The Effect Of Wnt/β-catenin Signaling Pathway On The Multidrug Resistence Of Breast Cancer Cells And Involved Mechanism

Background and Objective:Breast cancer is one of the most serious malignant tumor and harm to women’s health in the worldwide. Chemotherapy has an important role in the treatment of breast cancer. However, multidrug resistance (MDR) is a bottleneck of chemotherapy in the treatment of breast cancer. Currently, there is no effective treatment strategy. Therefore, the mechanism responsible for MDR and predicting of new biomarkers become more and more important. The mechanism of MDR in breast cancer is very complicated. So far, it is still unclear, although some literatures were reported after another. In our present study, based on the establishment of 5-fluorouracil (5-FU) inducing multidrug resistant breast cancer cell line before in our laboratory, we applied the technology of high-throughput microarray to investigate the gene expression profiles between the human breast cancer cell line MCF-7/5-FU with the parent cell line MCF-7, to analysis resistance-associated genes, to explore the molecular mechanism of Wnt/β-catenin signaling pathway on the multidrug resistance of breast cancer cells, trying to find a new biomarker, to further elaborate the molecular mechanism of MDR and providing a new theoretica basis for the targeted gene therapy. Methods:(1) Microarray experiments were performed using 22K human genome oligonucleotide microarrays containing 21,522 well-characteri-zed homo-sapiens genes to screen gene differential expressions in the human breast cancer cells line MCF-7/5-FU and its parent cell line MCF-7 cells. Some differentially expressed genes were verificated to microarray by quantitative real-time PCR (qRT-PCR). The distributions ofβ-catenin proteins in the cytoplasm and nucle were aetected by Western Blot.(2) Constructed the human GSK3B gene over-expression letiviral vector, GSK-3βover-expression on the MCF-7/5-FU cell line was established via infection of the high titer lentiviral particles, through the 293T cells packaging, purified. Then, the chemosensitivities to severl drugs were defined by MTS, and its related gene expressions mediated were detected by real-time PCR and Western Blot. Constructed the human CTNNB1 target gene RNAi letiviral vector,β-catenin low expression cell line was established via lentiviral infection. By MTS, real-time PCR and Western Blot, we detected drug sensitivities of breast cancer cells to various drugs and its related gene expressions mediated in the P-catenin gene low expression cell line.(3) Wnt/p-catenin signaling pathway associated proteins were detected by Western Blot in the various cell lines and different treatment groups. Wnt/β-catenin signaling pathway associated gene mRNA levels were detected by qRT-PCR in the various cell lines and different treatment groups. Cell cycle and apoptosis changes in the various cell lines arid different treatment groups were detected by flow cytometry. Nuclear translocations ofβ-catenin and NF-κB proteins were observed by immunofluorescence assay. The effect of GSK-3βon the activity of BCRP promoter was detected by Luciferase Reporter Assay. MatInspector program was used to search transcription factor binding sites and related literatures were colleted to screen the transcription factors in the regulation of BCRP by GSK-3β. At last, electrophoretic mobility shift assay (EMSA) and chromatin immuno-precipition (ChIP) assay were used to indentify the binding abilities of GSK-3βand NF-kB to BCRP promoter. Results:(1) A comprehensive, differentially gene expression profile was obtained for the resistance breast cancer cells. Comparison of the gene expression proflies of two cell lines, a series of genes expressed coincidentally in both gene chips were selected for drug-resistance. A total of 635 genes differentially expressed were screened out in the two cell lines. There were 217 genes upregulated and 418 genes downregulated in MCF-7/5-FU. The important genes upregulated are Bcl-2, BIRC5 (Survivin), ABCG2 (BCRP), LEF1; the important genes downregulated are GSK3B (GSK-3β) and CDH1 (E-cadherin). Different molecular functions involved in functional classification, mainly related to apoptosis, cytoskeleton and signal transduction, cell proliferation, differentiation and cycle regulation, membrane transport proteins, DNA repair, gene transcription and translation and cell adhesion and cell invasion and metastasis, and so on.(2) To show the reproducibility of the microarray analysis, five genes (e.g. GSK3B, CDH1, LEF1, ABCG2, and so on) were selected at random, and these changes were validated by qRT-PCR. The results were consistented with the differentially expressed genes in cDNA microarray.(3) Western Blot showed there is no significant change in the totalβ-catenin proteins between MCF-7/5-FU and MCF-7 cells. However, the corresponding protein in nucleus significantly increased in the MCF-7/5-FU cells comparing with parent cells MCF-7. MCF-7/5-FU cells after LiCl treatment, nuclear P-catenin protein increased more apparent.(4) The recombinants were successfully construced. After infecting MCF-7/5-FU cells, MCF-7/5-FU/GSK and MCF-7/5-FU/siCAT cell lines were obtained by pressure screening of puromycin and G418.(5) Infected with the letivirus of GSK-3βoverexpression or β-catenin interfering, MCF-7/5-FU can partially reverse drug resistance to chemotherapy. Compared with the control group, MCF-7/5-FU/GSK cells increased the sensitivity to 5-FU, THP, Mit and Taxol by 4.06,2.12, 3.01 and 2.00 times, respectively; MCF-7/5-FU/siCAT cells increased the sensitivity to 5-FU, THP, Mit, and Taxol by 3.80,2.52,2.62 and 2.06 times, respectively. The differences were statistically significant (p<0.05).(6) Flow cytometry analysis showed that the numbers of G0/G1 phase cells increased, S phase had no difference, apoptotic rate decreased in the MCF-7/5-FU cells comparing with parent cells MCF-7; the numbers of G0/G1 phase cells significantly decreased, S phase have no changes significantly, the apoptosis rates increased, the infected groups of overexpression of GSK-3P andβ-catenin siRNA comparing with the uninfected groups; the numbers of G0/G1 phase cells increased and apoptosis rate decreased, LiCl pretreatment group comparing with the untreated group.(7) Western Blot analysis showed that expressions of Survivin, Bcl-2 and BCRP increased, total protein expression levels ofβ-catenin and NF-κB did not change significantly in the MCF-7/5-FU cells comparing with parent cells MCF-7. However, the corresponding protein in nucleus significantly increased. Expressions of Survivin, Bcl-2 and BCRP decreased in the MCF-7/5-FU/GSK and MCF-7/5-FU/siCAT comparing with the control group. Expressions of Survivin, Bcl-2 and BCRP increased, total protein expression levels ofβ-catenin and NF-κB did not changes significantly in the MCF-7/5-FU/LiCl cells comparing with MCF-7/5-FU. Similarly, the corresponding nuclear protein significantly increased. Real-time PCR test results were (?)onsistent with the Western Blot.(8) By immunofluorescence assay, we found thatβ-catenin and NF-κB proteins occured significantly nuclear translocations from the cytoplasm to nucleus in the MCF-7/5-FU cells comparing with MCF-7 cells. It is more evident ofβ-catenin and NF-κB protein expressions in the nucleus, LiCl pretreatment group MCF-7/5-FU/LiCl comparing with untreatment group.(9) GSK-3βover-expression plasmids were cotransfected with BCRP promoter reporter plasmids. The promoter activities reduced significantly on the BCRP. Added NF-κB inhibitor, MG132, the BCRP promoter activities were more significantly reduced. After Adding LiCl, the promoter activities increased. In addition, EMSA showed that NF-κB was able to bind to the NF-κB (p50) binding site in the BCRP promoter. At last, chromatin immunoprecipition (ChIP) assay confirmed that NF-κB (p50) could directly bind to the NF-κB binding site in BCRP promoter. All these results demonstrated that GSK-3βcan regulate the expression of BCRP gene. This negative regulation of BCRP gene expression can be achieved possibly through inhibiting NF-κB activity. Conclusion:(1) In the present study, a widespread differential gene expression pattern was constructed in the multidrug resistant cells MCF-7/5-FU and parental cells MCF-7. The gene expression profiling shows that MDR is a complicated and multifactorial process and may involve some of all of the following changes:inhibition of apoptosis pathway, cytoskeleton, signal transduction, cell proliferation and differentiation, disturbance of cell cycle, membrane transport proteins, DNA repair, gene transcription and translation and cellular adhesion, cell invasion and metastasis, and other genetic changes. Further study of these MDR related genes may provide the insights into the molecular mechanism of MDR and cast light on the prediction-based cancer chemotherapy.(2) The overexpressions of GSK-3βandβ-catenin siRNA cell lines with MCF-7/5-FU/GSK and MCF-7/5-FU/siCAT were established. A good model was provided for the resistance mechanism research of breast cancer.(3) Wnt/β-catenin signaling pathway plays an important role in the multidrug resistance of breast cancer. Results showed that the role was implemented mainly by regulating the activity of NF-κB; on the one hand, affecting the cell cycle and apoptosis, on the other hand, regulating the expressions of several membrane proteins. This may be one of the most important mechanisms of Wnt/β-catenin signaling pathway mediated mutidrug resistance in the 5-FU induced breast cancer resistant cells, MCF-7/5-FU.(4) Intracellular transcription factor NF-κB played an important role in the Wnt/β-catenin pathway regulating cell growth and drug resistance. It provided a new idea to establish and improve the mechanisms of multidrug resistance for further gene analysis and therapy.(5) It provided an important clue that Wnt/β-catenin-mediated signaling pathway and its associated network illustrated the comprehensive mechanism of tumor resistance to chemotherapy. Wnt/β-catenin pathway related gene, such as GSK-3β,β-catenin, may be expected to be one of the important markers in the predicting chemotherapy effectiveness containing 5-FU and reversal of multidrug resistance.