All-trans retinoic acid regulates the expression of MMP-2 and TGF-β2 via RDH5 in retinal pigment epithelium cells

·AIM: To investigate the effect of all-trans retinoic acid(ATRA) on retinol dehydrogenase 5(RDH5), matrix metalloproteinase-2(MMP-2) and transforming growth factor-β2(TGF-β2) transcription levels, and the effect of RDH5 on MMP-2 and TGF-β2 in retinal pigment epithelium(RPE) cells.·METHODS: After adult RPE cell line-19(ARPE-19 cells) intervened with gradient concentrations of ATRA(0-20 μmol/L) for 24h, flow cytometry was used to detect the proliferation and apoptosis of cells in each group, and quantitative realtime polymerase chain reaction(q RT-PCR) was used to detect RDH5, MMP-2 and TGF-β2 m RNA expression. Then, after ARPE-19 cells transfected with three different si RNA targets for 48h, the RDH5 knockdown efficiency of each group and expression of MMP-2 and TGF-β2 m RNA within them was detected by q RT-PCR. ·RESULTS: Flow cytometry results showed that ATRA could inhibit the proliferation of RPE cells and promote the apoptosis of RPE cells, and the difference of apoptosis was statistically significant when the ATRA concentration exceeded 5 μmol/L and compared with the normal control group(P=0.027 and P=0.031, respectively). q RT-PCR results showed that ATRA could significantly inhibit the expression level of RDH5 m RNA(P<0.001) and promote the expression of MMP-2 and TGF-β2 m RNA(P=0.03 and P<0.001, respectively) in a dose-dependent manner, especially when treated with 5 μmol/L ATRA. The knockdown efficiency of RDH5 si RNA varies with different targets, among which RDH5 si RNA-435 had the highest knockdown efficiency, i.e., more than 50% lower than that of the negative control group(P=0.02). When RDH5 was knocked down for 48h, the results of q RT-PCR showed that the expressions of MMP-2 and TGF-β2 m RNA were significantly up-regulated(P<0.001).·CONCLUSION: ATRA inhibits the expression of RDH5 and promotes MMP-2 and TGF-β2, and further RDH5 knockdown significantly upregulates MMP-2 and TGF-β2. These findings suggest that RDH5 may be involved in an epithelial-mesenchymal transition of RPE cells mediated by ATRA.

CD71-mediated liposomal arsenic-nickel complex combined with all-trans retinoic acid for the efficacy of acute promyelocytic leukemia

Clinically,arsenic trioxide(ATO)was applied to the treatment of acute promyelocytic leukemia(APL)as a reliable and effective frontline drug.However,the administration regimen of AsⅢwas limited due to its fast clearance,short therapeutic window and toxicity as well.Based on CD71 overexpressed on APL cells,in present study,a transferrin(Tf)-modified liposome(LP)was established firstly to encapsulate AsⅢin arsenic-nickel complex by nickel acetate gradient method.The AsⅢ-loaded liposomes(AsLP)exhibited the feature of acid-sensitive release in vitro.Tf-modified AsLP(Tf-AsLP)were specifically taken up by APL cells and the acidic intracellular environment triggered liposome to release AsⅢwhich stimulated reactive oxygen species level and caspase-3 activity.Tf-AsLP prolonged half-life of AsⅢin blood circulation,lowered systemic toxicity,and promoted apoptosis and induced cell differentiation at lesion site in vivo.Considering that ATO combined with RA is usually applied as the first choice in clinic for APL treatment to improve the therapeutic effect,accordingly,a Tf-modified RA liposome(Tf-RALP)was designed to reduce the severe side effects of free RA and assist Tf-AsLP for better efficacy.As expected,the tumor inhibition rate of Tf-AsLP was improved significantly with the combination of Tf-RALP on subcutaneous tumor model.Furthermore,APL orthotopic NOD/SCID mice model was established by 60CO irradiation and HL-60 cells intravenously injection.The effect of co-administration(Tf-AsLP+Tf-RALP)was also confirmed to conspicuous decrease the number of leukemia cells in the circulatory system and prolong the survival time of APL mice by promoting the APL cells’apoptosis and differentiation in peripheral blood and bone marrow.Collectively,Tf-modified acid-sensitive AsLP could greatly reduce the systemic toxicity of free drug.Moreover,Tf-AsLP combined with Tf-RALP could achieve better efficacy.Thus,transferrinmodified AsⅢliposome would be a novel clinical strategy to improve patient compliance,with promising translation prospects.

Nano transdermal system combining mitochondria-targeting cerium oxide nanoparticles with all-trans retinoic acid for psoriasis

Psoriasis is an inflammatory skin disease that is intricately linked to oxidative stress.Antioxidation and inhibition of abnormal proliferation of keratinocytes are pivotal strategies for psoriasis.Delivering drugs with these effects to the site of skin lesions is a challenge that needs to be solved.Herein,we reported a nanotransdermal delivery system composed of all-trans retinoic acid(TRA),triphenylphosphine(TPP)-modified cerium oxide(CeO2)nanoparticles,flexible nanoliposomes and gels(TCeO_(2)-TRA-FNL-Gel).The results revealed that TCeO_(2)synthesized by the anti-micelle method,with a size of approximately 5 nm,possessed excellent mitochondrial targeting ability and valence conversion capability related to scavenging reactive oxygen species(ROS).TCeO_(2)-TRA-FNL prepared by the film dispersion method,with a size of approximately 70 nm,showed high drug encapsulation efficiency(>96%).TCeO_(2)-TRA-FNL-Gel further showed sustained drug release behaviors,great transdermal permeation ability,and greater skin retention than the free TRA.The results of in vitro EGF-induced and H2O2-induced models suggested that TCeO_(2)-TRA-FNL effectively reduced the level of inflammation and alleviated oxidative stress in HaCat cells.The results of in vivo imiquimod(IMQ)-induced model indicated that TCeO_(2)-TRA-FNL-Gel could greatly alleviate the psoriasis symptoms.In summary,the transdermal drug delivery system designed in this study has shown excellent therapeutic effects on psoriasis and is prospective for the safe and accurate therapy of psoriasis.

Effects of All-trans Retinoic Acid (ATRA) against Bacterial Infection in Chickens

In order to investigate the effects of all-trans retinoic acid （ATRA） against bacterial infection in chickens, 35 3-day-old AA broiler chickens were fed adaptively for two days and randomly divided into five groups, including Escherichia coli experimental group （ group 1 ）, Escherichia coli control group （group 2）, blank control group （ group 3 ）, PasteureUa experimental group （ group 4）, and PasteureUa control group （ group 5 ）. At 5 days of age, the chickens in group 1 and group 4 were drenched with 5 p.mol/kg ATRA for seven consecutive days according to their weight; the chickens in group 2, group 3 and group 5 were drenched with an equal volume of dimethyl sulfoxlde （DMSO）. The clinical symptoms and weight changes in each group were observed and recorded. Seven days later, the chickens were euthanized and dissected to determine the immune organ indexes. The results showed that there were significant differences in body weight between ATRA-administrated chickens and non-administrated chickens after bacterial infection （P 〈 0.05 ） ; moreover, the immune organ indexes of ATRA-administrated chickens exhibited significant differences compared with control group （P 〈 0.05 ）, indicating that ATRA could promote the development of immune organs of poultry, thereby enhancing the body immunity against bacterial infection.

All-trans retinoic acid(ATRA)inhibits insufficient radiofrequency ablation(IRFA)-induced enrichment of tumor-initiating cells in hepatocellular carcinoma

Objective:Local recurrence of hepatocellular carcinoma(HCC)after radiofrequency ablation(RFA)treatment remains a serious problem.Tumor-initiating cells(TICs)are thought to be responsible for tumor relapse.Here,we investigated the effect of the TIC differentiation inducer,all-trans retinoic acid(ATRA),on RFA and explored the potential molecular mechanisms.Methods:The proportions of CD133+and epithelial cell adhesion molecule(Ep CAM);TICs in recurrent HCC after RFA and primary HCC were first determined in clinic.Then,the effect of heat intervention or insufficient RFA(IRFA)on the malignant potential of HCC cells,including cell migration,sphere formation ability,tumor growth,the proportion of CD133+and Ep CAM+TICs and expression of stem cell-related genes,was evaluated in vitro and in vivo.Finally,the effect of ATRA on the tumor growth and the proportion of TICs was evaluated.Results:In clinical data,a higher proportion of CD133+and Ep CAM+TICs was found in recurrent tumors than in primary tumors.In vitro heat intervention promoted the cell migration and sphere formation ability.Additionally,it increased the proportion of CD133+and Ep CAM+TICs and the expression of stem cell-related genes.In addition,after IRFA the residual tumors in xenografts grew faster and had more TICs than untreated tumors.ATRA remarkably inhibited residual tumor growth after IRFA by elimination of TICs though the PI3 K/AKT pathway.Combination treatment with ATRA resulted in longer survival outcomes in mouse xenografts than RFA alone.Conclusions:ATRA,as a TIC differentiation inducer,could help to improve the effect of RFA treatment,which was partially attributed to its effect against TICs.The data indicated its potential as an alternative drug in the development of better therapeutic strategies for use in combination with RFA.

All-trans retinoic acid upregulates VEGF expression in glioma cells in vitro

All-trans retinoid acid （ATRA） is one of the most potent and most thoroughly studied differentiation inducers that induce the differentiation and apoptosis of glioma cells. However, the effect of ATRA on angiogenesis of glioma re- mains poorly understood. We examined the effect of ATRA on the expression of vascular endothelial growth fac- tor （VEGF） in different glioma cell lines and investigated the underlying mechanism, intending to partially reveal the effects of ATRA on angiogenesis of glioma. Glioma cells were treated by ATRA at 5 and 10 μmol/L. The VEGF mRNA transcript levels were determined by real-time RT-PCR and the protein levels of VEGF in glioma cells were evaluated by Western blotting assays. Moreover, hypoxia-inducible factor-1α （HIF-la） mRNA expression was analyzed by using real-time RT-PCR. After treatment with 5 and 10 μmol/L ATRA, the VEGF mRNA tran- script levels in glioma cells increased remarkably, compared with that in the control group, and the relative protein expression of VEGF was also up-regulated. Meanwhile, the HIF-la mRNA expression also increased. ATRA in- creases the expression of VEGF in glioma cells at both transcriptional and translational levels.

Effects of varied interferons in combination with all-trans retinoic acid ( ATRA ) on proliferation and differentiation of ATRA-resistent APL cell

Objective: To investigate the effects and mechanisms of interferon in combination with all-trans retinoic acid (ATRA) on proliferation and differentiation of ATRA-resistent APL cell. Methods:After MR2 cells (ATRA-resistance cell line) were treated with IFN-α, IFN-γand ATRA alone or IFN-αand IFN-γin combination with ATRA respectively. The cell proliferation was tested by MTT test and the cell differentiation was tested through light microscope by NBT test and flow cytometry (FCM). The expression of promyelocytic leukemia ( PML) protein was observed by indirect immune fluorescent method. Results: Both IFN-αand IFN-γcould inhibit the proliferation and induce the differentiation of MR2 cells to some extent. The effects were more obvious after both interferons in combination with ATRA respectively (P<0. 05). Moreover, the maturation of MR2 cells induced by IFN-γ+ ATRA group was more higher than that by IFN-α+ ATRA group (P<0. 05). Both interferons could induce the expressions of PML protein. Conclusion:Both interferons can inhibit MR2 cells proliferation, which may be related to the expression of PML protein induced by both interferons. The inducing differentiation effects of IFN-γ+ ATRA group on MR2 cells are more powerful than those of IFN-α+ATRA group, which may be related to the different signal transduction pathway of both interferons.

Apoptosis of Human Pancreatic Carcinoma Cells Induced By All-Trans Retinoic Acid and Interferon

Objective： To investigate the apoptosis of human pancreatic carcinoma PC3 cells induced by the combination of all-trans retinoic acid （ATRA） with interferon alpha （IFN-α）. Methods： PC3 cells were treated with ATRA and IFN-α. The inhibitory rate of PC3 cell proliferation was detected using MTT method. Cellular apoptosis was determined with flow cytometry. The percentage of PC3 cell apoptosis was assayed using TUNEL methods. Results： ATRA and IFN-α could inhibit cellular proliferation and induces cellular apoptosis of PC3 cells. The inhibitory effect was stronger when the ATRA and IFN-α were combined as a therapy. Conclusion： ATRA inhibits the proliferation of PC3 cells and induce the apoptosis of PC3 cells. The combination of IFN-α with ATRA may enhance these effects on PC3 cells.

Inhibition of matrix metalloproteinases expression in human dental pulp cells by all-trans retinoic acid

All-trans retinoic acid（ATRA） inhibits matrix metalloproteinase（MMP）-2 and MMP-9 in synovial fibroblasts, skin fibroblasts,bronchoalveolar lavage cells and cancer cells, but activates MMP-9 in neuroblast and leukemia cells. Very little is known regarding whether ATRA can activate or inhibit MMPs in human dental pulp cells（HDPCs）. The purpose of this study was to determine the effects of ATRA on the production and secretion of MMP-2 and-9 in HDPCs. The productions and messenger RNA（mRNA） expressions of MMP-2 and-9 were accessed by gelatin zymography and real-time polymerase chain reaction（PCR）, respectively. ATRA was found to decrease MMP-2 level in a dose-dependent manner. Significant reduction in MMP-2 mRNA expression was also observed in HDPCs treated with 25 mmol？L21ATRA. However, HDPCs treated with ATRA had no effect on the pattern of MMP-9 produced or secreted in either cell extracts or conditioned medium fractions. Taken together, ATRA had an inhibitory effect on MMP-2 expression in HDPCs,which suggests that ATRA could be a candidate as a medicament which could control the inflammation of pulp tissue in vital pulp therapy and regenerative endodontics.

Potential role of nuclear receptor ligand all-trans retinoic acids in the treatment of fungal keratitis

·Fungal keratitis(FK) is a worldwide visual impairment disease. This infectious fungus initiates the primary innate immune response and, later the adaptive immune response. The inflammatory process is related to a variety of immune cells, including macrophages, helper T cells, neutrophils, dendritic cells, and Treg cells, and is associated with proinflammatory, chemotactic and regulatory cytokines. All-trans retinoic acids(ATRA)have diverse immunomodulatory actions in a number of inflammatory and autoimmune conditions. These retinoids regulate the transcriptional levels of target genes through the activation of nuclear receptors.Retinoic acid receptor α(RAR α), retinoic acid receptor γ(RAR γ), and retinoid X receptor α(RXR α) are expressed in the cornea and immune cells. This paper summarizes new findings regarding ATRA in immune and inflammatory diseases and analyzes the perspective application of ATRA in FK.

Effect of All-trans Retinoic Acid on Liver Fibrosis Induced by Common Bile Duct Ligation in Rats

The aim of this study was to investigate the effect and possible mechanism of all-trans retinoic acid （ATRA） on liver fibrosis induced by common bile duct ligation （CBDL） in rats. Fifty-three female Wistar rats were randomly divided into 5 groups： sham operation group （group J, 5 animals） and groups A, B, C and D （12 animals in each group）. The rats in groups A, B, C and D were subjected to CBDL to induce liver fibrosis, while those in group J to sham operation. From the 3rd week the rats in groups B, C and D respectively received daily administration of ATRA via gas- tric tube at three different doses [0.1, 1.5 and 7.5 mg/kg body weight （BW）]. Animals were sacrificed at 6th week. Rats＇ liver tissues were observed for pathologic changes under a light microscope. The protein levels of type Ⅰ collagen （COLⅠ）, matrix metalloproteinase-2 （MMP2）, MMP13 and tissue inhibitors of metalloproteinase-1 （TIMP-1） in liver tissues were determined by immunohistochemical techniques. The expression levels of TGF-β1 and CTGF mRNA in liver tissues were detected by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR）. The results showed that loss of normal hepatic architecture and formation of obvious fibrosis were observed in group A, while ATRA treatment for 4 weeks notably alleviated the pathological changes of hepatocytes. The expres- sion of COL Ⅰ and TIMP-1 proteins in group A was increased, while decreased in ATRA-treated CBDL groups （P〈0.05）. ATRA （1.5 and 7.5 mg/kg BW） reduced the expression levels of COLⅠ protein more greatly than that of 0.1 mg/kg BW （P〈0.05）. ATRA treatment increased the protein levels of MMP2 and MMP13. The expression levels of TGF-β1 and CTGF mRNA in group A were increased. In comparison with group A, the mRNA levels of TGF-β1 and CTGF in ATRA-treated CBDL groups were significantly decreased （P〈0.05）. It was concluded that ATRA could inhibit CBDL-induced liver fibrosis in rats by suppressing the expression of TGF-β1 and CTGF so as to di- minish the inhibition of TIMP-1 on MMP2 and MMP13 and increase the activity of MMP2 and MMP13.

All-trans Retinoic Acid,Arsenic Trioxide,and Anthracycline-based Chemotherapy Improves Outcome in Newly Diagnosed Acute Promyelocytic Leukemia Regardless of FLT3-ITD Mutation Status

All-trans retinoic acid(ATRA)and pre-upfront arsenic trioxide(ATO)have revolutionized the therapy of acute promyelocytic leukemia(APL).However,internal tandem duplication of FMS-like tyrosine kinase 3(FLT3-ITD)mutations is associated with increased risk of relapse.The aim of this study was to analyze the prognostic impact of FLT3-ITD on APL patients who received remission induction with ATRA,idarubicin(IDA)and/or ATO,followed by ATRA plus ATO along with anthracycline,as consolidation therapy.A total of 72 patients newly diagnosed with APL were included in this study.83.3%of the patients achieved complete remission(CR)after induction therapy.FLT3-ITD mutations were detected in 16(22.2%)patients and closely related to bcr-3 PML-RARa transcript(P<0.001).The 5-year overall survival(OS)rate was 100%in both FLT3-ITDposltlve and FLT3-ITD^(negatlve)groups,and there was no significant difference in 5-year event-free survival(EFS)between the two groups(78.3%vs.83.3%,P=0.85).ATRA plus ATO and anthracycline-based chemotherapy achieved great outcome in newly diagnosed APL regardless of the FLT3-ITD mutation status.

Identification of target genes of transcription factor CEBPB in acute promyelocytic leukemia cells induced by all-trans retinoic acid

Objective:To indentify target genes of transcription factor CCA AT enhancer-binding protein P(CEBPB) in acute proinyelocytie leukemia cells induced by all-tram retinoie acid.Methods: A new strategy for high—throughput identification of direct target genes was established by combining chromatin immunoprecipitation(ChIP) with in vitro selection.Then,106 potential CKBPB binding fragments from the genome of the all-trans retinoie acid(ATRA)-treated NB4 cells were identified.Results:Of them,82 were mapped in proximity to known or previously predicted genes;7 were randomly picked up for further confirmation by ChlP-PCR and 3 genes (CALM,1TPR2 and 0RM2) were found to be specificaUy up-regulated in the ATRA-treated NB4 cells,indicating that they might lie the down-stream target genes of ATKA.Conclusions:Our results provided new insight into the mechanisms of ATRA-induced granulocytic differentiation.

MGMT is down-regulated independently of promoter DNA methylation in rats with all-trans retinoic acidinduced spina bifida aperta

O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day(E) 10, whereas normal control rats received the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2 A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2 A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methylation demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is independent of promoter DNA methylation.

VARIATION OF SERUM G-CSF LEVEL IN APL TREATED WITH ALL-TRANS RETINOIC ACID

Objective: To detect the level of serum G-CSF. from patients with acute promyelocytic leukemia pre- or post-treatment with ATRA and analyze the relationship between serum G-CSF and hyperleukocytosis. Methods: Enzyme-linked immunosorbent assay (ELISA) method was developed and used in detecting serum G-CSF. Linear correlation test and Spearman rank order correlation coefficient were used as the statistical analytical method. Results: The levels of serum G-CSF increased in 11.4% (4/35) of APL patients (equal of more than 0.095 ng/ml). It was also found that serum G-CSF level in 25 APL patients started to increase from the 6th day to 12th day and then gradually declined after treatment with ATRA. Both serum G-CSF and WBC numbers increase in 72% (18/25) patients; no obvious variation of WBC and increase of serum G-CSF and augmentation of WBC were seen in 12% (3/25) of the cases with APL. It was also demonstrated that serum G-CSF level was statistically related to the WBC number (r=0.275,P<0.05), promyelocytes (r=0.2015,P<0.05) or more matured granulocytes (r=0.2055P<0.05) by Spearman rank correlation analysis. Conclusion: The results of this study strongly indicate that G-CSF variation in patients with APL after treatment with ATRA plays an important role in hyperleukocytosis of WBC increase.

Downregulation of Astrocyte Elevated Gene-1 Expression Combined with All-Trans Retinoic Acid Inhibits Development of Vasculogenic Mimicry and Angiogenesis in Glioma

Objective This study aimed to investigate the effects of downregulating astrocyte elevated gene-1(AEG-1)expression combined with all-trans retinoic acid(ATRA)on vasculogenic mimicry(VM)formation and angiogenesis in glioma.Methods U87 glioma cells were transfected with AEG-1 shRNA lentiviral vectors(U87-siAEG-1)and incubated in a medium containing 20µmol/L ATRA.Matrigel-based tube formation assay was performed to evaluate VM formation,and the cell counting kit-8(CCK-8)assay was used to analyze the proliferation of glioma cells in vitro.Reverse transcription-quantitative polymerase chain reaction and Western blot analysis were used to investigate the mRNA and protein expression of related genes,respectively.Glioma xenograft models were generated via subcutaneous implantation of glioma cells in nude mice.Tumor-bearing mice received an intraperitoneal injection of ATRA(10 mg/kg per day).Immunohistochemistry was used to evaluate the expression of related genes and the microvessel density(MVD)in glioma xenograft models.CD34/periodic acid-Schiff double staining was performed to detect VM channels in vivo.The volume and weight of tumors were measured,and a tumor growth curve was drawn to evaluate tumor growth.Results A combination of ATRA intervention and downregulation of AEG-1 expression significantly inhibited the proliferation of glioma cells in vitro and glioma VM formation in vitro and in vivo.It also significantly decreased MVD and inhibited tumor growth.Further,the expression levels of matrix metalloproteinase(MMP)-2,MMP-9,vascular endothelial-cadherin(VE-cadherin),and vascular endothelial growth factor(VEGF)in glioma significantly decreased in vivo and in vivo.Conclusion Hence,a combinatorial approach might be effective in treating glioma through regulating MMP-2,MMP-9,VEGF,and VE-cadherin expression.

Effect of All-trans Retinoic Acid on Airway Inflammation in Asthmatic Rats and Its Mechanism

Summary: The inhibitive effects of all-trans retinoic acid (ARTA) on airway inflammation in asthmatic rats and its mechanism on the basis of the regulation of nuclear factor kappaB (NF-κB) were explored. Thirty-two SD rats were randomly divided into 4 groups: control group, asthma group, dexamethasone treatment group and retinotic acid treatment group. The total and differential cell counts in the collected bronchoalveolar lavage fluid (BALF) were measured. The pathological changes in lung tissues were estimated by scoring. The expression of NF-κB inhibitor (IκBa), NF-κB, intercellular adhering molecule-1 (ICAM-1) in lung tissue was detected by immunohistochemical method. The results showed that in the two treatment groups, the total cell counts and proportion of inflammatory cells in BALF were significantly reduced, but there was no significant difference in differential cell counts in BALF between them. The pathological changes in lung tissues in the treatment groups were significantly attenuated as compared with asthma group. Except the epithelial injury in retinotic acid treatment group was milder than in dexamethasone treatment group, the remaining lesions showed no significant difference between them. In the two treatment groups, the expression of IκBa was increased, while the expression of NF-κB and ICAM-1 decreased with the difference between the two groups being not significant. It was concluded that the similar anti-inflammatory effects and mechanism of ATRA on airway in asthmatic rats to those of dexamethasone were contributed to the increase of cytoplasmic IκBa content and suppression of NF-κB activation and expression.

The Effects of All-Trans Retinoic Acid on Vasculogenic Mimicry Formation Ability in CD133+ Glioma Stem Cells and Its Mechanisms

Objective: to investigate the effects of all-trans retinoic acid (ATRA) on vasculogenic mimicry formation in glioma stem cells. Methods: U87 stem cells were harvested through a suspension culture assay from the U87 cells, identified by CD133 and nestin, and counted by a flow cytometry. To investigate the VM formation ability of U87 stem cells with the treatment of various concentrations of ATRA, a Matrigel-based tube formation assay was used in the present study in vitro and tube-like structure (typical tube, TT;atypical tube AT) was observed and counted. Then the expressions of VEGF, VEGFR-2 and CD133 were measured throughout real time q-PCR, western blotting and immunofluorescence techniques. The data, presented as the mean ± standard deviation, were analyzed using SPSS software. One-way analysis of variance was used to compare groups and Fisher’s least significant difference tests were performed for subsequent comparisons between groups. P Results: Most of the harvested spheroid cells were positive for nestin and 88.4% were positive forCD133. The CD133+ U87 cells were cultured into tube like structure loaded on the top of Matrigel and the quantity of tubes was decreased under the treatment of ATRA. In addition, the expressions of VEGF, VEGFR-2 and CD133 were significantly reduced under the treatment of ATRA, particularly in the higher concentration groups (20 and 40 μmol, P Conclusions: ATRA may inhibit the establishment of VM differing from stem cells in glioma, and these effects may attribute to the effects of ATRA’s promotion of the differentiation of stem cells and/or down regulation of the expressions of proangiogenic factors VEGF and its receptor VEGFR-2. Thus, the results of the present study indicated a novel idea for the treatment of GBM and enriched the anti-glioma mechanisms of ARTA.

Identification of tumor invasion-related differentially expressed genes in different grades and all-trans retinoic acid-treated astrocytoma cell lines

BACKGROUND： Although several genetic aberrations and gene expressional changes have been shown to exist in tumors and different grades of astrocytomas, as well as in normal tissues, the gene profiling and genetic pathways associated with malignant transformation and progression remain unclear. OBJECTIVE： To identify differentially expressed genes related to tumor invasion from various grades and all-trans retinoic acid （ATRA）-treated astrocytoma cell lines by cDNA microarray. DESIGN, TIME AND SETTING： In vitro gene experiment was performed at the Department of Neurobiology, Third Military Medical University of Chinese PLA from January to October 2007. MATERIALS： Two different grades of astrocytoma cell lines CHG-5 （WHO grade II ） and SHG-44 （WHO grade IV） were developed by our laboratory; a cell differentiation-inducing agent ATRA and a human cDNA microarray technology were used to determine differentially expressed genes （City University of Hong Kong）. METHODS： Total RNA was extracted using the Trizol test kit. Reverse transcription was performed using Superscript 11 reverse transcriptase. The cDNA product （target DNA） was marked with fluorochromes Cy3 （normal SHG-44） and Cy5 （CHG-5 or ATRA-treated SHG-44）, followed by chip hybridization. MAIN OUTCOME MEASURES： Gene expression profiles of CHG-5 vs. SHG-44 and ATRA-treated vs. normal SHG-44 were performed to identify differentially expressed genes. Several of these genes were randomly selected for Northern Blot analysis. The identification of genes that were similarly regulated （overlapping） was performed by comparing gene expression profiles between CHG-5 and SHG-44 cells, and between SHG-44 cells with or without treatment with ATRA. RESULTS： No significant differences were observed between CHG5 and SHG-44 cell line morphology. Under confocal microscopy, GFAP staining intensity of CHG5 cells was greater than SHG-44 cells （t = 6.078 P = 0.004）. Growth curve analysis demonstrated that the speed of SHG-44 cell growth was greater than CHG5 cells. Flow cytometry analysis showed that the number of ATRA-treated SHG-44 cells at G0/G1 stage increased by 15%, compared with normal SHG-44 cells （P 〈 0.05）. A total of 31 known genes with altered expression were identified in this study. Among them, 20 genes were upregulated and 11 were downregulated in CHG-5 compared with SHG-44 cells, and ATRA-treated SHG-44 compared with untreated SHG-44 ceils. Four of these reported genes （CD151, G3BP, UGB, and CSTB） were shown to be involved in tumor invasion. Validation of a selection of differentially expressed genes was perfonlaed by Northern blot. CONCLUSION： A total of 31 known genes were demonstrated by cDNA microarray to relate to the malignant progression of astrocytomas, and four differentially expressed genes （CD151, G3BP, UGB, and CSTB） were shown to relate to tumor invasion.

Interon-gamma Enhances the Antitumor Effect of All-trans Retinoic Acid on Hepatocellular Carcinoma Cells by Inhibiting the Expression of Nuclear Factor-kappaB

Objective： To explore the combination effects of all-trans retinoic acid （ATRA） with interferon-gamma （IFN-γ） on human hepatocarcinoma cell line SMMC-7721 and the mechanism of action. Methods： SMMC-7721 cells were divided into treated group and control group. The cells were treated with ATRA or ATRA＋ IFN-γ in the former and added with PBS in the latter. The inhibition rate of SMMC-7721 cell proliferation was detected by MTT, the cell change in morphology was observed by electron microscope. The apoptosis was detected by flow cytometry and the expression changes of nuclear factor-kappaB （NF-κB） was analyzed by Western blotting when the SMMC-7721 cells were treated with ATRA and IFN-γ. Results： The SMMC-7721 cell proliferation was suppressed and apoptosis was induced after the cells were treated with ATRA treatment, and these effects were enhanced when ATRA was combined with IFN-γ. The expression of NF-κB was reduced after SMMC-7721 cell was treated with ATRA, and reduced significantly when the cells were treated with the combination of ATRA and IFN-γ. Conclusion： IFN-γ can enhance the inhibiting effects of ATRA on cell proliferation and inducing apoptosis on SMMC-7721 cell and these effects might be mediated by inhibiting the expression of NF-κB.