| 5-4A,a new retinoid,was developed by our institute. The induction of cell differentiation,anti-tumor effects,anti-invasion and its mechanism of action of 5-4A were studied in this paper. The results showed that the 5-4A significantly inhibited the growth and colony formation of human promyelocytic leukemia HL-60 cells, while the NBT reduction , the acid phosphatase activity and the phagocytic activity(the functional differentiation makers of granulocytes)were dramatically augmented when the cells were exposed to 5-4A at the concentration of 10-8-10-5mol/L. The NBT positive cells reached 90% at 10<sup>-6 mol/L after 6 days exposure. 42% of the whole cells exhibited morphological changes as metamyelocyte and banded or segment cells. The naphthol AS-D choroacetate esterase activity of the HL-60 cells induced by 5-4A shows that the cells differentiated along the granulocytic lineage.Studies also show that 5—4A has effects on the human histiocytic lymphoma cell line U937. when the cells were exposed to 5-4A at the concentration of 10-5-10-7mol/L, cell proliferaion was inhibited and the NBT reduction ability, the phagocytic activity in the cells were increased. Morphologically 80% of the cells differentiated along the monocytic lineage. Flow cytometry demonstrated that 5-4A as well as RA resulted in the arrest of cells in G1 phase and that the cell population in S and G2/M phase were decreased.The results also show that the cell proliferation and colony formaing efficiency of the human hepatocarcinoma Be17402 cell line were significantly inhibited when 5-4A were added at the concentration of 5x10-5mol/L for 4-6 days. The secretion of alpha-fetoprotein(AFP) and the activity of r-glutamyltranspeptidase(r-GT) were decreased, while the secretion of albumin was increased dramatically. It was also found that the cells treated with 5-4A changed morphologically. The ratio of nucleus and cytoplasm were decreased and the number of mitochondria and endoplasmin reticulum were increased. By using the [3H] precursor incorporation test, the inhibition of synthesis of protein and DNA was observed within two days after treated with 5-4Aat 5x10-5mol/L. These results suggest that 5-4A can induce the Be17402 cell differentiation.Being given orally to rats bearing chondrosarcoma at the dosage of 20mg/Kg for 20 days, this compound significantlly inhibited the growth of the chondrosarcoma and the inhibition rates reached 99.9%. In vitro studies demonstrate that 5-4A shows less cytotoxic effects on a varity of human cancer or normal cells. The EC50is above 10-4mol/L. Acute toxicity studies show that the LD50 of 5-4A for mice is 3.81g/Kg and it is lower than that of RA.The selective effects of 5-4h were observed between cancer cell line and the normal cell line. 5-4A can dramatically inhibit the colony formation efficiency of HL-60 cells, while it has little effect on the CFU-GM of bone marrow cells in mice at the same concentraion. These results demonstrate that 5-4A has a higher selectivity for cancer cells.The effect of 5-4h on the invasion of the tumor cells was also investigated. At the concentration of 8x10-5mol/L, 5-4A significantly inhibited the invastion of B16BL6 cells. The inhibition rate reached 70% within six hours. When the HT1080 cells were exposed to 5-4A at the concentration of 10-5-10-7 mol/L for 3 days, the collagenaseⅣactivity was significantly inhibited by 5-4A.The apoptosis induced by 5-4A in HL-60 cells was also studied. In agarose gel electrophoresis, DNA extracted from HL-60 cells treated with 10-6-10-8 mol/L of 5-4A show a typical DNA ladder and morphological changes as nuclear chronsome condensation as well as apoptotic body. In addition, the characteristic apoptotic DNA peak of cells was revealed by flow cytometry.Retioids are thought to act through nuclear retinoic acid receptors. which in turn act on some target gene. Our results show that 5-4A can induce differentiation of HL-60 cell but can not induce differentiation of HL-60/RA cell. By using a binding assay of RARα, the binding activity of 5-4A with RARαcan be observed but it is lower than that of RA. Northernbot and Dot blot analysis of total RNA with cDNA probes specific to RARs(RARα,RARβ,RARγ) and RXRαindicated that the RARexpression and RARγexpression in HL-60 were enhanced by 5- 4A, while the RXRa expression was not affected at the concentration of 5-4A (10-6mol/L) for 24 hours. Additional dot blot analysis exhibited that the c-myc expression was inhibited by 5-4A in HL-60 cells, while the c-fos expression was enhanced by 5-4A. These results demonstrated that 5-4A modulates oncogene expression and induces cell differentiation through RARs. Retinoic acid have a broad spectrum of biological activites in embryonic development and cell differentiation and proliferation. It shows significant therapeutic effects in the treatment of acute promyelocytic leukemia. This effect is thought to be the result from the interaction of retinoic acid with nuclear retinoic acid receptor. To investigate the retinoic acid receptor or screen for the retinoids by using the receptor is one of the central point in the differentiation therapy of cancer. As the concentration of retinoic acid receptor is very low in cells, it is hard to get enough receptor from the cells for screening of the new retinoids. The methods for obtaining the retinoic acid receptor by bioengineering technique is probably the only effective way for providing retinoic acid receptor.In this paper, the human RARαcDNA was cloned into the PT7-7 plasmids and the RARαexpression vector—PT7-7-RARαwas obtained. The recombinant protein was expressed in Escherichia coli. The sequence analysis shows that the RARαcDNA sequenceand the site in PT7-7 is the same as the PT7-RARαrecombinant vector reported in other paper. Western blotting assay shows that a specific band was observed in the site with a molecular mass of 53.0 KDa. By using the charcoal absorption hormone binding assay, the ligand-binding activity of the receptor was observed.For receptor binding assay, five retinoidswerechosen. The binding of retinoids to RARαwas investigated comparing to the ability of these compounds to induced differentiation of HL-60 cell line. The results show that the binding activity of these retinoids was well correlated to the NBT reduction and the acid phosphatase activity of HL-60 cell, which serve as differentiation marker in this assay. |
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