Enhancement of Allo-NK Cell Activity in Myeloid Leukemia Cell Line KG-1a after Combined with Aclacinomycin

Abstract

Abstract Objective: To investigate whether aclacinomycin can enhance the killing effects of allogeneic NK cells on acute myeloid leukemia and the molecular mechanisms, thus providing an experimental basis and a new thinking for the clinical treatment of acute leukemia. Methods: NK cells were isolated from the peripheral blood of healthy people by antibody-labeled immunomagnetic beads, the percentage of NK cells was detected by flow cytometry, and the inhibitory effects of aclacinomycin on the proliferation of KG-1a cells were detected by CCK-8 assay. In vitro killing assay was employed to detect the in vitro killing effects on NK-1a cells by NK cells with different effector/target ratios after treatment. Clone formation assay was used to detect the inhibitory effects of aclacinomycin in combination with allogeneic NK cells on the proliferation of KG-1a cells. Furthermore, the effects of aclacinomycin in combination with allogeneic NK cells in inducing the apoptosis of KG-1a cells was detected by flow cytometry, and changes in the expressions of IAP protein families and proteins associated with both intracellular and extracellular apoptosis signaling pathways after treatment of aclacinomycin in combination with allogeneic NK cells were analyzed by immunoblotting. Meanwhile, the differences in the expression levels of calreticulin, heat shock protein, adenosine triphosphate and high mobility protein-1 were detected. Results: The percentages of NK cells in the peripheral blood before and after isolation with anti-CD3-CD56+ antibody-labeled immunomagnetic beads were (15.2 ± 3.7)% and (84.9 ± 5.5)%, respectively. The results of CCK-8 assay showed that the inhibitory effects of aclacinomycin on the proliferation of KG-1a cells were increased in a time-dependent manner. At the same time, this study showed that after the co-culture of NK cells and KG-1a cells at the effector/target ratios of 10: 1 and 20: 1, the killing effects on KG-1a cells were not significant (P <0.05). After 72 hours of treatment at the ratio of 20: 1, aclacinomycin could significantly increase the killing effects of NK cells on KG-1a cells. The results of clone formation assay showed that at 72 hours after the treatment of acycloemycin in combination with allogeneic NK cells, the number of KG-1a cell clones was (11 ± 3), significantly lower than that in the aclacinomycin-alone group (68 ± 7) and the NK cell group (121 ± 12, P <0.05), the differences were statistically significant (P <0.001). The results of flow cytometry showed that the total apoptotic rate of KG-1a cells was increased significantly at 72 hours after the treatment of aclacinomycin in combination with allogeneic NK cells, and the results of immunoblotting showed that the expression of cIAP, XIAP and Bcl-2 were significantly down-regulated after the treatment of aclacinomycin in combination with NK cells, while the expressions of pro-apoptosis proteins Smac and caspase-3 were significantly up-regulated. Meanwhile, the expressions of immunogenic death-associated calreticulin, heat shock protein, adenosine triphosphate and high mobility protein-1 were significantly up-regulated. Conclusions: Aclacinomycin can significantly enhance the killing effects of allogeneic NK cells on the KG-1a cell line in vitro through such a molecular mechanism in which the expression levels of IAP protein family-related proteins are inhibited. Moreover, the mechanisms are associated closely with the activation of both intracellular and extracellular apoptosis signaling pathways and the activation of immunogenic death-associated proteins. DisclosuresNo relevant conflicts of interest to declare.