Polymethoxylated flavones (PMFs) are naturally occurring compounds that have biological effects on many cell types. We previously demonstrated that PMFs such as nobiletin potentiate the cytolytic activity of the human leukemic natural killer cell line KHYG-1 and increased level of the cytotoxic protein granzyme B (GrB) and the cytokine interferon-gamma (IFN-gamma). However, the precise mechanisms by which this occurs remain to be elucidated. In this study, we sought to identify and investigate the function of intracellular primary targets of the PMFs in KHYG-1 cells. Using affinity purification and mass spectrometry, we identified that 3'-hydroxy-4',5,6,7-tetramethoxyflavone (TMF) binds to the nuclear export factors Exportin-1 and -2 (XPO1 and XPO2) as TMF-binding proteins and demonstrated that nobiletin competes with TMF for XPO1 binding, suggesting that nobiletin also binds to XPO1. Treatment of KHYG1 cells with leptomycin B, a specific XPO1 inhibitor, increased the expression of GrB and IFN-gamma but did not potentiate lysis of specific target cells, suggesting that the cargo of XPO1 contributes to the expression of cytolytic genes but that this alone is insufficient to enhance cytolysis. Consistent with this, nobiletin and related PMFs induced the nuclear retention of NF-kappa B, a transcription factor that promotes GrB and IFN-gamma expression. PMFs also induced the nuclear retention of the tumor suppressor protein p53, a known XPO1 cargo protein, resulting in KHYG-1 cell cycle arrest. Collectively, these results suggest that PMFs modulate KHYG-1 function, at least in part, by inhibiting XPO1. (C) 2019 Elsevier Inc. All rights reserved.