A Small Peptide of SPARC Reverses Chemoresistance Figure 5. SPARC and Bcl2 both interact with caspase 8 with opposing effects. (A) In resistant MIP/5FU cells, caspase 8 interacts with Bcl2 and this is also observed in other resistant colorectal (RKO/5FU, RKO/CPT, RKO/CIS), pancreatic (MiaPaca/CPT) and breast cancer (MCF/CIS) cell lines (B). The Bcl2-caspase 8 interaction can be eliminated following exposure to rSPARC (100 ng/mL) (denoted as ‘‘*’’). C) The interaction between Bcl2- caspase 8 occurs at the N-terminus of caspase 8 as cells incubated with antibodies blocking the N-terminus of caspase 8 (N-term, 1.5–3 mg) prevented this Bcl2-caspase 8 interaction. D) Bcl2-caspase 8 interaction is abolished after exposure to 100 ng/mL rSPARC in resistant MIP/5FU and MiaPaca/CPT cells. (E) Chemoresistant MIP/5FU cells transfected with Bcl2 siRNA to reduce Bcl2 expression are now responsive to 1000 mM 5-FU (+), especially in combination with incremental concentrations of rSPARC (20–100 ng/ml). Results represent mean 6 s.e. (n = 3 independent studies). Student’s t-test, statistically significant when p,0.05. doi:10.1371/journal.pone.0026390.g005 SPARC resides within its N-terminus, but as well, that a synthetic peptide of this region is capable of chemosensitizing therapy- refractory cells. Mutations in the DED-domains of caspase 8 prevent interactions with Bcl2 and SPARC To identify the specific site within caspase 8 that interacts with Bcl2 or SPARC, mutations were introduced in three different regions of the N-terminus of caspase 8: the DEDI-domain (DEDIm), the putative binding region (PBm), and DEDII-domain (DEDIIm). Co-immunoprecipitation studies revealed that tran- sient overexpression of DEDIm and DEDIIm caspase 8 mutants interfered with the ability of Bcl2 to interact with caspase 8 in MIP/5FU and MiaPaca/CPT cells (Fig. 7A). In MIP/SP cells, overexpression of DEDIm-caspase 8 eliminated the interaction between SPARC-caspase 8, while mutations in the PB or DEDII- domains still allowed caspase 8 to interact with SPARC (Fig. 7B). These results suggest that the DEDI-domain of caspase 8 is the site of interaction with SPARC and Bcl2. Interestingly, we also noted that over-expression of caspase 8-mutants abolished the reduction in cell viability and increase in apopotosis that is normally conferred by caspase 8 in the presence of SPARC and 5-FU, in resistant cells (Fig. 7D–F). Similar observations were noted with chemosensitive MIP/SP and MIP/SP-N cells (Fig. 8A–C). Also, MIP/SP-Nmut cells failed to respond to 5-FU following transfection with either wild-type or mutant caspase 8 (Fig. 8D,E), thus supporting the N-terminus of SPARC as the site of interaction with caspase 8. The results reported in this study reveal how an interplay between SPARC, caspase 8 and Bcl2 may modulate apoptosis in response to cytotoxic agents. In addition, higher Bcl2 levels in human CRC (Fig. 8F) suggest that the ability of SPARC to interfere with Bcl2’s interaction with caspase 8 may be clinically relevant and can be targeted as a potential therapeutic. PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e26390