PML-RARα stabilized by zinc in human acute promyelocytic leukemia NB4 cells

Accepted Manuscript PML-RARα stabilized by zinc in human acute promyelocytic leukemia NB4 cells

Bo Zhu, Jia-yu Wang, Jun-jie Zhou, Feng Zhou, Wei Cheng, Yingting Liu, Jie Wang, Xiao Chen, Dian-hua Chen, Lan Luo, Zi-Chun Hua PII: DOI: Reference:

S0162-0134(17)30159-9 doi: 10.1016/j.jinorgbio.2017.07.007 JIB 10254

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

11 April 2017 3 July 2017 9 July 2017

Please cite this article as: Bo Zhu, Jia-yu Wang, Jun-jie Zhou, Feng Zhou, Wei Cheng, Ying-ting Liu, Jie Wang, Xiao Chen, Dian-hua Chen, Lan Luo, Zi-Chun Hua , PML-RARα stabilized by zinc in human acute promyelocytic leukemia NB4 cells, Journal of Inorganic Biochemistry (2017), doi: 10.1016/j.jinorgbio.2017.07.007

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ACCEPTED MANUSCRIPT PML-RARα stabilized by zinc in human acute promyelocytic leukemia NB4 cells Bo Zhua, Jia-yu Wanga, Jun-jie Zhoua, Feng Zhoua, Wei Chenga, Ying-ting Liua, Jie Wanga, Xiao Chena, Dian-hua Chena, Lan Luoa,*, Zi-Chun Huaa, b,* a

The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, PR China b

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Changzhou High-Tech Research Institute of Nanjing University and Jiangsu TargetPharma Laboratories Inc., Changzhou, PR China *

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Corresponding author: Zi-Chun Hua Address: School of Life Sciences, Nanjing University, 163 Xianlin Road, Nanjing 210023 (China) Phone: +86-25-8332-4605 Fax: +86-25-8332-4605 E-mail: [email protected]

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Lan Luo Address: School of Life Sciences, Nanjing University, 163 Xianlin Road, Nanjing 210023 (China) Phone: +86-25-8968-6657 Fax: +86-25-8968-6657 E-mail: [email protected]

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Abstract Acute promyelocytic leukemia (APL) is characterized and driven by the

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Previous studies have highlighted the importance of PML-RARα degradation in the treatment against APL. Considering the presence of two zinc fingers in the PML-RARα fusion protein, we explored the

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function of zinc homeostasis in maintaining PML-RARα stability. We demonstrated for the first time that zinc depletion by its chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) triggered PML-RARα degradation in NB4 APL cells via the proteasome pathway rather than the

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autophagy-lysosomal pathway. In contrast, autophagy protected TPEN-mediated PML-RARα degradation in NB4 APL cells. We further demonstrated that crosstalk between zinc homeostasis and

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nitric oxide pathway played a key role in maintaining PML-RARα stability in NB4 APL cells. These results demonstrate that zinc homeostasis is vital for maintaining PML-RARα stability, and zinc depletion by TPEN may be useful as a potential strategy to trigger PML-RARα degradation in APL

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cells. We also found that TPEN triggered apoptosis of NB4 APL cells in a time-dependent manner. The relationship between PML-RARα degradation and apoptosis triggered by TPEN deserves further study.

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Key words: zinc homeostasis; acute promyelocytic leukemia; PML-RARα degradation; nitric

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oxide; apoptosis

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1. Introduction Acute promyelocytic leukemia (APL) is a hematological malignancy characterized by the fusion of the N-terminus of the promyelocytic leukemia protein (PML) to the C terminal of the retinoic acid receptor (RARα), mostly due to a chromosomal translocation t(15;17) [1, 2]. The PML-RARα oncoprotein globally interferes with retinoic acid-dependent transcription, blocks myeloid maturation because of cell cycle arrest, and participates in the leukemogenesis of APL [3-5]. The PML-RARα

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fusion protein also heterodimerizes with PML, a tumor suppresser protein, and disrupts its anti-proliferation or pro-apoptotic function [6, 7]. APL is largely considered as a monogenic cancer driven by PML-RARα [8]. Both of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), two

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drugs exhibit extraordinary clinical activity, have been found to target PML-RARα and promote its degradation in APL cells [9-11]. Mouse modeling also highlights the importance of PML-RARα

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degradation in the therapy against APL [4, 12]. Hence, understanding the mechanism of PML-RARα degradation plays a key role in the treatment of APL. Furthermore, although the introduction of ATRA and ATO has largely improved the clinical outcomes of patients with APL, new drugs or strategies

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targeting PML-RARα are still in need, for drug resistance and side-effects induced by ATRA or ATO still exist in some patients [13, 14].

Zinc is an essential trace element that is vital for the functioning of numerous cellular processes in both

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normal and cancer cells [15-17]. It has been estimated that almost 3000 human proteins interact with zinc as their structural, catalytic or regulatory cofactors [18, 19]. However, the influence of zinc in protein science is even greater, and the function of zinc homeostasis in gene expression and regulation in human cells remains elusive. expression

or

degradation

remain

unknown

[20].

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PML-RARα

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The PML-RARα chimeric protein has two zinc fingers, but the effects of zinc homeostasis on N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) is a membrane-permeable zinc chelator (Fig. 1), which has been widely used in studies focused on zinc homeostasis in cell biology [21, 22]. Zinc depletion by TPEN has been reported to affecting proliferation and apoptosis in some kinds of

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solid tumor cells [23, 24]. However, most of the studies are focused on the toxicity of TPEN, little is known about the effects of zinc depletion on the degradation of a specific oncoprotein. In the present study, we explored the effects of zinc depletion by TPEN on PML-RARα degradation in NB4 APL

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cells.

2. Materials and methods 2.1. Chemicals and antibodies TPEN, cycloheximide (CHX), E64, pepstatin (PEP), chloroquine (CQ), zinc sulfate, sodium nitroprusside (SNP), 4′,6-diamidine-2-phenylindole dihydrochloride (DAPI), propidium iodide (PI), and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MG-132 was ordered from MedChem Express (Monmouth Junction, NJ, USA). Anti-RARα antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, 3

ACCEPTED MANUSCRIPT CA, USA). Microtubule-associated protein 1 light chain 3 beta (LC3B) and beclin-1 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin antibody was purchased from Abgent (San Diego, CA, USA). 4-Amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) was brought from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

2.2. Cell culture Human acute promyelocytic leukemia NB4 cells were grown in RPMI 1640 medium supplemented

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with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, and maintained in a humidified 5% CO2 atmosphere at 37°C. All media and reagents for

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cell culture were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

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2.3. RNA isolation and quantitative real-time PCR (qPCR) Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA)

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according to the manufacturer’s instructions. Subsequently, cDNA was synthesized using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). The qPCR assay was performed with SYBR Green Master Mix (Vazyme, Nanjing, China) on an ABI StepOnePlus TM real-time PCR system (Applied follows:

β-actin

F:

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Bio-systems, Foster City, CA, USA). The qPCR primer pairs for β-actin and PML-RARα were as 5'-CATCGAGCACGGCATCGTCA-3',

5'-TAGCACAGCCTGGATAGCAAC-3';

PML-RARα

F:

β-actin

R:

5'-GCAGAGGATGAAGTGCTACG-3',

PML-RARα R: 5'-AGGGCTGGGCACTATCTCTT-3'. Results were quantified using the comparative

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2-ΔΔCt method with β-actin as an internal control.

2.4. Western blot

Cells were harvested and lysed in a cell lysis buffer for western blotting and immunoprecipitation

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(Beyotime Institute of Biotechnology, Nantong, China) on ice for 1 h. Cell debris was removed by centrifugation at 12,000g for 10 min at 4°C. Protein concentration was determined using a Bicinchoninic acid assay Kit (Beyotime Institute of Biotechnology, Nantong, China). Equal amounts of

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protein for each sample were separated by 10% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes. PVDF membranes were blocked with TBST (Tris-buffered saline with Tween-20) containing 5% non-fat milk for 1 h at room temperature, and then incubated with primary antibodies at a dilution of 1:1000 at 4°C overnight. Washed with TBST for three times (10 min each), membranes were incubated with appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody for 1 h at room temperature. Following three washes with TBST, bands were revealed with an enhanced chemiluminescent reagent (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. Blots are semi-quantified by densitometry using the IMAGEJ software (NIH, Bethesda, Maryland).

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ACCEPTED MANUSCRIPT 2.5. CHX chase assay CHX, as an inhibitor of protein biosynthesis, is widely used to determine the stability of a given protein. The stability of PML-RARα protein was measured by CHX chase assay as previously described [25]. Briefly, about 1×106 NB4 cells were treated with 100 μg/ml CHX alone or in the presence of TPEN (5 μM) for indicated durations (0.5-24 h). Cells were harvested and PML-RARα protein levels in the whole cell lysates were analyzed by western blotting as described before. β-Actin was served as the

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loading control.

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2.6. Measurement of intracellular nitric oxide

Intracellular nitric oxide signaling was measured with DAF-FM DA by flow cytometry. In brief, NB4

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cells were treated with TPEN (5 μM) for 24 h, harvested, washed with phosphate-buffered saline (PBS), and then incubated with the probe DAF-FM DA (5 μM) at 37°C for 30 min in the dark. Washed with PBS for three times, cells were re-suspended in 600 μL PBS and analyzed by flow cytometry. DAF-FM DA.

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2.7. Detection of apoptosis

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Intracellular nitric oxide level was expressed by the relative geometric mean fluorescence intensity of

NB4 cells were treated with TPEN (5 μM) for indicated time periods (0-24 h), cell morphology was

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observed and photographed using an optional microscope. Cells were collected, fixed with 4%

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paraformaldehyde (10 min), stained with DAPI (2 μg/mL, 10 min), and then fluorescence microscopy analysis of apoptotic morphology such as chromatin condensation and nuclei fragmentation was performed with a Zeiss AX10 fluorescence microscope. Quantitative analysis of apoptosis was performed using Annexin V/PI staining by flow cytometry as previously described [26]. Annexin V+PI- represents early apoptotic cells, and Annexin V+PI+

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represents late apoptotic cells.

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2.8. Statistical analysis Data were analyzed using the Student’s t-test, or ANOVA with Dunnett's post-hoc test on a GraphPad Prism software package (Version 6.02, La Jolla, CA, USA). Significant differences were accepted when P<0.05.

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3. Results 3.1.

TPEN

induces

PML-RARα

degradation

in

a

time-dependent manner in NB4 APL cells

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NB4 cells were treated with TPEN (5 μM) for indicated durations and immunoblot analysis showed that PML-RARα protein level was significantly decreased in a time-dependent manner (Fig. 2A). It has been reported that PML-RARα expression can be regulated at the levels of transcription and protein

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stability [9, 27]. As shown in Fig. 2B, no obvious effect of TPEN on PML-RARα mRNA expression was found, suggesting that zinc depletion by TPEN may down-regulate PML-RARα protein level by

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promoting its degradation. To determine the effects of TPEN on PML-RARα protein stability, CHX chase assay was performed in NB4 APL cells. Semi-quantitative analysis revealed that TPEN treatment accelerated the degradation of PML-RARα protein in NB4 APL cells (Fig. 2C). These results indicated

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that zinc homeostasis plays a key role in maintaining PML-RARα protein stability in NB4 APL cells.

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3.2. TPEN inhibits constitutive autophagy in NB4 APL cells Both the autophagy-lysosomal pathway and the ubiquitin-proteasome system have been previously reported to mediate the degradation of PML-RARα in APL cells [9]. Recently, it has also been reported

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that PML-RARα enhances constitutive autophagic activity through inhibiting the Akt/mammalian target of rapamycin (Akt/mTOR) pathway in APL cells [28]. However, the regulatory relationship

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between PML-RARα protein stability and autophagy remains elusive. As shown in Fig. 3, decreased levels of autophagy-associated proteins beclin-1 and LC3B II, and decreased LC3B II/LC3B I ratio in NB4 cells treated with TPEN for indicated durations were detected by western blotting. These data

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indicated that zinc depletion by TPEN may inhibit constitutive autophagy in NB4 APL cells.

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3.3. Autophagy inhibitors enhance the degradation of PML-RARα triggered by TPEN in NB4 APL cells We further treated NB4 cells with TPEN alone or in the presence of CQ, a widely used inhibitor of autophagy by blocking auto-phagosome fusion and degradation, to explore the function of decreased basal autophagy in PML-RARα degradation triggered by TPEN. We found that CQ treatment significantly enhanced PML-RARα degradation triggered by TPEN in NB4 cells, although CQ used alone had no obvious effect on PML-RARα degradation (Fig. 4A, B). Differently, CQ treatment had no obvious effects on RARα degradation caused by TPEN (Fig. 4A, C). We also treated NB4 cells with TPEN in the presence of E64 and PEP, another two inhibitors of autophagy-lysosome pathway, and got similar results (Fig. 4D-F). These results suggest that constitutive autophagy may play a protective role in TPEN-triggered PML-RARα degradation in NB4 APL cells. 6

ACCEPTED MANUSCRIPT 3.4. TPEN-induced PML-RARα degradation in NB4 cells is mediated by proteasome Previous studies have shown the ubiquitin-proteasome pathway as an important mechanism involved in therapy-induced PML-RARα proteolysis [9, 29]. To explore the function of ubiquitin-proteasome pathway in TPEN-triggered PML-RARα degradation, we treated NB4 cells with TPEN alone or in the presence of MG-132, a widely used inhibitor of ubiquitin-proteasome pathway. We found that

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PML-RARα degradation triggered by TPEN at early stages (6-9h) could be reversed by MG-132 treatment in NB4 APL cells (Fig. 5A, B). These data indicated that zinc depletion by TPEN triggers PML-RARα degradation via the ubiquitin-proteasome pathway rather than the autophagy-lysosomal

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pathway. We also found that TPEN-triggered degradation of RARα could be almost completely reversed by MG-132 (Fig. 5A, C), indicating that TPEN may promote PML-RARα through affecting

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the stability of its RARα portion.

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3.5. TPEN triggers PML-RARα degradation depending on its

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zinc chelator activity

As a specific and effective zinc chelator, whether TPEN promotes PML-RARα degradation depending on its zinc chelator activity or not remains unknown. We found that PML-RARα degradation mediated by 5 μM TPEN could be partially reversed by exogenous 2.5 μM zinc supplement (Fig. 6), confirming

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that TPEN triggered PML-RARα degradation depending on its zinc chelator activity.

3.6. The crosstalk between zinc homeostasis and nitric oxide

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signaling is involved in TPEN-triggered PML-RARα

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degradation in NB4 cells The crosstalk between zinc signaling and nitric oxide pathway has been reported to be involved in many biological events [30-32]. Using the specific probe for nitric oxide, DAF-FM DA, we demonstrated that TPEN triggered PML-RARα degradation with decreased intracellular nitric oxide signaling (Fig. 7A, B). To explore the relationship of nitric oxide signaling and PML-RARα stability in NB4 cells under zinc depletion status, we treated NB4 cells with TPEN in the presence of SNP (a nitric oxide donor) or C-PTIO (a specific nitric oxide scavenger), and found that TPEN-triggered PML-RARα degradation could be partially reversed by SNP incubation (Fig. 7C, D). In contrast, the nitric oxide scavenger C-PTIO has no significant effect on TPEN-triggered PML-RARα degradation in NB4 cells (Fig. 7C, D). Similar results were observed for the degradation of RARα (Fig. 7C, E). These results indicated that the crosstalk between zinc signaling and nitric oxide is important for PML-RARα stability. 7

ACCEPTED MANUSCRIPT 3.7. Zinc depletion by TPEN induces apoptosis of NB4 APL cells The pro-proliferation or anti-apoptotic function of PML-RARα has been reported previously [6, 7, 33]. Whether PML-RARα degradation triggered by TPEN affects the viability of NB4 cells remains unknown. To explore the effects of zinc depletion by TPEN on the viability of NB4 cells, we treated cells with 5 μM TPEN for indicated time periods. After treated with TPEN for 12 or 24 h, NB4 cells

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exhibited morphological changes typical of apoptosis, including cell shrinkage, membrane blebbing, chromatin condensation and nuclei fragmentation (Fig. 8). Quantitative analysis by Annexin V/PI

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staining revealed that TPEN triggered apoptosis in NB4 cells in a time-dependent manner (Fig. 9).

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4. Discussion

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PML-RARα degradation is vital for the treatment of APL [9, 10]. Both of the two effective and unconventional drugs, ATRA and ATO, were first shown to exhibit extraordinary therapeutic effects and later found to promote the degradation of PML-RARα [27]. Understanding factors and against

APL.

In

previous

studies,

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mechanisms affecting PML-RARα stability in APL cells is important for developing future therapies both

of

the

ubiquitin-proteasome

system

and

the

autophagy-lysosomal pathway have been reported to mediate PML-RARα degradation in APL cells [29, 34-37]. Most of these studies are focused on therapy-induced degradation of PML-RARα by ATRA or

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ATO. New strategies and chemicals that trigger PML-RARα degradation are rarely investigated. In the

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present study, we demonstrated for the first time that zinc homeostasis regulated the stability of PML-RARα protein. Zinc depletion by TPEN triggered the degradation of PML-RARα protein in a proteasome-dependent way in NB4 APL cells. It has been reported that PML-RARα enhances constitutive autophagic activity via inhibiting the

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Akt/mTOR pathway in myeloid leukemia cells [28]. Consistently, we observed relatively high basal level of LC3B II, which is widely described as the marker of autophagy, in the t(15;17) positive NB4 APL cells. Zinc has been previously described as an inducer or mediator of autophagy in various cells,

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including hepatoma cells [38], astrocytes [39], breast cancer cells [40] and neurons [41]. In this study, we demonstrated that zinc depletion by TPEN inhibited expression of autophagy-associated protein markers beclin-1 and LC3B-II, indicating that zinc depletion may inhibit basal autophagy in NB4 APL cells. We further demonstrated that TPEN-triggered PML-RARα degradation could be enhanced by co-incubation of autophagy inhibitors, indicating that constitutive autophagy caused by PML-RARα may play a protective role in maintaining PML-RARα stability. Conversely, no obvious effect of autophagy inhibitors on TPEN-induced degradation of RARα was observed, suggesting that the protective effects of constitutive autophagy on PML-RARα might work through affecting the stability of its PML portion. In this study, we further demonstrated that TPEN-triggered PML-RARα degradation in NB4 cells could be reversed by MG-132 treatment, indicating that ubiquitin-proteasome system was involved in this biological process. It has been reported that ATRA activates PML-RARα degradation by nuclear feedback through proteasome interaction with the RARα portion, while ATO triggers its degradation by 8

ACCEPTED MANUSCRIPT the proteasome pathway via the PML portion and by the small ubiquitin-related modifier -mediated/ubiquitin-dependent pathway [9]. In the present study, we also demonstrated here that RARα degradation could be reversed by MG-132 treatment, indicating that TPEN may trigger PML-RARα degradation with proteasome interacting with the RARα portion, more similar to ATRA rather than ATO. Nitric oxide signaling has both pro- and anti-cancer effects due to its multifunctional effects on proliferation and apoptosis of cancer cells [42-44]. It has been reported that nitric oxide can promote zinc release from its intracellular storage in neurons [45]. Functions of the crosstalk between zinc signaling and nitric oxide pathway in protein science and cancer biology remain elusive. In the present

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study, we found a reduction in intracellular nitric oxide signaling in NB4 cells treated with TPEN, and demonstrated for the first that nitric oxide signaling negatively regulated PML-RARα degradation

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triggered by TPEN, indicating that the crosstalk between zinc homeostasis and nitric oxide pathway plays a key role in maintaining PML-RARα stability.

The anti-apoptotic function of PML-RARα has been reported previously [33]. As the most active single

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agent in the treatment of APL, ATO targets PML-RARα and leads to apoptosis of APL cells [14, 46]. In this study, we demonstrated that zinc depletion by TPEN accelerated degradation of the anti-apoptotic

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protein PML-RARα in a time-dependent manner. We further found that TPEN induced apoptosis of NB4 APL cells in a time-dependent manner. The relationship between PML-RARα degradation and apoptosis triggered by TPEN-mediated zinc depletion is unclear at present and deserves further

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investigations.

Zinc works as cofactor of many enzymes, and zinc signaling is involved in the regulation of multiple pathways. In the present study, we demonstrated the effects of zinc depletion on PML-RARα degradation and apoptosis in NB4 APL cells. The effects of zinc depletion on other zinc-dependent

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cellular processes remain unclear, and the selectivity of zinc depletion as a potential chemotherapy

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approach against APL deserves further investigation.

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5. Conclusion

In summary, we demonstrated that zinc depletion by TPEN accelerated PML-RARα degradation via the proteasome pathway and triggered apoptosis in NB4 APL cells. These in vitro data indicated that

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zinc depletion by TPEN may be a potential therapeutic strategy for APL.

Abbreviations APL

acute promyelocytic leukemia

ATRA

all-trans retinoic acid

ATO

arsenic trioxide

CHX

cycloheximide

C-PTIO

carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide

CQ

chloroquine

DAF-FM DA

4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate 9

ACCEPTED MANUSCRIPT 4′,6-diamidine-2-phenylindole dihydrochloride

LC3B

microtubule-associated protein 1 light chain 3 beta

mTOR

mammalian target of rapamycin

PBS

phosphate-buffered saline

PEP

pepstatin

PI

propidium iodide

PML-RARa

Promyelocytic leukemia protein -retinoic acid receptor alpha

PVDF

polyvinylidene fluoride

qPCR

quantitative real-time PCR

SNP

Sodium nitroprusside

TBST

Tris-buffered saline with Tween-20

TPEN

N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine

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DAPI

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The authors declare no conflict of interest.

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Conflict of interest

Acknowledgments

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This study was supported by grants from the Chinese National Natural Sciences Foundation (81630092,

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81421091), the National Key Research Program by Ministry of Science and Technology (2016YFC0902700, 2014CB744501).

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Figure legends

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Fig. 1. Structural representation of (A) N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine

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(TPEN) and (B) the bonds between TPEN and Zn 2+.

Fig. 2. PML-RARdegradation was induced by zinc chelator TPEN. (A) NB4 cells were incubated

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with 5 μM TPEN for indicated time periods (0-24 h), and the whole lysates were analyzed by western blotting for PML-RAR. (B) The mRNA level of PML-RARin NB4 cells treated with 5 μM TPEN

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for indicated time periods (0-24 h) was detected by qPCR. β-Actin was served as the reference gene. (C)

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NB4 cells were incubated with 100 μg/ml CHX for indicated time periods (0-24 h) with or without the presence of TPEN (5 μM), and the whole lysates were analyzed by western blotting for PML-RAR.

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Experiments were performed three times, and representative blots were shown. Data are represented as

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mean±SD. *P<0.05, **P<0.01 and ***P<0.005. N.D. means not detected.

Fig. 3. Autophagy was inhibited in NB4 cells treated with TPEN. NB4 cells were treated with TPEN (5 μM) for indicated time periods (0-24 h). Western blot was performed to determine the expression of beclin-1 and LC3B. (A) Representative blots from three independent experiments are shown. Semi-quantitative analysis of beclin-1 (B) or LC3B II (C) was performed, with the protein level of β-actin used as an internal control. (D) Fold change analysis of the ratio of LC3B II to LC3B I 14

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(compared to the control). Data are represented as mean±SD. *** P<0.005

Fig. 4. Autophagy inhibitors facilitated TPEN-triggered PML-RARα degradation in NB4 APL cells. NB4 cells were exposed to 5 μM TPEN alone or with the presence of 30 μM chloriquine (CQ) or

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10 μg/ml E64/pepstatin (PEP) for indicated time periods (0-12 h). Western blot was performed to

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determine the expression level of PML-RARα or RARα, with β-actin used as an internal control.

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Representative blots (A, D) and semi-quantitative analysis of protein level are shown (B, C, E, F). Experiments were performed three times. Data are represented as mean±SD. *P<0.05. NS stands for

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not significant.

Fig. 5. TPEN-triggered PML-RARα degradation in NB4 cells was reversed by MG-132 treatment.

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NB4 cells were treated with 5 μM TPEN with or without the presence of 1 μM MG-132 for the

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indicated durations (0-12 h) and then lysed. The lysates were analyzed for PML-RARα and RARα protein levels by western blotting. Representative blots (A) and semi-quantitative analysis are shown

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(B, C). β-Actin was served as an internal control. Results are expressed as the mean±SD of three

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independent experiments. *P<0.05, ** P<0.01. NS stands for not significant.

Fig.6. TPEN-triggered PML-RARα degradation in NB4 cells can be partially reversed by zinc supplement. NB4 cells were exposed to 5 μM TPEN alone or with the presence of 2.5 μM zinc sulfate for 24 h. Western blot was performed to determine the expression of PML-RARα and RARα. Representative blots (A) and semi-quantitative analysis of protein level (B, C) are shown. β-Actin was used as an internal control. Results are expressed as the mean±SD of three independent experiments.

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N.D. stands for not detected.

Fig.7. TPEN-triggered PML-RARα degradation in NB4 cells can be partially reversed by nitric oxide donor treatment. (A) NB4 cells were treated with 5 μM TPEN for 24 h, and then intracellular

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nitric oxide signaling were measured with DAF-FM DA staining by flow cytometry. (B) Relative

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intracellular nitric oxide level was expressed by the relative geometric mean fluorescence intensity of

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DAF-FM DA. (C) NB4 cells were treated with 5 μM TPEN with or without the presence of 500 μM SNP or 50 μM C-PTIO for 24 h. Western blot was performed to determine the expression of

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PML-RARα and RARα. Semi-quantitative analysis of PML-RARα and RARα protein level are shown

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in (D) and (E). β-Actin was used as an internal control. Results are expressed as the mean±SD of three

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independent experiments. ***P<0.005. N.D. stands for not detected.

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Figure 8. Apoptotic morphology of NB4 APL cells stimulated with TPEN. NB4 cells were treated with TPEN (5 μM) for indicated time periods (0-24 h), cell morphology was observed and recorded

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using an optical microscope (A). Cells were collected, fixed, stained with DAPI, and then

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photographed using a Zeiss AX10 fluorescence microscope. Scale bar=20 μm. A partially enlarged view was shown in the lower, left corner of each picture.

Figure 9. Quantitative analysis of apoptosis in NB4 cells treated with TPEN. NB4 cells were treated with TPEN (5 μM) for indicated time periods (0-24 h), collected, stained with Annexin V-EGFP/PI, and then analyzed by flow cytometry. Annexin V+PI- represents early apoptotic cells, and Annexin V+PI+ represents late apoptotic cells.

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ACCEPTED MANUSCRIPT Fig. 10. Schematic diagram of zinc chelator TPEN-induced PML-RARα degradation in NB4 APL cells. Zinc depletion by TPEN triggers PML-RARαdegradation via the proteasome pathway and induces apoptosis in NB4 APL cells. Autophagy may play a protective role in PML-RARα degradation triggered by TPEN. The crosstalk between zinc and nitric oxide pathway plays a key role in

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Graphical Abstract Zinc depletion triggers

α degradation via the proteasome pathway and induces apoptosis in NB4 cells. Autophagy may play a protective role in PML-RARα degradation triggered by zinc depletion. The

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crosstalk between zinc and nitric oxide pathway is important for PML-RARα stability.

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ACCEPTED MANUSCRIPT Highlights Zinc stabilized promyelocytic leukemia protein-retinoic acid receptor alpha (PML-RARα).

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Zinc depletion triggered PML-RARα degradation via the proteasome pathway.

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Autophagy inhibited PML-RARα degradation triggered by zinc depletion.

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Crosstalk between zinc and nitric oxide signaling stabilized PML-RARα.

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Zinc depletion induced apoptosis in NB4 cells.

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