
==== Front
Cancer Biol Ther
Cancer Biol Ther
Cancer Biology & Therapy
1538-4047
1555-8576
Taylor & Francis

39286953
10.1080/15384047.2024.2402588
2402588
Version of Record
Research Article
Research Paper
EX527, a sirtuins 1 inhibitor, sensitizes T-cell leukemia to death receptor-mediated apoptosis by downregulating cellular FLICE inhibitory protein
R. GUO ET AL.
CANCER BIOLOGY & THERAPY
Guo Rongqi a b c *
Wei Yihui d *
Du Yating a b c *
Liu Luyue e
Zhang Haoqi f
Ren Ruiying a b c
Sun Ruili b c
Zhang Tingting f
Xiong Xiwen g
Zhao Lijun b c
Wang Hongfei b c
Guo Xiaofang c f
https://orcid.org/0000-0001-6360-140X
Zhu Xiaofei a b c
a Department of Clinical Laboratory, The Third Affiliated Hospital of Xinxiang Medical University, Xinxiang, China
b Henan Key Laboratory of Immunology and Targeted Drugs, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China
c Xinxiang Key Laboratory of Tumor Microenvironment and Immunotherapy, Xinxiang Medical University, Xinxiang, China
d Henan Red Cross Blood Center , Xinxiang, China
e Departments of Laboratory Medicine, Zhoukou Central Hospital , Zhoukou, China
f Department of Microbiology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, China
g School of Forensic Medicine, Xinxiang Medical University , Xinxiang, China
CONTACT Xiaofang Guo guoxiaofang_1981@126.com Department of Microbiology, School of Basic Medical Sciences, Xinxiang Medical University, No.601, Jinsui Road, Xinxiang, Henan, China
Xiaofei Zhu zhuxf@xxmu.edu.cn Department of Clinical Laboratory, The Third Affiliated Hospital of Xinxiang Key Laboratory of Tumor Microenvironment and Immunotherapy, Xinxiang Medical University, Xinxiang, China
* These authors contributed equally.

17 9 2024
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© 2024 Xinxiang Medical University. Published with license by Taylor & Francis Group, LLC.
2024
Xinxiang Medical University
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Death receptor-mediated extrinsic apoptosis system had been developed as a promising therapeutic strategy in clinical oncology, such as TRAIL therapy. However, multiple studies have demonstrated that TRAIL resistance is the biggest problem for disappointing clinical trials despite preclinical success. Targeting cellular FLICE inhibitory protein (cFLIP) is one strategy of combinatorial therapies to overcome resistance to DR-mediated apoptosis due to its negative regulator of extrinsic apoptosis. E × 527 (Selisistat) is a specific inhibitor of SIRT1 activity with safe and well tolerance in clinical trials. Here, we show that E × 527 could strengthen significantly activation of rhFasL-mediated apoptotic signaling pathway and increased apoptotic rate of T leukemia cells with high expression of cFLIP. Mechanically, Inhibition of SIRT1 by E × 527 increased polyubiquitination level of cFLIP via increasing acetylation of Ku70, which could promote proteosomal degradation of cFLIP protein. It implied that combinatorial therapies of E × 527 plus TRAIL may have a potential as a novel clinical application for TRAIL-resistant hematologic malignancies.

KEYWORDS

EX527
sirtuins 1
T leukemia
apoptosis
cellular flice-inhibitory protein
National Natural Science Foundation of China 10.13039/501100001809 The Key Scientific Research Projects of Universities in Henan Province Natural Science Foundation of Henan 242300420119 This work was supported by the National Natural Science Foundation of China under Grant No.[81373135 and No.81771690]; The Key Scientific Research Projects of Universities in Henan Province under [Grant No. 21zx011]. Natural Science Foundation of Henan [242300420119].
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pmcIntroduction

Pro-apoptosis is one of important mechanism in most of anti-cancer therapy, especially chemotherapy and radiotherapy. Apoptosis can be initiated essentially through the mitochondrial-dependent intrinsic pathway and death receptor (DR)-mediated extrinsic pathway. The latter was triggered upon binding of ligands with their cognate death receptors at the cells’ surface, such as TRAIL/TRAILR, FasL/Fas.1,2 As a tumor suppressor, the DR-mediated apoptosis system exerts tumor cell destruction via FasL and TNF-related apoptosis inducing ligand (TRAIL) in tumor immunosurveillance.3 The binding of ligand to receptors induces the formation of receptor microaggregates and activation. The activated receptor recruits the adapter molecule Fas-associated death domain (FADD) through their death domains and the initiator procaspase 8 by the death effector domain (DED) of FADD to form the death-inducing signaling complex (DISC), and subsequent proteolytic autocleavage of procaspase-8 to active caspase 8, which dissociates from the DISC to proceed with the activation of the caspase cascade.4–6 Therefore, the DR-mediated apoptosis system had been developed as a promising therapeutic strategy in clinical oncology, such as TRAIL therapy. However, multiple studies have demonstrated that TRAIL resistance is the biggest problem for disappointing clinical trials despite preclinical success.7,8

It had been evident that one of important reasons for resistance of DR-mediated anticancer therapy is overexpression of intracellular anti-apoptotic proteins cellular-FLIP (c-FLIP) strikingly in some solid and the majority of hematologic malignancies,9–11 including hepatocellular cancers,9,12 colorectal cancers,13 adult T-cell leukemia/lymphoma,14 Hogkins lymphoma,15 chronic lymphocytic leukemia,16 Burkitt’s lymphoma,17 non-Hodgkin’s lymphoma.18 Moreover, high levels of c-FLIP correlate with more aggressive tumors.19 In response to DR ligation, overexpression of all c-FLIP isoform (mainly long and short c-FLIP isoforms, named c-FLIPL and c-FLIPS) have been reported to be present at the DISC plays an antiapoptotic role by the replacement of procaspase-8 from the DED filament or form heterodimers with procaspase-8 in the DED filaments that break the chain growth, thereby preventing subsequent dimerization and activation of procaspase-8.20–22 Therefore, one strategy for the sensitization of DR-mediated anticancer therapies is targeting c-FLIP to overcome resistance to DR-mediated apoptosis by downmodulation of c-FLIP expression levels or direct pharmacological targeting of c-FLIP proteins.

In clinical trial data, it was indicate that histone deacetylase (HDAC) inhibitors, mainly targeting class I and II HDACs, have clinical activity and response in hematologic malignancies as a single agent,23 such as vorinostat, entinostat and sodium butyrate in T cell leukemia, which of mechanism was closely associated with downregulating c-FLIP isoforms.24–26 Therefore, the combination of HDAC inhibitors and TRAIL might be a powerful tool to sensitize DR-mediated anticancer therapies in hematologic malignancies. Though the pro-survival role of SIRT1, a class III HDAC, have been reported in hematologic malignancies,27 SIRT1 inhibitor has not been evaluated in T cell leukemia of preclinical and clinical trials. As a specific SIRT1 inhibitor, E×527 (Selisistat) has much lower potency against other member of sirtuins family, such as SIRT2 and SIRT3, but also does not inhibit class I/II HDAC activity at concentrations up to 100 μM.28,29 In addition, E×527 exerts an inhibitory effect on SIRT1 activity without affecting SIRT1 expression on both mRNA and protein levels.30 More importantly, in clinical trials, E×527 have been confirmed to be safe and well tolerated by healthy volunteers and patients.31,32

In this study, we investigated the effect of E×527 on sensitizing DR-mediated apoptosis of T leukemia cells and explored its possible pharmacological mechanism. The results demonstrated that the expression levels of SIRT1 and cFLIP in T leukemia cells was significantly and positively associated with rhFasL-mediated apoptosis. E×527 strengthened significantly activation of rhFasL-mediated apoptotic signaling pathway and increased apoptotic rate of T leukemia cells. Inhibition of SIRT1 by E×527 or knockdown of SIRT1 disrupted polyubiquitination level of cFLIP via increasing acetylation of Ku70. It implied in this study that SIRT1 inhibitor E×527 may have a potential as a novel clinical application drug for TRAIL-resistant hematologic malignancies by combining with TRAIL to augment sensitivity to TRAIL and consequently reduce morbidity and mortality from this type of resistant cancers.

Methods and materials

Plasmids and reagents

The pcDNA3.1-SIRT1 and SIRT1 H363Y mutant expression vector was kindly provided by professor Melanie Ott, Gladstone Institute of Virology and Immunology, University of California. Flag-pcDNA3 cFLIP expression vector was kindly provided by professor Xin Lin, the Institute for Immunology, Tsinghua University School of Medicine. Flag-pcDNA3 Ku70 expression vector was constructed as described.33 Recombinant human Fas Ligand were purchased from Cell Signaling Technology (5452). EX-527 were purchased from Selleck Chemicals (S1541).The following Abs were used for immunoblot analysis or immunoprecipitation: anti-SIRT1 (ab32441; Abcam), anti-Ku70 mouse and Rabbit Ab (ab202022, ab83501; Abcam), anti-Acetylated-Lysine (9441; Cell Signaling Technology), anti-cFLIP (sc5276; Santa Cruz Biotechnology), anti–β-actin (60008–1; Proteintech), rabbit IgG (A7016,Beyotime Biotechnology), anti-Flag mouse and Rabbit Ab (66008-3-Ig,20534-1-AP; proteintech), anti-Bid (2002; Cell Signaling Technology), anti-PARP/caspase-3/cleaved caspase-3 (9915; Cell Signaling Technology), Caspase-8 (9746; Cell Signaling Technology), anti-Ubiquitin (sc8017; Santa Cruz Biotechnology).

Microarray analysis

SIRT1 and cFLIP expression level of adult T-cell leukemia (ATL) patients, asymptomatic carriers, and normal CD4+ T cells was analyzed by the Gene Expression Omnibus database (GSE55851). For each gene, the raw RNA-Seq readcount were normalized with Z-scores standardization. The heatmaps were generated in GraphPad Prism with the normalized data of readcount.

Cell culture and transfection

293T were cultured in Dulbecco’s modified Eagle’s medium, MT-2, MT-4, C8166, Jurkat of various T leukemia cells were maintained in RPMI 1640 medium. Both mediums were supplemented with 10% fetal bovine serum (FBS), 4 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin under humidified conditions with 5% CO2 at 37°C. Transfection plasmids into 293T was performed with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. At 48 h after transfection, the cells were used for further experiments.

RNA interference

The siRNAs used for the targeted silencing of SIRT1 (SiRNA1: 5′-GGAUAGGUCC AUAUACUUU-3′; SiRNA2: 5′-CCACCUGAGUUGGAUGAUA-3′) and the scrambled siRNA(SCR: 5′-CUUCCCGAAAACUUGAGAC-3′) as a control was synthesized by Takara Biomedical Technology. Transfection siRNA into 293T, MT-2 and MT-4 was performed with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. At 48 h after transfection, the cells were used for further experiments.

Cell viability assay

The in vitro cytotoxicity of suspension cultured cells was performed by CellTiter 96® A Queous One Solution Reagent (G3582, Promega (Beijing) Biotechnology) according to the manufacturer’s instructions. In brief, cells were seeded in 96-well plates and incubated in 37°C for 24 h, and then exposed to different concentrations of rhFasL for 16 h. The One Solution Reagent was added to each well and incubated for another 4 h at 37°C. The absorbance at 490 nm was measured using an ELISA reader (Thermo Fisher). Growth inhibition was calculated as a percentage of the nontreated controls.

Annexin V-staining apoptosis assay

The FasL-induced apoptotic cells were measured by an Annexin V-FITC/PI staining kit(C1062M, Beyotime Biotechnology). After different concentration of rhFasL or E×527 treatment, cells were harvested, washed twice with PBS, and centrifuged at 1000 rpm for 5 min. The cell pellets were resuspended in 500 μl binding buffer containing of 10 μl Annexin V-FITC and 5 μl PI, incubated at room temperature for 15 min, and then analyzed for fluorescence with flow cytometer.

Immunoblot and immunoprecipitation assay

For Immunoblot assay, 293T cells were collected by scraping or centrifugation, immediately lysed in a mild RIPA buffer (10 mM Tris-HCl PH 7.4, 1% Triton X-100, 0.25% Sodium deoxycholate, 0.1% SDS, 160 mM NaCl, 5 mM EDTA, 50 mM NaF, 10% Glycerol, 1 mM Na3VO4, 5 mM Sodium Pyrophoshate, 1 μg/ml Aprotinin, 10 μg/ml leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride) for 30 min at 4°C. he protein concentration of extracts was determined by Bradford assay. Equal amounts of protein samples were subjected to 6–15% SDS-PAGE according to the molecular weight of target proteins and transferred onto PVDF membrane. The membranes were incubated successively with various primary antibodies and horseradish peroxidase-conjugated secondary antibodies at appropriate dilutions. The specific bands were visualized with ECL Plus Western Blotting Detection System according to the manufacturer’s recommendation.

For immunoprecipitation assay performed by Capturem™ IP & Co-IP Kit (Code No.635721, Clontech), according to the manufacturer’s instructions. In brief, whole cell extracts from cells exposed to different treatment were prepared and incubated with anti-cFLIP, anti-Flag or anti-Ku70 antibody, respectively. The eluted sample were boiled, electrophoresed on SDS-PAGE gels, and analyzed by Western blotting.

Statistical analysis

All values in this article were given as mean ± SEM, unless stated otherwise. All experiments were reproduced at least three independent times, and results shown are representative. Statistical significance was calculated by two-tailed unpaired t test using GraphPad Prism software. Statistical significance was set based on p values; *, p < .05; **, p < .01; ***, p < .001; ****, p < .0001.

Results

High expression of cFLIP associated with resistance to FasL-mediated apoptosis in T leukemia cells

By Annexin V and PI-staining assay as showed in Figure 1a, it was showed that apoptotic rates of MT-2 cells were 6.74 ± 1.3%, 7.05 ± 1.5%, 7.9 ± 2.2%, respectively, with a dose-escalation of rhFasL treatment from 5 to 20 ng/ml, which was significantly lower than that in Jurkat (p < .0001). For examples, apoptotic rate of Jurkat treated with 5 ng/ml and 20 ng/ml rhFasL reached to 49.2 ± 0.5% and 79.2 ± 0.3%, respectively. Meanwhile, In T leukemia cells demonstrated in Figure 1b, the expression level of SIRT1 and cFLIPL/S in MT-2 and MT-4 was also upregulated significantly (p < .001). Then, cell viability in T leukemia cells were detected under a dose-escalation of rhFasL treatment as showed in Figure 1c. Difference of cell viability between MT-2/MT-4 and Jurkat cells was very significant when concentrations of FasL reached to 10 ng/ml (p < .001). Survival rate of MT-2, MT-4 and Jurkat were 85 ± 2.7%, 81 ± 3.1% and 41 ± 2.1%, respectively. Even at 100 ng/ml FasL treatment, the survival rate in MT-2 and MT-4 did not show a significant reduction, while only 25 ± 1.06% of survival rate was determined in Jurkat. Figure 1. SIRT1 and cFLIP expression in ATL patients and T leukemia cells associated with resistance to rhFasL-mediated apoptosis. (a) MT-2, MT-4 and Jurkat cells were treated with indicated concentrations of rhFasL for 16h at the indicated concentration. Representative flow cytometry graph and Bar graph of the percentages of apoptotic cells. (b) Representative Western blot of SIRT1, cFLIP and β-Actin in T leukemia cells and Jurkat cells. Bar graph of fold changes in cFLIP/β-Actin and SIRT1/β-Actin were calculated by normalizing to that in Jurkat according to gray value. (c) Cell viability assay after rhFasL treatment in MT-2, MT-4 and Jurkat cells. Percentage of viable cells is expressed relative to control cells Jurkat. (d) The heatmaps of SIRT1 and cFLIP. each row of RNA-Seq raw readcount were normalized with Z-scores standardization. Expression levels of SIRT1 and cFLIP genes in normal CD4+ T cell, asymptomatic carriers, smoldering-type AT, chronic-type ATL and acute-type ATL. Values represent a mean of three experiments. *p < 0.05, **p < 0.01.

By analyzing Gene-expression profiling of different subtypes ATL patients, asymptomatic carriers, and normal CD4+ T cells in the Gene Expression Omnibus database as showed in Figure 1d, it was found the expression level of SIRT1 and cFLIPL in asymptomatic carriers was significantly increased compared with healthy control (p < .01). However, in other distinct clinicopathologic subtypes, there was no significance because of complicated development of ATL. Interesting, the expression level of cFLIPS was significantly upregulated in acute subtype (p < .01), the most common subtype in clinic, with an identical trend of expression SIRT1 (p = .065).

This data showed that high expression of cFLIP was a key reason for resistance to Fas/FasL-mediated apoptosis in T leukemia cells and may be an immune escape mechanism in asymptomatic carriers ATL patients to develope symptomatic ATL.

Knockdown SIRT1 increased FasL-mediated apoptosis in T leukemia cells by donwregulating cFLIP

In order to further evaluate the correlation between high expression SIRT1 and cFLIP in FasL-mediated apoptosis of T leukemia cells, SIRT1 was knocked down by siRNA technology and the apoptotic rate of MT-2 and MT-4 cells were detected. As showed in Figure 2a, the FasL-mediated apoptosis were significantly increased in SIRT1-knockdown MT-2 and MT-4 cells compared with that in scrambled siRNA (SC)-transfected control (p < .05). It was demonstrated that the efficacy of SIRT1 knockdown in siRNA2-transfected cells was better than that in siRNA1-transfected cells. Compared with 4.3 ± 1.51% and 6.5 ± 2.01% of apoptotic rates in SC-transfected control cells, apoptotic rates in siRNA2-transfected cells reached to 8 ± 2.51% and 16.2 ± 1.65%, respectively. Concurrently, SIRT1 knockdown caused a downregulation of cFLIPL, which was more obvious in siRNA2-transfected cells as showed in Figure 2b. Therefore, siRNA2 and MT4 cells were chosen for subsequent experiments. As expected and showed in Figure 2c, SIRT1 knockdown decreased significantly survival rate of MT-4 to 70 ± 6.53% when treated with 10 ng/ml rhFasL (p < .05), while to 58 ± 4.25% at 20 ng/ml rhFasL compared with an 83 ± 3.73% survival rate of control cells (p < .01). these results implied that expression level of SIRT1 may be a positive correlation to that expression of cFLIP in T leukemia cells. Figure 2. Cell viability and apoptosis in SIRT1-knockdown T leukemia cells. Two SIRT1 siRNA (siRAN1 and siRNA2) or scrambled control (SC) were transfected transiently into MT-2 and MT-4 cells for 48h, then cells were treated with or without rhFasL for 16 h. (a) Representative flow cytometry graph and Bar graph of the percentages of apoptotic cells in MT-2 and MT-4 cells. (b) Representative Western blot of SIRT1, cFLIP and β-Actin in MT-2 and MT-4 cells. Bar graph of fold changes in cFLIP and SIRT/β-Actin were calculated by normalizing to that in SC group according to gray value. (c) Percentage of viability in MT-4 cells without (wt) or with SIRT1 siRNA2 transfection (kd) is expressed relative to control cells Jurkat. Values represent a mean of three experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

SIRT1 inhibitor E×527sensitize T leukemia cells to FasL-mediated apoptosis

As a deacetylation enzyme, the function of SIRT1 can be arrested by inhibitors, such as EX527, a SIRT1 specific inhibitor. In our experiment showed in Figure 3c, E×527 could decrease the expression levels of cFLIPL and cFLIPS in a dose-dependent manner, which have no effect on SIRT1 expression. It demonstrated that a significant decline of cFLIP was at 20 μM E×527 treatment. Then, the pro-apoptotic effect of E×527 combined with rhFasL on MT-4 was evaluated through Annexin V and PI-staining assay. As showed in Figure 3b, apoptotic rate of MT-4 was significantly raised to 25.2 ± 1.73% by an administration of 20 μM E×527 plus 20 ng/ml rhFasL compared with a 15.6 ± 1.43% or 16.5 ± 2.13% apoptotic rate in FasL or E×527 treatment, respectively. The FasL-mediated apoptosis signaling was also detected as illustrated in Figure 3a. Compared with control, the fragments of cleaved caspase-8 in combination 20 μM E×527 with 20 ng/ml rhFasL was more than that in any single treatments. Especially, the 19kD and 17kD fragment of caspase-3, an apoptotic effector activated by caspase-8, in MT-4 cells treated with 20 μM E×527 plus rhFasL was significantly higher than that in any single treatments (p < .01). Moreover, the 89kD fragment of poly ADP-ribose polymerase (PARP) was reasonably increased in MT-4 with a combination of E×527 and rhFasL treatment, which was higher than that with single rhFasL treatment (p < .01). In addition, the fragments of Bid cleaved (tBid), another substrate for activated caspase-8, in combination treatment was more than that in any single treatments. These results showed that inhibition of SIRT1 deacetylation function could downregulate cFLIP to sensitize activation of FasL-mediated apoptosis pathway. Figure 3. EX527 sensitized rhFasL-induced apoptosis in T leukemia cells. MT-4 cells were treated continuously with 10 or 20 ng/ml rhFasL for 16 h after pretreated with or without 20 μM EX527 for 36 h, then protein lysates were immunoblotted. (a) Representative Western blot of effector proteins in FasL-mediated apoptosis signaling pathway including caspase-8,3, Bid and PARP. Bar graph of fold changes in cleaved caspase-8,3 Bid and PARP/β-Actin were calculated by normalizing to that in untreated control cells or 20 ng/ml single rhFasL treated cells according to gray value. (b) The apoptotic rate of MT4 cells after treatment indicated. Representative flow cytometry graph and Bar graph of the percentages of apoptotic cells. (c) Cells were treated with indicated concentrations of EX527 for 24 h, then protein lysates were immunoblotted. Representative Western blot of SIRT1, cFLIP and β-Actin. Bar graph of fold changes in cFLIPL or cFLIPS/β-Actin were calculated by normalizing to that in untreated control cells according to gray value. Values represent a mean of three experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

SIRT1 contributed to stabilization cFLIP protein via deacetylating Ku70

Due to SIRT1 function, we wondered if cFLIP was the substrate of SIRT1. Coimmunoprecipitation assays based on Flag-specific antibody revealed that cFLIPL not only interacted with Ku70, one known protein with stabilizing cFLIP, but also with SIRT1 as depicted in Figure 4a. Simultaneously, in another coimmunoprecipitation assays based on Ku70-specific antibody as showed in Figure 4b, it confirmed that Ku70 also interacted with SIRT1 and cFLIPL. Interesting, as showed in Figure 4c, SIRT1 knockdown not only caused a significant decrease in the expression levels of cFLIPL and cFLIPS, but also a significant increase in acetylation of Ku70 in MT-4 leukemia cells. Therefore, it was reasonable to suppose that the statue of Ku70 acetylation could be influenced by SIRT1.The protein levels of Ku70 and Ku70 acetylation were examined after transfection of 293T cells with SIRT1-wild and H363Y mutant expression vector (a site-mutation in 363rd amino acid, which lead to loss of deacetylase activity). As showed in Figure 5a, acetylation lysine of Ku70 in mutant group was higher than that in wild group (p < .05), but expression level of Ku70 have no significant change, as same as siRNA2-knockdown did. Considering to exclude interference of other proteins, it was confirmed by co-immunoprecipitation assays that the acetylation levels of Ku70 was increased after transfection of 293T cells with SIRT1 H363Y mutant expression vector as demonstrated in Figure 5b. Moreover, not only knockdown of SIRT1 but also E×527 treatment obviously disrupted the binding of cFLIP to Ku70 as demonstrated in Figure 5c, D. Expectedly, it illustrated in Figure 5e that polyubiquitination of cFLIP isoforms were significantly increased in cells treated with E×527 as similar as MG132 did, a cell-permeable proteasome inhibitor, which was also confirmed in T leukemia cells transfected with SIRT1 siRNA2. These results were suggested that SIRT1 could prevent cFLIP protein from ubiquitination-mediated degradation by deacetylating Ku70. Figure 4. SIRT1 interacted with Ku70/cFLIP and were related to acetylation level of Ku70. (a) Protein lysates from 293T cells transfected transiently with 2 ug Flag-tagged cFLIP expression vector were immunoprecipitated (IP) with anti-Flag or control IgG anti-bodies, and then immunoblotted (IB) for the presence of Ku70, SIRT1 and cFLIP. (b) Protein lysates from 293T cells transfected transiently with 3ug Flag-tagged Ku70 expression vector were immunoprecipitated (IP) with rabbit anti-Ku70(R) or control IgG antibodies, and then immunoblotted (IB) for the presence of Flag, SIRT1 and cFLIP. (c) Acetylation level of Ku70 in SIRT1-knockdown MT4. Representative Western blot of SIRT1, cFLIP, Ku70 and β-Actin. Bar graph of fold changes in cFLIPL/β-Actin, Acetylated-Ku70/Ku70 and SIRT1/β-Actin were calculated by normalizing to that in SC group according to gray value. Values represent a mean of three experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. SIRT1 contributed to stability of cFLIP via Ku70 deacetylation. (a) 4 ug wild-type or H363Y mutant SIRT1 expression vector or 20 μM SIRT1 siRNA2 was transfected transiently into 293T cells for 48 h, then protein lysates were immunoblotted. Representative Western blot of acetylated-Ku70, total Ku70 andβ-Actin. Bar graph of fold changes in acetylated Ku70/total Ku70 were calculated by normalizing to that in control group according to gray value. (b) Protein lysates from 293T cells transfected transiently with 4 ug wild-type or H363Y mutant SIRT1 expression vector were immunoprecipitated(IP) with anti-Ku70(R) antibody, and then immunoblotted (IB) for the presence of acetylation lysine, Ku70 and SIRT1. (c) Protein lysates from 293T cells transfected transiently with 20 μM SIRT1 siRNA2 or scrambled siRNA were immunoprecipitated (IP) with anti-Ku70 (R), and then immunoblotted (IB) for the presence of Ku70 (M, mouse Ab), SIRT1 and cFLIP. (d) Protein lysates from 293T cells transfected transiently with 2 ug Flag-tagged cFLIP expression vector before 10 μM EX527 treatment for 24 h, were immunoprecipitated (IP) with anti-Flag antibodies, and then immunoblotted (IB) for the presence of Ku70, SIRT1 and cFLIP. (e) 293T cells were transfected transiently with 2.5 ug Flag-tagged cFLIP expression vector for 48 h, then treated with 20 μM EX527 for 6h or 10 μM MG132 for 8 h or proceed SIRT1 siRNA2 transfection for 48h. Protein lysates were immunoprecipitated(IP) with anti-cFLIP antibodies, and then immunoblotted (IB) using anti-ubiquitin antibody. Values represent a mean of three experiments. Values represent a mean of three experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

One of the classic hallmarks of cancer is evasion of apoptotic-mediated cell death signaling, which promotes tumor growth and metastasis.33 Therefore, targeting the cellular apoptosis pathway had been proposed as a rational and effective strategy of anticancer therapies. For several decades, targeting death receptor (DR)-mediated extrinsic apoptosis pathway, such as TRAIL anticancer therapies, has demonstrated tremendous promise in preclinical studies. However, TRAIL resistance disappointed clinical trials despite preclinical success.2,7,21 In this study, we evaluated the effect of SIRT1 inhibitor E×527 on sensitizing DR-mediated apoptosis in T leukemia cells and explored its possible pharmacological mechanism. E×527 could strengthen significantly activation of rhFasL-mediated apoptotic signaling pathway and increased apoptotic rate of T leukemia cells. Mechanically, Inhibition of SIRT1 by E×527 increased polyubiquitination level of cFLIP via increasing acetylation of Ku70, which could lead to proteosomal degradation of cFLIP protein. It implied that combinatorial therapies of E×527 plus TRAIL may have a potential as a novel clinical application for TRAIL-resistant hematologic malignancies.

SIRT1 is one of class III HDACs(sirtuins) that requires nicotinamide adenine dinucleotide (NAD) as a co-factor to function deacetylation of substrates. As an epigenetic regulator, the expression level of SIRT1 is contradictory in different types of cancer, especially in solid cancer. However, it was reported that in most of the hematologic malignancies, SIRT1 was overexpressed. For an example, in leukemia lymphoblasts, it was reported that SIRT1 was the only overexpressed significantly HDAC.34,35 In our study, it was interesting that there was positive correlation between expression of SIRT1 and cFLIPL by analyzing Gene-expression profiling of T cells from any subtypes of ATL patients and asymptomatic carriers. High expression levels of both were observed significantly in asymptomatic carriers compared with normal healthy persons. Interesting, the expression level of cFLIPS was significantly upregulated in acute subtype, the most common subtype in clinic, with an identical trend of expression SIRT1. Similarly, in T leukemia cells, overexpression levels of SIRT1 and cFLIPL was also determined significantly in MT2, MT4 and C8166 compared with Jurkat. This reason for differentiation of expression may be attributed to similarity of MT2, MT4 and C8166 with asymptomatic carriers ATL cells, which was consistent with previous studies.36,37 Indeed, in T leukemia cells MT2 and MT4, the survival rates were significantly higher than that in Jurkat after rhFasL treatment(above 10 ng/ml). The same phenomena were also observed in apoptotic rate. When we transiently knocked down SIRT1 in MT2 and MT4, the expression level of cFLIPL decreased significantly. The apoptotic rate increased by approximately 50% and the survival rate decreased by about 20% in SIRT1-knockdown MT4 cells after rhFasL treatment. These results suggested that there was closely positive correlation between SIRT1 and c-FLIPL, and overexpression levels of both may be involved in the resistance to FasL-mediated apoptosis in T leukemia cells and may be an immune escape mechanism in asymptomatic carriers ATL patients to develope symptomatic ATL.

The cellular FLICE inhibitory proteins (c-FLIP) are key regulators of extrinsic apoptosis, which antagonized activation of caspases by being recruited to the DISC and replaced procaspase-8 in the DED filaments at high concentrations.38 c-FLIP is reportedly overexpressed in a wide variety of cancers and is associated with poor prognosis, which was regarded as one of important reason for anticancer therapy resistance, including targeting death receptor (DR) therapy.9,10,39 Therefore, one strategy for the development of anticancer therapies is to influence c-FLIP activity to overcome resistance to DR-mediated apoptosis by downmodulation of c-FLIP expression levels or direct pharmacological targeting of c-FLIP proteins.7 Histone deacetylase (HDAC) inhibitors, such as vorinostat and entinostat, in combination with TRAIL and chemotherapeutics, has been extensively studied in several cancers and might be a potential new anticancer therapies, which have revealed HDAC inhibitors can downmodulate c-FLIP through the effects on Ku70-mediated regulation of c-FLIP.40–42

However, these HDAC inhibitors mainly targeted class I and II HDACs. There were few reports to exploit the roles of class III HDACs inhibitors in death receptor (DR)-mediated therapy. One of SIRT1 inhibitor, Amurensin G, had showed sensitize apoptosis of TRAIL-resistant leukemic cells, but its specificity of pharmacological targets and pharmacological mechanisms need to be disputed, which was active compound identified from methanol extracts of V. amurensis with SIRT1 inhibitory activity (35% inhibition at 30 ug/ml). Actually it is a resveratrol trimer.43,44 As a specific SIRT1 inhibitor, E×527 (Selisistat) has much lower potency against other member of sirtuins family, such as SIRT2 and SIRT3, but also does not inhibit class I/II HDAC activity at concentrations up to 100 μM.(28,29) More importantly, in clinical trials, E×527 have been confirmed to be safe and well tolerated by healthy volunteers and patients.31,32 In our study, E×527 could not only induce cFLIP downregulation as same as SIRT1 knockdown did, but also sensitize T leukemia cells to rhFasL-induced apoptosis. The activation of extrinsic apoptotic pathway triggered by Fas-FasL engagement was significantly augmented in E×527-treated T leukemia cells. The apoptotic rate of T leukemia cells treated with combination of E×527 plus rhFasL increased by approximately 10% compared with any single treatments. A significantly increase in activation of caspase-8, cleavage of its down-stream effectors caspase-3, Bid and PARP was observed in combination treatment group compare with any single treatments. These data demonstrated that inhibition of SIRT1 activity by E×527 could sensitize FasL-mediated apoptosis in T leukemia cells, which was closely associated with downregulation of cFLIP.

Considering Ku70-mediated regulation of c-FLIP, we speculated that Ku70 may be also a substrate and deacetylated by SIRT1. By overexpression of Flag-tagged cFLIP or Flag-tagged Ku70 protein, it was demonstrated that SIRT1 could interact with the complex of cFLIP/Ku70. Moreover, no matter in SIRT1 knockdown or overexpression SIRT1 H363Y mutant vector (loss of deacetylase activity) cells, the higher expression level of acetylated lysine of Ku70 was detected in our experiments. And it was confirmed by co-IP assay that acetylated Ku70 was significantly increased when SIRT1 lost its deacetylase activity. As expected, the formation of Ku70/cFLIP complex was significantly decreased when SIRT1 knockdown or E×527treatment. The turnover of c-FLIP is strongly regulated by the ubiquitin-dependent degradation rate.45 Sequentially, the polyubiquitination level of cFLIP was also increased after treatment of EX-527 and knockdown of SIRT1, respectively, which showed the same result as MG132 did, a cell-permeable proteasome inhibitor. This results implied that Ku70 may be also a substrate of SIRT1 and SIRT1 could regulate c-FLIP protein by Ku70 deacetylation, which explain partly the mechanism that high expression of SIRT1 in T leukemia cells contributed to maintain high level of cFLIP protein for resisting FasL-mediated apoptosis.

In general, it was demonstrated in our study that high expression level of SIRT1 in T leukemia cells may be play a key role in resisting FasL-mediated apoptosis by deacetylating Ku70, resulting in stabilization of cFLIP to be prevented from degradation. Therefore, SIRT1 inhibitor E×527 could sensitize DR-mediated apoptosis (Figure 6). The combinatorial therapies of anticancer based on TRAIL and E×527 might be of special interest for future clinical application for TRAIL-resistant hematologic malignancies. Figure 6. Schematic overview of SIRT1 role in T leukemia cells during FasL-mediated apoptosis. In T leukemia cells, high expression of cFLIP inhibit Fas/FasL-initiated extrinsic apoptotic signaling through inactivation of caspase-8, which is one of reasons for resistance to FasL-mediated apoptosis. SIRT1 interact with cFLIP/Ku70 complex and prevent their disruption via deacetylase Ku70. When inhibition of SIRT1 activity by E×527 increased acetylation of Ku70, cFLIP/Ku70 complex disrupt, which cause cFLIP polyubiquitination and sequentially induce degradation via the proteasome. The downregulation of cFLIP relieved inhibitory effect on Fas/FasL-initiated extrinsic apoptotic signaling. caspase-8 activated can activate executioner caspases-3 by cleaving pro-caspase-3 directly or indirectly by caspase-8-mediated cleavage of Bid, which induce the intrinsic mitochondrial-mediated apoptotic pathway. Caspase-3 further act on apoptotic-related substrate to induce cellular apoptosis.

Acknowledgments

The authors are grateful to prof. Bo Yang from Laboratory of Infection and Immunology and Lichen Zhang from Laboratory of Genetic Regulators in the Immune System, Xinxiang Medical University, for technical assistance.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Rongqi Guo, Yating Du, Yihui We performed experiment design, operation and analyzing data; Luyue Liu performed flow cytometry experiments and analyzing data; Xiaofang Guo and Xiaofei Zhu conceived the hypothesis, supervised experiments design and wrote the manuscript; Lijun Zhao and Hongfei wang performed GEO microarray analysis. Haoqi Zhang, Ruili Sun, Ruiying Ren, Tingting Zhang assisted with the experiments and data collection. Xiwen Xiong discussed experiment design and paper writing. All authors have read and approved the submitted and final versions of the manuscript for publication.

IRB or ethical review board approval

This research does not require IRB or ethics review board approval.

Data availability statement

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.
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