Session Speaker

A New Lytic Peptide for the Treatment of Cancer
Claudia Szczepanski, Rolf Bjerkvig, Olav Tenstad, Lene Nybø and Lars Prestegarden
Norway

Background: A current approach for cancer treatment is the development and use of cationic peptides. Such peptides, which are known to have significantly higher toxicity to bacteria than to normal mammalian cells, have also been shown to exhibit a broad spectrum of cytotoxic activity against cancer cells. Most membrane active peptides bind rapidly to the plasma membrane of cancer cells and disrupt it, leading to cell death. The exact mechanisms of action have not been fully understood yet. However, the outer part of the phospholipids within the membrane of cancer cells has a slightly more negative charge than in normal cells, which is mainly due to phosphatidylserine, which can make up 3-9% of the total membrane phospholipids of their outer leaflet. In this work we have designed 3 peptides that showed potential therapeutic efficacy against a number of cancer cell types.

Methods: Based on the fact that currently a high number of tumour suppressor genes is known, including their mode of action, we designed 96 novel peptides with potential tumour suppressor activity in silico, which were then screened in vitro on U87 glioma cells for biological activity by high throughput MTS assays. Three of these novel mimetic peptides showed considerable anti-tumour activity, whereby one peptide exhibited a particularly outstanding and durable tumour suppressor efficacy. This peptide was studied further with respect to its anti-tumour efficacy both in vitro and in vivo. To avoid proteolytic degradation, which is often the case for small proteins, the design and 3D structure of the peptide were modified without loosing biological activity. Using radioactive labelled peptide its distribution and pharmacokinetic profile was determined in vivo.

Results: Time lapse confocal microscopy revealed that the peptide induced cell death at a concentration > 10 μg/ml within minutes after application and continued to work progressively for an exposure time of 6h, resulting in 80 to 94% cell death at 35μg/ml, depending on the resp ective cancer type and cell line tested. Both electron and atomic force microscopy revealed holes in the plasma membrane with a subsequent degradation of cell membrane components. Using different viability assays on a number of tumour cell lines, including 5 osteosarcoma, 6 glioma and 4 breast cancer cell lines, the peptide showed severe cytolytic action at a concentration of > 15μg/ml. In contrast, a number of normal cell lines, were significantly less sensitive to treatment. The pharmacokinetic profile of the peptide, including its half- life and systemic toxicity as studied in vivo, the IC 50 doses for the respective cell lines at various time points were determined in vitro. Based on these results, 4T1 murine breast carcinomas were initiated in BALB/c mice. At a tumour size of 1cm2 the mice were treated by a single-shot local bolus injection of 600μg/ 100μl of peptide. This led to a significant reduction of tumour size within 2-3 days post injection and reduced tumour re-growth in the following 4 weeks. Histological evaluation revealed severe necrosis in the tumour treatment group.

Conclusions: A new stable lytic peptide with high anti-tumour efficacy was developed that shows resistance towards proteolytic degradation. Compared to a number of normal cell lines, the peptide showed significant toxic effects on a number of tumour cell types in vitro. Moreover, its pharmacological profile and distribution was delineated in vivo.