Date of Award

Fall 12-2013

Degree Type

Thesis

Degree Name

MS Biology

Department

Biology

Advisor

Allan Blake, Ph.D.

Committee Member

Constantine Bitsaktsis, Ph.D.

Committee Member

David Sabatino, Ph.D.

Committee Member

Jane Ko, Ph.D.

Keywords

cancer, cell therapy, GRP78, siRNA

Abstract

Cancer is a complex genetic disease that is driven by genetic mutations resulting in chronic, inappropriate cell proliferation. Many current cancer therapies lack specificity towards tumor tissues, ultimately leading to adverse side effects and limited clinical efficacy. Recently, selective cancer cell therapy has examined a well-characterized cell surface marker that is preferentially expressed on tumor cells. The over-expression of an endoplasmic reticulum chaperone protein, 78-kDa glucose-regulated-protein (GRP78), has been observed on the surface of cancer cells, but not on normal tissues. By selectively targeting GRP78 with short-interfering RNA (siRNA), potent GRP78 silencing is anticipated by the RNA interference (RNAi) pathway. Silencing GRP78 expression is expected to compromise cell signaling activity, ultimately resulting in cancer cell death. Towards this effect, I have shown that linear, branched and hyper-branched GRP78-targeting siRNA constructs elicited significant reductions in GRP78 protein expression (40 – 60%) in a cell culture model of human liver cancer, the HepG2 cell. The siRNA-hyper-branched A-1-2 construct produced 18% more knockdown when compared to its linear siRNA controls (siRNA-GRP78-1 + GRP78-2). This result highlights the synergistic effects of the novel A-1-2 construct, which combines two different GRP78-targeting siRNA motifs within a single molecular structure. A greater reduction in GRP78 expression resulted in greater apoptotic cell death. The novel siRNA motifs, specifically V-branched and A-1-2 hyper-branched, elicited the most cell death, 11 -12%, revealing that they may enhance cell apoptosis by conferring stable and potent siRNA structures. Effectively targeting cell surface GRP78 with siRNA ­provides a selective approach for compromising human cancer cell activity.

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