| Background and Objective:Chemotherapy plays a vital role in breast cancer treatment. However, the therapeutic benefits of chemotherapy can be attenuated because of drug resistance involving primary drug resistance and acquired drug resistance. The mechanism responsible for multidrug resistance (MDR) in breast cancer is multiple complex, and to date, is still unclear. To further explore the molecular mechanism responsible for MDR and new biomarkers predicting the response to 5-Fu, in our present study, we first time established a 5-Fu induced multidrug resistant breast cancer cell line MCF-7/5-Fu to identify drug resistance related proteins and to construct the signal transduction net mediating MDR, to provide new biomarkers and evidence for resistance reversion.Methods:(1) MCF-7 cells were exposed in stepwise escalating concentration of 5-Fu to develop the resistant cell line, MCF-7/5-Fu. Biological and molecular characteristics of the cells were studied through MTT assay, flow cytometry after Annexin-V and PI double stain, real-time PCR and Western-blot; (2) The global protein profiles between MCF-7/5-Fu and its parental MCF-7 cells were compared using proteomic approach that combines 2-DE and MALDI-TOF-MS and ESI-Q-TOF-MS. Then some of the differential expressed proteins were validated by Western-blot; (3) 14-3-3σoverexpression and endogenously silenced by RNAi cell lines were established using LipofectAMINE Plus, respectively. MTT assay, Western-blot and immunoprecipitation were used to determine the role of 14-3-3σon the sensitivity of MCF-7/5-Fu cells to anticancer drugs and the related moleculars change; (4) MTT assay was used to determine the growth rate of MCF-7 cells at 0,12,24, 48h time point after 5-Fu treatment. And Western-blot was used to observe related moleculars change after the same treatment.Results:(1) The multidrug resistant cell line induced by 5-Fu was successfully established, whose IC50 to 5-Fu is 5.21 fold higher than that of MCF-7 (p<0.001), and showed more antiapoptotic ability to 5-Fu. And the resistance index of MCF-7/5-Fu cells to MX, cDDP, ADM and TAXOL was3.37(p<0.01),3.21 (p<0.01),2.03 (p<0.05) and 1.81 (p<0.05) respectively, and to VP-16 and THP was 1.35 (p>0.05) and 1.06 (p>0.05) respectively. The resistance may be related to the overexpression of BCRP, but not to MDR1 or MRP1. (2) Differentially expressed proteins between MCF-7/5-Fu and MCF-7 cells were identified: 12 proteins involving EF-TU were up-regulated and 18 proteins involving 14-3-3σ,HSC70,MnSO and CK8 were down-regulated in MCF-7/5-Fu cells. The expression levels of some proteins in Western-blot validation were consistent with the results in proteomic analysis. (3) The level of Akt phosphorylation (p-Akt) in MCF-7/5-Fu cells was increased and protein level of p53 was downcreased. NF-kB-p50, surviving and Bcl-2 at the downstream signal pathway of p-Akt and p53 were up-regulated and changes were associating with the 14-3-3σdownregulation. (4) 5-Fu treatment can induce the up-regulation of 14-3-3σand p53 synergisticly in MCF-7 cells, and down-regulation of p-Akt, NF-kB-p50, Survivin and Bcl-2, time-dependently. The phenomenon was accompanied with the anticancer effect of 5-Fu.Conclusion:(1) The multidrug resistant breast cancer cell line MCF-7/5-Fu was first time established and MDR related protein profile was constructed. (2) 14-3-3a can enhance the sensitivity of MCF-7/5-Fu cells to 5-Fu, MX and cDDP via inhibiting the phosphorylation of Akt via physical binding and upregulating p53 expression, leading to the expression decrease or function inhibition of NF-kB-p50, Survivin, Bcl-2 and BCRP. (3)5-Fu can induce 14-3-3σand p53 expression, inhibiton Akt phosphorylation, and then NF-kB-p50,Survivin,Bcl-2 downregulation to exert its anticancer effect. |
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