A Small Peptide of SPARC Reverses Chemoresistance Figure 7. The DEDI-domain of caspase 8 is critical for its interaction with Bcl2. Vectors containing mutations in specific domains (DEDI, putative binding(PB), DEDII) of caspase 8 were transiently transfected into (A) MIP/5FU, MiaPaca/CPT or (B) MIP/SP cells, to determine the ability of the mutant proteins to co-IP with Bcl2 or SPARC. C) Basal levels of caspase 8 gene expression between various cancer cell lines. D–F) The effect of SPARC on cell viability (D, E) or apoptosis (F) in cells overexpressing mutant forms of caspase 8 was assessed. Cells were transiently transfected with wild-type caspase 8 (Csp8), mutants (DEDIm, PBm, and DEDIIm), or empty-vector (EV, control) for 48 h, followed by exposure to 100 ng/mL rSPARC for 48 h, and treated with 0 (&) or 500 mM (%)5-FU for an additional 24 h. D–E) In resistant cells, exposure to rSPARC and 5-FU resulted in decreased cell viability following transfection with EV-control: MIP/5FU: 90.1462.80 (SPARC) vs. 69.8964.64% (SPARC+5-FU) (p,0.005); MiaPaca/CPT: 108.7766.85 (SPARC) vs. 73.9864.46% (SPARC+5-FU) (p,0.005). Over-expression of wild-type caspase 8 in resistant cells further decreased cell viability following exposure to rSPARC and 5-FU, in comparison to EV-controls: MIP/5FU: 69.8964.64 (SPARC + 5-FU) vs. 59.2163.66% (SPARC + 5-FU + caspase 8) (p = 0.03); MiaPaca/CPT: 73.9864.46 (SPARC + 5-FU) vs. 46.7862.01% (SPARC + 5-FU + caspase 8) (p,0.005). However, over-expression of mutant forms of caspase 8 abolished the reduction in cell viability seen in the presence of rSPARC. Results represent mean 6 s.e. (n = 3 independent studies). *p,0.05, Student’s t-test. doi:10.1371/journal.pone.0026390.g007 Figure 8. Interaction between the N-terminus of both caspase 8 and SPARC are required to reduce cell viability and enhance apoptosis. A–B) Cell viability further decreased in MIP/SP and MIP/SP-N after transfection with wild-type caspase 8 and 5-FU treatment, relative to empty vector (EV, control): MIP/SP: 77.263.8 (EV) vs. 59.161.5% (caspase 8) (p,0.005); MIP/SP-N: 68.864.8 (EV) vs. 50.663.6% (caspase 8) (p = 0.04). Transfection with any of the caspase 8 mutants eliminated their response to 5-FU, not only in terms of cell viability, but apoptosis (C). In cells overexpressing mutant SP-N (MIP/SP-Nmut1), transfection with caspase 8 was incapable of decreasing cell viability (D) or increasing apoptosis (E). Results represent mean 6 s.e. (n = 3 independent studies). *p,0.05, Student’s t-test. F) Bcl2 expression of clinical specimens of human CRCs obtained from the same individual following primary tumor resection (chemotherapy-na¨ıve) and disease recurrence post-chemotherapy (chemotherapy- resistant) (paraffin-embedded 6 mM sections). doi:10.1371/journal.pone.0026390.g008 PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e26390