Synergistic Inhibition Of Breast Cell Proliferation By Several Drugs And The Underlying Mechanisms

Aim:To examine the synergistic inhibitions of several anticancer drugs on MCF-7breast cancer cells and delineate their combined mechanisms of action.Methods:Anti-proliferation effects of gefitinib,sorafenib,everolimus,cladribine,dasatinib,vorinostat,and fludarabine phosphate,alone or in combination,on MCF-7cells were designed according to Chou–Talalay method and analyzed with MTT assay.The combination index（CI）and drug reduction index（DRI）were calculated based on the median-effect principle.Cell-cycle perturbation and cell apoptosis induction of the combination drugs were examined by Flow cytometry.The generation of reactive oxygen species（ROS）,loss of mitochondrial membrane potential,and expressions of Bcl-2 and Caspase-3 were determined by immunofluorescence.Results:1.Treatment with anti-cancer drugs,such as gefitinib,sorafenib,everolimus,cladribine,dasatinib,vorinostat,and fludarabine phosphate alone,inhibited the growth and proliferation of breast cancer MCF-7 cells.Within a certain range,the following drug combinations showed synergistically inhibitoty effects on breast cancer cells:cladribine and gefitinib,cladribine and dasatinib,vorinostat and gefitinib,and vorinostat and dasatinib.In contrast,the following combination of drugs showed antagonistic inhibitory effects on breast cancer cells:cladribine and sorafenib,vorinostat and sorafenib,gefitinib and fludarabine,gefitinib and sorafenib,and sorafenib and everolimus.2.The combined treatment of cladribine with either gefitinib（15～60μΜ）or dasatinib（9～72μΜ）for 48 h exhibited a synergistic growth inhibition in MCF-7 cells.The DRI values of cladribine and gefitinib ranged from 3.12 to 4.02 and 2.48 to 3.69,respectively.The DRI values of cladribine and dasatinib ranged from 1.948～4.065 and2.763～5.415,respectively.Similarly,combination of vorinostat with either gefitinib（4.125～33μΜ）or dasatinib（1.5～24μΜ）exhibited a synergistic growth inhibition in MCF7 cells upon treatment for 48 h.The DRI values of vorinostat and gefitinib ranged from 2.703 to 3.697 and 2.655～5.933,respectively.The DRI values of vorinostat and dasatinib ranged from 2.337 to 4.177 and 2.167 to 3.907,respectively.3.Cell cycle analysis indicated that combined treatment with cladribine and gefitinib,cladribine and dasatinib,vorinostat and gefitinib and orvorinostat and dasatinib blocked the breast cancer cells in G₂/M phase.4.We found that treatment with cladribine,gefitinib,dasatinib,and vorinostat alone led to increased ROS,decreasedΔΨm,downregulation of apoptosis-related protein Bcl-2and upregulation of Caspase-3 expression in breast cancer cells.Notably,the combined treatment with these drugs showed significant synergy in these biological changes.Conclusion:1.All the aboved drugs could inhibit the proliferation of MCF-7 cells alone,.Combinations of cladribine and gefitinib,cladribine and dasatinib,vorinostat and gefitinib,and vorinostat and dasatinib showed synergistic inhibitions in MCF-7 cells.2.The four groups of drugs could modulat cell cycle distribution and induce apoptosis in MCF-7 cells in synergy.3.The four groups of synergistic anti-cancer drug combinations induced apoptosis in MCF-7 cells by regulating the expression of apoptosis-related proteins,promoting the production of ROS,and destroying the mitochondrial function,thus altogether inhibiting the proliferation of tumor cells.