A Small Peptide of SPARC Reverses Chemoresistance Figure 6. Augmentation of apoptosis by peptide-NT. Sensitive MIP101 or RKO and 5-FU resistant MIP/5FU and RKO/5FU cells were incubated with peptide-NT 100 ng/ml +/2 5-FU 5 mM for 24 hrs and assayed for (A) caspase 3/7 levels, or (B) cell viability by MTT assays. (C) Immunoblots showing the ability of peptide-NT to interfere with the interaction between caspase 8 and Bcl2: MIP/5FU cells were incubated with peptide-NT or scramble control peptides 1 or 2 for 24 hrs; cell pellets were collected 24 hrs later for co-immunoprecipitation studies. doi:10.1371/journal.pone.0026390.g006 Discussion Apoptosis occurs as a result of a series of well coordinated events that classically involve either activation of the extrinsic or intrinsic pathways and members of the Bcl2 protein family. We and others have previously shown that high levels of SPARC, a matricellular protein known to influence cell growth and apoptosis, to be associated with increased apoptosis in ovarian, pancreatic and CRCs [4,6,7], and that its exogenous exposure in-vivo promotes greater tumor regression in CRCs that had become refractory to conventional chemotherapies [4]. Recently, we identified a potential mechanism that allows SPARC to augment the apoptotic cascade, enhancing the effect of cytotoxic agents used in the treatment of malignancies [8]: SPARC promotes the activation of the extrinsic pathway of apoptosis by interacting with the N- terminus of caspase 8, with subsequent involvement of the intrinsic pathway, via Bid, to enhance apoptosis [8]. As an extension to these earlier observations, the current study demonstrates that in cancer cells that have become resistant to chemotherapy, the N- terminus of caspase 8 interacts instead with Bcl2 to restrict apoptosis. However, this inhibitory effect on apoptosis is reversible in the presence of higher levels of SPARC, either following exogenous exposure to this protein or its forced endogenous over- expression. Importantly, we not only identify the site interacting with caspase 8 as the N-terminus domain of SPARC, but that a synthetic peptide of this region (51aa in length) is responsible for conferring SPARC’s apoptotic activity. In fact, our results demonstrate that tumor xenografts of cells over-expressing only the N-terminus domain of SPARC, and not others, experienced the most dramatic tumor regression in response to chemotherapy. SPARC is expressed in many different cell types [18] and plays a complex role in tumorigenesis. While some studies report a positive association between high SPARC and more aggressive tumors [19,20], several studies, including our own, support the view that it functions in part as a tumor suppressor in neuroblastomas, leukemias, colorectal, ovarian, pancreatic, lung, and breast cancers [3,4,6,7,21,22]. These divergent actions of SPARC are puzzling, but may be explained by differences in the biological activities of the various proteolytic products of SPARC. Specifically, previous studies have demonstrated that SPARC undergoes proteolysis by a variety of proteases, such as metalloproteinases (MMPs), elastases, cathepsins, and serine proteases [23]. These degradation products have differing biological activities [10]: for example, a small peptide containing only 20 amino acids (aa 21–40) of the N-terminus domain of SPARC had previously been shown to inhibit endothelial cell spreading and bFGF-induced cell migration [24]; and the native SPARC protein is also known for its ability to inhibit angiogenesis [10], yet release of a proteolytic product containing the Cu2+ binding sequence KGHK (lysine-glycine-histidine-lysine) opposes the activity of the full-length protein by instead, stimulating angiogenesis in-vitro and in-vivo [25,26]. Therefore, differences in tumor-specific protease expression may account for the differences in SPARC’s biological behavior in tumorigenesis and different cancers. This also helps explain our findings that the effect of the recombinant peptide of SPARC’s N-terminus domain on tumor PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e26390