Figure 1

Par-4 sensitizes colon cancer cells to 5-FU-induced apoptosis and inhibits NFκB activity in an AKT1-dependent manner. (A) Western analysis of Par-4 protein in HT29 cells transfected with empty vector (WT) or pCB6+-par-4. Clone 4 was used for subsequent Par-4 overexpression studies. (B) 5-FU treatment of Par-4- but not empty vector-transfected HT29 cells increases caspase-3 activity. Results are the mean ± S.D. of 4-5 independent experiments. *Significantly different from vehicle (P < 0.05, Student's t-test). (C) AKT inhibition promotes Par-4 binding to NFκB. Native HT29 and SW480 cells were treated with an anti-AKT1 shRNA, and lysates without (total) or with Par-4-immunoprecipitation (IP) were immunoblotted for p65, p50, AKT1, PKA, Par-4 and actin. Results are representative of 4-5 independent experiments. (D) Inhibition of AKT1 by ISC-4 promotes apoptosis. Native HT29 and SW480 cells were treated for 24 h with ISC-4 and subjected to an MTA viability assay. Decrease in OD reading corresponds to decreased viability. Results are the mean ± S.D. of 4-5 independent experiments. (E) Par-4 binding in native HT29 cells treated for 24 h with sub-lethal ISC-4 doses. Lysates without or with Par-4-immunoprecipitation were immunoblotted for p50, 14-3-3, PKA and actin. Results are representative of 4-5 independent experiments. (F) Effects of Par-4 overexpression (Par-4 plasmid) and AKT1 inhibition (pshAkt1) on NFκB transcriptional activity in HT29 cells. *Significantly different from empty vector-transfected cells (Vec only) by ANOVA and Tukey post-hoc test (P < 0.01). Negative (Neg) and positive controls (Pos) are reporter plasmids without promoter or with SV40 promoter sequences, respectively. Results are the mean ± S.D. of 4 independent experiments.