Effect of a Holliday junction binding peptide, wrwycr, in human cells

Cancer is a life threatening disease characterized by uncontrolled growth and spread of abnormal cells. Extensive research in the field for the last fifty years led to the discovery of diverse classes of drugs called chemotherapeutics. The mode of action of many of these drugs exploits the fact that cancer cells replicate much faster than normal cells. Different classes of drugs target different proteins in the DNA replication and repair pathways and kill cells by interfering with these indispensable life processes. Two major obstacles to successful cancer chemotherapy are the significant cytotoxicity of the chemicals to normal cells and the high probability of resistance. Combination therapies, in which two or more different drugs are used, may help avoid both these problems.;In this study I investigated the effects of a hexapeptide molecule (sequence wrwycr) in cancer cells versus normal cells with particular emphasis on the cytotoxicity of the peptide, and DNA damage resulting from exposure. Peptide wrwycr was originally isolated in our laboratory in a combinatorial peptide library screen for inhibitors of phage lambda site-specific recombination. Subsequently it was discovered that the peptide stabilizes a transient DNA intermediate, the Holliday junction (HJ) generated in the process of recombination and disrupts the equilibrium of the reaction. Extensive in vitro and ex vivo characterization of the peptide has revealed that it belongs to the class of broad spectrum antibacterial that induces its bactericidal effect via DNA damage. Since HJs (and other branched DNA substrates) are produced in both prokaryotes and eukaryotes, I tested the effects of wrwycr on human cells by studying two cancer cell lines, U2OS, HeLa and a non-cancerous lung fibroblast, IMR-90 as a control. This is a preliminary step towards exploring the anticancer potential of this peptide.;Our results indicate that the effect of wrwycr in cultured human cells is cell-type dependent. Like natural, cationic, hydrophobic antibacterial peptides, wrwycr can enter cancer cells more efficiently than normal cells. U2OS, an osteosarcoma cell line is more sensitive to wrwycr-induced cytotoxicity than HeLa, a cervical cancer cell line. In U2OS cells, wrwycr produces a non-linear dose-dependent cytotoxicity partly via apoptosis. Concurrent administration of a sublethal dose of etoposide with wrwycr potentiates cell death in U2OS and induces the DNA double strand break response. More specifically, wrwycr interferes with recovery from intra-S arrest in synchronized U2OS cells in a linear, dose-dependent manner with no significant killing. This suggests that the exponential increase in cell death upon linear increase in wrwycr dose could be due to the interaction of peptide wrwycr with other cellular targets generating different forms of damage like ER stress, membrane rupture or mitochondrial damage.;HeLa cells show more signs of cell cycle arrest than cell death with wrwycr treatment. This becomes more evident upon simultaneous treatment with etoposide. Interestingly, IMR-90 cells are the most resistant to wrwycr lethality and DNA damage. This can be attributed only partly to lesser uptake inside these cells. Taken together, we have starting evidence that wrwycr has potential to be used as an anticancer drug by itself or in combination therapy. Much more basic and clinical research needs to be done before this long term goal can be achieved.