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| Author(s): |
Ana Carolina Pavanelli
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD) |
| Defense date: | 2015-03-27 |
| Examining board members: |
Maria Aparecida Nagai;
Maria Lucia Hirata Katayama
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| Advisor: | Maria Aparecida Nagai |
| Abstract | |
The SPARC (secreted protein, acidic, cysteine-rich) gene encodes a 42kDa protein that belongs to a family of matricellular proteins, which interact with cell-surface receptors, growth factors and the ECM (extracellular matrix) components. SPARC plays a role in tissue remodeling, cell migration, angiogenesis, embryonic development, tumorigenesis and chemiosensitivity. Docetaxel, which is an antimicrotubulin agent, is an effective chemotherapeutic drug for the treatment of advanced breast cancer. However, a considerable proportion of breast cancer patients do not respond positively to docetaxel. The mechanisms of docetaxel resistance are poorly understood. In a previous study, we identified SPARC as differentially expressed in mammary epithelial cells expressing different levels of HER-2. In the present study, we evaluate the effects of SPARC over-expression on the sensitivity of breast cancer cells to docetaxel. MCF7 cells were transfected with the expression vector pCMV6-SPARC or pCMV6-Neo. Real time PCR, western blot and immunofluorescence were used to characterize the clones over-expressing SPARC. Proliferation was not significantly affected by SPARC over-expression. However, we observed an increased sensitivity to docetaxel when comparing the MCF7 control cells with the MCF7 cells overexpressing SPARC, indicating that SPARC expression has chemosensitive properties in breast cancer cells. We used cDNA microarray to evaluate the expression profile of MCF7 cells with SPARC overexpression in comparison with MCF7 control cells before and after docetaxel treatment for 24h.We have identified several differentially expressed genes potentially involved in SPARC-mediated chemosensibility to docetaxel. Seventy of the highly expressed genes were selected from all treatments and several molecular networks were identified using the Ingenuity Pathway Analysis (IPA). After manual annotation, six genes, SPANXA1, ALDH1A3, TPM4, TARP, XAF1, and WNT5A, potentially associated with SPARC mediated chemiosensitivity were selected and validated by qPCR. Using label-free approach for quantitative proteomic analysis, we further evaluated the proteomic changes in the comparison of the MCF7 cells control and over-expressing SPARC before and after docetaxel treatment. Among the molecular interaction networks, cytoskeleton remodeling pathway was the top pathway map observed in the comparison between MCF7 cells over-expressing SPARC and the control cells before docetaxel treatment and GTP metabolism pathway was the top pathway map observed in the comparison between MCF7 cells over-expressing SPARC and the control cells after docetaxel treatment. Transcriptome and proteome integrated data, resulted in forty commonly up and down regulated genes, being the WNT pathway represented among them. Our findings suggest that SPARC expression can influence Docetaxel sensitivity in MCF7 cells by modulating genes involved in diverse biologic process that might be related to drug sensitivity (AU) | |
| FAPESP's process: | 12/03920-0 - Evaluation of the transcriptome and proteome of breast cancer cells with different expression profile of SPARC (secreted protein acidic and rich in cysteine ) in the presence and absence of docetaxel |
| Grantee: | Ana Carolina Pavanelli |
| Support Opportunities: | Scholarships in Brazil - Master |