recombinant peptide containing this smaller region alone is capable of conferring greater apoptosis and tumor regression in vivo. In addition, while we previously demonstrated an interaction between SPARC and caspase 8 in potentiating the apoptotic cascade [8], this study invokes Bcl2, an anti-apoptotic member of the intrinsic/mitochondrial pathway of apoptosis, as an important component in this interaction with caspase 8 and SPARC. This network of interactions affects the apoptotic cascade which then influences drug sensitivity, therapy response and reversal of drug resistance. Results Effect of different SPARC domains on apoptosis We previously showed that exposure to high levels of SPARC enhances apoptosis and significantly reduces cell viability in CRC cells that have become resistant to chemotherapy [4,8]. Although a number of biological activities (such as inhibition of angiogenesis and proliferation) have already been ascribed to smaller proteolytic cleavage products of SPARC [10], it is not known if any of them also induce apoptosis similar to the native protein. Therefore, in order to investigate this possibility, sensitive and resistant CRC MIP101 cells were transiently transfected with vectors containing only the N-terminus (SP-N), the follistatin-like (SP-F), or the C-terminus (SP-C) domains of SPARC, or mutant domains (SP-Nmut1, SP-Nmut2, SP-Fmut1, SP-Fmut2) (Fig. 1A). In the chemotherapy na¨ıve (MIP101) and resistant cells (to CPT- 11, MIP/CPT; or to 5-FU, MIP/5FU) examined, transient over- expression of SP-N reduced cell viability (Fig. 1B–D) in response to chemotherapy. In sensitive cells, such as MIP101, this represented an additional decrease in viability of 37.1863.65% (p,0.005) following transfection with SP-N (Fig. 1B), in comparison to 5-FU- treated ZEO (empty vector)-transfected cells. Even more signifi- cant is the observation that SP-N also decreased cell viability in the chemo-resistant cells examined (Fig. 1C, D). Based on these initial results suggesting that a recombinant protein containing only the N-terminus domain of SPARC was capable of diminishing cell viability in not only sensitive CRC cells, but also in their chemo-resistant counterparts, we decided to validate this observation by mutating the N-terminus domain, and found that the mutant forms no longer promoted a chemosensitiz- ing effect. In resistant MIP/5FU cells, transient over-expression of two different mutants of SP-N (SP-Nmut1, SP-Nmut2) failed to increase their sensitivity to 5-FU (Fig. 1D), while over-expression of wild-type SP-N was able to decrease cell viability by 24.560.8% (p,0.005) in response to 5-FU. This negative effect of the N-terminus domain of SPARC on cell viability led us to further evaluate them using stable clones of MIP101 cells over-expressing the N-terminus (MIP/SP-N), the follistatin-like (MIP/SP-F), or the extracellular (MIP/SP-C) domains. MIP/SP and MIP/SP-N cells showed the greatest decrease in cell viability and proliferation after 5-FU treatment, suggesting enhanced chemosensitivity in comparison to control MIP/ZEO cells (Fig. 1E). This reduction in cell viability resulting from overexpression of the N-terminus domain of SPARC was associated with a 20-fold increase in the percentage of MIP/SP-N cells undergoing apoptosis in response to 5-FU (Fig. 1F). A similar but less dramatic response is seen with MIP/SP cells (Fig. 1F), while no significant increase in apoptosis were observed in MIP/ SP-F or MIP/SP-C following exposure to 5-FU. N-terminus domain of SPARC and apoptosis We previously reported that SPARC-mediated apoptosis involved the activation of the extrinsic pathway, via caspase 8 A Small Peptide of SPARC Reverses Chemoresistance [8]. Given the above results, the possibility that the N-terminus domain of SPARC may engage in a similar mechanism to enhance apoptosis was investigated. Following exposure to 5- FU, caspase 8 activity (based on its cleaved product, 43 kDa) was mainly observed in MIP/SP-N cells (Fig. 2A). There was also higher expression of cleaved-BID and earlier activation of caspase 3 in MIP/SP-N cells (by 8 hrs after exposure to 5-FU), suggesting a similar involvement of the extrinsic pathway in cells over-expressing the N-terminus domain. To further validate the involvement of caspase 8 and Bid in the N-terminus-mediated apoptotic events, siRNA knock-down studies were undertaken which revealed that the absence of caspase 8 eliminated the apoptotic response following treatment with chemotherapy in MIP/SP and MIP/SP-N cells, as measured by cell viability assays (Fig. 2B) and caspase 3/7 activity (Fig. 2C). Similarly, in the absence of Bid following siRNA knock-down, MIP/SP and MIP/SP-N cells also demonstrated a diminished caspase 3/7 response to chemotherapy (Fig. 2D). Interestingly, while intrinsically high SPARC-expressing HCT116 cells were also no longer responsive to chemotherapy following caspase 8 and Bid gene-silencing, cells overexpressing the FS- and EC-domains of SPARC were unaffected by caspase 8 or Bid knock-down (Fig. 2B–D). Tumor xenografts overexpressing the N-terminus domain of SPARC are more chemosensitive The results of the in-vitro studies indicate that the N-terminus domain of SPARC alone is capable of enhancing chemosensitivity. This, together with previous in-vivo studies demonstrating greater tumor regression of xenografts of MIP/SP cells to chemotherapy [4] led us to examine if tumor xenografts overexpressing the N- terminus domain of SPARC also had a heightened sensitivity to chemotherapy. Indeed, in-vivo, MIP/SP-N cells were also more sensitive to chemotherapy than xenografts of MIP/ZEO control cells: xenografts of tumors over-expressing either the N-terminus domain or SPARC were the most responsive to 5-FU treatment, as tumors remained ,400 mm3 after 41 days of treatment, while saline-treated animals harbored tumors greater than 1144.16181.3 mm3 (Fig. 3A,B). Tumor xenografts of cells overexpressing the other domains of SPARC did not differ in size between the treatment and control groups. There was also a significantly longer tumor doubling times for MIP/SP and MIP/ SP-N saline-treated xenografts, in comparison to MIP/ZEO xenografts (p,0.005; Fig. 3C). Xenografts of MIP/SP-C had a similar tumor doubling time as MIP/ZEO (p = 0.07), while MIP/ SP-F had a faster growth rate (p,0.005). The extent of tumor regression correlated with a greater percentage of cells undergoing apoptosis in xenografts of MIP/SP (22.262.4% vs 4.263.1% in control, p = 0.01) and MIP/SP-N (31.363.2% vs 4.360.9% in control, p,0.005) cells following treatment with 5-FU in comparison to their saline-treated counterparts (Fig. 3D, E). Caspase 3 (cleaved) expression was also highest in tumors of MIP/SP-N and MIP/SP cells (Figure S1). No differences were detected in xenografts of the other cell lines, despite 5-FU treatment. Angiogenesis also appeared to be affected, as xenografts of MIP/SP and MIP/SP-N cells showed significantly fewer CD31+ staining than xenografts of MIP/ZEO, MIP/SP-F or MIP/SP-C (p,0.05, Fig. 3F). These findings demonstrate that a fragment of SPARC containing only its N-terminus domain has similar biological activities as the full-length protein in relation to its ability to promote apoptosis while inhibiting angiogenesis [4,16]. PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e26390