Book of abstract 2008

Electrogene therapy with p53 alone or in combination with
electrochemotherapy using cisplatin reduces the survival of human colon
carcinoma cells
Alenka Grošel1, Maja Čemažar2, Gregor Serša2
1Dept. of Clinical Laboratory Diagnostics, Institute of Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia;
2Dept. of Experimental Oncology, Institute of Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia
Electroporation is an established and successful method for delivering either
chemotherapeutic drugs, such as cisplatin and bleomycin (electrochemotherapy), or nucleic
acids (gene electrotransfer) into cells in vitro and in vivo. The tumour suppressor gene p53
plays a pivotal role in maintaining genome integrity and in regulating cell proliferation,
differentiation and apoptosis. In more than 50% of human cancer, p53 is mutated or its
function is otherwise impaired.
Our study aimed to evaluate electrogene therapy with p53 alone or combined with
electrochemotherapy using cisplatin in two human colon carcinoma cell lines, HT-29 and
LoVo, each with a different p53 status.
To evaluate the cytotoxic effect of combined treatment, the cells were treated with plasmid
DNA encoding wild type p53, cisplatin or electroporation, or combinations of these
treatments. We determined the status of the p53 gene in the cell lines immunocytochemically
and by using p53 gene sequencing. The permeabilization of cells after application of different
electric pulses was measured by the propidium iodide fluorescence, and the cytotoxicity
of the treatments by colony forming assay. Morphological evidence of apoptotic death
was obtained by observing changes in cells stained with an acridine orange/ethidium
bromide mixture up to 24 hours after treatment. The interaction between the treatments
was calculated assuming that they have independent mechanisms of action.
The results of our study show that electrogene therapy with p53 alone had a synergistic
cytotoxic effect in both colon carcinoma cell lines HT-29 (p53 mt, homozygous) and
LoVo (p53 wt) regardless of the p53 status. Electrogene therapy in combination with
electrochemotherapy also had a synergistic cytotoxic effect in both cell lines; The effect
was more pronounced in HT-29 cells, where survival of cells after electrogene therapy
combined with electrochemotherapy was significantly lower (P=0.001) than with
electrogene therapy with p53 alone. Cytological analysis of cells exposed to treatments
combined with cisplatin showed a greater proportion of apoptotic cell death.
In conclusion, our study showed that electrogene therapy with p53 alone or in combination
with electrochemotherapy synergistically reduced the survival in both human colon
carcinoma cell lines.
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