Caveolin-1, a Novel Ku70-Interacting Protein Figure 4. Chemotherapeutic drugs promote the Ku70-Cav-1 interaction. (A) HCT116 cells were treated with either etoposide or 5-FU for 24 hours. Untreated cells were used as control. Cell lysates were then immunoprecipitated with an antibody probe specific for caveolin-1 and immunoprecipitates subjected to immunoblotting analysis with anti-Ku70, anti-Bax and anti-caveolin-1 IgGs. (B) HCT116 colon cancer cells were transfected with HA-tagged wild type Ku70. One day after transfection, cells were treated with 5-FU for 24 hours. Untreated cells were used as control. Cells were then subjected to immunofluorescence analysis using antibody probes specific for the HA tag (green) and caveolin-1 (red). Nuclei were detected by DAPI staining (Blue). Yellow staining in the merged images shows co-localization between caveolin-1 and Ku70 only after 5-FU treatment (see arrows). Representative images are shown. doi:10.1371/journal.pone.0039379.g004 knockdown of caveolin-1 protein expression by itself in untreated cells failed to promote the conformational change of Bax (Figures 5C and 5D). Since our results show that chemothera- peutic drugs stimulate the binding of caveolin-1 with Ku70 and the interaction of Bax with Ku70 is known to prevent the activation of Bax, our data suggest that caveolin-1 forms a complex with Ku70 that inhibits the activation of Bax. The dissociation of caveolin-1 from Ku70 is not sufficient to activate Bax but potentiates Bax activation induced by chemotherapeutic drugs. Consistent with these results, chemotherapeutic drug- induced translocation of Bax to mitochondria was enhanced in HCT116 cells in which caveolin-1 protein expression was reduced by siRNA, as compared to HCT116 cells expressing scrambled siRNA (Figure 5E). We conclude that binding of caveolin-1 to Ku70 limits the release of Bax from Ku70 and the activation of Bax induced by chemotherapeutic drugs. Enhancement of Apoptosis Induced by the Knockdown of Caveolin-1 Protein Expression is Dependent upon the Expression of Bax If knockdown of caveolin-1 protein expression potentiates apoptosis in colon cancer cells by enhancing the release of Bax from Ku70, one would not expect potentiation of apoptosis by knockdown of caveolin-1 in HCT116 cells lacking Bax expression. To directly test this possibility, we took advantage of HCT116 Bax (2/2) isogenic cells [31], which do not express Bax (data not shown). Caveolin-1 protein expression was knockdown by siRNA in HCT116 and HCT116 Bax cells (Figure 6A). Cells were then treated with either 5-FU or etoposide and apoptosis was quantified by DAPI staining. We demonstrate that knockdown of caveolin-1 protein expression by siRNA potentiated 5-FU-induced apoptosis in HCT116 cells by 81% but only by 46% in HCT116 Bax cells (Figure 6B). Similarly, knockdown of caveolin-1 protein expression by siRNA enhanced etoposide-induced apop- tosis in HCT116 cells by 84% but only by 10% in HCT116 Bax cells (Figure 6D). Consistent with these data, the enhanced activation of caspase 3 by downregulation of caveolin-1 expression following treatment with either 5-FU or etoposide observed in HCT116 cells was dramatically inhibited in HCT116 Bax cells (Figures 6C and 6E, respectively). Thus, Bax expression plays a central role in the potentiation of chemotherapeutic drug- induced apoptosis following knockdown of caveolin-1 in HCT116 cells. The fact that we observed apoptosis after 5-FU treatment even in HCT116 Bax cells transfected with scrambled siRNA (Figures 6B and 6D) suggests that apoptosis in these cells can also be Bax-independent, confirming previously reported data showing Bax-independent apoptosis in these cells [32,33]. Finally, the 46% increase in apoptosis induced by 5-FU found in caveolin-1 siRNA- PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e39379