Effects Of PPARγ And Its Agonist On Colorectal Cancer And Its Correlation With MMPs And TIMPs

Peroxisome proliferator-activated receptors(PPARs)are ligand-dependent transcription factor belonging to the nuclear receptor superfamily.Three subtypes of PPARs(α,β,γ)have been identified.PPARγhas many biological functions such as participate in cell growth,development,metabolism,and differentiation.It was recently reported that PPARγagonist could inhibit cell proliferation,induce cell cycle arrest and apoptosis,inhibit angiogenesis.PPARγagonist has a potential antitumor effect and became a new focus.PPARγis activated after binding to ligand,then the heterodimers bind to the peroxisome proliferator response elements(PPREs)in the promoter regions of target genes,and regulate transcription of target genes.PPARγligands include synthetic ligands and natual ligands.Natual ligands are prostaglandin-derived compounds such as 15d-PGJ₂ which is widely used.Synthetic ligands are thiazolidinediones such as rosiglitazone and troglitazone.The mechanisms of PPARγagonists antitumor effects:1.inhibition of cell proliferation Several studies reported that PPARγagonists may inhibit the expression of cyclinD1,cyclinB,cyclinE,CDK4 and CDK2,up-regulate p21 and p27,inhibit cell proliferation and growth.2.induction of apoptosis PPARγagonists could up-regulate the expression of apoptosis protein BAX and BAD,caspases were activiated which leading to apoptosis.3.induction of differentiation The activation of PPARγinitiated the apoptosis proseure at the same time the transcription and expression of differential genes started,which could reverse tumor cells to normal cells.4.inhibition of angiogenesis PPARγagonists may inhibit endothelial cells proliferation,induce endothelial cells apoptosis,down-regulate endothelial cell growth factors.5.decrease tumor cell invasion.PPARγagonists may suppress some tumor cells growth,there was few reports about PPARγagonists how to affect tumor cell invasion.MMPs can degradate almost all of extracellular matrix component,remodel cell adhesion, promote the synthesis and releasing vascular growth factor,participate in tumor immunity.MMPs play critical role in tumor invasiveness and metastsis.Our studies detected the expression of PPARγand MMPs,TIMPs in colorectal cancer,and explored the antitumor effect of PPARγagonist in HCT-8,at the same time,investigated the relation between PPARγand MMPs,TIMPs.The results show as follows:1.The expression of PPARγmRNA and protein in colorectal tumorThe exprssion of PPARγmRNA and protein were detected in 38 cases of colorectal tumor tissues and 20 cases of surrouding normal tissues by immunohistochemistry and RT-PCR.The results showed that the positive rate of PPARγprotein was significantly higher in colorectal tissue than in surrouding normal tissue(p＜0.05).It have not been found that there was correlation between the expression of PPARγprotein and the type of colorectal tumor. PPARγmRNA in colorectal tumor tissue increased significantly than that in relative normal tissue(p＜0.05).This suggested that the detection of PPARγmRNA and protein may serve as a molecular marker in colorectal tumor.2.The expression of MMP-2,MMP-7,MMP-9,TIMP-1,TIMP-2 protein in colorectal tumor and the correlation with PPARγexpressionThe exprssion of MMP-2,MMP-7,MMP-9,TIMP-1,TIMP-2 protein were detected by immunohistochemistry in 38 cases of colorectal tumor tissues and 20 cases of surrouding normal tissues.Results showed the positive rate of MMP-2,MMP-7,MMP-9,TIMP-1 protein were significantly higher in colorectal tissues than in surrouding normal tissueit(p＜0.01).The positive rate of TIMP-2 in colorectal tumor and surrouding normal tissues had no significant difference.The positive correlation between PPARγand MMP-7 was observed in colorectal tumor,and the negative corrlation between PPARγand MMP-2,MMP-9,TIMP-1 was observed,too.These results suggested that PPARγmay affect the exprssion of MMP-2,MMP-7,MMP-9,it may have close relationship with the development of colorectal tumor.3.growth inhibition of rosiglitazone on HCT-8 cellsHCT-8 cells were cultivated in DMEM(high glucose).The expression of PPARγmRNA and protein were detected in HCT-8 cells by immunocytochemistry and RT-PCR.It was confirmed that there was PPARγmRNA and protein expression in HCT-8 cells.HCT-8 cells were treated with different concentration of rosiglitazone(vehicle,1.25μmol/L,2.5μmol/L,5μmol/L,10μmol/L,100μmol/L)for 48 hours.MTT assays were performed to measure cell proliferation.The results revealed that rosiglitazone inhibit the HCT-8 cells growth in a dose dependent manner.The cell cycle and apoptosis were detected by flow cytometry.The percentage of cells in G0/G1 significantly increased after 5μmol/L,10μmol/L,100μmol/L rosiglitazone treatment(p＜0.05). The percentage of apoptosis increased after 10μmol/L,100μmol/L rosiglitazone treatment(p＜0.01).These results indicated that rosiglitazone may inhibit the growth of HCT-8 cells,induce apoptosis,lead to G1 arrest.4.effects of rosiglitazone on the expression of MMP-2,MMP-7,MMP-9,TIMP-1,TIMP-2 proteinThe expression of MMP-2,MMP-7,MMP-9,TIMP-1,TIMP-2 protein was detected in HCT-8 cells by immnocytochemistry after adding different dosage of rosiglitazone.The results showed the exprssion of MMP-2,MMP-7 MMP-9 significantly decreased as the dosage of rosiglitazone increased,in controvase,the exprssion of TIMP-1 increased as the dosage of rosiglitazone increased,but the difference was not significant(p＞0.05).The expression of TIMP-2 protein in different groups was not significant.The results suggested that rosiglitazone may regulate the expression of MMP-2,MMP-7 and MMP-9.5.the effects of rosiglitazone on the activity of MMP-2,MMP-9The activity of MMP-2,MMP-9 in HCT-8 cells afer rosiglitazone treatment were assayed by zymography.The results showed that 10μmol/L,100μmol/L rosiglitazone can significantly decrease the activity of MMP-2,and 5μmol/L,10μmol/L,100μmol/L rosiglitazone can decrease the activity of MMP-9 significantly(p＜0.05).These indicated rosiglitazone may regulate the expression of MMP-2,MMP-9.6.rosiglitazone inhibit the growth of HCT-8 xenograft tumorFirst the nude mouse HCT-8 xenograft model was established, rosiglitazone(10mg·kg¹)was intaken each day.Then the length and width were measured and the mean volume was caculated,tumor growth curve was drown. It was found that rosiglitazone could inhibit the growth of HCT-8 xenograft tumor,the rate of tumor inhibition was 28.57%.7.effects of rosiglitazone on the expression of MMP-2,MMP-7,MMP-9,TIMP-1,TIMpo2 in xenograft tumorMouse were sacrificed after 21 days of treatment with rosiglitazone.The exprssion of MMP-2,MMP-7,MMP-9,TIMP-1,TIMP-2 in xenograft tumor was detected by immunohistochemistry.The results showed the expression of MMP-2,MMP-7,MMP-9 decreased significantly in treatment group than in vehicle group.No significant differences in TIMP-1 and TIMP-2 expression were detected when compared with vehicle group.These suggested PPARγmay regulate the expression of MMPs.Our studies displaved that PPARγhad high level expression in colorectal tumor.PPARγagonist rosiglitazone may inhibit HCT-8 cell growth in vitro and vivo.There was close relationship between PPARγand MMPs,TIMPs,which may affect colorectal tumor development,invasion,metastasis.PPARγagonist may become a new method of tumor prevention and treatment.