Therapeutic Effects Of All-trans Retinoic Acid Research Articles

ATRA Could Correct the Defective S1P-Mediated Cytoskeletal Reorganization in Proplatelet Formation of ITP

Introduction Sphingosine-1-phosphate (S1P) is now emerging as a vital lipid mediator. Activation of sphingosine kinase (SphK) produces intracellular S1P, which in turn can be secreted out of the cell and act extracellularly by binding to S1P receptors (S1PR). Recent studies suggest that the "inside-out" signaling by S1P in megakaryocytes (MKs) plays a critical role in proplatelet formation (PPF) (Blood, 2013; J EXP MED, 2012). PPF requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases which can be activated by S1P, including Rac1 and Cdc42, have been shown to be master regulators of cytoskeletal rearrangements. The pathogenesis mechanisms of immune thrombocytopenia (ITP) are not entirely understood. Our previous data indicated that impaired PPF contributed to the development of thrombocytopenia in ITP. To further explore the underlying mechanism of impaired PPF in ITP, we found that S1P-mediated microtubule reorganization is defective in PPF of ITP. All-trans retinoic acid (ATRA), which has demonstrated to be a promising option for ITP patients in our previous study (Lancet Haematology, 2017), could correct the altered microtubule reorganization and promote PPF. Methods Thirty consecutive patients with primary ITP and 20 healthy donors were enrolled in our study. MKs were isolated from bone marrow samples, and they were collected again after ITP patients received ATRA therapy. MK mRNA sequencing by microarray was used to assess the difference of gene expression between ITP and controls. Microtubule regrowth assay was performed to observe microtubule dynamic behavior. In this assay nocodazole was first used to induce complete depolymerization of microtubule network, followed by drug washout to allow microtubule regrowth over time. ATRA was added to the culture medium of MKs to determine the mechanism of ATRA in correcting impaired PPF. Additionally, ITP mice model was established to observe the therapeutic effects of ATRA in PPF. Pf4-Cre/loxP system was used to specifically knock down gene of MKs. Results S1P concentration in bone marrow from ITP patients was lower compared to healthy donors. MKs mRNA sequencing demonstrated that S1P synthetase SphK2 and S1P receptor S1PR1 gene were downregulated while S1P lyase (SPL) gene was upregulated in ITP patients, which caused abnormal S1P signaling. Furthermore, we observed that PPF capacity of MKs in patients with ITP was reduced. Pharmacological disruption of S1PR1 blocked PPF, exogenous S1P corrected impaired PPF. Collectively, deregulation of S1P signaling was associated with impaired PPF in ITP. To verify the downstream role of S1P in regulating PPF, the Rho GTPases detection of MKs revealed a decrease in Cdc42 and Rac1 levels from ITP patients. Immunofluorescence of the differentiated MKs showed that the expression and distribution of β1 tubulin were abnormal from ITP patients. Early PPs from MKs of healthy donors displayed a well-organized tubulin bundles resembling bunches of grapes. In contrast, in MKs from ITP patients, tubulin was disorganized in thick bundles. In addition, TEM analysis of the MKs showed an irregular distribution of granules, tortuous membranes and impaired proplatelet structure. In microtubule regrowth assay, MKs from ITP patients had significantly lower microtubule regrowth at 10 min post-nocodazole washout compared with controls. Together, microtubule alteration resulted in impaired PPF in ITP. We tested whether S1P pathway were required for microtubule reorganization, both SphK2-/- and S1PR1-/- mice displayed significantly reduced S1P, Cdc42 and Rac1, altered microtubule architecture and defective PPF. Taken together, abnormal S1P pathway accounted for impaired microtubule reorganization in ITP. Next, we explored the effect of ATRA on microtubules reorganization in ITP patients, our data showed that in vitro treatment with ATRA restored microtubules structure by upregulating S1P and activating Rho GTPases. In vivo studies showed that ARTA could rescue the impaired PPF in both patients and mice model with ITP. Conclusions The MKs of ITP patients displayed defective cytoskeletal reorganization regulated by S1P pathway. ATRA restored cytoskeletal structure and corrected impaired PPF by upregulating S1P and activating Rho GTPases. It sheds light on a novel mechanism of ITP pathogenesis and provides a basis for the therapeutic potential of ARTA in ITP patients. Disclosures No relevant conflicts of interest to declare.

Lipid-polymer nanoparticles with CD133 aptamers for targeted delivery of all-trans retinoic acid to osteosarcoma initiating cells

Osteosarcoma, a common type of bone cancer in children, and represents an aggressive and fetal cancer worldwide. Osteosarcoma initiating cells are considered to be a subpopulation of cancer cells which contribute to the progression, recurrence, metastasis and multi-drug resistance of osteosarcoma. CD133 is considered to be one marker for osteosarcoma initiating cells. All-trans retinoic acid (ATRA), an active metabolite of vitamin A under the family retinoid, is an up-and-coming drug which was able to effectively treat various cancer initiating cells. Nevertheless, there have been no research that reported the activity of ATRA against osteosarcoma initiating cells. In this research, we hereby examined the potential activity of ATRA in osteosarcoma initiating cells, and developed lipid-polymer nanoparticles with CD133 aptamers for targeted ATRA delivery to osteosarcoma initiating cells. Using the cytotoxicity assay, colony formation assay, tumorsphere formation assay and flow cytometry, the therapeutic effect of ATRA and ATRA-loaded lipid-polymer nanoparticles conjugated with CD133 aptamers (ATRA-PLNP-CD133) against osteosarcoma initiating cells were investigated. The results showed that ATRA exerted potent activity towards osteosarcoma initiating cells. ATRA-PLNP-CD133, which showed a size of 129.9 nm and a sustained release of ATRA during 144 h, was demonstrated to efficiently and specifically promote the ATRA delivery to osteosarcoma initiating cells, and achieve superior therapeutic efficacy in osteosarcoma compared with ATRA and non-targeted nanoparticles. This is the first report of the therapeutic efficacy of ATRA towards osteosarcoma initiating cells, and the increased ATRA delivery by nanoparticles to osteosarcoma initiating cells using CD133 aptamers. ATRA-PLNP-CD133 represent an up-and coming approach for the therapy of osteosarcoma initiating cells.

Efficacy of Immunomodulatory Therapy with All-Trans Retinoid Acid for Adults with Chronic Immune Thrombocytopenia

Abstract 3335 Objective:Immune thrombocytopenia (ITP) is an immune-mediated disorder characterized by antibody- and cell-mediated platelet destruction and impaired platelet production. In addition of antibody-mediated platelet clearance, T-cell is also involved in platelet destruction or marrow suppression. During immune responses CD4+ T cells can differentiate into a range of cell types, including T helper type 1 (Th1), Th2, Th17 and regulatory T cells (Treg). The goal of the present study is to investigate the therapeutic effects of all-trans-retinoic acid (ATRA) plus prednisone treatment on adult refractory idiopathic thrombocytopenic purpura (RITP) and to further explore the underlying mechanisms. Methods:All 35 patients were treated with ATRA at fixed dose of 10 mg/tid plus prednisone and were started after discontinuation of other previous ITP treatment. Peripheral blood samples were collected from 35 RITP patients and 20 healthy subjects. The concentrations of the peripheral blood CD 4+ T cells (Th1, Th2, TH17, TREG) were analyzed by flow cytometry, the levels of cytokines were confirmed by enzyme-linked immunoassay (ELISA), and the expression of T-bet, GATA-3, RORγt, FOXP3 were detected by semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) in 35 RITP patients before and after treatments, with 20 normal individuals as respective controls. Results:The overall response rate of ATRA plus prednisone treatment in RITP patients was 54.3%, 28.6%(n=10) of patients with a complete response and 25.7%(n=9) of patients with a partial response. Among the 19 responders, the mean platelet count was 34+13×106/ml before ATRA therapy and 106+29×106/ml after treatment. No severe adverse effects happened. The levels of regulory T cells were significantly increased in patients after ATRA plus prednisone treatment (P < 0.05); the levels of Th1, Th2, Th17 were not significantly improved (P >0.05) in effective patients after treatments in flow cytometry analysis. The concentrations of IL-10, TGF-β was increased (P<0.05), whereas IFN-γ, IL-4, IL-17 factor showed no obvious change in the effective groups after treatment (P > 0.05). The expression levels of Foxp3 enhanced dramatically after treatment in real-time RT-PCR analysis (P < 0.05). However, the concentrations of T-bet, GATA-3 and ROR-γ showed no obvious change after the therapy in real-time RT-PCR analysis(P > 0.05). Conclusions:54.3% of RITP patients recovered after ATRA plus prednisone treatments. The therapeutic effects of ATRA plus prednisone may be involved in the increased levels of regulory T cells in peripheral blood through increased expression of TGF-β, IL-10, and Foxp3. In comparison, the therapeutic effects may not be attributed to the expression of TH1, TH2, TH17, IFN-γ, IL-4, IL-17, T-bet, GATA-3, RORγt. Disclosures:No relevant conflicts of interest to declare.

Therapeutic effects of all trans-retinoitc acid combined with transarterial chemoembolization on Walker-256 hepatoma in rats

In order to investigate the inhibitory effects of all trans-retinoic acid (ATRA) on differentiation and apoptosis of Walker-256 hepatocellular carcinoma cells and the therapeutic effects of ATRA combined with transarterial chemoembolization (TACE) on rat Walker-256 transplanted hepatocarcinoma, Walker-256 hepatocarcinoma cell lines were treated with ATRA at different concentrations. After culture for 48 h, the inhibitory rate of cell proliferation was determined by MTT assay; the changes of Fas and Bcl-2 mRNA expression were determined by RT-PCR, and the expression levels of Caspase3 and Caspase8 proteins were detected by Western blot. Twenty-seven Wistar rat models of hepatocarcinoma were set up successfully by implanting Walker-256 cell lines. The tumor volume at the 11th day after implantation (V(preoperation)) was measured by magnetic resonance imaging (MRI). The 27 rats were randomly and equally divided into three groups, and the therapy scheme was performed as follows: group A (ATRA 0.1 mg+mitomycin 0.05 mL+lipiodol 0.05 mL+gelfoam powder 0.025 mg); group B (mitomycin 0.05 mg+lipiodol 0.05 ml+gelfoam 0.025 mg; group C (0.9% NaCl 0.2 mL). After another 11 days, MRI was performed once again to measure the tumor volume (V(postoperation)). The expression of factor and Ki VIII -67 in the tumor tissues was detected by immunohistochemistry. The results showed that ATRA could suppress proliferation of Walker-256 cell lines. After treatment of Walker-256 cell lines with ATRA, the expression of Fas mRNA was significantly up-regulated and the Bcl-2 mRNA was significantly down-regulated by ATRA at the concentration of 10 mumol/L as compared with the control group (P<0.05). After treatment with 10 mumol/L ATRA for 48 h, the Caspase3 and Caspase8 were significantly activated as compared with the control group (P<0.05). Significant difference existed in growth rate among the three groups (P<0.01) and between either two groups (P<0.05). The expression rate of factor VIII and Ki-67 was gradually increased from group A, group B to group C. The study suggests that ATRA could inhibit the proliferation of Walker-256 cells and the effectiveness of the combined therapy (ATRA+TACE) for treating transplanted hepatoma of rats is superior to that of TACE alone.

Retinoic Acid Attenuates β-Amyloid Deposition and Rescues Memory Deficits in an Alzheimer's Disease Transgenic Mouse Model

Recent studies have revealed that disruption of vitamin A signaling observed in Alzheimer's disease (AD) leads to beta-amyloid (Abeta) accumulation and memory deficits in rodents. The aim of the present study was to evaluate the therapeutic effect of all-trans retinoic acid (ATRA), an active metabolite of vitamin A, on the neuropathology and deficits of spatial learning and memory in amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic mice, a well established AD mouse model. Here we report a robust decrease in brain Abeta deposition and tau phosphorylation in the blinded study of APP/PS1 transgenic mice treated intraperitoneally for 8 weeks with ATRA (20 mg/kg, three times weekly, initiated when the mice were 5 months old). This was accompanied by a significant decrease in the APP phosphorylation and processing. The activity of cyclin-dependent kinase 5, a major kinase involved in both APP and tau phosphorylation, was markedly downregulated by ATRA treatment. The ATRA-treated APP/PS1 mice showed decreased activation of microglia and astrocytes, attenuated neuronal degeneration, and improved spatial learning and memory compared with the vehicle-treated APP/PS1 mice. These results support ATRA as an effective therapeutic agent for the prevention and treatment of AD.

ID1 and ID2 are retinoic acid responsive genes and induce a G0/G1 accumulation in acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) is uniquely sensitive to treatment with all-trans retinoic acid (ATRA), which results in the expression of genes that induce the terminal granulocytic differentiation of the leukemic blasts. Here we report the identification of two ATRA responsive genes in APL cells, ID1 and ID2. These proteins act as antagonists of basic helix-loop-helix (bHLH) transcription factors. ATRA induced a rapid increase in ID1 and ID2, both in the APL cell line NB4 as well as in primary patient cells. In addition, a strong downregulation of E2A was observed. E2A acts as a general heterodimerization partner for many bHLH proteins that are involved in differentiation control in various tissues. The simultaneous upregulation of ID1 and ID2, and the downregulation of E2A suggest a role for bHLH proteins in the induction of differentiation of APL cells following ATRA treatment. To test the relevance of this upregulation, ID1 and ID2 were overexpressed in NB4 cells. Overexpression inhibited proliferation and induced a G0/G1 accumulation. These results indicate that ID1 and ID2 are important retinoic acid responsive genes in APL, and suggest that the inhibition of specific bHLH transcription factor complexes may play a role in the therapeutic effect of ATRA in APL.

Id1 and Id2 Are Retinoic Acid Responsive Genes and Induce a G0/G1 Arrest in Acute Promyelocytic Leukemia Cells.

Acute promyelocytic leukemia (APL) is uniquely sensitive to treatment with all-trans retinoic acid (ATRA), which overcomes the differentiation arrest and induces terminal granulocytic differentiation of the leukemic blasts. In 98% of the cases of APL, the leukemic cells express a promyelocytic leukemia (PML)- retinoic acid receptor a (RARa) fusion protein as a result of a t(15;17) chromosome translocation. Previously, we have identified Id1 and Id2 as direct retinoic acid target genes being upregulated after ATRA stimulation in the APL cell line NB4 as well as in primary leukemic cells from APL patients. Id (inhibitor of DNA-binding) proteins act as antagonists of basic helix-loop-helix (bHLH) transcription factors by trapping them in heterodimeric complexes, thereby inhibiting DNA-binding and gene transactivation. Various bHLH proteins are pivotal in the control of differentiation and proliferation in various tissues (like muscle and nerve). We have studied the expression pattern of E2A, an ubiquitously expressed bHLH protein, which is generally considered as a promiscuous heterodimerization partner of other, more tissue restricted bHLH proteins. The expression of E2A was high in untreated APL cells and strongly downregulated upon ATRA stimulation. The simultaneous upregulation of Id1 and Id2, and the downregulation of E2A suggest a role for bHLH proteins in the induction of differentiation of APL cells upon treatment with ATRA. To assess the importance of Id1 and Id2 induction for the neutrophilic differentiation of the cells, we have overexpressed both proteins in the APL cell line NB4 using amphotropic retroviral transduction. Ectopic expression of Id1 and Id2 resulted in respectively 27% (n=3, SD= 9%) and 48% (n=3, SD=25%) inhibition of clonogenic growth in semi-solid medium, compared to vector-transduced control cells. Apart from the reduction in the number of colonies, overexpression of Id1 and Id2 did not alter the ATRA sensitivity of APL cells. NB4 liquid cultures revealed that Id1 and Id2 overexpression resulted in inhibition of proliferation and an increase of the percentage of cells in G0/G1, without having an effect on differentiation or apoptosis. These results indicate that Id1 and Id2 are important retinoic acid responsive genes in APL, and suggest that the inhibition of specific bHLH transcription factor complexes may play a role in the therapeutic effect of ATRA in APL.

Retinoids (All-trans and 9-cis Retinoic Acid) Stimulate Production of Macrophage Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor by Human Bone Marrow Stromal Cells

Retinoic acids (RAs) exert pleiotropic effects on cellular growth and differentiation. All-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), a stereoisomer of ATRA, induce differentiation of leukemic cell lines and cells from patients with acute myelogenous leukemia (AML) in vitro. Despite information on the effects of RAs on hematopoietic cells, little is known about how RAs act on the hematopoietic microenvironment, especially on bone marrow stromal cells. Based on recent observations that various cytokines produced mainly by bone marrow stromal cells regulate hematopoiesis, we analyzed the effects of RAs on cytokine production by these cells. ATRA or 9-cis RA treatment of human bone marrow stromal cell line KM101, which produces macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) constitutively, enhanced mRNA levels of both cytokines in a dose-dependent manner. Both RAs also stimulated M-CSF production from primary cultures of human bone marrow stromal cells. Both retinoic acid receptor (RAR)-alpha and retinoid X receptor (RXR)-alpha were expressed constitutively in KM101 cells. ATRA did not affect the expression of either receptor, whereas 9-cis RA increased RXR-alpha mRNA expression in a dose-dependent manner, but did not affect levels of RAR-alpha mRNA. These findings may have important biologic implications for both the role of RAs in hematopoiesis and the therapeutic effects of ATRA on the hematopoietic microenvironment in patients with acute promyelocytic leukemia (APL).