Release of nucleophosmin from the nucleus: Involvement in aloe-emodin–inducedhuman lung non small carcinoma cell apoptosisHong-Zin Lee1*, Chun-Hsiung Wu2 and Shen-Peng Chang2

1School of Pharmacy, China Medical University, Taichung, Taiwan2Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan

Aloe-emodin (1,8-dihydroxy-3-(hydroxymethyl)-anthraquinone) isone of the active constituents from the root and rhizome of Rheumpalmatum. Our previous study has demonstrated that aloe-emodininduced a significant change in the expression of lung cancer cellapoptosis-related proteins compared to those of control cells. How-ever, the molecular mechanisms underlying the biological effectsof aloe-emodin still remain unknown. Based on these reasons, wewere interested in the change of aloe-emodin–induced total proteinexpression by the proteomics technique during aloe-emodin–in-duced lung cancer cell apoptosis. Our study applied 2D electro-phoresis to analyze the proteins involved in aloe-emodin–inducedapoptosis in H460 cells. We found that the release of nucleophos-min from the nucleus to the cytosol and the degradation of nu-cleophosmin were associated with aloe-emodin–induced H460 cellapoptosis. Our study also demonstrated that the gene expression ofnucleophosmin remained unchanged after treatment with aloe-emodin. The aloe-emodin–caused increase in the amount of pro-form and fragment of nucleophosmin in cytoplasm may be one ofthe important events for aloe-emodin–induced H460 cell apoptosis.© 2004 Wiley-Liss, Inc.

Key words: aloe-emodin (1,8-dihydroxy-3-(hydroxymethyl)-anthra-quinone); 2D electrophoresis; human lung non small cell carcinomacell line H460; nucleophosmin (nucleolar phosphoprotein B23); geneexpression; proteomics

Aloe-emodin (1,8-dihydroxy-3-(hydroxymethyl)-anthraquinone) isone of the active constituents from the root and rhizome of Rheumpalmatum. Our previous study demonstrated that 24 hr of contin-uous exposure to 40 �M of aloe-emodin induced a typical apo-ptosis on lung carcinoma cell line H460. Aloe-emodin–inducedapoptosis was characterized by nuclear morphological changes andDNA fragmentation.1 Many proteins, such as PKC, bcl-2, caspaseand the MAP kinase family members, have been demonstrated tobe involved in aloe-emodin–induced apoptosis.2 However, themolecular mechanisms underlying the biological effects of aloe-emodin still remain unknown.

Nucleophosmin is a nucleolar phosphoprotein that accumulates inthe nucleoplasm of cells. It is significantly more abundant in tumorand proliferating cells than in normal resting cells.3,4 The putativefunction of nucleophosmin is ribosome assembly and transport.5,6

Recently, interest in nucleophosmin has been growing due to its rolein DNA repair and in cancer. Nucleophosmin is also a mobile nucle-olar protein that shuttles between nucleoli and cytoplasm or shiftsfrom the nucleoli to the nucleoplasm when cells are exposed to certainanticancer drugs.6,7 Recently, evidence has demonstrated that nucleo-phosmin is downregulated during drug-induced apoptosis of cancercells.8,9 Hsu and Yung10 indicated that there was a decrease in thelevel of cellular nucleophosmin protein and the appearance of itsdegraded product (25 kDa) during TPA-induced differentiation inhuman myelogenous leukemia K562 cells. Therefore, nucleophosminis one of the key elements in the regulation of nucleolar function forcellular differentiation and apoptosis in cancer cells.

Proteomics is now generally accepted as a method to analyzetotal protein expression and elucidate cellular processes at themolecular level.11,12 Therefore, proteome analysis allowed theidentification of marker proteins that are involved in the inductionof apoptotic cell death in cancer cells. Since identification ofproteins differentially expressed in apoptotic cells could lead tonew insights into the pathway or help to investigate the keydeterminants for aloe-emodin–induced apoptosis, this study ap-

plied 2D gel electrophoresis to analyze the proteins involved inaloe-emodin–induced apoptosis at the protein level in H460 cells.We demonstrated that the release of nucleophosmin from thenucleus to cytosol and the degradation of nucleophosmin wereinvolved in aloe-emodin–induced apoptosis of H460 cells.

Material and methodsMaterials

Aloe-emodin (1,8-dihydroxy-3-(hydroxymethyl)-anthraquinone),antipain, aprotinin, dithiothreitol (DTT), EDTA, ethyleneglycol-bis-(�-aminoethyl ether)-N,N,N�,N�-tetraacetic acid (EGTA), leupep-tin, pepstatin, phenylmethylsulfonyl fluoride and Tris were pur-chased from Sigma Chemical Company (St. Louis, MO, USA);anti-mouse IgG peroxidase-conjugated secondary antibody werepurchased from Amersham (Buckinghamshire, United Kingdom).Nucleophosmin antibody was purchased from Zymed Laboratories(South San Francisco, CA, USA); Enhanced chemiluminescent(Renaissance) detection reagents were obtained from NEN LifeScience Products (Boston, MA, USA).

Cell cultureH460 cells were grown in monolayer culture in Dulbecco’s

modified Eagle’s medium (DMEM; Life Technologies, Rockville,MD, USA) containing 5% FBS (HyClone, Logan, UT, USA), 100U/ml penicillin and 100 �g/ml streptomycin (Gibco BRL, Rock-ville, MD, USA) and 2 mM glutamine (Merck, Darmstadt, Ger-many) at 37°C in a humidified atmosphere comprised of 95% airand 5% CO2. When H460 cells were treated with 40 �M aloe-emodin, the culture medium containing 1% FBS was used. All datapresented in this report are from at least 3 independent experimentsshowing the same pattern of expression.

Protein preparationProtein was extracted as previously described.1 Adherent and

floating cells were collected at the indicated times and washedtwice in ice-cold PBS. Cell pellets were resuspended in lysis buffer(Tris-HCl 50 mM [pH 7.5], NaCl 150 mM, EGTA 1 mM, DTT 1mM, PMSF 1 mM, sodium orthovanadate 1 mM, sodium fluoride1 mM, aprotinin 5 �g/ml, leupeptin 5 �g/ml, antipain 5 �g/ml, 1%Nonidet P-40, 0.25% sodium deoxycholate [Sigma]) for 30 min at

Abbreviations: Aloe-emodin, 1,8-dihydroxy-3-(hydroxymethyl)-anthra-quinone; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate; DTT, dithiothreitol; EGTA, ethyleneglycol-bis-(�-aminoethylether)-N,N,N�,N�-tetraacetic acid; EST, expressed sequence tags; H460cell, human lung non small carcinoma cell line; IEF, isoelectric focusing;IgG, immunoglobulin; IPG, immobilized pH gradients; Mw, molecularweight; PKC, protein kinase C; pl, isoelectric point; PMSF, phenylmeth-ylsulfonyl chloride; TBST, Tris-buffered saline with Tween; TPA, 12-O-tetradecanoylphorbol-13-acetate; Vh, voltage hour.

Grant sponsor: National Science Council; Grant numbers: NSC 92-2320-B-039-019, NSC 93-2320-B-039-025; Grant sponsor: China MedicalCollege Grant, Republic of China; Grant number: CMU 92-P-04.

*Correspondence to: School of Pharmacy, China Medical University,91, Hsueh-Shih Road, Taichung, 40402, Taiwan. Fax: �886-4-22039203.E-mail: hong@mail.cmu.edu.tw

Received 3 June 2004; Accepted 1 September 2004DOI 10.1002/ijc.20676Published online 28 October 2004 in Wiley InterScience (www.

interscience.wiley.com).

Int. J. Cancer: 113, 971–976 (2005)© 2004 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

4°C. Lysates were clarified by centrifugation at 100,000 g for 30min at 4°C and the resulting supernatant was collected, aliquotted(50 �g/tube) and stored at –80°C until assay. The protein concen-trations were estimated with the Bradford method.13

Western blot analysisSamples were separated by 12% of SDS-PAGE (Bio-Rad, Her-

cules, CA, USA). The SDS-separated proteins were equilibrated intransfer buffer (Tris-HCl 50 mM [pH 9.0–9.4], glycine 40 mM,0.375% SDS [Bio-Rad], 20% methanol [Merck]) and electrotrans-ferred to Immobilon-P Transfer Membranes (Millipore, Bedford,MA, USA). The blot was blocked with a solution containing 5%nonfat dry milk in Tris-buffered saline (Tris-HCl 10 mM, NaCl150 mM [Sigma]) with 0.05% Tween 20 (TBST; Merck) for 1 hr,washed and incubated with antibodies to �-actin (1:5,000 [Sigma];the detection of �-actin was used as an internal control in all of thedata from the Western blotting analysis) and nucleophosmin (1:250; Zymed Laboratories, Inc., South San Francisco, CA). Thesecondary antibody consisted of a 1:20,000 dilution of horseradishperoxidase (HRP)-conjugated goat anti-mouse IgG (Amersham,Buckinghamshire, United Kingdom). The enhanced chemilumi-nescent (NEN Life Science Products, Boston, MA, USA) detectionsystem was used for immunoblot protein detection.

Two-dimensional gel electrophoresisThe proteins (250 �g) were dissolved in a rehydration buffer (urea

9.8 M, CHAPS 0.5%, DTT 10 mM, Biolytes (BioRad, Hercules, CA)0.2% and a trace of bromphenol blue) to a final volume of 300 �l. Thesamples were added to the 17-cm IPG strips (pH 3–10, linear, Readys-trip; BioRad), which were rehydrated for 12 hr. After rehydration, thestrips were focused for 60,000 Vh, starting at 250 V and graduallyraising the voltage to 10,000 V. Once the IEF was completed, thestrips were equilibrated in 6 M urea containing 2% SDS, 0.375 M Tris(pH 8.8), 20% glycerol and 130 mM DTT. The 2D electrophoresiswas performed using 12% SDS-PAGE. Gels were stained with stan-dard silver staining protocol.

Database searchesMass analysis was carried out according to the TurboSequest

software (ThermoFinnigan, San Jose, CA) using the ThermoFinni-gan LCQ DECA XP mass spectrometer. Comparisons of thesequences of protein spot 1 (Fig. 1) and nucleophosmin withnucleotide and protein sequences in the GenBank database (http://www.ncbi.nlm.nih.gov) were done using the NCBI BLAST pro-gram (http://www.ncbi.nlm.nih.gov/BLAST).

Immunolocalization of nucleophosminH460 cells were seeded at a density of 5 � 104 cells per well

onto 12-well plates 48 hr before drug treatment. After treatment,cells were fixed with 3.7% formaldehyde (Merck) for 15 min andpermeabilized with 0.1% Triton X-100 (Merck). Cells were thenincubated 1 hr at 37°C with nucleophosmin antibody dilute 1:50 inTBST solution. The cells were washed 3 times with TBST andincubated for 30 min at 37°C with fluorescein-conjugated anti-mouse IgG antibody diluted 1:50 in TBST. The cells were thenwashed with TBST and examined by fluorescence microscopy(Olympus IX 70; Olympus, Hamburg, Germany).

Extraction of total RNATotal RNA was isolated from control or aloe-emodin–treated H460

cells with the RNeasy Mini kit (Qiagen, Valencia, CA) according tothe manufacturer’s instructions. RNA concentration was quantifiedusing a spectrophotometer at a wavelength of 260 nm.

Reverse transcriptase–PCRcDNA was prepared by reverse transcription of 1.5 �g of total

RNA. Nucleophosmin and �-actin transcripts were determined byreverse transcriptase–PCR (RT-PCR) with the RNA PCR kit (In-vitrogen Life Technologies, Carlsbad, CA). The nucleophosminprimers were 5�-ATGAATGACGAAGGCAGTCC-3� and 5�-GGCAATAGAACCTGGACAACA-3�, which amplify a 746-bp

product, and the primers of �-actin (GenBank accession no.G15871) were 5�-ACAAAACCTAACTTGCGCAG-3� and 5�-TCCTGTAACAACGCATCTCA-3�. The amplification was per-formed with 1 denaturing cycle at 95°C for 5 min, then the 95°Cfor 1 min, 55°C for 1 min, as well as 72°C for 1 min were done for30 cycles, and 1 final extension at 72°C for 10 min. RT-PCRproducts were separated by electrophoresis on 2% agarose gel andvisualized by ethidium bromide staining.

ResultsIdentification of differentially expressed proteins by 2D gel

The major purpose of our study was to investigate how manyproteins decreased, disappeared or were newly developed aftertreatment with 40 �M aloe-emodin for 24 hr. The protein sampleswere run on parallel 2D gels and visualized using silver staining.Many protein spots were found varying in intensity between thecontrol-cell–loaded and the aloe-emodin–treated cell–loaded gels,except for the pH range of 4.75–6.5 and the molecular weightrange of 29–95 kDa (Fig. 1). In aloe-emodin–treated cells, theprotein expression is significantly decreased compared to the con-trol cells. In the inset area, (pH range of 4.2–5.9 and molecularweight below 19 kDa), 3 proteins were expressed in aloe-emodin–treated cells, whereas diminished or no expression was detected incontrol cells (Fig. 1; inset). One of these altered proteins wascharacterized by mass spectrometry. Using the amino acid se-quences of protein spot 1 (Table I) as the query in a basic localalignment search tool (BLAST) of the EST database at the NCBI,protein spot 1 was identified as nucleophosmin (pI/molecularweight [Mw]: 4.64/32,575.02, GenBank accession no. A32915).

The effect of aloe-emodin on the expression of nucleophosmin inH460 cells

Based on the difference between the theoretical molecular weight(32.575 kDa) from the GenBank database and the apparent molecularweight (about 18 kDa) determined by 2D gel, we proposed that adegradation of nucleophosmin might have occurred during aloe-emodin–induced apoptosis in H460. To obtain further support for theinduction of fragmentation of nucleophosmin by aloe-emodin inH460 cells, monoclonal anti-nucleophosmin, which reacts with thec-terminus of nucleophosmin, was used to detect the proform ofnucleophosmin (32.575 kDa) in this study. In 2D gel, the protein at 18kDa was not found in aloe-emodin–treated cells by Western blotanalysis (Fig. 2). However, comparing the control and aloe-emodin–treated cells, we found nucleophosmin to be markedly increased at 32kDa in the aloe-emodin–treated samples (Fig. 2b). The differentialexpression of nucleophosmin was also analyzed by Western blotanalysis of 1D gel. Aloe-emodin–treated samples had a significantincrease in the expression of nucleophosmin (32 kDa) (Fig. 3). Toelucidate whether the increased nucleophosmin protein level is re-quired during drug-induced apoptosis in lung carcinoma H460 cells,we used various apoptosis-inducing compounds, such as baicalein,emodin and luteolin. After treatment with baicalein (50 �M, 24 hr),emodin (50 �M, 16 hr) or luteolin (30 �M, 24 hr), the protein levelsof nucleophosmin (32 kDa) were significantly increased by Westernblotting analysis (Fig. 3).

Intracellular localization of nucleophosminTo analyze the intracellular localization of nucleophosmin, im-

munostaining with an antibody against nucleophosmin was per-formed. Immunostaining studies verified the altered expression ofnucleophosmin in aloe-emodin–treated cells compared to control(Fig. 4). Fluorescence in the control cells and in most aloe-emodin–treated cells exhibited diffuse staining throughout nucleus(Fig. 4; arrow), while in a portion of the aloe-emodin–treated cellsthe fluorescence staining was low in the nucleus and most intensein the cytoplasm (Fig. 4; arrow).

Analysis of nucleophosmin mRNA by RT-PCRAccording to the results of proteome or Western blotting, aloe-emodin

induced an increase in the nucleophosmin protein level. To elucidate

972 LEE ET AL.

whether the expression of nucleophosmin mRNA is involved in aloe-emodin–induced apoptosis, RT-PCR techniques were used. After H460cells were treated with 40 �M aloe-emodin for the indicated time inter-vals, there were no changes in the expression of nucleophosmin mRNA(Fig. 5). This result indicated that aloe-emodin had no significant effect onthe mRNA level of nucleophosmin in H460 cells.

Discussion

Many proteins, such as protein kinase C, mitogen-activatedprotein kinase, bcl-2 and caspase family members, have beendemonstrated to be involved in aloe-emodin–induced apoptosis ofH460 cells in our previous studies.1,2 However, the molecularmechanisms underlying the biological effects of aloe-emodin re-main unknown. Therefore, detailed knowledge of the molecularchanges within apoptotic cells may provide a better understanding

of apoptosis and may help to investigate the key determinants.Proteomics, the large-scale screening of proteins, has been used foranalyzing the changes in total proteins of H460 cells duringaloe-emodin–induced apoptosis in the present study. Our studyfound changes in many protein spots in the 2D gel during aloe-emodin–induced H460 cell apoptosis.

Comparing the proteome of the control and aloe-emodin–treatedcells, we found a markedly increasing protein spot (pI about 4.7and Mw about 18 kDa) in aloe-emodin–treated samples. Analysiswith mass spectrometry and the EST database at the NCBI re-vealed that this protein was nucleophosmin (pI/Mw: 4.64/32,575.02, GenBank accession no. A32915). Therefore, we con-sidered that the cleavage of nucleophosmin should be producedduring aloe-emodin–induced apoptosis in H460 cells. In our study,the proform (pI about 5 and Mw about 35 kDa) of nucleophosmin

FIGURE 1 – Two-dimensional electrophoresis maps of control or aloe-emodin–treated H460 cells. Cells were incubated with or without 40 �Maloe-emodin in the presence of 1% serum for 24 hr. Proteins were separated on a pH 3–10 IPG-strip (17 cm) in the first dimension and on a12% SDS-polyacrylamide gel in the second dimension. Staining of the protein spots was accomplished by silver nitrate. Magnified views of gelsfrom the control cells or from cells treated with aloe-emodin are from the section with pH range 4.2–5.9 and molecular weight below 19 kDa.

TABLE I – MS/MS ANALYSIS OF TRYPTIC FRAGMENTS FROM SPOT 1 IN ALOE-EMODIN-TREATED SAMPLES

z MH� Xcorr Reference ()Sequence

3 2,929.4 5.35 gi�107221 (–)TVSLGAGAKDELHIVEAEAMNYEGSPIK2 2,145.5 2.49 gi�107221 (–)DELHIVEAEAMNYEGSPIK2 1,569.5 3.43 gi�107221 (–)VDNDENEHQLSLR2 2,928.7 3.18 gi�107221 (–)TVSLGAGAKDELHIVEAEAMNYEGSPIK1 745.0 1.2 gi�107221 (–)VTLATLK

973ALOE-EMODIN–INDUCED APOPTOSIS IN H460 CELLS

was detected with the nucleophosmin antibody. Aloe-emodin in-duced a significant increase in the protein level of the proform ofnucleophosmin in 2D gel by Western blotting analysis. Aloe-emodin–treated H460 cells also revealed increases in the proteinlevel of nucleophosmin (35 kDa) in 1D gel. Nucleophosmin is amajor nucleolar phosphoprotein that is 20 times more abundant incancer cells than in normal cells.14 The abundance of nucleophos-min is directly proportional to cell proliferation.15 Downregulationof nucleophosmin is associated with anticancer drug–induced ap-optosis in various human cancer cells.8,9 Therefore, our results arenot consistent with previous studies in which nucleophosmin wasfrequently overexpressed in a variety of human malignancies anddownregulated in drug-induced apoptosis cells. Since aloe-emodininduced the change in the expression of nucleophosmin, we fo-cused on the expression of nucleophosmin in aloe-emodin–in-duced H460 cell apoptosis. Our study also found that aloe-emodinhad no significant effect on the mRNA level of nucleophosmin inH460 cells. Therefore, a direct relationship between nucleophos-min-translocation and apoptosis was determined by immunostain-ing in our study. We found nucleophosmin (32 kDa) to be mark-edly translocated from the nucleus to the cytoplasm, but not fromnucleoli to the nucleoplasm during aloe-emodin–induced H460cell apoptosis. This result suggested that a certain degree of nu-cleophosmin-translocation from nucleoli to the cytoplasm shouldbe necessary for the increasing in the proform of nucleophosminduring aloe-emodin–induced H460 cell apoptosis.

In addition, nucleophosmin is a mobile nucleolar protein thatshuttles between nucleoli and nucleoplasm or nucleoli and cyto-plasm.6 Based on the shuttle action, nucleophosmin may recruit

protein factors into nucleoli for various functions.16,17 In the past,many investigators found that nucleophosmin shifts from nucleolito the nucleoplasm when cells are exposed to certain anticancerdrugs.18,19 Previously, many reports suggested that the nucleolus isone of the target areas of anticancer drugs, such as Adriamycin,actinomycin D, mitoxantrone and camptothecin. These anticancerdrugs localize in nucleoli or have effects on nucleoli, includingaltering nucleolar structure and inhibiting its function.20,21 Basedon the above observations, we proposed that nucleophosmin shiftsfrom nucleoli to the cytoplasm when cells are exposed to aloe-emodin. First, aloe-emodin might induce the destruction of nucle-oli structure and function during H460 cell apoptosis. Second, thedepletion of nucleophosmin from nucleoli or inhibition of theshuttling of nucleophosmin from cytoplasm to nucleus may beresponsible for the increase in nucleophosmin during aloe-emod-in–induced H460 cell apoptosis. However, the cause–effect rela-tionship between nucleophosmin-translocation and apoptosis re-mains to be investigated.

It is noteworthy that the fragment of nucleophosmin (pI about4.7 and Mw about 18 kDa) was not observed in 2D gel by Westernblotting analysis. This seemed to indicate that the commercialantibody of nucleophosmin, which reacted with the C-terminal ofnucleophosmin, could not recognize the fragment at the N-terminalof nucleophosmin in our study. The fragment, with a predicted Mwof about 18 kDa, at the N-terminal of nucleophosmin, was ob-served in 2D gel during aloe-emodin–induced H460 cell apoptosis.This result is consistent with another observation that a degradedproduct of nucleophosmin was detected during the TPA-induceddifferentiation of K562 cells.10 To further investigate the relation-ship between the increased nucleophosmin protein level and H460cell apoptosis, we examined the effect of emodin, luteolin orbaicalein, which have been demonstrated to be apoptosis-inducingagents in a series of our studies, on the expression of nucleophos-min. The amount of nucleophosmin induced by emodin, luteolin orbaicalein was more intense than that found in untreated cells ofH460 cells. This result indicated that the increase, not downregu-lation, in the protein level of nucleophosmin is involved in drug-induced H460 cell apoptosis.

In summary, we applied 2D electrophoresis to analyze theproteins involved in aloe-emodin response at the protein level inH460 cells. The release of nucleophosmin from the nucleus tocytosol and the degradation of nucleophosmin were observedduring aloe-emodin–induced H460 cell apoptosis. We also dem-onstrated that the gene expression of nucleophosmin remainedunchanged after treatment with aloe-emodin. The aloe-emodin–caused increase in the amount of proform and fragment of nucleo-phosmin may be involved in aloe-emodin–induced H460 cellapoptosis.

FIGURE 2 – The effect of aloe-emodin on the ex-pression of nucleophosmin in H460 cells. Aloe-emodin–induced expression of nucleophosmin wasdetected by Western blotting of 2D gel. H460 cellswere incubated without (a) or with (b) 40 �M aloe-emodin in the presence of 1% serum for 24 hr.Proteins were separated on a pH 3–10 IPG-strip (17cm) in the first dimension and on a 12% SDS-polyacrylamide gel in the second dimension. The2D-separated proteins were electrotransferred to Im-mobilon-P transfer membranes and then probed withnucleophosmin antibody as described in Materialand methods. Results are representative of 3 inde-pendent experiments.

FIGURE 3 – The effect of aloe-emodin, baicalein, emodin or luteolinon the expression of nucleophosmin of H460 cells. Drug-inducedexpression of nucleophosmin was detected by Western blotting of 1Dgel. Cell lysates were analyzed by 12% SDS-PAGE and then probedwith nucleophosmin antibody as described in Material and methods.Lane 1, 3, 5 and 7, control cells (DMSO); Lane 2, aloe-emodin(AE)-treated cells (40 �M, 24 hr); Lane 4, emodin (E)-treated cells (50�M, 16 hr); Lane 6, baicalein (B)-treated cells (50 �M, 24 hr); Lane8, luteolin (L)-treated cells (30 �M, 24 hr); Lane 9, positive control(HeLa cells). Results are representative of 3 independent experiments.

974 LEE ET AL.

References

1. Lee HZ. Protein kinase C involvement in aloe-emodin- and emodin-induced apoptosis in lung carcinoma cell. Brit J Pharmacol 2001;134:1093–103.

2. Yeh FT, Wu CH, Lee HZ. Signaling pathway for aloe-emodin-induced apoptosis in human H460 lung nonsmall carcinoma cell. IntJ Cancer 2003;106:26–33.

3. Chan WY, Liu QR, Borjigin J, Busch H, Rennert OM, Tease LA,Chan PK. Characterization of the cDNA encoding human nucleophos-min and studies of its role in normal and abnormal growth. Biochem-istry 1989;28:1033–9.

4. Feuerstein N, Spiegel S, Mond JJ. The nuclear matrix protein,numatrin (B23), is associated with growth factor-induced mitogenesisin Swiss 3T3 fibroblasts and with T-lymphocyte proliferation stimu-lated by lectins and anti-T cell antigen receptor antibody. J Cell Biol1988;107:1629–42.

5. Schmidt-Zachmann M, Hugle-Dorr B, Franke WW. A constitutivenucleolar protein identified as a member of the nucleoplasmin family.EMBO J 1987;6:1881–90.

6. Borer RA, Lehner CF, Eppenberger HM, Nigg EA. Major nucleolarproteins shuttle between nucleus and cytoplasm. Cell 1989;56:379–90.

7. Chan PK, Chan FY. A study of correlation between NPM-transloca-tion and apoptosis in cells induced by Daunomycin. Biochem Phar-macol 1999;57:1265–73.

8. Liu WH, Yung BY. Mortalization of human promyelocytic leukemiaHL-60 cells to be more susceptible to sodium butyrate-induced apo-ptosis and inhibition of telomerase activity by down-regulation ofnucleophosmin/B23. Oncogene 1998;17:3055–64.

9. Wu HL, Hsu CY, Liu WH, Yung BY. Berberine-induced apoptosis ofhuman leukemia HL-60 cells is associated with down-regulation ofnucleophosmin/B23 and telomerase activity. Int J Cancer 1999;81:923–9.

10. Hsu CY, Yung BY. Involvement of nucleophosmin/B23 in TPA-induced megakaryocytic differentiation of K562 cells. Br J Cancer2003;89:1320–6.

11. Bogaerdt AJ, Ghalbzouri AE1, Hensbergen PJ, Reijnen L, Verkerk M,

FIGURE 4 – Immunofluorescence localizationof nucleophosmin in control or aloe-emodin–treated H460 cells. Cells were incubated with orwithout 40 �M aloe-emodin in the presence of1% serum for 24 hr. After washing with PBS,cells were fixed and stained with nucleophosminantibody as described in Material and methods.(a) Control cells (0.1% DMSO). (b) 40 �Maloe-emodin–treated cells. (c) Cells treated with40 �M aloe-emodin, but not stained with nu-cleophosmin antibody. (d) Magnified view of asection of (b). Results are representative of 3independent experiments.

FIGURE 5 – The effect of aloe-emodin on theexpression of nucleophosmin mRNA of H460cells. Aloe-emodin–induced expression of nucleo-phosmin mRNA was detected by RT-PCR. H460cells were incubated with 0.1% DMSO or 40 �Maloe-emodin in the presence of 1% serum for 1, 2,4, 8, 16 and 24 hr. RNA samples were preparedfrom control or aloe-emodin–treated cells. PCRproducts run on a 2% agarose gel. Lane 1, marker;Lanes 2, 4, 6, 8, 10 and 12, control cells; Lanes 3,5, 7, 9, 11 and 13, aloe-emodin–treated cells. Up-per panel: The band represents nucleophosmin(746 bp). Lower panel: The band represents �-actin(241 bp).

975ALOE-EMODIN–INDUCED APOPTOSIS IN H460 CELLS

Kroon-Smits M, Middelkoop E, Ulrich MMW. Differential expres-sion of CRABP-II in fibroblasts derived from dermis and subcutane-ous fat. Biochem Biophys Res Commun 2004;315:428–33.

12. Dallmann K, Junker H, Balabanov S, Zimmermann U, Giebel J,Walther R. Human agmatinase is diminished in the clear cell type ofrenal cell carcinoma. Int J Cancer 2004;108:342–7.

13. Bradford MM. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein using the principle of protein-dyebinding. Anal Biochem 1976;72:248–54.

14. Chan WY, Liu QR, Borjigin J, Busch H, Rennert OR, Tease LA, ChanPK. Characterization of the cDNA encoding human nucleophosminand studies of its role in normal and abnormal growth. Biochemistry1989;28:1033–9.

15. Feuerstein N, Mond JJ. “Numatrin,” a nuclear matrix protein associ-ated with induction of proliferation in B lymphocytes. J Biol Chem1987;262:11389–97.

16. Valdez BC, Perlaky L, Henning D, Saijo Y, Chan PK, Busch H.Identification of the nuclear and nucleolar localization signals of the

protein p120. Interaction with translocation protein B23. J Biol Chem1994;269:23776–83.

17. Perlaky L, Valdez BC, Busch H. Effect of cytotoxic drugs on trans-location of nucleolar RNA helicase RH-II/Gu. Exp Cell Res 1997;235:413–20.

18. Finch RA, Revankar GR, Chan PK. Nucleolar localization of nucleo-phosmin/B23 requires GTP. J Biol Chem 1993;268:5823–7.

19. Finch RA, Chan PK. ATP depletion affects NPM translocation andexportation of rRNA from nuclei. Biochem Biophys Res Commun1996;222:553–8.

20. Nakamura T, Imai H, Tsunashima N, Nakagawa Y. Molecular cloningand functional expression of nucleolar phospholipid hydroperoxideglutathione peroxidase in mammalian cells. Biochem Biophys ResCommun 2003;311:139–48.

21. Mi Y, Thomas SD, Xu X, Casson LK, Miller DM, Bates PJ. Apo-ptosis in leukemia cells is accompanied by alterations in the levels andlocalization of nucleolin. J Biol Chem 2003;278:8572–9.

976 LEE ET AL.