A Small Peptide of SPARC Reverses Chemoresistance Figure 2. N-terminus domain of SPARC activates caspase 8 in response to treatment with 5-FU. A) Changes in the expression of proteins involved in the apoptotic pathway following incubation with 5-FU at different time points in MIP101 cells stably overexpressing SPARC domains by immunoblotting. B–D) The contribution of caspase 8 or Bid in inducing SP-N-associated apoptosis were compared with various cell lines after transient transfection with caspase 8 and Bid siRNA (or scramble control) for 48 h, followed by no treatment (grey bars) or incubation with 5-FU (white or orange bars) for 24 h. Cell viability after caspase 8 siRNA transfection was assessed by WST assay (B), while apoptosis after caspase 8 (C) or Bid (D) siRNA transfection was assessed by caspase 3/7 assay. Results represent mean 6 s.e. (n = 3–4 independent studies). *p,0.05, Student’s t-test, in comparison to untreated controls. doi:10.1371/journal.pone.0026390.g002 The N-terminus of SPARC interacts with caspase 8 and prevents its interaction with Bcl2 The ability of SPARC to promote greater apoptosis in-vitro and in- vivo following exposure to cytotoxic agents [4] results, in part, from its interaction with caspase 8 leading to the activation of the extrinsic pathway of apoptosis [8]. A similar interaction was detected with the N-terminus domain of SPARC, but not the FS- or EC-domains, following co-immunoprecipitation studies using antibodies against caspase 8 and His6 (to detect fusion proteins of the NT, FS or EC- domains) (Fig. 4A). To further validate this observation, cells transiently overexpressing the wild-type NT-domain effectively co- immunoprecipitated caspase 8, while this interaction was absent with mutant proteins SP-Nmut1, and SP-Nmut2 (Fig. 4B). Our finding of an interaction between caspase 8 and SPARC, and previous reports of an interaction between caspase 8 and Bcl2 in neuroblastomas [17], led us to examine if such an interaction also occurs in cancer cells and whether it can be influenced by SPARC to facilitate apoptosis in response to chemotherapy. Our findings were interesting in that an interaction between caspase 8 and Bcl2 was detected in chemoresistant MIP/5FU cells, but not in sensitive MIP/ZEO and MIP/SP cells (Fig. 5A). This pattern of interaction was replicated in other chemotherapy-resistant cancer cells (Fig. 5B): CRC RKO/5FU, RKO/CPT, RKO/CIS; pancreatic MiaPaca/CPT; and breast cancer MCF-7/CIS. Even more exciting is the observation that when RKO/5FU cells were incubated with recombinant SPARC (rSPARC), the interaction Figure 3. Xenografts overexpressing SP-N have greater tumor regression and apoptosis in response to 5-FU. A,B) Comparison of tumor size in xenografts from MIP/SP-N, MIP/SP-F, MIP/SP-C, and MIP/ZEO cells with or without treatment with 5-FU; ‘‘+’’ = 5-FU-treatment; ‘‘2’’ = saline-treatment, (n = 14/group; mean 6 s.e). C) Tumor doubling times of xenografts following 5-FU treatment. (D) Percentage of apoptotic cells detected by TUNEL staining [representative images in (E)]. (F) number of CD31-positive stained blood vessels in the tumor xenografts of (&) saline or (%) 5-FU-treated animals. Student’s t-test * p,0.05, ** p,0.01. doi:10.1371/journal.pone.0026390.g003 PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e26390