| Background:Peroxisome proliferator activated receptor (PPARy) is the member of the nucleus receptor superfamily. PPARy is originally known as the important regulatory factor in adipocyte differentiation.Recent studies evidenced that PPARy was expressed in variety tumor cells and participate in the regulation of proliferation differentiation and apoptosis of tumor cells. Some natural or synthetic ligands such as 15-Deoxy-12,14-prostaglandin J2 (15d-PGJ2, the natural ligand) or fatty acid derivatives or thiazolidinediones (TZD) can activate PPAR gamma to regulate many downstream gene including adipocyte differentiation and lipid storage gene expression. PPAR gamma agonists,as the factor which restraining or preventing the development of malignancies,could inducing tumor cell apoptosis or differentiation.Accordingly PPAR gamma agonists may become a therapeutic method for phymatology in the future. Retinoic acid receptor (RXR) belongs to the steroid/thyroid hormone receptor superfamily, and the transcription factor can be activated by ligands to regulate various aspects of life in multicellular animals.This ligands is the most successful differentiation-inducing deagents in clinical now.The retinoic acid receptor has plenty of natural and synthetical retinol ligand, including natural ligand 9-cis retinoic acid is the most efficient ligands, which activate RXR by eliminating inhibition.In the beginning PPAR gamma is used to combine with RXR alpha in a heterologous dimer in order to activate downstream gene transcriptional activity,and then caused a series of apoptosis-related gene to expression, promote the tumor cell apoptosis or differentiation.Purpose:This paper used the human osteosarcoma cell line MG-63 as the target cell to investigate the influence of 15d-PGJ2 and 9-cisRA on the viability of MG-63 cell,and to explore the possibilities mechanism of PPAR gamma combined with RXR alpha to inhibit tumor growth. Expect to find out a new target in the treatment of tumor.Methods:1.Test the anti-proliferative effect in MG-63 cell treated with 15d-PGJ2 or 9-cisRA or both of them by MTT assay.2,MG-63 cell treated with 15d-PGJ2 or 9-cisRA or both of them was used to study the effects of cell cycle and apoptosis by flow cytometry.3,Detected the expression of PPARγand RXRαin the presence of 15d-PGJ2 or 9-cisRA or both by RT-PCR.4,Detected the expression of apoptosis-related gene caspase3,p53 and Bax/bcl-2 by immunocytochemistry to investigate the possibilities mechanism of PPAR gamma combined with RXR alpha induced cell apoptosis.Results:1,MTT assay showed that uesd 15d-PGJ2 or 9-cisRA separately took inhibitory effect on the growth of MG-63 in a dose-dependent manner,and each concentration of the drugs group had significant difference compared with the control group(p<0.05). After treated for 48h the drugs got the best inhibit effect. There were no significances compared the inhibit effect of 48h,72h and 96h(P>0.05). the combination group inhibitory effect was superior to those of 15d-PGJ2 or 9-cisRA(P<0.05).2, Flow cytometry showed that the proportion of G1 phase increased and the S phase decreased when treated with 15d-PGJ2 or 9-cisRA or 15d-PGJ2 combined with 9-cisRA,and associated with the increased apoptosis rate. The proportion of G1 and apoptosis rate of combination group was obviously higher than tthat in 15d-PGJ2 or 9-cisRA grou(P<0.05).3,RT-PCR used to detect the PPARy mRNA and RXRa Mrna,and reversely transcript the stable internal standard segementβ-actin at the same time, then the ratio of two area light degree (IA) was used as the relative expression of mRNA of the receptors.There is the amount of PPARy mRNA expression in each group:in control group is 0.94±0.23,in 5μM 9-cisRA group is 1.85±0.44,in 20μM 15d-PGJ2 group is 2.06±0.35,in combination group is 2.44±0.36; The amount of RXRa mRNA expression in each group:in control group is 1.06±0.16,in 5μM 9-cisRA group is 2.13±0.12,in 20μM 15d-PGJ2 group is 1.78±0.31,in combination group is 2.33±0.47. There were significant differences in eath drug group compared with control group(P<0.05);And the expressions in combination group were higher than 15d-PGJ2 group or 9-cisRA group(P<0.05).4, Immunocytochemistry demonstrated that compared with control group, in the drug intervention group the expression of caspase3 and Bax were increased.However the expression of p53 and bcl-2 was decreased(p<0.05).And the effect of the combination group were stronger than 15d-PGJ2 group or 9-cisRA group(P<0.05).Conclusions:1,15d-PGJ2(ligands of PPARy) and 9-cisRA(ligands of RXRa) were combined or used separately could inhibit the growth of MG-63 cell.And resulting in the restriction of MG-63 cell in its G0/G1 phase of the cell cycle and enhancing the apoptosis of MG-63 cell. The combination group were stronger than 15d-PGJ2 or 9-cisRA used separately.The results show that the two drugs have additive effect.2,15d-PGJ2 and 9-cisRA induced the apoptosis of MG-63 cell may be realized by increased PPARy mRNA and RXRa mRNA level and then up-regulation of Bax and caspase3 expressions and down-regulation of bcl-2 and p53 expressions.3,PPARy and RXRa may be a novel promising strategy for the treatment of malignant tumors. |
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