Intrinsic Sensitivity of Tumor Cells to Bleomycin as an Indicator of Tumor Response to Electrochemotherapy

Abstract

Electrochemotherapy (ECT) involves the use of locally applied electric pulses to increase delivery of chemotherapeutic drugs into cells in tissues. ECT with bleomycin (BLM) is a very effective local treatment, but different tumors have different response rates to ECT. The aim of our study was to compare the responsiveness of SA-1 and EAT tumors to BLM and ECT in vitro and in vivo, in order to find possible reasons for the observed difference in response rate. The difference in sensitivity to ECT in vitro between the SA-1 and EAT cells was 10-fold and was the same as the difference in sensitivity to chronic BLM exposure, as measured by tetrazolium-based colorimetric (MTT) assay. This difference in sensitivity between SA-1 and EAT to ECT was also reflected in tumor cure rate. A six-times lower dose of BLM was needed to obtain local tumor control in SA-1 than in EAT tumors. Therefore, we suggest that the difference in sensitivity to BLM and ECT predominantly reflects the difference in intrinsic sensitivity of the cells to BLM.Key words: Electrochemotherapy -Bleomycin -Electroporation -Experimental tumor -Mice Bleomycin (BLM) is a water-soluble glycopeptidic antibiotic with limited antitumor effectiveness, and is currently used only in combined chemotherapeutic schedules in the treatment of cancer.1) The major reason for its limited antitumor effectiveness is the hampered transport of BLM through the plasma membrane. However, once inside the cell, BLM is highly cytotoxic, inducing single and double strand DNA breaks.2) Different approaches have been tested to increase the antitumor effectiveness of BLM, [3][4][5] mainly with the aim of facilitating entry of the drug into the cells. Electropermeabilization (electroporation), i.e., a technique for introduction of molecules into cells by exposure of the cells to intense electrical pulses, proved to be very effective. Under specific conditions, electropermeabilization is a reversible process which does not impair cell viability. 6)Use of electropermeabilization to increase BLM uptake into the cells and consequently to increase the antitumor effectiveness was demonstrated in vitro, in vivo and also in clinical trials.7-21) This treatment was termed electrochemotherapy (ECT) and was also extended to other chemotherapeutic drugs. [22][23][24] Its application to cisplatin has proven to be very effective. 23,24) Preclinical studies of ECT with BLM were performed on a variety of transplantable and spontaneous tumors in immunocompetent and immunodeficient mice. [9][10][11][12][13][14][15][16][17][18] In all of these studies ECT proved to be an effective antitumor treatment, inducing a prolonged tumor growth delay compared to BLM treatment only. In addition, ECT results in tumor cures. In these preclinical studies a certain degree of variation in responsiveness to ECT was observed among the tumor types treated, as well as in the first clinical trials on basal cell carcinoma, malignant melanoma, head and neck squamous cell carcinoma and breast adenocarcinoma. 7-24)The aim of our...

Electroporation of human microvascular endothelial cells: evidence for an anti-vascular mechanism of electrochemotherapy

“…This is consistent with a report of relatively little antivascular action of cisplatin in vivo (Clements et al, 1999). In contrast, the present result for bleomycin indicates that HMEC-1 cells show an equal, or enhanced, sensitivity to that reported for tumour cells with the maximum value of ˜2 00-fold more sensitive than tumour cell lines (Table 2) (Hait et al, 1994;Dirix et al, 1997;Cemazar et al, 1998a). At present, only Dirix et al have reported on the sensitivity of two different endothelial cell lines to bleomycin (Dirix et al, 1997).…”

“…Increased drug uptake and potentiation of cytotoxicity has been demonstrated in vitro for several tumour cell lines treated with cisplatin, bleomycin, peplomycin and others, following exposure to electric pulses (Orlowski et al, 1988;Sersa et al, 1995;Kambe et al, 1996;Cemazar et al, 1998a;Kuriyama et al, 1999). When used in combination with application of electric pulses the drug dosage can be greatly reduced for equivalent or higher cytotoxic effect, compared to drug treatment alone (Orlowski et al, 1988;Sersa et al, 1995;Kambe et al, 1996;Cemazar et al, 1998a;Kuriyama et al, 1999). When electrochemotherapy is performed in vivo, the tumour perfusion and intravascular concentration of the drug are additional factors to be considered in achieving optimal tumour cytotoxicity.…”

“…Although the direct comparison of cell sensitivity to the combination of electroporation and bleomycin or cisplatin would require that the experiments would be done in the same study, according to those studies there is some evidence for endothelial cells being more sensitive to bleomycin than tumour cells. When electroporation was combined with bleomycin, cell cytotoxicity was assessed using MTT assay, while clonogenic assay was used in most of the studies testing combination of electroporation and cisplatin (Tables 1, 2) (Sersa et al, 1995;Kambe et al, 1996;Cemazar et al, 1998a).…”

Therapeutic Applications of Electroporation

“…For example, many immunizations are based on the delivery of protein drugs and are often done by injection. Current effort in the area of drug delivery include the development of targeted delivery in which the drug is only active in the target area of the body (for example, in cancerous tissues) and sustained release formulations in which the drug is released over a period of time in a controlled manner from a formulation Cemaẑar et al (1998); ; Serŝa et al (1993). This is achieved by combining phenomenon of electroporation with the application of drugs.…”

Radiosensitizing Effect of Electrochemotherapy in a Fractionated Radiation Regimen in Radiosensitive Murine Sarcoma and Radioresistant Adenocarcinoma Tumor Model

“…The principal mechanism of this therapy is increased permeabilization of cell membranes, resulting in increased binding of the chemotherapeutic drugs to DNA; binding to DNA was increased fourfold for bleomycin and twofold for cisplatin with appropriate electric-field distribution in the tumors (8)(9)(10)(11)(12)(13). Electrochemotherapy is already being used in clinical settings, and the results of published clinical trials have demonstrated that electrochemotherapy has excellent antitumor effects in different cutaneous and subcutaneous tumors, predominantly in palliative care, resulting in approximately 80% objective responses of the treated tumor nodules (14)(15)(16)(17)(18).…”