| Objective: To investigate the relationship between the expression of PPARγand RXRαand the inhibitory effect of pseudolaric acid B, ligand of PPARγand 9-cis-RA, ligand of RXRαon growth of human leukemia cell line HL-60 in vitro.Methods1 The Growth inhibition of a combination of PLAB and 9-cis RA on HL-60 cells was analyzed by cell viability using MTT assay.2 Microscopic structure changes and ultrastructural feature of cells were observed by light microscopy and transmission electric microcopy; Nuclear morphological assessment of apoptotic cells and dead cells were observed by fluorescence microscopy.3 The cell cycle state and apoptotic rate were examined by flow cytometry analysis (FCM).4 The DNA ladder was revealed by agarose gel electrophoresis.5 The mRNA expression of RXRα, PPARγ,CyclinD1 and CDK4 was semi-quantified by reverse transcription PCR(RT-PCR).Results1 HL-60 cells were significantly inhibited by 9-cisRA,,PLAB and a combination of PLAB and 9-cis RA. The hightest inhibitory rate of 9-cisRA combined treatment with PLAB for 96 hours was 54.57 %; 9-cisRA combined treatment with PLAB inhibited the growth of HL-60cells in a dose and time-dependment manner.2 The obvious morphological changes of cells treated with 9-cisRA combined with PLAB were detected by light microscopy. HL-60 cells became sphericalin shape and detached. Apoptotic cells were identified by the presence of cells shrinkage, plasma membrane blebbing and final separation of apoptotic bodies. Apoptotic cells nuclear chromatin condensation and fragmentation shaped bright-pearl structure by fluorescence microscopy. Apoptotic cells organelles degeration, chromatin margination and cup-shaped compact structure were revealed by electricmicrocopy.3 10μmol/L 9-cisRA combined with 0.1μmol/L PLAB could induced apoptosis of HL-60 cells in a time-dependment manner. After 96 hours treatment with 10μmol/L 9-cisRA combined with 0.1μmol/L PLAB, 54.7 % apoptotic cells were detected and a typical subdiploid peak was observed by flow cytometry,which is higher than that of 9-cis-RA or PLAB alone.4 The DNA ladder were revealed by agarose gel electrophoresis. The treatment of HL-60 cells with 10μmol/L 9-cisRA combined with 0.1μmol/L PLAB for 72, 96 and 120 hours respectively, after 72 hours induced internucleosomal DNA fragmentation in the form of a laddering pattern. The DNA ladders and their intensity increased in a time-dependment manner.5 The down-regulation expression of CyclinD1 and CDK4 mRNA occurred from 96 hours and decreased continuously while the RXRαand PPARγexpression conversely showed a significant increase. Compared to single 9-cis-RA or PLAB,combination therapy was more effective in the down-regulation expression of CyclinD1 and CDK4 mRNA and the up-regulation expression of RXRαand PPARγmRNA .Conclusion1 9-cisRA combined with PLAB has a potent growth-inhibitory effect on HL-60 cells in a dose-and time-dependent manner.2 Activation of PPARγexerts a negative regulation effect on the growth of human leukemia HL-60 cells in vitro. Activation of RXRαhas a synergic action with PPARγagonist on the growth inhibition of HL-60 cells. 3 Activation of PPARγinduces cell cycle arrest and apoptosis in HL-60 cells, which is associated with down-regulation about the expression of CDK4 and CyclinD1. These results suggest that PPARγmight be a novel therapeutic target for the human leukemia.
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