Eur. J. Biochem.253, 7662770 (1998) FEBS1998

Induction of necrosis by zinc in prostate carcinoma cells and identificationof proteins increased in association with this induction

Kazuhiro IGUCHI1, Michiko HAMATAKE 2, Ryoji ISHIDA3, Yoshiko USAMI1, Tetsuo ADACHI1, Hajime YAMAMOTO4, Kiyoshi KOSHIDA4,Tadao UCHIBAYASHI5 and Kazuyuki HIRANO1

1 Department of Pharmaceutics, Gifu Pharmaceutical University, Japan2 Department of Pharmacy, Aichi Cancer Center Hospital, Japan3 Laboratory of Chemotherapy, Aichi Cancer Center Research Institute, Japan4 Toyama Prefectural Center Hospital, Toyama, Japan5 Department of Urology, School of Medicine, Kanazawa University, Japan

(Received 3 February1998) 2 EJB 98 0163/1

Zinc exhibits inhibitory effects on apoptosis, and a deficiency in this metal generally causes this typeof cell death to occur. In the present study, we found that exposure to zinc results in necrosis of prostatecarcinoma cells. When zinc acetate was added to LNCaP or PC-3 cells in monolayer culture, they beganto detach from the culture dishes, and viability was lost after 428 h. Most of the cell death was found tobe due to necrosis as determined by double staining with fluorescein-isothiocyanate-labeled annexin Vand ethidium bromide, and by detection of hypodiploid cells. Associated with the induction of necrosiswas an increase in low molecular-mass proteins, identified by HPLC analysis to be thymosinβ10, parathy-mosin and GAGE in LNCaP cells, and thymosinβ4, parathymosin and metallothionein in PC-3. The timecourse of the increase of thymosinβ10 in LNCaP cells and thymosinβ4 in PC-3 cells was consistentwith that of appearance of cell detachment and dead cells. These results indicate that zinc can inducenecrosis and suggest that production of proteins includingβ-thymosins is involved in induction of pro-cesses leading to cell detachment.

Keywords:necrosis ; zinc; thymosin; annexin; cell detachment.

Zinc is an essential trace element and plays an important swelling with apparently intact nuclei, and no DNA fragmenta-tion [10].role as a component of an essential cofactor for many enzymes

Apoptosis is an active process for negative selection of cellsinvolved in macro molecule synthesis or protein metabolism [1].which become unnecessary for maintaining tissue function dur-Intracellularly, it is bound to ligands [2]. Heavy metals such asing embryogenesis and development of adult tissues [11]. Thiszinc and cadmium enhance expression of various genes includ-form of cell death is also involved in elimination of self-reactiveing metallothionein, protooncogenes and heat-shock protein [3,T-cells and low reactivity B lymphocytes [12]. The former is4], this expression presumably being mediated by enhancedtriggered by recognition of self antigens by self-reactive thymo-binding of zinc-finger transcription factor to metal-response ele-cytes. Failure to eliminate cells by apoptosis may result in tumorments (MREs) [5]. A deficiency of zinc leads to thymic atrophygrowth and development of auto-immune disease [13]. Necrosis,in laboratory animals, and male hypogonadism accompanied byin contrast, is thought to be a passive degenerative phenomenoninhibition of sperm formation and a lack of secondary sex char-induced by toxic agents [14].acteristics [6, 7].

There is evidence that the intracellular zinc concentrationApoptosis and necrosis are two distinct types of cell deathinfluences apoptosis, and rats or mice on a zinc-deficient dietcharacterized by differences in morphology [8]. Apoptosis fea-have an elevated level of such ‘programmed’ cell death [15, 16].tures high molecular-mass DNA fragmentation, abnormal chro-The zinc-chelators,N,N,N′,N′-tetrakis (2-pyridylmethyl)ethyl-mosome condensation, a reduced membrane electric potentialenediamine and1,10-phenanthroline, trigger apoptosis in humanin mitochondria, formation of apoptotic bodies and a shift inthymocytes and chronic lymphatic leukemia cells [17, 18]. Inmembrane phospholipids so that anionic phosphatidylserine,contrast, the addition of zinc to cell cultures prevents the induc-which is normally located in the inner plasma membrane, be-tion of apoptosis byγ irradiation, hormones or serum depletioncomes exposed on the cell membrane [8]. Although internucleo-[19221]. Thus, at present, it is believed that an increase in intra-somal fragmentation is a hallmark of apoptosis, recent studiescellular zinc blocks apoptosis induced by various stimuli. Itshave revealed that cellular DNA is not always fragmented intoeffects on necrosis, however, are largely unknown. We reportnucleosomal ladders in apoptotic cells, thus apparently depend-here that zinc can induce necrosis in prostate carcinoma cells,ing on cell type [9]. Necrosis is defined by mitochondriain association with an increase in low molecular-mass proteinssuch as thymosinβ4, thymosinβ10, parathymosin, GAGE andCorrespondence toK. Hirano, Department of Pharmaceutics, Gifu

Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu, Japan metallothionein.502-8585

Fax: 181 58 237 5979.E-mail : hirano@gifu-pu.ac.jp MATERIALS AND METHODSAbbreviation.MRE, metal-response element.

Cell culture. LNCaP and PC-3 cells, derived from humanEnzymes.Endoproteinase Asp-N (EC 3.4.24.33) ; endoproteinaseGlu-C (EC 3.4.21.19). adenocarcinoma of the prostate, were routinely cultured in

767Iguchi et al. (Eur. J. Biochem. 253)

Fig.2. Effects of zinc on cell viability and cell detachment.LNCaPand PC-3 cells were cultured on 60-mm dishes and exposed to 500µMand 200µM zinc, respectively. Cell viability and detachment from disheswere examined at the indicated times. Cell viability was examined by the

Fig. 1. Effects of zinc and copper on the growth of human prostatictrypan blue exclusion assay, and cells were counted in a hemocytometer.cell lines.The cells were cultured in the presence of increasing concen-Floating LNCaP cells were almost inviable, although 40% of these weretrations of zinc (d) or copper (s). On day 3 of treatment, the living celldead in PC-3 case.population was determined as described in Materials and Methods. Data

presented are the mean6SD values from four different incubations.* P,0.01 versus control.

Analysis of N-terminal amino acid sequences.AfterHPLC, the collected proteins in each peak were evaporated todryness and cleaved with cyanogen bromide in 70% formic acid

RPMI-1640 medium containing10% fetal calf serum under a or digested with endoproteinase Glu-C or endoproteinase Asp-N.humidified atmosphere of 5% CO2 in air. After separation by C18 reverse-phase HPLC, the amino acid

Cell proliferation. Cell proliferation was evaluated by mea-sequences of the peptides were determined using an Appliedsurement of the fluorescence intensity in the presence of alamarBiosystems Protein Sequencer model 473A.blue (Wako Pure Chemical Industries) [22]. Cells were seeded Analysis of apoptosis by annexin staining.The level ofin 96-well multidishes (Costar Corporation) at a density ofapoptosis was determined with an apoptosis detection kit (R &2.53103 cells (LNCaP)/well or13103 cells (PC-3)/well in cul- D systems). Briefly, after zinc treatment, floating cells andture medium, and after one day, zinc acetate was added to thetrypsinized cells were collected and washed twice with NaCl/Pi,culture medium. After 72 h, 20µl alamar blue was added to eachand 10 mM Hepes/sodium hydroxide, pH 7.4,140 mM sodiumwell, and the plates were incubated for 4 h. The fluorescencechloride, 2.5 mM calcium chloride (buffer A) at once. The cellsintensity was measured using the Cytofluor 2350 with excitationwere dissolved in buffer A at13106 cells/ml, and propidiumat 530 nm and emission at 590 nm. iodide and fluorescein-isothiocyanate-labeled annexin V were

HPLC of intracellular proteins. LNCaP and PC-3 cells added. The samples were incubated for15 min, and analyzed(50260% confluent) in100-mm culture dishes were exposed towithin 1 h on a FACScan (Becton-Dickinson) using LYSYS-2.200µM (LNCaP) or 120 µM (PC-3) zinc acetate for the indi- Analysis of apoptosis and necrosis in terms of DNAcated times. The cells were washed with1.47 mM KH2PO4, content. Cells were washed with NaCl/Pi and fixed with three8.1 mM Na2HPO4, 2.68 mM KCl,137 mM NaCl, pH 7.4 (NaCl/ volumes of 70% ethanol at 4°C for 1 h. After washing twicePi) sonicated in 20 mM Tris/HCl, pH 7.4, and centrifuged forwith NaCl/Pi, they were suspended in NaCl/Pi containing propid-60 min at 4°C and1050003g. The supernatants were subjectedium iodide and RNase at final concentrations of 50µg andto reverse-phase HPLC (µBondasphere, 5µm, C18, 30 nm, 0.5 mg/ml, respectively, and incubated for15 min at room3.9 mm3150 mm) using an acetonitrile gradient at a flow ratetemperature. The samples were then washed with NaCl/Pi andof 1 ml/min. Proteins were monitored by measuring absorbanceanalyzed on a FACScan.at 210 nm.

SDS/polyacrylamide gel electrophoresis.For electro-RESULTSphoresis, 20% polyacrylamide gels were used according to the

methods of Laemmli [23], and proteins were detected by silverEffects of zinc on cell growth in prostate carcinoma cells.Since Zn21 is essential for cell growth and is enriched in thestaining.

768 Iguchi et al. (Eur. J. Biochem. 253)

Fig. 3. Flow cytometric analysis of annexin-V-fluorescein and pro-Fig.4. DNA fluorescence histograms of propidium-iodide-stainedpidium-iodide-stained cells.LNCaP cells were cultured in the absencecells. The cell lines and zinc-treatment conditions were the same as(A) or presence (C) of 500µM zinc, and PC-3 cells were cultured in thedescribed for Fig. 3.absence (B) or presence (D) of 200µM zinc for 24 h prior to being

stained.

To confirm that zinc selectively induces necrosis, the numberof hypodiploid cells (apoptotic cells) was examined by flow

prostate [1], its influence on growth of the prostate carcinomacytometry. Hypodiploid cells can be induced by agents causingcells, LNCaP and PC-3 was examined. Copper is generally moreapoptosis but not necrosis, and contain less DNA than normalcytotoxic than zinc, so copper was used as a control metal. WhenG1 cells [26, 27]. LNCaP or PC-3 cells exhibited typical cellLNCaP and PC-3 cells were exposed to various concentrationspopulations which correspond to cells in G1, S and G2-M phase,of either zinc acetate or cupric acetate for 72 h, their growth wasrespectively (Fig. 4A and B). 24 h after treatment with zinc,inhibited by both metals in a dose-dependent manner (Fig.1). cells contained less DNA than in Fig. 2C did not increaseAnother metals such as Ca21 and Mg21 did not influence cell (Fig. 4C and D), which means that zinc dose not inducegrowth in this dose (data not shown). The effects were moreapoptosis.pronounced in the zinc acetate case, and were presumably ex-erted by zinc itself, since addition of an equivalent amount ofIncrease and identification of intracellular proteins in zinc-EDTA completely canceled the growth inhibition. Moreover, theinduced necrotic cells.To investigate the process of inductioneffect was found to be cytocidal. The growth inhibition was alsoof necrosis in more detail, quantitative or qualitative changes inobserved by the lactate dehydrogenase assay (data not shown).proteins in Zn21-treated LNCaP and PC-3 cells were assessed byAfter addition of zinc at 500µM or 200µM, LNCaP and PC-3 reverse-phase HPLC analysis. When extracts from zinc-treatedcells, respectively, began to detach from culture dishes and dieLNCaP cells were compared with those of non-treated cells, newafter 428 h. For LNCaP cells, more than 90% of the cells wereprotein peaks 226 appeared at positions eluting after retentionreleased from the dish after 24 h and most were not viable, whiletimes of15220 min (Fig. 5). In the zinc-treated PC-3 cells, the17% of the PC-3 cells were detached and 40% of these wereamounts of proteins corresponding to peaks 3 and 4 (Fig. 5)dead (Fig. 2). were significantly elevated. SDS/PAGE analysis showed the

molecular masses of proteins of peaks126 to range over 5215 kDa (data not shown). Proteins responsible for peaks 226 inZinc-induced necrosis.To determine whether zinc-induced cell

death was apoptosis or necrosis, cells were double-stained with the LNCaP cells and peaks 3 and 4 in the PC-3 case (Fig. 5)were increased 428 h after the addition of zinc, and withpropidium iodide and fluorescein-isothiocyanate-labeled an-

nexin V. Apoptotic cells exclude dyes such as trypan blue or increasing concentrations of this metal (1002150 µM).To identify the increased proteins in the zinc-treated cells,propidium iodide, while necrotic cells do not. Thus, the latter

but not the former cells were stained with propidium iodide. they were purified, hydrolyzed with endoproteinase Glu-C,endoproteinase Asp-N or cyanogen bromide, and the peptideFluorescein-isothiocyanate-labeled annexin V has been used to

detect apoptotic cells since annexin binds phosphatidylserine ex- fragments were analyzed by N-terminal amino acid sequencing.Proteins corresponding to peaks126 on the HPLC chromato-posed to the outer membrane in apoptotic cells [24, 25]. When

LNCaP or PC-3 cells were exposed to zinc (Fig. 3), numbers of gram (Fig. 5) for LNCaP cells were identified as prothymosinA,thymosinβ10, parathymosin and GAGE, respectively. Similarlynecrotic cells increased, while the lower right population of

cells, which represents apoptotic cells having positive annexin protein peaks from zinc-treated PC-3 cells were found to be pro-thymosinA, thymosinβ10, thymosinβ4, parathymosin and met-V staining, did not increased.

769Iguchi et al. (Eur. J. Biochem. 253)

induce apoptosis in some cell lines [35, 36]. We confirmed thatzinc causes early, not late apoptosis in human T-cell leukemialine, MOLT 4, where cells were positive for detection of annexinstaining and an apoptotic-specific mitochondria protein (data notshown). Thus, possible responses to zinc exposure includeapoptosis and necrosis, which can occur depending on thecell.

There is accumulating evidence that apoptosis and necrosisare interconvertible [37239]. H2O2 triggers necrosis orapoptosis depending on the oxidant concentration [37]. Palombaet al. [38] showed that 3-aminobenzamide, a inhibitor of poly-(ADP-ribose) polymerase prevents necrosis and inducesapoptosis in H2O2-treated cells. ATP depletion results in a switchfrom apoptosis to necrosis in staurosporin and CD95-inducedapoptotic Jurkat cells [39]. Apoptosis can be also modulated byprotease inhibitors or the protooncogene, bcl-2 [40, 41]. It isthus conceivable that zinc can increase the susceptibility of cellsso that necrosis rather than apoptosis is induced by insult,through inhibition of poly(ADP-ribose)polymerase or depletionof ATP, or some unknown mechanism.

A parallel increase in necrotic cells, cell detachment andβ-thymosin was evident after exposure of cells to zinc (Figs1and 2), thereby suggesting a relationship among these events. Itshould be noted that most floating cells lost their viability inLNCaP cells, albeit the figure being only about 40% for thePC-3 cells. A similar positive relationship between detachmentand apoptotic cell death has been noted in human epithelial cellsfrom familial adenoma and carcinoma [42]. Floating cells ex-hibit a high degree of apoptosis (36299%), while only123%

Fig. 5. Reverse-phase HPLC profile of intracellular proteins in of spread cells undergo this form of cell death. Detachment fromLNCaP and PC-3 cells in the absence (A) or presence (B) of zinc. the extracellular matrix is known to trigger apoptosis of epithe-The cells were treated with 200µM (LNCaP) or 120 µM (PC-3) zinc lial and endothelial cells, but not fibroblasts [43, 44]. The datafor 3 days and subjected to HPLC according to methods given in

suggest that floating cells are prone not only to apoptosis butMaterials and Methods. In the zinc-treated cells, new peaks 226 in thealso necrosis.LNCaP cells, peaks 3 and 4 in the PC-3 cells were significantly elevated.

As the amounts of thymosinβ10 and thymosinβ4 increasein zinc-induced necrotic LNCaP and PC-3 cells, it would leadto disruption of the F-actin cytoskeleton, since theseβ-thymo-allothionein. Thymosinβ10, parathymosin and GAGE were par-sins are major G-actin-sequestering proteins. This would be ex-ticularly increased in LNCaP cells, and thymosinβ4, parathy-pected to lead to a rounding of cells and enhanced detachment.mosin and metallothionein were increased in PC-3 cells. LevelsIndeed, overexpression of the thymosinβ10 gene has been re-of prothymosinA were not elevated in either cell line. Amongported to cause susceptible cells to undergo apoptosis [29]. Thethese proteins, we focused on thymosinβ10 and thymosinβ4amounts of metallothionein and GAGE were also elevated. Met-because they are actin-sequestering proteins so presumablyallothionein is a major metal-inducible protein which is cysteine-interact with actin stress fibers [28, 29]. Both were increased 5 hrich and binds heavy metals such as zinc or cadmium [45].after exposure of LNCaP and PC-3 cells to zinc, at the timeGAGE, belonging to the melanoma-associated antigen family, iswhen cell death became prominent. Therefore,β-thymosinsan antigen on a melanoma cell line that is recognized by cyto-seems to linked to necrosis.toxic T lymphocytes [46]. It is expressed in a number of tumorsof different histological origins, but not in normal tissues exceptfor in testis. [46]. Nothing is known about the function of this

DISCUSSION protein. Although it is not known whether either or both of theseproteins are involved in apoptosis and necrosis, metallothioneinZinc has been shown to inhibit apoptosis in murine or humanis presumed to contribute to influx, retention or detoxificationthymocytes treated with dexamethasone orγ irradiation [19, 20],of zinc [45].and in various tumor cells exposed to tumor necrosis factor or

In the present study, we have shown that zinc inducesantitumor drugs [21, 30]. In many studies, zinc did not protectnecrosis associated with enhanced production of low molecular-against the initial stage of apoptosis but rather influenced latermass proteins in prostate carcinoma cells. The significance ofevents such as DNA fragmentation or chromosomal condensa-this evidence for the induction of necrosis, demonstrating mosttion [19, 21, 31]. Ca21/Mg21-dependent endonuclease, a candi-pronounced alterations of thymosinβ4 and thymosinβ10,date for production of DNA oligonucleosomal ladders, is inhib-GAGE, metallothionein and parathymosin remains to be clari-ited by zinc [16, 32]. Zinc has been shown to inhibit the activityfied. As suggested by Palomba et al. [38], if the same initialof caspase-3 [33], and more recently an endonuclease was founddamage can induce both apoptosis and necrosis, depending onto cause DNA fragmentation in apoptotic cells. The enzyme issubsequently occurring events the mechanisms underlying zinc-activated by caspase-3 [34].induced cell death may basically be the same for both types ofThe present study revealed that zinc may also inducecell death.necrosis in prostate carcinoma cells at the same or slightly lower

concentrations as applied in previous studies. Although there are This work was supported by a Grant-in-aid for Cancer Researchfrom the Ministry of Education, Science, Sports and Culture of Japan.many examples of inhibition, the metal has been reported to

770 Iguchi et al. (Eur. J. Biochem. 253)

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