and an anti-Apaf-1 antibody (Figure 5F). These results suggested that caspase-3s prevent the apoptosome assembly by sequestrat- ing procaspase-3. Discussion A variety of mechanisms has been described to account for the resistance of breast cancer cells as well as other tumor types to anticancer agents. Indeed, patients often develop drug resistance towards drugs of unrelated structures and activities. To fight against chemotherapy, cancer cells are able to develop various mechanisms of resistance such as activation of survival pathways or apoptosis resistance. Apoptosis resistance can reflect resistance to chemotherapeutic treatments [1]. Caspase-3 is a major pro- apoptotic protein and is a key enzyme in drug-induced apoptosis of tumor cells [9]. In contrast, caspase-3s overexpression in breast carcinomas might be indicative of chemoresistance to neoadjuvant cyclophosphamide-containing treatment [6]. Thus, it was suggest- ed that caspase-3s might be an apoptosis protagonist which could be a new predictive marker of apoptosis resistance. To evaluate this hypothesis, the ability of caspase-3s to counteract apoptosis induced by various pro-apoptotic chemotherapeutic drugs was evaluated in this study with the use of breast tumor cell lines which are deficient or proficient for caspase-3. The present study demonstrates that caspase-3s, generated by alternative splicing of caspase-3 pre-mRNA, negatively interferes with caspase-3 activation and apoptosis in breast cancer cells exposed to cytotoxic drugs. This effect appears to involve an interaction of caspase-3s with procaspase-3 that prevents its recruitment in the apoptosome and its activation. Our results confirm and extend the observation that procaspase- 3 expression in MCF-7 cells, which do not express spontaneously this protease, enhances the cell ability to undergo cell death in response to etoposide, methotrexate and other cytotoxic drugs [30]. Caspase-3 activation was confirmed by immunoblot showing the proteolytic cleavage of the protein. The cleavage of a-fodrin and lamin A, which was described as caspase-3 targets, also argues for a role of caspase-3 in these apoptotic pathways. In addition, siRNA-mediated down-regulation of caspase-3 in HBL100 decreased the apoptotic response to the drugs used, whereas caspase-3s expression inhibition increases cell death induced by the treatment in HBL100 and MDA-MB-231. It is important to note that there are no differences between HBL100 and MDA- MB-231 cells in expression of other apoptotic factor analyzed in this study. In MCF-7 cells, caspase-3 is absent and caspases-4 and - 10 are downregulated. Nevertheless, as caspase-4 is an inflamma- tory caspase and caspase-10 is only involved in extrinsic apoptosis pathway, both these proteins have no influence on the studied apoptosis pathway. In apoptosis involving the mitochondrial pathway, procaspase-3 is activated by caspase-9, subsequently to apoptosome formation [31]. Thus, it was of interest to precise the interaction between caspase-3s, pro-caspase-3 and/or caspase-9. Indeed, the ability of caspase-3s to interact with pro-caspase-3 and/or caspase-9 might, at least in part, contribute to explain its anti-apoptotic activity. We observed that procaspase-3 or caspase-3s expressed in MCF-7 cells interacted with active caspase-9 when these cells were exposed to apoptotic stimuli. This interaction was abrogated when caspase-3s and procaspase-3 are expressed simultaneously. These observa- tions could suggest that interaction of active caspase-9 with a catalytically inactive caspase-3s could stimulate caspase-9 activity or activation at the apoptosome level explaining why, in MCF-7, the overexpression of caspase-3s alone increased slightly apoptosis rate. However this situation was observed only in MCF-7, which is Caspase-3s Inhibits Apoptosome Assembly deficient for caspase-3, and not in HBL100 as caspase-3s is expressed with procaspase-3 (Table S3). Nevertheless, in human tissues, caspase-3s is always expressed with procaspase-3. Taken together, these results strongly suggested that caspase-3s prevent the apoptosome assembly by sequestrating procaspase-3. These data evocate the mechanism of caspase-9 gene which also possesses a splice variant with antagonist apoptotic properties to the principal transcript. Indeed,, casp9-c, an alternative splice variant of caspase-9, functions as an endogenous apoptotic inhibitor by interfering with the Apaf-1/procaspase-9 interaction [32]. Similarly, forced expression of the caspase-8 alternative splice variant, caspase-8L, protected hematopoietic cells from extrinsic apoptosis by preventing CD95 to connect to the caspase cascade [33]. In conclusion, our study indicates that caspase-3s could have its anti-apoptotic function by sequestration of procaspase-3, inhibit- ing the interaction of procasase-3 with caspase-9 and apoptosome, thus counteracting the activation of the apoptotic machinery (Figure 6). Our data support that the investigation of caspase-3s in breast tumors might contribute to explain certain forms of drug resistance, and to develop more adapted and efficient treatments. Supporting Information Figure S1 Control of transfection by Western Blot assay with anti-GFP antibody in MCF-7 cell line. Procaspase-3- GFP (ProC3-GFP) and caspase-3s-YFP (C3s-YFP) were stably overexpressed in single transfection and in co-transfection. GFP vector (GFP) was used as control. b-actin was used as loading control. (TIF) Figure S2 Control of procaspase-3 and capsase-3s extinction after siRNA transfection. A. Endogenous procas- pase-3 was inactivated in HBL100 cells by siRNA transfection and the expression was monitored every 24 hours by western blot analysis from 24 to 96 hours after transfection. The transfection of siRNA induced the complete extinction of procaspase-3 from 48 to 96 hours after transfection. Caspase-3s expression was not affected by procaspase-3 siRNA transfection. B. Endogenous caspase-3s was inactivated in HBL100 and MDA-MB-231 cells by siRNA transfection and the expression was monitored every 24 hours up to 96 hours by real time quantitative PCR. The transfection of siRNA induced the complete extinction of caspase-3s from 48 to 96 hours after transfection. Nonetheless, procaspase-3 expression was not down-regulated by caspase-3s siRNA transfection, but its expression was strongly increased. C. The extinction of endoge- nous caspase-3s induced a strong increase of the endogenous procaspase-3/caspase-3s ratio. Double arrows indicate the optimal time lapse for cytotoxicity assay. (TIF) Primers and probes sequences used in this study. Table S1 (DOC) Table S2 Chemotherapeutic agents and doses used in this study to induce apoptosis. (DOC) Table S3 The cells (MCF7: deficient for caspase-3; HBL100: proficient for caspase-3) were incubated for 48 H in drug- containing medium and apoptosis was detected by flow cytometry. *Data represent the percentage (6 Standard Deviation) of apoptotic cells in each treatment conditions (mean of 3 experiments). CT: control, 5FU: 5- Fluorouracil, BLM: Bleomy- cine, CDDP: Cisplatin, EPI: Epirubicin, DOC: Docetaxol, VIN: Vincristine, STAU: Staurosporine. For the different drugs, the PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e29058