| The peroxisome proliferator activated receptors(PPARs) are a subgroup ofligand-activated nuclear receptors responsible for the regulation of cellular eventsranging from glucose and lipid homeostasis to cell differentiation and apoptosis.Currently, the 3 known forms of PPAR areα,γ,δ. The most widely studiedform among PPAR is PPAR r, which is expressed in a wide variety of cell types,including adipocytes, moarcophages, and others. Significant evidence from manyexperimental systems suggestes that PPARγis over-expressed and PPARγligands induce terminal differentiation, cell growth inhibition, apoptosis andinhibit invasion in several cancer cells including breast, prostate, colorectal, renal,pancreatic, and lung cancer.Thiazolidinediones (TZDs) are pharmaceutical PPARγligands, which canactivate PPARγstrongly. GLP02 is also a new agonist, which was synthesizedby Professor Guo Zongru research team. This section of paper expounded possiblemolecular mechanisms underlying the ROZ and GLP02-induced growth inhibition,differentiation, and apoptosis in colon cancer, breast cancer and melanoma cells.1. Anti-cancer effects of ROZ and GLP02 in human colorectal cancer cell lineHT-29PPARγexpression was detected in HT-29 cells by RT-PCR, Western Blotand immunohistochemistry methods. The data showed that ROZ could induce theincrease both in PPARγmRNA and protein levels in a dose-dependent manner.GLP02 increased the PPARγexpression at lower dose, while decreased thePPARγexpression at higher dose.ROZ and GLP02 suppressed the colony formation. The inhibition of GLP02 is stronger than that of ROZ. ROZ and GLP02 significantly and dose-dependentlyinhibited DNA and protein synthesis.The data of FCMs demonstrated that ROZ and GLP02 reduced the proportionof cells in G1 phase. In addition, cells accumulated in G2+S phase. Treatmentwith ROZ and GLP02 for 72h resulted in typical DNA fragmentation in agaroseelectrophoresis and the proportion of sub-G1 cells increased by FCMs analysis.The data of Western Blot showed that the Bcl-2 protein level decreasedremarkably by treatment with ROZ and GLP02.No consistent change in the expression of total ERK was detected by treatmentwith ROZ, while phosphorylated ERK expression was persistently enhanced byROZ dose-dependently.On the contrary, GLP02 decreased the expression of ERKand p-ERK significantly.Our data in vivo strongly supported our results in vitro. The ROZ and GLP02visibly inhibited the tumor growth of HT-29 cells in nude mice as measured bytumor size and tumor weight. The extensive fibrosis of the tumors was striking bythe ROZ and GLP02 treatment. The effect of GLP02 was stronger than that ofROZ.2. Anti-cancer activities of combination of ROZ with ATRA in humancolorectal cancer cell line HT-29PPARγacts as ligand-senstitive transcription factor and regulates genetranscription by binding as a heterodimer with retinoid X receptors to specificresponse elements(PPREs) in the promoter regions of target genes. Presented inthis paper was the evaluation of growth inhibition and differentiation inductionmechanisms of a colorectal cancer cells by the activation of two currently usedtherapeutics ATRA and ROZ. The combination of RXR and PPARγagonist onHT-29 cells demonstrated a cooperative effect on growth inhibition and differentiation induction with the eventual conclusion of increased antitumorefficacy in a nude mouse xenograft tumor model.Combination of ROZ with ATRA could increase the effect of growth inhibition.FCMs results came out that percentage of apoptotic HT-29 cells increasedmarkedly after combination treatment with ROZ and ATRA. ATRA had noobvious effect on Bcl-2 protein level and ROZ could decrease the Bcl-2 proteinlevel slightly, simultaneously Bcl-2 expression markedly decreased by treatmentcombination of ATRA with ROZ.The activity of AKP is the hall marker of differentiation in HT-29 cells. ROZ orATRA alone could increase the activity of AKP slightly, while the activity ofAKP was elevated significantly when HT-29 cells were treated by combination.The tumor formed by HT-29 cells after treatment of ATRA was the same asthat of control group in size, and the tumor formed by HT-29 cells after treatmentof ROZ was half of control group in size. The tumor was notably smaller when thecells were treated by the combination of ROZ with ATRA, and the tumor size wasonly 10% of that of control group.FCMs data showed that the combination of ROZ with ATRA caused thedecrease of G1+G2 phase cell percentage and the elevation of S phase cellpercentage.ROZ had no influence on ERK protein level and increased the p-ERK level.ROZ combined with ATRA could upregulate the level of ERK phosphorylation inlower dose, while downregulate the level of p-ERK in higher dose.3. Anti-cancer effects of ROZ and GLP02 in breast cancer cell line MCF-7ROZ could increase PPARγmRNA and protein expression, while GLP02could decrease the PPARγexpression. GLP02 decreased the PPARγexpression in a dose-dependent manner. ROZ and GLP02 inhibited the colony formation and dose-dependently inhibitedDNA synthesis.The effect of GLP02 is stronger than that of ROZ.FCMs results demonstrated that ROZ induced cell cycle accumulation in G1and G2 phase, while downregulated S phase. GLP02 increased the proportion ofcells in G2 and S phase. Treatment with ROZ and GLP02 for 72h inducedapoptosis in MCF-7 cells by FCMs analysis. The data of Western Blot showedthat the Bcl-2 protein level decreased by treatment with ROZ and GLP02.At the dosage lower than the one which could induce apoptosis, both ROZ andGLP02 induced accelerated cellular senescence(ACS) in p53 wild-type cancer cellline MCF-7 cells, and simulated the expression of p53.No consistent change in the expression of total ERK was detected by treatmentwith ROZ, while phosphorylated ERK expression was increased. On the contrary,GLP02 increased the expression of ERK and dereased the level of ERKphosphorylation significantly.The ROZ markedly inhibited the tumor growth of HT-29 cells in nude mice asmeasured by tumor size and tumor weight. The inhibition effect of ROZ was equalto that of ADM.4. Anti-cancer effects of ROZ and GLP02 in melanoma cell linePPARγexpression was detected in A375 cells by RT-PCR, Western Blot andimmunohistochemistry assays. The data revealed that ROZ could increase PPARγprotein expression in a dose-dependent manner. GLP02 increased the PPARγexpression at lower dose, while decreased the PPARγexpression at higher dose.In vitro, both ROZ and GLP02 were found to inhibit the proliferation of cancercells and the colony formation ability of A375 cells was inhibited significantly byROZ and GLP02.ROZ and GLP02 induced the differentiation of melanoma. The cells were induced to change from small, dense unregular shape to large, elongated dentritic ones.Consistent with the morphological changes, increased melanin content and tyrosinaseactivity were observed in A735 cells.ROZ and GLP02 also suppressed A375 cells to invade through reconstitutedbasement membrane.FCMs data demonstrated ROZ caused accumulation in G1 and G2 phase, butGLP02 arrested in S phase.A375 cells were induced apoptosis by treatment of ROZ and GLP02 and bothof compounds simulated the expression of P53 and suppressed the expression ofBcl-2.In nude mouse model, A375 xenograft tumor responed to the treatment ofGLP02 very well. The tumor size and weight were reduced after treatment ofGLP02.Both ROZ and GLP02 also exerted inhibitory effect on the relaxation activityof DNA Topo I in vitro, resulting in the electrophoretically measuredaccumulation of supercoiled monomers followed by ethidium bromide staining.In summary, our results had shown that PPARγexpressed and wasfunctionally active in colon, breast and melanoma cancer. PPARγactivation byits ligand of ROZ and GLP02 resulted in antiproliferative, apoptotoic,differentiation and anti-invasion activities through different mechanism. Itsuggested the possibility of utilizing PPARγagonists such as ROZ or GLP02for the treatment of cancers. The relationship of PPARγand cancer is not elucidated yet. A number ofimportant questions need to be addressed. Most importantly, many of the anticancereffects of PPARγligands are probably not mediated by PPARγ. Molecular targetsof TZDs and other PPARγligands in breast cancer cells are still poorly defined.PPARγpathway interacts with other signal transduction pathways which is importantin breast cancer, and the interaction with ER and EGFR transduction pathways needsto be studied in more detail.In the present study, we found that forced overexpression of PPARγleaded to thechanges in differentiation, proliferation, cell cycle, apoptosis and invasion of cancercells.The NIH3T3, MCF-7, MAD-MB-231 cells were stably transfected with anexpression vector pcDNA3.1(+) either lacking an insert or harboring the cDNA forthe full length of hPPARγ2. Analysis of the levels of PPARγmRNA and protein inthe stably transfected cells were performed by RT-PCR and Western Blot. Expressionof PPARγwas increased dramatically in the clones harboring the hPPARγ2-containing expression vector. ROZ could upregulate the PPARγexpression ofcancer cells without harboring the cDNA of hPPARγ2. For the cancer cellstransfected with hPPARγ2, ROZ increased the PPARγ2 level of NIH3T3 anddecreased the PPARγ2 level of MDA-MB-231 and increased the level at low dosagewhile decreased the level of MCF-7 at high dosage.1. NIH3T3 cells and Py2-NIH3T3 cells1.1 After transfected with hPPARγ2, morphology of NIH3T3 changed.Py2-NIH3T3 cells was more sensitive to ROZ than NIH3T3 cells. 1.2 There are no difference in cell-cycle between NIH3T3 cells and Pγ2-NIH3T3cells. ROZ induced cell cycle arrest in G1 in both cells. The cells transfectedwere not prone to induction of apoptosis and the expression of P53 declined inthe transfected cells. ROZ had no influence on the P53 level, but increased theexpression of P53 in cells transfected with hPPARγ2. Expression of P21increased in the transfected cells and ROZ increased the P21 level in both cells.1.3 The adhesive ability of Pγ2-NIH3T3 cells with basement membranecomponents (FN) was markedly enhanced and E-selection diminshedaccordingly. ROZ decreased the adhesive ability in both cells. ROZ alsoreduced the expression ofβ-cat in NIH3T3 cells, while reduced theexpression of E-cadherin and E-selectin exceptβ-cat in transfected cells.1.4 After transfected with hPPARγ2, the chemotactic migration rate of NIH3T3cells increased significantly and ROZ suppressed this migration.1.5 hPPARγ2 did not exert effect on the expression of ERK and p-ERK inNIH3T3 cells. In untransfected cells ROZ stiumlated the ERK phosphorylationwhich was supressed by ROZ in transfected NIH3T3 cells.1.6 The JNK and p-JNK level were increased after transfected in NIH3T3. ROZhad no effect on JNK level in NIH3T3, but could degraded phosphorylation ofJNK in transfected cells.1.7 The P38 and p-P38 level were decreased after transfecting. ROZ onlydiminished P38 level in NIH3T3 cells slightly, but could decreased both P38and p-P38 level in transfected cells.1.8 hPPARγ2 induced tumor formation of NIH3T3 cells.2. MDA-MB-231cells and Pγ2-MDA-MB-231 cells2.1 After transfected with hPPARγ2, morphology of MDA-MB-231 changed.Pγ2-MDA-MB-231 cells was more sensitive to ROZ than MDA-MB-231 cells.2.2 There were no difference in cell-cycle between MDA-MB-231 cells andPy2-MDA-MB-231 cells. In MDA-MB-231 cells, ROZ induced cell cycle arrest in G2 phase, but induced cell cycle arrest in G1 phase in Pγ2- MDA-MB-231cells.2.3 The cells transfected was prone to induction of apoptosis. The expression ofBcl-2 increased and that of bax decreased in the transfected cells. ROZ had noinfluence on the Bcl-2 and bax level, but reduced the expression of Bcl-2 andincreased the expression ofbax in cells transfected with hPPARγ2.2.4 Expression of P16 increased in the transfected cells and ROZ increased the P16level in both cells. Cyclin E level did not proceed changes after transfected andROZ suppressed the expression in both cells.2.5 The adhesive ability of Pγ2-MDA-MB-231 cells with basement membranematrigel was markedly decreased. ROZ decreased the adhesive ability inMDA-MB-231 cells but did not exert effect on Pγ2-MDA-MB-231 cells.2.6β-cat level was increased slightly and ICAM level had no changes intransfected cells. ROZ had no influence on the expression ofβ-cat andICAM in MDA-MB-231 cells, but suppressed expression of that in transfectedcells.2.7 After transfected with hPPARγ2, the ability of MDA-MB-231 cells to invadethrough reconstituted basement membrane markedly increased, while treatmentwith ROZ, invasive potential of MDA-MB-231 cells and Pγ2-MDA-MB-231cells both diminished.2.8 hPPARγ2 increased the expression of p-ERK, JNK, p-JNK and P38, p-P38 inMDA-MB-231 cells. ROZ enhanced the p-ERK level in MDA-MB-231 cellsand did not produce a marked effect on others. In transfected cells, ROZreduced the expression of above proteins except ERK.2.9 After transfected with hPPARγ2, the mRNA level of BRCA1 decreased.2.10 hPPARγ2 did not influence mRNA level of TGFβ1,TGFβ2,TGFβ3. ROZcould stimulate the TGFβ1 expression in both cells and supressed TGFβ2 andTGFβ3 expression only in transfected cells, hPPARγ2 had no effect on TGFβRâ… level and increased TGFβRâ…¡and p-TGFβRâ…¡protein expression. TGF βRâ… , TGFβRâ…¡and p-TGFβRâ…¡levels were heighten by treatment of ROZ inMDA-MB-231 cells. TGFβRâ…¡and p-TGFβRâ…¡level were diminished bytreatment of ROZ in transfected cells, while TGFβRâ… level was still heighten.2.11 After transfected with hPPARγ2, the mRNA level of IGF1R increased andthat of IGFBP3 did not change. ROZ could suppress expression of abovemRNA in both cells.2.12 hPPARγ2 stimulated COX-2 expression and had no influence on TopoI. ROZcould suppressed expression of above mRNA in both cells.2.13 hPPARγ2 promoted the tumor formation of MDA-MB-231 cells.3. MCF-7 cells and Pγ2-MCF-7 cells3.1 After transfected with hPPARγ2, morphology of MCF-7 changed. Pγ2-MCF-7cells was more sensitive to ROZ than MCF-7 cells.3.2 After transefected, the cells accumulated in G1 phase increased and S phasedecreased slightly. ROZ induced cell-cycle arrest in G1+G2 phase in both cells.3.3 The cells transfected was prone to induction of apoptosis.3.4 The expression of P53 increased slightly and that of Bcl-2 and bax did notchange in the transfected cells. ROZ had no influence on the P53 and Bcl-2level, but reduced the expression of Bcl-2 and increased the expression of P53and bax in cells transfected with hPPARγ2.3.5 MCF-7 cells was induced differentiation easily, but it became difficult intransfected cells. The ability of differentiation was increased at low dose anddecreased at high dose by ROZ in both cells.3.6 MCF-7 cells had little invasive potential. After transfected with hPPARγ2,The ability of MCF-7 cells to invade through reconstituted basement membranemarkedly increased, while treatment with ROZ, invasive potential ofPγ2-MCF-7 cells decreased.3.7 The adhesive ability of Pγ2-MCF-7 cells with basement membrane components(Matrigel) was markedly decreased. ROZ decreased the adhesive ability in both cells, hPPARγ2 had no influence on E-cad,β-cat,β2-mg level, but couldincrease integrinα2 level. ROZ did not exert effect on above mRNA levelexceptβ-cat in untransfected cells, while could diminished that in transfectedcells.3.8 hPPARγ2 up-regulated the p-ERK, JNK, and p-P38 expression in MCF-7cells, but down-regulated that of p-JNK. ROZ reduced the p-ERK, JNK, p-JNK,P38 level in untransfected MCF-7 cells and JNK, p-JNK and p-P38 level intransfected cells.3.9 hPPARγ2 did not cause the changes of TGFβ2,TGFβ3 mRNA level.hPPARγ2 increased TGFβRâ… , TGFβRâ…¡and p-TGFβRâ…¡proteinexpression. TGFβRâ… were heighten by ROZ in MCF-7 cells. TGFβRâ…¡andp-TGFβRâ…¡level were supressed by ROZ in transfected cells, simultaneouslyTGFβRâ… level was still heighten.3.10 After transfected with hPPARγ2, the expression of EGF did not change, whilethat of EGFR increased. ROZ could reduced EGF level and EGFR only intransfected cells.3.11 Transfected with hPPARγ2, the expression of ERαreduced. ROZ had noeffect on ERαlevel in untransfected cells, but the expression of ERαcouldbe decreased by ROZ in transfected cells.3.12 After transfected with hPPARγ2, the mRNA level of COX-2 did not change,which was suppressed by ROZ in transfected cells.In summary, hPPARγ2 could cause the chang of cell morphology and theadhersion ability, increase tumor formation abilities of cells, ability of invasion andmotility, and decrease differentiation ability, hPPARγ2 interacted with MAPK signalpathway, growth factor pathway and cell-cyclin related protein. So hPPARγ2 couldrepresent a novel therapeutic target for cancer and its agonists could become a newkind of drugs for cancer therapy. At first, cDNA of hPPARγ2 was obtained from pMD18-T/hPPARγ2, hPPARγ2was expressed in prokaryocyte and purified successfully.Secondly, the anti-hPPARγ2 monoclonal antibodies (mAbs) 1B4, 3H2, 3H10,10D6 and 10D8 were produced by immunization with purified hPPARγ2. Thespecificity of the antibodies was identified by ELISA and Western Blot assay. Theresults showed that they were all specific to hPPARγ2.The antibodies obtained provide us more useful tools for following researches andsettle the bases of screening new drugs and mechanism.
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