Lambertianic Acid Sensitizes Non-Small Cell Lung Cancers to TRAIL-Induced Apoptosis via Inhibition of XIAP/NF-κB and Activation of Caspases and Death Receptor 4

Abstract

Lambertianic acid (LA) is a biologically active compound from the leaves of Pinus koraiensis. In the present study, apoptotic mechanisms of LA plus TNF-related apoptosis-inducing ligand (TRAIL) were elucidated in non-small cell lung cancer cells (NSCLCs). Cytotoxicity assay, flow cytometry, immunoprecipitation, and Western blotting were performed. Here, combined treatment of LA and TRAIL increased cytotoxicity, sub-G1 population, cleaved poly (ADP-ribose) polymerase (PARP), and caspase3/8/9 in A549 and H1299 cells compared to LA or TRAIL alone. Furthermore, combined treatment of LA and TRAIL significantly decreased antiapoptotic proteins such as B-cell lymphoma 2 (Bcl-2), Fas-like inhibitor protein (FLIP), and X-linked inhibitor of apoptosis protein (XIAP), and enhanced the activation of proapoptotic proteins Bid compared to LA or TRAIL alone. In addition, combined treatment of LA and TRAIL upregulated the expression of Death receptor 4 (DR4) and downregulated phosphorylation of nuclear factor κ-light-chain-enhancer of activated B cells (p-NF-κB), inhibitory protein of kB family (p-IκB), and FLIP in A549 and H1299 cells along with disrupted binding of XIAP with caspase3 or NF-κB. Overall, these findings suggest that lambertianic acid enhances TRAIL-induced apoptosis via inhibition of XIAP/NF-κB in TRAIL resistant NSCLCs.

Keywords: NF-κB; TRAIL; XIAP; apoptosis; lambertianic acid; non-small cell lung cancer.

Figure 1

Cytotoxicity of the combination of lambertianic acid (LA) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in A549 and H1299 non-small cell lung cancer cells. (a) Chemical structure of LA. Molecular weight = 316.43. (b) Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded onto 96 well microplates and treated with various concentrations of LA (0, 5, 10, 20 μM) and TRAIL (0, 20, 40, 80 ng/mL) for 24 h. The cytotoxic effects of LA (20 μM) and TRAIL (20 ng/mL) on TRAIL in A549 and H1299 cells. Data represent means ± SD. * p< 0.05, *** p < 0.001 versus LA-treated control (n = 3). (c) A549 cells were treated with LA (20 μM) and/or TRAIL (20 ng/mL) for 24 h and then stained with crystal violet. Data represent means ± SD. *** p < 0.001 versus TRAIL alone (n = 4).

Figure 2

Combined effect of LA and TRAIL on the sub-G1 population and apoptotic proteins in A549 and H1299 non-small cell lung cancer cells. (a) Cells were treated with LA (20 μM) and/or TRAIL (20 ng/mL) for 24 h. The treated cells were fixed with 70% ethanol, stained with propidium iodide (PI) and analyzed by flow cytometry with or without with caspase inhibitors (pan caspase inhibitor; z-VAD-fmk (80 μM), caspase-8 inhibitor; z-IETD-fmk (50 μM)). Bar graphs show quantification of cell cycle population (%). Data represent means ± SD. *** p < 0.001 versus TRAIL alone, # p < 0.05, ### p < 0.001 versus LA+TRAIL treated control. (n = 3)). (b) Cells were treated with LA (20 μM) and/or TRAIL (20 ng/mL) for 24 h. Cell lysates were prepared and subjected to Western blotting for procaspase-8,9,3, Pro-PARP, cleaved caspase-8,9,3, and cleaved-PARP. (c) Cells were treated with LA (20 μM) and/or TRAIL (20 ng/mL) for 24 h. The cells were stained using FITC-Annexin V/PI dye and early and late apoptotic portions were detected by flow cytometry.

Figure 3

Combined effect of LA and TRAIL on antiapoptotic and proapoptotic proteins in A549 and H1299 non-small cell lung cancer cells. (a,b) Cells were treated with LA (20 µM) and/or TRAIL (20 ng/mL) for 24 h. Cell lysates were prepared and subjected to Western blotting for Bid, Bcl-2, and XIAP.

Figure 4

Combined effect of LA and TRAIL on the expression of DR4, p-NF-κB, p-IκB, and FLIP in A549 and H1299 cells. Cells were treated with LA (20 µM) and/or TRAIL (20 ng/mL) for 24 h and cell lysates were prepared and subjected to Western blotting. (a) Combined effect of LA and TRAIL on the expression of DR4 and DR5 in A549 and H1299 cells. (b) Combined effect of LA and TRAIL on the expression of p-NF-κB and p-IκB in A549 and H1299 cells. (c) Combined effect of LA and TRAIL on the expression of FLIP, DcR1, and DcR2 in A549 and H1299 cells.

Figure 5

Combined effect of LA and TRAIL on XIAP interaction with caspase3 and NF-κB in A549 non-small cell lung cancer cells. (a) XIAP interacts with caspase3 and NF-κB in the STRING database. Red text (interaction score). (b) A549 cells were treated with LA (20 µM) and/or TRAIL (20 ng/mL) for 24 h. Immunoprecipitation (IP) was performed with lysates from A549 cells using anti-caspase3 and anti-NF-κB antibodies. Western blot analysis was performed to detect XIAP in whole cell lysates. Western blot analysis was performed to detect XIAP, Pro-cas3, and NF-κB in input lysates. Input lysates indicated that the 5% pre-immunoprecipitated samples and β-actin levels were confirmed as being equivalent to protein loading.