Mechanisms underlying reversed TRAIL sensitivity in acquired bortezomib-resistant non-small cell lung cancer cells

Abstract

Aim: The therapeutic targeting of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors in cancer, including non-small cell lung cancer (NSCLC), is a widely studied approach for tumor selective apoptotic cell death therapy. However, apoptosis resistance is often encountered. The main aim of this study was to investigate the apoptotic mechanism underlying TRAIL sensitivity in three bortezomib (BTZ)-resistant NSCLC variants, combining induction of both the intrinsic and extrinsic pathways. Methods: Sensitivity to TRAIL in BTZ-resistant variants was determined using a tetrazolium (MTT) and a clonogenic assay. A RT-qPCR profiling mRNA array was used to determine apoptosis pathway-specific gene expression. The expression of these proteins was determined through ELISA assays and western Blotting, while apoptosis (sub-G1) and cytokine expression were determined using flow cytometry. Apoptotic genes were silenced by specific siRNAs. Lipid rafts were isolated with fractional ultracentrifugation. Results: A549BTZR (BTZ-resistant) cells were sensitive to TRAIL in contrast to parental A549 cells, which are resistant to TRAIL. TRAIL-sensitive H460 cells remained equally sensitive for TRAIL as H460BTZR. In A549BTZR cells, we identified an increased mRNA expression of TNFRSF11B [osteoprotegerin (OPG)] and caspase-1, -4 and -5 mRNAs involved in cytokine activation and immunogenic cell death. Although the OPG, interleukin-6 (IL-6), and interleukin-8 (IL-8) protein levels were markedly enhanced (122-, 103-, and 11-fold, respectively) in the A549BTZR cells, this was not sufficient to trigger TRAIL-induced apoptosis in the parental A549 cells. Regarding the extrinsic apoptotic pathway, the A549BTZR cells showed TRAIL-R1-dependent TRAIL sensitivity. The shift of TRAIL-R1 from non-lipid into lipid rafts enhanced TRAIL-induced apoptosis. In the intrinsic apoptotic pathway, a strong increase in the mRNA and protein levels of the anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) and B-cell leukemia/lymphoma 2 (Bcl-2) was found, whereas the B-cell lymphoma-extra large (Bcl-xL) expression was reduced. However, the stable overexpression of Bcl-xL in the A549BTZR cells did not reverse the TRAIL sensitivity in the A549BTZR cells, but silencing of the BH3 Interacting Domain Death Agonist (BID) protein demonstrated the importance of the intrinsic apoptotic pathway, regardless of Bcl-xL. Conclusion: In summary, increased sensitivity to TRAIL-R1 seems predominantly related to the relocalization into lipid rafts and increased extrinsic and intrinsic apoptotic pathways.

Keywords: Bcl-2 family; TRAIL; bortezomib; cytokines; lipid rafts; resistance; sensitization.

Figure 1

Acquired BTZ-resistant A549 cells demonstrate reversed phenotypic TRAIL sensitivity. (A) The clonogenic outgrowth was significantly reduced in both the H460BTZR (not measurable) and A549BTZR cells after exposure to 100 ng/mL TRAIL for 6 h; (B) Western blot analysis of caspase and PARP cleavage shows the enhanced cleavage already after 6 h in the A549BTZR cells; (C) BID and XIAP were reduced in the A549BTZR cells after exposure to 100 ng/mL TRAIL for 6 and 24 h; (D) the ZVAD (100 µg/mL) completely reduced the TRAIL-enhanced apoptosis after 24 h exposure as determined with Flow Cytometry; (E) Incubation with 30 μM necrostatin for 24 h did not result in reduced TRAIL-induced apoptosis as determined with Flow Cytometry; (F) Expression of TRAIL in A549BTZR cells as determined with Flow cytometry. Similarly, the TRAIL expression was not different between parental and resistant variants of H460 and SW1573 cells. The values are the means ± SEM and the western blots depicted represent at least three independent experiments. Similarly, the effect of TRAIL was significantly higher in A549BTZR compared to A549 cells. ^**P < 0.001; ^*P < 0.01. BTZ: Bortezomib; TRAIL: TNF-related apoptosis-inducing ligand; TNF: tumor necrosis factor; BID: BH3 Interacting Domain Death Agonist; XIAP: X-linked inhibitor of apoptosis protein.

Figure 2

Basal protein levels were differently expressed in the A549 and A549BTZR cells. (A) Western blot analysis was used to detect the basal expression levels of several proteins in the A549 and A549BTZR cells. The blots are representative of at least three separate experiments. β-actin was used as a loading control; (B) Scans showing the ratio A549BTZ/A549. ^*P < 0.01.

Figure 3

Enhanced secretion of OPG by A549BTZR did not induce TRAIL sensitivity. (A) With the use of ELISA, the secreted OPG levels were measured in the supernatant of the different cell lines; ^*significantly different (P < 0.01). The secretion of OPG by both SW1573 variants was 0.41 pg/mL; (B) Cell death was measured in the TRAIL-resistant A549 and SW1573 cells after incubation with 900 pg/mL OPG for 4 h followed by the addition of 100 ng/mL TRAIL for 24 h. The effect of TRAIL was significantly higher than that of OPG (^*P < 0.01). The values are the means of at least three independent experiments ± SEM. OPG: Osteoprotegerin; TRAIL: TNF-related apoptosis-inducing ligand; TNF: tumor necrosis factor.

Figure 4

Different secretion products from the A549BTZR cells did not trigger TRAIL sensitivity. (A) Western blot analysis was used to detect the basal expression levels of caspase-1, -4 and -5 in A549 and A549BTZR cells; (B) Supernatant of the A549 and A549BTZR cells was used to determine the cytokine secretion. The cytokine secretion levels detected in the A549BTZR supernatant were compared with the secretion by A549 cells, which was set to 1 (calculated from the means); (C) Cell death (subG1) was measured in A549 cells exposed to 100 ng/mL TRAIL for 24 h in supernatant collected from the H460 and A549BTZR cells. As a control, H460 and A549BTZR cells were exposed to 100 ng/mL TRAIL for 24 h in fresh medium; (D) Cell death (SubG1) in the H460 and SW1573 cells was determined after exposure to 100 ng/mL TRAIL for 24 h in culture medium or supernatant collected from A549 cells. The values are the means ± SEM of at least three independent experiments, while the blots are representative of at least 3 separate experiments. The effect of TRAIL treatment on H460 and A549BTZR cells on induction of apoptosis (C and D) was significant (P < 0.001). TRAIL: TNF-related apoptosis-inducing ligand; TNF: tumor necrosis factor.

Figure 5

A549BTZR TRAIL-induced apoptosis was mediated via TRAIL-R1, which was redistributed into lipid rafts. (A) Cell death was determined in the A549 and A549BTZR cells after exposure to 100 ng/mL TRAIL, 50 ng/mL 4C7 (TRAIL-R1-specific TRAIL variant), or 50 ng/mL D269HE195R (TRAIL-R2-specific TRAIL variant) for 24 h. The effect of TRAIL and TRAIL-R1 was significant in A549BTZR cells compared to A549 parental (P < 0.0001); (B) Western blot analysis of the TRAIL-R1 and -R2 expression levels in different cellular fractions, with fractions 5-7 and fractions 8-10 representing the lipid and non-lipid rafts, respectively. The values are the means ± SEM of at least three independent experiments, while blots are also representative of at least 3 separate experiments. TRAIL: TNF-related apoptosis-inducing ligand; TNF: tumor necrosis factor.

Figure 6

The intrinsic apoptotic pathway was involved in the TRAIL-induced apoptosis in the A549BTZR cells, though reduced levels of Bcl-xL were not a determinant. (A) The silencing of Bcl-xL, as shown by western blot analysis, resulted in enhanced cell death in the A549 and SW1573 cells after 24 h exposure to 100 ng/mL TRAIL; (B) A549BTZR cells were transfected with a control puromycin vector or a Bcl-xL-containing vector, as shown by western blot analysis; (C) Cell death was measured in the A549BTZR cells and the transfected variants after incubation with 100 ng/mL TRAIL for 24 h; (D) Western blot analysis was used to detect the cleavage of caspase-8, -9, -3, and PARP after exposure to 100 ng/mL TRAIL for several time intervals in the A549BTZR and A549BTZR-Bcl-xL cells; (E) Cell death was determined in the H460 and H460-Bcl-xL cells after exposure to 10 ng/mL TRAIL for 24 h; (F) Cell death was measured in A549BTZR and A549BTZR-Bcl-xL cells after silencing of BID. ^***P < 0.001; ^**P < 0.01; ^*P < 0.05. TRAIL: TNF-related apoptosis-inducing ligand; TNF: tumor necrosis factor; Bcl-xL: B-cell lymphoma-extra large; BID: BH3 Interacting Domain Death Agonist.