Hsp90 plays a key role in conformational maturation and stability of client proteins in transducing proliferative and anti-apoptotic signals [
10]. In the present study we found that Hsp90 inhibitor 17-AAG and GA or Hsp90 knockdown induced c-FLIP
L down-regulation which subsequently resulted in cancer cell apoptosis. Although DR5 is an important death receptor in the extrinsic apoptotic pathway, in the above experiments, we failed to detect DR5 up-regulation (data no shown). This indicates that c-FLIP
L may primarily contribute to Hsp90 inhibition-induced apoptosis in lung cancer cells.
Currently, more than one hundred client proteins of Hsp90 have been identified, and they are degraded when Hsp90 multi-chaperone complex activity is disrupted by the inhibitors. When we inhibited Hsp90 expression using siRNA technique and examined the c-FLIP
L level, we found c-FLIP
L was reduced in the cells whose Hsp90 was knocked down, suggesting that c-FLIP
L is a client protein of Hsp90. It is reported when Hsp90 is inhibited, it recruits E3 ligase (e.g. CHIP) to degrade the client proteins [
13‐
15]. When we knocked down CHIP, c-FLIP
L degradation was inhibited after treatment with 17-AAG, which indicated that CHIP modulated c-FLIP
L degradation in the NSCLC cell lines. Next, we performed immunoprecipitation experiments in Calu-1 cell lines. We found that CHIP was co-precipitated with c-FLIP
L in the precipitated complex. We also found that Hsp90 was precipitated at the same time, hinting that c-FLIP
L is the client protein of Hsp90 and CHIP modulates c-FLIP
L degradation through the Hsp90 chaperone complex. Itch is reported as an E3 ligase of c-FLIP
L in the mouse cells upon exposure to TNFα [
7]. However, it has also been reported that some agents induced c-FLIP
L degradation independently of Itch [
17‐
19]. Additionally, we found that CHIP promotes the ubiquitination of c-FLIP, indicating CHIP regulates c-FLIP
L degradation through uibiquitination when the cells were treated with 17-AAG.
It has been investigated that the clinically used anti-inflammatory drug celecoxib induces c-FLIP
L down-regulation and facilitates cancer cell apoptosis alone or in combination with other agents [
16]. We found that 17-AAG and celecoxib had synergistic effects in promoting c-FLIP
L down-regulation and cellular apoptosis. In addition, overexpression of c-FLIP
L could rescue cancer cells from apoptosis. These data suggest that c-FLIP
L down-regulation plays important role in Hsp90 inhibition-induced apoptosis in NSCLC cells.