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Abstract
Effective treatments for cancer are still needed, both for cancers that do not respond well to current therapeutics and for cancers that become resistant to available treatments. Herein we investigated the effect of a structure-selective d-amino acid peptide wrwycr that binds replication fork mimics and Holliday Junction (HJs) intermediates of homologous recombination (HR) in vitro, and inhibits their resolution by HJ-processing enzymes. We predicted that treating cells with HJ-binding compounds would lead to accumulation of DNA damage. As cells repair endogenous or exogenous DNA damage, collapsed replication forks and HJ intermediates will accumulate and serve as targets for the HJ-binding peptides. Inhibiting junction resolution will lead to further accumulation of DNA breaks, eventually resulting in amplification of the damage and causing cell death. Both peptide wrwycr and the related wrwyrggrywrw entered cancer cells and reduced cell survival in a dose- and time-dependent manner. Early markers for DNA damage, γH2AX foci and 53BP1 foci, increased with dose and/or time exposure to the peptides. DNA breaks persisted at least 48 h, and both checkpoint proteins Chk1 and Chk2 were activated. The passage of the cells from S to G2/M was blocked even after 72 h. Apoptosis, however, was not induced in either HeLa or PC3 cells. Based on colony-forming assays, about 35% peptide-induced cytotoxicity was irreversible. Finally, sublethal doses of peptide wrwycr (50–100 µM) in conjunction with sublethal doses of several DNA damaging agents (etoposide, doxorubicin, and HU) reduced cell survival at least additively and sometimes synergistically. Taken together, the results suggest that the peptides merit further investigation as proof-of-principle molecules for a new class of anti-cancer therapeutics, in particular in combination with other DNA damaging therapies.
Highlights
Cancer is a consortium of life threatening diseases with no definitive treatment
To investigate if peptide-induced cell death occurs via apoptosis, we examined caspase-3 activation as a late marker of apoptosis in PC3 cells treated with wrwycr over a time course of 12–48 h (Figure 3A)
Peptide wrwycr induces DNA-damage sensors To further characterize peptide wrwycr- or wrwyrggrywrwinduced DNA damage in cells, we examined the activation of H2AX, a variant of H2A histone protein activated by phosphorylation at Ser139 following ds DNA damage
Summary
Therapies targeted to individual cancer types may be the ideal treatment, this goal remains distant and general therapies are still useful. Many tumor cells bear mutations that reduce the efficacy of repair mechanisms and the responses to accumulating damage. Many of the effective anti-cancer therapies currently in use, including radiation, are DNA damaging agents [1,2,3,4] that generate double strand DNA breaks (DSBs). The resulting DNA damage overwhelms the DNA repair capacity of the tumor cells, leading to cell cycle arrest and cell death. The option of using DNA damaging agents that induce specific damage in conjunction with different DNA repair inhibitors is being explored to improve the cancer treatment [5,6,7,8]
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