LBH589 Donwregulates c-FLIP through Promoting Ubiqitin/proteasome-mediated http://www.selleckchem.com/products/lapatinib.html Degradation Given the critical role of c-FLIP downregulation in mediating enhancement of TRAIL-induced apoptosis by LBH589 as demonstrated above, we further addressed how LBH589 decreased c-FLIP levels. Because c-FLIP proteins are known to be regulated by ubiquitin/proteasome-mediated degradation [23], [25], we then determined whether the observed downregulation of c-FLIP by LBH589 would be mediated via this process. Thus, we first examined whether LBH589 promotes c-FLIP degradation. To this end, we treated Panc-1cells with either DMSO or LBH589 for 4 h and then washed away the drug followed by refilling the cells with fresh medium containing the protein synthesis inhibitor CHX.

At the indicated times post CHX, the cells were harvested for Western blotting to analyze c-FLIP degradation rate. As presented in Fig. 6A, the reduction or degradation rate of FLIPL in LBH589-treated cells was apparently faster than that in DMSO-treated control cells, indicating that LBH589 indeed facilitates c-FLIP degradation. Next, we treated cells with LBH589 in the absence and presence of the proteasome inhibitor MG132 and then compared c-FLIP modulation under these conditions. As presented in Fig. 6B, LBH589 decreased c-FLIP levels in the absence of MG132, but not in the presence of MG132, suggesting that LBH589-induced c-FLIP degradation is proteasome-dependent. By immunoprecipitation/Western blotting, we also detected the highest levels of ubiqutinated FLIPL in cells treated with LBH589 plus MG132 compared to cells exposed to LBH589 alone or MG132 alone (Fig.

6C), indicating that HNK increases c-FLIP ubiquitination. Taken together, we conclude that LBH589 induces ubiquitin/proteasome-mediated c-FLIP degradation, leading to downregulation of c-FLIP in human pancreatic cancer cells. Figure 6 LBH589 reduces c-FLIP levels through ubiquitin/proteasome-mediated protein degradation. Discussion Human pancreatic cancer tumors or cell lines exhibit heterogeneous responses to TRAIL. Some of these tumors or cell lines are intrinsically insensitive to TRAIL-induced apoptosis [17], [18]. In this study, we have presented a novel finding that the histone deacetylase inhibitor LBH589 effectively augments TRAIL-induced apoptosis in human pancreatic cancer cells including those resistant to TRAIL-induced apoptosis.

Given that LBH589 shows anticancer activity in preclinical pancreatic cancer models [28] as well that the tumor-selective TRAIL is a potential cancer therapeutic protein and is being tested in phase I clinical trials, our findings warrant further evaluation on the combination of LBH589 and Drug_discovery TRAIL as a potential therapeutic regimens against pancreatic cancer in animal models and in clinical trials.