NDRG2 contributes to cisplatin sensitivity through modulation of BAK-to-Mcl-1 ratio

Abstract

The downregulation of N-Myc downstream-regulated gene 2 (NDRG2) is known to be associated with the progression and poor prognosis of several cancers. Sensitivity to anti-cancer may be associated with a good prognosis in cancer patients, and NDRG2, which is induced by p53, sensitizes the cells to chemotherapy. However, the unique function of NDRG2 as an inducer of apoptosis under chemotreatment has not been sufficiently studied. In this study, we investigated the role of NDRG2 in chemo-sensitivity, focusing on cisplatin in U937 histiocytic lymphoma, which has the loss-of-functional mutation in p53. NDRG2 promoted the sensitivity to cisplatin through the modulation of the BAK-to-Mcl-1 ratio. The degradation of Mcl-1 and increase in BAK were mediated by JNK activation and the eIF2α/p-eIF2α pathway, respectively, which depended on PKR activation in NDRG2-overexpressed U937 (U937-NDRG2) cells. NOX5 was highly expressed in U937-NDRG2 cells and contributed to ROS production after cisplatin treatment. ROS scavenging or NOX5-knockdown successfully inhibited the sensitivity of U937-NDRG2 cells to cisplatin. Taken together, these findings indicate that NDRG2 contributed to the increased sensitivity to ciplatin through the modulation of Bak-to-Mcl-1 ratio regulated by NOX5-ROS-PKR pathway; therefore, we suggest that NDRG2 may be a molecular target for improving the efficacy of drug treatment in cancer patients.

Fig. 1. NDRG2 sensitized U937 cells to cisplatin through the modulation of the BAK-to-Mcl-1 ratio.

a U937-Mock and -NDRG2 cells were incubated with cisplatin (10 μM, 20 h). The cells were stained with Annexin V/PI and analyzed by flow cytometry. b The cells were stained with MitoTracker Red CMXRos, and those with depolarized mitochondria were analyzed by flow cytometry. c Bcl-2 family proteins were analyzed by immunoblotting using the indicated antibodies. The normalized BAK or Mcl-1 band intensity with α-tubulin was acquired using Image J software to determine the fold induction. d U937-Mock and U937-NDRG2 cells treated with cisplatin were immunostained with anti-active Bax and analyzed using a confocal microscope. Active Bax was labeled with a FITC-conjugated secondary antibody (green), and the mitochondria and nuclei were stained with Mitotracker CMXRos (red) and DAPI (blue), respectively. U937-Mock cells infected with the shMcl-1 lentivirus (e) or U937-NDRG2 cells infected by the shBAK lentivirus (f) were treated with cisplatin and the apoptotic cells were analyzed by Annexin V/PI staining. Ctrl: vehicle treatment or not infected group. **p < 0.01, ***p < 0.005 by t-tests. Data are presented as mean ± SEM

Fig. 2. JNK activation contributed to the sensitivity of U937-NDRG2 cells to cisplatin through the induction of Mcl-1 degradation.

a JNK phosphorylation kinetics in the cells treated with cisplatin at the indicated time points. b U937-NDRG2 cells were treated with cisplatin in the presence or absence of 5 μM SP600125 (a JNK inhibitor), and then the levels of p-JNK, Caspase, PARP, Mcl-1 and BAK protein were determined by immunoblotting. c, d The apoptotic cell death and mitochondrial depolarization of cisplatin-treated U937-NDRG2 cells were analyzed by flow cytometry. e U937-NDRG2 cells treated with cisplatin in the presence or absence of SP600125 were stained with anti-active Bax antibody (green), MitoTracker Red CMXRos (red), and DAPI (blue). The fluorescent pattern was visualized by confocal microscopy. Ctrl: vehicle-treated group. ***p < 0.005 by t-test. Data are presented as mean ± SEM

Fig. 3. The eIF2α/p-eIF2α pathway was involved in the upregulation of BAK in U937-NDRG2 cells treated with cisplatin.

a U937-Mock and U937-NDRG2 cells were treated with cisplatin for the indicated time, and the levels of p-eIF2α (Ser51), ATF4, and CHOP proteins were determined by immunoblotting. b, c U937-NDRG2 cells were incubated with cisplatin in the presence or absence of salubrinal (40 μM). Apoptosis and depolarization of the mitochondrial membrane were analyzed using flow cytometry. d The levels of the indicated proteins were determined by immunoblotting. ***p < 0.005 by t-test. Data are presented as mean ± SEM

Fig. 4. Activation of PKR was necessary for the sensitivity of U937-NDRG2 cells to cisplatin.

a The phosphorylation of PKR was determined by immunoboltting in U937-Mock and U937-NDRG2 cells treated with cisplatin. b, c U937-NDRG2 cells were treated with cisplatin in the presence or absence of the PKR inhibitor C16 (250 nM). Apoptosis and mitochondrial potential were analyzed using flow cytometry. d Levels of the indicated proteins were determined by immunoblotting. ***p < 0.005 by t-test. Data are presented as mean ± SEM

Fig. 5. ROS produced in cisplatin-treated U937-NDRG2 cells induced PKR activation.

a U937-Mock and U937-NDRG2 cells were treated with cisplatin, and then the level of ROS was determined using DCFH-DA staining. b, c U937-NDRG2 cells were treated with cisplatin in the presence or absence of a ROS scavenger, NAC (10 μM). Apoptosis and the mitochondrial potential were analyzed using flow cytometry. d Levels of the indicated proteins were determined by immunoblotting. Ctrl: vehicle-treated group. ***p < 0.005 by t-test. Data are presented as mean ± SEM

Fig. 6. The upregulation of NOX5 expression was essential for the higher sensitivity of U937-NDRG2 cells to cisplatin.

a Human NOX5 expression was confirmed by quantitative RT-PCR. b U937-NDRG2 cells were transfected with scramble- or shNOX5-lentivirus. The knockdown efficiency of NOX5 expression was confirmed by immunoblotting, and apoptotic cell death was then analyzed by Annexin V/PI staining. c NOX5-knockdown cells were stained with DCFH-DA and ROS levels were determined by flow cytometry. d Active Bax (green) in the indicated experimental groups was visualized by confocal microscopy. U937-NDRG2 cells were treated with cisplatin for 12 h. e Levels of the indicated proteins were determined by immunoblotting. Ctrl: vehicle treatment or not infected group. ***p < 0.005 by t-test. Data are presented as mean ± SEM

Fig. 7

Diagrammatic representation of the proposed apoptotic pathway that mediates the sensitivity to cisplatin in NDRG2-expressing U937 cells