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Connective Tissue Oncology Society |
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| 2001 CTOS Annual Meeting—Oral Presentations Young
Investigator Award Winner OBJECTIVE: Abnormalities in signal transduction pathways are common in soft tissue sarcomas. Because these are rare tumors that also respond poorly to standard chemotherapy and bear a 50% 5-year mortality rate, we investigated the possible therapeutic benefits of the tyrosine kinase inhibitor STI-571 in soft-tissue sarcomas. The objectives of our laboratory investigation are (1) to characterize the anti-tumor activity of STI-571 alone and with doxorubicin, in vitro; (2) to determine whether the inhibition of signal transduction molecules by STI-571 causes cell cycle arrest, induction of apoptosis, inhibition of the invasive phenotype and/or abrogation of angiogenesis; (3) to determine the anti-tumor activity of STI-571 alone and in combination with doxorubicin in human tumor xenografts. METHODS: We initially screened 15 soft-tissue sarcoma cell lines for expression of PDGF-R. Western blot analysis identified 12 that expressed high levels and 3 that expressed low levels. We have investigated the efficacy of STI-571 (0.4-20micromolar) alone and combined with doxorubicin(0.1-10micromolar) in the rhabdomyosarcoma cell line A204. Kit, PDGF-R, Map kinase, and Akt-1 signaling were evaluated by immunoblot. Proliferation assays were performed with a 48 hour exposure to incremental drug concentrations and viability assessed by MTS reagent. TUNEL assay and cell cycle analysis were performed by flow cytometry. Soft agar colony formation assay was performed to assay for anchorage independent growth. Endothelial cell proliferation rate in response to conditioned media from STI-571 treated A204 cells will be assessed. Zymogen invasion assay will be performed as an in vitro assessment of the invasive phenotype. SCID mice will be injected with A204 cells prior to treatment with doxorubicin alone, STI-571 alone, or the combination. RESULTS: Sarcoma cells treated with doxorubicin, STI-571, or the combination, exhibit a 40%, 20%, and 60% absolute reduction in percent viable cells, respectively. STI-571 increased the relative percentage of cells in G1 (by 57%) and decreased S-phase (by 63%) when compared to untreated cells. Apoptosis increased from 4% to 15% after treatment. Doxorubicin treatment did not induce cell cycle arrest, but induced apoptosis from a baseline of 5% to 50%. STI-571 treatment resulted in a decrease (48%) in the number of colonies established by soft agar growth. Endothelial cell proliferation assay, Zymogen invasion assay, and in vivo therapy with STI-571 experiments are ongoing. CONCLUSION: In vitro results suggest an additive, non-overlapping effect of doxorubicin+STI-571, indicating a potentially useful combination. Together, these efforts should result in new mechanism-driven therapies that are efficacious in soft-tissue sarcoma.
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