BASIC MECHANISMS OF ELECTROCHEMOTHERAPY
Maja Cemazar and Gregor Sersa
Institute of Oncology Ljubljana, Department of Experimental Oncology, Zaloska cesta 2,
SI-1000 Ljubljana, Slovenia
E-mail: mcemazar@onko-i.si; gsersa@onko-i.si
Abstract: Electrochemotherapy consists of chemotherapy followed by local application of electric pulses to the tumor to increase drug delivery into cells in tumors. Drug uptake can be increased by electroporation only for drugs having impeded transport through the plasma membrane. Among many drugs which have been tested so far, only cisplatin and bleomycin have found their way from preclinical testing to clinical trials. In vitro studies demonstrated a several-fold increase of their cytotoxicity by electroporation of cells. In vivo, electroporation of tumors after local or systemic administration of either of the drugs i.e. electrochemotherapy, proved to be an effective antitumor treatment. Electrochemotherapy studies using either bleomycin or cisplatin in several tumor models elaborated treatment parameters for effective local tumor control. In veterinary medicine, electrochemotherapy proved to be effective in primary tumors in cats, dogs and horses. In clinical studies, electrochemotherapy was performed on accessible tumor nodules of different malignancies in progressive disease. All clinical studies provided evidence that electrochemotherapy is an effective treatment for local tumor control in patients with different types of cancer.
INTRODUCTION
Treatments for cancer may be divided into different categories based on their goals and mode of action. Very often, the different types of treatment are used in combination, either simultaneously or sequentially. In general, cancer treatment includes three major treatment modalities: surgery and radiation, which are local treatment modalities and chemotherapy, which is a systemic treatment modality. Chemotherapy, a systemic treatment modality for cancer, is effective for drugs which readily cross the plasma membrane and are cytotoxic once they reach their intracellular targets. However, among the chemotherapeutic drugs which are very cytotoxic, there is some having hampered transport through the plasma membrane. These drugs are good candidates for electrochemotherapy. Electrochemotherapy is a local treatment combining chemotherapy and application of electric pulses to the tumor. In electrochemotherapy, the optimal antitumor effectiveness is achieved when
electric pulses are given at the time of the highest extracellular concentration of the hydrophilic chemotherapeutic drug, thereby increasing its transport through the plasma membrane towards the intracellular targets (1-4).
PRECLINICAL DATA
In vitro studies
Electroporation proved to be effective in facilitating transport of different molecules across the plasma membrane for different biochemical and pharmacological studies. However, when using chemotherapeutic drugs, this facilitated transport increases intracellular drug accumulation with the aim to increase their cytotoxicity. Since electroporation can facilitate drug transport through the cell membrane only for molecules which are poorly permeant or non-permeant, suitable candidates for electrochemotherapy are limited to those drugs that are hydrophilic and/or lack a transport system in the membrane. Several chemotherapeutic drugs were tested in vitro for potential application
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in combination with electroporation of cells. Among the tested drugs, only two were identified as potential candidates for electrochemotherapy of cancer patients. The first is bleomycin, which is hydrophilic and has very restricted transport through the cell membrane, but its cytotoxicity can be potentiated up to several 1000 times by electroporation of cells. A few hundred internalized molecules of bleomycin are sufficient to kill the cell. The second is cisplatin, whose transport through the cell membrane is also hampered. Early studies suggested that cisplatin is transported through the plasma membrane mainly by passive diffusion, while recent studies have demonstrated that transporters controlling intracellular copper homeostasis are significantly involved in influx (Ctr1) and efflux (ATP7A and ATP7B) of the cisplatin (5). Electroporation of the plasma membrane enables greater flux and accumulation of the drug in the cells, which results in an increase of cisplatin cytotoxicity by up to 80-fold (1-4). This promising preclinical data obtained in
Electric pulse generator
Electric pulses: 8 x 0.1 ms; 1300 V/cm,
1 Hz or 5 kHz
Tumor
drug injection
Figure 1. Protocol of electrochemotherapy of experimental tumors presented schematically. The drug is injected either intravenously or intratumorally at doses which do not usually exert an antitumor effect. After an interval which allows sufficient drug accumulation in the tumors, electric pulses are applied to the tumor either by plate or needle electrodes. The electrodes are placed in such a way that the whole tumor is encompassed between the electrodes, providing good electric field distribution in the tumors for optimal electroporation of cells in the tumors.
vitro on a number of different cell lines has paved the way for testing these two drugs in electrochemotherapy in vivo on different tumor models.
In vivo studies
Bleomycin and cisplatin were tested in an electrochemotherapy protocol in animal models in vivo (Figure 1). Extensive studies in different animal models with different types of tumors, either transplantable or spontaneous, were performed. The antitumor effectiveness of electrochemotherapy was demonstrated on tumors in mice, rats, hamsters, cats, dogs, horses and rabbits. Tumors treated by electrochemotherapy were either subcutaneous or located in muscle, brain or liver, being sarcomas, carcinomas, gliomas or malignant melanoma (1-4,6).
In these studies, different factors controlling antitumor effectiveness were determined:
•	The drugs can be given by different routes of administration, they can be injected either intravenously or intratumorally. The prerequisite is that, at the time of application of electric pulses to the tumor, a sufficient amount of drug is present in the tumor. Therefore, after intravenous drug administration into small laboratory animals ( for example 4 mg/kg of cisplatin or 0.5 mg/kg bleomycin), only a few minutes interval is needed to reach the maximal drug concentration in the tumors. After intratumoral administration, this interval is even shorter and the application of electric pulses has to follow the administration of the drug as soon as possible (within a minute) (1-4).
•	Good antitumor effectiveness may be achieved by good tissue electroporation. Electroporation of the plasma membrane is obtained if the cell is exposed to a sufficiently high electric field. This depends on the electric field distribution in the tissue which is controlled by the electrode geometry
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Figure 2. Example of good antitumor effectiveness in SA-1 tumors after electrochemotherapy with cisplatin. Cisplatin was given intravenously (4 mg/ kg), 3 min thereafter 8 electric pulses were applied to the tumor with plate electrodes. Electric pulses were applied in two directions; 4 pulses in one and the other 4 in the perpendicular direction. Eight days after the treatment good antitumor effectiveness of electrochemotherapy with cisplatin is evident, compared to the single treatments with cisplatin or electric pulses.
and tissue composition. The electric field distribution in the tissue and cell electroporation can be improved by rotating the electric field. Surface tumours can be effectively treated by plate electrodes, whereas appropriate electric field distribution in the deeper parts of the tumour is assured by using needle electrodes (7-9). • The antitumor effectiveness depends on the amplitude, number, frequency and duration of the electric pulses applied. Several studies in which parallel plate electrodes were used for surface tumors showed that amplitude over distance ratio above 1000 V/cm is needed for tumor electroporation, and that above 1500 V/cm, irreversible changes in the normal tissues adjacent to the tumor occur. So, the window for effective and safe electrochemotherapy is between 1000-1500 V/cm. In most studies, the amplitude over distance ratio of 1300 V/cm induced good antitumor effectiveness without suboptimal electroporation of the tissue or
damage to the tissue due to irreversible cell permeabilisation (8). For other types of electrodes, the electric field distribution and thus, also the necessary amplitude of electric pulses, need to be determined by numerical calculations. Repetition frequencies of the pulses for electrochemotherapy are either 1 Hz or 5 kHz with equal effect if the concentration of drug present in the tumor is high enough. The minimal number of pulses used is 4; most studies use 8 electric pulses of 100 ps (1,4,8,10-12).
All the experiments conducted in vivo in animals provided sufficient data to demonstrate that electrochemotherapy with either bleomycin or cisplatin is effective in the treatment of solid tumors, using drug concentrations which have no or minimal antitumor effect without application of electric pulses. A single treatment by electrochemotherapy already induces partial or complete regression of tumors, whereas treatment with bleomycin or cisplatin alone or application of electric pulses alone has no or minimal antitumor effect (Figure 2).
Mechanisms of action
The principal mechanism of electrochemotherapy is electroporation of cells in the tumors, which increases the drug effectiveness by enabling the drug to reach the intracellular target. This was demonstrated in studies which measured the intratumoral drug accumulation and the amount of drug bound to DNA. Basically, the amounts of bleomycin and cisplatin in the electroporated tumours were up to 2-4 fold higher than in those without application of electric pulses (1-4).
Besides membrane electroporation, which facilitates drug transport and its accumulation in the cell, other mechanisms that are involved in the antitumor effectiveness of electrochemotherapy were described. The application of electric pulses to tissues induces a transient, but reversible reduction of blood flow (13,14).
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Restoration of the blood flow in normal tissue is much faster than that in tumors (14,15). The vascular lock in the tumor induces drug entrapment in the tissue, providing more time for the drug to act.
Thecytotoxiceffectofelectrochemotherapy is not limited only to tumor cells in the tumors. Electrochemotherapy also acts on stromal cells, including endothelial cells in the lining of tumor blood vessels, which undergo cell death (16). Consequently, by vascular-disrupting action of electrochemotherapy, a cascade of tumor cell death occurs due to long-lasting hypoxia in the affected vessels. This represents yet another mechanism involved in the antitumor effectiveness of electrochemotherapy, i.e. a vascular-disrupting effect (17-19). This vascular-disrupting action of electrochemotherapy is important in clinical situations where haemorrhagic tumor nodules need to be treated (20).
A difference in the antitumor effectiveness of electrochemotherapy was observed between immunocompetent and immunodeficient experimental animals, indicating on involvement of the immune response in antitumor effectiveness (21). Due to massive tumor antigen shedding in organ ism s after electroch em otherapy, systemic immunity can be induced and also up-regulated by additional treatment with biological response modifiers like IL-2, GM-CSF and TNF-a (22-24).
To sum up, the electrochemotherapy protocol was optimized in preclinical studies in vitro and in vivo, and basic mechanisms were elucidated. In addition to the electroporation of cells, vascular lock leading to drug entrapment in tumors, a vascular- disrupting effect and involvement of the immune response were also demonstrated. Based on all this data, electrochemotherapy with bleomycin and cisplatin was promptly evaluated in clinical trials and is now in routine use in human and veterinary oncology.
PERSPECTIVES
Knowledge about the mechanisms involved in the antitumor effectiveness of electrochemotherapy opened new possibilities for the application of electric pulses or electrochemotherapy in the treatment of cancer.
The chemotherapeutic drugs which increase effectiveness of radiation therapy are radiosensitizing drugs. These include bleomycin and cisplatin. Recently, some new drugs and chemicals were used in combination with electric pulses in preclinical studies, such as Mitomycin C, Ruthenium compounds and Calcium. The results of the studies in mice shown positive effects (25-27). Since drug delivery induced by electroporation is site-specific, it could be used for tumor-specific delivery of radiosensitizing drugs. By increased radiosensitizing drug delivery into tumors and not in the surrounding normal tissue, the therapeutic index of tumor irradiation is increased. In our studies, we combined electrochemotherapy with bleomycin or cisplatin with radiotherapy and demonstrated a good potentiation of the sarcoma tumor radiation response: 1.9-fold for electrochemotherapy with bleomycin and 1.6- fold for electrochemotherapy with cisplatin (28,29). The radiosensitizing effect of electrochemotherapy with cisplatin was also demonstrated in breast cancer and with bleomycin in a fractionated radiation regime which makes this treatment potentially available also in the clinic (30,31).
The application of electric pulses was shown to modulate tumor blood flow. Both reduced blood flow and lowered partial oxygen pressure (pO2) in the tumors are consequences of the applied electric pulses (32). The reduced pO2 can activate bioreductive drugs to exhibit a cytotoxic effect on hypoxic cells (33). In well- oxygenated cells, the drug remains inactive. On the other hand, tumor hypoxia induced by application of electric pulses can improve therapeutic conditions for
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the use of hyperthermia since tumor cells are more sensitive to heat in sub-optimal physiological conditions (34). Electrochemotherapy is an effective cytoreductive treatment; however, its curative effect depends on the permeabilisation of possibly all cells in the tumour. Since permeabilisation of every single cell in the tumour is virtually impossible, electrochemotherapy could be combined with other cytoreductive treatments that should have a systemic component. This can be achieved by a combination of electrochemotherapy with electrotransfer of different therapeutic genes acting either locally or sistemically, such as p53, IL-2; GM-CSF or IL-12. The results of the studies demonstrate positive results, further supporting this concept (35-38).
Finally, electrochemotherapy with cisplatin or bleomycin is also successfully used in veterinary medicine. It was used to treat different tumors, such as mammary adenocarcinoma,	fibrosarcoma,
cutaneous mast cell tumor, hemangioma, hemangiosarcoma, perianal tumors, neurofibroma and sarcoids in dogs, cats, hamsters, rabbits and horses. Recent reports demonstrated successful treatment of different neoplasms in companion animals and sarcoids in horses either of electrochemotherapy alone or in combination with other treatment, mainly surgery (39-47). Hopefully, electrochemotherapy will be broadly used in veterinary medicine for the treatment of different malignancies, both in primary and metastatic disease. In conclusion, electroporation in electrochemotherapy has already been very well exploited; however, there are new biomedical applications of electroporation in cancer treatment that still need testing and development.
ACKNOWLEDGEMENT
This research was funded by research grants from Slovenian Research Agency
and was conducted in the scope of the EBAM European Associated Laboratory (LEA) and COST Action TD1104.
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