Synergistic Growth Inhibition by the Combination of Acyclic Retinoid Plus Vitamin K2 in HL-60 Cells
Authors: Kitagawa Junichi , Kasahara Senji , Hara Takeshi , Kanemura Nobuhiro , Shimizu Masahito , Tsurumi Hisashi , Moriwaki Hisataka 
Published: April 2020

Abstract
Introduction: All-trans retinoic acid (ATRA) inhibits the proliferation and induces terminal differentiation in APL cells by binding to retinoic acid receptor-a (RARa). The activation of retinoid X receptor (RXR) also plays an essential role. These receptors form heterodimeric complex (RAR/RXR) and thereby regulate the expression of target genes. We have recently reported that the accumulation of phosphorylated forms of RXRa (p-RXRa), which loses heterodimeric activity with RAR, is associated with the abnormal cell growth and the resistance to retinoic acid in APL cells (Kanemura N. et al. Leuk Res. 2008). Acyclic retinoid (ACR) inhibits cell growth and induces apoptosis in liver cancer cells by inhibiting the phosphorylation of RXRa. ACR acted synergistically to inhibit growth of these cancer cells when combined with vitamin K2 (VK2). In order to develop a new effective regimen for APL treatment, we investigated whether the combination of ACR plus VK2 exerts synergistic growth-inhibitory effects on HL-60 cells.
Materials and Methods: HL-60 cells were treated with the indicated concentrations of ACR (0.01 – 50 mM) or ATRA (0.01 – 50 mM) in the presence or absence of VK2 (1 mM) for 72 hours. The cells were also treated with the indicated concentrations of VK2 (0.01 – 50 mM) in the presence or absence of ACR (1 mM) for same period and the cell numbers were then determined by using the Trypan Blue dye exclusion method. To determine whether the combined effects of ACR plus VK2 were synergistic, the combination index-isobologram was calculated and used in the combination assays. The induction of apoptosis by the combination of ACR plus VK2 was examined using a fluorescent annexin V/propidium iodide staining. Cell cycle analysis was performed by determinng the BrdU-labeled cells using flow cytometry. The effects of the combination of ACR plus VK2 on the expression levels of phosphorylated (i.e. activated) form of ERK (p-ERK) and p-RXRa proteins were examined by Western blot analysis.
Result: ACR and ATRA dose-dependently inhibited proliferation in HL-60 cells. Although VK2 (1 mM) alone could not inhibit the growth of HL-60 cells, the combination of various concentrations of ACR plus 1 mM VK2 synergistically inhibited the growth of these leukemic cells. On the other hand, no synergistic growth inhibition was observed when the cells were treated with the combination of ATRA plus VK2. The induction of apoptosis was significantly increased by the combined treatment with ACR plus VK2 (38.0% to 46.6%, p&lt;0.05). Cell cycle analysis indicated that the percentage of G0/G1 phase cells was significantly increased by the treatment with ACR alone (32.5% to 62.3%, p&lt;0.05) or VK2 alone (27.8% to 52.8%, p&lt;0.05), respectively. The expression of p-ERK protein was decreased when the cells were treated with VK2 alone or the combination of ACR plus VK2. Treatment of these leukemic cells with ACR alone or combination of ACR plus VK2 also caused a marked decreased in the expression level of p-RXRa. This decrease was most apparent when the cells were treated with a combination of ACR plus VK2.
Discussion: The present study indicated that the combination of ACR plus VK2 caused the synergistic growth inhibition and induction of apoptosis in HL-60 cells, whereas the combination of ATRA plus VK2 did not exert such beneficial effects. The synergism might be associated with the induction of G0/G1 phase of cell cycle arrest and the inhibition of the expression of p-ERK and p-RXRa proteins caused by the combination of these agents. Among these findings, restoration of the function of RXRa by inhibiting its aberrant phosphorylation plays a critical role to exert synergistic anti-cancer effects.
Conclusion: The combination of ACR plus VK2 might hold promise as a clinical modality for the treatment of APL due to their synergism. Targeting the RXRa phosphorylation might be one of the effective strategies for the therapy of certain types of human leukemia.