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.

Study of Bcl-2 siRNA Enhancement of Sensitivity of HL-60 Cells to All Trans Retinoic Acid

OBJECTIVE To study whether siRNA targeting against the Bcl-2 gene can enhance sensitivity of HL-60 cells to all trans retinoic acid(ATRA). METHODS siRNA,which is a leading sequence selected by previous experiments,was transferred into HL-60 cells.At 6 h after transfection,the cells were cultured with ATRA.The cell growth of the HL-60 cells was measured by the MTT assay at 24, 48,72 h.The level of the Bcl-2 protein and ROS(reactive oxygen species)as well as membrane potential of the mitochondria were determined by flowcytometry. RESULTS siRNA significantly increased the inhibitory effect of ATRA on growth of the HL-60 cells.The combination of siRNA with ATRA resulted in a decrease in the Bcl-2 protein level and an increase in the ROS level as well as significantly lowering the mitochondrial membrane potential of the HL-60 cells(P<0.05). CONCLUSION Effective siRNA targeting of Bcl-2 increases the sensitivity of HL-60 leukemic cells to ATRA by inhibiting the expression of the Bcl-2 protein.

A practical strategy to subcutaneous administered in-situ gelling co-delivery system of arsenic and retinoic acid for the treatment of acute promyelocytic leukemia

Arsenic trioxide(ATO) combined with all trans retinoic acid(ATRA) is the first choice for the treatment of low and medium risk acute promyelocytic leukemia(APL). Clinical studies reported that the combination of ATO and ATRA could achieve a significant curative effect. However, the retinoic acid syndrome, serious drug resistance and the short half-life in vivo which lead to frequent and large dose administration limit the application of ATRA. In addition, the preparations of arsenic are conventional injections and tablets in clinic, which has poor patients’ compliance caused by frequent long-term administration and serious side effects. In order to overcome the above limitations, a phospholipid phase separation gel(PPSG) loaded with ATO and ATRA was developed. ATO + ATRA-PPSG(AAP), as a biodegradable sustained-release delivery system, was the first achievement of co-delivery of hydrophilic ATO and lipophilic ATRA with high drug loading which is the main problem in the application of nano preparation. The prepared PPSG displayed high safety and biocompatibility. The drug in PPSG was released slowly and continuously in vivo and in vitro for up to 10 d, which could reduce the side effects caused by the fluctuation of blood drug concentration and solve the problem of the long treatment cycle and frequent administration. In vivo pharmacokinetics depicted that PPSG could improve the bioavailability, decrease the peak concentration, and prolong the t 1/2 of ATO and ATRA. Particularly, AAP significantly inhibited the tumor volume, extended the survival period of tumor-bearing mice, and promoted the differentiation of APL cells into normal cells. Therefore, ATO + ATRA-PPSG not only could co-load hydrophilic ATO and lipophilic ATRA according to the clinical dosage, but also possessed the sustained-release and long-acting treatment effect which was expected to reduce administration time and ameliorate compliance of patients. Thus, it had great potential for clinical transformation and application.

THE DIFFERENTIATION INDUCING EFFECT OF TANSHINONE AND RETINOIC ACID ON HUMAN CERVICAL CARCINOMA CELL LINE（ME180） IN VITRO

The cervical carcinoma cell line, ME180 cells were treated with tanshinone (Tan) or retinoic acid (RA) in DMSO (final concentration 0.02%, V/V) on 4 successive days. The cells treated with the same concentration of DMSO alone served as control. Morphological studies with light and transmission electron microscopy showed that the cells treated with both Tan and RA became welldifferentiated. The cellular growth and proliferation were suppressed (as revealed by cell counting. [3H]-thymidine uptake and colony-forming assay). The number of nuclear organizer regions(AgNORs) in cells reduced and the distribution type returned nearly to normal type. The tumorigenicity in nude mice was reduced. The cell RNA dot hybridization showed that the expression of c-myc and c-Ha-ras mRNA was inhibited markedly. All above results showed that Tan and RA could reverse some malignant Phenotype and possessed differentiation inducing activity on ME180 cell line. No significant difference was observed between the cells treated with Tan and RA.

Growth inhibition of gastric cancer cells by all-trans retinoic acid through arresting cell cycle progression

Objective To investigate the mechanism of all-trans retinoic acid (ATRA) on the regulation of the cell cycle in gastric cancer cells. Methods The protein level was detected by Western blot. Immunoprecipitation was used in protein kinase activity determination. Cell growth and cell cycle phase were examined by MTT assay and flow-cytometric analysis, respectively.Results ATRA could effectively induce G0/G1 arrest and inhibit cell growth in certain human gastric cancer cell lines. ATRA might induce p21WAF1/CIP1 expression in ATRA-sensitive cell lines through p53-dependent and p53-independent pathways. Induction of p21WAF1/CIP1 caused decrease in CDK4 and CDK2 activities independent of CDK4 and CDK2 protein expression levels. In addition, the dephosphorylated form of Rb protein increased because of the down-regulation of CDK4 and CDK2 activities by ATRA. Conclusions Growth inhibition on gastric cancer cells by ATRA occurs through the regulation of relevant proteins leading to the arrest of cell cycle progression.

Mechanism of inhibition on activator protein 1 activity by all trans retinoic acid in gastric cancer cells

To determine the mechanism of all trans retinoic acid (ATRA) on growth inhibition in human gastric cancer cells Methods Gastric cancer cell lines: MGC80 3, BGC 823, SGC 7901 and MKN 45 CAT assay, Northern blot, Western blot, gene transfection and MTT assay Results ATRA can inhibit the activator protein 1 (AP 1) activity in ATRA sensitive cell lines, but not in ATRA resistant cell line, and the anti AP 1 activity of ATRA is mediated by its receptor, retinoic acid receptor α (RARα) ATRA can also inhibit the expression of cJun and cFos One of the mechanisms for ATRA to inhibit the growth of gastric cancer cells may be through its inhibitory effect on the AP 1 activity and its influence on up regulation of RARα expression The inhibition of cJun and cFos expressions by ATRA may also contribute to the anti AP 1 activity Conclusions ATRA inhibits the growth of gastric cancer cells through the regulation of AP 1 activity This action is mediated by RARα

Effect of all-trans retinoic acid on growth of xenograft tumor and its metastasis in nude mice

Objective To study the effect of all trans retinoic acid on growth of xenograft tumor and its metastasis in nude mice Methods Human gastric cancer BGC 823 and MKN 45 cells were inoculated into spleen subcapsule of nude mice, respectively The nude mice were subsequently administered with all trans retinoic acid every other day Food consuming and body weight of nude mice were measured weekly Six weeks later, the nude mice were killed Xenograft tumors in spleen and metastatic tumors in liver were pathologically examined Microvessel density in the tumors was detected immunohistochemically, and serum carcinoembryonic antigen was measured by radioimmunoassay Results After the nude mice were fed with all trans retinoic acid, the growth of splenic tumor and its liver metastasis were inhibited and the metastatic rates decreased by 50% (BGC 823) and 33 3% (MKN 45), respectively The microvessel density in splenic and hepatic tumors reduced by 28 58% and 35 47% (BGC 823), 19 45% and 14 52% (MKN 45), respectively The concentration of carcinoembryonic antigen decreased by 50 24% (BGC 823) and 48 10% (MKN 45) Conclusion All trans retinoic acid may effectively inhibit the growth of xenograft tumor in spleen and its metastasis to liver in nude mice, which can be corroborated by the decrease of carcinoembryonic antigen and microvessel density

Hemostatic abnormalities associated with acute promyelocytic leukemia and corrective effects of all-trans-retinoic acid or arsenic trioxide treatment

Objective To study in vivo effect of all trans retinoic acid (ATRA) or arsenic trioxide (As 2O 3) on the expression of tissue factor (TF) and the hemostatic disorders, a series of parameters were measured in bone marrow blasts and plasma from acute promyelocytic leukemia (APL) patients Methods The plasma variables were measured by ELISA or chromogenic study The TF transcription was assessed using reverse transcription polymerase chain reaction technique (RT PCR) Results The blast cell procoagulant activity (PCA), TF antigen of APL cell lysates, as well as the transcription of APL TF mRNA elevated at diagnosis, were reduced after ATRA or As 2O 3 therapy The plasma level of platelet α granular membrane protein 140, soluble fibrinomonomer complex, thrombomo^dulin, tissue plasminogen activator and D dimer significantly increased, fibrinogen, antigen level of protein C, plasminogen, α2 plasminogen inhibitor and plasminogen activator inhibitor decreased at diagnosis, were restored to normal after complete remission but protein C activity and protein S remained elevated in ATRA group Conclusions There existed activation of platelets and consumption of anticoagulants as well as activation of coagulation and fibrinolytic system before treatment Both ATRA and As 2O 3 therapy down regulated the expression of TF mRNA, decreased the PCA and TF level in APL cells, inhibited coagulation activation, secondary hyperfibrinolysis and recorrected other hemostatic abnormalities, thus greatly improved the bleeding symptom in early stage of the treatment

Oral encapsulated transforming growth factorβ1 reduces endogenous levels:Effect on inflammatory bowel disease

BACKGROUND TreXTAM®is a combination of the key regulatory cytokine transforming growth factor beta(TGFβ)and all trans retinoic acid(ATRA)microencapsulated for oral delivery to immune structures of the gut.It is in development as a novel treatment for inflammatory bowel disease(IBD).AIM To measure TGFβlevels in blood and tissue after oral administration of encapsulated TGFβ.METHODS Animals were orally administered encapsulated TGFβby gavage.Levels of drug substance in blood and in gut tissues at various times after administration were measured by ELISA.RESULTS We made the surprising discovery that oral administration of TreXTAM dramatically(approximately 50%)and significantly(P=0.025)reduced TGFβlevels in colon,but not small intestine or mesenteric lymph nodes.Similarly,levels in rat serum after 25 d of thrice weekly dosing with either TreXTAM,or microencapsulated TGFβalone(denoted as TPX6001)were significantly(P<0.01)reduced from baseline levels.When tested in the SCID mouse CD4+CD25-adoptive cell transfer(ACT)model of IBD,oral TPX6001 alone provided only a transient benefit in terms of reduced weight loss.CONCLUSION These observations suggest a negative feedback mechanism in the gut whereby local delivery of TGFβresults in reduced local and systemic levels of the active form of TGFβ.Our findings suggest potential clinical implications for use of encapsulated TGFβ,perhaps in the context of IBD and/or other instances of fibrosis and/or pathological TGFβsignaling.