This study shows that electrochemotherapy with cisplatin reduces tumour blood flow instantaneously and persists for several days. This reduction in tumour blood flow was reflected in reduced tumour oxygenation recovering to the pretreatment level within two days. The time course of reduced tumour blood flow and tumour oxygenation correlated with time course of antitumour effectiveness, tumour necrosis and apoptosis induced by electrochemotherapy with cisplatin. Therefore, the data indicate that antitumour effectiveness of electrochemotherapy is not only due to increased cytotoxicity of cisplatin due to electroporation of tumour cells, but also due to anti-vascular effect of electrochemotherapy, which results in reduced tumour blood flow and oxygenation.
Increased cytotoxicity of the drugs by electroporation is the predominant mechanism involved in antitumour effectiveness of electrochemotherapy. This notion is supported by the data in vitro
demonstrating that exposure of tumour cells to electric pulses increases cytotoxicity of bleomycin several 100-fold, and as in this study, cisplatin cytotoxicity 10-fold (Jaroszeski et al, 2000
). Among several other known anti-neoplastic drugs that were tested, these two drugs were shown to be the best candidates for electrochemotherapy, because they are hydrophilic and have good antitumour effectiveness once their access to cytosol is facilitated by electroporation (Sersa et al, 1995
; Mir and Orlowski 1999
). Presumption, that increased cytotoxicity of the drugs is the main mechanism involved in antitumour effectiveness of electrochemotherapy, is supported by the data demonstrating that electroporation causes increased bleomycin as well as cisplatin accumulation in the tumours (Belehradek et al, 1994
; Cemazar et al, 1999
). Specifically, in SA-1 tumours, as used in this study, the amount of platinum bound to DNA and platinum content in the tumours treated by electrochemotherapy was approximately two times higher compared to cisplatin treatment alone (Cemazar et al, 1999
). All these data support the notion that electroporation either in vitro
or in vivo
is the predominant mechanism involved in antitumour effectiveness of electrochemotherapy.
In view of our previous study, demonstrating that electrochemotherapy with cisplatin induced more than a 20-fold increase in cell kill in SA-1 tumours, compared with cisplatin treatment alone, we proposed that, in addition to electroporation of tumour cells that induces approximately 10-fold increase in cytotoxicity in SA-1 cells in vitro
, other mechanism may be involved in antitumour effectiveness of electrochemotherapy (Cemazar et al, 1999
). Additional mechanisms involved in antitumour effectiveness of electrochemotherapy with cisplatin were proposed: immune response (Mir et al, 1992
; Sersa et al, 1997
), anti-vascular effect due to cytotoxic action on endothelial cells (Cemazar et al, 2001
) and tumour blood modifying effect of electrochemotherapy (Sersa et al, 1999b
Electroporation is a universal process, therefore application of electric pulses to solid tumours would be expected to increase permeability of cell membrane of all cells that are treated. Potential targets include endothelial cells lining the tumour vasculature. Damage to endothelial cells may lead to obstruction of blood flow and ischaemic death of tumour cells lining the obstructed blood vessel. This phenomenon, described as anti-vascular or vascular-targeted therapy, gained high interest and has been exploited in several studies (Chaplin et al, 1998
; Denekamp et al, 1983
). In the first step to confirm that electrochemotherapy has an anti-vascular effect, we determined in our previous study sensitivity of human microvascular endothelial cells (HMEC-1) to bleomycin and cisplatin, with and without in vitro
electroporation of cells (Cemazar et al, 2001
). HMEC-1 cells were moderately sensitive to continuous exposure to cisplatin, but showed greater sensitivity to bleomycin. However, electroporation of cells increased cytotoxicity by ~10-fold for cisplatin and ~5000-fold for bleomycin.
The above mentioned is supported by the observations that electrochemotherapy with bleomycin resulted in complete shut down of tumour blood flow observed by 12
h after the treatment, and correlated with antitumour effectiveness and the extent of tumour necrosis (Sersa et al, 1999b
). In the present study, the antitumour effectiveness of electrochemotherapy with cisplatin could also in part be ascribed to its anti-vascular effect. Electrochemotherapy induced significant reduction in tumour perfusion, stabilizing at 40% pretreatment level up to the 5th day after treatment. The data on contrast enhanced MRI support these observations, 24
h after the electrochemotherapy tumours were significantly less enhanced than after the treatment with cisplatin or electric pulses.
Reduced tumour blood flow is expected to result in lower oxygen tension. By using EPR oximetry we were able to follow the time course of oxygen tension in the tumours. Electrochemotherapy induced profound and long lasting pO2
reduction, which is expected to cause some additional cell kill in the tumours due to the prolonged hypoxia. The onset and time course of tumour oxygenation changes correlated with tumour blood flow reduction, and resulted in gradual increase in extent of tumour necrosis, that reached approximately 90% of the tumour area, three days after treatment. That coincided with the time when the tumours started to reduce in the size and ceased to grow. The tumours started to regrow only 7 days after the treatment. The resulting antitumour effect was 9.4 days tumour growth delay, which is significantly better than the effect of single treatments, cisplatin (0.5 days tumour growth delay) and application of electric pulses (0.8 days tumour growth delay). The effect was less pronounced than in electrochemotherapy with bleomycin, which is consistent with in vitro
data on endothelial cells sensitivity (Sersa et al, 1999b
; Cemazar et al, 2001
The proportion of apoptotic cells increased only after electrochemotherapy with cisplatin. Cisplatin is known to induce apoptotic cell death, therefore it can be speculated that electrochemotherapy, which increases cisplatin delivery into the cells, also stimulates apoptotic cell death (Meyn et al, 1995
; Jordan and Carmo-Foncesca, 2000
). In histological sections, apoptotic cells were observed in non-necrotic regions, however necrotic regions represented a major part in the sections of tumors treated with electrochemotherapy, demonstrating that necrosis is a dominant way of cell death induced by electrochemotherapy. In tumours treated with cisplatin only apoptosis was not observed in the histological sections and also not detected by use of ELISA test for determination of mono and oligonucleosomes in the cytoplasmic fraction of cell lysates. The cisplatin dose used in experiments was very low and is most probably the reason for the absence of apoptosis. However, further studies are needed to fully understand the mechanisms of cell death induced by electrochemotherapy with cisplatin. It could be that higher amounts of cisplatin in the cells combined with electroporation might more efficiently trigger signalling pathways that lead to cell death. When response of the tumours to electrochemotherapy using bleomycin or cisplatin is compared macroscopically, tumours after electrochemotherapy with cisplatin respond to the treatment much slower and usually there is no ulceration of the tumours.
In conclusion, this study provides data on physiological changes in the tumours, as a consequence of electrochemotherapy with cisplatin and correlates these effects with antitumour effectiveness and histological changes in the tumours. The results indicate that reduction in tumour blood flow and tumour oxygenation correlate with extensive tumour necrosis and induction of apoptosis of the tumours treated by electrochemotherapy with cisplatin. The data support the evidence that the antitumour effectiveness of electrochemotherapy is not only due to increased drug delivery to the cells, but also to anti-vascular effect of electrochemotherapy, which results in reduced tumour blood flow and oxygenation.