APR-246/PRIMA-1MET inhibits thioredoxin reductase 1 and converts the enzyme to a dedicated NADPH oxidase

Abstract

The low-molecular-weight compound APR-246 (PRIMA-1(MET)) restores wild-type conformation and function to mutant p53, and triggers apoptosis in tumor cells. We show here that APR-246 also targets the selenoprotein thioredoxin reductase 1 (TrxR1), a key regulator of cellular redox balance. APR-246 inhibited both recombinant TrxR1 in vitro and TrxR1 in cells. A Sec-to-Cys mutant of TrxR1 was not inhibited by APR-246, suggesting targeting of the selenocysteine residue in wild-type TrxR1. Preheated APR-246 and its conversion product methylene quinuclidinone (MQ) were much more efficient TrxR1 inhibitors than APR-246 itself, indicating that MQ is the active compound responsible for TrxR1 enzyme inhibition. TrxR1 inhibited by MQ was still functional as a pro-oxidant NADPH oxidase. Knockdown of TrxR1 caused a partial and reproducible attenuation of APR-246-induced tumor cell death independently of p53 status. Cellular TrxR1 activity was also inhibited by APR-246 irrespective of p53 status. We show that APR-246 can directly affect cellular redox status via targeting of TrxR1. Our findings provide an explanation for the previously observed effects of APR-246 on tumor cells lacking mutant p53.

Figure 1

Inhibition of TrxR1 in vitro by APR-246. (a) Preheated APR-246 and MQ efficiently inhibited TrxR1 according to the DNTB (Ellman) assay. (b) Kinetics of TrxR1 inhibition by indicated concentrations of APR-246, preheated APR-246 and MQ. (c) NADPH oxidase activity for TrxR1 treated with APR-246, preheated APR-246 and MQ as assessed by the juglone assay. (d) Sec-to-Cys variants of TrxR1 are resistant to inhibition by APR-246, preheated APR-246 or MQ. Results are means±S.E., n=3–6

Figure 2

Inhibition of TrxR1 activity in living cells. (a) APR-246 inhibited activity of TrxR1 in H1299, H1299-His175, Saos-2, Saos-2-His273 and BL41tsp53 cells. Results are means±S.E., n=4. (b) Treatment with APR-246 reduced the expression of TrxR1 in H1299-His175 cells according to the western blot analysis

Figure 3

siRNA knockdown of TrxR1 inhibits APR-246-induced cell death. (a) Two different siRNAs against TrxR1 (TrxR1-siRNA-1 and TrxR1-siRNA-2) inhibited TrxR1 expression in H1299 and H1299-His175 cells for at least 72 h. (b) H1299-His175 cells treated either with scrambled siRNA or a combination of scrambled siRNA and APR-246, or with TrxR1-siRNA-2 and APR-246. DNA content was assessed by flow cytometry. (c) Quantification of the sub-G1 cell population. Data are means±S.E., n=4

Figure 4

siRNA knockdown of TrxR1 inhibits generation of ROS induced by treatment with APR-246. (a) H1299-His175 cells treated either with scrambled siRNA or with a combination of scrambled siRNA and 50 μM of APR-246 or with TrxR1-siRNA-2 and 50 μM of APR-246. ROS production was estimated by DCF staining and assessed by flow cytometry. (b) Quantification of ROS levels in H1299 and H1299-His175 cells. Data are means±S.E., n=4. MFI, mean fluorescence intensity