Killer effect of natural killer cells combined with tamoxifen on breast cancer cells in vitro and its mechanism / 吉林大学学报(医学版)

ABSTRACT

Objective:To investigate the synergistic killer effect of natural killer cells(NK cells) combined with tamoxifen(TAM) on breast cancer cells(BCC)through the experiment in vitro,and to explore its mechanism.Methods:Three kinds of BCC with different receptor expression levels were selected for the experiment.Blank control group, different concentrations of TAM groups and different time groups were set up.MTT assay was used to detect the inhibitory rates of proliferation of cells,and the final experiment concentration of 5 μmol·L-1 was determined.The cells were divided into natural-release group,largest-release group,TAM group,NK cells group, and combined-experimental group(BCC+NK cells+TAM),and the synergistic killer effect of NK cells combined with TAM in different effector-target ratios were detected with Calcein-AM release assay.In ELISA assay the cells were divided into blank control group (NK cells),NK cells+TAM group, NK cells+BCC group and combined-experimental group,and the levels of IFN-γ and TNF-α in the NK cells in various groups were measured.In flow cytometry detection the cells were divided into blank control group (NK cells),NK cells+TAM group,NK cells+ BCC group, and combined-experimental group,and the expression levels of NKp46,CD158a,CD158b,CD158b2,and CD158e were determined;while the cells were divided into blank control group (BCC),BCC+TAM group,BCC + NK cells group, and combined-experimental group,and the expression levels of the MICA,ULBP1 and ULBP2 were detected.Results: The MTT assay results showed that the inhibitory rates of proliferation of 3 kinds of BCC had obvious time-and concentration-dependence (P<0.05).The Calcein-AM release assay results showed that the killing-rates of BCC in TAM groups were increased with the increase effector-target ratios of compared with NK cells group;and the killing-rate in combined experimental group was obviously higher than those in NK cells and TAM groups(P<0.05).The ELISA assay results showed that the levels of TNF-α and IFN-γ of NK cells in various experimental groups with BCC or not were increased compared with blank control group(P<0.05 or P<0.01);the levels were significantly increased when combined with TAM (P<0.05).The flow cytometry results showed that the NKp46 expression levels in various experimental groups were elevated compared with blank control group(P<0.05);the expression levels of CD158a, CD158b,CD158b2, and CD158e were significantly decreased(P<0.05);the expression levels MICA,ULBP1, and ULBP2 in BCC were significantly increased (P<0.05).Conclusion:The NK cells combined with TAM has the synergistic killer effect on the BCC in vitro.The synergetic mechanism may be as follows TAM could increase the secretion of TNF-α and IFN-γ of NK cells to enhance their cytotoxicity;TAM also could up-regulate the expression levels of activating receptors and activating ligands,and down-regulate the expression levels of inhibitory receptors to increase the killing ability of NK cells.