Caveolin-1, a Novel Ku70-Interacting Protein Figure 6. Knockdown of caveolin-1 protein expression fails to enhance apoptosis induced by chemotherapeutic drugs in the absence of Bax expression. HCT116 and HCT116 Bax cells were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. In (A), expression of endogenous caveolin-1 was detected by immunoblotting analysis 48 hours after transfection using an antibody probe specific for caveolin-1. Immunoblotting with anti-b-actin IgGs was done to show equal loading. In (B–E), 24 hours after transfection, cells were treated with either 5-FU (25 mg/ml) for 48 h (B and C) or etoposide (Etop.; 800 mM) for 24 h (D and E). Untreated cells were used as control. In (B and D), cells were stained with DAPI and the number of cells showing nuclear condensation was quantified. Values represent mean 6 SEM; *P,0.001. In (C and E), 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. doi:10.1371/journal.pone.0039379.g006 localization of WRA-Ku70 with caveolin-1 in HCT116 cells (Figure S6). Interestingly, overexpression of wild type Ku70, but not WRA-Ku70, promoted a ,40% decrease in apoptosis induced by either 5-FU or etoposide (Figures 7B and 7C). In support to this data, we show in Figure 7D that wild type Ku70, in contrast to WRA-Ku70, prevented the activation of caspase 3 by either 5-FU or etoposide and that overexpression of wild type Ku70, but not WRA-Ku70, limited the Ku70/Bax dissociation promoted by 5-FU in HCT116 cells (Figure 7E). These results support our conclusion that the interaction of Ku70 with caveolin- 1 limits the chemotherapeutic drug-induced release of Bax from Ku70 and therefore Bax-dependent apoptosis. Discussion Apoptosis is the process of programmed cell death adopted by multicellular organisms for proper development and maintenance of adult individuals. Defective apoptosis has been linked to pathological conditions. Excessive apoptosis may lead to atrophy whereas reduced apoptosis may lead to uncontrolled cell proliferation and cancer. Apoptosis is also a major cytotoxic effect of chemotherapy. Thus, understanding, at the molecular level, how apoptosis is regulated will not only bring insight into a fundamental biological event but also contribute to the develop- ment of more efficient therapeutic interventions for the treatment of cancer. 5-fluorouracil is a chemotherapeutic drug that has been the cornerstone of treatment for patients with colorectal cancer for almost four decades. 5-FU blocks the enzyme thymidylate synthase and inhibits both RNA and DNA synthesis. In sensitive cells, 5-FU induces apoptosis. Unfortunately, tumor responses to 5-FU is limited by toxicity to tissues containing highly proliferative cell populations [34]. In this report, we show that knockdown of caveolin-1 protein expression in colon cancer cells enhances apoptosis induced by 5-FU and etoposide. Mechanistically, our data reveal a novel anti-apoptotic mechanism in which the binding of caveolin-1 to Ku70 limits the dissociation of Bax from Ku70 and the activation of Bax induced by apoptotic stimuli. Consistent with this conclusion, we show that the enhancement of 5-FU- PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e39379