The Effects Of Bortezomib In Combination With Epirubicin On The Proliferation And Apoptosis Of The Breast Cancer Cells

Background:Breast cancer is a kind of malignant neoplasms originated from the mutative epithelium of the breast tissue. Recently, much more attention has been paid to the treatment of the Ubiquitin Proteasome Pathway (UPP) in malignant tumors. It has been proved that the ubiquitin-proteasome pathway plays a lot of important roles in cancer including the regulation of tumor growth through multiple targets impacting cell cycle progression and apoptosis, cell adhesion, invasion, and metastasis. As the first UPP inhibitor, Bortezomib had shown a promising antitumor activity in a number of preclinical cancer cell-line models in vivo vitro, such as lung, colon and prostate cancer as well; and it had also been used as an alternative selection to chemotherapy resistant multiple myeloma. Besides, dozens and hundreds of clinical studies indicated the efficacy of bortezomib in the treatment of several other malignant tumors.Objective:The purpose for this study is to characterize the effects of bortezomib in breast cancer MCF-7cell lines on viability, cell cycle and apoptosis. Considering the important role of the epidermal growth factor receptor (EGFR) and transforming growth factor β1(TGF-β1), and their signaling transduction pathways in breast cancer, the effects and possible mechanisms of bortezomib on these pathways were also investigated.Methods:Human breast cancer cell lines, MCF-7, were cultured with Epirubicin (with the concentration of0.05,0.25,0.50,1.00and5.00μg/mL, and named as A1, B1, C1, D1, E1respectively) alone and plus Bortezmib (with the concentration of0.01,0.10,1.00,5.00and10.00μg/mL, named as A2, B2, C2, D2, E2respectively). The combination of Bortezomib and Epirubicin are listed below:Epirubicin0.05μg/mL+Bortezomib0.01,0.10,1.00,5.00,10.00μg/mL, named as A1A2, A1B2, A1C2, A1D2, A1E2; others as the former:B1A2, B1B2, B1C2, B1D2, B1E2; C1A2, C1B2, C1C2, C1D2, C1E2; D1A2, D1B2, D1C2, D1D2, D1E2; E1A2, E1B2, E1C2, E1D2, E1E2, altogether25groups. All of them were incubated for24,48and72hours, respectively. Then the effects of Bortezomib and Epirubicin either alone or together on the proliferation and viability of breast cancer cells were studied by the way of MTT assay. MCF-7cells were cultured with Bortezomib0.10μg/mL plus Epirubicin0.50μg/mL, Bortezomib0.10μg/mL plus Epirubicin0.25μg/mL, and Bortezomib plus Epirubicin with the same concentration alone for24,48and72hours, respectively. Then the cells stained with propidium iodide (PI) or double stained with Annexin V-PI were detected by the means of flow cytometry, especially for cell apoptosis.Results:Bortezomib caused neither a time-dependent nor a dose-dependent reduction in cell viability and proliferation in MCF-7cell lines with the dose>0.10μg/mL (B2、C2、D2、E2group)(P<0.05).Bortezomib caused a nor time-dependent neither dose-dependent reduction in cell viability and proliferation in MCF-7cell lines (P<0.05), when the dose>0.10μg/mL (B2、C2、D2、E2group) Epirubicin inhibited the proliferation and viability of MCF-7cells in a dose-dependent and time-dependent way(P<0.05), and induced apoptosis in MCF-7breast cancer cells with the dose>0.25μg/mL (B1、C1、D1、E1group). In addition, when Bortezomib and Epirubicin were put together, the inhibition effect was enhanced compared with administrated alone (P<0.05), and the inhibition effect would be maximal at the moment when the cells were treated with both for48hours. Bortezomib made the cell cycles stop at G2-M stage, while Epirubicin made them stop at S stage. Apoptosis of MCF-7cells at24hours were9.28%、10.50%、14.10% (B2,B1、C1group) and12.50%.17.80%(B1B2、C1B2group) compared with1.74%(control)(P<0.01); and11.40%、23.70%、30.60%(b2、b1、c1), and25.20%、40.70%(b1b2、c1b2) compared with4.13%(control) at48hours(P<0.01). The combination of Bortezomib and Epirubicin could induce apoptosis of MCF-7cells at very low concentrations indicating a synergistic effect on them. Moreover, the RT-PCR results showed Bortezomib alone and combined with Epirubicin had declined the expression of EGFRmRNA, though insignificant (P>0.05); Epirubicin treated the cells alone make no difference. There was no significances on the expression of TGF-β1mRNA as the cells treated with Bortezomib and Epirubicin alone or in combination.each or both (P>0.05).Conclusion:Bortezomib may have some inhibiting effects on MCF-7cells. In addition, Bortezomib and Epirubicin have a synergistic effect over the inhibition of MCF-7human breast cancer cells. The research shows that the dose of Epirubicin can be decreased if applied together with Bortezomib without changing their sensitivity and chemotherapeutic effects thereby improve the adverse effects of the drugs. Besides, Bortezomib was able to inhibit the cell cycles and promote apoptosis of MCF-7cells, alone or together with Epirubicin. Furthermore, to some extent, Bortezomib may decrease the invasion and metastatic ability of MCF-7cells, which indicated Bortezomib may show superiority in the treatment of breast cancer.