Valproic acid in combination with all-trans retinoic acid and intensive therapy for acute myeloid leukemia in older patients.

The outcome of patients with acute myeloid leukemia who are older than 60 years has remained poor because of unfavorable disease characteristics and patient-related factors. The randomized German-Austrian AML Study Group 06-04 protocol was designed on the basis of in vitro synergistic effects of valproic acid (VPA) and all-trans retinoic acid with chemotherapy. Between 2004 and 2006, 186 patients were randomly assigned to receive 2 induction cycles with idarubicin, cytarabine, and all-trans retinoic acid either with VPA or without (STANDARD). In all patients, consolidation therapy was intended. Complete remission rates after induction tended to be lower in VPA compared with STANDARD (40% vs 52%; P = .14) as a result of a higher early death rate (26% vs 14%; P = .06). The main toxicities attributed to VPA were delayed hematologic recovery and grade 3/4 infections, observed predominantly during the second induction cycle. After restricting VPA to the first induction cycle and reducing the dose of idarubicin, these toxicities dropped to rates observed in STANDARD. After a median follow-up time of 84 months, event-free and overall survival were not different between the 2 groups (P = .95 and P = .57, respectively). However, relapse-free-survival was significantly superior in VPA compared with STANDARD (24.4% vs 6.4% at 5 years; P = .02). Explorative subset analyses revealed that AML with mutated Nucleophosmin 1 (NPM1) may particularly benefit from VPA. This trial was registered at www.clinicaltrials.gov as #NCT00151255.

Retinoic acid receptor-ß gene reexpression and biological activity in SHI-1 cells after combined treatment with 5-aza-2'-deoxycytidine and all-trans retinoic acid.

BACKGROUND: This study was conducted to determine the antineoplastic activities of 5-aza-2'-deoxycytidine (decitabine; DAC) and all-trans retinoic acid (ATRA), administered either alone or in combination, on in vitro cultured SHI-1 cells as well as their effects on the expression of the tumor suppressor gene p16(INK4a) (p16) and the retinoic acid receptor (RAR)-ß. METHODS: Cell growth inhibition, differentiation and apoptosis were determined in SHI-1 cells treated with DAC and/or ATRA, and the combination index of the two compounds was calculated. Methylation of the p16 and RAR-ß genes in SHI-1 cells was detected by methylation-specific polymerase chain reaction (PCR). Real-time quantitative reverse transcriptase PCR was used to detect mRNA expression of the p16 and RAR-ß genes, and Western blot analysis was performed for protein expression. RESULTS: The drug combination had a synergistic effect on growth inhibition, differentiation and apoptosis of SHI-1 cells, and the effects of DAC and ATRA were dependent on time. DAC, either alone or in combination with ATRA, induced demethylation of the genes p16 and RAR-ß, whereas ATRA alone had no effect on methylation. The RAR-ß gene was reexpressed following DAC-ATRA combination treatment, and both agents had no effect on p16 expression. CONCLUSION: The results revealed that DAC used in combination with ATRA has significant clinical potential in the treatment of acute monocytic leukemia.

Moderate vitamin A supplementation in obese mice regulates tissue factor and cytokine production in a sex-specific manner.

Vitamin A (vitA) regulates obesity, insulin resistance, inflammation, dyslipidemia and hemostasis through its metabolites retinaldehyde (Rald) and retinoic acid (RA) produced in endogenous enzymatic reactions. Combination of at least 3 of these conditions leads to development of metabolic syndrome (Msyn) and, consequently, type 2 diabetes and/or cardiovascular disease. Although many foods are fortified with vitA, it remains unknown what conditions of Msyn are influenced by moderate dietary vitA supplementation. A family of aldehyde dehydrogenase 1 (Aldh1) enzymes is a key contributor to obesity via sex- and fat depot-specific production of RA in adipose tissue. Therefore, we studied effects of moderate vitamin A supplementation of an obesogenic high-fat (HF) diet (4 IU vitA/g and 20 IU vitA/g HF diet) on multiple conditions and mediators of Msyn in wild-type (WT, C57Bl/6) and Aldh1a1(-/-) mice. We found that mild vitamin A supplementation did not influence obesity, fat distribution, and glucose tolerance in males and females of the same genotype. In contrast, multiplex analysis of bioactive proteins in blood showed moderately increased concentrations (10-15%) of inflammatory IL-18 and MIP-1γ in vitA supplemented vs. control WT males. Marked decrease (28-31%) in concentrations of lymphotactin and tissue factor, a key protein contributing to thrombogenesis during injury, was achieved by vitA supplementation in WT females compared to control WT females. Aldh1a1 deficiency reduced obesity, insulin resistance, suppressed many pro-inflammatory cytokines, and abolished the effects of vitA supplementation seen in WT mice. Our study revealed specific inflammatory and pro-thrombotic proteins in plasma regulated by dietary vitamin A and the critical role of endogenous vitA metabolism in these processes. The sex-specific decrease of plasma tissue factor concentrations by moderate dietary vitA supplementation could potentially reduce pro-thrombotic states in obese females.

Wnt/beta-catenin and retinoic acid receptor signaling pathways interact to regulate chondrocyte function and matrix turnover.

Activation of the Wnt/beta-catenin and retinoid signaling pathways is known to tilt cartilage matrix homeostasis toward catabolism. Here, we investigated possible interactions between these pathways. We found that all-trans-retinoic acid (RA) treatment of mouse epiphyseal chondrocytes in culture did increase Wnt/beta-catenin signaling in the absence or presence of exogenous Wnt3a, as revealed by lymphoid enhancer factor/T-cell factor/beta-catenin reporter activity and beta-catenin nuclear accumulation. This stimulation was accompanied by increased gene expression of Wnt proteins and receptors and was inhibited by co-treatment with Dickkopf-related protein-1, an extracellular inhibitor of Wnt/beta-catenin signaling, suggesting that RA modulates Wnt signaling at Wnt cell surface receptor level. RA also enhanced matrix loss triggered by Wnt/beta-catenin signaling, whereas treatment with a retinoid antagonist reduced it. Interestingly, overexpression of retinoic acid receptor gamma (RARgamma) strongly inhibited Wnt/beta-catenin signaling in retinoid-free cultures, whereas small interfering RNA-mediated silencing of endogenous RARgamma expression strongly increased it. Small interfering RNA-mediated silencing of RARalpha or RARbeta had minimal effects. Co-immunoprecipitation and two-hybrid assays indicated that RARgamma interacts with beta-catenin and induces dissociation of beta-catenin from lymphoid enhancer factor in retinoid-free cultures. The N-terminal domain (AF-1) of RARgamma but not the C-terminal domain (AF-2) was required for association with beta-catenin, whereas both AF-1 and AF-2 were necessary for inhibition of beta-catenin transcriptional activity. Taken together, our data indicate that the Wnt and retinoid signaling pathways do interact in chondrocytes, and their cross-talks and cross-regulation play important roles in the regulation of cartilage matrix homeostasis.

Liposomal delivery of hydrophobic RAMBAs provides good bioavailability and significant enhancement of retinoic acid signalling in neuroblastoma tumour cells.

Retinoid treatment is employed during residual disease treatment in neuroblastoma, where the aim is to induce neural differentiation or death in tumour cells. However, although therapeutically effective, retinoids have only modest benefits and suffer from poor pharmacokinetic properties. In vivo, retinoids induce CYP26 enzyme production in the liver, enhancing their own rapid metabolic clearance, while retinoid resistance in tumour cells themselves is considered to be due in part to increased CYP26 production. Retinoic acid metabolism blocking agents (RAMBAs), which inhibit CYP26 enzymes, can improve retinoic acid (RA) pharmacokinetics in pre-clinical neuroblastoma models. Here, we demonstrate that in cultured neuroblastoma tumour cells, RAMBAs enhance RA action as seen by morphological differentiation, AKT signalling and suppression of MYCN protein. Although active as retinoid enhancers, these RAMBAs are highly hydrophobic and their effective delivery in humans will be very challenging. Here, we demonstrate that such RAMBAs can be loaded efficiently into cationic liposomal particles, where the RAMBAs achieve good bioavailability and activity in cultured tumour cells. This demonstrates the efficacy of RAMBAs in enhancing retinoid signalling in neuroblastoma cells and shows for the first time that liposomal delivery of hydrophobic RAMBAs is a viable approach, providing novel opportunities for their delivery and application in humans.

Enhancement in alpha-tocopherol succinate-induced apoptosis by all-trans-retinoic acid in primary leukemic cells: role of antioxidant defense, Bax and c-myc.

We investigated the possible mechanisms of All-trans retinoic acid (ATRA)-promoted apoptosis induced by alpha-tocopherol succinate (alpha-TS) in freshly isolated leukemic cells obtained from chronic myeloid leukemic patients. alpha-TS at 50 microM concentration significantly decreased superoxide dismutase (SOD) activity and reduced glutathione (GSH) by 29% and 25%, respectively, and increased lipid peroxidation level by 33%. Though 10 microM ATRA did not affect these parameters, it further significantly enhanced alpha-TS-induced changes. Bax expression in the leukemic cells was increased by treatment with ATRA, alpha-TS, and their combination to 40%, 240%, and 320%, respectively, without any change in Bcl2 and p53 expression. C-myc was down regulated by treatment with ATRA, alpha-TS and their combination to 22%, 48.5%, and 52%, respectively. In conclusion, the data reveal that enhancement of alpha-TS-induced apoptosis by ATRA in leukemic cells was through up regulation of Bax and lipid peroxidation, and down regulation of c-myc and GSH.

Improved synthesis of histone deacetylase inhibitors (HDIs) (MS-275 and CI-994) and inhibitory effects of HDIs alone or in combination with RAMBAs or retinoids on growth of human LNCaP prostate cancer cells and tumor xenografts.

We have developed new, simple, and efficient procedures for the synthesis of two promising histone deacetylase inhibitors (HDIs), CI-994, (N-(2-aminophenyl)-4-acetylaminobenzamide), and MS-275 (N-(2-aminophenyl)4-[N-(pyridine-3-yl-methoxycarbonyl)aminomethyl]benzamide) from commercially available acetamidobenzoic acid and 3-(hydroxymethyl)pyridine, respectively. The procedures provide CI-994 and MS-275 in 80% and 72% overall yields, respectively. We found that the combination of four HDIs (CI-994, MS-275, SAHA, and TSA) with retinoids all-trans-retinoic acid (ATRA) or 13-cis-retinoic acid (13-CRA) or our atypical retinoic acid metabolism blocking agents (RAMBAs) 1 (VN/14-1) or 2 (VN/66-1) produced synergistic anti-neoplastic activity on human LNCaP prostate cancer cells. The combination of 2 and SAHA induced G1 and G2/M cell cycle arrest and a decrease in the S phase in LNCaP cells. 2+SAHA treatment effectively down-regulated cyclin D1 and cdk4, and up-regulated pro-differentiation markers cytokeratins 8/18 and pro-apoptotic Bad and Bax. Following subcutaneous administration, 2, SAHA or 2+SAHA were well tolerated and caused significant suppression/regression of tumor growth compared with control. These results demonstrate that compound 2 and its combination with SAHA are potentially useful agents that warrant further preclinical development for treatment of prostate cancer.

Retinoic acid inhibits neuronal voltage-gated calcium channels.

Retinoic acid is the active metabolite of vitamin A and regulates several important cellular processes by activating retinoic acid receptors (RAR) and retinoid X receptors (RXR). These receptors generally act as transcription factors, though non-genomic actions of both retinoic acid and the receptors have also been reported. One such nongenomic effect includes the modulation of Ca2+ levels during homeostatic synaptic plasticity in the hippocampus. Retinoic acid can thus affect Ca2+ signaling and can potentially control both synaptic plasticity and neuronal firing. However, whether retinoic acid can regulate voltage-gated Ca2+ channels (either via genomic or nongenomic actions), which are fundamental to these processes, has not yet been studied in detail. Here we demonstrate the effects of retinoic acid on the biophysical properties of voltage-gated Ca2+ channels in cultured invertebrate motorneurons. Overnight exposure to physiological concentrations of retinoic acid significantly inhibited the voltage-gated Ca2+ current (ICa) in an isomer-dependent manner. Specifically, all-trans retinoic acid (atRA), but not 9-cis RA (9cRA), depolarized the voltage of half-maximal activation of ICa. AtRA also reduced the rate of channel activation and delayed recovery from inactivation. We provide evidence that both L-type and non-L-type voltage-gated Ca2+ channels are affected by atRA, as both nifedipine-sensitive and nifedipine-resistant ICa were inhibited in these neurons. These effects of retinoic acid are thought to be at least partially mediated by the retinoid receptors, as treatment of the neurons with synthetic RAR and RXR agonists produced a similar inhibition of ICa.

Adhesion molecules and Differentiation Syndrome: phenotypic and functional analysis of the effect of ATRA, As2O3, phenylbutyrate, and G-CSF in acute promyelocytic leukemia.

BACKGROUND AND OBJECTIVES: Differentiation Syndrome (DS) is a treatment complication which can occur in patients treated with acute promyelocytic leukemia (APL) with all transretinoic acid (ATRA) or As(2)O(3), and is characterized by enhanced leukocyte transmigration. As(2)O(3), Phenylbutyrate (PB) and G-CSF are known to potentiate ATRA effects. Our aim was to analyze the changes in expression and function of adhesion molecules induced by ATRA, As(2)O(3), G-CSF and PB, and their association. DESIGN AND METHODS: APL blasts and NB4 cells were treated with ATRA, As(2)O(3), PB, G-CSF or their association and the expression of adhesion molecules was determined by flow cytometry. Cell adhesion was evaluated in vitro using Matrigel and for the in vivo analysis, Balb-c mice were injected with NB4 cells pre-treated with ATRA, As(2)O(3), ATRA+G-CSF or ATRA+As(2)O(3). In addition, CD54 and CD18 knock-out mice were injected with NB4 cells and concomitantly treated with ATRA. In both models, the MPO activity in the lungs was determined 6 hours after the injection of the cells. RESULTS: In NB4 and APL blasts, ATRA and As(2)O(3) increased CD54 expression, but no synergism was detected. CD11b and CD18 were also up-regulated by ATRA in primary cells. PB and G-CSF had no effect, but the latter potentiated ATRA-induced CD18 up-regulation. These changes were accompanied by increased adhesion to Matrigel and to lung microvasculature, and reversed by anti-CD54, anti-CD18 antibodies. In CD54 and CD18 knock-out mice the ATRA effect was canceled. INTERPRETATION AND CONCLUSIONS: The use of As(2)O(3), PB and G-CSF in association with ATRA should not aggravate DS in APL.

Effects of an HMG-CoA reductase inhibitor, simvastatin, on human myeloma cells.

To look for new candidates for agents to use in maintenance therapy for myeloma patients, the growth inhibitory effects of a 3-hydroxy-3-mehtylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin), simvastatin, was analyzed using human myeloma cell lines. Several investigations have indicated growth reduction in certain lineages of cancer cells including one report on myeloma, and inhibitory effects of statins on GTPases and involving MAP-kinases. Most (12 out of 13) myeloma lines examined showed growth inhibition when cultured with various concentrations (1-30 microM) of simvastatin in a dose-dependent manner. Simvastatin in combination with other biological response modifiers such as ATRA or DEX had additional effects on growth. In addition, anti-oxides prevented the simvastatin-induced growth inhibition and apoptosis. Furthermore, myeloma cells treated with simvastatin clearly showed inactivation of various MAP-kinase pathways such as ERK1/2, MEK1/2, JNK, and p38. Based on these findings, statins may be suitable for clinical usage in maintenance therapy for myeloma patients.

Integrative analysis of mRNA and miRNA array data reveals the suppression of retinoic acid pathway in regulatory T cells of Graves' disease.

CONTEXT: It is well known that regulatory T cells (Tregs) are abnormal in Graves' disease (GD) and play crucial roles in the breakdown of immune tolerance and GD development. However, there are controversies about whether the quantity and/or function of Tregs is aberrant in GD. The molecular mechanism of Tregs abnormality and its effects on GD development was still unclear, until now. OBJECTIVE: MiRNAs play important roles in the function and development of the immune system including Tregs. To reveal the Tregs abnormality and its molecular mechanism in GD, we systematically studied the quantity and immunosuppressive function as well as the differential expression profiles of miRNA and mRNA of Tregs in newly diagnosed patients with GD using TaqMan miRNA array and mRNA microarray. RESULTS: Our results showed that the quantity and immunosuppressive function of Tregs in initial patients with GD was significantly decreased. More importantly, the retinoic acid (RA) pathway was markedly suppressed and its agonist, all-trans retinoic acid, could notably improve the quantity and immunosuppressive function of Tregs from patients with GD in vitro. In addition, many other pathways including protein ubiquitination and circadian rhythm were also significantly regulated in Tregs of GD. CONCLUSIONS: This integrative study first revealed the expression profiles of mRNA/miRNA in Tregs of initial GD and RA pathway might play important roles in GD development. Our results implied that all-trans RA, which had been used for a long time in the clinical setting, had potential value in the treatment of GD and was worthy of additional study.

PPARgamma-active triterpenoid CDDO enhances ATRA-induced differentiation in APL.

Acute promyelocytic leukemia (APL) is associated with oncogenic PML-RARalpha that acts as a dominant negative transcriptional repressor of retinoic acid (RA) receptor target genes by recruiting histone deacetylase (HDAC). The peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear receptor family that forms heterodimers with retinoid X receptor (RXR). In addition to RAR targets, PML-RARalpha silence a wide range of nuclear receptor target genes including PPARgamma targets. All-trans-retinoic acid (ATRA), a ligand for the RA receptor (RAR), restores normal retinoid signaling and induces terminal differentiation of APL cells; however, APL cells can develop resistance to ATRA. Using ATRA sensitive NB4 and ATRA-resistant derivative MR2 cell lines, we demonstrate that PPARgamma ligand 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO) enhances pro-apoptotic and differentiating effects of ATRA in ATRA-sensitive NB4 cells and partially reverses ATRA resistance in MR2 cells. The CDDO/ATRA combination synergistically induces RARbeta2 expression both in ATRA-sensitive and -resistant APL cells. RARbeta2 MrNA induction by CDDO/ATRA was mediated in part by enhanced H3-Lys9 acetylation in the RARbeta2 promoter which in turn increased the affinity of RARbeta for betaRARE. PPARgamma specific inhibitor T007 and silencing of PPARgamma by siRNA diminished CDDO-induced maturation and RARbeta2 mRNA along with PPARgamma induction indicating that PPARgamma activation is at least partially responsible for the RARbeta2 transcription and maturation induction. In an in vivo mouse model of APL, CDDO derivative CDDO-methyl ester markedly enhanced ATRA-induced maturation and extended the survival of mice. In summary, these results provide rationale for the combined targeting of RAR and PPARgamma nuclear receptors in the therapy of APL.

Acute effects of dietary retinoic acid on ocular components in the growing chick.

When the eyes of chicks are induced to grow toward myopia or hyperopia by having them wear spectacle lenses or diffusers, opposite changes take place in the retina and choroid in the synthesis and levels of all-trans Retinoic Acid (RA). To explore whether RA plays a causal role in the regulation of eye growth, we fed young chicks RA (doses 0.5 to 24 mg/kg) either twice a day or on alternate days or only once. Refractive error was measured with a Hartinger refractometer; ocular length, lens-thickness and choroidal thickness were measured by A-scan ultrasound. The amount of RA present in ocular tissues was determined using HPLC. Oral delivery of RA effectively increased RA in ocular tissues within 8h. During the first day after feeding RA at levels above 8 mg/kg, the rate of ocular elongation tripled, the choroid thickened and lens thickening was inhibited. The day following a dose of RA, the rate of ocular elongation was inhibited and the lens thickened more than normal. Nonetheless, the cumulative effect of repeated doses was that the eye became longer and the lens became thinner than normal, with no net change in refractive error. The rate of elongation was also increased by feeding 13-cis RA, and was reduced by citral, an inhibitor of RA synthesis. Surprisingly, birds fed RA while being kept in darkness also had normal refractive errors despite increased ocular elongation, and birds wearing either +6D or -6D spectacle lenses compensated normally for the lenses despite the enhanced ocular elongation caused by the RA. These results suggest that RA may act at the level of a coordinated non-visual regulatory system which controls the growth of the various ocular components, arguing that emmetropization does not depend entirely on vision.

Retinoid-mediated stimulation of steroid sulfatase activity in myeloid leukemic cell lines requires RARalpha and RXR and involves the phosphoinositide 3-kinase and ERK-MAP kinase pathways.

All-trans retinoic acid and 9-cis-retinoic acid stimulate the activity of steroid sulfatase in HL60 acute myeloid leukemia cells in a concentration- and time-dependent manner. Neither of these 'natural retinoids' augmented steroid sulfatase activity in a HL60 sub-line that expresses a dominant-negative retinoic acid receptor alpha (RARalpha). Experiments with synthetic RAR and RXR agonists and antagonists suggest that RARalpha/RXR heterodimers play a role in the retinoid-stimulated increase in steroid sulfatase activity. The retinoid-driven increase in steroid sulfatase activity was attenuated by inhibition of phospholipase D (PLD), but not by inhibitors of phospholipase C. Experiments with inhibitors of protein kinase C (PKC) show that PKCalpha and PKCdelta play an important role in modulating the retinoid-stimulation of steroid sulfatase activity in HL60 cells. Furthermore, we show that pharmacological inhibition of the RAF-1 and ERK MAP kinases blocked the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells and, by contrast, inhibition of the p38-MAP kinase or JNK-MAP kinase had no effect. Pharmacological inhibitors of the phosphatidylinositol 3-kinase, Akt, and PDK-1 also abrogated the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells. These results show that crosstalk between the retinoid-stimulated genomic and non-genomic pathways is necessary to increase steroid sulfatase activity in HL60 cells.

Novel retinoids with receptor selectivity and functional selectivity.

Retinoids mediate a number of physiological pathways through their effects on cellular growth and differentiation. Upon binding to retinoic acid receptors (RARs) and retinoid X receptors (RXRs), retinoids regulate cellular processes by directly modulating the expression of responsive genes. The wide-ranging effects of retinoid action are attributable to two main factors-the ubiquitous distribution of several subtypes and isoforms of RARs and RXRs, and the ability of these receptors to regulate numerous genes upon ligand activation. The broad range of effects mediated by retinoids means not only that they have many potential therapeutic applications but also that non-selective retinoids are associated with a high incidence of adverse effects. The design of retinoids that are receptor-selective and function-selective is a strategy that is proving successful in developing novel retinoids that offer not only good efficacy but also good tolerability. Tazarotene, a receptor-selective retinoid indicated for the topical treatment of psoriasis, is at the forefront of this new generation of retinoids. In the near future, other receptor-selective retinoids may prove useful for the treatment of other dermatological diseases, cancer, and diabetes.