A natural product with potent anti-tumor activity against a variety of different tumor cell lines is present in media from short-term cultures of bonnethead shark (Sphyrna tiburo) epigonal cells. Preliminary data suggest that the inhibitory factor is a protein, tentatively termed Sphyrna tiburo epigonal protein-1 (STEP-1), and that it appears to act by blocking DNA synthesis and inducing apoptosis in proliferating cells. The broad, long-term Objectives of this project are to isolate the active factor and purify it to a form that can be produced through recombinant technology.
The specific aims of the current project are to characterize the molecular mechanism of action of STEP-1 and to purify bioactive STEP-1. Once purified, the potency and molecular mechanism of action of STEP-1 will be characterized, and the toxicity, pharmacokinetics, and the in-vivo anti-tumor activity will be evaluated. Purification of bioactive STEP-1 will be accomplished by employing various biochemical and immunoaffinity purification strategies that will utilize a novel high throughput flow cytometric screening technique termed Flow Cytometric High Content Screening (FC-HCS). Protein sequence information of purified STEP-1 will also be obtained. Methods used to determine the mechanism of action of STEP-1 will include FACS, immunoblot, Northern blot and other molecular analyses of apoptosis and cell cycle associated proteins. Toxicity and pharmacokinetics of purified STEP-1 protein will be evaluated using hematopoietic progenitors in CFU assays and testing in a battery of standard preclinical toxicity assays following standard ISO methods. Pharmacologic studies will be performed in mice injected with STEP-1. If these studies demonstrate that STEP-1 has potent anti-tumor activity with acceptable toxicity and pharmacokinetic properties, it will provide the justification to proceed with future studies involving aimed at producing recombinant STEP-1 for further testing as an anti-cancer agent in phase I clinical trials.
| Walsh, Catherine J; Luer, Carl A; Bodine, A B et al. (2006) Elasmobranch immune cells as a source of novel tumor cell inhibitors: Implications for public health. Integr Comp Biol 46:1072-1081 |
