Differential bortezomib sensitivity in head and neck cancer lines corresponds to proteasome, nuclear factor-kappaB and activator protein-1 related mechanisms

Abstract

Head and neck squamous cell carcinomas (HNSCC) exhibit constitutive activation of transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), which are modulated by the proteasome and promote resistance to cell death. HNSCC show variable sensitivity to the proteasome inhibitor bortezomib in vitro as well as in murine xenografts and patient tumors in vivo, and the mechanisms are not well understood. To address this question, the sensitivities of nine HNSCC cell lines to bortezomib were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and the potential relationship between the sensitivity and bortezomib effects on biological processes was examined in HNSCC lines of differential bortezomib sensitivity. The most sensitive cell line (UM-SCC-11B) underwent cell death at 10(-9) mol/L in vitro and tumor regression at a maximally tolerated dose of bortezomib in a murine xenograft model. The differential sensitivity between UM-SCC-11A and UM-SCC-11B cells corresponded to differences in the extent of suppression of proteasome activity, ubiquitinated protein degradation, and NF-kappaB and AP-1 activation. Lower concentrations of bortezomib transiently increased NF-kappaB and sustained AP-1 activation in UM-SCC-11A cells. AP-1 reporter activity and cell density of UM-SCC-11A were suppressed when bortezomib was combined with c-Jun NH(2)-terminal kinase and p38 kinase pathways inhibitors. Thus, the differential sensitivities to bortezomib corresponded to dissimilar effects on the proteasome, NF-kappaB and AP-1 activities. Inhibition of c-Jun NH(2)-terminal kinase and p38 pathways blocked AP-1 activity and enhanced the antitumor effects. These findings revealed molecular mechanisms of bortezomib sensitivity and resistance, which are under development as biomarkers for clinical trials in patients with HNSCC.

Fig. 1. Sensitivity of UM-SCC cell lines and cultured normal human keratinocytes to bortezomib in vitro

(A) Nine UM-SCC cell lines and human normal keratinocytes were plated at 5X10³ cells per well in quadruplicates in the 96 well microplate. The next day, cells were exposed to bortezomib at different concentrations (day 0). Cells were labeled with MTT reagent for four hours at day one, two and three after treatment, and the optical density of labeled cells was measured the following day. The data are presented as one representative of repeated experiments showing mean+standard deviation calculated from quadruplicates. The IC₅₀ shown was calculated using the data from day 2. (B) Nuclear extracts were harvested from cultured UM-SCC cells, and NF-κB p65 binding activity was measured using a TransAM NF-κB binding kit with 10 μg of nuclear extract in each well. The data represent the mean plus standard deviation from triplicates. (C) Cytotoxic effects of bortezomib in UM-SCC-11 A and -11B cells were tested by trypan blue assay. UM-SCC-11A (left panel) and -11B cells (right panel) were plated in the T-25 flask in triplicates, and treated with bortezomib at 10^–9 and 10^–8M 48 hours later. At the each time point, both attached and detached cells were collected, stained with trypan blue dye, and counted. Total viable cells were calculated and presented.

Fig. 2. Anti-tumor activity of bortezomib in human UM-SCC tumor xenograft model

(A) UM-SCC-11A and -11B cells were injected subcutaneously at the dosage of 1.5x10⁷ cells/mouse over the flanks of immunodeficient BALB/c SCID mice. When the mean tumor volume reached ~0.3 cm³ in each tumor-bearing group, intraperitoneal injections of bortezomib (2.0 mg/kg/dose) were administered on a Monday, Wednesday, Friday schedule, with the time of each dose indicated by arrows. Tumors were measured, and tumor volume was calculated and presented as Mean+SE. Vehicle for compound delivery was DMSO in sterile PBS. (B) The tumors from UM-SCC-11B xenograft models were harvested 24 hours after bortezomib treatment, and H&E and TUNEL stainings were performed on the frozen sections. The photographs of controls (left panels) and bortezomib treated tumors (right panels) were taken under the light microscopy with 200X magnification.

Fig. 3. Bortezomib induced cell cycle arrest and apoptosis in UM-SCC-11A and -11B cells

UM-SCC-11A (A) and -11B cells (B) were treated with bortezomib at different concentrations, and harvested at 12, 24 and 96 hours after treatment. 10⁵ cells were collected and stained with propidium iodide for apoptosis and cell cycle analysis using cycleTEST Plus DNA reagent kit. Fluorescence intensity of 10,000 cells in each sample was measured by flow cytometry.

Fig. 4. Bortezomib differentially affected NF-κB and AP-1 reporter activities in UM-SCC-11A and -11B cells

UM-SCC-11A and -11B were transiently transfected with NF-κB (A–D) or AP-1 (E, F) luciferase reporter constructs. After transfection, cells were treated with bortezomib at 10^–8M for different time points. Cell lysates were harvested and analyzed for luciferase activity using Tropix Dual-Light reporter assay system. Each value were adjusted to controls and represented as the mean luciferase activity ± standard deviation from triplicates. For the positive controls, the cells were also co-transfected with DN IκBαMutant plasmid with a NF-κB luciferase reporter construct, and treated with 2000U/ml of human recombinant TNF-α for 24 hours (C and D).

Fig. 5. Bortezomib differentially inhibited the proteasome activity and protein ubiquitination in UM-SCC 11A and 11B cells

UM-SCC-11A (A) and -11B (B) cells were treated with bortezomib at different concentration, and the cell lysates were harvested 4 hours after treatment. The proteasome activity was measured, and the data were calculated and presented as the mean + standard deviation from the triplicates. Cells were treated with bortezomib at 10^–8 M and the cell lysates were harvested at different time points. The protein ubiquitination was assessed by the Western blot analysis with anti-ubiquitin antibody (C).

Fig. 6. Chemical JNK inhibitor SP600125 significantly inhibits AP-1 reporter activity and synergizes with bortezomib to inhibit cell proliferation in UM-SCC-11A cells

(A) UM-SCC-11A cells were plated in 24 well plates overnight and transfected with AP-1 and LacZ reporter constructs. After transfection, the cells were treated with 10 μM of SP600125 or SB203580 for four hours, and then treated with 10^–8M of bortezomib for 24 hours. The cells were harvested and reporter activity was measured as described previously. (B) UM-SCC-11A cells were plated in 96 well plates overnight, and chemical inhibitors were added four hours before bortezomib treatment. Then cells were labeled with MTT reagent for four hours at day one, three and five after treatment, and optical density of labeled cells was measured the following day.