A Small Peptide of SPARC Reverses Chemoresistance Figure 4. The site of interaction between SPARC and caspase 8 is at the N-terminus of SPARC. Co-localization of the N-terminus of SPARC with caspase 8 was determined by co-immunoprecipitation (IP) studies using whole protein extracts. A) Only the recombinant N-terminus containing His-tagged fusion protein from MIP/SP-N cells co-IP with caspase 8 in a reciprocal fashion. B) Mutations in the N-terminus domain of SPARC (SP- Nmut1, SP-Nmut2) abolishes this interaction with caspase 8, and only the wild-type SP-N interacted with caspase 8. As additional controls, neither the wild type nor the mutant FS domain (SP-Fmut1, SP-Fmut2) interacted with caspase 8. doi:10.1371/journal.pone.0026390.g004 between caspase 8 and Bcl2 was abolished (denoted by ‘‘*’’, Fig. 5B). Based on these observations, we proceeded to determine the region of caspase 8 that interacts with Bcl2, by incubating chemotherapy-resistant cells with antibodies targeting either the N- or C-terminus of caspase 8, and observed that the interaction could only be abolished when antibodies blocked the N-terminus of caspase 8 (Fig. 5C, Figure S2C). In addition, this caspase 8-Bcl2 interaction appeared to localize to the membrane fraction, and in line with previous results, was eliminated following exposure to rSPARC (Fig. 5D). However, co-incubation with rSPARC in the presence of anti-SPARC antibodies again restored this caspase 8- Bcl2 interaction (Fig. 5D). These observations indicate that interactions involving Bcl2, caspase 8 and SPARC exist in cancer cells. In order to assess the significance of the interaction, the expression of Bcl2 was modulated by decreasing its expression in chemoresistant MIP/ 5FU cells. Only following a reduction in Bcl2 expression by siRNA transfection was there a decrease in cell viability in response to 5- FU (p = 0.04) or SPARC alone (p = 0.003), and these results were even more significant when the treatment included a combination of 5-FU with escalating concentrations of SPARC (Fig. 5E). These results support the biological importance of a Bcl2-caspase 8- SPARC interaction in modulating chemosensitivity. A synthetic peptide of the N-terminus of SPARC enhances apoptosis by inhibiting the interaction between caspase 8 and Bcl2 In order to confirm that the N-terminus of SPARC did indeed confer greater chemosensitivity by augmenting apoptosis by interfering with the interaction between caspase 8 and Bcl2, we generated a synthetic peptide of the N-terminus of SPARC (peptide-NT), tagged with a TAT peptide (YGRKKRRQRRR) to facilitate its intracellular uptake. Caspase 3/7 assays confirmed ,40–60% increase in apoptotic activity in not only sensitive MIP and RKO CRC cells, but as well the resistant MIP/5FU and RKO/5FU cells following incubation with peptide-NT and 5 mM 5-FU for 24 hrs (Fig. 6A). Also in line with previous observations: (1) cell viability also diminished significantly following exposure to this novel combination (Fig. 6B), and (2) immunoprecipitation studies revealed peptide-NT’s ability to disrupt the interaction between caspase 8 and Bcl2 (Fig. 6C). These exciting results not only support our findings that the apoptosis-conferring region of PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e26390