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.

All-trans retinoic acid alleviates transmissible gastroenteritis virus-induced intestinal inflammation and barrier dysfunction in weaned piglets

Background Transmissible gastroenteritis virus(TGEV)is one of the main pathogens causing severe diarrhea of pig-lets.The pathogenesis of TGEV is closely related to intestinal inflammation.All-trans retinoic acid(ATRA)is the main active metabolite of vitamin A,which has immunomodulatory and anti-inflammatory properties.However,it is unclear whether ATRA can alleviate TGEV-induced intestinal inflammation and barrier dysfunction in piglets.This study aimed to investigate the effects of ATRA on growth performance,diarrhea,intestinal inflammation and intesti-nal barrier integrity of TGEV-challenged piglets.Methods In a 19-d study,32 weaned piglets were randomly divided into 4 treatments:Control group(basal diet),TGEV group(basal diet+TGEV challenge),TGEV+ATRA5 group(basal diet+5 mg/d ATRA+TGEV challenge)and TGEV+ATRA15 group(basal diet+15 mg/d ATRA+TGEV challenge).On d 14,piglets were orally administered TGEV or the sterile medium.Results Feeding piglets with 5 and 15 mg/d ATRA alleviated the growth inhibition and diarrhea induced by TGEV(P<0.05).Feeding piglets with 5 and 15 mg/d ATRA also inhibited the increase of serum diamine oxidase(DAO)activ-ity and the decrease of occludin and claudin-1 protein levels in jejunal mucosa induced by TGEV,and maintained intestinal barrier integrity(P<0.05).Meanwhile,5 mg/d ATRA feeding increased the sucrase activity and the expres-sions of nutrient transporter related genes(GLUT2 and SLC7A1)in jejunal mucosa of TGEV-challenged piglets(P<0.05).Furthermore,5 mg/d ATRA feeding attenuated TGEV-induced intestinal inflammatory response by inhibit-ing the release of interleukin(IL)-1β,IL-8 and tumor necrosis factor-α(TNF-α),and promoting the secretion of IL-10 and secretory immunoglobulin A(sIgA)(P<0.05).Feeding 5 mg/d ATRA also down-regulated the expressions of Toll-like receptors and RIG-I like receptors signaling pathway related genes(TLR3,TLR4,RIG-I,MyD88,TRIF and MAVS)and the phosphorylation level of nuclear factor-κB-p65(NF-κB p65),and up-regulated the inhibitor kappa B alpha(IκBα)protein level in jejunal mucosa of TGEV-challenged piglets(P<0.05).Conclusions ATRA alleviated TGEV-induced intestinal barrier damage by inhibiting inflammatory response,thus improving the growth performance and inhibiting diarrhea of piglets.The mechanism was associated with the inhibi-tion of NF-κB signaling pathway mediated by TLR3,TLR4 and RIG-I.

Inhibition of all-trans retinoic acid on MDM2 gene expression in astrocytoma cell line SHG-44

Objective To investigate the impact of all-trans retinoic acid （ATRA） on MDM2 gene expression in astrocytoma cell line SHG-44, and to provide basic data for further research on the progression mechanism and gene therapy of human astrocytoma. Methods The differential expressions of MDM2 gene and protein in SHG-44 cells were detected by cDNA microarray and Western blot, respectively, before and after treatment of ATRA. The expressions of MDM2 protein in WHO grade Ⅱ and grade Ⅳ astrocytomas were determined by immunohistochemical streptavidin-peroxidase method. Some differentially expressed genes were selected randomly for Northern blot analysis. Results The intensity ratio of ATRA-treated to untreated SHG-44 cell was 0.37 in the cDNA microarray, suggesting that the expression of MDM2 gene was down-regulated in SHG-44 cells after treatment with ATRA. Some genes differentially expressed in the microarray were confirmed by Northern blot. Western blot demonstrated that the optical density ratios of MDM2 to β-actin in ATRA-treated and untreated SHG-44 were 14.02±0.35 and 21.40±0.58 （t = 24.728, P = 0.000）, respectively, suggesting that the expression of MDM2 protein was inhibited in ATRA-treated SHG-44 cells. Moreover, the percentages of MDM2-positive protein were 24.00% （6/25） and 56.52% （13/23） （x^2 = 5.298, P = 0.021） in WHO grade Ⅱ and grade Ⅳ astrocytomas, respectively, suggesting that the expression of MDM2 protein may increase along with the elevation of astrocytoma malignancy. Conclusion ATRA can inhibit MDM2 gene expression in SHG-44 cells, and MDM2 is related to astrocytoma progression.

THE STUDY ON POST-REMISSION THERAPIES IN PATIENTS WITH APL AFTER COMPLETE REMISSION INDUCED BY ALL-TRANS RETINOIC ACID

As to determine the effect of post-remission therapy in prolonging survival and duration of remission after complete remission, 50 patients with APL In complete remission Induced by retinolc acid (RA) were divided into three groups randomly: (A) 30 cases, treated by alternate chemotherapy with RA; (B) 10 cases, with RA alone; (C) 10 cases, only with chemotherapy.The survival curves showed mat Group A had the survival time more than 1 year In 87. 4 %, more than 2 year in 80.7%. 26/30 cases were survival and still in remission, the survival curve tend to be a plateau at 16 months. In Group B more than 1 year in 45. 7%. In Group C, more than 1 year In 50%. (Keplan-Meler x2 = 8. 93 P <0. 01).This result showed that the alternate chemotherapy with RA for post- Induction remission therapy could be useful to Improve long-term survivors and to prolong the duration of remission.

Synthesis and anti-tumor activity of all-trans retinoic acid derivatives

A series of retinoate and retinamide derivatives were designed, synthesized, and their anti-tumor activities were investigated in NB4 by MTT and flow cytometry assays （FCM）. All compounds showed cytotoxicity, especially compounds la and ld exhibited a higher cytotoxicity than other derivatives and all-trans rethaoic acid （ATRA）. Furthermore, compound 1d could induce NB4 cell lines differentiation efficiently. O 2009 Fei Hu Chert. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

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.

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.

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.

THE EFFECT OF ALL-TRANS RETINOIC ACID ON GAP JUNCTIONAL INTERCELLULARCOMMUNICATION AND CONNEXIN 43 GENE EXPRESSION IN GLIOMA CELLS

To illuminate the regulating effect of all trans retinoic acid (ATRA ) on gap junctional intercellular communication (GJIC) and connexin 43 (Cx43) ge ne expression in glioma cells, which is tissue and organ specific. Method. Rat C6 glioma cells were exposed to ATRA at a concentration of 1, 10, 10 0 μmol/L respectively, and the GJIC function of the cells was examined with scr ape loading dye transfer assay 24 hours, 48 hours and 72 hours after ATRA treat ment. The effect of ATRA on Cx43 gene expression was measured with semiquantitat ive reverse transcription polymerase chain reaction (RT PCR) 24 hours after ATR A exposure. Results. The GJIC function of C6 glioma cells was significantly increased by ATR A at each concentration applied. The dye passed 4 to 5 rows of cells from the sc raping edge in ATRA treated cells, but only 1 or 2 rows in the control. The augm ent effect was observed 24 hours after each concentration ATRA treatment, and la sted till 72 hours after treatment with 1μmol/L and 10μmol/L ATRA. Forty eigh t hours after exposed to 100μmol/L ATRA, the enhancement of GJIC was less obvi ous. There was no significant increase induced by ATRA on the transcription of C x43 gene, as demonstrated by semiquantitative RT PCR. Conclusion. ATRA turned out to be a potent enhancer on GJIC function in C6 gliom a cells, and the enhancement effect was most probable at post transcriptional l evel.

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.

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.

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.

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.

Apoptosis of Glioblastoma U251 Cells Induced by Carmustine Combined All-trans Retinoic Acid via Regulating Cyclin E and p27kip 1

The effect and mechanism of carmustine（BCNU） combined with all-trans retinoic acid（ATRA） on the apoptosis of human glioblastoma U251 cells were investigated by means of 3-（4,5-dimethylthiazol-2-yl）-2,5-diphe- nyltetrazolium bromide（MTT） assay, flow cytometry, reverse transcription-polymerase chain reaction（RT-PCR） and Western blot analysis. The results show that BCNU or ATRA shows time- and dose-dependent inhibition effects on human glioblastoma U251 cells and the combination of BCNU with ATRA shows an synergistic inhibition effect on human glioblastoma U251 cells, and the combined BCNU and ATRA can significantly inhibit the proliferation of human glioblastoma U251 cells, and induce the apoptosis of them, making the cells arrest in the stage of G1 phase, the stage of S and G2 phases decline, the rate of the apoptosis of human glioblastoma U251 cells increase, the corresponding mRNA expression of cyclin E and cyclin-dependent kinase 2（CDK2） downregulated and the correspon- ding mRNA expression of p27kip 1 unregulated. In addition, the combined BCNU and ATRA reduced the protein expression of nuclear factor kappa B（NF-κB）. Taken together, these results suggest that the treatment of human glioblastoma U251 cells with a combination application of ATRA and BCNU can exert synergistic effect, the course of this kind of combination chemotherapy may likely be associated with multiple molecular mechanisms for apoptosis, furthermore, the cyclin E and p27kip 1 should be considered as novel targets for controlling the growth of glioblastoma cells.

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.

EXPERIMENTAL STUDY OF THE DIFFERENTIATION HPV16 SUBGENES-IMMORTALIZED HUMAN ENDOCERVICAL CELLS INDUCED BY ALL-TRANS-RETINOIC ACID

Objective： To investigate the differentiation-inducing effects of all-trans-retinoic （ATRA） to HPV16 subgenesimmortalized human l：ndocervical cells （H8 cell） in vitro. Methods： HPV16 subgenes-immortalized human endocervical cells （H8 cells） were cultured in vitro. After treated with ATRA, the proliferation of immortalized human endocervical cells was measured by MTT assay; morphological changes were observed using M and TEM; cell cycle was analyzed by FCM; expression of Ki67 was tested using immunocytochemistry and the activity of telomerase was tested using PCR-ELISA. Results： ATRA could inhibit proliferation of H8 cells significantly and induce their morphodifferentiation. According to FCM, H8 cells accumulated in G1 phase and expression of Ki67 and activity of telomerase reduced significantly after treatment with ATRA. Conclusion： ATRA could induce the differentiation of H8 cell line obviously, which might be achieved by inhibiting proliferation, blocking cell cycle, and reducing activity of telomerase.

EXPRESSION OF IL-6, sgp130, IL-8 AND THEIR RECEPTORS INACUTE PROMYELOCYTIC LEUKEMIA DURING ALL-TRANSRETINOIC ACID INDUCTION TREATMENT

Objective: To evaluate the expression and its clinical significance of interleukin 6 (IL-6), soluble glycoprotein 130 (sgp130), interleukin 8 (IL-8) and type A interleukin 8 receptor (IL-8RA) in acute promyelocytic leukemia (APL) patients during all-trans retinoic acid (ATRA) induction treatment. Methods: Plasma and bone marrow mononuclear cell (MNC) culture supernatant IL-6, sgp130, IL-8 concentration of 18 cases with APL were kinetically measured in vivo and in vitro (ELISA). Bone marrow MNC IL-8RA was measured by flow cytometry after being cultured with ATRA (10?6mmol/L). Results: Plasma IL-6, sgp130, IL-8 levels were higher than normal (P<0.05), IL-6, spg130 levels correlated with white blood cell (WBC) counts (P<0.05) while IL-8 levels correlated with body temperature (P<0.05) at initial diagnosis. After 72-hour incubation with ATRA, concentration of IL-6 of bone marrow MNC culture supernatant did not change, that of sgp130 mildly decreased, and IL-8 significantly decreased while the positive rate of IL-8RA on bone marrow MNC increased. During ATRA treatment, plasma IL-6 changes were correlated with WBC counts. Peak levels of IL-6 and WBC were lower in patients who received intermittent therapy than those who received continuous therapy. Plasma IL-6 and IL-8 were increased when complicated with infection and IL-8 seemed more sensitive. Conclusion: Plasma IL-6, sgp130, IL-8 levels may reflect patients' responsiveness to ATRA treatment, and could be used to predict hyperleukocytosis and intercurrent infection. ATRA induces APL cell differentiation possibly via sgp130 signal transducer chain. Measurement of sgp130 had certain meaning to prognosis.

Induction of apoptosis and change of bcl-2 expression in macrophage Ana-1 cells by all-trans retinoic acid

Macrophage cells play an important role in the initiation and regulation of the immune response. All-trans retinoic acid (ATRA) and its natural and synthetic analogs (retinoids) affect a large number of biological processes.Recently , retinoids have been shown promise in the therapy and prevention of various cancers. However, many interesting questions related to the activities of retinoids remain to be answered: (Ⅰ) Molecular mechanisms by which retinoids exert their effects; (Ⅱ) why the clinical uses of retinoids give undesirable side effects of varying severity with a higher freqllency of blood system symptoms; (Ⅲ)little is known for its impacts on macrophage cells etc. We set up this experiment, therefore, to examine the apoptosis of ATRA on macrophage Ana-1 cell line. Apoptosis of the cells was quantitated, after staining cells with propidium iodide (PI), by both accounting nuclear condensation and flow cytometry. When the cells were treated with ATRA at or higher than 1 μM for more than 24 h, significant amount of the apoptotic cells was observed. Induction of apoptosis of Ana-1 cells by ATRA was in time- and dose-dependent manners, exhibiting the similar pattern as the apoptosis induced by actinomycin D (ACTD). ATRA treatment of Ana-1 cells also caused the changes of the mRNA levels of apoptosis-associated gene bcl-2, as detected by Northern blot analysis. The temporal changes of bcl-2 expression by ATRA was also parallel to that by ACTD. In conclusion,ATRA can induce apoptosis in macrophage cells, which may be helpful in understanding of immunological functions retinoids.