ABSTRACT
The MCF-7 and MDA-MB-435 cells were treated with 1 and 10 μmol/L VRB. The cell adhesion was tested by MTS, the invasion was detected by Transwell, secretion of TGF-β was detected using ELISA, the activities of MMP-2 and MMP-9 were detected by zymography, the expression of proteins, including E-cadherin (E-CAD), N-cadherin (N-CAD), MMP-2, and MMP-9, p-JNK and, p-Akt was evaluated by Western blotting, RT-PCR was used to detect E-CAD, N-CAD, MMP-2, and MMP-9 genes, and dual-luciferase reporter assay was used to validate the activities of AP-1 and NF-κB. Results After being treated with 1 and 10 μmol/L VRB, for MCF-7 and MDA-MB-435, the adhesion ability was decreased by 34.8% and 66.8%, 42.6% and 72.3%; The metastatic ability was decreased by 44.4% and 72.2%, 47.7% and 86.4%; The secretion of TGF-β was reduced by 28.2% and 52.1%, 39.0% and 55.1%, significantly; The mRNA expression levels of E-CAD were increased by 86.5% and 181.6%, 116.6% and 160.7%; while the levels of N-CAD were decreased by 33.7% and 74.1%, 57.6% and 76.9%; The gene expression of MMP-2 was decreased by 71.6% and 88.4%, 57.4% and 69.4%; The gene expression of MMP-9 was decreased by 15.0% and 84.0%, 22.1% and 73.0%, respectively. The protein expression of E-CAD was up-regulated while the protein expression of N-CAD, MMP-2, MMP-9, p-JNK, and p-Akt were down-regulated significantly; And dual-luciferase reporter assay validated VRB could inhibit the transcriptional activity of AP-1 and NF-κB by 27.7% and 68.2%, 34.8% and 71.4%, 18.4% and 44.8%, 24.4% and 51.9%, respectively. Conclusion VRB could inhibit the metastasis of breast cancer MCF-7 and MDA-MB-435 cells via down-regulation of inhibition and blocking of signaling pathway correlated with metastasis and epithelial-mesenchymal transition.
ABSTRACT
AIM: Gene transduction with a recombined murine stem cell retroviral vector has been investigated to find an effective way of gene transduction and to offer theory and experimental basis for the recombined murine stem cell retroviral vector used for gene transduction. METHODS: 1. Construction of retrovirus vector: EC1-4 gene (repeats 1-4 of cadherin-5 extracellular domain) and mutant (Ser 222A) MEK1 gene were cloned into retrovirus vector pMSCV after cut by Bgl Ⅱ and EcoR 1 restriction endonuclease. 2. Obtaining CD41 + cells and cell culture: Cells expressing CD34 + from cord blood were isolated. The inducement of cells expressing CD41 from CD34 + cells was performed by using TPO and cells CD41 + were selected by FACS. NIH 3T3 cells were cultured in high sugar DMEM medium and U937 in RPMI 1640 medium. UT7 cells which is cytokine-dependent cell line were grown in Iscove's modified Dulbeco's medium supplemented by GM-CSF. 3. Determination of viral titers: Retroviral vectors were transferred to packaging cell line 293. Retroviral containing supenatant was collected after transfection. The viral titers was tested on infection of NIH 3T3 cells by FACS analysis. 4. Western blot: Transduced CD41 +,UT7,U937 and MDA-MB-435 cells were analyzed by western blot to examine expression of transduced genes. RESULTS: A packaging cell line 293 produces high-titer MEK1 pMSCV retroviruses (3.1?10 7) and EC1-4 pMSCV retroviruses (1.0?10 8). With 8-folds dilution retroviruses,60.73% GFP positive cells have been obtained in MEK1 pMSCV transduced UT7 cells,72.56% in U937 cells and 30.57% in CD41 + cells,respectively. GFP positive cells have reached up 97.54 % in EC1-4 pMSCV transducted MDA-MB-435 cells. Phosphorylated MEK1 has been decreased in experiment group when TPO has stimulated CD41 + and UT7 cells or serum has stimulated U973 cells. This indicates that is a dominant negative effect of mutation MEK gene. EC1-4 gene transduced MDA-MB-435 cells have expressed EC1-4. CONCLUSION: The recombined murine stem cell retrovirus can effectively mediate gene transduction of CD41 +,UT7,U937 and MDA-MB-435 cells,and transduced genes can be stably expressed.