Cellular Damage and Altered Carbohydrate
Expression in P815 Tumor Cells Induced by
Direct Electric Current: An InVitro Analysis
VenõÂcio F.Veiga,1yCarla Holandino,2,3yMarcio L. Rodrigues,1
Marcia A. M. Capella,3Salatiel Menezes,4and Celuta S. Alviano1*
1Instituto de Microbiologia Professor Paulo de Go¨es,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
2Departamento de Medicamentos-Faculdade de Farma¨cia,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
3Instituto de BiofõÂsica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, Brazil
4Institut de Recherche sur la Peau, Paris, France
Treatment with direct electric current (DC) can inhibit tumor growth in several systems. To evaluate the cellular reactions generated by this treatment, we stimulated mouse mastocytoma P815 cells with DC and examined their viability and ultrastructural characteristics, as well as the effect of DC on surface carbohydrate expression. DC treatment affected cell viability and caused marked altera-tions in vital structures of P815 cells. Alteraaltera-tions varied depending on the duration of stimulation and polarity of electrode. Anodic and cathodic treatments caused decrease in cell viability, although the latter was more effective in generating cell lysis. DC stimulation also induced changes such as membrane damage, alterations in cell shape and chromatin organization, mitochondrial swelling and condensation, cytoplasmic swelling, and matrix rarefaction. Stimulation of P815 cells without contact with electrodes produced no alterations, suggesting that this contact might be essential for the occurrence of the cellular modi®cations. DC treatment also altered the membrane distribution of anionic sites of P815 cells, as well as the surface carbohydrate exposition, involving a diminished binding of Concanavalin A to the cell surface after cathodic stimulation, and an increased binding of sialic acid- and fucose-speci®c lectins after anodic treatment. In this work we describe important cellular targets for the action of DC, which may contribute to the understanding of the mechanisms by which DC supresses several kinds of tumors. Bioelectromagnetics 21:597±607, 2000.
# 2000 Wiley-Liss, Inc.
Key words: electric stimulation; cancer treatment; in vitro tumor cell inhibition
INTRODUCTION
Direct electric current (DC) has been shown to induce a large variety of biological events, such as tendon healing [Nessler and Mass, 1987], treatment of nonunion fractures [Basset et al., 1964; Brighton et al., 1977; 1995], skin repair [Carley and Wainapel, 1985; Lee et al., 1993] and antimicrobial effects [Bolton et al., 1980; Chu et al., 1988]. Humphrey and Seal [1959] demonstrated that DC also inhibits the growth of experimental tumors in mice, which raised the possibility of the use of electric current for tumor treatment. Since then, a number of investigators reported the inhibition of tumor growth in animals and humans either by the application of direct electric current [Chou et al., 1997; Grif®n et al., 1994; NordenstroÈm, 1994a; Schauble et al., 1977] or of short intense electric pulses in association with
chemother-apeutic agents, a treatment known as electroche-motherapy [Jaroszeski et al., 1997; Mir et al., 1998; Ramirez et al., 1998]. Although treatment with DC has
ß2000 Wiley-Liss,Inc.
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Contract grant sponsors: Financiadora de Estudos e Projetos (FINEP); Conselho Nacional de Desenvolvimento Cientõ®co a TecnoloÂgico (CNPq); Programa de Apoio a NuÂcleos de ExceleÃncia (PRONEX).
yV.F. Veiga and C. Holandino have contributed equally to this work.
*Correspondence to: VenõÂcio F. Veiga, Universidade Federal do Rio de Janeiro, Centro de CieÃncias da SauÂde, Instituto de Micro-biologia Professor Paulo de GoÂes, Ilha do FundaÄo, 21941-590, Rio de Janeiro, RJ, Brazil. E-mail: immgceu@microbio.ufrj.br Received 23 March 1999; Final revision received 2 February 2000
been shown to be ef®cient as a therapy for local cancer [NordenstroÈm, 1994b; Xin et al., 1997; Kuanhong, 1994], little is known about the cellular targets of DC, and a detailed and reproducible methodology has not been established.
Kulsh [1997] hypothesized that the primary mechanism that could explain tumor inhibition caused by electrotherapy would involve the inhibition of ribonucleotide reductase (RR), an enzyme responsible for converting building blocks of RNA into those of DNA in the cell division process. The molecule contains a stable tyrosyl free radical in its active site, which is essential for enzymatic activity [Graslund et al., 1982]. Such free radicals can be neutralized or destroyed by free-¯oating electrons, which are easily available from DC. According to this theory, para-meters such as polarity of electrode are inconse-quential, since free-¯oating electrons are generated independently of electrode polarity.
In this work, we show that several cellular alterations are detected in target tumor cells (P815) when they are subjected to anodic or cathodic stimulation, but not when they are treated with DC without contact with electrodes. Our results indicate that the nature of stimulation induces different cellular effects, which raises the possibility of the existence of alternative mechanisms by which DC would inhibit tumor growth in vitro.
MATERIAL AND METHODS Chemicals
Culture media reagents were purchased from Gibco BRL (New York, NY). Hydrochloric acid and sodium hydroxide were from Merck (Rio de Janeiro, RJ, Brazil). Phosphate buffered saline (PBS; 8.0 g/l NaCl, 0.2 g/l KCl, 2.17 g/l Na2HPO4.7H2O, 0.2 g/l
KH2PO4) and phosphate buffer (10.3 g/l KH2PO4,
20.3 g/l Na2HPO4.7H2O) components were from
Sigma (Richmond, CA), as well as ¯uorescein iso-thiocyanate-labeled lectins and the reagents used in electron microscopy.
Cell Culture
Mouse mastocytoma P815 cells were purchased from Rio de Janeiro's Cell Bank (CBRJ, catalogue number CR054). Cells were grown at 37C, in 25 cm2
culture ¯asks containing D-MEM medium supplemen-ted with 10% fetal bovine serum (FBS). pH was controlled by adding 3 g/l N-(2-hydroxyethyl)-piper-azine-N0-(2-ethanesulfonic acid) (HEPES), and 0.2 g/l
NaHCO3 to the medium composition, as previously
described [Freshney, 1994]. The initial inoculum was
5 104 cells/ml, and cells were subcultured every 2
days and maintained in the log phase of growth. DC Stimulation
P815 cells were collected by centrifugation, washed, and suspended (1.0 106 cells/ml) in two different treatment media, which were phosphate buffered saline (PBS, pH 7.4) or phosphate buffer (pH 7.4), both at 308 mOsm. These cellular suspen-sions were then distributed in a system of three acrylic chambers (2 ml of cell suspension per chamber), connected in series by ®lter-paper bridges, and ®tted with platinum electrodes in their extremities (Figure 1). In these conditions, an electric ®eld of approxi-mately 1 V/cm was generated. In this system, cell suspensions can be exposed directly to the anodic reactions (AC), cathodic reactions (CC), and electric current without contact with the electrodes, in the intermediary chamber (IC). Cell suspensions were treated with DC of 2 mA, which was a value of DC similar to those previously used for cancer treatment [David et al., 1985; Marino et al., 1986], for 0 (control), 2, 4, 6, 8, and 10 min, using a DC source (KLD Biossistemas Equipamentos EletroÃnicos Ltda, Brazil; model ETM 901). Treatments were perfomed at room temperature, which was monitored with a thermometer. The highest temperature achieved was 25.4C, after 10 min of stimulation. Alterations in pH
after DC treatments were monitored by measuring the cell suspension pH after each period of treatment. Cell Viability
To evaluate the effect of DC in cell lysis, the number of cells remaining after DC treatment was determined in a Neubauer chamber, and the number of nonviable, lysed cells was considered to be the
Fig. 1. Schematic representation ofthe acrylic chambersused for treatment of P815 cellswith DC.Chambersare connectedinseries by filter-paper bridges, and fittedwith platinum electrodesin their extremities. In this system, cell suspensions can be exposed directly to the anodic reactions (AC) or cathodic reactions (CC) or to electric current without contact with the electrodes, inthe inter-mediarychamber (IC).Internalvolume:3 cm3.
difference between the initial number of cells added to the chamber and the number of remaining cells. Viability of P815 cells which were not lysed by DC treatment was measured immediately after each period of stimulation by determining the number of trypan blue stained cells, which were also considered as nonviable, in a Neubauer chamber [Patterson, 1979]. To evaluate the in¯uence of pH on cell viability, cells were suspended in PBS and the pH adjusted to the same values obtained after DC treatment. The cells were incubated for 2±10 min at the same temperature used in DC treatment, and cell viability was deter-mined as described.
Electron Microscopy
Ultrastructural alterations in PBS-suspended P815 cells after DC treatment were evaluated by transmission electron microscopy. Cells were ®rst centrifuged and resuspended in a 2.5% glutaraldehyde solution. After 2 h of incubation in this solution, cells were washed in 0.1 M cacodylate buffer (pH 7.2) containing 0.2 M sucrose and post-®xed in 1% osmium tetroxide for 45 min. After ®xation, they were pre-included in 1.5% agar, dehydrated with ethanol and acetone, and embedded in Epon [Lewis and Knight, 1991]. Ultrathin sections were prepared with a diamond knife in an KLB ultramicrotome, collected on 300 mesh cooper grids, counterstained with uranil acetate and lead citrate, and examined under a Phillips EM-301 electron microscope. The membrane negative charge pro®le in DC-treated cells was examined by the ruthenium red method as described elsewhere [Luft, 1971a,b].
Lectin Binding
The cell surface carbohydrate expression of DC-treated and unDC-treated cells was analyzed by the binding of FITC-labeled lectins to P815 surface. Cells were ®xed in 4% paraformaldehyde in PBS for 1 h at room temperature. Fixed cells were washed twice in PBS and incubated sequentially for 30 min in PBS
contain-ing 150 mM NH4Cl and then in 1% bovine serum
albumin in PBS for 1 h. Cells were rinsed in PBS and then incubated in the presence of FITC±Concanavalin agglutinin (ConA), ±Limulus polyphemus agglutinin (LPA), ±Ulex I agglutinin (UEA), ±Wheat germ agglutinin (WGA) or±Peanut agglutinin (PNA) for 1 h at room temperature. After incubation, the cells were washed three times in PBS and used for analysis (n 5000) in an EPICS ELITE ¯ow cytometer (Coulter Electronics, Hialeah, Fla.) equipped with a 15 mW argon laser emitting at 488 nm. Control cells were ®rst analyzed to determine their auto¯uores-cence. To determine the optimum concentration of each lectin, P815 cells were previously titrated with nonlabeled lectins, and the minimum lectin concentra-tion required for agglutinaconcentra-tion was used. The speci®c sugar residues recognized by the lectins and their concentrations are shown in Table 1.
RESULTS Cell Viability
The in¯uence of DC treatment on P815 cell viability is shown in Figure 2A. Cells were treated in PBS or phosphate buffer (see buffer composition in Chemicals), and results of ®ve independent experi-ments are shown as means SD. Cathodic stimulation of P815 cells suspended in PBS (a) clearly induced cell lysis (P < 0.05) in a time-dependent process. The alterations in the percentage of the trypan blue±stained remaining cells (nonlysed) were not signi®cant. Cell suspension pH after this stimulation progressively augmented from 7.4 to 10.5. In contrast, the anodic stimulation (b) caused a time-dependent increase in the percentage of trypan blue±stained cells (P < 0.05), indicating that the cell viability was affected, although the occurrence of cell lysis was diminished if com-pared with the effects generated by the cathodic stimulation. Anodic stimulation caused a pH decrease from 7.4 to 6.5. Cells treated in the intermediary
TABLE 1. Lectin Speci®city and Optimum Concentrationafor Binding to P815 Cell Surface
Optimum concentration
Lectin Sugar speci®city (mg/ml)
ConA a-D-mannose, a-D-glucose 4
LPA N-acetylneuraminic acid 8
UEA L-fucose 50
WGA N-acetylneuraminic acid, chitobiose 1
PNA b-D-galactose (1-3)-D-N-acetylgalactosamine 50
aOptimum concentration of each lectin was determined by titration of P815 cells with nonlabeled
lectins. In ¯ow cytometry with FITC-labeled lectins the minimum lectin concentration required for cell agglutination was used.
Fig. 2. Effect of DC treatment (A) andmediumpH (B) onviabilityof P815 cells.The cellsuspensions were treatedwith DC (2 mA) for 0 (control),2,4,6,8, and10 min, usinga DC source.Black bars repre-sent the percentage of cells that were stained by trypan blue (nonviable, nonlysed cells), and white bars, nonviable, lysed cells. Alterations in the cell suspension pH after each treatment are shown in the bottom of each graphic. A: Cathodic stimulation of P815 cells suspended in PBS (a) or phos-phate buffer (d), anodic treatment of P815 cells suspended in PBS (b) or phosphos-phate buffer (e), and treatment of cells suspended in PBS (c) or phosphate buffer (f) without contact with the electrodes are shown. B: The influence of pH over cellviability is demonstrated by the artificialreproduction of thepHprofilesgeneratedincathodic(a)oranodic(b)stimulation.Resultsoffiveindependentexperi-ments, expressedasmeans SD, are shown.
chamber (c and f) did not evidence changes in suspension pH or cell viability in the buffer solutions tested.
To evaluate the in¯uence of the ionic composi-tion of the treatment medium, PBS was replaced by phosphate buffer (Figure 2A, d±f). In this model, no signi®cant alterations in cell viability or suspension pH were detected after DC treatment of P815 cells with cathodic or anodic stimulation.
To investigate whether changes in cell viability were due to alterations in cell suspension pH, the pH of PBS buffer was adjusted, with HCl or NaOH, to those obtained during treatment with DC (Figure 2B). The pH adjustment mimicking the cathodic and anodic treatments is shown in a and b, respectively. Extremely higher pHs (10.5) caused cell lysis (P < 0.05), while lower pHs did not in¯uence cell viability.
Electron Microscopy
Transmission electron microscopy was used to determine the ultrastructural cell changes induced by DC. In these experiments, P815 cells were treated for 6 or 8 min, ranges where the most signi®cant alterations in cell number and viability were observed. Periods of 10 min were not included in these experiments, because they induce complete cell lysis after cathodic stimulation. Untreated P815 cells evidenced typical cell surface ®lopodia, rare vacuole, eccentric large nucleus, and typical mitochondria (not shown).
Several cellular effects were observed after exposure of P815 mastocytes to DC, many of them being similar for both cathodic and anodic stimula-tions. After a 6 min cathodic treatment, several P815 cells presented intense cytoplasmic membrane damage, with marked alterations in cell shape (Figure 3). Many cells lost their typical cell surface ®lopodia. After the same period of anodic stimulation, cells approved essentially similar to those treated with cathodic stimulation (not shown), although presenting a less intensive cell membrane damage. After the 6 min exposition of P815 cells to cathodic or anodic elec-trodes, chromatin appeared condensed adjacent to the inner membrane of the nuclear envelope, disappearing from other parts of the nucleus (shown for cathodic stimulation in Figure 4A). For both anodic and cathodic-treated cells, P815 mitochondria became more electron-dense (Figure 4A), as a result of mito-chondrial condensation.
However, different effects were observed when cells were treated for longer periods. After an 8 min cathodic treatment, P815 mitochondria showed orga-nelle swelling and, in some cases, membrane rupture with unfolding of cristae (Figure 4B). Cells that were treated for the same period in the anodic compartment
presented condensed mitochondria, with a dense matrix and an expanded outer compartment including the envelope and the intracristal spaces (Figure 4C). The 8 min treatment also caused dramatic cell damage in cells treated in the cathodic chamber with intense occurrence of cell lysis (not shown), while the anodic treatment promoted bleb formation, loss of ®lopodia,
Fig. 3. Transmissionelectronmicrographsofthin sectionsof P815 cells after DC (cathodic) treatment.After 6 minof stimulation,P815 cells presented severe cytoplasmic membrane damage with markedalterationsin cell (arrows).Many cellslost their typicalcell surfacefilopodia.Scalebar,2 mm.
Fig. 4. Effect of DC treatment on P815 mitochondria and nucleus. After a 6 min stimulation, chromatin condensation was observed and mitochondria (indicated as M) became more electron-dense (Aöscale bar, 1 mm). Longer duration of DC treatment (8 min) caused mitochondrial swelling (indicated as MS) and membrane rupture with unfolding of the cristae after cathodic treatment (Bö scale bar, 0.5 mm), and expansion of the outer compartment, including the envelope and the intracristal spaces, after anodic treatment (CöScalebar,0.5 mm).
apparent cytoplasmic swelling, matrix rarefaction and chromatin margination and condensation (Figure 5). Cells treated in the intermediary chamber were similar to untreated cells after 6 or 8 min of DC treatment.
Prior studies suggest that cell membrane is a key target for the effects caused by electric treatment [Capella and Menezes, 1992; Holandino et al., 1998]. Cell membrane alterations induced by DC were evaluated by cationic ligand ruthenium red [Luft, 1971a,b], which binds to the cellular surface mem-brane, a highly negatively charged compartment. After incubation with ruthenium red dye untreated P815 cells showed a homogeneous distribution of the membrane aniogenic sites (not shown). After a 6 min cathodic stimulation, P815 cells lost a large number of anionogenic sites throughout the membrane (Figure 6A), while P815 cells that were treated for 6 min with anodic stimulation also presented loss of anionic sites, but restricted to speci®c regions (Figure 6B). Cells treated in the intermediary chamber behaved similarly to control cells (not shown).
Lectin Binding
The effects of DC treatment on surface carbohy-drate expression in P815 cells were analyzed following the interaction of these cells with FITC-conjugated lectins of different speci®cities (Table 1). In these experiments, cells were treated for 4 min. This period of treatment was chosen because longer periods of DC treatment induced signi®cant alterations of cell number and viability (Figure 2), hindering the use of ¯ow cyto¯uorimetry. Figure 7 shows that cathodic stimulation of P815 cells caused a signi®cantly
decreased binding of FITC-ConA to cell surface (P < 0.05), indicating a diminished exposition of mannose and/or glucose residues. In contrast, anodic stimulation generated a signi®cantly increased level of binding of FITC-LPA and -UEA (P < 0.05) to cell surface, indicating that sialic acid and fucose residues had their exposition increased.
DISCUSSION
In this work we evaluate the effects of in vitro treatment with DC on cell integrity and surface alterations of P815 tumoral cells. In previous studies, we have demonstrated that treatment of bacterial and tumoral cells in a single chamber can induce several cellular effects, such as alterations in cell surface composition and cell viability [Capella and Menezes, 1992; Holandino et al., 1998; 2000]. The experimental approach used in the present study allowed us to assess
Fig. 5. Transmission electron micrograph of thin sections of P815 cellsafter DC (anodic) treatment for 8 min.Anodic stimulation pro-moted bleb formation (arrows), loss of filopodia, apparent cyto-plasmic swelling, condensation of chromatin, and matrix rarefaction.Scalebar,1 mm. C.
Fig. 6. Transmissionelectronmicrographsofthin sectionsof P815 cellsafterincubationwithrutheniumred dye. A.6 mincathodic sti-mulation, causingloss of a large numberof anionic sites (arrows). Scalebar,0.5 mm. B.A 6 minanodicstimulationalsocausedlossof anionogenic sites, although limited to specific regions (arrows). Scalebar,0.5 mm.
relevant alterations induced by each independent electrolytic reaction.
Our results show that cathodic stimulation of P815 cells in PBS caused cell lysis simultaneous to pH increase of the reaction medium. In contrast, when cells were treated in phosphate bufferÐa stronger buffering system that does not allow pH variations as consequence of DC treatmentÐneither of these effects were observed. Superoxide, hydroxyl groups, and molecular hydrogen and oxygen are major products of cathodic electrochemical reactions occuring in aqueous solution. The production of hydroxyl groups in these reactions is probably a major contributing
factor in the pH increase, which, in turn, causes cell lysis, as shown in Figure 2B. Superoxide radicals can also in¯uence the occurrence of cell lysis, since they are generated by cathodic reactions [Forman and Fridovich, 1972] and are ef®cient agents in destructing target cells [Saran et al., 1998; Zhou and Petty, 1993]. We believe, however, that the expressive induction of cell lysis by the cathodic stimulation observed in our experimental system is mainly regarded as an effect of pH increase. These results are consistent with those presented by Morris et al. [1992].
Importantly, the same did not occur when P815 cells were treated with the anodic stimulation. The
Fig. 7. Effect of DC treatment on surface carbohydrate expression of P815 mastocytes.Cells were treated with DC, incubated in the presence of PBS (control), or FITC-conjugated ConA, UEA, LPA, WGA or PNA, and analyzed by flow cytofluorimetry. Autofluorescence inherent in unstained cells (&), and the fluorescence detected after cathodic ( ), anodic ( ) or intermediary (&) stimulation are shown. Fluorescence intensity (y-axis) is represented on a liner scale. Results of five indepen-dent experiments, expressedasmeans SD, are shown.
anodic treatment greatly diminished cell viability, which did not occur when P815 cells were incubated in solutions presenting the same pHs as those observed after exposure to the anodic electrode. This observa-tion excludes the possibility of pH-induced cell death caused by anodic reactions and strongly indicates its electrochemical nature. Replacement of PBS by phosphate buffer for treatment of P815 cells with DC, which means absence of chloride ions, resulted in levels of cell viability similar to untreated cells. Anodic electrochemical reactions occurring in the presence of chloride ions typically produce chlorine [Samuelsson and JoÈnsson, 1980], which is a powerful oxidant. We believe that chlorine production is one of the key factors in the decrease of cell viability after P815 anodic stimulation. The ability of oxidant agents to promote membrane injury has been described [Johnson et al., 1980], as well as the contribution of chlorine to tissue destruction [Samuelsson and JoÈns-son, 1980; Samuelsson et al., 1980].
Morris et al. [1992] have demonstrated that treatment of tumor in mice yields different results depending on whether anodic or cathodic stimulation is used. They have showed that cells in the region of the anode die prior to sacri®ce of the animals, but do not exhibit signs of disruption. In contrast, cathodal cells are disrupted, and the typical cellular features cannot be identi®ed. Interestingly, the clinical use of DC for the treatment of cancer has been shown to be more effective when the anodic electrode is inserted into the tumor [Schauble et al., 1977; David et al., 1985; Grif®n et al., 1994], and in addition the use of the cathode in the tumor treatment was speci®cally avoided in Nordenstrom's experiments [Nordenstrom, 1985], which described the possibility of disease aggravation promoted by cathodic stimulation.
The use of transmission electron microscopy made possible the analysis of the cellular effects caused by exposure of P815 mastocytes to cathodic and anodic electrodes. The major effects observed were chromatin condensation, cytoplasmic membrane damage, loss of cell surface ®lopodia, altered mito-chondria, matrix rarefaction, and bleb formation. In other mammalian systems, electron microscopic analysis indicated that cellular exposure to oxidizing agents caused similar effects to those generated by DC treatment, such as mitochondrial swelling and decreased cell viability [Robb and Connor, 1998]. These effects were directly associated with the consequent cell death. Additionally, P815 cells treated with both anodic and cathodic stimulations showed condensation and margination of chromatin, which are typical features of irreversible injury leading to cell death [Ghadially, 1982]. Previous studies have also
shown that changes in cell shape and ®lopodia may be sensitive indicators of cellular stress [Saxena et al., 1974; Bell et al., 1979]. For instance, Mollenhauer and co-workers [Mollenhauer et al., 1990] have demon-strated that loss of cell surface ®lopodia was the earliest cellular response of cultured mammalian cells to the exposition to toluene, a low molecular weight aromatic hydrocarbon capable of well-described toxic effects to mammalian cells.
The surface conformation of P815 cells was dramatically changed after electrode exposure. Ruthe-nium staining showed that cathodic treatment caused loss of anionogenic sites in parallel with the occur-rence of cell lysis. When P815 anodic-treated cells were analyzed by the same method, membrane damage and loss of anionogenic sites in speci®c regions were observed, but cell lysis was rarely detected. In previous studies, Capella and Menezes [1992] used the cationized ferritin technique and determination of the zeta potential to demonstrate that treatment of Escherichia coli with electricity also promoted loss of membrane negative charges. According to these authors and others [Zimmerman, 1982], the cell membrane is an important target of electric stimula-tions that funcstimula-tions as a dielectric, affecting the internal components of the cell because of the in¯uence of external electric variations. In the current work, we have found similar results after stimulation of P815 cells with DC.
An additional alteration observed at the cell surface of DC-treated P815 cells was the altered carbohydrate expression. Cathodic stimulation caused a decreased binding of FITC-ConA to the cell surface, indicating that i) the lectin accessibility to mannose and/or glucose residues was impaired or ii) the expression of ConA receptors was diminished. As reviewed by Poo [1981], the carbohydrate expression on the cell membrane can be modi®ed after exposure to an electric ®eld. Effects such as accumulation of membrane components at a local region of the cell membrane are expected and could result from a preferential degradation of the preexisting components in the membrane which would re¯ect internalization or shedding of surface components. Accordingly, it has been demonstrated that surface ConA receptors can be redistributed in living muscle cells after exposure to an electric ®eld [Poo and Robinson, 1977], which could modify the accessibility of an external ligand such as the ConA lectin. Additionally, it has been shown that the threshold ®eld required to induce diffusion of ConA receptors in the membrane of these cells is between 1.0 and 1.5 V/cm [Poo et al., 1978], which is very close to the electric ®eld strength used in this work. Previous studies by our group showed that
treatment of bacterial cells with DC also caused an altered expression of mannose residues [Holandino et al., 1998], but a mechanism that would explain this observation is still unknown.
Exposure of P815 cells to the positive electrode caused an augmented binding of FITC-LPA and -UEA to the cell surface, suggesting that the anodic treatment can alter sialic acid and fucose surface exposition. Since the period of stimulation used in our study (4 min) to evaluate the effect of DC on the P815 carbohydrate expression is probably not suf®cient to induce glycoprotein synthesis, this unexpected obser-vation may be a result of an incorporation of newly synthesized glycoproteins into the membrane, a phenomenon similar to those previously reviewed by Poo [1981]. According to this author and others [Gross, 1988; Jaffe, 1977], heterogeneous glycopro-teins of varying charge properties can be differently distributed at the cell surface after exposition to an electric ®eld varying from 1 to 12 V/cm [Poo et al., 1978; Zagyansky and Jard, 1979], with the duration of exposure varying from 3 to 24 h [reviewed by Poo, 1981]. Since sialic acids are negatively charged car-bohydrates, glycoconjugates presenting these acidic sugars are expected to present a negative net charge, which may be involved in the augmented exposure of sialic acid±containing receptors at the outer mem-brane after the anodic stimulation. These structures should naturally occur at the P815 cell surface, since glycoproteins with their oligosaccharide moiety pre-senting structures, such as, the fucosylated and/or sialylated molecules Lex3, Ley, sialyl Lex, and sialyl
Leaare frequently regarded as tumor-associated
anti-gens [Hakomori, 1989; Hakomori and Kannagi, 1983; Fukushi et al., 1985; Zenita et al., 1988; Magnani et al., 1982].
Common phenomena occurring in malignant tumors are changes in the composition and structure of cell surface glycoconjugates [Roth et al., 1996]. Carbohydrate-mediated interactions are involved in the adhesion of cancer cells to endothelial cells, which is correlated with metastasis [Kannagi, 1997]. Altered carbohydrate exposition of P815 cells after treatment with sublethal doses suggests the importance of determining the optimum dose for treatment of tumors with DC in order to eliminate the possibility of enhancement of carbohydrate-mediated metastasis promoted by DC stimulation.
The cellular mechanisms by which DC treatment causes cancer regression are largely unknown. DC has been shown to destroy a signi®cant amount of tumor tissue in large tumors and to prolong survival of mice [Morris et al., 1992]. The use of DC was also effective for the treatment of mouse and rat ®brosarcomas
[Chou et al., 1997]. Our work shows several cellular effects generated by in vitro treatment of tumor cells with DC. These effects vary from alterations in vital organelles to an altered cell surface organization, and these modi®cations may be linked to the occurrence of cell death. Stimulation of P815 cells in the inter-mediary chamber, where they have no contact with electrodes, consistently failed to produce cellular alterations in the experiments performed in this study. We demonstrated that the effects of DC stimulation in the viability of tumoral cells are related with electrode polarity, since different results are obtained if P815 cells are stimulated with anodic or cathodic reactions. Contrary to what has been hypothesized by other authors [Kulsh, 1997], the selection of speci®c positions and electrodes for the delivery of DC current may represent an alternative and ef®cient method for the treatment of localized tumors.
ACKNOWLEDGMENT
We thank Dr Maria Angelica Borges for revising this manuscript.
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