Direct Current Decreases Cell Viability but not
P-Glycoprotein Expression and Function in
Human Multidrug Resistant Leukemic Cells
Carla Holandino,1,2Venicio F.Veiga,3Marcio L. Rodrigues,5,6Marcelo M. Morales,2Marcia A. M. Capella,2,4and Celuta S. Alviano3*
1Departamento de Medicamentos-Faculdade de Farma¨cia, 2Instituto de Biofisica Carlos Chagas Filho, 3Instituto de Microbiologia Professor Paulo de Goes,
4Departamento de Bioquimica Medica, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, Brazil
5Disciplina de Biologia Celular, UNIFESP, SP-Brazil 6Disciplina de Microbiologia Me¨dica, Faculdade de Medicina,
Universidade Esta¨cio de Sa¨, RJ-Brazil
Inhibition of tumor growth induced by treatment with direct current (DC) has been reported in several systems. In the current work, the cellular effects generated by the DC treatment of the human leukemic K562 cell line and its vincristine-resistant derivative K562-Lucena 1 were analyzed by trypan blue staining and transmission electron microscopy. DC stimulation induced cell lysis, alterations in shape, membrane extraction or discontinuity, and intense vacuolization of some cells. In addition, treatment of K562 and K562-Lucena 1 cells caused a marked decrease in viability. Since multidrug resistance is a major factor contributing with failure of chemotherapy in many tumors, the expression and function of P-glycoprotein (P-gp) in K562-Lucena 1 cells were also studied. The expression of mdr1, the gene encoding P-gp, was analyzed by reverse transcription polymerase chain reaction, which showed that this gene was equally expressed in either treated or untreated cells. These results were con®rmed by ¯ow cytometry with a monoclonal anti P-gp antibody and the Rhodamine 123 extrusion method, which revealed that P-gp surface expression and function were unaltered after DC treatment. Our results suggest that DC treatment does not affect P-gp in human leukemic cells, but affects their viability by mechanisms that would involve clear cellular effects, but also additional targets, whose relevance in dc treated tumoral cells is currently discussed. Bioelectromagnetics 22:470±478, 2001. ß 2001 Wiley-Liss, Inc.
Key words: DC; electric stimulation; human leukemic cells; multidrug resistance; tumor growth inhibition
INTRODUCTION
It has been demonstrated that direct current (DC) inhibits the growth of experimental tumors in several systems, which raises the possibility of the use of electric current as an alternative therapy for cancer. Accordingly, several reports describe the inhibition of tumor growth in animals and humans by the applica-tion of DC [Humphrey and Seal, 1959; Schauble et al., 1977; David et al., 1985; Chudomel et al., 1989; Heiberg et al., 1991; Xin, 1993; Grif®n et al., 1994; Nordenstrom, 1994; Li et al., 1997; Chou et al., 1997; Xin et al., 1997a,b; Yen et al., 1999; Turler et al., 2000].
Few reports discussing the mechanisms by which DC would block cell proliferation are available in the current literature [Kulsh, 1997; Kurokawa et al., 1997].
In previous studies using the mouse mastocytoma P815 cells, our group described dc induced cellular effects, such as decreased viability and proliferation
ß2001Wiley-Liss,Inc.
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Contract grant sponsors: Financiadora de Estudos e Projetos (FINEP); Conselho Nacional de Desenvolvimento Cienti®co e Tecnologico (CNPq), Fund~ao de Amparo a Pesquisa no Estado do Rio de Janeiro (FAPERJ); and Programa de Apoio a Nucleos de Excel^encia (PRONEX).
*Correspondence to: Celuta S. Alviano, Universidade Federal do Rio de Janeiro, Centro de CieÃncias da Saude, Instituto de Microbiologia Professor Paulo de Goes, Ilha do Fund~ao, 21941-590, Rio de Janeiro, RJ, Brazil.
E-mail: [email protected]
Received for review 18 September 2000; Final revision received 9 January 2001
[Holandino et al., 2000], and changes in the cells, such as modi®ed membrane conformation, alterations in cell shape and chromatin organization, and mitochon-drial swelling and condensation [Veiga et al., 2000]. Additionally, the effects of DC treatment in human leukemic cell lines has been evaluated by Kurokawa et al. [1997], who described an apoptosis inducing activity of DC. Finally, tumor inhibition promoted by DC could also involve a decreased ribonucleotide reductase activity [Kulsh, 1997]. This enzyme, which is responsible for converting building blocks of RNA into those of DNA in the cell division process, has been shown to be linked with malignant transformation and tumor cell proliferation [Mau and Powis, 1992; Szek-eres et al., 1994; SzekSzek-eres et al., 1997], which makes it an attractive target for cancer therapy.
Multidrug resistance (MDR) is a major factor contributing to the failure of chemotherapy in many tumors. MDR phenomenon is directly associated with the overexpression of the mdr1 gene [Nooter and Herweijer, 1991], which encodes the surface tran-smembrane molecule P-glycoprotein (P-gp). P-gp, found in high levels in multidrug-resistant cells, func-tions as an energy dependent pump capable of trans-locating chemotherapeutic agents from the inner to the outer compartments of tumor cells, which allows them to accumulate less drug than the drug sensitive cells from which they are derived [Juliano and Ling, 1978; Gottesman and Pastan, 1988; Altenberg et al., 1994].
In this work, we analyzed the effects of DC treatment in human leukemic K562 cell line and its vincristine resistant derivative K562-Lucena 1. We report that DC stimulation affects essential targets in a segment of the cell population, as observed by trans-mission electron microscopy. Additional effects of DC stimulation are suggested, since cell viability and total cell number were signi®cantly decreased after electric treatment. In our experimental conditions, analysis of P-gp expression and function by using reverse trans-cription polymerase chain reaction (RT-PCR) and ¯ow cytometry revealed that this molecule is not affected, which excluded the possibility of an involvement of P-gp with the viability of K562-Lucena 1 cells promoted by DC treatment.
MATERIALS AND METHODS Cell Culture
The human leukemic K562 cell line and its vincristine-resistant derivative K562-Lucena 1 were used in this work. These cell lines were kindly provid-ed by Dr Vivian Rumjanek (Departamento de Bio-quimica MeÂdica, Universidade Federal do Rio de
Janeiro, Brazil). Cells were grown at 37C in 25 cm2
culture ¯asks containing Dulbecco's modi®ed Eagle's medium (D-MEM) medium supplemented with 10% fetal bovine serum (FBS). pH control was achieved by adding 3 g/liter N-(2-hydroxyethyl)-piperazine-N0
-(2-ethanesulfonic acid) (HEPES), and 0.2 g/liter NaHCO3
to the medium composition, as previously described [Freshney, 1994]. The initial inoculum was 5 104 cells/ml, which were subcultured every 2 (K562)
or 3 (K562-Lucena 1) days, as described elsewhere [Trindade et al., 1999], and maintained in log phase growth.
DC Stimulation
Human cells were collected by centrifugation, washed, and suspended (1.0 106 cells/ml) in
phos-phate buffered saline (PBS, pH 7.4) at 308 mOsm. The cellular suspensions 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 [Veiga et al., 2000]. In these conditions, an electric ®eld of approximately 1 V/cm was generated. In this system, cell suspensions can be exposed directly to the cathodic reactions (CC), anodic reactions (AC), 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 similar to those previously used [David et al., 1985; Marino et al., 1986; Holandino et al., 2000; Veiga et al., 2000], for 0 (control), 2,4,6,8, and 10 mins, using a dc source (KLD Biossistemas Equipamentos EletroÃnicos Ltda, Brazil Ðmodel ETM 901). Treatments were performed at room temperature, which was monitored with a thermometer. The highest temperature achieved was 25.4C, after 10 min of stimulation. Room
tempera-tures, were chosen based on previous experiments by our group, which produced results similar to those obtained when cells were treated at 37C (data not
shown). Cell Viability
To evaluate the effect of DC in cell lysis, the number of remaining cells after DC treatment was determined in a Neubauer chamber, and the number of nonviable, lysed cells were considered to be the difference between the initial number of cells added to the chamber and the number of remaining cells. Cell viability of human leukemic 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]. This method has been used to measure viability in
K562 [Trindade et al., 1999] and other cell lines [Capella et al., 2000; Veiga et al., 2000]. In the speci®c case of K562 cells, the trypan blue method was compared with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, which has been classically used to measure cell viability. Since very similar results were generated using the K562-Lucena 1 cell line (G. S. Trindade, personal commu-nication), the trypan blue assay was chosen. We also evaluated the effect of DC treatment on the total number of K562-Lucena 1 cells cultivated for 0, 24, 48, and 72 h after stimulation in D-MEM supplemen-ted with FBS. Leukemic cells (106) were suspended in
2 ml of PBS and treated for 6min with DC. Aliquots of 400 ml of the cell suspensions were taken, washed in PBS, suspended in 2 ml of culture media and added to a 24 well plate. Aliquots of 25 ml of the cell culture were then taken after 24 h intervals and the total number of cells determined in a Neubauer chamber. Electron Microscopy
Ultrastructural alterations in leukemic cells after dc treatment were evaluated by transmission electron microscopy (TEM). 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 preincluded in 1.5% agar, dehydrated with ethanol and acetone, and emb-edded in Epon [Lewis and Knight, 1991]. Ultrathin sections were prepared with a diamond knife in KLB ultramicrotome, collected in 300 mesh cooper grids, counterstained with uranyl acetate and lead citrate, and examined under a Zeiss 900 transmission electron microscope operating at 80 kV.
mdr1 Expression
The occurrence of possible alterations on mdr1 mRNA expression induced by a 6min DC treatment was analyzed by RT-PCR. Total RNA was extracted from K562-Lucena 1 cells using the Trizol Reagent (Gibco BRL, Grand Island, NY, USA), a one step phenol-guanidinium isothiocyanate based procedure [Chomczynski and Sacchi, 1987]. To eliminate con-tamination with genomic DNA, the isolated RNA was treated with RNAse-free DNAse I (1 U/ml) for 1 h at 37C. The total RNA concentration was determined
by measuring the absorbance of the preparation at 260 nm with a spectrophotometer (Hitachi model U-1100, Japan).
First strand complementary DNA (cDNA) synth-esis was performed in a 20 ml reaction containing 500 ng of total RNA, 50 U murine leukemia virus
reverse transcriptase, 20 U RNase inhibitor, 2.5 mM oligo (dT)16, 2 ml of 5 fold concentrated ®rst strand
buffer (250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2), 1 mM dNTP, and diethyl pyrocarbonate
(DEPC) treated water. The reaction was performed at 37C for 80 min and 99C for 5 min in water bath. The
negative control (reverse transcriptase replaced with DEPC treated water) was included with all reverse transcription reactions. The resultant cDNA was diluted in 20 ml of DEPC treated water and stored at 20C. PCR technique was used to amplify the
synthesized cDNA. The following solution was employed in PCR reaction: 0.2 mM/l of dNTP, 50 mM/l of KCl, 10 mM/l of Tris-Cl (pH 8.3), and 1.5 mM/l of MgCl2plus 2.5 U of thermostable DNA
polymerase (Taq polymerase; Gibco BRL, Grand Island, NY), and 0.2 mM/l of the b-actin and mdr-1 sense and antisense primers.
The primers used for PCR ampli®cation were designed based on the published nucleotide sequence of human mdr1 and b-actin [Ponte et al., 1984; Clen et al., 1990]. For mdr1, the sense primer (50
-CACACCTGGGCATCGTGT-30) is located in exon 26
and the antisense primer (50
-GCTGACGTGGCTT-CATCC-30) is located in exon 27, corresponding to
nucleotides 3332±3344 and 3597±3614, respectively, of human mdr1 sequence. For human b-actin, the sense primer (50-GTTGCTATCCAGGCTGTG-30) and the
antisense primer (50-CTGTACGCCAACACAGTG-30)
are located in the different exons, and they correspond to nucleotides 441±458 and 918±935, respectively, of the human b-actin sequence. Semiquantitative PCR was performed with 34 cycles of denaturation (94C,
1 min), annealing (51C, 1 min), and extension (72C,
1 min).
The PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining (0.5 mg/ml) under ultraviolet light. The gels were photographed with a camera using positive/negative ®lm (type 667; Polaroid, Cambridge, MA). The photographs were scanned and the densito-metric analysis made by the use of image analysis software (Sigma Gel version 1.1, 1995, Jandel Scienti®c). To control variations in RNA quality and RT ef®ciency between different samples, the mdr1 bands density were divided by the respective b-actin bands density and expressed in arbitrary units [Morales et al., 2000]. The possibility of occurrence of differe-ntial gene expression was statistically analyzed using the independent Student's t test (P<0.05).
The semiquantitative method of RT-PCR was validated in preliminary experiments. Firstly, the opti-mal PCR conditions that yielded a single band on agarose gel electrophoresis were determined for each
gene in the same reaction tube. Secondly, to deter-mine whether the method was semiquantitative, serial quantities of total RNA (62.5, 125, 250, 500, 1000, and 2000 ng) extracted from Lucena cells were used for RT-PCR ampli®cation for both genes in the same reaction tube. Thirdly, experiments were performed to determine the optimal number of PCR cycles that yielded PCR products in the linear phase of ampli®ca-tion. Finally, ensure that the reactions were consistent, PCR reactions were performed at least twice. Only one of this reaction was included for ®nal densitometric analysis, and the selection was arbitrary. All reaction included a negative control (cDNA replaced with DEPC treated water). The identity of the ampli®cation was con®rmed by determination of the molecular size on agarose gel electrophoresis with 100 bp DNA molecular marker (Gibco BRL, Grand Island, NY). P-gp Surface Exposition
To evaluate the in¯uence of DC treatment on the surface exposition of P-gp in K562-Lucena 1 cells, ¯ow cytometry with a mouse anti P-gp monoclonal antibody (Antibody JSB1-Kamiya Biomedical Co., Seattle, WA) was used. After 6min of DC treatment, cells were ®xed in 4% paraformaldehyde in PBS for 1 h at room temperature and washed twice in PBS. Washed cells were then incubated sequentially for 30 min in PBS containing 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 the P-gp antibody for 1 h at room temperature. After incubation, the cells were washed three times in PBS and then incubated in the presence of a ¯uorescein isothiocyanate labeled (FITC) antimouse antibody for 1 h at room temperature. Cells were again washed and used for analysis (n 5000) in an EPICS ELITE ¯ow cytometer (Coulter Electronics, Hialeah, FL) equipped with a 15 mW argon laser emitting at 488 nm. Control cells, which were not incubated in the presence of the P-gp antibody, were ®rst analyzed.
P-gp Function
To evaluate the effects of DC treatment on P-gp function, the Rhodamine 123 (Rh 123) extrusion method [Neyfalkh, 1988] was used. K562-Lucena 1 cells (106cells) were treated for 6min with DC, the
maximum period of treatment for which cell lysis was not signi®cant. PBS was replaced by 500 ml of D-MEM, followed by incubation of cells with 200 ng/ml of Rh 123 for 30 min at 37C. Cells were
then centrifuged, washed in cold PBS, and incubated again for 30 min at 37C in D-MEM. As a negative
control, K562-Lucena 1 cells were incubated in low temperature, conditions in which P-gp activity is
blocked in K562 cells [Denis-Gay et al., 1995]. Alternatively, DC treated cells were incubated for 24 h in D-MEM supplemented with FBS, HEPES, and NaHCO3, and assayed for Rh 123 as described above.
Cells were washed twice in PBS and analyzed in the EPICS ELITE ¯ow cytometer equipped with a 15 mW argon ion laser emitting at a 488 and 585 nm band ®lter.
RESULTS Cell Viability
The in¯uence of DC treatment on the viability of K562 cell line and its vincristine resistant derivative K562-Lucena 1 were analyzed as previously described [Veiga et al., 2000]. The cellular modi®cations induced by DC were also evaluated, by using TEM. For this analysis, the 6min stimulation was used, since this period generated the most signi®cant alterations in cell number and viability in others systems [Holandino et al., 2000; Veiga et al., 2000]. Figure 1 shows the results of ®ve independent experiments (means SD) obtained when K562-Lucena 1 were exposed to DC treatment. Cathodic stimulation induced signi®cant cell lysis (P <0.05) after periods of treatment higher than 4 min (Fig. 1A), with the maximum levels of lysis reached after the 10 min treatment. Alterations in the number of trypan blue-stained cells were not sig-ni®cant, if compared with untreated cells. The majority of the population presented no ultrastructural altera-tions, although some cells with severe alterations in shape (Fig. 1B), with complete membrane extraction and intense vacuolization, were seen. Anodic treat-ment of K562-Lucena 1 cells also induced lysis, but not so intensively as the cathodic treatment (Fig. 1C). A continuous increase of trypan blue-stained cells was observed, which indicates membrane damage. This observation was con®rmed by TEM, which revealed the occurrence of cells with membrane discontinuity and matrix rarefaction (Fig. 1D), effects that were very similar to those previously obtained with P815 cells [Veiga et al., 2000]. Cells that were treated in the intermediary chamber, which had not been directly exposed to electrodes, presented no signi®cant altera-tions (Fig. 1E, F), when compared with untreated cells (not shown). Cathodic, anodic, and intermediary treat-ments caused similar effects when the parental cell line K562 was used (not shown).
K562-Lucena 1 cells were stimulated with DC for 6min and subsequently cultivated for periods varying from 0 to 72 h. Comparison between the number of untreated and DC treated cells revealed that the total cell number was diminished after stimulation
of leukemic cells in the cathodic, anodic, or intermedi-ary chambers (Fig. 2). These results were observed immediately, although they were more accentuated after 72 h. This may suggest that DC treatment in¯uences the rates of cell division in the K562-Lucena 1 line. Interestingly, these results revealed that DC also affected cells treated in the IC, although they did not present ultrastructural alterations.
mdr1 Expression
To determinate whether DC modulates the mdr1 gene, its expression was analyzed by semiquantitative RT-PCR in control or dc treated cells (Fig. 3). In these experiments, RNA analysis was performed with K562-Lucena 1 cells immediately after electric treatment or cultivated for 24 h after DC stimulation, systems that
Fig. 1. Cellular effects presented by K562-Lucena 1 cells after DC treatment.To evaluate the influ-ence of DC onviability (A,C,E), cell suspensionswere treated with DC (2 mA) for 0 (control), 2,4,6,8, and10 min, using a DC source.The effects of DC on cellviabilityafter stimulation of K562-Lucena1 cellsin the cathodic (A), anodic (C), or intermediary (E) chambers are shown.Black bars represent the percentage of cellsthat were stained by trypan blue (nonviable, nonlysed cells) andwhite bars, nonviable, lysed cells. Results of five independent experiments (means SD) were statistically analyzed using the Student'st test (P <0.05).The ultrastructural features of representative DC trea-ted cells were also analyzed, with some cells showing complete membrane extraction andintense vacuolization aftercathodic treatment (B), andmembrane discontinuityandmatrixrarefaction after anodic stimulation (D), whereas cells that were treated in the intermediary chamber presented no significant alteration (F).V, vacuoles; M, mitochondria; N, nucleus.Scalebarsrepresent 2mm.
would be able to detect acute or succeeding effects. Expression of this gene was apparently identical in all systems, as demonstrated by a representative PCR experiment showed in Figure 3A. This observation was con®rmed (P<0.05) by densitometric analysis (Fig. 3B).
P-gp Surface Expression and Function
The mouse monoclonal JSB-1 P-gp antibody was used in ¯ow cytometry experiments to evaluate whether DC treatment would alter P-gp surface
Fig. 2. Effectofdctreatmentonthetotalnumberof K562-Lucena1 cells cultivated for periodsvarying from 0 to 72 h after stimulation. The totalnumberof cellstreatedinthe cathodic ( ), anodic ( ), or intermediary (&) chambers, as well as the number of untreated cells (&), are shown.Resultsare expressedasmeans SDof five independent experiments. Statistical analysis, which were per-formedusingthe Student'st test (P <0.05).
Fig. 3. RT-PCR determinationofmdr1expressionin K562-Lucena 1cells.PCR products (A) are essentially similar in untreated (con-trol) or treated cells proceeded from the cathodic (CC), anodic (AC), or intermediary (IC) chambers Densitometric analysis (B) as also shown. Compares control with dc treated cells, no signifi-cant alterations (P > 0.05, asinferred from analysis using the Stu-dent'st test), were observed.
Fig. 4. P-gpsurfaceexpressionin K562-Lucena1cells.Flow cyto- metrywithaP-gpantibodyshowedthatuntreated(control)ortrea-ted cells proceeded from the cathodic (CC), anodic (AC), or intermediary (IC) chambers comparably express P-gp on their surface. Similar results are observed when K562-Lucena 1 cells are analyzed immediately after electric treatment (A-0 h) or culti-vated for 24 hafterdc stimulation (B).
expression. For this purpose, cells were analyzed immediately after DC treatment or after a postincuba-tion of 24 h following stimulapostincuba-tion with DC, as described for the PCR analysis. Cells that were treated with DC and immediately analyzed by ¯ow cytometry presented a surface P-gp expression comparable to that of untreated cells (Fig. 4A). The same was observed for cells cultivated in D-MEM for 24 h after DC treatment (Fig. 4B), indicating that later effects on the surface expression of P-gp were not detected.
P-gp function was analyzed by the Rh 123 extr-usion method [Neyfalkh, 1988]. In this analysis, cells that present functional P-gp will extrude Rh 123, which ®nally generates non¯uorescent cells. Cells with an impaired P-gp function would remain stained and a shift of the peak corresponding to the generation of ¯uorescent cells would be observed. Cells that were incubated on ice, conditions in which P-gp activity is blocked in K562 resistant cells [Denis-Gay et al., 1995], signi®cantly retain rhodamine 123 (Fig. 5A, E). In contrast, rhodamine ef¯ux was not affected in cells stimulated in the cathodic, anodic, or intermediary chamber (Fig. 5B±D, F±H).
DISCUSSION
Growth inhibition of tumoral cells promoted by treatment with electric current has been described by a number of authors [Humphrey and Seal, 1959; Schauble et al., 1977; Grif®n et al., 1994; Holandino et al., 2000]. The use of electric current for tumor treatment can be performed either by the application of DC [Nordenstrom, 1994; Chou et al., 1997; Xin et al., 1997a; Yen et al., 1999] or of short intense electric pulses in association with chemotherapeutic agents, a treatment known as electrochemotherapy [Jaroszeski et al., 1997; Mir et al., 1998; Ramirez et al., 1998].
Multidrug resistance remains as one of the main obstacles for successful therapies of human malig-nancies. Tumor cells demonstrate a wide variety of mechanisms that allow them to survive in the presence of the cytotoxic drugs used for chemotherapy. These mechanism of course, diminishes the therapeutic effe-ctiveness of such treatment [Trindade et al., 2000]. In this context, an undesirable effect of DC treatment of multidrug resistant cells would be an upregulation of mdr genes and consequent enhancement of resistance, which would limit the use of this therapy. To evaluate the relationship between dc treatments and mdr1 regulation, we analyzed the expression and function of its product, P-gp. Our results indicate that, in all levels of analysis, P-gp is not in¯uenced by the action of DC, corroborating with its use for the treatment of cancer cells.
Previous results demonstrated that DC treatment can acutely alter the distribution of surface glycopro-teins [Veiga et al., 2000], which was not the case for P-gp in this work. Cultivation of K562-Lucena 1 cells after DC treatment revealed that P-gp expression was equally unaltered, which lead us to conclude that this molecule is really not susceptible to the action of DC. A related result was obtained after exposure of H9, a human T cell leukemia line, to an electric ®eld of 750 mV/cm, which inhibited the expected upregulation of mdr1 by treatment with 1-b-D -arabinofuranosylcy-tosine (AraC) in other system [Walter et al., 1997].
Mechanisms that would explain DC induced cancer regression are not well established, although some reports characterized possible targets that can be affected by DC. In a previous work, our group demonstrated the acute effects observed after treat-ment of P815 cells with DC, which included affected cell viability and marked alterations in vital structures [Holandino et al., 2000; Veiga et al., 2000]. As mentioned above, DC treatment also in¯uenced the surface distribution of carbohydrates in the P815 model, suggesting an altered glycoprotein pro®le me-diated by electric stimulation. Additionally, the ability
Fig. 5. P-gpfunctionality (A) Control.Autofluorescenceinherentin unstained K562-Lucena 1 cells. (B) Incubation of K562-Lucena 1 with Rh123 immediatelyafter DCtreatment (0 h) orcultivated after DC stimulation (24 h).Usingthe Rh123 extrusionmethod, cellsthat wereincubatedonice (negativecontrol) inthepresenceofthisdye became fluorescent (Aand E), asindicatedby the shift ofthe fluor-escence peak to the right. Contrarily, rhodamine efflux was not affected in cells stimulated in the cathodic (B, F), anodic (C, G), or intermediarychambers (D,H).
of electric ®elds of affecting the cell biochemistry and biology of leukemia cells has been described [Walter et al., 1997]. For instance, DC treatment induced apoptosis in leukemic cell lines [Kurokawa et al., 1997], suggesting the occurrence of an additional mechanism that could, at least in part, explain the antitumoral activity of DC.
In the current work, we show that DC stimulation induced cell lysis, alterations in cell shape, membrane extraction or discontinuity and intense vacuolization of some cells. As previously described by our group in another model [Veiga et al., 2000], cell lysis was more expressive after cathodic stimulation, a result that may be explained by the generation of superoxide radicals, which are commonly detected after cathodic reactions [Forman and Fridovich, 1972]. In contrast, anodic reactions promoted a gradual decrease in cell viability, as demonstrated by the augmented number of trypan blue stained cells.
Although some cells were affected in vital targets after cathodic or anodic stimulations, the majority of the population did not present the effects described above. The existence of additional targets for the antitumoral activity of DC could therefore be sug-gested, since the electric treatment caused a decrease in the total cell number of K562-Lucena 1 cells that were treated with DC and subsequently cultivated in D-MEM supplemented with FBS. These results are in accordance with the well described growth inhibition of tumoral cells promoted by dc [Chou et al., 1997; David et al., 1985]. Since the majority of the population analyzed in this experiment was apparently unaffected, we suggest the existence of mechanisms of tumor growth inhibition different from those described by microscopy analyses, although additional experi-mental information is needed to conclude it. Interest-ingly, cells that were not directly exposed to electrodes (intermediary chamber) were identical to untreated cells, except for the modi®ed total cell number pro®le. This observation may indicate that cellular alterations occur after exposition to an electric ¯ow, even in the absence of a direct contact of target cells with electrodes.
We still have not found the target for the action of dc in cells that did not present ultrastructural altera-tions, although a relationship between growth inhibi-tion of apparently unaltered cells with a ribonucleotide reductase (RR) de®ciency may occur. RR is the rate limiting enzyme in the de novo synthesis of deox-ynucleotide triphosphates [Kuo and Kinsella, 1998; Myette et al., 1998]. For this reason, RR plays a pivotal role in the DNA replication, which, as a consequence, makes the enzyme a key target for cancer chemother-apeutic agents [Szekeres et al., 1997]. This enzyme,
which is linked with malignant transformation and tumor cell proliferation [Szekeres et al., 1994], contains a stable tyrosyl free radical which is essential for its activity [Graslund et al., 1982; Mau and Powis, 1992]. Such free radicals are easily inactivated in the presence of electric current, making this enzymatic activity susceptible to inhibition by DC [Kulsh, 1997]. This would be an alternative mechanism that could explain the DC induced tumor regression which would not involve the ultrastructural alterations observed in other systems [Ghadially, 1982; Holandino et al., 2000; Veiga et al., 2000]. Although further biochem-ical studies are necessary to con®rm this theory, we believe that, as proposed by Kulsh [1997], a hypothesis involving the growth inhibition of cancer cells mediated by the inactivation of RR after DC treatment may provide an explanation for the ®ndings described here, and previously by other authors.
ACKNOWLEDGMENTS
We thank Dr Vivian Rumjanek for the cell lines and primary antibodies used in this work, Dr Pedro Persechini for the use of the ¯ow cyto¯uorometer, and FlaÂvio H. Paraguassu-Braga for the help with cell culture.
REFERENCES
Altenberg GA, Vanoye CG, Han ES, Deitmer JW, Reuss L. 1994. Relationships between Rhodamine 123 transport, cell volume, and ion-channel function of P-glycoprotein. J Biol Chem 269(10):7145±7149.
Capella LS, Alcantara JS, Moura-Neto V, Lopes AG, Capella MAM. 2000. Vanadate is toxic to adherent-growing multi-drug resistant cells. Tumor Biol 21:54±62.
Chomczynski P, Sacchi N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyonate-phenol-chloro-form extraction. Anal Biochem 162:156±159.
Chou CK, Mc Dougall JA, Ahn C, Vora N. 1997. Electrochemical treatment of mouse and rat ®brosarcomas with direct current. Bioelectromagnetics 18:14±24.
Chudomel V, Soucek J, Hruba A, Jerabek J, Schwarz J, Smetana K. 1989. Positive effect of direct current on cytotoxicity of human lymphocytes. Neoplasma 36(5):573±582.
Clen CJ, Clark D, Ueda K, Pastan I, Gottesman MM, Roninson IB. 1990. Genomic organization of the human multidrug resistance (MDR-1) gene and origin of P-glycoproteins. J Biol 265:506±514.
David SL, Absolom DR, Smith CR, Gams J, Herbert MA. 1985. Effect of low level direct current on in vivo tumor growth in hamsters. Cancer Res 45:5625±5631.
Denis-Gay M, Petit JM, Ratinaud MH. 1995. Rhodamine 123: is it an appropriate dye to study P-glycoprotein activity in adriamycin-resistant K562 cells? Anticancer Res 15(1): 121±126.
Forman HJ, Fridovich I. 1972. Electrolytic univalent reduction of oxygen in aqueous solution demonstrated with superoxide dismutase. Science 175:339.
Freshney RI. 1994. Culture of animal cells: a manual of basic technique. In New York: Wiley-Liss. 80±83.
Ghadially FN. 1982. Ultrastructural pathology of the cell and matrix. In London: 2nd Ed. 1±12.
Gottesman MM, Pastan I. 1988. The multidrug transporter, a double-edged sword. J Biol Chem 265:12163±12166. Graslund A, Ehrenberg A, Thelander L. 1982. Characterization of
the free-radical of mammalian ribonucleotide reductase. J Biol Chem 257:5711±5715.
Grif®n DT, Dodd NJ, Moore JV, Pullan BR, Taylor TV. 1994. The effects of low-level direct-current therapy on a preclinical mammary carcinoma: tumor regression and systemic biochemical sequel. Br J Cancer 69:875±878.
Heiberg E, Nalesnik WJ, Janney C. 1991. Effects of varying potential and electrolytic dosage in direct current treatment of tumors. Acta Radiol 32(2):174±177.
Holandino CQ, Veiga VF, Capella MAM, Menezes S and Alviano CS. 2000. Damage induction by direct electric current in tumoral target cells (P815). Indian J Exp Biol 38:554±558. Humphrey CE, Seal EH. 1959. Biophysical approach toward tumor
regression in mice. Science 130:388±389.
Jaroszeski MJ, Gilbert RA, Heller R. 1997. In vivo antitumor effects of electrochemotherapy in a hepatoma model. Biochim Biophys Acta 1334:15±18.
Juliano RL, Ling V. 1978. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 455:152±162.
Kulsh J. 1997. Targeting a key enzyme in cell growth: a novel therapy for cancer. Med Hypotheses 49:297±300.
Kuo ML, Kinsella TJ. 1998. Expression of ribonucleotide reductase after ionizing radiation in human cervical carcinoma cells. Cancer Res 58(10):2245±2252.
Kurokawa M, Sakagami H, Kokubu F, Noda H, Takeda M, Adachi M. 1997. Induction of apoptotic cell death by direct-current treatment in human leukemic cell lines. J Cancer Res Clin Oncol 123:370±376.
Lewis PR, Knight DP. 1991. Staining methods for sectioned material. In: Audrey M. Glauert, editor. Amsterdam, New York, Oxford: North-Holland Publishing Company. pp 2±47. Li K, Xin Y, Gu Y, Xu B, Fan D, Ni B. 1997. Effects of direct
current on dog liver: possible mechanisms for tumor electrochemical treatment. Bioelectromagnetics 18(1):2±7. Marino AA, Morris D, Arnold T. 1986. Electrical treatment of
Lewis lung carcinoma in mice. J Surg Res 41:198±201. Mau BL, Powis G. 1992. Inhibition of cellular thioredoxin
reduc-tase by diaziquone and doxorubicin. Relationship to the inhibition of cell proliferation and decreased ribonu-cleotide reductase activity. Biochem Pharmacol 43(7): 1621±1627.
Mir LM, Glass LF, Sersa G, Teissie J, Domenge C, Miklavcic D, Jaroszeski MJ, Orlowski S, Reintgen DS, Rudolf Z, Belehradek M, Gilbert R, Rols M-P, Belehradek J Jr, Bachaud JM, DeConti R, Stabuc B, Cemazar M, Coninx P, Heller R. 1998. Effective treatment of cutaneous and subcutaneous malignant tumours by electrochemotherapy. Br J Cancer 77:2336±2342.
Morales MM, Capella MAM, Sanches MV, Lopes AG, Guggino WB. 2000. Modulation of the mdr-1b gene in the kidney of rats subjected to dehydration or a high-salt diet. Eur J Physiol 439:356±362.
Myette MS, Elford HL, Chitambar CR. 1998. Interaction of gallium nitrate with other inhibitors of ribonucleotide reductase: effects on the proliferation of human leukemic cells. Cancer Lett 129(2):199±204.
Neyfalkh AA. 1988. Use of ¯uorescent dyes as molecular probes for the study of multidrug resistance. Exp Cell Res 174:168± 176.
Nooter K, Herweijer H. 1991. Multidrug resistance (mdr) genes in human cancer. Br J Cancer 63:663±669.
Nordestrom BWE. 1994. Electrostatic ®eld interference with cellular and tissue function, leading to dissolution of metastases that enhances the effect of chemotherapy. Eur J Surg Suppl 574:93±109.
Patterson MK Jr. 1979. Measurement of growth and viability of cells in culture. In: Jakoby WB, Pastan IH, editors. Methods in enzymology, Vol. 58. New York: Academic Press. pp 141±152.
Ponte P, Ng SY, Engel J, Gunning P, Kedes L. 1984. Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta-actin cDNA. Nucleic Acids Res 12(3):1687±1696.
Ramirez LH, Orlowski S, Bindoula G, Dzodic R, Ardouin P, Bognel C, Belehradek J Jr, Munck J-N, Mir LM. 1998. Electrochemotherapy on liver tumours in rabbits. Br J Cancer 77:2104±2111.
Schauble MK, Habal MB, Gullick HD. 1977. Inhibition of experimental tumour growth in hamsters by small direct currents. Arch Pathol Lab Med 101:249±297.
Szekeres T, Fritzer-Szekeres M, Elford HL. 1997. The ezyme ribonucleotide reductase: target for antitumor and anti-HIV therapy. Crit Ver Clin Lab Sci 34(6):503±528.
Szekeres T, Gharehbaghi K, Fritzer M, Woody M, Srivastava A, van't Riet B, Jayaram HN, Elford HL. 1994. Biochemical and antitumor activity of trimidox, a new inhibitor of ribonucleotide reductase. Cancer Chemother Pharmacol 34(1):63±66.
Trindade GS, Capella MAM, Capella LS, Affonso-Mitidieri OR, Rumjanek VM. 1999. Differences in sensitivity to UVC, UVB, and UVA radiation of a multidrug-resistant cell line overexpressing P-glycoprotein. Photochem Photobiol 69(6):694±699.
Trindade GS, Farias SLA, Rumjanek VM, Capella MAM. 2000. Methylene blue reverts multidrug resistance: sensitivity of multidrug resistant cells to this dye and its photodynamic action. Cancer Lett 151:161±167.
Turler A, Schaefer H, Schaefer N, Wagner M, Maintz D, Qiao JC, Hoelscher AH. 2000. Experimental low-level direct current therapy in liver metastases: in¯uence of polarity and current dose. Bioelectromagnetics 21(5):395±401.
Veiga VF, Holandino C, Rodrigues ML, Capella MAM, Menezes S, Alviano CS. 2000. Cellular damage and altered car-bohydrate expression in P815 tumor cells induced by direct electric current: an in vitro analysis. Bioelectromagnetics 21(8):597±607.
Walter RJ, Shtil AA, Roninson IB, Holian O. 1997. 60-Hz electric ®elds inhibit protein kinase C activity and multidrug resist-ance gene (MDR1) up-regulation. Radiat Res 147:369±375. Xin Y, Xue F, Ge B, Zhao F, Shi B, Zhang W. 1997a. Electro-chemical treatment of lung cancer. Bioelectromagnetics 18(1):8±13.
Xin Y, Xue F, Zhao F. 1997b. Effectiveness of electrochemical therapy in the treatment of lung cancers of middle and late stage. Chin Med J 110(5):379±383.
Xin YL. 1993. Traditional and Western medical treatment of 211 cases of late stage lung cancer. Chung Ku Chung Hsi I Chieh Ho Tsa Chih 13(3):135±213.
Yen Y, Li JR, Zou BS, Rojas F, Yu J, Chou CK. 1999. Electro-chemical treatment of human KB cells in vitro. Bioelec-tromagnetics 20(1):34±41.
