In this study, we analyzed effects of the flavonoid quercetin on TRAIL-mediated apoptosis in human glioma cells. Both TRAIL and quercetin are in clinical testing and have been shown to be of minimal toxicity.9,30
Many cancer cells are resistant to TRAIL therapy.1,31,32
We therefore aimed at sensitizing TRAIL-resistant glioma cells with quercetin. In this study, we demonstrated that combined application of TRAIL and quercetin strongly reduced viability of U251, LN229, U87-MG, and A172 glioma cells but failed to do so in U373 cells. All our experiments on cell cultures were conducted with concentrations of quercetin that have been reached in clinical trials up to 400 μM plasma concentration of quercetin was achieved.30
First, we demonstrated that viability of glioma cells was not affected 24 h after a single dose of quercetin. This finding is similar to results in various carcinoma cell lines.33,34
We then determined viability of our set of glioma cells after TRAIL treatment and detected an effect only at a high TRAIL concentration of 500 ng/ml. Our results match those of previously published data that demonstrated moderate TRAIL sensitivity of U87-MG and A172 cells but high resistance to this treatment in U251, LN229, and U373 cells.10,11
Upon combined treatment with quercetin and TRAIL, U87-MG, U251, A172, and LN229 cells exhibited strongly enhanced apoptosis, whereas U373 cells proved completely resistant. Our data parallel those achieved by sensitizing glioma cells to TRAIL with the protein synthesis inhibitor cycloheximide, to which U373 cells were also insensitive.11,35
The strong effect of simultaneous application of TRAIL and quercetin demonstrates a synergistic action of combined treatment. Our results are in line with previous reports because combined treatment with TRAIL and quercetin has been successfully tested in other cell lines.33,34
The resistance of U373 cells is not fully understood. It has been reported that U373 cells express only low levels of the initiator caspase-8, thereby leading to an insufficient activation of DISC, resulting in inhibition of the extrinsic apoptotic pathway.10
Inactivation of p53, which is involved in the intrinsic apoptotic pathway, seems not to be of major importance to quercetin-TRAIL–mediated apoptosis. This is supported by TP53
mutations in both the sensitive U251 and the completely resistant U373 cell lines.36
The IAPs are an important family of apoptosis regulating proteins, with survivin and XIAP as prominent members. Because survivin and XIAP block apoptosis at the level of effector caspases, a point where multiple signaling pathways converge, strategies targeting survivin to remove its inhibitory effect seem to be useful to overcome the resistance of cancer cells. Suppression of survivin has been demonstrated upon quercetin treatment in H460 lung cancer cells.34
We analyzed the effect of quercetin in four glioma cell lines on expression of survivin and XIAP. Survivin expression was strongly reduced in three of four glioma cell lines, whereas XIAP was reduced only in U87-MG. Notably, the U373 glioma cell line, which could not be sensitized to TRAIL-mediated apoptosis, showed suppression neither of survivin nor of XIAP upon treatment with quercetin. The role of survivin in quercetin-TRAIL–mediated apoptosis was demonstrated by ectopic expression. Cells with vector-driven overexpression of survivin had a strongly reduced rate of apoptosis after combined TRAIL-quercetin treatment. These findings indicate that suppression of survivin is a key mechanism through which quercetin enhances TRAIL-mediated apoptosis. The previous demonstration of augmentation of TRAIL-induced apoptosis by cycloheximide may also directly connect to survivin prevalence,10,11
because survivin has a short half-life of 30 min, making this protein very susceptible to inhibition of protein synthesis. In contrast, XIAP expression was reduced in only one of four cell lines upon quercetin treatment. With the exception of U87-MG, the other cell lines did not show XIAP reduction, paralleling the lacking response of such treatment in non-small-cell lung cancer cells.34
Low prevalence of both IAPs, survivin and XIAP, may explain the high rate of apoptosis of U87-MG upon quercetin-TRAIL treatment ().
We identified two potential mechanisms leading to quercetin-induced changes of expression in survivin. Pretreatment with MG132 significantly inhibited quercetin-induced downregulation of survivin in U251 and U87-MG glioma cells, suggesting that quercetin may promote proteasome-mediated degradation of survivin. Proteasome-mediated degradation of survivin as a mechanism of regulation has already been reported.34,37
It has been demonstrated that survivin protein was unstable (half-life ~ 30 min) and easily ubiquitinized, followed by degradation through the proteasome.29
Akt, also referred to as Rac or protein kinase B, promotes cell survival and blocks apoptosis.38
Activation of phosphoinositide 3-kinase/Akt pathway generates phosphatidylinositol-3,4,5-triphosphate, which in turn binds to a domain of serine/threonine kinase Akt, resulting in recruitment of Akt to the cell membrane. A conformational change of Akt results in phosphorylation of residues Thr-308 and Ser-473 by the upstream kinases 3-phosphoinositide dependent protein kinase-1 (PDK-1) and PDK-2. Akt regulates apoptosis via the direct phosphorylation and inactivation of Bid, caspase 9, the Fork-head transcription factors, and nuclear factor-κB.39,40
The Akt-survivin pathway has been implicated in the resistance of cancer cells to therapeutics and TRAIL.41,42
Quercetin has been shown to inhibit the Akt-1 pathway in HepG2 cancer cells.43
In line with previous reports, in the present study we demonstrated that the inhibitory effect of quercetin on survivin expression appears to result from suppression of Akt activity by quercetin, because overexpression of phosphorylated Akt inhibited quercetin-mediated survivin suppression in U87-MG cells (). Other research groups have also reported that elevated Akt activity protects cells from TRAIL-induced apoptosis,41,44
which is in line with our results because Akt regulates survivin expression.
In conclusion, we demonstrated that quercetin effectively enhances TRAIL-mediated cytotoxicity by suppressing survivin via increased proteasomal degradation and via repressing phosphorylated Akt known to coregulate survivin protein levels.