Abstract
Introduction and objectives: Bag1 (Bcl‐2 associated athanogene‐1) is a family of proteins conserved throughout evolution, promoting cell survival and inhibiting apoptosis under stressful conditions. They are found overexpressed in several types of tumors and associated with poor prognosis. Intriguingly, the nuclear isoform of the Bag‐1 proteins, Bag‐1L, also exhibits pro‐apoptotic properties through an unknown mechanism. In extensive deletion mutagenesis studies we identified sequences in the Bag‐1L protein that exert pro‐apoptotic action. The objective of the study is to characterize the mechanism of action of these sequences and to explore their use as candidate peptides for prostate cancer therapy.
Materials and Methods: In vitro inhibition of tumor cell growth was analyzed in clonogenic assay where prostate cells were transfected with plasmids expressing different Bag‐1L peptides. In vivo growth inhibition was analysed by the injection of prostate cells stably expressing the Bag‐1L peptides into the flank of athymic nude mice and analyzing their effect on tumor size in vivo. Histological analysis for apoptosis was carried out on the tumor samples. For analyzing the interaction partners of the Bag‐1L peptides, the peptides were generated as fusion proteins with glutathione S transferase (GST), immobilized on glutathione agarose beads and allowed to interact with lysates from prostate cells. Specifically bound proteins were then eluted and analysed by mass spectrometry and by Western blotting.
Results: Overexpression of a 68 amino acid sequence of Bag‐1L reduced the growth of 22Rv.1 prostate cancer cells but not of benign prostate hyperplasia cells in a clonogenic assay. Stable overexpression of this peptide in the 22Rv.1 prostate cancer cells also reduced cell growth in vivo in athymic nude mice. Histological analysis revealed massive apoptosis in the tumor cells expressing the peptide. Further deletion analysis of the 68 amino acid peptide showed that the N‐terminal 40 amino acids but not the C-terminal 29 amino acids are sufficient for the growth inhibitory action. Protein‐protein interaction studies using GST‐pull down assay demonstrated that the growth inhibitory function of the peptides correlated with their ability to bind glucose regulated proteins 75 and 78 (GRP75 and GRP78). GRP75 and GRP78 have been implicated in tumor formation through their action in the mitochondria and in the endoplasmic reticulum in the regulation of the unfolded protein response.
Conclusions: We have therefore identified Bag1‐L peptides which when expressed in prostate tumor cells reduce the growth in vitro as well as in a mouse model of tumor formation. The action of the peptides is to induce apoptosis and it is correlated with their ability to bind the molecular chaperones GRP75 and GRP78.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A216.
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