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Introduction

Acute promyelocytic leukemia (APL) is characterized by specific chromosomal translocations, typically t(15;17), which results in the formation of the promyelocytic leukemia-retinoic acid receptor-α (PML-RARα) fusion gene (1,2). PML-RARα fusion protein forms homo/heterodimers that sequestrate RXR and/or PML proteins in a large protein complex and disrupt the retinoic acid (RA) signal pathway. This specific oncogenic lesion determines characteristic cell morphology and clinical presentations, and it also determines the unique response to the treatment with all-trans retinoic acid (ATRA) or arsenic agents (3,4). Both drugs have been demonstrated to target the PML/RARα oncoprotein for proteasome-mediated degradation. Clinically, ATRA induces complete remissions in ~90% of newly diagnosed APL, but many patients eventually experience a relapse and develop ATRA-resistance (5,6). Arsenic trioxide is also shown to be effective in the treatment of APL, especially in relapsed APL with ATRA-resistance (7,8).

Arsenic trioxide has dual effects of inducing differentiation and apoptosis of APL cells. However, there are issues of availability and cost of arsenic trioxide that limit its general applications. The development of oral form of arsenic drug may promote its applications in APL. Arsenic sulfide (As4S4), also known as realgar, is an oral arsenic formulation. This oral arsenic drug has been shown to have similar effect and safety to intravenous arsenic trioxide in the treatment of newly diagnosed and relapsed/refractory APL or ATRA-resistance (9). The therapeutic action of As4S4 is closely associated with its function of inducing apoptosis. Although it is known that As4S4 induces cell apoptosis through degrading PML-RARα fusion protein (10), the definitive molecular mechanisms of action of As4S4 remain unclear and require further investigations.

In the present study, we used a comparative proteomic approach to screen and identify proteins that are differentially expressed in APL cells induced by As4S4. By using two-dimensional gel electrophoresis (2-DE) followed by a matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis, we identified prohibitin (PHB) among the differentially expressed proteins. PHB was significantly upregulated in ATRA-resistance APL cells (NB4-R1) by As4S4 treatment. Further studies of PHB-knockdown and PHB-overexpression indicate a functional role of PHB in As4S4-induced apoptosis of NB4-R1 cells.

Materials and methods

Cell culture

The ATRA-resistance human APL cell line (NB4-R1), received from Shanghai Institute of Hematology, (Shanghai, China) was maintained in cultures with RPMI-1640 medium (Gibco-BRL, Carlsbad, CA, USA) supplemented with 10% heated-inactivated fetal bovine serum (FBS) at 37ºC in a humidified incubator containing 5% CO2.

Cell viability assay

Cytotoxicity of As4S4 (Xi'an Traditional Chinese Drug Company, Xi'an, China) was assessed by using MTT assay (Sigma, St. Louis, MO, USA) (11). The absorbance was measured at 570 nm using a universal microplate reader (Model ELx800; BioTek Instruments, Inc., Winooski, VT, USA). Experiments were performed in triplicate.

Apoptosis evaluation

Transmission electron microscopy (TEM) and flow cytometric analysis (FCM) were performed to evaluate cell apoptosis. After the various treatments, the cell samples were examined under a JEM-100SX electron microscope (JEOL, Ltd., Tokyo, Japan) and were analyzed in a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA) and CellQuest software, respectively. All experiments were performed in triplicate.

2-DE and image analysis

Total cellular proteins were prepared from NB4-R1 cells before and after As4S4 treatment. Protein extraction was performed by sonication in a sample buffer (SB) containing 40 mM Tris base, 8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1% (w/v) dithiothreitol (DTT), 1 mM EDTA and protease inhibitor cocktail (Roche Diagnostics Ltd., Mannheim, Germany). For nuclei enrichment cells were dissolved in 200 μl of lysis buffer [10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, in the presence of protease inhibitor cocktail (Sigma), 20 ng/μl DNase and 20 ng/μl RNase] and incubated on ice for 30 min. After incubation, NP-40 (Roche) was added at final concentration of 0.5% (v/v). After centrifugation at 14,000 rpm for 30 min at 4ºC, the supernatant was used for analysis with the protein concentration determined by the Bradford method with a commercial Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA) (12).

2-DE was performed as described by Görg et al (13). Briefly, 140 μg of protein (for silver nitrate staining gels) or 1.4 mg of protein (for coomassie brilliant blue staining gels) was diluted to 350 μl with rehydration solution and applied onto 18 cm (pH 3–10) not linear immobilized pH gradient dry strip (Amersham Pharmacia Biotech, Uppsala, Sweden). After the strips were rehydrated, isoelectric focusing was performed in the IPGphor system (Amersham Pharmacia Biotech) according to the manufacturer's protocol (14). The strips were equilibrated for 15 min in a solution containing 6 M urea, 2% (w/v) SDS, 20 mM DTT, 30% (w/v) glycerol and 50 mM Tris-HCl (pH 8.8). A second equilibration was also carried out for 15 min in the same solution except for DTT replaced by 100 mM iodoacetamide. The second dimension was performed on 13% SDS-polyacrylamide gradient gels using the PROTEAN XI Cell (Bio-Rad Laboratories) at 20 mA/gel for 40 min.

Silver nitrate staining according to the protocol of Lelong et al (15), and coomassie brilliant blue R-250 (0.05% brilliant blue) was used for the analytical and preparative gels. The 2-DE images were acquired using Image scanner (Amersham Pharmacia Biotech). Gel images were analyzed by the ImageMaster 2D Platinum software (Amersham Pharmacia Biotech). Spot detection and normalization were performed by the automated software tools.

MALDI-TOF MS and MALDI-TOF MS/MS analysis

Differentially expressed spots were manually excised from 2-DE gels. Gel pieces were destained and digestion. In-gel digestion was done according to the protocol of Granvogl et al (16).

MALDI-TOFMS analysis was performed on a Bruker REFLEX III MALDI-TOF-MS (Bruker-Franzen, Bremen, Germany). Peptides were desalted by C18 ZipTips (Millipore, Billerica, MA, USA) and co-crystallized with a solution of 0.5 mg/ml α-cyano-4-hydroxycinnamic acid dissolved in acetonitrile/0.1% (v/v) trifluoroacetic acid (TFA) in H2O (1:1) pre-spotted with a thin layer of 10 mg/ml α-cyano-4-hydroxycinnamic acid dissolved in ethanol/acetonitrile/0.1% (v/v) TFA in H2O (49.5:49.5:1). Monoisotopic peptide masses were used to search the database, allowing a peptide mass accuracy of 0.3 Da and one partial cleavage. The proteins were identified by peptide mass fingerprinting (PMF) searching, against the Swiss-Prot databases and NCBI databases, using the search program Mascot (http://www.matrixscience.com).

The protein spots which were not identified by MALDI-TOF-MS were analyzed by MA LDI-TOF MS/MS. MALDI-TOF MS/MS analysis was performed in LIFT mode. Precursor ions were selected manually. MS/MS spectra were acquired with a minimum of 4000 and a maximum of 8000 laser shots using the instrument calibration file. The precursor mass window was set automatically after the precursor ion selection. Spectra baseline subtraction, smoothing (Savitsky-Golay) and centroiding was performed by FlexAnalysis software (version 3.0; Bruker Daltonik GmbH, Bremen, Germany).

Western blot analysis

Cell protein extracts were prepared following standard procedures. The protein samples (~20 mg) were separated by SDS-PAGE. After SDS-PAGE, proteins were transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA, USA). The filters were washed, blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (25 mM Tris, pH 7.4, 136 mM NaCl, 2.6 mM KCl and 0.5% Tween-20) for 1 h, and incubated overnight with mouse anti-PHB antibody diluted to 1:700 (Abcam, Cambridge, MA, USA) at room temperature. After washing three times with TBST buffer, the membranes were incubated with the secondary HRP-conjugated goat anti-mouse IgG Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:10,000 dilution. Mouse anti-GAPDH antibody (Santa Cruz Biotechnology) was used to ensure equal loading of samples.

Quantitative real-time PCR (qRT-PCR)

The total RNA from cells was isolated with TRIzol (Life Technologies, Rockvile, MD, USA) and reverse-transcribed to cDNA by using the PrimeScript™ RT reagent kit (Takara Bio, Dalian, China). The cDNA was studied using a CFX96 real-time PCR system (Bio-Rad Laboratories) with SYBR-Green PCR Master Mix (Takara) to determine the transcriptional expression of PHB gene. PCR products were electrophoresed on 1.5% agarose gels. The GAPDH was used for normalization, relative gene expression was calculated by the 2−ΔΔCt method.

Knockdown and overexpressing of PHB

Lentiviral vector-mediated shRNA targeting human PHB mRNA (named pGCSIL-GFP-PHB) was previously described (17). The target sequences on the human PHB gene (GeneBank accession number NM_002634) for RNAi were designed using an internet application system as follows: 5′-GAGTTCACAGAAGCGGTGGAA3′. A shRNA which had no significant homology to any known human gene (5′-TTCTCCGAACGTGTCACGT-3′) was used as a negative control. Oligonucleotides were ligated into the AgeI and EcoRI sites of pGCSIL-GFP vector (BD Biosciences, San Jose, CA, USA) to generate a pGCSIL-GFP-PHB, which was then transformed into E. coli. Positive recombinant clones were selected by PCR (upstream primer: 5′-CCTATTTCCCATGATTCCTTCATA-3′; downstream primer: 5′-GTAATACGGTTATCCACGCG-3′) and DNA sequencing. The recombinant lentivirus vector was produced by co-transfecting 293T cells with the lentivirus expression plasmid and packaging plasmids (pHelper 1.0 and pHelper 2.0) with Lipofectamine 2000 (Invitrogen). Infectious lentivirus vector was harvested at 48 h post-transfection and then concentrated. The infectious titer was determined by the GFP-tagged positive rate in 293T cells. NB4-R1 cells were cultured at a density of 6×105/well in 6-well plates and infected with lentivirus in RPMI-1640 media containing 10% FBS and 8 μg/ml of polybrene (Sigma), at the multiplicity of infection (MOI) 20, according to the pre-experimental results. After 48 h of culture, the transduction efficiency was ascertained on the basis of GFP expression under a fluorescence microscope. The knockdown efficiency of PHB was analyzed by real-time quantitative PCR and western blot analysis. NB4-R1 cells transfected with vector containing pGCSIL-GFP-PHB were designated as PHB-knockdown (KD).

The PHB gene overexpression vector (named pEGFP-N1-3FLAG-PHB) was also established. Briefly, the cDNA fragment of PHB was amplified using a PCR-based approach (upstream primer: 5′-CCGCTCGAGATGGCTGCCAAAGT GTTTG; downstream primer: 5′-GGGGTACCGTCTGGGG CAGCTGGAGGAG) from a cDNA library. The PCR fragment of confirmed sequences was ligated into the XhoI and KpnI sites of overexpression vector pEGFP-N1-3FLAG (BD Biosciences). The resultant construct, pEGFP-N1-3FLAG-PHB, was transformed into E. coli. Positive recombinant clones were selected by PCR and DNA sequencing (upstream primer: 5′-CGCAAATGGGCGGTAGGCGTG-3′; downstream primer: 5′-CGTCGCCGTCCAGCTCGACCAG-3′). The expression of PHB was analyzed by real-time quantitative PCR and western blot analysis. The NB4-R1 cell clone transfected with the vector containing pEGFP-N1-3FLAG-PHB were designated as PHB-overexpression (OE).

Statistical analysis

The results are expressed as mean ± standard deviation values of three experiments performed in duplicate. Statistical analysis was carried out by one-way analysis of variance. Newman-Keuls test was used for the identification of statistically significant differences in spot volume percentage among different samples. Differences were considered statistically significant when P<0.05.

Results

As4S4 inhibits the growth of ATRA-resistant NB4-R1 cells

We started with MTT assay to evaluate the cytotoxicity of As4S4 on ATRA-resistant NB4-R1 cells. The results demonstrated that As4S4 inhibited the growth of NB4-R1 cells in a dose-and time-dependent manner (Fig. 1). The IC50 values of As4S4 were determined at 43.04±0.11 μM for 12 h, 25.07±0.27 μM for 24 h, 9.70±0.13 μM for 48 h and 6.38±0.09 μM for 72 h in culture. The concentration of 25 μM, the IC50 of As4S4 at 24 h, was chosen for subsequent experiments.

Figure 1 Dose- and time-dependent inhibition of NB4-R1 cells as determined by the MTT assay. The results are expressed as a percentage of viable cells compared with control cells. Each value represents the mean ± SD of triplicate experiments.

As4S4 induces apoptosis of NB4-R1 cells

As4S4-induced apoptosis was assessed by using TEM and FCM analysis. The NB4-R1 cells treated with As4S4 showed morphological features of cytoplasmic vacuolization, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies (Fig. 2A). The apoptotic cells were quantified by FCM assay for Annexin V+ cells. The percentage of apoptotic cells was significantly increased with As4S4 treatment for 24 and 48 h (Fig. 2B).

Figure 2 Evaluation of apoptosis of NB4-R1 cells induced by As4S4. (A) Ultrastructural changes. Cells treated with As4S4 for 24 h showed cytoplasmic vacuolization (Vac) and chromatin condensation, and cells treated with As4S4 for 48 h showed nuclear fragmentation and formation of apoptotic bodies (Apo). Original magnification, ×5000. (B) Flow cytometric analysis of NB4-R1 cells apoptosis induced by As4S4. The percentage of apoptotic cells (Annexin V+) was 2.7, 33.4 and 44.5% in control, As4S4 24 and 48 h, respectively.

PHB is an upregulated protein induced by As4S4

We next used proteomic approaches to screen and identify proteins that were differentially expressed following As4S4 treatment. The comparison of 2-DE protein profiles of NB4-R1 cells at 0 h with that at 24 and 48 h As4S4 treatment were performed, and 22 protein spots with at least a 2-fold increase or decrease in density were selected for further analysis (Fig. 3A and B).

Figure 3 Selection and identification of upregulated PHB in NB4-R1 cells induced by As4S4. (A) Representative silver-stained 2-DE maps of NB4-R1 cells untreated (R0), treated with As4S4 for 24 h (R24) and 48 h (R48). Circles indicate the protein spots identified by MS or MS/MS. (B) The amplified image of differential expression protein spot U8 in R0, R24 and R48. Spot U8 showed increase in intensity at R24 and R48. (C) MALDI-TOF-MS analysis of differential protein spot U8. Spot U8 was identified as prohibitin (PHB) according to its MALDI-TOF-MS mass spectrum. (D) Protein sequence of PHB is shown and matched peptides are underlined. (E) Western blot analysis of PHB protein levels with PHB/GAPDH ratio as control for the comparison of the grayscale. (F) RT-PCR analysis of PHB mRNA levels. The grayscale ratio of PHB/GAPDH is provided for comparison. *P<0.05; **P<0.01.

These spots were cut out, followed by in-gel trypsin digestion and MALDI-TOF MS analysis. The protein spots which were not identified by MALDI-TOF-MS were further analyzed by MALDI-TOF MS/MS. PMF and peptide amino acid sequence were analyzed for protein identification using the Mascot search program. Fig. 3C showed the PMF of spot U8 analyzed by MALDI-TOF-MS. spot U8 was identified as prohibitin (PHB) and the corresponding protein sequence is shown in Fig. 3D. The annotation of the 22 identified proteins is shown in Table I.

Table I Identification of differentially expressed protein spots by MALDI-TOF-MS and MALDI-TOF-MS/MS.

Table I

Identification of differentially expressed protein spots by MALDI-TOF-MS and MALDI-TOF-MS/MS.

Spot	Protein name	NCBInr ID no.	Function classification	Mr (Da)		pI		Peptides (MALDI/MS)		Sequence coverage (%)	Protein expressionb R24/R48
				Theor.	Observ.	Theor.	Observ.	Match	Total
D1	Poly C binding protein 1 (PCBP1)	gi|6754994	Regulates gene expression	37474	43062	6.66	7.83	17	28	52	0.57/0.19
D2	Acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A)	gi|5453880	Cell proliferation, differentiation, apoptosis	28568	30123	3.99	3.88	8	14	31	0.70/0.42
D3a	SET/protein phosphatase 2A inhibitor (SET/I2PP2A)	gi|170763500	Multitasking protein	33469	41249	4.23	4.01	7	13	27	0.34/0.10
D4	Eukaryotic translation initiation factor 4H isoform 1 (eIF4H-1)	gi|11559923	Protein synthesis	27368	32661	6.67	7.16	14	29	48	0.64/0.20
D5	60S acidic ribosomal protein P2 (RPP2)	gi|4506671	Protein synthesis	11658	16831	4.42	4.13	7	20	77	0.40/0.30
U1	High mobility group protein B1 (HMGB1)	gi|4504425	Signal transduction	24878	29744	5.62	6.88	11	20	48	4.58/2.95
U2	Transgelin-2 (TAGLN2)	gi|4507357	Not be determined	22377	20417	8.41	5.58	15	19	56	2.50/6.07
U3	Eukaryotic translation initiation factor5A) (eIF5A-1	gi|183448388	Protein synthesis, cellular growth, differentiation and proliferation	16821	16949	5.08	7.37	9	31	52	2.46/10.14
U4	Transcription factor(TF)	gi|388307	Transcription	20700	22567	6.28	5.49	2	43	12	6.18/19.98
U5	α-tubulin	gi|37492	Cellular motility and transportation	50126	22567	5.02	5.49	3	26	9	6.18/19.98
U6	Histone H2B type 1-M (H2B1M)	gi|4504263	Transcription, DNA repair	13981	16949	10.31	7.37	12	31	67	2.12/15.87
U7	Rho GDP dissociation inhibitor β 2 (RhoGDI2)	gi|56676393	Signal transduction and regulates Rho GTPases	22974	24685	5.10	7.01	8	33	54	5.31/16.83
U8	Prohibitin (PHB)	gi|4505773	Cell proliferation, tumor suppressor	29786	31560	5.57	5.37	13	14	61	2.18/3.68
U9	Ribosomal phosphoprotein P0 (RPP0)	gi|4506667	Protein synthesis and apoptosis	34252	39054	5.71	6.27	14	19	46	16.16/22.4
U10	Heat shock 27 kDa protein (HSP27)	gi|4504517	Stress resistance	22768	28891	5.98	6.34	11	18	46	2.77/1.79
U11	Elongation factor 1-β (EF-1-β)	gi|18203449	Protein synthesis	24748	32071	4.50	4.38	6	13	37	1.53/2.84
U12	Keratin-2	gi|47132620	Proliferation and keratinization	65393	18903	8.07	6.21	11	38	25	4.23/14.82
U13	ERP29	gi|5803013	Protein processing	28975	31332	6.77	5.89	12	28	42	1.30/5.06
U14	β-actin (ACTB)	gi|4501885	Cellular motility	41710	14843	5.29	8.26	10	20	23	1.90/13.48
U15	GTPase-activating protein	gi|62911375	Increase GTP hydrolysis	23439	27647	5.21	5.25	6	17	30	1.70/3.27
U16a	Neuropolypeptide h3	gi|913159	Serine protease inhibitor	20913	66684	7.42	5.88	-	-	31	0.95/3.18
U17	Proteasome β 4 subunit (PSMB4)	gi|22538467	Proteolysis	29185	28177	5.72	5.56	13	25	35	1.32/3.42

{ label (or @symbol) needed for fn[@id='tfn1-ijo-47-06-2286'] } D, downregulation; U, upregulation; pI, isoelectric point; Mr, molecular weight.

a The spot is identified by MALDI-TOF-MS and MALDI-TOF-MS/MS;

b As₄S₄-treated (R24 and R48) divided by untreated (R0); all values are statistically significant, P<0.05.

PHB was identified from the spot U8, which was upregulated induced by As4S4. The increase in PHB protein was confirmed by western blot analysis. As shown in Fig. 3E, there was a 2.0- and 3.9-fold increase in PHB protein with As4S4 for 24 and 48 h, respectively. At mRNA level, PHB expression was increased by 1.8- and 3.2-fold with As4S4 for 24 and 48 h, respectively (Fig. 3F). The results indicate an upregulation of PHB gene expression at both mRNA and protein levels.

Generation of PHB-overexpression and PHB-knockdown NB4-R1 cells

To investigate whether PHB plays a functional role in NB4-R1 cell apoptosis, we used the PHB gene overexpressing vector (pEGFP-N1-3FLAG-PHB) to generate PHB-overexpression NB4-R1 cells (OE group). The PHB-overexpression efficiency was then validated by qRT-PCR and western blot analysis, respectively. Our results showed that PHB expression in OE group was increased by 67.8% at mRNA level and 45.8% at protein level (Fig. 4A and B). Similarly, the RNA interference vector (pGCSIL-GFP-PHB) of PHB gene was used to generate PHB-knockdown NB4-R1 cells (KD group). Our results showed that PHB expression was reduced by 83.5% at mRNA level and 89.7% at protein level, respectively (Fig. 4C and D).

Figure 4 RT-PCR and western blot analysis of PHB overexpression and knockdown in NB4-R1 cells. (A) RT-PCR analysis of PHB overexpression. (B) Western blot analysis of PHB overexpression. (C) RT-PCR analysis of PHB knockdown. (D) Western blot analysis of PHB knockdown. **P<0.01. CON, control (NB4-R1 cells); NC, negative control (NB4-R1 cells transfected with empty vector); KD, PHB-knockdown NB4-R1 cells; OE, PHB-overexpressing NB4-R1 cells.

PHB-overexpression promotes NB4-R1 apoptosis and PML-RARα fusion protein degradation

Our results showed after 48 h of transfection, the percentages of apoptotic cells in OE group was increased by 3.8-fold in comparison with the parental NB4-R1 cells (26.73±6.53 vs. 7.11±1.02%, P<0.01) (Fig. 5A), and the PML-RARα fusion protein was reduced by 1.5-fold in comparison with the control (34.21±3.81 vs. 51.31±8.55%, P<0.01) (Fig. 5B).

Figure 5 Effect of PHB overexpression on the apoptosis and the expression of PML-RARα protein in NB4-R1 cells induced by As4S4. (A) FCM analysis of the apoptosis of the control NB4-R1 cells, OE cells, NB4-R1 cells treated by As4S4 and OE cells treated by As4S4, respectively. (B) Western blot analysis of the expression of PML-RARα fusion protein. **P<0.01. CON, control (NB4-R1 cells untreated); OE, PHB-overexpression NB4-R1 cells; As4S4, NB4-R1 cells treated by As4S4; OE+As4S4: PHB-overexpressing NB4-R1 cells treated by As4S4.

The response of the OE cells to As4S4 was evaluated in comparison with parental NB4-R1 cells. OE cells showed an increase in As4S4-induced apoptosis. With As4S4 at the concentration of 25 μM for 48 h, the apoptotic cells in NB4-R1 and OE cells were 48.33±9.84 and 58.71±11.74%, respectively (Fig. 5A). PML-RARα fusion protein was assessed by western blot analysis, and the results showed that As4S4 treatment led to greater reduction of PML-RARα protein in OE cells than that in NB4-R1 cells. In comparison with untreated NB4-R1 cells, As4S4 treatment reduced PML-RARα protein by 51.0 and 76.9% in NB4-R1 and OE cells, respectively (the grayscale ratios of PML-RARα/GAPDH: 25.14±2.87 and 11.86±2.99%, P<0.05) (Fig. 5B).

PHB-knockdown reduces As4S4-induced apoptosis and degradation of PML-RARα protein

PHB-knockdown NB4-R1 cells (KD) was evaluated in comparison with parental NB4-R1 cells. With no As4S4 treatment, there was no significant difference in the baseline apoptotic cells between KD and NB4-R1 cells. Similarly, no significant difference was seen between KD and the NB4-R1 in the expression of PML-RARα fusion proteins, as determined by PML-RARα/GAPDH (53.16±7.83 vs. 49.78±1.89%) (Fig. 6A and B).

Figure 6 Effect of PHB knockdown on the apoptosis and the expression of PML-RARα protein in NB4-R1 cells induced by As4S4. (A) FCM analysis of the apoptosis of NB4-R1 and KD cells untreated, and treated by As4S4, respectively. (B) Western blot analysis of the expression of PML-RARα fusion protein. The grayscale ratio of PML-RARα/GAPDH is provided for comparison. **P<0.01. CON, control (NB4-R1 cells untreated); KD, PHB-knockdown NB4-R1 cells; As4S4: NB4-R1 cells treated by As4S4; KD+As4S4: PHB-knockdown NB4-R1 cells treated by As4S4.

The KD cells were then used to examine its response to As4S4 treatment. As4S4-induced apoptosis was evaluated with As4S4 at the concentration of 25 μM for 48 h. In comparison with parental NB4-R1 cells, the KD showed a lesser degree of cellular apoptosis. The percentages of apoptotic cells in NB4-R1 and KD were determined to be 45.17±5.43 and 22.16±3.92%, respectively (Fig. 6A). Thus, there was a 2.0-fold less As4S4-induced apoptosis in KD than that in parental NB4-R1.

PML-RARα fusion protein of KD cells by western blot analysis

By using the grayscale ratios of PML-RARα/GAPDH, the levels of PML-RARα protein were determined to be 49.78±1.89% in the untreated cells, and 24.21±1.73 and 37.95±7.79% in As4S4-treated NB4-R1 and KD cells, respectively. Using the untreated cells as the baseline, As4S4 lowered PML-RARα protein by 51.3 and 23.7% in NB4-R1 and KD cells, respectively (Fig. 6B). The results indicate that KD cells presented with a lesser degree of As4S4-induced PML-RARα degradation, ~50% of that in parental NB4-R1 cells.

Discussion

Arsenic agents have been proved highly effective in the treatment of APL. It is particularly useful for relapsed/refractory APL with ATRA-resistance (18). As4S4, is a new and promising oral arsenic formulation. A multicenter study in China has shown that a complete remission (CR) rate of 99.1% and a disease-free survival (DFS) rate of 98.1% at 2 years were achieved in 108 APL cases treated with an oral As4S4 combined with ATRA (19, 20). In the present study, we demonstrated that As4S4 inhibited the growth and induced apoptosis of ATRA-resistant NB4-R1 cells. The result is consistent with previous findings (21,22). By using comparative proteomic approach, we identified PHB was significantly upregulated during As4S4-induced NB4-R1 apoptosis. As PHB is of particular interest, further experiments were performed to modulate the gene expression, either PHB overexpression or PHB knockdown. The results with modulation of PHB expression implicate its activity in promoting As4S4-induced apoptosis.

PHB was selected in this study for its diverse roles in the regulation of proliferation, apoptosis and gene transcription (23–27). PHB proteins have been found to localize in the mitochondria, nucleus and plasma membrane of mammalian cells. PHB is implicated in diverse cellular processes, including mitochondrial biogenesis, cell death and replicative senescence. A functional role for PHB as a regulator of transcription has been shown for its interactions with p53, E2F and Rb (28–30). PHB has been associated with various types of cancer. The role of PHB in cancer cell proliferation or tumor suppression is considered controversial. PHB was shown to be necessary for the activation of C-Raf by the oncogene Ras in HeLa cells (31). However, many reports have shown evidence that PHB has antitumorigenic activity in prostate, gastric and ovarian cancer (32–35). PHB overexpression was shown to result in the inhibition of prostate cancer cell growth and the knockdown of PHB by siRNA accelerates tumor growth (33).

In the present study, stable clones of KD (PHB-knockdown NB4-R1 cells) and OE (PHB-overexpression NB4-R1 cells) were established and used to determine the cellular response to As4S4. The results showed that PHB overexpression enhanced apoptosis of NB4-R1 cells, and reduction of PML-RARα fusion protein. Although PHB knockdown had no significant effect on baseline apoptosis and PML-RARα fusion protein, a downregulation of PHB was associated with an attenuated apoptosis and lesser reduction of PML-RARα protein in the cells treated with As4S4. These results strongly support that PHB has antitumorigenic activity.

The effects of PHB on cellular processes may be due to its subcellular localization in different type cells. The subcellular localization of PHB has been shown to affect cell fate, specifically apoptosis (36). PHB has been shown with an increased level on the cell membrane that facilitates tumorigenesis through its interaction with c-Raf induced by the Ras oncogene (37,38), whereas increased levels of PHB in the nucleus induce apoptosis by increasing the transcriptional activity of p53 and its translocation to the cytoplasm (39). We have found the increased levels of PHB, either modulated by As4S4 or by PHB overexpression vectors, in the nucleus locations of APL cells.

The PML-RARα fusion protein is the key molecule that drives APL cells. This fusion protein also serves as the therapeutic target of ATRA and arsenic agents (40). While ATRA induces APL to undergo differentiation by targeting the RARα moiety, arsenic agents induce apoptosis through SUMO-1-mediated degradation of the PML moiety of the fusion protein (41). However, other molecules involved in the process remain to be identified. In this study, we showed a close relationship of upregulation of PHB with reduction of PML-RARα during As4S4-induced apoptosis. Consistently, PHB knockdown experiments showed a reduced degradation of PML-RARα protein. These results indicate that PHB is involved in the APL cell apoptosis. However, the biochemical pathway of PHB activity in relation to PML-RARα remains the subject of investigations.

In conclusion, PHB was identified among the upregulated proteins associated with As4S4-induced apoptosis of NB4-R1 cells. The experiments with modulation of PHB expression indicate that PHB overexpression enhances apoptosis and degradation of PML-RARα fusion protein, and consistently PHB knockdown attenuated the cellular response to As4S4 treatment.

Acknowledgements

The present study is supported by a research grant from the Natural Science Foundation of China (NSFC, grant no. 30701133), the Shaanxi Province Science and Technology Development Fund (SPSTDF, grant no. 2012KTCL03-12). The authors thank Dr Qunling Zhang from Shanghai Institute of Hematology for providing the NB4-R1 cell line; Dr Xinyang Wang from the First Affiliated Hospital, Xi'an Jiaotong University for their technological assistance; and Dr Byron Song from Univerity of Tronto, Ontario, Canada for critically reading the manuscript.

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References