Background
Retinoid receptors are retinoid ligand-activated transcription factors that are divided into retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Both RARs and RXRs have three isoforms, including RARα/β/γ and RXRα/β/γ. These proteins are encoded by distinct loci and exist as alternatively spliced variants [
1]. Active retinoid receptors consist of RAR/RXR heterodimers, which activate the transcription of many target genes by binding retinoic acid responsive elements in promoter and/or enhancer regions. They exert many essential and potent biological functions with respect to the regulation of cell proliferation, differentiation, apoptosis, and autophagy [
2-
4]. Accordingly, retinoids and their receptors are also widely involved in the pathogenesis of many diseases, especially cancers [
5]. A typical example is that of acute promyelocytic leukemia (APL), a unique subtype of acute myeloid leukemia (AML). Almost all APL patients carry chromosome translocations involving
RARα
, most of which are t(15;17). This causes fusion of the
promyelocytic leukemia (
PML) gene to the
RARα gene and expression of a
PML-RARα fusion gene, leading to impaired retinoid signaling and pathogenesis of APL. Importantly, all-trans retinoic acid (ATRA) and arsenic trioxide target the PML-RARα fusion protein to induce differentiation and/or apoptosis of leukemia-initiating cells [
6-
10]. Besides APL, some other types of cancer also present with aberrant expression of RARs. For example, the expression of RARα/β and RXRα/β are down-regulated in pancreatic ductal adenocarcinoma, which is associated with poor patient survival outcomes [
11].
The mechanisms regulating the expression of RARs are not fully understood. ATRA can directly target RARα to ubiquitin-proteasome degradation in APL and non-APL cells [
12], while activation of c-Jun N-terminal kinase (JNK) can contribute to RAR dysfunction by phosphorylating RARα at Thr181, Ser445, and Ser461. This induces RAR degradation through the ubiquitin-proteasome pathway, pointing to JNK as a key mediator of aberrant retinoid signaling in lung cancer cells [
13]. Additionally, JNK activation by oxidative stress also suppresses retinoid signaling through proteasomal degradation of RARα in hepatic cells [
14]. More recently, pharicin B, a novel natural
ent-kaurene diterpenoid derived from
Isodon pharicus leaves, was reported to rapidly stabilize RARα protein in various AML cell lines and primary leukemic cells from AML patients [
15].
Oridonin, another
ent-kaurene diterpenoid isolated from
Rabdosia rubescens, has a variety of biological effects, such as anti-inflammatory, anti-viral, and anti-bacterial functions, as well as anti-tumor effects on different cancers including liver [
16], prostate [
17], breast [
18], and leukemia [
19]. Accumulating evidence illustrates that oridonin has extensive anti-tumor effects involving regulation of the cell cycle, apoptosis, autophagy, and differentiation [
20-
22]. Previously, we reported that oridonin could induce ROS-initiated apoptosis and enhance ATRA-induced differentiation in APL cells. Interestingly, the differentiation-enhancing effect of oridonin was accompanied by increased levels of RARα protein [
23]. In this work, we further investigated the mechanisms underlying oridonin stabilization of RARα protein.
Methods
Cells
NB4/GFP and NB4/GFP-MAD cells were generous gifts from F. Besancon (Hôpital St. Louis, Paris, France). Construction of the two cell lines was described previously by Komura et al. [
24]. NB4, NB4/GFP, and NB4/GFP-MAD cells were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT, USA). COS-7 and 293 T cells were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, USA), supplemented with 10% FBS in a humidified incubator at 37°C with 5% CO
2/95% air (v/v).
Reagents and antibodies
Oridonin (purity >99.5%) was purchased from Xi’an Haoxuan Biotechnique, China. It was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM and stored at −20°C. Both N-acetyl-l-cysteine (NAC) and ATRA were purchased from Sigma-Aldrich. Recombinant human tumor necrosis factor (TNFα) was obtained from Peprotech (Rocky Hill, NJ, USA). Cycloheximide was purchased from Sigma-Aldrich. ERK inhibitor PD98059, p38 inhibitor SB203580, JNK inhibitor SP600125, and NF-κB inhibitor Bay 11–7082 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). When cells were treated with these reagents, matching concentrations of vehicle were used as the control and the final concentration of DMSO was kept at or below 0.1% in all experiments.
Antibodies recognizing p65, IκBα, and RARα were purchased from Santa Cruz Biotechnology. Antibodies recognizing phospho-IκBα (Ser32/Ser36), phospho-p65, IκB kinase beta (IKKβ), phospho-IKKα/β, phospho-ERK1/ERK2, ERK1/ERK2, phospho-p38, p38, phospho-JNK, and JNK were purchased from Cell Signaling Technology (Beverly, MA, USA).
Western blot
Equal amounts of protein extracts were loaded onto a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system, electrophoresed, and transferred to nitrocellulose membranes (Amersham). After blocking with 5% (w/v) nonfat milk in PBS for 2 hours at room temperature, the membranes were incubated with specific antibodies overnight, followed by incubation with horseradish peroxidase-linked secondary antibody (Cell Signaling Technology) for 1 hour at room temperature. The signals were detected by the chemiluminescence phototope-HRP kit (Millipore), according to the manufacturer’s instructions. β-actin was probed as an internal control. All experiments were repeated three times, and similar results were obtained.
RNA extraction and real-time quantitative RT-PCR
The cells were lysed, and total RNA was isolated using a TRIzol kit (Invitrogen). Then, the RNA was treated with DNase (Promega). Complementary DNA was synthesized according to the manufacturer’s instructions. Real-time quantitative RT-polymerase chain reactions (PCRs) for RARα, retinoic acid receptor beta (RARβ), CCAAT/enhancer binding protein-beta (C/EBP-β), retinoic acid-induced genes E (RIG-E) and interferon regulatory factor 1 (IRF-1), were performed with SYBR Green PCR Master Mixture Reagents (Applied Biosystems) on the Applied Biosystems 7300 real-time RT-PCR system. The specific primers used as follows: 5′-TCTGTGAGAAACGACCGAAAC-3′ and 5′-TGAGGGTGGT GAAGCCG-3′ for RARα gene, 5′-AGTTTGATGGAGTTGGG TGGAC-3′ and 5′-GATGCTGCCATTCGGTTTG-3′ for RARβ, 5′-TCAGCACCC TGCGGAACTT-3′ and 5′-AAGTGCCCCAGTGCCAAAG-3′ for C/EBPβ, 5′-AGG GAGACCGTGTCAGTA GGG-3′ and 5′-CGGAAGTGGCAGAAACCCC-3′ for RIG-E, and 5′-ATGAGACCCTGGCTAGAG-3′ and 5′-AAGCATCCGGTAC ACTCG-3′ for IRF-1. The primers were synthesized by Sangon Biotech (Shanghai, China). All experiments were performed in triplicate. Data were normalized to the housekeeping gene β-actin, and the relative abundance of transcripts was calculated by the comparative ΔΔCT method.
Redox diagonal electrophoresis
The samples were prepared in 1× SDS sample buffer without any reducing agent and loaded onto 10% SDS-PAGE gels. After the first dimension, non-reducing electrophoresis, the entire lane containing the separated proteins was excised and soaked for 20 min in SDS sample buffer containing 100 mM dithiothreitol to reduce any disulfide bonds present between proteins or within proteins. The gel lane was then rotated 90 degrees and placed horizontally on top of a large-format, 1.5-mm-thick 10% acrylamide gel. Under these conditions, the proteins that do not form disulfide bond electrophorese identically in both dimensions and form a diagonal after the second dimension. In contrast, proteins that contain intra-chain disulfide bond lie above this diagonal, while those that form inter-disulfide bond fall below the diagonal. Finally, immunoblot was performed to identify the dots containing RARα.
Detection of intracellular ROS level
The cells were incubated with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probes/Invitrogen) in PBS for 30 min at 37°C while protected from light. The fluorescence intensity, which resulted from the oxidation of the dye, was measured by fluorescence-activated cell sorting (FACS) to determine the level of ROS. The experiments were performed in triplicate.
Plasmid construction and transfection
Pairs of complementary shRNA oligonucleotides against catalase (5′-AGATGATCTACT CAGAAAT-3′), p65 (5′-GATGAGATCTTCCTACTGT-3′), and non-targeting control NC (5′-TCCCGTGAATTGGAATCCT-3′) were synthesized by Sangon Biotech (Shanghai, China), annealed, and ligated into the pSIREN-RetroQ Vector (Clontech Laboratories) between the BamHI and EcoRI sites. A full-length cDNA of human RARα was amplified from NB4 cells by PCR and cloned into the virus expression vector, pMSCV-puro (Clontech Laboratories). shRNA/cDNA-carrying retroviruses were produced in 293 T cells and used to infect NB4 or COS-7 cells. Forty-eight hours after transfection, cells were selected with puromycin (Sigma-Aldrich).
Immunofluorescence assay
The cells, which were treated as described in the text, were collected onto slides and fixed with 4% paraformaldehyde. After permeabilization with methanol and blocking with 2% (w/v) bovine serum albumin in PBS, the cells were incubated overnight with the antibody against p65. Then, the cells were stained with FITC-labeled anti-rabbit IgG for 1 hour. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Eugene, OR). The stained cells were visualized by fluorescence microscopy (Olympus BX51; Olympus, Tokyo, Japan).
Patient samples
Patient samples were collected after obtaining informed consent under a procurement protocol that was approved by the Ethics Committee of Rui-Jin Hospital affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China. Mononuclear cells were isolated from bone marrow of AML patients using Ficoll-Hypaque liquid (Pharmacia, Piscataway, NJ, USA) and standard procedures.
Statistical analysis
Results were derived from at least three independent experiments and expressed as the mean ± standard deviation. The Student’s t-test was used for statistical analysis. P < 0.05 was considered to be statistically significant.
Discussion
In this study, we report that the natural diterpenoid, oridonin, induces a moderate production of cellular ROS that activates upstream of the NF-κB signaling pathway to cause nuclear translocation of p65, which is responsible for oridonin-stabilized RARα protein. These findings indicate that moderate oxidative stress induced by oridonin may change the intrinsic mechanisms that regulate RARα protein stability through the NF-κB signaling pathway, which provides a new perspective of oridonin as a candidate anti-neoplastic drug.
The modulation of RARα by ATRA during APL treatment has stimulated considerable interest in RARα metabolism and its potential therapeutic mechanism [
37]. ATRA activates RARα signaling with subsequent effects on differentiation, while at the same time steady-state RARα protein levels are markedly reduced [
12]. RARα, as the receptor for ATRA, is required for its action; therefore, RARα degradation is thought to be an inbuilt resetting mechanism to make ATRA signaling self-limiting. Therefore, it is possible that stabilizing the RARα protein can optimize this signaling, which indicates that RARα could be a potential target for cancer therapeutics. Recently, several studies have demonstrated that some compounds, such as lithium chloride (LiCl) [
38], granulocyte-colony stimulating factor [
38], STI571 [
39], di-
tert-butyl-benzohydroquinone [
40], Pharicin B [
15], and oridonin [
23], which are capable of attenuating ATRA-induced loss of RARα protein, have been shown to enhance ATRA-induced differentiation. However, the underlying mechanism of RARα accumulation has not been fully described. In this work, we used oridonin as a probe to show that a moderate level of oxidative stress can stabilize RARα protein through the nuclear translocation of p65. Further investigation is needed to test whether this mechanism can be extended to other small molecules with similar RARα-stabilizing ability. In addition, because RARα is an essential transcriptional and homeostatic regulator of a plethora of physiological processes, numerous investigations have established correlations between down-regulation of RARα and malignant progression. In addition to APL, this has been observed in cervical carcinoma [
41], skin tumors [
42], motor neuron disease [
43], and breast cancer [
44]. In this context, stabilizing RARα may permit optimized use of retinoids in cancer prevention and treatment, which warrants further investigation.
It is now widely accepted that a moderate degree of ROS can play an important role in determining cell fate through the modulation of cellular signaling and gene expression [
45,
46]. For example, elevated but sub-lethal levels of ROS can modulate the differentiation of various types of cells, such as hematopoietic cells [
47,
48], neurons [
49], embryonic stem cells [
50], osteoclasts [
51], and cardiac stem cells [
52]. However, little is known regarding the molecular targets of ROS. Here, we found that moderately increased levels of ROS are crucial for oridonin-induced RARα stabilization, which may account for the anti-neoplastic mechanism of oridonin. It is tempting to suggest that this newly identified mechanism may underlie similar differentiation effects of some natural diterpenoids. Nevertheless, attention should be paid to the cell type, as well as to the extent and duration of ROS increase, as these factors can determine the precise consequences of the cellular response to oxidative stress. For instance, a relatively high concentration of H
2O
2 (0.1 mM) can suppress retinoid signaling through the proteasomal degradation of RARα [
14].
The NF-κB family is a group of transcriptional factors consisting of p65 (RelA), RelB, c-Rel, p50/p105, and p52/p100. In the classical NF-κB signaling pathway, the p50/p65 dimer is sequestered in the cytoplasm by IκΒα. After stimulation, IκΒα is phosphorylated and consequently degraded through the proteasomal pathway. Thus, the p50/p65 dimer is released, translocates to the nucleus, and activates target genes [
53]. In this report, we revealed that oridonin stabilizes RARα protein by inducing nuclear translocation of p65, which was evidenced by the use of the ROS scavenger, NAC, the NF-κB inhibitor, Bay 11–7082, IκΒα (A32/36) over-expression, and p65 knockdown. Moreover, we tested whether TNFα, a classical activator of NF-κB signaling, modulates stability of RARα protein. As expected, TNFα treatment also strongly increased RARα expression, which may account, at least in part, for TNFα-induced differentiation in some leukemia cells [
54,
55]. Previous studies indicated that oridonin mainly activates the upstream of the NF-κB signaling pathway, while its inhibitory effect is due to the direct interference of NF-κB DNA binding activity [
56-
59]. Leung et al. demonstrated that oridonin decreased the DNA binding activity of NF-κB without interfering with p65 translocation [
59]. Of note, the exact mechanisms by which activated NF-κB stabilizes RARα protein require further investigation.
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Competing interests
The authors declare that they have no competing interest.
Authors’ contributions
Conceived and designed the experiments: HY, YLW. Performed all the experiments and analyzed the data: YC, WW. Contributed reagents/materials/analysis tools: NZ, QY, WBX, WJY. Wrote the manuscript: YC, WW, GQC, YLW. Revised the manuscript: GQC, HY, YLW. All authors read and approved the final manuscript.