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26 ACTIVATION OF THE APOPTOTIC PATHWAY BY SPARC IN RESPONSE TO CHEMOTHERAPY MJ Tang, IT Tai
A major obstacle in cancer therapy is the development of chemotherapy resistance. Our laboratory noted that secreted protein acidic and rich in cysteine (SPARC) was differentially expressed in chemotherapy resistant colorectal cancer cells. SPARC is developmentally regulated and has been found to be differentially expressed in many tumours. In ovarian cancers, upregulation of SPARC is associated with increased numbers of apoptotic cells. Our lab has found that resistant MIP101 colon cancer cells exposed to SPARC enhances apoptosis in vitro. In our animal tumour models, the combination of SPARC and the chemotherapy reagent, 5-fluorouracil (5-FU) resulted in a significantly greater tumour regression, relative to 5-FU treatment alone. Therefore, the objective of this study is to understand how SPARC modulates apoptosis to increase sensitivity of colorectal cancer cells to chemotherapy, to further determine the mechanism of SPARC-induced apoptosis.
METHODS: Cell viability in response to chemotherapy was measured through a clonogenic assay. Total RNA was isolated for RT-PCR to determine differentially expressed genes involved in apoptosis in response to SPARC modulation. Our empty vector control (MIP/ZEO), overexpressing SPARC (MIP-SP), and 5-FU resistant (5FUR) cells were used for all studies. Proteins were isolated at 0 h, 4 h, 8 h and 12 h after treatment with 1000 ÁM 5-FU, to determine the activation of the apoptotic pathway. Colocalization of SPARC with members of the apoptotic pathway were determined through immunoprecipitation, immunoblotting and immunocytochemistry.
RESULTS: The activation of the intrinsic pathway was detected earlier in MIP-SP cells, with increased caspase 9 expression soon after 5-FU treatment. However, MIP/ZEO underwent activation only 4 h after treatment. There was also the recruitment of the extrinsic pathway based on increased caspase 8 activation following 5-FU exposure in MIP-SP cells, while MIP/ZEO or 5FUR cells did not show any significant activation of caspase 8. Expression of a receptor, TRAIL-R1, was seen in MIP-SP cells soon after exposure to 5-FU, while this expression was only seen at 12 h in our control MIP/ZEO cells, and little expression was noted in 5FUR cells. Preliminary results also showed that SPARC can be detected in the nucleus, cytosol and membrane, but colocalized with TRAIL-R1 and caspase 8 in the membrane fraction only.
CONCLUSION: Our preliminary results reveal that SPARC enhances apoptosis in therapy-refractory MIP101 colon cancer cells exposed to chemotherapy by activating the extrinsic pathway of apoptosis while further enhancing the effect of chemotherapy through the intrinsic pathway.
ACTIVATION OF THE APOPTOTIC PATHWAY BY SPARC IN RESPONSE TO CHEMOTHERAPY
MJ Tang, IT Tai