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. 2014 Dec;35(12):2660-9.
doi: 10.1093/carcin/bgu186. Epub 2014 Sep 3.

Induction of Nur77-dependent apoptotic pathway by a coumarin derivative through activation of JNK and p38 MAPK

Yuqi Zhou  1 , Wei Zhao  1 , Guobin Xie  1 , Mingfeng Huang  1 , Mengjie Hu  1 , Xin Jiang  1 , Dequan Zeng  1 , Jie Liu  1 , Hu Zhou  2 , Haifeng Chen  1 , Guang-Hui Wang  3 , Xiao-Kun Zhang  4
Affiliations

Induction of Nur77-dependent apoptotic pathway by a coumarin derivative through activation of JNK and p38 MAPK

Yuqi Zhou et al. Carcinogenesis. 2014 Dec.

Abstract

Coumarins are plant-derived natural products with a broad range of known pharmacological activities including anticancer effects. However, the molecular mechanisms by which this class of promising compounds exerts their anticancer effects remain largely unknown. We report here that a furanocoumarin named apaensin could effectively induce apoptosis of cancer cells through its activation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Apoptosis induction by apaensin in cancer cells was suppressed by chemical inhibitors of JNK and p38 MAPK. Inhibition of the expression of orphan nuclear receptor Nur77 by small interfering RNA (siRNA) approach also abrogated the death effect of apaensin. Molecular analysis demonstrated that JNK activation was required for the nuclear export of Nur77, a known apoptotic event in cancer cells. Although p38 MAPK activation was not involved in Nur77 nuclear export, it was essential for Nur77 mitochondrial targeting through induction of Nur77 interaction with Bcl-2, which is also known to convert Bcl-2 from an antiapoptotic to a proapoptotic molecule. Together, our results identify a new natural product that targets orphan nuclear receptor Nur77 through its unique activation of JNK and p38 MAPK and provide insight into the complex regulation of the Nur77-Bcl-2 apoptotic pathway.

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Figures

Fig. 1.
Fig. 1.
Induction of apoptosis by apaensin. (A) Structure of four coumarin derivatives and their apoptotic effect. NIH-H460 cells treated with 10 μM of isooxypeucedanin (a), oxyalloimperatorin (b), apaensin (c) and demethylfuropinarine (d) for 6h were analyzed for the cleavage of PARP by western blotting. (B) Apoptotic effect of apaensin revealed by DAPI staining. NIH-H460 cells treated with apaensin (10 μM) for 6h were subjected to DAPI staining. Upon apaensin treatment, about 43.6% of cells displaying apoptotic nuclear morphology cells, while 7.8% of cells showed apoptotic nuclear morphology in the absence of treatment. (C) Dose dependent effect of apaensin. NIH-H460 cells or MCF-7 cells treated with apaensin (2.5, 5 and 10 μM) for 3h were analyzed by western blotting. (D) Time-course analysis. NIH-H460 cells or MCF-7 cells treated with apaensin (10 μM) for 1, 3 and 6h were analyzed by western blotting. One of three to five similar experiments is shown.
Fig. 2.
Fig. 2.
Role of apaensin activation of p38 MAPK and JNK. (A) Activation of p38 MAPK and JNK by apaensin. NIH-H460 cells treated with 5 μM of apaensin for 0, 0.5, 1, 3, 6h were analyzed for the activation of JNK and p38 MAPK by western blotting using antibodies recognizing phosphorylated (P-) JNK and p38 MAPK. The levels of β-actin were used for loading control. Right panels represent densitometric analyses of relative pJNK/JNK and P-p38/p38 MAPK ratio. (B) Role of p38 MAPK, JNK and LMB. NIH-H460 cells were incubated for 3h with apaensin (10 μM), in the presence or absence of SP600125 (10 μM), SB203580 (10 μM) or LMB (10ng/ml), and analyzed by western blotting for PARP cleavage. (C) Nur77 expression is required for the death effect of apaensin. NIH-H460 cells were transfected with control or Nur77 siRNA for 48h, then treated with or without 5 μM apaensin for the indicated time. Lysates prepared were analyzed for PARP cleavage and Nur77 expression by western blotting. One of three to five similar experiments is shown.
Fig. 3.
Fig. 3.
Induction and regulation of Nur77 nuclear export by apaensin. (A) Effect of apaensin on the subcellular localization of Nur77. NIH-H460 cells were treated with apaensin (5 μM) in the presence or absence of SP600125 (10 μM) or SB203580 (10 μM) for 3h. The subcellular localization of Nur77 was examined by immunostaining using anti-Nur77 antibody and revealed by immunofluorescence microscopy. About 95% of control cells showed exclusive nuclear staining of Nur77, while approximately 80–85% of apaensin-treated cells displayed diffused distribution of Nur77 shown. (B) Effect of apaensin on the subcellular localization of Nur77 revealed by cellular fractionation. Nuclear and cytoplasmic fractions were prepared from NIH-H460 cells treated with apaensin (5 μM for 1 and 3h or 10 μM for 1 and 2h) and subjected to western blotting analysis using anti-Nur77 antibody. The purity of cellular fraction was confirmed by using anti-PARP and anti-α-tubulin antibodies. One of three similar experiments is shown. (C) JNK phosphorylation of Nur77. NIH-H460 cells treated with 5 μM apaensin in the presence or absence of 10 μM SP600125 or 10 μM SB203580 were analyzed for Nur77 expression by western blotting using anti-Nur77 antibody. (D) JNK activation is required for induction of Nur77 nuclear export. Nuclear and cytoplasmic fractions were prepared from NIH-H460 cells treated with apaensin (5 μM for 3h) in the presence or absence of SP600125 (10 μM) and subjected to western blotting analysis using anti-Nur77 antibody. The purity of cellular fraction was confirmed by using anti-PARP and anti-α-tubulin antibodies. One of three similar experiments is shown.
Fig. 4.
Fig. 4.
Effect of apaensin on Nur77 mitochondrial targeting and BAX activation. (A) Induction and regulation of Nur77 mitochondrial targeting by apaensin. NIH-H460 cells treated with apaensin (5 μM) for 3h in the presence or absence of SP600125 (10 μM), SB203580 (10 μM) or BIRB796 (1 μM). Endogenous Nur77 was immunostained with anti-Nur77 antibody. Cells were co-stained with Mito-tracker that recognizes the mitochondria and with DAPI to visualize the nuclei. Fluorescent microscopy was used to determine the overlap of Nur77 and Mito-tracker. About 95% of control cells showed exclusive nuclear staining of Nur77, and approximately 65% of apaensin-treated cells displayed colocalization with mitochondria shown. Most of cells displayed exclusive nuclear staining of Nur77 upon cotreatment of apaensin with SP600125, whereas 50–55% of cells exhibited cytoplasmic Nur77 incapable of colocalizing with Mito-tracker when cotreated with apaensin and p38 MAPK inhibitor (SB203580 or BIRB796). (B) Apaensin activation of p38 MAPK is required for Nur77 mitochondrial targeting. Nuclear, cytosolic and mitochondrial fractions prepared from NIH-H460 cells treated with apaensin (5 μM for 3h) in the presence or absence of SP600125 (10 μM) or SB203580 (10 μM) were subjected to western blotting analysis using anti-Nur77 antibody. The purity of cellular fractions was confirmed by using anti-PARP antibody, anti-α-tubulin and Hsp60 antibody. (C) Induction of Nur77 colocalization with Bcl-2 by apaensin. NIH-H460 cells were treated with apaensin (5 μM) for 3h, and the localization of Nur77 and Bcl-2 was examined by immunostaining and revealed by immunofluorescence microscopy. Approximately 75% apaensin-treated cells displayed colocalization with Bcl-2 shown. (D) Induction of BAX activation by apaensin. NIH-H460 cells treated with apaensin (5 μM) for 6h were examined for BAX activation by antibody against BAX (6A7). Cells were also stained with DAPI to visualize the nucleus. About 85% of apaensin-treated cells displayed various degrees of BAX staining shown.
Fig. 5.
Fig. 5.
Role of p38 MAPK in Nur77-Bcl-2 interaction. (A) Induction of Nur77 interaction with Bcl-2 by apaensin and its inhibition by SB203580. Myc-Bcl-2 (2 μg) and Flag-Nur77 (2 μg) alone or together were transfected into MCF-7 cells. After 16h, cells were treated with apaensin (5 μM) in the presence or absence of SB203580 (10 μM) for 3h. Cell lysates were immunoprecipitated by using monoclonal anti-Flag antibody. Lysates and immunoprecipitates were examined by immunoblotting using anti-Flag and anti-Myc antibody. The same membranes were blotted with anti-Flag antibody to determine immunoprecipitation specificity and efficiency. Input represents 5% of cell lysates used in the co-immunoprecipitation assays. (B) Role of apaensin activation of JNK and p38 MAPK in the regulation of the Nur77-Bcl-2 apoptotic pathway. Our results demonstrate that apaensin activation of the Nur77-Bcl-2 apoptotic pathway involves its activation of JNK and p38 MAPK. While apaensin activation of JNK promotes Nur77 nuclear export through phosphorylation of Nur77, apaensin activation of p38 MAPK induces interaction of cytoplasmic Nur77 with Bcl-2. The coordinated action of JNK and p38 MAPK and perhaps other undefined apaensin-regulated cellular factors results in Nur77 mitochondrial targeting, leading to apoptosis of cancer cells.
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