regression was superior to SPARC: in addition to enhancing apoptosis in response to 5-FU, it also reduced the rate of tumor growth by , 40% in comparison to SPARC-over-expressing tumors. Our findings indicate that while SPARC’s N-terminus domain inhibits tumor growth, the follistatin-like (FS-) domain alone may have growth-promoting properties, as xenografts overexpressing recombinant proteins of this fragment had significantly shorter doubling times than control xenografts. These tumors were also unresponsive to 5-FU treatment. Taken together, these observations suggest that the efficacy of the native SPARC protein on tumor regression may be blunted when its proteolysis results in the release of different fragments with opposing biological effects. These observations again reflect SPARC’s intricate biology, as potential proteolytic products may have opposing effects on tumor growth, progression and response to therapy. This is an area that requires further investigations, in order to allow a better understanding of SPARC’s effect in different types of cancers. Based on the results of the current study, another dimension has been added to the multifaceted biological behavior of SPARC, by demonstrating the pro-apoptotic activity of the N- terminus domain of SPARC. In addition, we further confirm the involvement of Bid in SPARC-mediated apoptosis as cells overexpressing only the N-terminus of SPARC (MIP/SP-N) are capable of activating Bid, and this effect was easily abolished with Bid-siRNA in MIP/SP-N cells only. More importantly, we show that the ability of either SPARC or its smaller N-terminus domain to promote apoptosis results from their interference in Bcl2’s interaction with caspase 8, thereby leading to the activation of the extrinsic pathway (Fig. 9). Our assessment of a potential triad involving SPARC-caspase 8-Bcl2 in regulating apoptosis was based on earlier observations that Bcl2 binds to caspase 8 to inhibit caspase 8-mediated apoptosis in Fas- resistant neuroblastomas [17]; reports of SPARC’s ability to promote apoptosis by decreasing the ratio of Bcl2 and BAX in autosomal dominant polycystic kidney cells [27], together with our recent observations that decreasing SPARC expression by siRNA diminishes chemosensitivity [28] and that this effect is based on SPARC’s ability to interact with caspase 8 to promote apoptosis [8]. Bcl2 has long been associated with drug resistance since its discovery as a proto-oncogene in non-Hodgkin’s B-cell lymphomas [29], and over the years, it has been extensively studied as a potential target for cancer therapy [30]. Recently, the differential compartmentalization of Bcl2 and NRAS has been reported to influence disease states, such as myelodysplas- tic syndromes and acute myeloid leukemia [31]. In their mouse model, the physical interaction of Bcl2 and mutant NRAS affected the apoptotic machinery. These novel interactions between Bcl2 and other proteins, as demonstrated by us and others, highlight the complex mechanisms utilized by cancer cells to evade cell death, leading to disease progression and drug resistance. In this study, we demonstrate that Bcl2 and caspase 8 interact in cancer cells that have become refractory to therapy, thus providing another mechanism by which Bcl2 promotes survival and drug resistance. In addition, our in-vitro and in-vivo results reveal that the N-terminus domain of SPARC, and its synthetic peptide, are highly efficacious in modulating and enhancing apoptosis, in part, through its ability to dissociate the interaction between Bcl2 and caspase 8. These exciting findings point to the possibility that this smaller N-terminus peptide, based on its ability to interfere with the Bcl2-caspase 8 interaction, can be exploited as a potential therapeutic in disease states, such as cancer, where augmentation of the apoptotic cascade is clinically beneficial. A Small Peptide of SPARC Reverses Chemoresistance Materials and Methods Cell Lines, Reagents Colorectal (MIP101, HCT 116, RKO), pancreatic (MiaPaCa) and breast (MCF-7) cancer cells (sensitive and resistant) were maintained in DMEM media as previously described [4]. HCT116, RKO, MiaPaCa and MCF-7 were obtained from ATCC. MIP101 cells were kind gifts from Dr. Lan Bo Chen [32]. Also, please refer to Table S1 for a list of abbreviations. For resistant cells, media were also supplemented as follows: MIP/ 5FU, 500 mM 5-FU; MIP/CPT, 10 mM CPT-11; HCT116/5FU, 10 mM 5-FU; RKO/5FU, 25 mM 5-FU; RKO/CPT, 18 mM CPT-11; cisplatin (CIS) (RKO/CIS), 30 mM CIS; MiaPaca/CPT, 100 mM CPT-11; MCF7/CIS, 30 mM CIS. MIP101 transfected with empty vector (MIP/ZEO) or stably transfected with SPARC (MIP/SP) underwent selection with Zeocin (Invitrogen). Mammalian expression vectors containing the N-terminus (SP- N), follistatin-like (SP-F), and extracellular (SP-C) domains of SPARC were kind gifts from Dr. W. Schiemann (National Jewish Medical and Research Centre; Denver, CO). The recombinant proteins were myc/His6–tagged [33]. MIP101 cells stably transfected with SP-N (MIP/SP-N), SP-F (MIP/SP-F), or SP-C (MIP/SP-C) were also generated and selected for hygromycin- resistance. Overexpression of these recombinant proteins in cells were confirmed by ELISA (Figure S3). Of note, previous studies utilizing tagged-SPARC (or related peptides) have demonstrated similar biological activity as the native, un-tagged proteins [33,34], therefore, myc or His6–tagged proteins were used in these studies. Recombinant human SPARC (rSPARC), were generously provided by Dr. Desai (Abraxis BioScience Inc., USA). Human peptide of the N-terminus domain of SPARC (Peptide sequence: APQQEALPDETEVVEETVAEVTEVSVGANPVQVEVGEFD- DGAEETEEEVVA) was synthesized by AnaSpec (USA) with a TAT sequence (YGRKKRRQRRR) added to its amino-terminus, to facilitate its intracellular entry. Site-directed Mutagenesis SPARC (SP), SP-N, SP-F, and caspase 8 containing plasmids were used for site-directed mutagenesis, using GeneTailor Site- Directed Mutagenesis System (Invitrogen) as per manufacturer’s protocol. Mutant constructs were verified by DNA sequencing. Please refer to Table S2 for a list of primers used. Transfection studies Plasmids. In order to assess if the smaller peptides representing the different domains of SPARC had an effect on cell viability, cells were transiently transfected with constructs containing: SPARC (SP), SPARC’s N-terminus domain (SP-N), mutant FS-domain (SP-F), EC domain (SP-C); mutant N-terminus domain (SP-Nmut1, SP-Nmut2), FS-domain (SP-Fmut1, SP- Fmut2); wild-type caspase 8 or mutant DEDI-domain (DEDIm), putative-binding-domain (PBm), DEDII-domain (DEDIIm); or empty-vector control (ZEO) for 48-hours. Depending on the study, cells were then exposed to 100 ng/mL SPARC, and/or 5– 1000 mM 5-FU (MIP101-related cells; with the higher concentrations of 5-FU used in the resistant MIP/5FU cells as their IC50 was .18-fold higher than sensitive MIP101 cells), 50– 100 mM CPT-11 (MIP101 related cells), 18–36 mM CPT-11 (RKO-related cells), 100–200 mM CPT-11 (MiaPaca-related cells), or 60 mM CIS (MCF7-related cells) for 24–48-hours. Stable cell lines overexpressing the different SPARC domains were also generated and subjected to similar treatment protocols. Cell viability and proliferation were assessed by WST reagent (Roche) at 450 nM [8] at the completion of the study. PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e26390