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NF-κB axis induces fludarabine resistance by suppressing TXNIP expression in acute myeloid leukemia cells
Biochemical and Biophysical Research Communications xxx (2018) 1e8
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TLR4/NF-kB axis induces fludarabine resistance by suppressing TXNIP expression in acute myeloid leukemia cells Hangsak Huy a, b, Tae-Don Kim a, b, Won Sam Kim a, Dong Oh Kim a, Jae-Eun Byun a, c, Mi Jeong Kim a, Young-Jun Park a, b, Suk Ran Yoon a, b, Ji-Yoon Noh a, Jungwoon Lee a, Kyoo-Hyung Lee d, Inpyo Choi a, b, **, Haiyoung Jung a, * a
Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong-gu, Daejeon, 34141, Republic of Korea Department of Functional Genomics, University of Science and Technology, Yuseong-gu, Daejeon, 34113, Republic of Korea c Department of Biochemistry, School of Life Sciences, Chungbuk National University, Cheongju, 28644, Republic of Korea d Hematology and Oncology Sections, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 October 2018 Accepted 7 October 2018 Available online xxx
Overcoming drug resistance is one of key issues in treating refractory acute myeloid leukemia (AML). The Toll-like receptor 4 (TLR4) signaling pathway is involved in many aspects of biological functions of AML cells, including the regulation of pro-inflammatory cytokine products, myeloid differentiation, and survival of AML cells. Thus, targeting TLR4 of AML patients for therapeutic purposes should be carefully addressed. In this regard, we investigated the possible role of TLR4 as a regulatory factor against fludarabine (FA) cytotoxicity activity. Here, we identified the differential expression of TLR4 and CD14 receptors in AML cell lines and examined their relationship to FA sensitivity. We found that the stimulation of TLR4 with lipopolysaccharide (LPS) in a TLR4-expressing cell line, THP-1, increased cell viability under FA treatment condition and showed that TLR4 stimulation overcame FA sensitivity through the activation of NF-kB, which subsequently upregulated several anti-apoptotic genes. The inhibition of TLR4/NF-kB signaling could partially or completely reverse LPS-induced cell survival under FA treatment conditions. Interestingly, we found that the expression of thioredoxin-interacting protein (TXNIP), a well-known tumor suppressor, was induced by FA treatment; however, it was suppressed by LPS treatment. Furthermore, the expression level of TXNIP was critical for FA-induced cytotoxicity or LPS-induced FA resistance of THP-1 cells. Our data suggest that TXNIP plays an important role in FA-induced cytotoxicity and TLR4/NF-kB-mediated FA resistance of AML cells. Therefore, TXNIP may be a potential therapeutic target for AML treatment. © 2018 Elsevier Inc. All rights reserved.
Keywords: Fludarabine TLR4 NF-kB TXNIP AML
1. Introduction Acute myeloid leukemia (AML) is a heterogeneous clonal disorder disease, characterized by uncontrolled proliferation of immature abnormal blast cells that commonly occurs in adult patients. Up to 60% of adult patients with AML were diagnosed with genetic abnormalities [1] and, subsequently, multiple dysregulated
* Corresponding author. ** Corresponding author. Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong-gu, Daejeon, 34141, Republic of Korea. E-mail addresses: [email protected] (I. Choi), [email protected] (H. Jung).
genes, such as p53, BCL2 family, and nuclear factor NF-kB, that led to tumor maintenance and chemoresistance [2]. However, despite advanced understanding of the molecular pathogenesis of AML, chemotherapeutic treatment remains a major challenge due to diverse prognostic outcomes with high rate of mortality in overall survival (<50%) and low relapse-free survival [3]. Fludarabine (FA), a purine nucleoside analogue, is an anti-metabolite small molecule that kills the malignant cells by inhibiting RNA/DNA synthesis and DNA repair through multiple mechanisms [4]. FA is an essential component in therapy for chronic lymphocytic leukemia (CLL) and AML [5]. In an in vitro study using AML lines and primary cells, the cytotoxicity effect of FA was significantly elevated when combined with other agents such as DNA alkylating agents [6] or histone
https://doi.org/10.1016/j.bbrc.2018.10.047 0006-291X/© 2018 Elsevier Inc. All rights reserved.
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deacetylase inhibitors (HDACI) [7,8]. Regimens containing FA have generated remarkable improvement of relapse-free survival in AML patients [9]. Toll-like receptors (TLRs) that play a critical role in initiating innate and adaptive immune response are expressed not only in immune cells but also in malignant cells, including hematopoietic malignancies [10]. TLRs recognize both pathogen-associated molecule patterns (PAMPs) and damage-associated molecule patterns (DAMPs), and activation of TLRs is involved in normal hematopoiesis and specific hematologic pathologies [11]. In contrast to the antitumor activity of TLRs agonists against various cancer cells, including leukemia, negative feedback associated with cancer survival, proliferation, metastasis, and drug resistance were also observed [11]. Some receptor complexes, such as TLR1/2, TLR2/6 or TLR9, play important roles in protecting cells from death induced by FA in CLL primary cells [12]. Interestingly, higher expression of TLRs was observed in AML patients without responding to chemotherapy treatment compared to patients with complete remission who had low expression of TLRs [10]. In particular, the expression of TLR4 was observed in some types of primary AML cells [10]; however, its role against chemotherapeutic drugs such as FA and its underlying mechanism upon activation in these cells have not fully elucidated yet. Thioredoxin-interacting protein (TXNIP) is well known as a tumor suppressor gene (TSG) and plays a crucial role in mediation of cell apoptosis and cycle arrest of tumor cells [13e15]. However, TXNIP was frequently downregulated in various cancer including AML and the decreased TXNIP expression in AML cells was mainly due to epigenetic silencing caused by the polycomb repressive complex 2 (PRC2) that directly mediated TXNIP silencing [16]. Reactivation of TXNIP in cancer cells by anti-cancer drugs or epigenetic modification agents increased anti-tumor effect of TXNIP [13,16,17]. However, the function of TXNIP on FA-induced apoptosis of AML cells is not clearly elucidated yet. In this study, we found that TLR4 inhibited FA-induced cytotoxicity by inducing NF-kB activity and its target genes, which were implicated in modulation of cell apoptosis and drug resistance in a TLR4-expressing AML line. In addition, we revealed that the expression of TXNIP was regulated by FA or LPS treatment and was important for FA-induced cytotoxicity or LPS-induced cell survival of AML cells. Altogether, our data suggest that TLR4/NF-kB induces FA resistance by suppressing TXNIP expression, and TXNIP may be a new potential therapeutic target for AML treatment.
from Santa Cruz Biotechnology. FACS antibodies, TLR4-APC (ab155343) was purchased from Abcam; and CD14-FITC (555397), IgG-FITC (349041) and IgG-APC (554686) from BD Biosciences. 2.3. Cell viability and apoptosis analysis Cell viability and apoptosis analysis were performed in the same experimental condition with 1 h of prior-stimulation with LPS (1 mg/ml), followed by 24 h of treatment with FA, unless otherwise stated. For TLR4 inhibition, cells were prior incubated with 30 mg/ ml LPS-RS (RS) for 4 h or 10 mM IAXO for 2 h before LPS or FA treatment. For IKK/NF-kB inhibition, cells were prior-incubated with celastrol (Cel) 0.5 mM for 2 h before LPS or FA treatment. Cell viability was accessed using Cell Counting Kit-8 (CCK-8) from Dojindo Molecular Technologies, Inc. Briefly, 2 104 cells/100 ml were seeded in 96-well plate and pre-incubated for 24 h at 37 C. Cells were then pre-stimulated and treated with LPS and FA. Then, 10 ml CCK-8 was added to each well and incubated for 3 h at 37 C in the cell culture incubator. Plates were measured at O.D. 450 nm using a SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices). Apoptotic cells were assessed using AnnexinV/PI kit (556547, BD Pharmigen) and FACSCanto II (BD Biosciences) following the manufacturer's instructions. 2.4. Quantitative real-time PCR
Cell lines used in this study, including HL-60, THP-1, CCRF-CEM and MOLT-4 were obtained from Korea cell line bank; JURKAT and 293T lines were from American Type Culture Collect (ATCC). Leukemia and 293T lines were cultured in RPMI 1640 medium and DMEM (WELGENE Inc., Gyeongsan, Korea) supplemented with 10% fetal serum albumin and antibiotic-antimycotic (Gibco) at 37 C in a humidified atmosphere of 5% CO2 in air.
mRNA expression was analyzed by quantitative real-time PCR (qPCR). Briefly, total RNA was extracted using RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. 1 mg of each RNA sample was used for the reverse-transcriptase PCR using a First-Strand cDNA synthesis kit (FSK-101, Toyobo). cDNAs were diluted 1:10 and submitted to qPCR using the SYBR Premix ExTaq (Takara Bio) and Applied Biosystems ViiA 7 Real-Time PCR system (Thermo Fisher Scientific). The human primer sequences were as follows: GAPDH forward 50 -GAGTCAACGGATTTGGTCGT-30 and reverse 50 -TTGATTTTGGAGGGATCTCG-3’; TLR4 forward 50 TCATTGGTGTGTCGGTCCTC-30 and reverse 50 -CTGCCAGGTCTGAGCAATCT-3’; CD14 forward 50 -CTCTGTCCCCACAAGTTCCC-30 and reverse 50 -GGATTCCCGTCCAGTGTCAG-3’; XIAP forward 50 GGCATTTCCAGATTGGGGCT-30 and reverse 50 -TTTGTAGACTGC GTGGCACT-3’; cIAP2 forward 50 -GTCACTCCCAGACTCTTTCCA-30 and reverse 50 -GGGCTGGCATGAGACTTCTT-3’; Survivin forward 50 ACGACCCCATGCAAAGGAAA-30 and reverse 50 -CTGGTAAGCCCGGGAATCAA-3’; MLIAP forward 50 -CCTGCTCCGGTCAAAAGGAA-30 and reverse 50 -GCTCAAGAACCCACCACGC-3’; BCL2 forward 50 CCTTTGTGGAACTGTACGGC-30 and reverse 50 -CCGGCCAACAACATGGAAAG-3’; BCL2A1 forward 50 -CTGCAGTGCGTCCTACAGAT-30 and reverse 50 -TTGTGGGCCACTGACTCTAC-3’; BCL-xl forward 50 GCTTTGAACAGGATACTTTTGTGGA-30 and reverse 50 -AGGGAGGCTAAGGGGTAAGG-3’; BCL-w forward 50 -CCTGACCCGTGAGATCCCTA-30 and reverse 50 -CCACCAGTGGTTCCATCTCC-3’; MCL-1 forward 50 -CGACTTTTGGCCACCGGC-30 and reverse 50 -TAGCCAGTCTTTTGTCC-3’; and TXNIP forward 50 -AAGACCAGCCAACAGGTGAG-30 and reverse 50 -AGGAAGCTCAAAGCCGAACT-3’. GAPDH was used for normalization of target genes.
2.2. Reagents and antibodies
2.5. Western blot
Reagents acquired from Sigma-Aldrich were fludarabine (F9813) and LPS (L2880); from InvivoGen, celastrol (anti-cls) and LPS-RS (tlrl-rslps); and Adipogen Life Sciences, IAXO-101 (IAXO) (IAX600-001). Western blot antibodies PARP (9542), caspase-3 (9665), NF-kB p65 (8242), phospho-NF-kB p65 (3033), IkB-a (2682), p-IkBa (2859), BCL2A1 (14093), cIAP2 (3130), and TXNIP (14715) were purchased from Cell Signaling Technology; b-actin (sc-47778) was
Protein expression and phosphorylation were analyzed by Western blot. Protein lysates were extracted using 0.1% NP40 lysis buffer supplemented with protease and phosphatase inhibitors (Calbiochem) as previously described [18,19]. Briefly, all lysates were quantified and loaded at the same amount on 8e15% SDSPAGE gels before transferring to PVDF membranes (Millipore). Membranes were blocked with 5% skim milk and incubated with
2. Materials and methods 2.1. Cell lines and culture
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primary antibodies overnight at 4 C. Bound proteins were detected using peroxidase-conjugated anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch). Proteins were then visualized using EzWestLumiOne (ATTO) or SuperSignal West Pico Chemilumicesent (Thermo Scientific) substrate and WSE-6200 LuminoGraph II Imaging System (ATTO). b-actin was used as a loading control.
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3.2. LPS increases anti-apoptotic activity through the TLR4/NF-kB signaling
293T cells were transiently co-transfected using PLUS and Lipofectamine (Invitrogen) with cytomegalovirus-Renilla luciferase construct (pRL-CML) (Promega) and TXNIP-Luciferase reporter plasmid (pTXNIP-luc) as previously described [20], with or without HA tagged p65 and HA tagged IkB-a plasmid. A total of 100 ng of each plasmid DNA was used in this study. Transfected cells were cultured for 48 h before lysis and detection using a luciferase assay kit (E1500, Promega) according to the manufacturer's recommendations. Firefly luciferase and Renilla luciferase activity were measured by Dual Luciferase Reporter Assay System (Promega) on a Luminometer (Turner Designs). Luciferase activity was expressed as the value of the mean fluorescence intensity (MFI).
Next, we investigated the roles of NF-kB on LPS-induced FA resistance in THP-1 cells, because it is well known as a downstream component of TLR signaling and a transcription factor for many genes, including members of the inhibitor of apoptosis protein (IAP) and B-cell lymphoma-2 (BCL-2) families [21]. Moreover, NF-kB was reported as a target molecule of FA in human T-cell leukemia virus type 1 (HTLV-1)-infected cells [22]. As expected, TLR4 stimulation accelerated IkB-a phosphorylation or degradation, and phosphorylated NF-kB (p65) in a time- and concentration-dependent manner (Fig. 2AeB). Interestingly, some NF-kB target genes were highly induced by LPS treatment, including BCL2A1 (>1000 fold), cIAP2 (>50 fold), and BCL-xl (>5 fold) (Fig. 2C). In addition, the expression of these target genes under LPS treatment were observed at transcriptional and post-translational levels in a time- and concentration-dependent manner (Fig. 2DeE). However, TLR4 blocked with LPS-RS, a TLR4 antagonist, attenuated the LPS-induced phosphorylation of IkB-a and NF-kB and the expression of NF-kB target genes (Fig. 2FeG). These results implied that LPS might induce FA resistance by activating TLR4/NF-kB signaling pathways.
2.7. Electroporation
3.3. Inhibition of TLR4 or NF-kB restores FA-induced cytotoxicity
2.6. TXNIP luciferase assay
Amaxa nucleofector kit (VCA-1003) and Nucleofactor 2b device from Lonza were applied for knockdown of TXNIP in THP-1 cells. TXNIP siRNA and control RNAi were acquired from Santa Cruz Biotechnology. A total of 1 10^6 cells/sample was electroporated with 200 nM of siRNA using the V-01 program and cultured for 24 h before another 24 h of treatment with FA. 2.8. Statistical analysis All experiments were performed three times with triplication in each sample and representative data are shown, unless otherwise stated. Data collected from each experiment were expressed as the means ± SD. For statistical analysis of the data, p values were analyzed using an unpaired t-test in the Prism program. A p value < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 3. Results 3.1. TLR4-dependent resistance to FA-induced cytotoxicity in AML cells To investigate the function of TLR4 signaling on FA-induced apoptosis in leukemia cells, we examined the expression of TLR4 and CD14 in leukemia cell lines including AML cells (HL-60 and THP-1) and acute lymphocytic leukemia (ALL) cells (CCRF-CEM, JURKAT and MOLT-4). Remarkably, THP-1 cells were observed to have the highest expression of TLR4 and CD14 receptors and genes compared to other leukemia lines (Fig. 1AeB). Next, to identify the effect of TLR4 stimulation on FA-induced cytotoxicity, we stimulated the TLR4 receptor with lipopolysaccharide (LPS), a TLR4 agonist. LPS significantly increased the cell viability of THP-1 cells but not in other cell lines (Fig. 1C). To understand the function of TLR4 in FA-induced apoptosis of THP-1 cells, we analyzed apoptotic phenotypes using AnnexinV/PI staining (Fig. 1D) and immunoblotting of cleaved poly(ADP-ribose) polymerase (PARP) and caspase-3 (Fig. 1E) by LPS treatment. These results indicated the negative role of TLR4 signaling against FA-induced cytotoxicity in TLR4-expressing cells.
To prove the effect of TLR4/NF-kB signaling on FA-induced cytotoxicity, we applied IAXO, an antagonist for CD14/TLR4 and celastrol, an IKK/NF-kB inhibitor. Notably, prior incubation with IAXO significantly attenuated LPS activity against FA-induced cytotoxicity and restored FA sensitivity in THP-1 cells (Fig. 3AeC). Consistent with this, prior incubation with celastrol completely inhibited LPS-induced phosphorylation of IkB-a and NF-kB and recovered FA cytotoxicity against LPS activity (Fig. 3DeF). These results showed that the TLR4/NF-kB axis is a critical mediator of LPS-induced FA resistance in AML cells. 3.4. FA-induced cytotoxicity depends on TXNIP expression We and other research groups previously reported that TXNIP was a tumor suppressor and regulator of oxidative stress in malignant cells [18,23]. TXNIP expression is upregulated by various environmental stresses, transforming growth factor-b and vitamin D3 [13]. Moreover, several epigenetic modifiers, such as LAQ-824, SAHA and DZNep, have been reported to induce TXNIP expression and mediate oxidative stress-induced apoptosis in malignant cells [7,16,17]. Here, we found that FA induced TXNIP expression in THP1 cells in a time- and concentration-dependent manner at transcriptional and post-transcriptional levels (Fig. 4AeB), and its expression was partially blocked from LPS-induced down-regulation by TLR4 inhibition (Fig. 4C). Interestingly, FA-induced TXNIP expression was strongly suppressed by LPS treatment either prior to or after FA treatment, and its expression was parallel to the phosphorylation of IKB-a and NF-kB (Fig. 4DeE). Moreover, inhibition of NF-kB by celastrol prevented TXNIP down-regulation induced by LPS treatment (Fig. 4F). To gain direct evidence that NF-kB was indeed a negative regulator of TXNIP expression, we performed luciferase assays using 293T cells. Notably, luciferase activity of TXNIP promoter was dramatically decreased by p65 overexpression or rescued by IkB-a overexpression (Fig. 4G). Collectively, these findings suggested that NF-kB was a negative and upstream regulator of TXNIP expression. To validate the role of TXNIP expression in FA-induced cytotoxicity, we suppressed TXNIP expression with TXNIP siRNA (Fig. 4H) and analyzed the apoptosis of THP-1 cells (Fig. 4I). These results showed that TXNIP played an important role in FA-induced cytotoxicity of THP-1 cells.
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Fig. 1. TLR4-dependent resistance to FA-induced cytotoxicity in AML cells. A, Representative histograms of surface receptors analyzed by FACS. Gray-filled lines are receptors stained positively with anti-TLR4 or anti-CD14 antibodies, and non-filled lines indicate negative staining with anti-IgG control antibodies. B, TLR4 and CD14 mRNA expression. C, Cell viability. Apoptotic cells (D) and protein marker (E) of THP-1 cells. #, denoted for RNA amplification value below detection; FL, full length; CL, cleavage protein. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
4. Discussion In this study, we reported the expression of TLR4 and CD14 in AML lines, but not ALL lines. Notably, only THP-1 cells were protected from FA-induced cytotoxicity upon TLR4 stimulation with LPS, revealing it to be cell-context dependent. Further, we confirmed a critical role of TLR4/NF-kB activation against FAinduced cytotoxicity in THP-1 cells, and we found that TXNIP gene expression was a common target for TLR4/NF-kB activation and FA induction. NF-kB is a key player downstream of all TLRs, making it more attractive for therapeutic drug development for treatment of various diseases [24,25]. Constitutive NF-kB activation was observed in AML blasts and other hematopoietic cancers [26]. In AML cells, aberrant activation of NF-kB was the result of mutation or activation of critical oncogenes such as CCAAT/enhancer-binding protein alpha, Ataxia Telangiectasia Mutated, runt-related
transcription factor 1, or fms-like tyrosine 3, which directly or indirectly interact with NF-kB and activate its pathway [27]. In human myeloid monocytic-leukemia THP-1 cells, NF-kB activation following TLR4 stimulation by LPS was reported [28] and contributed to increase in expression of multiple genes, including various pro-inflammatory cytokines and TLR4, which played roles in inflammatory disease [29]. However, the role of TLR4/NF-kB against anti-cancer drugs as FA was not clear. Here, we showed that NF-kB was activated by TLR4 stimulation and played a key role in determining cell viability or apoptosis in response to FA treatment. The activation of NF-kB was associated with IkB-a phosphorylation and high expression of downstream targets, anti-apoptotic genes including BCL2A1, cIAP2 and BCL-xl, after TLR4 stimulation. Consistently, TLR4 antagonists, LPS-RS or IAXO, attenuated TLR4 signaling and maintained FA sensitivity, which is associated with decreased NF- kB activation and target gene expression. Strikingly, NF-kB activity against FA-induced cytotoxicity was completely
Please cite this article in press as: H. Huy, et al., TLR4/NF-kB axis induces fludarabine resistance by suppressing TXNIP expression in acute myeloid leukemia cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.047
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Fig. 2. LPS increases anti-apoptotic activity through TLR4/NF-kB signaling pathway. Protein phosphorylation in a time-dependent (A) and concentration-dependent manner (B). C, mRNA expression after 4 h of LPS treatment. D, mRNA and protein expression after LPS (1 mg/ml) treatment. E, mRNA expression. Protein (F) and mRNA (G) expression after incubation with TLR4 antagonist (RS) and LPS. ns means not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Please cite this article in press as: H. Huy, et al., TLR4/NF-kB axis induces fludarabine resistance by suppressing TXNIP expression in acute myeloid leukemia cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.047
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Fig. 3. Inhibition of TLR4 or NF-kB restores FA-induced cytotoxicity. Representative cytograms (A) and data (B) of apoptotic cells after incubation with TLR4/CD14 antagonist (IAXO), LPS and FA. C, Cell viability. D, Protein phosphorylation analysis after 2 h of prior incubation with IKK/NF-kB inhibitor (Cel), followed by LPS treatment. Representative cytograms (E) and data (F) of apoptotic cells after incubation with Cel, LPS and FA. **P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Please cite this article in press as: H. Huy, et al., TLR4/NF-kB axis induces fludarabine resistance by suppressing TXNIP expression in acute myeloid leukemia cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.047
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Fig. 4. FA-induced cytotoxicity depends on TXNIP expression. A, TXNIP expression in a time-dependent manner with FA incubation. B, TXNIP mRNA and protein expression in a concentration-dependent manner with LPS (1 h) treatment. C, TXNIP expression after 4 h of prior incubation with TLR4 antagonist (RS), followed by 1 h with LPS treatment. D, Protein expression and phosphorylation after prior incubation with LPS (1 mg/ml), followed by FA treatment. E, TXNIP mRNA and protein expression after prior incubation with FA (25 mM/ml), followed by LPS (1 mg/ml) treatment. F, TXNIP expression after 2 h of prior incubation with celastrol (Cel), followed by 1 h with LPS treatment. G, Luciferase promoter assay analysis. H, Transfection efficiency confirmed by Western blotting 2 days after electroporation. I, Representative data of apoptotic cells. ns means not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
blocked by the pre-incubation with the IKK/NF-kB inhibitor celastrol. These results highlighted the critical role of NF-kB signaling as a survival regulator of TLR4 stimulation. In comparison to the previous report, the NF-kB/XIAP pathway is the survival signal of some human T-cell leukemia (ATL) lines and was indeed inhibited by FA treatment [22]. Moreover, the increase of FA activity by HDACI was the result of the suppression of the NF-kB/XIAP pathway and subsequently induced apoptosis in AML cells [30]. However, we could not find the induction of XIAP expression at the time point of TLR4 stimulation in this study (Fig. 2C). Thus, targeting NF-kB with BCL2A1 and/or cIAP2 would be an alternative target for treatment of AML with constitutive TLR4 or NF-kB activation, and the combination of FA and NF-kB inhibitor would be useful. Also, further investigation of BCL2A1 and cIAP2 in AML cells would be interesting, as they have not well studied compared to BCL2 or XIAP [2,31]. Although NF-kB was regulated following TLR4 stimulation with LPS, we could not exclude other possible factors, such as AKT, ERK, or c-JUN, as their activation was modulated by LPS or celastrol treatment [32,33] and observed in our experiment (data not shown).
TXNIP, which is known as a TSG, is frequently downregulated in various cancer cells [23] and plays a crucial role in mediation of cell apoptosis and cycle arrest [13,14]. In cDNA sequencing data from AML blasts of 270 patients, mutation of TXNIP was relatively infrequent [34]; thus, decreasing TXNIP in AML cells might be induced mainly by epigenetic silencing. In AML primary cells and lines, TXNIP expression was reported to be 11-fold and 5-fold lower compared to normal blasts, and this low expression was silenced by the PRC2 protein complex [16]. In addition, a pronounced expression of TXNIP in AML cells was upregulated when treated with a HDACI, 3-Deazaneplanocin A, which suppressed PRC2 and was associated with anti-tumor activity of TXNIP [16]. Here, we reported that the expression of TXNIP was dramatically upregulated by FA treatment at both the protein and RNA levels (Fig. 4). Moreover, we also confirmed that silencing of TXNIP by siRNA significantly decreased FA-induced cytotoxicity in THP-1 cells (Fig. 4). These findings suggest a role for TXNIP in FA-induced cytotoxicity in THP-1 cells. TLR4 or NF-kB activation downregulated TXNIP expression and suppressed FA-induced cytotoxicity, and pretreatment of TLR4 or NF-kB inhibitor could prevent down-regulation of
Please cite this article in press as: H. Huy, et al., TLR4/NF-kB axis induces fludarabine resistance by suppressing TXNIP expression in acute myeloid leukemia cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.047
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TXNIP and FA sensitivity (Fig. 4). In our previous report, we showed the correlation between TXNIP and NF-kB, and TXNIP inhibited NFkB activity via physical interaction with p65 [18]. Furthermore, we also showed that the expression of TXNIP was reduced by TNF-amediated NF-kB activation while overexpression of TXNIP robustly suppressed NF-kB activation [35]. In Fig. 4G, we showed that overexpression of NF-kB suppressed TXNIP promoter activity, while co-transfection with an IKB-a plasmid rescued TXNIP expression. These results suggest that NF-kB is an upstream regulator of TXNIP, and NF-kB may directly or indirectly regulate the transcription of TXNIP. Further study is needed to prove the transcriptional regulation of TXNIP by NF-kB. In conclusion, we unveil a novel role of the TLR4/NF-kB axis against FA-induced cytotoxicity through suppression of TXNIP and expression of anti-apoptotic genes in THP-1 cells. From these results, we suggest that TXNIP may be a new therapeutic target for AML therapy. Acknowledgments This work was supported in part by the National Research Council of Science and Technology (NST) grant (No. CRC-15-02KRIBB), and the KRIBB Research Initiative Program from the Korea government (MSIP).
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Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.10.047.
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TLR4/NF-kB axis induces fludarabine resistance by suppressing TXNIP expression in acute myeloid leukemia cells Hangsak Huy a, b, Tae-Don Kim a, b, Won Sam Kim a, Dong Oh Kim a, Jae-Eun Byun a, c, Mi Jeong Kim a, Young-Jun Park a, b, Suk Ran Yoon a, b, Ji-Yoon Noh a, Jungwoon Lee a, Kyoo-Hyung Lee d, Inpyo Choi a, b, **, Haiyoung Jung a, * a
Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong-gu, Daejeon, 34141, Republic of Korea Department of Functional Genomics, University of Science and Technology, Yuseong-gu, Daejeon, 34113, Republic of Korea c Department of Biochemistry, School of Life Sciences, Chungbuk National University, Cheongju, 28644, Republic of Korea d Hematology and Oncology Sections, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 October 2018 Accepted 7 October 2018 Available online xxx
Overcoming drug resistance is one of key issues in treating refractory acute myeloid leukemia (AML). The Toll-like receptor 4 (TLR4) signaling pathway is involved in many aspects of biological functions of AML cells, including the regulation of pro-inflammatory cytokine products, myeloid differentiation, and survival of AML cells. Thus, targeting TLR4 of AML patients for therapeutic purposes should be carefully addressed. In this regard, we investigated the possible role of TLR4 as a regulatory factor against fludarabine (FA) cytotoxicity activity. Here, we identified the differential expression of TLR4 and CD14 receptors in AML cell lines and examined their relationship to FA sensitivity. We found that the stimulation of TLR4 with lipopolysaccharide (LPS) in a TLR4-expressing cell line, THP-1, increased cell viability under FA treatment condition and showed that TLR4 stimulation overcame FA sensitivity through the activation of NF-kB, which subsequently upregulated several anti-apoptotic genes. The inhibition of TLR4/NF-kB signaling could partially or completely reverse LPS-induced cell survival under FA treatment conditions. Interestingly, we found that the expression of thioredoxin-interacting protein (TXNIP), a well-known tumor suppressor, was induced by FA treatment; however, it was suppressed by LPS treatment. Furthermore, the expression level of TXNIP was critical for FA-induced cytotoxicity or LPS-induced FA resistance of THP-1 cells. Our data suggest that TXNIP plays an important role in FA-induced cytotoxicity and TLR4/NF-kB-mediated FA resistance of AML cells. Therefore, TXNIP may be a potential therapeutic target for AML treatment. © 2018 Elsevier Inc. All rights reserved.
Keywords: Fludarabine TLR4 NF-kB TXNIP AML
1. Introduction Acute myeloid leukemia (AML) is a heterogeneous clonal disorder disease, characterized by uncontrolled proliferation of immature abnormal blast cells that commonly occurs in adult patients. Up to 60% of adult patients with AML were diagnosed with genetic abnormalities [1] and, subsequently, multiple dysregulated
* Corresponding author. ** Corresponding author. Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong-gu, Daejeon, 34141, Republic of Korea. E-mail addresses: [email protected] (I. Choi), [email protected] (H. Jung).
genes, such as p53, BCL2 family, and nuclear factor NF-kB, that led to tumor maintenance and chemoresistance [2]. However, despite advanced understanding of the molecular pathogenesis of AML, chemotherapeutic treatment remains a major challenge due to diverse prognostic outcomes with high rate of mortality in overall survival (<50%) and low relapse-free survival [3]. Fludarabine (FA), a purine nucleoside analogue, is an anti-metabolite small molecule that kills the malignant cells by inhibiting RNA/DNA synthesis and DNA repair through multiple mechanisms [4]. FA is an essential component in therapy for chronic lymphocytic leukemia (CLL) and AML [5]. In an in vitro study using AML lines and primary cells, the cytotoxicity effect of FA was significantly elevated when combined with other agents such as DNA alkylating agents [6] or histone
https://doi.org/10.1016/j.bbrc.2018.10.047 0006-291X/© 2018 Elsevier Inc. All rights reserved.
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deacetylase inhibitors (HDACI) [7,8]. Regimens containing FA have generated remarkable improvement of relapse-free survival in AML patients [9]. Toll-like receptors (TLRs) that play a critical role in initiating innate and adaptive immune response are expressed not only in immune cells but also in malignant cells, including hematopoietic malignancies [10]. TLRs recognize both pathogen-associated molecule patterns (PAMPs) and damage-associated molecule patterns (DAMPs), and activation of TLRs is involved in normal hematopoiesis and specific hematologic pathologies [11]. In contrast to the antitumor activity of TLRs agonists against various cancer cells, including leukemia, negative feedback associated with cancer survival, proliferation, metastasis, and drug resistance were also observed [11]. Some receptor complexes, such as TLR1/2, TLR2/6 or TLR9, play important roles in protecting cells from death induced by FA in CLL primary cells [12]. Interestingly, higher expression of TLRs was observed in AML patients without responding to chemotherapy treatment compared to patients with complete remission who had low expression of TLRs [10]. In particular, the expression of TLR4 was observed in some types of primary AML cells [10]; however, its role against chemotherapeutic drugs such as FA and its underlying mechanism upon activation in these cells have not fully elucidated yet. Thioredoxin-interacting protein (TXNIP) is well known as a tumor suppressor gene (TSG) and plays a crucial role in mediation of cell apoptosis and cycle arrest of tumor cells [13e15]. However, TXNIP was frequently downregulated in various cancer including AML and the decreased TXNIP expression in AML cells was mainly due to epigenetic silencing caused by the polycomb repressive complex 2 (PRC2) that directly mediated TXNIP silencing [16]. Reactivation of TXNIP in cancer cells by anti-cancer drugs or epigenetic modification agents increased anti-tumor effect of TXNIP [13,16,17]. However, the function of TXNIP on FA-induced apoptosis of AML cells is not clearly elucidated yet. In this study, we found that TLR4 inhibited FA-induced cytotoxicity by inducing NF-kB activity and its target genes, which were implicated in modulation of cell apoptosis and drug resistance in a TLR4-expressing AML line. In addition, we revealed that the expression of TXNIP was regulated by FA or LPS treatment and was important for FA-induced cytotoxicity or LPS-induced cell survival of AML cells. Altogether, our data suggest that TLR4/NF-kB induces FA resistance by suppressing TXNIP expression, and TXNIP may be a new potential therapeutic target for AML treatment.
from Santa Cruz Biotechnology. FACS antibodies, TLR4-APC (ab155343) was purchased from Abcam; and CD14-FITC (555397), IgG-FITC (349041) and IgG-APC (554686) from BD Biosciences. 2.3. Cell viability and apoptosis analysis Cell viability and apoptosis analysis were performed in the same experimental condition with 1 h of prior-stimulation with LPS (1 mg/ml), followed by 24 h of treatment with FA, unless otherwise stated. For TLR4 inhibition, cells were prior incubated with 30 mg/ ml LPS-RS (RS) for 4 h or 10 mM IAXO for 2 h before LPS or FA treatment. For IKK/NF-kB inhibition, cells were prior-incubated with celastrol (Cel) 0.5 mM for 2 h before LPS or FA treatment. Cell viability was accessed using Cell Counting Kit-8 (CCK-8) from Dojindo Molecular Technologies, Inc. Briefly, 2 104 cells/100 ml were seeded in 96-well plate and pre-incubated for 24 h at 37 C. Cells were then pre-stimulated and treated with LPS and FA. Then, 10 ml CCK-8 was added to each well and incubated for 3 h at 37 C in the cell culture incubator. Plates were measured at O.D. 450 nm using a SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices). Apoptotic cells were assessed using AnnexinV/PI kit (556547, BD Pharmigen) and FACSCanto II (BD Biosciences) following the manufacturer's instructions. 2.4. Quantitative real-time PCR
Cell lines used in this study, including HL-60, THP-1, CCRF-CEM and MOLT-4 were obtained from Korea cell line bank; JURKAT and 293T lines were from American Type Culture Collect (ATCC). Leukemia and 293T lines were cultured in RPMI 1640 medium and DMEM (WELGENE Inc., Gyeongsan, Korea) supplemented with 10% fetal serum albumin and antibiotic-antimycotic (Gibco) at 37 C in a humidified atmosphere of 5% CO2 in air.
mRNA expression was analyzed by quantitative real-time PCR (qPCR). Briefly, total RNA was extracted using RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. 1 mg of each RNA sample was used for the reverse-transcriptase PCR using a First-Strand cDNA synthesis kit (FSK-101, Toyobo). cDNAs were diluted 1:10 and submitted to qPCR using the SYBR Premix ExTaq (Takara Bio) and Applied Biosystems ViiA 7 Real-Time PCR system (Thermo Fisher Scientific). The human primer sequences were as follows: GAPDH forward 50 -GAGTCAACGGATTTGGTCGT-30 and reverse 50 -TTGATTTTGGAGGGATCTCG-3’; TLR4 forward 50 TCATTGGTGTGTCGGTCCTC-30 and reverse 50 -CTGCCAGGTCTGAGCAATCT-3’; CD14 forward 50 -CTCTGTCCCCACAAGTTCCC-30 and reverse 50 -GGATTCCCGTCCAGTGTCAG-3’; XIAP forward 50 GGCATTTCCAGATTGGGGCT-30 and reverse 50 -TTTGTAGACTGC GTGGCACT-3’; cIAP2 forward 50 -GTCACTCCCAGACTCTTTCCA-30 and reverse 50 -GGGCTGGCATGAGACTTCTT-3’; Survivin forward 50 ACGACCCCATGCAAAGGAAA-30 and reverse 50 -CTGGTAAGCCCGGGAATCAA-3’; MLIAP forward 50 -CCTGCTCCGGTCAAAAGGAA-30 and reverse 50 -GCTCAAGAACCCACCACGC-3’; BCL2 forward 50 CCTTTGTGGAACTGTACGGC-30 and reverse 50 -CCGGCCAACAACATGGAAAG-3’; BCL2A1 forward 50 -CTGCAGTGCGTCCTACAGAT-30 and reverse 50 -TTGTGGGCCACTGACTCTAC-3’; BCL-xl forward 50 GCTTTGAACAGGATACTTTTGTGGA-30 and reverse 50 -AGGGAGGCTAAGGGGTAAGG-3’; BCL-w forward 50 -CCTGACCCGTGAGATCCCTA-30 and reverse 50 -CCACCAGTGGTTCCATCTCC-3’; MCL-1 forward 50 -CGACTTTTGGCCACCGGC-30 and reverse 50 -TAGCCAGTCTTTTGTCC-3’; and TXNIP forward 50 -AAGACCAGCCAACAGGTGAG-30 and reverse 50 -AGGAAGCTCAAAGCCGAACT-3’. GAPDH was used for normalization of target genes.
2.2. Reagents and antibodies
2.5. Western blot
Reagents acquired from Sigma-Aldrich were fludarabine (F9813) and LPS (L2880); from InvivoGen, celastrol (anti-cls) and LPS-RS (tlrl-rslps); and Adipogen Life Sciences, IAXO-101 (IAXO) (IAX600-001). Western blot antibodies PARP (9542), caspase-3 (9665), NF-kB p65 (8242), phospho-NF-kB p65 (3033), IkB-a (2682), p-IkBa (2859), BCL2A1 (14093), cIAP2 (3130), and TXNIP (14715) were purchased from Cell Signaling Technology; b-actin (sc-47778) was
Protein expression and phosphorylation were analyzed by Western blot. Protein lysates were extracted using 0.1% NP40 lysis buffer supplemented with protease and phosphatase inhibitors (Calbiochem) as previously described [18,19]. Briefly, all lysates were quantified and loaded at the same amount on 8e15% SDSPAGE gels before transferring to PVDF membranes (Millipore). Membranes were blocked with 5% skim milk and incubated with
2. Materials and methods 2.1. Cell lines and culture
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primary antibodies overnight at 4 C. Bound proteins were detected using peroxidase-conjugated anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch). Proteins were then visualized using EzWestLumiOne (ATTO) or SuperSignal West Pico Chemilumicesent (Thermo Scientific) substrate and WSE-6200 LuminoGraph II Imaging System (ATTO). b-actin was used as a loading control.
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3.2. LPS increases anti-apoptotic activity through the TLR4/NF-kB signaling
293T cells were transiently co-transfected using PLUS and Lipofectamine (Invitrogen) with cytomegalovirus-Renilla luciferase construct (pRL-CML) (Promega) and TXNIP-Luciferase reporter plasmid (pTXNIP-luc) as previously described [20], with or without HA tagged p65 and HA tagged IkB-a plasmid. A total of 100 ng of each plasmid DNA was used in this study. Transfected cells were cultured for 48 h before lysis and detection using a luciferase assay kit (E1500, Promega) according to the manufacturer's recommendations. Firefly luciferase and Renilla luciferase activity were measured by Dual Luciferase Reporter Assay System (Promega) on a Luminometer (Turner Designs). Luciferase activity was expressed as the value of the mean fluorescence intensity (MFI).
Next, we investigated the roles of NF-kB on LPS-induced FA resistance in THP-1 cells, because it is well known as a downstream component of TLR signaling and a transcription factor for many genes, including members of the inhibitor of apoptosis protein (IAP) and B-cell lymphoma-2 (BCL-2) families [21]. Moreover, NF-kB was reported as a target molecule of FA in human T-cell leukemia virus type 1 (HTLV-1)-infected cells [22]. As expected, TLR4 stimulation accelerated IkB-a phosphorylation or degradation, and phosphorylated NF-kB (p65) in a time- and concentration-dependent manner (Fig. 2AeB). Interestingly, some NF-kB target genes were highly induced by LPS treatment, including BCL2A1 (>1000 fold), cIAP2 (>50 fold), and BCL-xl (>5 fold) (Fig. 2C). In addition, the expression of these target genes under LPS treatment were observed at transcriptional and post-translational levels in a time- and concentration-dependent manner (Fig. 2DeE). However, TLR4 blocked with LPS-RS, a TLR4 antagonist, attenuated the LPS-induced phosphorylation of IkB-a and NF-kB and the expression of NF-kB target genes (Fig. 2FeG). These results implied that LPS might induce FA resistance by activating TLR4/NF-kB signaling pathways.
2.7. Electroporation
3.3. Inhibition of TLR4 or NF-kB restores FA-induced cytotoxicity
2.6. TXNIP luciferase assay
Amaxa nucleofector kit (VCA-1003) and Nucleofactor 2b device from Lonza were applied for knockdown of TXNIP in THP-1 cells. TXNIP siRNA and control RNAi were acquired from Santa Cruz Biotechnology. A total of 1 10^6 cells/sample was electroporated with 200 nM of siRNA using the V-01 program and cultured for 24 h before another 24 h of treatment with FA. 2.8. Statistical analysis All experiments were performed three times with triplication in each sample and representative data are shown, unless otherwise stated. Data collected from each experiment were expressed as the means ± SD. For statistical analysis of the data, p values were analyzed using an unpaired t-test in the Prism program. A p value < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 3. Results 3.1. TLR4-dependent resistance to FA-induced cytotoxicity in AML cells To investigate the function of TLR4 signaling on FA-induced apoptosis in leukemia cells, we examined the expression of TLR4 and CD14 in leukemia cell lines including AML cells (HL-60 and THP-1) and acute lymphocytic leukemia (ALL) cells (CCRF-CEM, JURKAT and MOLT-4). Remarkably, THP-1 cells were observed to have the highest expression of TLR4 and CD14 receptors and genes compared to other leukemia lines (Fig. 1AeB). Next, to identify the effect of TLR4 stimulation on FA-induced cytotoxicity, we stimulated the TLR4 receptor with lipopolysaccharide (LPS), a TLR4 agonist. LPS significantly increased the cell viability of THP-1 cells but not in other cell lines (Fig. 1C). To understand the function of TLR4 in FA-induced apoptosis of THP-1 cells, we analyzed apoptotic phenotypes using AnnexinV/PI staining (Fig. 1D) and immunoblotting of cleaved poly(ADP-ribose) polymerase (PARP) and caspase-3 (Fig. 1E) by LPS treatment. These results indicated the negative role of TLR4 signaling against FA-induced cytotoxicity in TLR4-expressing cells.
To prove the effect of TLR4/NF-kB signaling on FA-induced cytotoxicity, we applied IAXO, an antagonist for CD14/TLR4 and celastrol, an IKK/NF-kB inhibitor. Notably, prior incubation with IAXO significantly attenuated LPS activity against FA-induced cytotoxicity and restored FA sensitivity in THP-1 cells (Fig. 3AeC). Consistent with this, prior incubation with celastrol completely inhibited LPS-induced phosphorylation of IkB-a and NF-kB and recovered FA cytotoxicity against LPS activity (Fig. 3DeF). These results showed that the TLR4/NF-kB axis is a critical mediator of LPS-induced FA resistance in AML cells. 3.4. FA-induced cytotoxicity depends on TXNIP expression We and other research groups previously reported that TXNIP was a tumor suppressor and regulator of oxidative stress in malignant cells [18,23]. TXNIP expression is upregulated by various environmental stresses, transforming growth factor-b and vitamin D3 [13]. Moreover, several epigenetic modifiers, such as LAQ-824, SAHA and DZNep, have been reported to induce TXNIP expression and mediate oxidative stress-induced apoptosis in malignant cells [7,16,17]. Here, we found that FA induced TXNIP expression in THP1 cells in a time- and concentration-dependent manner at transcriptional and post-transcriptional levels (Fig. 4AeB), and its expression was partially blocked from LPS-induced down-regulation by TLR4 inhibition (Fig. 4C). Interestingly, FA-induced TXNIP expression was strongly suppressed by LPS treatment either prior to or after FA treatment, and its expression was parallel to the phosphorylation of IKB-a and NF-kB (Fig. 4DeE). Moreover, inhibition of NF-kB by celastrol prevented TXNIP down-regulation induced by LPS treatment (Fig. 4F). To gain direct evidence that NF-kB was indeed a negative regulator of TXNIP expression, we performed luciferase assays using 293T cells. Notably, luciferase activity of TXNIP promoter was dramatically decreased by p65 overexpression or rescued by IkB-a overexpression (Fig. 4G). Collectively, these findings suggested that NF-kB was a negative and upstream regulator of TXNIP expression. To validate the role of TXNIP expression in FA-induced cytotoxicity, we suppressed TXNIP expression with TXNIP siRNA (Fig. 4H) and analyzed the apoptosis of THP-1 cells (Fig. 4I). These results showed that TXNIP played an important role in FA-induced cytotoxicity of THP-1 cells.
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Fig. 1. TLR4-dependent resistance to FA-induced cytotoxicity in AML cells. A, Representative histograms of surface receptors analyzed by FACS. Gray-filled lines are receptors stained positively with anti-TLR4 or anti-CD14 antibodies, and non-filled lines indicate negative staining with anti-IgG control antibodies. B, TLR4 and CD14 mRNA expression. C, Cell viability. Apoptotic cells (D) and protein marker (E) of THP-1 cells. #, denoted for RNA amplification value below detection; FL, full length; CL, cleavage protein. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
4. Discussion In this study, we reported the expression of TLR4 and CD14 in AML lines, but not ALL lines. Notably, only THP-1 cells were protected from FA-induced cytotoxicity upon TLR4 stimulation with LPS, revealing it to be cell-context dependent. Further, we confirmed a critical role of TLR4/NF-kB activation against FAinduced cytotoxicity in THP-1 cells, and we found that TXNIP gene expression was a common target for TLR4/NF-kB activation and FA induction. NF-kB is a key player downstream of all TLRs, making it more attractive for therapeutic drug development for treatment of various diseases [24,25]. Constitutive NF-kB activation was observed in AML blasts and other hematopoietic cancers [26]. In AML cells, aberrant activation of NF-kB was the result of mutation or activation of critical oncogenes such as CCAAT/enhancer-binding protein alpha, Ataxia Telangiectasia Mutated, runt-related
transcription factor 1, or fms-like tyrosine 3, which directly or indirectly interact with NF-kB and activate its pathway [27]. In human myeloid monocytic-leukemia THP-1 cells, NF-kB activation following TLR4 stimulation by LPS was reported [28] and contributed to increase in expression of multiple genes, including various pro-inflammatory cytokines and TLR4, which played roles in inflammatory disease [29]. However, the role of TLR4/NF-kB against anti-cancer drugs as FA was not clear. Here, we showed that NF-kB was activated by TLR4 stimulation and played a key role in determining cell viability or apoptosis in response to FA treatment. The activation of NF-kB was associated with IkB-a phosphorylation and high expression of downstream targets, anti-apoptotic genes including BCL2A1, cIAP2 and BCL-xl, after TLR4 stimulation. Consistently, TLR4 antagonists, LPS-RS or IAXO, attenuated TLR4 signaling and maintained FA sensitivity, which is associated with decreased NF- kB activation and target gene expression. Strikingly, NF-kB activity against FA-induced cytotoxicity was completely
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Fig. 2. LPS increases anti-apoptotic activity through TLR4/NF-kB signaling pathway. Protein phosphorylation in a time-dependent (A) and concentration-dependent manner (B). C, mRNA expression after 4 h of LPS treatment. D, mRNA and protein expression after LPS (1 mg/ml) treatment. E, mRNA expression. Protein (F) and mRNA (G) expression after incubation with TLR4 antagonist (RS) and LPS. ns means not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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Fig. 3. Inhibition of TLR4 or NF-kB restores FA-induced cytotoxicity. Representative cytograms (A) and data (B) of apoptotic cells after incubation with TLR4/CD14 antagonist (IAXO), LPS and FA. C, Cell viability. D, Protein phosphorylation analysis after 2 h of prior incubation with IKK/NF-kB inhibitor (Cel), followed by LPS treatment. Representative cytograms (E) and data (F) of apoptotic cells after incubation with Cel, LPS and FA. **P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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Fig. 4. FA-induced cytotoxicity depends on TXNIP expression. A, TXNIP expression in a time-dependent manner with FA incubation. B, TXNIP mRNA and protein expression in a concentration-dependent manner with LPS (1 h) treatment. C, TXNIP expression after 4 h of prior incubation with TLR4 antagonist (RS), followed by 1 h with LPS treatment. D, Protein expression and phosphorylation after prior incubation with LPS (1 mg/ml), followed by FA treatment. E, TXNIP mRNA and protein expression after prior incubation with FA (25 mM/ml), followed by LPS (1 mg/ml) treatment. F, TXNIP expression after 2 h of prior incubation with celastrol (Cel), followed by 1 h with LPS treatment. G, Luciferase promoter assay analysis. H, Transfection efficiency confirmed by Western blotting 2 days after electroporation. I, Representative data of apoptotic cells. ns means not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
blocked by the pre-incubation with the IKK/NF-kB inhibitor celastrol. These results highlighted the critical role of NF-kB signaling as a survival regulator of TLR4 stimulation. In comparison to the previous report, the NF-kB/XIAP pathway is the survival signal of some human T-cell leukemia (ATL) lines and was indeed inhibited by FA treatment [22]. Moreover, the increase of FA activity by HDACI was the result of the suppression of the NF-kB/XIAP pathway and subsequently induced apoptosis in AML cells [30]. However, we could not find the induction of XIAP expression at the time point of TLR4 stimulation in this study (Fig. 2C). Thus, targeting NF-kB with BCL2A1 and/or cIAP2 would be an alternative target for treatment of AML with constitutive TLR4 or NF-kB activation, and the combination of FA and NF-kB inhibitor would be useful. Also, further investigation of BCL2A1 and cIAP2 in AML cells would be interesting, as they have not well studied compared to BCL2 or XIAP [2,31]. Although NF-kB was regulated following TLR4 stimulation with LPS, we could not exclude other possible factors, such as AKT, ERK, or c-JUN, as their activation was modulated by LPS or celastrol treatment [32,33] and observed in our experiment (data not shown).
TXNIP, which is known as a TSG, is frequently downregulated in various cancer cells [23] and plays a crucial role in mediation of cell apoptosis and cycle arrest [13,14]. In cDNA sequencing data from AML blasts of 270 patients, mutation of TXNIP was relatively infrequent [34]; thus, decreasing TXNIP in AML cells might be induced mainly by epigenetic silencing. In AML primary cells and lines, TXNIP expression was reported to be 11-fold and 5-fold lower compared to normal blasts, and this low expression was silenced by the PRC2 protein complex [16]. In addition, a pronounced expression of TXNIP in AML cells was upregulated when treated with a HDACI, 3-Deazaneplanocin A, which suppressed PRC2 and was associated with anti-tumor activity of TXNIP [16]. Here, we reported that the expression of TXNIP was dramatically upregulated by FA treatment at both the protein and RNA levels (Fig. 4). Moreover, we also confirmed that silencing of TXNIP by siRNA significantly decreased FA-induced cytotoxicity in THP-1 cells (Fig. 4). These findings suggest a role for TXNIP in FA-induced cytotoxicity in THP-1 cells. TLR4 or NF-kB activation downregulated TXNIP expression and suppressed FA-induced cytotoxicity, and pretreatment of TLR4 or NF-kB inhibitor could prevent down-regulation of
Please cite this article in press as: H. Huy, et al., TLR4/NF-kB axis induces fludarabine resistance by suppressing TXNIP expression in acute myeloid leukemia cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.047
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H. Huy et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
TXNIP and FA sensitivity (Fig. 4). In our previous report, we showed the correlation between TXNIP and NF-kB, and TXNIP inhibited NFkB activity via physical interaction with p65 [18]. Furthermore, we also showed that the expression of TXNIP was reduced by TNF-amediated NF-kB activation while overexpression of TXNIP robustly suppressed NF-kB activation [35]. In Fig. 4G, we showed that overexpression of NF-kB suppressed TXNIP promoter activity, while co-transfection with an IKB-a plasmid rescued TXNIP expression. These results suggest that NF-kB is an upstream regulator of TXNIP, and NF-kB may directly or indirectly regulate the transcription of TXNIP. Further study is needed to prove the transcriptional regulation of TXNIP by NF-kB. In conclusion, we unveil a novel role of the TLR4/NF-kB axis against FA-induced cytotoxicity through suppression of TXNIP and expression of anti-apoptotic genes in THP-1 cells. From these results, we suggest that TXNIP may be a new therapeutic target for AML therapy. Acknowledgments This work was supported in part by the National Research Council of Science and Technology (NST) grant (No. CRC-15-02KRIBB), and the KRIBB Research Initiative Program from the Korea government (MSIP).
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Transparency document [23]
Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.10.047.
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Please cite this article in press as: H. Huy, et al., TLR4/NF-kB axis induces fludarabine resistance by suppressing TXNIP expression in acute myeloid leukemia cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.047