 and 39.25%+/-3.09), compared to the control group (86.50%+/-9.20 and 100%+/-3.87, p<0.001 and p<0.0001) and to the estradiol group (87.00%+/-6.79 and 86.10%+/-7.0, p<0.001). Apart from their blocking effect on the cell cycle, retinoids also induce the apoptotic pathway.     

The effect of retinol and retinoic acid on the testicular phospholipase a activity in retinol-deficient rats.

Both phospholipases A1 and A2 activities (EC 3.1.1.4) at pH 7.4 were found to be significantly decreased in retinol-deficient rat testes supplemented with retinoic acid as compared to retinol-fed controls using 1-acyl-2-[1-(14)C]-oleoyl-sn-glycero-3-phosphocholine as substrate. However, little or no difference was observed in phospholipase A1 activity at pH 3.0 in both groups of rats.     

Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line

The ability of retinoic acid (RA) to modulate acetylcholinesterase (AChE) activity in a human neuroblastoma cell line (LN-N-5) was examined. The specific activity of AChE was significantly increased 3 days after exposure of LA-N-5 to RA and reached its maximum values after 9 or more days of culturing. Dose-response experiments demonstrated that large increases of AChE occurred at RA concentrations between 10(-7) and 10(-6) M with maximum AChE values detected at 10(-6)-10(-5) M. Increased AChE activity paralleled neurite outgrowth in LA-N-5 cultures. These findings demonstrate that RA can regulate specific AChE activity in human neuroblastoma cells in a manner consistent with neuronal maturation.