Abstract 3850: Production of recombinant lunasin peptides with enhanced anticancer activity using transient expression in tobacco

Epidemiological observations suggest a correlation between high levels of soybean product consumption and lowered incidence and mortality due to prostate, breast and colon cancer. Lunasin, a 43-44 amino acid peptide derived from soybean, has been implicated as a significant source of this anticancer activity. Lunasin contains nine consecutive aspartic acid residues at the C-terminus that bind to hypoacetlyated core histones, a minimal internal RGD cell adhesion motif and a helical region exhibiting structural homology to conserved sequences of chromatin binding proteins. Initial studies demonstrated that lunasin can prevent the transformation of mammalian cells by chemical carcinogens or viral oncogenes, and inhibits chemically-induced tumors in a mouse skin cancer model. These results suggest that lunasin may be useful as a cancer chemoprevention agent. More recent studies have demonstrated that lunasin can inhibit the proliferation of several established human cancer cell lines in vitro and in vivo, suggesting that lunasin may also be useful as a cancer therapeutic. Lunasin's anticancer effects are currently limited to specific cancer cell types and require 10-100 µM concentrations of peptide. We have now established a transient expression system based on the Tobacco Mosaic Virus vector, Geneware®, for large-scale production of modified forms of recombinant lunasin in tobacco. Our initial studies revealed that we could not detect any significant protein accumulation using constructs expressing lunasin peptides alone. We modified our strategy by expressing lunasin peptides as C-terminal fusions to green fluorescent protein (GFP) that includes a linker sequence containing a thrombin cleavage site. We have demonstrated that this system can produce GFP-lunasin at levels >100 mg/kg fresh weight tissue and demonstrated that the lunasin peptide can be recovered by proteolytic cleavage with thrombin. We have used this system to produce a modified form of lunasin that contains an N-terminal RGD domain sequence that preferentially binds to the αvβ6 integrin that is highly expressed in a number of epithelial-derived carcinomas. In vitro studies using cancer cell lines expressing the αvβ6 integrin demonstrate that the RGD-lunasin peptide is >10-fold more active in inhibiting cancer cell proliferation than the natural form of lunasin. Moreover, RGD-lunasin inhibits the proliferation of some cancer cell lines that are insensitive to 100 µM lunasin. These results demon