NF-κB inhibitor with Temozolomide results in significant apoptosis in glioblastoma via the NF-κB(p65) and actin cytoskeleton regulatory pathways

Abstract

Glioblastoma (GBM) is the most malignant brain tumor characterized by intrinsic or acquired resistance to chemotherapy. GBM tumors show nuclear factor-κB (NF-κB) activity that has been associated with tumor formation, growth, and increased resistance to therapy. We investigated the effect of NF-κB inhibitor BAY 11-7082 with Temozolomide (TMZ) on the signaling pathways in GBM pathogenesis. GBM cells and patient-derived GBM cells cultured in 3D microwells were co-treated with BAY 11-7082 and TMZ or BAY 11-7082 and TMZ alone, and combined experiments of cell proliferation, apoptosis, wound healing assay, as well as reverse-phase protein arrays, western blot and immunofluorescence staining were used to evaluate the effects of drugs on GBM cells. The results revealed that the co-treatment significantly altered cell proliferation by decreasing GBM viability, suppressed NF-κB pathway and enhanced apoptosis. Moreover, it was found that the co-treatment of BAY 11-7082 and TMZ significantly contributed to a decrease in the migration pattern of patient-derived GBM cells by modulating actin cytoskeleton pathway. These findings suggest that in addition to TMZ treatment, NF-κB can be used as a potential target to increase the treatment's outcomes. The drug combination strategy, which is significantly improved by NF-κB inhibitor could be used to better understand the underlying mechanism of GBM pathways in vivo and as a potential therapeutic tool for GBM treatment.

Figure 1

Representative images of the GBM tumor cells cultured in the PEGDA microwells. (a–c) LN229, U87 and patient-derived GBM cells were cultured in the microwells for 7 days, respectively. After day 7, Bay 11-7082 and TMZ were applied either alone or in combination onto the cell spheroids. Control group did not receive any treatment. The cells were cultured with or without drugs additional 7 more days. The images were taken on Day 1, Day 4 and Day 7 after the drug application to observe the disruption in the spheroids. Dotted black lines represent the edge of the tumor spheroid. Scale bars, 200 µm. (d) Bar graph showing trypan blue staining for cell viability of LN229, U87 and patient-derived GBM cells. (e–g) Spheroid size quantification was done using ImageJ for LN229, U87 and Patient-derived GBM cells, respectively. Two-tailed t-test followed by Wilcoxon test were done (GraphPad Prism v5). Data represent the mean ± SD of three biological replicates. * p < 0.05 and ** p < 0.01.

Figure 2

NF–kB activity in LN229, U87 and patient-derived GBM cell lines. (a) NF–kB p65 subunit activity in LN229, U87 and patient-derived GBM cell lines, respectively. The cells cultured with or without drugs for 7 days were collected from the microwells and subjected to ELISA. Data represent the mean ± SD of three biological replicates. * p < 0.05 and ** p < 0.01. (b) Representative immunoblots. LN229, U87 and patient-derived GBM cells were cultured with or without drugs for 7 days, lysed and immunoblotted with the indicated antibodies. Quantification of the fold-changes in protein levels (bottom panel). Data were normalized to B-actin. Data represent the mean ± SD of three biological replicates. * p < 0.05, ** p < 0.01.

Figure 3

The effect of Bay 11-7082 and TMZ on signaling pathways in patient-derived GBM cells. (a) Heat map presentation of RPPA analysis showing the changes in the protein expression. RPPA was performed on lysates treated with Bay 11-7082 and TMZ alone or in combination. All relative protein level data points were normalized to the control group. Red and green indicate up and down regulations, respectively in the heat map. The samples were run in duplicate (n = 3). (b) Fold change of the selected proteins relative to the control group via RPPA. Data represent the mean ± SD of two biological replicates. (* p < 0.05, ** p < 0.01, Wilcoxon rank sum test). (c) Analysis of down-regulated RPPA proteins shows a significant activation in numerous Enrichr KEGG pathways. The pathways were sorted by p value ranking. (d) Representative immunoblot validation of significantly altered proteins involved in different KEGG pathways. Patient-derived GBM cells were cultured with or without drugs for 7 days, lysed and immunoblotted with the indicated antibodies. Quantification of the fold-changes in protein levels (right panel). Data were normalized to B-actin. Data represent the mean ± SD of three biological replicates. * p < 0.05, ** p < 0.01.

Figure 4

Apoptosis assay (TUNEL). (a) Fluorescent images of TUNEL ( +) cells in patient-derived GBM cells. TUNEL assay was performed on cells treated with Bay 11-7082 and TMZ in combination or alone in the microwells. Cells were collected from the microwells, trypsinized and replated into 8-well chamber slides. TUNEL ( +) cells (green) with ring-like nuclear stain are indicated with red arrows. Nuclei were counterstained with DAPI (blue). (b) Numbers of TUNEL ( +) cells are presented as mean ± SD of three biological replicates. * p < 0.05 and ** p < 0.01. X20 objective. Scale bars, 100 µm.

Figure 5

Changes in the actin cytoskeleton and migration pattern in patient-derived GBM cells co-treated with Bay 11-7082 and TMZ or single drug treated in the microwells. (a) Upper panel; representative images of the patient-derived GBM cells co-treated with Bay 11-7082 and TMZ or single drug treated at day 1, stained with phalloidin (green) and DAPI (blue). Bottom panel; representative images of the patient-derived GBM cells co-treated with Bay 11-7082 and TMZ or single drug treated at day 7, stained with phalloidin and DAPI. Scale bars, 100 µm. (b) Intensity of staining obtained with phalloidin was measured in each cell using ImageJ and displayed as box-plots with 5 to 95% confidence intervals. A two-way ANOVA with Dunnett’s multiple comparisons test was performed to determine statistical relevance. Three biological replicates (n = 30). ** p < 0.01, *** p < 0.001. (c) Representative immunoblots show the levels of FAK pTyr397 and total FAK in patient-derived GBM cell lysates co-treated with Bay 11-7082 and TMZ or single drug treated for 7 days in the microwells. The levels of the proteins were quantified using ImageJ (right panel). Data were normalized to B-actin. Data represent the mean ± SD of three biological replicates. ** p < 0.01.

Figure 6

Cell migration of patient-derived GBM cells by wound healing assay. (a) Patient-derived cells were co-treated with Bay 11-7082 and TMZ or single drug treated in the microwells, trypsinized and replated in 24-well plates. After they reached to their confluency, a scratch wound was formed with a 200-μl tip and cells were incubated for the next 48 h. Images were taken (4x) at 0 h, 24 hr and 48 hr. Scale bars, 100 µm. (b) The wound width was measured with ImageJ and the average wound width was shown. Data represent the mean ± SD of three biological replicates. * p < 0.05 and ** p < 0.01 (one-way ANOVA with Tukey’s post hoc test).

Figure 7

Proposed schematic of the signaling pathways involved in Bay 11-7082 and TMZ-mediated inhibition in GBM patient-derived cells. The effect of combined therapy of Bay 11-7082 and TMZ was achieved through the inhibition of Src/FAK/Vinculin, which regulate the cytoskeleton organization through MAPKs, JNK and PI3K/AKT signaling pathways. Exposure to both Bay 11-7082 and TMZ also leads to receptor-mediated activation of Bax but not Bcl-2 in the subsequent inhibition of the downstream NF-κB transcription factor. Inhibition of NF-κB, in turn, causes cell death.