Soy Isoflavones Inhibit The Invasion And Metastasis Of Human Breast Cancer MCF-7Cells Via The Activation Of PPARγ

As one of the common malignant tumors in women, breast cancer is a severe threat tolife. Dietary and nutritional factors play a pivotal role in the genesis and progression ofbreast cancer. A systematic review and meta-analysis of epipidemiological studies hasdemonstrated that the consumption of soybeans and its product is inversely associated withthe risk of human breast cancer.Soy isoflavones as one of the phytochemicals existed insoybeans mainly contain geniszein and daidzein,and these two components of soyisoflavones were regared as the main active substances of the anti-cancer actions ofsoybeans. A great number of in vitro and in vivo experimental studies have demonstratedthat the antioxidative effects,regulation of cell cycle progression,induction of cellapoptosis,inhibition of invasion and metastasis as well as the tumor angiogenesis wereinvolved in the underlying mechanisms for the anti-carcinogenic effects of soyisoflavones.It is well known that tumor metastasis is the major cause of death of cancerpatients,so the cancer therapy has been focused on the field of the control of tumormetastasis.The underlying mechanism for tumor metastasis is complicated and influencedby many factors.Besides the interactions between the tumor cells and their surroundingmicroenvironment,and the regulation by the whole body, the intrinsic properties of thetumor cells such as adhesion, invasion and migration were also responsible for tumormetastasis. Although some experimental studies have confirmed that soy isoflavones couldinhibit the invasion and metastasis of breast cancer cells,the precise mechanism for theinhibitory effects of soy isoflavones have not been fully elucidated.Peroxisome proliferator-activated receptors （PPARs） belong to the nuclear receptorssuperfamily, and include three subtypes: PPARα, PPARβ and PPAR γ. PPAR γ has beenwidely studied,once activated by a ligand,it acted as a transcription factor and modulated the expression of genes by binding to the peroxisome proliferator responsive element in thepromoter area of target genes,and then regulated many important biological processes.Agreat many of studies have demonstrated that PPARγ was an important target for theprevention and therapy of some chronic diseases such as diabetes, metabolic syndrome andatherosclerosis.Nowadays, thiazolidinediones as one kind of PPARγ ligands such asrosiglitazone，pioglitazone, troglitazone,etc,have been applied to clinic in the chronicdiseases mentioned above. Recently,many studies found that PPARγ widely expressed inmany kinds of tumor cells originated from different organs,so PPARγ may be associatiedwith the genesis and progression of tumor.A lot of experimental studies have demonstratedthat some ligands for PPARγ such as TZDs,15d-PGJ2,etc,exerted anti-cancer actions inmany kinds of tumor cells originated from different organs through inhibition of cellproliferation，metastasis and tumor angiogenesis as well as induction of apoptosis,thereforethe ligands for PPARγ have potential clinical value for the prevention and therapy oftumor,especially for control of tumor metastasis and recurrence.Since genistein and daidzein were proved to be natural ligands for PPARγ and this wascorrelated with the hypolipidemic and anti-diabetic effects of soy isoflavones, the roles ofPPARγ signaling pathway in the diverse effects of soy isoflavones gradually attracted moreattention. In our previous studies,we found that soy isoflavones inhibited the proliferationof breast cancer MCF-7cells through regulation the expression of cell cycle-relatedmolecules such as cyclin B and p21via the activation of PPARγ.Based on the aboveevideces,we hypothesized that soy isoflavones could inhibit the invasion and metastasis ofbreast cancer through the activation of PPARγ.In the present study, the effects of genistein and daidzein,the two main components ofsoy isoflavones,and PPARγ agonist rosiglitazone（as a posistive control）alone or incombination with GW9662，a selective inhibitor of PPARγ on the ability of adhesion,invasion and migration of tumor cells, the expression of FAK, uPA, MMP-2andMMP-9,and cytoskeleton of human breast cancer MCF-7cells were examined with CCK-8assay, the Millicell Chamber membrane invasion culture system, immunocytochemicalstaining and immunocytochemical staining,so as to investigate the effects of soyisoflavones on the invasion and metastasis of breast cancer cells and its correlation with the PPARγ signaling pathway.The main results and conclusions were summarized as the following:1. After treatment with genistein or daidzein at the concentration of8×10^－5mol/L,orrosiglitazone at the concentration of2×10^－5mol/L respectively for48hours, the adhesionrate of breast cancer MCF-7cells significantly decreased as compared with that of control（P＜0.05）, but the inhibitory effects of genistein, daidzein and rosiglitazone on celladhesion could be blocked significantly by GW9662at the dosage of1×10^－5mol/L（P＜0.05）.2. The invasive ability of MCF-7cells through the reconstituted basement membraneof Millicell chamber significantly decreased after treatment with genistein, daidzein androsiglitazone respectively for48hours,but GW9662significantly mitigated the inhibitoryeffects of genistein, daidzein and rosiglitazone on the invison of breast cancer MCF-7cells.3. After treatment with genistein, daidzein and rosiglitazone respectively for48hours,the number of MCF-7cells passing through fibronectin of Millicell chamber significantlydecreased in comparison with control, but the inhibitory effects of genistein, daidzein androsiglitazone on the migration of MCF-7cells could be reversed significantly by GW9662.4. Immunofluorescence staining assay showed that cytoskeleton changes of MCF-7cells was characterized by the shorter microtubules, collapsed or even disappearedfilaments after treatment with genistein, daidzein and rosiglitazone respectively for48hours,but GW9662could alleviate cytoskeleton damage of MCF-7cells induced bygenistein, daidzein and rosiglitazone.5. Immunocytochemical staining assay showed that the immunopositive stainingwith brown colour of FAK, uPA, MMP-2and MMP-9in MCF-7cells distributed cellmembrane and cytoplasm, and the expression of all these four molecules in MCF-7cellssignificantly decreased after treatment with genistein, daidzein and rosiglitazonerespectively for48hours,but GW9662obviously attenuated the inhibitory effects ofgenistein, daidzein and rosiglitazone on the expression of tumor metastasis-relatedmolecules.The above results demonstrate that genistein and daidzein inhibit the invasion andmetastasis of human breast cancer MCF-7cells in vitro through down-regulation of FAK,uPA,MMP-2and MMP-9expression and damaging cytoskeleton of MCF-7cells byactivating the PPARγ signaling pathway.