Caveolin-1, a Novel Ku70-Interacting Protein Figure 7. WRA Ku70 fails to interact with caveolin-1, to protect against chemotherapeutic drug-induced apoptosis and to limit the 5-FU-induced dissociation of Bax from Ku70. HCT116 cells were transfected with either wild type Ku70-HA (WT Ku70) or WRA Ku70-HA (WRA Ku70) and treated with different concentrations of either 5-FU (for 24 h in A and E; for 48 h in C and D) or etoposide (for 24 h in C and D). Untreated cells were used as control. In (A), cell lysates were immunoprecipitated with an antibody probe specific for caveolin-1 and immunoprecipitates subjected to immunoblotting analysis with anti-HA, anti-Bax and anti-caveolin-1 IgGs. In (B), total expression of WT Ku70-HA and WRA Ku70-HA was detected by immunoblotting analysis with anti-HA IgGs before drug treatment. In (C), cells were stained with DAPI and the number of cells showing nuclear condensation was quantified. Values represent mean 6 SEM; *P,0.001. In (D), cells were subjected to immunoblotting analysis with antibody probes specific for cleaved caspase 3. Immunoblotting with anti-b-actin IgGs was done to show equal loading. In (E), cell lysates were immunoprecipitated with and antibody probe specific for Bax and immunoprecipitates subjected to Western blotting analysis with anti-HA and anti- Bax IgGs. doi:10.1371/journal.pone.0039379.g007 induced apoptosis after knockdown of caveolin-1 protein expres- sion is dramatically prevented in the absence of Bax expression. Therefore, caveolin-1 acts as an anti-apoptotic molecule in colon cancer cells by inhibiting Bax-dependent cell death. Overexpression of caveolin-1 was linked to drug-resistance of cancer cells, including colon cancer cells [35,36]. Our findings contribute to explain, at the molecular level, the role of caveolin-1 in drug resistance of cancer cells. Increased levels of caveolin-1 may promote resistance to chemotherapeutic drugs by bolstering the inhibitory action of caveolin-1 on the Ku70-Bax complex. In fact, in the presence of elevated caveolin-1 levels, the release of Bax from Ku70 induced by chemotherapeutic drugs would be further inhibited. This mechanism would also be consistent with a proposed scenario in which caveolin-1 expression is lost in the initial phases of cancer development but returns to high levels at later stages when the tumor has acquired multi-drug resistance properties (reviewed in [37]). Caveolin-1 was recently shown to modulate the cellular response to DNA damage by regulating both homologous recombination (HR) and non-homologous end joining (NHEJ) repair pathways [38]. Interestingly, Ku70 was originally discov- ered as a molecule involved in the repair of DNA double-strand breaks by non-homologous end joining (NHEJ). Since we demonstrate in this report that Ku70 is a novel caveolin-1 binding protein, our findings suggest that caveolin-1 may regulate DNA repair through modulation of Ku70 functions which are indepen- dent of apoptosis. How caveolin-1 regulates Ku70 in the context of DNA repair mechanisms is currently under investigation. Overexpression of Ku70 has been shown to protect against apoptosis [22,25]. Our data contribute to explain this previous finding at the molecular level. We confirmed that overexpression of wild type Ku70 inhibits both 5-FU- and etoposide-induced apoptosis of colon cancer cells. More importantly, we show that a mutant form of Ku70 that cannot interact with caveolin-1 (WRA PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e39379