β-Thujaplicin Enhances TRAIL-Induced Apoptosis via the Dual Effects of XIAP Inhibition and Degradation in NCI-H460 Human Lung Cancer Cells.

Saki Seno,Takahiro Oyama,Aoi Terabayashi,Minori Kimura,Takehiko Abe,Yuki Yashiro,Ryoko Takasawa,Ryutaro Kimura,Mio Takahashi,Hideaki Abe,Kanae Adachi,Sei-Ichi Tanuma

doi:10.3390/medicines8060026

Abstract

Background: β-thujaplicin, a natural tropolone derivative, has anticancer effects on various cancer cells via apoptosis. However, the apoptosis regulatory proteins involved in this process have yet to be revealed. Methods: Trypan blue staining, a WST-8 assay, and a caspase-3/7 activity assay were used to investigate whether β-thujaplicin sensitizes cancer cells to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Additionally, western blotting was performed to clarify the effects of β-thujaplicin on X-linked inhibitor of apoptosis protein (XIAP) in NCI-H460 cells and a fluorescence polarization binding assay was used to evaluate the binding-inhibitory activity of β-thujaplicin against XIAP-BIR3. Results: β- and γ-thujaplicins decreased the viability of NCI-H460 cells in a dose-dependent manner; they also sensitized the cells to TRAIL-induced cell growth inhibition and apoptosis. β-thujaplicin significantly potentiated the apoptosis induction effect of TRAIL on NCI-H460 cells, which was accompanied by enhanced caspase-3/7 activity. Interestingly, β-thujaplicin treatment in NCI-H460 cells decreased XIAP levels. Furthermore, β-thujaplicin was able to bind XIAP-BIR3 at the Smac binding site. Conclusions: These findings indicate that β-thujaplicin could enhance TRAIL-induced apoptosis in NCI-H460 cells via XIAP inhibition and degradation. Thus, the tropolone scaffold may be useful for designing novel nonpeptidic small-molecule inhibitors of XIAP and developing new types of anticancer drugs.

Highlights

Since β-thujaplicin acts as a chelator of zinc ions [1], it may play an important role in the regulation of X-linked inhibitor of apoptosis protein (XIAP)-mediated apoptosis
Evaluation of the Combinatorial Effects of Drugs The combinatorial effects of TNF-related apoptosis-inducing ligand (TRAIL) and β- or γ-thujaplicin were evaluated using an excess-over-Bliss additivism (EOBA) model [22,23] with the following Formula: EOBA = C − [A + B − (A × B)], where C is equal to the fractional inhibition of both drugs simultaneously, A is equal to the fractional inhibition of drug A, and B is equal to the fractional inhibition of drug B, while fractional inhibition is equal to 1.0 minus the viability
We further investigated β-thujaplicin as a cotreatment, whether it could promote activation of caspase-3/7, which occurs during TRAIL-induced apoptosis in NCI-H460 cells. β-Thujaplicin cotreatment with TRAIL was shown to significantly enhance the increase in caspase-3/7 activity in NCI-H460 cells

Summary

Introduction

This research is the first to demonstrate that β-thujaplicin enhances TRAIL-induced apoptosis in NCI-H460 cells. As EOBA values >0.1 denote a synergistic combination [22,23], the data indicated that cotreatment with a relatively low concentration of TRAIL and 10 μM β- or γ-thujaplicin produced a synergistic antiproliferative effect on NCI-H460 cells (Figure 3c). Potentiating Effect of β-Thujaplicin on TRAIL-Induced Cell Death in NCI-H460 Cells Is Accompanied by Enhanced Caspase-3/7 Activity

Results

Conclusion