Figure 1. Response of a Panel of Lung Cancer Cell Lines to Smac Mimetic In Vitro and In Vivo.
(A) A panel of 50 non-small-carcinoma-cell lung cancer cell lines was tested for responsiveness to a Smac-mimetic treatment alone. IC50s were determined for each cell line based on cell survival as measured by ATP levels in live cells using Cell Titer-glo (Promega). IC50 determination was based on concentrations of compound 3 that yielded half-maximal luminescence relative to untreated cells.
(B) Treatment of selected group of cell lines to 100 nM of compound 3 and to compound 4 (negative control compound with similar structure by differing in function group configuration as described in Li et al., 2004). Smac-mimetic-sensitive cell lines (HCC44 and HCC461) and Smac-mimeticresistant cell lines (HCC827 and H2009) were chosen. Each graphical representation for IC50s and cell survival indicates the mean ± SD of at least three independent testing conditions.
(C) In vitro pull-down utilizing a biotinylated form of compound 3. The biotinylated compound was able to pull down protein bands at around 50 kDa and at around 70 kDa not seen in control lanes (avidin beads only), which were cut out of the gel and analyzed by mass spectroscopy. Proteins identified are indicated.
(D) In vivo response of mouse xenografts of HCC461 cells to compound 3. Harlan athymic nude mice were injected subcutaneously with HCC461 cells in a matrigel randomly separated into treatment groups (n = 5) and given six intravenous injections of compound 3 or saline every other day. Tumors were measured twice perweek until the end of the experiment. In the compound 3 treatment group, 2/5 (40%) remained tumor-free at the end of the experiment.
(E) In vivo response of mouse xenografts of a Smac-mimetic-resistant HCC15 cells to compound 3. Conditions were identical to those for HCC461 xenografts. For tumor size measurements, graphical representations indicate the mean ± SEM of five individual samples per condition.