| Background: Breast cancer is the most commonly diagnosed type of cancer, thesecond leading cause of cancer-related deaths in women. Invasion and metastasis are themost lethal characteristics of breast cancer: almost all of patients die from long-termrecurrence. It is well known that several steps are necessary in cancer metastasis，suchas angiogenesis, invasion, embolism and adherence, etc. However, a growing area ofinterest is the association of cancer metastasis with epithelial-mesenchymal transition(EMT). EMT is defined by the loss of epithelial characteristics and the acquisition of amesenchymal phenotype and is essential in embryonic development and inflammation.The transformation has implications in onset of breast cancer to metastasis, andincreases evidences to support hypothesis of cancer stem cell.EMT can increase breast cancer cell abilities of migration and invasion in vivo andin vitro. Recent evidences indicate that EMT is associated to chemo-resistance andcirculating tumor cells in breast cancer. Thus, the reversal of EMT may be a potentialmethod to improve the prognosis of breast cancer patients. There are several hallmarks,such as E-cadherin, catenin (epithelial marker) and N-cadherin, fibronectin, vimentin(mesenchymal marker). Dysregulation of these proteins is provoked by transcriptionrepressors involving Snail/SNAI1, Slug/SNAI2. TGF-β signalling as well as Wnt-,Notch-, Hedgehog-, and NF-κB-dependent pathways can induce and maintain EMT,considered as potential targets on EMT reversal. However, there is a more interestingapproach to block this phenotype transition.Elemene (1-methyl-1-vinyl-2,4-diisopropenyl-cyclohexane), an active componentof herb medicine Curcuma wenyujin, is a novel anti-cancer drug. The extract ofelemene is a mixture of α-, β-and δ-elemene, with β-elemene (ELE) as the maincomponents, which accounts for60~72%of the three isoforms. The anti-cancermechanism of ELE is that (i) it induces apoptosis as well as protective autophagy in human non-small-cell lung cancer A549cells;(ii) it can down-regulate the expressionof survivin, Bcl-xL and Mta-1and induce the apoptosis of T24cells at a molecular level;(iii) it participates the regulation on phosphorylation of multiple members of the kinasefamilies inducing apoptosis in tumour cells, such as in the Ras/Raf/MEK/ERK cascadeand AKP. However, it is unknown that ELE affects EMT in human breast cancer cells.We reported that ELE up-regulated expression of the oestrogen receptor alpha(ERalpha) located breast cell membrane though repressing the acitivity of theRas/MARK/ERK pathway. A previous report showed that ERalpha inhibited theexpression of Snail though metastasis related protein3(MTA3), and regulated the EMTwith TGF-β signaling together. This study plans to establish a breast cancer EMT model,cleared the effect on breast cancer cells and to detect the breast cancer tissue analysedthe relation between EMT and clinical pathological characters. At last, we explored thereversal effect of ELE on breast cancer cells.Objective:1. To identify the effect EMT induced by TGF-β1on breast cancercells;2. To detect the relations in the expression of E-cadherin and Snail and clinicalpathological characters of breast cancer patients.3. To explore the reversal effect onbreast cancer cells EMT with ELE.Methods:1. We cultivate breast cancer cell line MCF-7and induce EMT withTGF-β1, and then identify the EMT though cell shape and protein expression’s change,and then examine the cell abilities of migration and invasion by wound healing assayand transwell assay.2. We detect the expression of E-cadherin and Snail in breastcancer tissue by immunity-histochemistry, and analysis the relation in the expression ofE-cadherin and Snail with clinical pathological characters and prognosises of breastcancer patients.3. We treat the breast cancer cell EMT model with ELE and detect thechange in protein expression and cell abilities of migration and invasion, identified thereversal effect on EMT. We explore the molecular mechanism of the reversal effectthough TGF-β/Smads signal pathway and transcription factor Snail.Results:1. Treating with10ng/ml TGF-β1for24hours, the breast cancer cell lineMCF-7changed to the fusiform shape and the disappeared cell link. We find that theexpression of E-cadherin and β-catenin were down-regulated, whereas N-cadherin andvimentin were up-regulated. The increase of cell abilities of migration and invasion wasconfirmed by wound healing assay and transwell assay.2. The immunity-histochemistryshowed the expression of E-cadherin and Snail in breast cancer tissue.103breast cancerpatients were enrolled and the relation in the expression of E-cadherin and Snail and clinical pathological characters of breast cancer patients was researched. The resultsshowed low expression of E-cadherin and high expression of Snail was related withclinical pathological malignancy characters and prognosis of breast cancer patients.3.40μg/ml ELE can blocks breast cancer cell EMT induced by TGF-β1, further ELE wasable to inhibit TGF-β1-mediated up-regulation of mRNA and protein expression ofnuclear transcription factors (SNAI1and SNAI2), in part through decreasing expressionand phosphorylation of Smad3, a central protein in TGF-β1signalling pathway.Conclusions：1. The breast cancer cells can be induced to a EMT by TGF-β1andcell abilities of migration and invasion were increased.2. The expression of E-cadherinand Snail were related to clinical prognosis of breast cancer patients.3. ELE blocksEMT induced by TGF-β1in breast cancer cells and the mechanism is inhibitingTGF-β1-mediated up-regulation of mRNA and protein expression of nucleartranscription factors (SNAI1and SNAI2), in part through decreasing expression andphosphorylation of Smad3. |
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