The Cytotoxicity Of Natural Killer Cells Combined With Tamoxifen Against Breast Cancer Cells In Vitro And Its Mechanism

Objective:To investigate the synergistic killer effect of natural killer(NK)cells combined with tamoxifen(TAM)against breast cancer cells(BCC)in vitro,and to explore its mechanism.Methods:1.Selected three human BCC lines with different surface receptors.They were MCF-7(ER+,PR+,HER-2–),BT-474(ER+,PR+,HER-2+)and MDA-MB-231(ER–,PR–,HER-2–).MTT assay was used to detect the anti-proliferation effects of TAM against BCC and NK cells.2.Calcein-AM release assay was used to detect the synergistic cytotoxicity of NK cells combined with TAM.3.Flow cytometry was used to detect the expression of activating-receptors NKp44,NKp46 and NKG2D and inhibitory-receptors CD158 a,CD158b,CD158b2 and CD158 e on NK cell,and detect activating-ligands MICA,ULBP1 and ULBP2 on BCC.Meanwhile,we detected the release of perforin and granzyme B of NK cells in different situations.4.ELISA assay was used to test the level of TNF-α and IFN-γ of NK cells.Results:1.TAM could inhibit the proliferation of MCF-7 and BT-474,and the anti-proliferation effect against MCF-7 was stronger than that against BT-474,P<0.05.It also could inhibit the proliferation of MDA-MB-231,but the effect was weaker than that against MCF-7 and BT-474,P<0.05.TAM against three BCC depended on concentration and time,P<0.05.2.The final concentration of TAM was 5μM for 24 h.TAM has no apparent anti-proliferation effect on NK cells,so they could be used simultaneously;Moreover,They had synergistic effects against BCC,and the effect of combination-group was obviously higher than that of NK group and TAM group,P<0.05.3.The expression of activating receptor NKp46 and ligands MICA,ULBP1 and ULBP2 increased when NK cells combined with TAM;In contrast,TAM could down-regulated the expression of inhibitory receptors CD158 a,CD158b,CD158b2 and D158 e,P<0.05.Due to the high basic level,the expression of perforin and granzyme B had no difference between different groups.4.Regardless of whether BCC excited,TAM could enhance the expression of NK cells cytokines TNF-α and IFN-γ,P<0.05.Conclusion:1.TAM could inhibit the proliferation of two hormone-receptor-positive BCC,and the anti-proliferation effect against HER-2–cell was stronger than that against HER-2+ cell.It also could inhibit the proliferation of hormone-receptor-negative BCC,but the effect was weaker than that against hormone-receptor-positive BCC.The anti-proliferation effects of TAM against three BCC have obvious concentration-time dependence.2.NK cells combined with TAM have synergistic killer effect against BCC in vitro.As the increase of effector-target ratios,the effects also increased.3.The synergetic mechanism may be as follows: 1)TAM could up-regulate the expression of activating receptors and ligands,meanwhile down-regulate the expression of inhibitory receptors to increase the killing ability of NK cells;2)TAM also could increase the secretion of TNF-α and IFN-γ of NK cells to enhance their cytotoxicity.