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C.R. Wagner
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-052 - Targeting Oncogenic Eukaryotic Protein Translation in Thoracic Malignancies with Small-Molecule Inhibitors (ID 3094)
09:30 - 09:30 | Author(s): C.R. Wagner
- Abstract
Background:
Hyperactivation of cap-mediated translation can induce oncogenic transformation by enhancing the translation of a subset of mRNAs involved in the genesis, maintenance and progression of cancer.In this investigation, disabling the eIF4F complex by disrupting the eIF4E-mRNA-cap interaction is evaluated as a therapy for mesothelioma and non-small cell lung cancer (NSCLC).
Methods:
Cell lines and culture. H513 and H2373 were from American Type Culture Collection (ATCC) and cultured in either RPMI 1640 containing 10% calf serum supplemented with 10% calf serum and maintained at 37[o]C. Cell proliferation assay. 5000 cells were seeded into wells of 96 well plates. Following overnight incubation cells were treated with varying doses of cpd 267, 272 or pemetrexed for 72 h. Viable cells were counted employing Cell Counting Kit 8 (Dojindo). Cap-affinity assay. Cell lysate was mixed with 50 mL of a 50% mixture of m[7]GTP-Sepharose resin with and without 400 mM of cpd 267 for 2 h at 4[o]C to capture eIF4E and eIF4G. The captured bound proteins were eluted and prepared for immunoblot analysis. Immunoblot analysis. Protein samples were separated by SDS-PAGE and transferred to Hybond PVDF membrane. Blots were probed for eIF4E and eIF4G (both from Cell Signaling and diluted 1:1000). Detection was carried out using ECL Plus Western Blotting System (Amersham) to visualize the bands of interest.
Results:
Mesothelioma and NSCLC cells were treated with small-molecule inhibitors [compounds 267 and 272] that mimic the cap structure that displace capped mRNAs from the eIF4F complex resulting in suppression of cap-dependent translation of malignancy-related proteins. Treatment with the compounds resulted in a dose dependent decrease in cell viability. Combination therapy of the compounds with cytotoxic agents further decreased cell survival. Binding to a synthetic cap-analogue was employed to assess the strength of eIF4F complex activation in lysates exposed to the compounds.
Conclusion:
These novel compounds reduce cancer cell proliferation,reduce eIF4F complex formation and sensitizes mesothelioma and NSCLC cells to cytotoxic agents.
