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Electrochemical Therapy to Treat Cancer: in vitro and in vivo
evaluations
Journal: Transactions on Biomedical Engineering Manuscript ID: Draft
Manuscript Type: Paper Date Submitted by the
Author: n/a
Complete List of Authors: Tello, Marcos; PUCRS, Electrical Engineering Holandino, Carla; UFRJ, Pharmacy
Teixeira, Cesar; UFRJ, Pharmacy Oliveira, Luciana; UFRGS, Veterinary
Parise, Orlando; Sirio Libanês Hospital, Oncology
Buzaid, Antônio; Beneficiência Portuguesa Hospital, Oncology Oliveira, Rosemari; UFRGS, Veterinary
Zanella, Rodrigo; UFRGS, Electrical Engineering Cardona, Augusto; PUCRS, Mathematics Bittencourt, Hélio; PUCRS, Mathematics Schuck, Jr., Adalberto; UFRGS, PPGEE - DELET Morales, Marcelo; UFRJ, Pharmacy
Veiga, Venicio; UFRJ, Pharmacy
Silva, Isaac; PUCRS, Electrical Engineering
TIPS: Cancer Treatment, Electrochemical Therapy, in vitro experiments, in vivo treatment
For Review Only
Abstract— Electrochemical Therapy (EChT) is a form of local treatment for cancer. EChT uses a low-level direct electric current (DC current) for tumor destruction. The DC current flowing through a pair of electrodes inserted in the tumor induces chemical changes into the cancer cells promoting a local destructive electrolysis. The aim of this paper is to present the evaluations and results attained by our research group from both the in vitro and in vivo applications of EChT to treat tumors. In
vitro experiments indicate: dramatic reduction in cancer cell viability especially around the anodic electrode, apoptosis in the anodic region and necrosis in the cathodic area. In vivo results indicate: EChT in combination with chemotherapeutic drugs is better than EChT and chemotherapy alone; EChT effectiveness Dose [Dose (Coulomb) = Current (Ampère) x time (second)] is in the range of 20.8 to 23.9 C/cm3; EChT applied to small tumors is more effective than the tumors of large dimensions; no systemic side effects were verified; and EChT is indicated for superficial, not-operable or chemotherapy-resistant tumors.
Index Terms—Cancer Treatment, Electrochemical Therapy, in
vitro experiments, in vivo treatment.
Manuscript received May 30, 2011.
M. Telló is with Pontific Catholic University of Rio Grande do Sul, Porto Alegre, RS, Brazil (corresponding author, phone: +55 51 33824421; fax: +55 51 33824992; e-mail: tello@pucrs.br).
C. Holandino, C. A. A. Teixeira, M. M. Morales and V. F. Veiga are with Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil (e-mail: cholandino@gmail.com).
L. O. Oliveira, R. T. Oliveira, R. Zanella and A. Schuck Jr. are with Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil (e-mail: luoliv@gmail.com).
O. Parise is with the Sirio Libanês Hospital, São Paulo, SP, Brazil (e-mail: oparise@uol.com.br).
A. C. Buzaid is with the Oncology Center, Hospital São José da Beneficência Portuguesa, São Paulo, SP, Brazil (e-mail: buzaidac@yahoo.com.br).
A. V. Cardona, H. R. Bittencourt and I. N. L. da Silva are with Pontific Catholic University of Rio Grande do Sul, Porto Alegre, RS, Brazil (e-mail: acardona@pucrs.br).
I. INTRODUCTION
ChT implies that tumor area is treated with direct current. The current flowing through electrodes into the cancer region starts an electrolytic process, which cause chemical changes in the cell environment [1]-[2]. Electrochemical products of electrolysis are formed destroying cancer cells by creating toxic products in the vicinity of the electrodes.
In the EChT process, anodic electrochemical reactions are characterized by the production of oxygen, chlorine and protons, while hydrogen and hydroxide ions are released at the cathode [3]. As a result of this process, anodic acidification and cathodic alcalinization occur, as well as electroosmotic movement of water from the anode to the cathode, which promotes dryness and hydration in the anode and cathode zones, respectively [4]. Necrosis is mainly induced in tissues exposed to alkaline environment, while apoptosis occurs in acidic environment. Reports on the anti-tumor effect of direct current dates to the end of the 19th century. However, Prof. Björn Erik Wilheim Nordenström is considered a pioneer in the treatment of tumors using direct current in patients. Prof. Nordenström, in the late of 1970s, started to treat primary lung cancers using EChT, where 20 patients with 26 lung tumors were treated. Regression was observed in 12 out of the 26 tumors and no signs of regrowth were detected after 2-5 years follow-up period [1].
Since 1987 the EChT has been used in China for the treatment of malignant and benign tumors, and so far it has been applied to more than 20,000 patients [3]. Despite the Chinese clinical data, the essential preclinical studies and clinical trials are missing [5]. At present in the world there are several research groups studying and applying the EChT, and an interesting EChT review can be found in [6].
It’s important to point out that the prevalent mechanism of cancer cell destruction by EChT is not yet completely understood. In an effort to understand the EChT mechanisms our research group initiated in 2000 the in vitro studies [7], evaluating the effects of the EChT treatment in HL60 cells, treated separately with the cathode and anode. The cathode treatment mostly caused the cell lysis; the anode treatment however caused irreversible damage to the membrane. Cell
Electrochemical Therapy to Treat Cancer: in
vitro
and in vivo evaluations
Marcos Telló, Carla Holandino, Cesar A. A. Teixeira, Luciana O. Oliveira, Orlando Parise, Antonio
C. Buzaid, Rosemari T. Oliveira, Rodrigo Zanella, Augusto V. Cardona, Hélio R. Bittencourt,
Adalberto Schuck Jr., Marcelo M. Morales, Venicio F. Veiga and Isaac N. L. da Silva
E
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analysis showed that necrosis is the main mechanism in celldeath. The anode treatment induced apoptosis in its typical pattern. Additionally, the research evaluated the cell reactions caused by the EChT in the mastocytoma P815 cells. The results show that the treatment affected the cell feasibility and produced deep alterations in the cells’ vital structures. Such alterations varied depending on the stimulus length and on the electrode’s polarity. The anode and cathode treatments caused a decrease in cell feasibility although the cathode treatment proved to be more effective in the creation of cell lysis. The EChT also induced such changes as membrane damage, alterations in cell shape and in the formation of chromatin, mitochondrial swelling and condensation, and cytoplasm swelling. In the next step, our team began in vivo EChT applications. Firstly, evaluations considering mice Ehrlich ascites tumor cells implanted into dorsal region of mice were done. In the mice experiments the results applying only direct current; only chemotherapeutic drugs and only direct current associated with chemotherapeutic drugs were considered separately. After mice experiments the EChT was applied to treat dogs and cats. Hitherto more than one hundred animals with different types of cancer were treated by our research group. We agree that in veterinary medicine, very few clinical trials with EChT were done worldwide so far. EChT experiments were conducted in animals that acquired the malignant tumor during their normal lives, without human interference. The aim of our study was to standardize experiments in animals, to establish an adequate dose-response relationship and to determine the response that each different type of tumor presents after EChT. We started in vivo EChT evaluations treating canine mammary neoplasms. Nowadays, we are treating different types of tumors in mammary gland, skin and subcutaneous tissues of pet dogs and cats.
The structure of this paper also presents the EChT in vitro comparison with the in vivo experimental results.
II. IN VITROEXPERIMENTS
Our group has previously introduced an experimental system which allows a study of the effects of cathodic and anodic reactions separately [7]. Additionally, the use of this model made it possible to analyze not only the electrolytic reactions, but also the effects of the DC current in the absence of the electrodes. Using this approach, the cellular suspensions were distributed in a 24-well plate (2 ml of cell suspension per chamber), connected in series by filter-paper bridges moisted with saline solution (for proper electric conduction), and with platinum electrodes fitted to the plate lid at both extremities. In this system, cell suspensions can be exposed directly and independently to the anodic or cathodic reactions or to DC current without contact with the electrodes (Fig. 1). The electrodes were introduced in the selected wells to guarantee a contact surface of 60 mm2 with the cell suspension. The distance between the electrodes was 8 cm and an electric field of approximately 2.5 V/cm was generated as a result. The
24-well plate was adapted to a DC current source whose intensity was monitored by an amperemeter and the cell suspensions were treated with DC current at different exposure intensity and time.
Fig 1: Schematic representation of the 24-well plate used for treatment of cells with direct electric current. Wells are connected in series by filter-paper bridges moisted with saline solution, and fitted with platinum electrodes in their extremities. In this system, cell suspensions can be exposed directly to the anodic flow in the anodic well (AW) or cathodic flow in the cathodic well (CW) or to direct electric current without contact with the electrodes, in the intermediary well (IW). The 24-well plate was adapted to a DC current source whose intensity was monitored by an amperemeter. Control cells are exposed to the same conditions, except for the use of DC current.
Several types of cancer were studied with this model, such as P815 (mouse mastocytoma) [7], K562 (human myelogenous leukemia cell line) and its multidrug-resistant variant Lucena-1 [8], HL-60 (human promyelocytic leukemia cell line) [9] and B16F10 (multidrug resistant melanoma cell) [10]. DC current produced a similar extension of immediate cell killing in all studies, with an average DL50 (Median Lethal Dose) of 6 minutes of treatment with 2.0 mA. No significant differences between the susceptibility of the different cell types to anodic or cathodic treatments were found. In addition, DC current treatment seemed to impair cell proliferation capability, since cell numbers could not be restored 48 h after the treatment [8]. Conversely, the electroionic flow alone was not able to produce cell death, which corroborates the key role of electrolytic products in DC current antitumoral activity.
However, marked morphological differences can be observed depending on the polarity of the flow applied. The cathodic treatment induces an intense cell swelling, culminating in extensive lysis, as can be observed by optical microscopy, and the evident decrease in the total cell number. On the other hand, the anodic treatment causes different damage, such as: cell shrinking, cell lysis, loss of cytoplasm, nuclear condensation and membrane blebbing. These characteristics are suggestive of apoptosis, a hypothesis that
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was strengthened by the observation of a typical pattern ofDNA fragmentation in treated HL60 cells [9].
Focusing on the promising proapoptotic effects of anodic treatment, a new study has recently started with a human lung adenocarcinoma cell line (A549), aiming to produce evidence data for further clinical trials with lung cancer patients. A new experimental system was validated in this study using 96-well plates. This model had a higher electrode surface contact and solution volume relationship than those of the 24-well plate. The 96-well plate system had 6mm2 and 0.2 mL of surface contact and solution volume, respectively. The comparison between both systems indicated that the A549 cell damage induced by the anodic flow was proportional to the electrode contact surface (Fig. 2 and 3). In clinical terms, these results call the attention for the importance of a controlled depth of insertion of electrodes for the standardization of the technique.
Fig. 2: A549 lung cell viability by the MTT method after anodic DC current treatment in the 96-well model with different intensities and exposure time.
Fig. 3: A549 lung cell viability by the MTT method after anodic DC current treatment in the 24-well model with 2.0 mA.
Cell viability changes induced by different anodic flow treatments combining time of exposure with DC current intensity were evaluated by MTT assay. This experiment indicated no difference in A549 cell viability after immediate anodic flow stimulation (Fig. 4, time 0, dark blue bars). However, after 24 hours in fresh culture medium cell viability
rates raised differently. As shown in Fig. 4, higher dose/lower treatment time interferes with cell viability increase rates, when compared to untreated cells (Fig. 4, time 24h, light blue bars). This result suggests that higher dose/lower exposure time regimens can be more effective in controlling tumor growth than lower dose/higher exposure time approaches.
Fig. 4: A549 cell viability by the MTT method. The cell viability was evaluated immediately (0h) and 24 hours after three different exposures to anodic flow.
In accordance with previous observations of other DC current-treated cell lines, the A549 lung cells presented important morphological changes, which are suggestive of apoptosis. As can be seen in Fig. 5, immediately after anodic treatment, line B cells showed membrane projections, also known as “blebs” (blue arrows), cytoplasmatic loss and loss of monolayer confluence. In Fig. 5, four hours after anodic treatment, most line C cells presented a rounded shape, with dramatic cytoplasmic loss and nuclear condensation (red arrows), morphological features of apoptotic bodies.
Fig. 5: A549 lung cell stained by Giemsa method. Line A – control untreated cells; Line B – cells immediately after 2 mA anodic treatment; Line C – cells four hours after the same treatment.
New studies are being carried out to consolidate the apoptosis induction by anodic treatment. Besides, these studies also focus on characterization of the biochemical events involved in this apoptosis triggering, such as caspase-3 activity, a key enzyme in this process.
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III. IN VIVOEXPERIMENTSThe EChT in vivo experiments conducted here specifically evaluated neoplasm cells implanted into dorsal region of mice, dogs and cats with spontaneous mammary and head & neck neoplasms. The EChT procedures and the main in vivo results will be presented.
EChT Procedures: All the animals were placed under
general anesthesia for EChT. With the animals anesthetized two or more needle electrodes [90% Platinum (Pt) + 10% Rhodium (Rh)], with 1.0 mm diameter and 80 mm length were inserted 1.0 to 3.0 cm apart in the cancerous region. The needle electrodes were connected to a DC current source. An electronic device was designed to operate as DC current source. The equipment developed was designed to deliver an electric current of a pre-determined dose (charge) in “Coulombs” [Dose in Coulomb = Current (Ampère) x time (second)]. All data of interest is transferred to a lap-top connected to the equipment, and could be visualized on the microcomputer screen. The current available values of the DC current were in the range up to 150 mA.
A. EChT in Mice
EChT evaluations considering mice Ehrlich ascites tumor cells implanted into dorsal region of mice were done. Fig. 6 shows mice EChT session [2].
Fig. 6. EChT session.
The mice were divided into control group and experimental group (N = 35). Thus, 7 groups were formed with 5 mice in each group. The mice in experimental group were divided in 6 subgroups: subgroup 1 received direct current alone (EChT); subgroup 2 received Bleomicyn alone (8 UI/m2), subgroup 3 received Cisplatin alone (0.8 mg/kg), subgroup 4 received Bleomicyn combined with direct current; subgroup 5 received Cisplatin combined with direct current and in subgroup 6 the electrodes polarity were changed during the treatment. When tumor mass reached approximately 15 mm in diameter it was exposed to direct current - 10 mA for 22 minutes using two Pt/Rh needle electrodes inserted into the tumor mass in parallel to each other with 10 mm apart. In the subgroup 6, after 11 minutes of the treatment beginning the electrodes polarity was changed and in this new electrode configuration
the direct current was applied during 11 minutes. Fig. 7a and 7b show the obtained results for subgroup number 5 (EChT + Cisplatin) and number 4 (EChT + Bleomicyn), respectively.
Fig. 7a. ■ Control group; ● Cisplatin; ○ EChT; □ EChT + Cisplatin [2].
Fig. 7 b. ■Control group; ● Bleomicyn; ○ EChT; □EChT + Bleomicyn [2].
In the mice experiments the Bleomicyn and Cisplatin effects were potentialized by direct current. Changing the electrodes polarity the anti tumoral effect of direct current is not observed (result not shown).
B. EChT in dogs and cats with spontaneous mammary
neoplasm
Our research group started the studies applying EChT to treat dogs and cats in October of 2001. EChT experiments using dogs and cats were approved by the Veterinary Hospital of the Federal University of Rio Grande do Sul. The EChT experiments were conducted in vivo (animals that acquired the malignant tumor during their lives that means spontaneous cancer). At the beginning of the EChT evaluations, six mongrel dogs with mammary cancer were used. After EChT treatments, three dogs were submitted to euthanasia. Necropsies were done to evaluate possible systemic effects caused by EChT. No alterations were found in adjacent or distant organs. Considering our good results with mongrel dogs, we extended the new anti-cancer therapy to pet dogs with mammary carcinoma. The animals accepted for EChT had recurrent, progressive disease, or the owner refused standard treatment, such as surgery or systemic chemotherapy. Selection of patients included clinical exams, thoracic
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radiographs, complete blood count, biochemical profile andcytopathological exams. Before treatment, the owners signed the informed consent. After the treatment the dogs were kept at the hospital for two to four hours for clinical observation then were sent home. After that, they were weekly examined to evaluate the treatment and possible side effects.
Fig. 8a and 8b show cytopathological exams of dog’s mammary gland before and after EChT, respectively.
Fig. 8a – Before EChT. Fig. 8b – After EChT. Before EChT there was a group of neoplastic cells (Fig. 8a) and after EChT the cytopathological exams revealed the existence of cellular debris and macrophage cells, as well as the absence of neoplastic cells (Fig. 8b).
A second stage of our experiment consisted of EChT of dogs and cats with different types of tumors in mammary, skin and subcutaneous tissues. The treatment was stopped when cytopathological exams of the treated region showed normal cells or absence of neoplastic cells.
Up to now our research group treated more than one hundred pet dogs and cats that had different types of cancer with EChT.
C. EChT in dogs and cats with spontaneous head and
neck neoplasm
Other in vivo EChT experiment was conducted. Specifically, a sequential and non comparative study, dogs and cats presenting a malignant spontaneous head and neck neoplasm were clinically evaluated, staged and treated by EChT or EChT associated with chemotherapy in a veterinary hospital. Survival analysis was assessed by Kaplan-Meier and compared by Log-rank test. The obtained results were: Twenty-eight animals (14 dogs – 9 male, 5 female; and 14 cats – 7 male, 7 female) were treated by EChT (15 cases) or EChT in combination with chemotherapy (13 cases – Bleomycin 10 cases, Doxorubicin, Cisplatin and Carboplatin 1 case each). Tumors were 16 cases of squamous cell carcinoma, 4 cases of melanoma, 2 cases of mast cell tumor, 1 case of epulis, mixosarcoma, osteosarcoma, fibrosarcoma, mucinous carcinoma and transmissible veneral tumor, with stages T1 = 10 cases, T2a = 6 cases, T2b = 2 cases, T3a = 5 cases, and T3b = 5 cases; N0 = 22 cases, N1 = 3 cases, N2 = 1 case, and N3 = 2 cases. The mean electrical charge/tumor volume was 38.50 C/cm3. Two animals died during the treatment. Fig. 9 shows an EChT head and neck treatment session.
Fig. 9 – EChT head & Neck Neoplasm.
Our data suggests that EChT and EChT in combination with chemotherapy are potentially effective treatment for head and neck tumors of dogs and cats and deserves further exploration (see Fig. 11).
IV. STATISTICAL ANALYSIS
Dogs and Cats with different types of neoplastic diseases except head and neck neoplasm were statistically evaluated: In
vivo EChT statistical analyzes were performed considering almost all treated animals. The total number of animals statistically evaluated (animals submitted to the EChT) was 79 (147 tumors), having different types of neoplastic diseases. Basically, the statistical interest was related to the treatment response as function of the applied Dose (C/cm3).
Receiver Operational Curve (ROC) was constructed to plot the true-positive rate (sensitivity) as a function of the false-positive rate (specificity) [11]. The statistical analyses applied ROC curves, true-positive means CR (Complete Response or absence of cancer cells) and false-positive means PR (Partial Response or existence of cancer cells) or NR (Non-Response to the EChT treatment).
A ROC curve describes the inherent predictive ability of the experiment. Each point along the curve corresponds to the sensitivity and specificity for the test threshold (see Fig. 10). ROC curve is a 1*1 unit (or 100*100%) diagram. In this diagram it was considered 1-specificity so that the upper left corner of the graph is the ideal point. The lengths of abscissa and ordinates were normalized to one and the maximum area of the plot was one (by definition). The ROC curve starts at P(0,0) and ends at P(1,1), and the diagonal line separates the area in two halves having 0.5 units each. So, the area under the ROC curve lies between 0.5 and 1. According to [11], a higher Area Under Curve (AUC) indicates better diagnostic test (the point P(0,1)) and represents the “perfect model”, where the true- and false-positive are correctly classified.
The SPSS software version 11.5 was used to perform the statistical analysis. Fig. 10 shows ROC curve relating EChT response to Dose imparted into cancer region.
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ROC CurveDiagonal segments are produced by ties.
1 - Specificity 1.00 .75 .50 .25 0.00 S e n s it iv it y 1.00 .75 .50 .25 0.00
Fig. 10. EChT ROC curve.
Fig. 10 allows us to identify the dose that maximizes the probability of Complete Response (CR). CR means absence of cancer cells. In order to determine the optimum dose from ROC curve the Square Euclidean Distance to the ideal point (0, 1) was calculated. According to the ROC curve model and the Square Euclidean Distance the “ideal/optimum” dose is situated between 20.8 and 23.9 C/cm3.
Dogs and Cats with head and neck neoplasm: see III IN
VIVO EXPERIMENTS, regarding dogs and cats with head &
neck neoplasm. By univariated analysis, factors impacting on survival were T stage (p = 0.04) and charge/tumor volume (p < 0.01). Survival analysis was assessed by Kaplan-Meier and compared by Log-rank test. Fig. 11 shows the obtained results.
Fig. 11. EChT + chemotherapy head and neck tumors rate C/cm3.
V. ECHTCURVES
ROC curve model indicated that the ideal/optimum dose is situated between 20.8 and 23.9 C/cm3. So, EChT curves were developed (curves for different times of EChT sessions). These curves permit to determine the direct current level that must be imparted into the cancer region function of tumor volume and the desired total dose to be applied. Fig. 12 shows one of the referred curves.
Fig. 12. EChT Curves
Curves of Fig. 12 were obtained from electrostatics equation and EChT Dose curves constitutes an important tool to treat cancer.
VI. ECHTELECTRODES EVALUATIONS
The EChT electrodes deterioration through use was evaluated by Scanning Electron Microscopy (SEM) and Electric Dispersion Scanning (EDS) techniques. Two needle electrodes were examined (old and new electrodes). Fig. 13a and 13b shows the SEM image from new and old electrodes, respectively.
Fig. 13a. New electrode. Fig. 13b. Old electrode.
Fig. 13. Images from SEM.
SEM and EDS indicated:
• The new electrode presented 9% of Rhodium and 91% of Platinum
• The old electrode presented ~0% of Rhodium
These results explain the fact that older electrodes lose the mechanical rigidity.
VII. RESULTS
EChT induced A549 lung cancer cell death in a dose dependent manner, corroborating results of previous works with other cell lines. The variation of electrode contact surface with the electrolytic medium resulted in dramatic effects on cell killing ability of DC current, which calls the attention for the importance of this parameter for EChT protocol
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development. Finally, important apoptotic-like morphologicalfeatures were observed in treated A549 cells, just as previously observed in HL60 cell line.
Response to EChT in dogs and cats included inflammation and necrosis of the neoplasms, with abundant macrophages in early response. In a few days after treatment, the nodules showed an increased volume due to edema, followed by development of foci of necrosi. Subsequently, the volume of the tumors reduced gradually. After destruction of the neoplastic mass, of the skin healed by second intention. Destruction of neoplastic cells was confirmed at the end of the treatment by cytopathological exams. Dogs with mammary tumor with positive lymph node had poor response to therapy and shorter survival time. In many of these cases the relief of pain was considered a good result. When treatment of early stage tumors was done, better results were obtained. In many patients (dogs and cats) EChT was successfully associated with either surgery or chemotherapy. In human beings EChT is still in process of evaluation.
VIII. CONCLUSIONS
The in vitro studies are an important tool for the controlled analysis of many aspects of EChT. These studies strongly suggested the occurrence of apoptosis as a possible mechanism of cell death induced by DC current, which is an important achievement in the characterization of EChT mechanisms of antitumoral activity.
Our results with dogs and cats indicate that EChT is effective in lower stage tumors, has low cost, can be repeated or associated with other cancer therapies and has high benefit/cost ratio for the patient. Its use as unique method of treatment or adjuvant to surgery or chemotherapy can improve quality of life and prolongate survival time for many cancer patients. Before treating dogs and cats with EChT some criteria should be observed. The patient should undergo appropriate staging tests (diagnosis of type of tumor, evaluation of lymph nodes, thoracic radiographs and abdominal ultrasounds). This information can assist the decision for appropriate treatment for each patient.
REFERENCES
[1] B. E. W. Nordenström, Biologically closed electric circuits – clinical,
experimental and theoretical evidence for an additional circulatory system. Sweden: Nordic Medical Publications, 1983.
[2] O. Parise, L. O. Oliveira, M. Telló, R. Zanella, R. T. Oliveira, C. C. F. Silva, M. A. Gioso and A C. Buzaid, “Treatment of spontaneous head and neck tumors of cats and dogs with electrolysis and electrochemotherapy”, in Multidisciplinary Head and Neck Cancer
Symposium (ASTRO, ASCO, AHNS), Rancho Mirage, CA, 2007. [3] M. Telló, A. Raizer, A. C. Buzaid, C. Domenge, G. A. D. Dias, H. D.
Almager, L. Oliveira, P. Farber, R. T. Oliveira and V. D. Silva, The use
of electrical current to treat cancer (O uso da corrente elétrica no tratamento do câncer). Porto Alegre, Brazil: Edipucrs, 2004. (in Portuguese)
[4] Y. Xin, F. Xue, B. Ge, F. Zhao, B. Shi and W. Zhang, “Electrochemical treatment of lung cancer”, Bioelectromagnetics, vol. 18(1), pp. 8-13, 1997.
[5] C. K. Chou et alli, “Electrochemical treatment of localized tumors with direct current”, in 2nd International Conference on Bioelectromagnetism, Melbourn, Australia, 1998, pp. 19-20.
[6] S. V. Cardoso, M. V. Caliari, M. C. F. Aguiar and G. D. Cassali, “Immunohistochemical staining of metallothionein in canine mammary tumors: better survival with higher expression”, Oncology Reports, vol. 12(6), pp. 1317-1321, 2004.
[7] V. F. Veiga, C. Holandino, M. L. Rodrigues, M. A. M. Capella, S. Menezes and C. S. Alviano, “Cellular damage and altered carbohydrate expression in P815 tumor cells induced by direct electric current: an in vitro analysis”, Bioelectromagnetics, vol. 21(8), pp. 597-607, 2000. [8] C. Holandino, V. F. Veiga, M. L. Rodrigues, M. M. Morales, M. A. M.
Capella and C. S. Alviano, “Direct current decreases cell viability but not P-glycoprotein expression and function in human multidrug resistant leukemic cells”, Bioelectromagnetics. vol. 22(7), pp. 470-478, 2001.
[9] V. F. Veiga, L. Nimrichter, C. A. A. Teixeira, et alli, “Exposure of human leukemic cells to direct electric current: generation of toxic compounds inducing cell death by different mechanisms”, Cell
Biochemistry and Biophysics, vol. 42(1), pp. 61-74, 2005.
[10] V. E. B. Campos, C. A. A. Teixeira, V. F. Veiga, E. Ricci Jr. and C. Holandino, “L-Tyrosine-loaded nanoparticles increase the antitumoral activity of direct electric current in a metastatic melanoma cell model”,
International Journal of Nanomedicine, vol. 5, pp. 961-971, 2010. [11] H. J. Koch and P. Hau, “ROC analysis as an additional method to
characterize time to event data”, Pathology and Oncology Research, vol. 11(1), pp. 50-52, 2005. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
