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P1.01 - Poster Session 1 - Cancer Biology (ID 143)
- Event: WCLC 2013
- Type: Poster Session
- Track: Biology
- Presentations: 1
- Coordinates: 10/28/2013, 09:30 - 16:30, Exhibit Hall, Ground Level
P1.01-008 - Inhibition of Binding at Exon 4 of Osteopontin Increases Apoptosis and Apoptotic Protein Expression in Non-Small Cell Lung Cancer (ID 2885)
09:30 - 16:30 | Author(s): C. Goparaju
Osteopontin (OPN) is a ubiquitous extracellular protein associated with a wide range of normal and pathologic functions. It is a central regulator of the malignant phenotype in non-small cell lung cancer (NSCLC) through binding of cell surface receptors, but underlying mechanisms are poorly understood. Among OPN’s critical roles in NSCLC progression, is preventing apoptosis related to oxidative stress by activating alternative survival pathways. While OPN’s central RGD domain is considered important to this function, we hypothesize that exon 4, in the amino terminus, which is present in OPN’s A and B isoforms, but not in the C isoform, is essential to this process. We sought to determine the impact of inibition of binding between NSCLC cells and OPN exon 4 on apoptosis and apoptotic protein expression in NSCLC.
A 16 amino acid peptide mimicking the central sequence of OPN exon 4 and a scrambled sham were constructed. Competitive binding assays had previously determined that the OPN exon 4 peptide binds the NSCLC cell surface and inhibits OPN binding. Two NSCLC cell lines with wt p53, A549 (moderate endogenous OPN) and H460 (high endogenous OPN), were placed in serum-free media with OPN exon 4 peptide or scrambled peptide for 48 hours. Cells were then evaluated by TUNEL assay or harvested, lysed and protein expression measured using Human Apoptosis Array (R&D, Minneapolis, MN).
Exon 4 peptide treatment resulted in significant increases in apoptosis in both cell lines (Fig). A similar pattern of change in apoptotic protein expression was seen in both cell lines, with significant increases in Bax, cleaved Caspase-3, CytC, Hsp32, Pon2, Cdnk1, and p53-pS15, p53-pS46, and p53-pS392, while Survivin and Claspin expression were significantly decreased (Fig). Notably no significant change was seen in Bclx, Bcl2, and pro-Caspase 3 expression. Scrambled peptide had no effect on apoptosis or protein expression. Figure 1
Inhibition of binding between OPN exon 4 and NSCLC cells significantly increased the rate of apoptosis and expression of proteins which modulate apoptosis associated with oxidative stress, including several key phosphorylated p53 variants. These data implicate OPN exon 4 interactions in NSCLC progression and resistance mechanisms and may explain the importance of OPN’s A and B isoforms as opposed to isoform C in NSCLC pathogenesis.