Chalcone skeleton promotes transcription of gp91‐phox gene but inhibits expression of gp91‐phox protein, and hydroxyl groups in hydroxychalcones participate in the stable expression of gp91‐phox protein

Abstract

The effects of chalcone and butein on the induction of the superoxide anion (O2‐)‐generating system were studied in U937 cells by all‐trans retinoic acid (RA). The chalcone skeleton, a common structural motif in them, significantly enhanced the transcription of gp91‐phox in an epigenetic manner. In contrast, chalcone and butein showed opposite effects on the induction of the O2‐‐generating activity by RA and the expression of gp91‐phox protein. Chalcone inhibited, whereas butein promoted, the induction of O2‐‐generating activity by RA and the expression of gp91‐phox protein. These data raise the possibility that modification of the chalcone skeleton could produce more effective differentiation‐promoting agents.

Sulforaphane displays the growth inhibition, cytotoxicity and enhancement of retinoic acid-induced superoxide-generating activity in human monoblastic U937 cells

“…Human monoblastic leukemia U937 cells (RCB0435) were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. Cells were grown in RPMI-1640 culture medium containing 10% FBS and 5 μg/mL plasmocin as described (Kikuchi et al, 2018a;Kikuchi et al, 2018b;Kikuchi et al, 2019). Cells (1.0 x 10 6 ) in 5 mL of culture medium were incubated in the absence or presence (1, 2 or 5 μM) of sulforaphane at 37°C.…”

“…Cells (1.0 x 10 6 ) in 5 mL of culture medium were incubated with 1 μM RA in the absence or presence of sulforaphane (1 or 2 μM) for 48 hr at 37°C. O 2 -was quan-tified by measuring chemiluminescence (CL) using Diogenes-luminol CL probes as described (Kikuchi et al, 2011;Kikuchi et al, 2018a;Kikuchi et al, 2019). Cells (1 x 10 6 cells) were washed by PBS, and resuspended in 1 mL PBS containing 1 mM MgCl 2 , 0.5 mM CaCl 2 , 5 mM glucose and 0.03% bovine serum albumin.…”

“…Total RNA was isolated from the cells. Semiquantitative RT-PCR was performed as described using specific sense and antisense primers of five components essential for the O 2 --generation system: p22-phox, gp91-phox, p40-phox, p47-phox and p67-phox (Kikuchi et al, 2011;Kikuchi et al, 2018a;Kikuchi et al, 2019). Human GAPDH gene was used as internal controls.…”

“…Fifty μL of loading buffer [0.5 M Tris-HCl (pH 6.8) containing 5% SDS, 20% glycerol] was added to the solubilized membrane fraction. Immunoblotting was carried out as described Kikuchi et al, 2011, Kikuchi et al, 2018a, Kikuchi et al, 2019. Data analyses were performed using a luminescent image analyzer STAGE-5100.…”

“…On various stimuli, these five proteins are assembled on the membrane (formation of NADPH oxidase), resulting in O 2 --generation. Human monoblastic leukemia U937 cells differentiate to macrophage-like cells by all-trans retinoic acid (RA), and aquire the O 2 --generating activity (Kikuchi et al, 1994;Kikuchi et al, 1996;Kikuchi et al, 2010;Kikuchi et al, 2011;Kikuchi et al, 2018a;Kikuchi et al, 2019). Of course, gene expression of each the five essential components for the O 2 --generation activity (p22-phox, gp91phox, p40-phox, p47-phox and p67-phox) is significantly up-regulated.…”