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. 2016;15(4):559-72.
doi: 10.1080/15384101.2015.1136038.

TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance in multiple myeloma through NF-кβ inhibition

Prahlad V Raninga  1   2 , Giovanna Di Trapani  1 , Slavica Vuckovic  3   4 , Kathryn F Tonissen  1   2
Affiliations

TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance in multiple myeloma through NF-кβ inhibition

Prahlad V Raninga et al. Cell Cycle. 2016.

Abstract

Multiple myeloma (MM) is a B-cell malignancy characterized by an accumulation of abnormal clonal plasma cells in the bone marrow. Introduction of the proteasome-inhibitor bortezomib has improved MM prognosis and survival; however hypoxia-induced or acquired bortezomib resistance remains a clinical problem. This study highlighted the role of thioredoxin reductase 1 (TrxR1) in the hypoxia-induced and acquired bortezomib resistance in MM. Higher TrxR1 gene expression correlated with high-risk disease, adverse overall survival, and poor prognosis in myeloma patients. We demonstrated that hypoxia induced bortezomib resistance in myeloma cells and increased TrxR1 protein levels. Inhibition of TrxR1 using auranofin overcame hypoxia-induced bortezomib resistance and restored the sensitivity of hypoxic-myeloma cells to bortezomib. Hypoxia increased NF-кβ subunit p65 nuclear protein levels and TrxR1 inhibition decreased hypoxia-induced NF-кβ p65 protein levels in the nucleus and reduced the expression of NF-кβ-regulated genes. In addition, higher TrxR1 protein levels were observed in bortezomib-resistant myeloma cells compared to the naïve cells, and its inhibition using either auranofin or TrxR1-specific siRNAs reversed bortezomib resistance. TrxR1 inhibition reduced p65 mRNA and protein expression in bortezomib-resistant myeloma cells, and also decreased the expression of NF-кβ-regulated anti-apoptotic and proliferative genes. Thus, TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance by inhibiting the NF-кβ signaling pathway. Our findings demonstrate that elevated TrxR1 levels correlate with the acquisition of bortezomib resistance in MM. We propose considering TrxR1-inhibiting drugs, such as auranofin, either for single agent or combination therapy to circumvent bortezomib-resistance and improve survival outcomes of MM patients.

Keywords: NF-кβ; Thioredoxin reductase; auranofin; bortezomib; drug resistance; hypoxia; multiple myeloma.

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Figures

Figure 1.
Figure 1.
Increased TrxR1 expression in MM patients correlates with decreased overall survival and poor prognosis. (A) TrxR1 expression in high-risk and low-risk MM patients was determined from the gene expression profiling data deposited in the gene expression omnibus database (GSE4581). Unpaired student t test was performed. P < 0.05 (compared to low-risk patients) (B) Overall survival of high-risk and low-risk patients receiving Total Therapy 2 and 3 was estimated by generating the Kaplan-Meier curve. (C) Overall survival was estimated in the patients with high and low TrxR1 expression levels receiving Total Therapy 2 and 3 by generating the Kaplan-Meier curve.
Figure 2.
Figure 2.
TrxR1 is upregulated in hypoxic myeloma cells and its inhibition overcomes hypoxia-induced bortezomib-resistance in myeloma cells. (A, B) RPMI8226 (A) and U266 (B) cells were cultured under normoxia (Nrx, 21% O2) and hypoxia (Hx, 1% O2) for 24 hours. Both cell lines were treated with bortezomib (0–40 nM) for 24 hours under normoxia and hypoxia and cell proliferation was analyzed by CellTiter blue assays. (C, D) RPMI8226 and U266 cells were cultured under hypoxia for indicated time periods. Total cell extracts were prepared, and HIF-1α and TrxR1 protein expression was analyzed by western blot analysis. β-tubulin was used as a loading control. Western blots are the representative of 3 independent experiments. (E) Hx-RPMI8226 and Hx-U266 cells were treated with auranofin (0–4 μM) for 24 hours under hypoxia (1% O2) and protein was extracted. The TrxR1 activity was analyzed by measuring the NADPH-dependent reduction of DTNB by TrxR1 enzyme and normalizing against the protein concentration. (F, G) RPMI8226 (F) and U266 (G) cells were cultured under normoxia (21% O2) and hypoxia (1% O2) for 24 hours. Both cell lines were treated with auranofin (0–8 μM) for 24 hours under normoxia and hypoxia and cell proliferation was analyzed by CellTiter blue assay. (H) Hx-RPMI8226 and Hx-U266 cells were treated with auranofin (2 μM) and bortezomib (10 nM) either alone or in combination for 24 hours under hypoxia. Cell proliferation was analyzed by CellTiter Blue assays. Values indicate mean ± SEM (n = 3). One-way ANOVA followed by Tukey's post-test were employed. *, P < 0.05.
Figure 3.
Figure 3.
TrxR1 inhibition overcomes hypoxia-induced bortezomib resistance by inhibiting NF-кβ signaling pathway. (A) U266 cells were cultured under hypoxia (1% O2) for indicated time periods. NF-κβ subunit p65 protein levels were analyzed by western blot analysis. (B) U266 cells were cultured under hypoxia for 24 hours and subsequently treated with auranofin (4 μM) for 4 hours, and cell proliferation was analyzed. (C) U266 cells were cultured under hypoxia for 0, 2, and 4 hours with or without auranofin (4 μM) treatment. NF-кβ subunit p65 protein levels were analyzed by western blot analysis. Lamin B1 (nuclear fraction) and β-tubulin (cytosolic fraction) were used as loading controls. Western blots are the representative of 3 independent experiments. (D) Hypoxic U266 cells were treated with 4 μM AF for 4 hours under hypoxia (1% O2). Expression of indicated NF-κβ-regulated genes was analyzed by RT-qPCR and normalized against L32. Values indicate mean ± SEM (n = 3). Unpaired student t test was employed. *, P < 0.05.
Figure 4.
Figure 4.
TrxR1 protein expression in the parent and bortezomib-resistant myeloma cells. (A-D)Parent and bortezomib-resistant RPMI8226 and U266 cells (RPMI8226-BR and U266-BR) were treated with indicated concentrations of bortezomib for 24 hours and cell proliferation (A, B) and caspase-3 activity (C, D) were measured. One-way ANOVA followed by Tukey's post-test was employed. *, P < 0.05 (compared to the 0nM bortezomib treatment) (E) TrxR1 protein levels in the parent and bortezomib-resistant myeloma cells were analyzed by western blot. β-tubulin was used as a loading control. Western blots are the representative of 3 independent experiments.
Figure 5.
Figure 5.
TrxR1 inhibition induces apoptosis in bortezomib-resistant myeloma cells. (A, B, C) RPMI8226-BR and U266-BR cells were treated with indicated concentrations of auranofin for 24 hours. The TrxR1 activity (A), cell proliferation (B), and apoptosis were assessed (C). Values indicate mean ± SEM of 3 independent experiments performed in triplicate. For caspase-3 activity assay (n = 3). One-way ANOVA followed by Tukey's post-test was employed. *, P < 0.0001 (compared to the 0 μM auranofin treatment). (D-G) RPMI8226-BR and U266-BR cells were transfected with 100 nmol/L of either control or TrxR1 specific siRNA. TrxR1 protein levels (48 hours) were analyzed by western blot (D, F). Cell viability was determined by using Trypan blue exclusion method 48 hours post-transfection (E, G). β-tubulin was used as a loading control. Western blots are the representative of 3 independent experiments. Values indicate mean ± SEM (n = 3). Unpaired student t test was employed. *, P < 0.05 (compared to the siControl).
Figure 6.
Figure 6.
TrxR1 inhibition sensitizes bortezomib-resistant myeloma cells to bortezomib and reverses chemoresistance. (A) PBMCs, RPMI8226-BR and U266-BR cells, and (B) parent RPMI8226 and U266 cells were treated with 2 μM auranofin (AF) and 10 nM bortezomib (Btz) either alone or in combination. Cell proliferation was analyzed by CellTiter Blue assay 48 hours post-treatment. (C, D) RPMI8226-BR and U266-BR cells were treated with 2 μM auranofin (AF) and 10 nM bortezomib (Btz) either alone or in combination. Apoptosis was assessed by measuring caspase-3 and caspase-9 activity 24 hours post-treatment in RPMI8226-BR (C) and U266-BR (D) cells. E, RPMI8226-BR and U266-BR cells, and F, parent RPMI8226 and U266 (F) cells were transfected with 100 nmol/L of either control or TrxR1 specific siRNA. Cells were treated with or without 10 nM bortezomib 4 hours post-transfection for 48 hours. Cell viability was measured by Trypan blue exclusion method. Values indicate mean ± SEM of 3 independent experiments performed in triplicate. For caspase-3 and 9 activity assay (n = 3). One-way ANOVA followed by Tukey's post-test was employed. * (compared to the untreated cells), Δ (compared to either AF alone or Btz alone panel B, C); (compared to either siTrxR1 alone or Btz alone panel D, E), P < 0.05.
Figure 7.
Figure 7.
TrxR1 inhibition suppresses NF-кβ signaling pathway in bortezomib-resistant myeloma cells. (A) NF-кβ subunit p65 protein levels were analyzed in the nuclear and cytosolic fractions of parent and bortezomib-resistant (BR) U266 and RPMI8226 cells by western blot analysis. (B) U266-BR cells were treated with the indicated concentrations of AF for 24 hours and with 4 μM AF for the indicated time periods. Expression of phospho-Iкβα was analyzed by western blot analysis. (C) NF-кβ p65 mRNA expression in U266-BR and RPMI8226-BR cells treated with or without 4 μM AF for 24 hours was analyzed by real time-qPCR (RT-qPCR). NF-кβ p65 mRNA expression was normalized against L32 housekeeping gene. (D) U266-BR cells were treated with AF (0–4 μM) for 24 hours. Nuclear, cytosolic, and total cell extracts were prepared and p65 protein levels were analyzed by western blot analysis. Lamin B1 and β-tubulin were used as loading controls for nuclear and cytosolic fractions, respectively. Western blots are the representative of 3 independent experiments. (E, F) U266-BR (E) and RPMI8226-BR (F) cells were treated with or without 4 μM AF for 24 hours. Expression of indicated NF-кβ-regulated genes was analyzed by RT-qPCR and normalized against L32. Values indicate mean ± SEM (n = 3). Unpaired student t test was employed. *, P < 0.05.
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