150 - Use of phenylarsine oxide-affinity chromatography to identify common cellular targets of cancer-active electrophiles

Abstract

Trivalent arsenicals, such as arsenic trioxide, and 3-bromopyruvate (3BP) are thiol-reactive electrophiles that have shown remarkable ability to selectively impair the viability of cancer cells both in vitro and in vivo. However, the broad reactivity of these substances, potentially forming adducts with many different proteins and altering numerous cellular pathways, has precluded a clear identification of the pivotal mechanisms underlying their preferential anticancer activities. Furthermore, the possibility that these chemically-distinctive substances may act in part by targeting the same protein(s) is unknown. The study described here employed Saccharomyces cerevisiae, which reproduces several metabolic characteristics of cancer cells, as a model to examine the cytotoxicity and protein binding partners of phenylarsine oxide (PAO), a representative trivalent arsenical, and 3BP. Both PAO and 3BP abolished cellular growth. In addition, up to 10% of the yeast proteome bound immobilized PAO in a dithiothreitol-sensitive manner. Among the most abundant PAO-binding proteins were glycolytic enzymes and the vacuolar-ATPase which play key roles in tumorigenesis. Moreover, binding of the yeast proteins to the immobilized PAO was inhibited by about 50% by prior incubation with 3BP demonstrating that a substantial fraction of protein can bind both PAO and 3BP. The proteins binding uniquely to PAO and those binding both PAO and 3BP, identified by LC-MS/MS, will be reported and discussed in the contexts of cellular viability and cancer.