Investigational New Drugs

https://doi.org/10.1007/s10637-017-0552-y

PRECLINICAL STUDIES

Histone deacetylase inhibitor chidamide induces growth

inhibition and apoptosis in NK/T lymphoma cells
through ATM-Chk2-p53-p21 signalling pathway
Jianan Zhou 1 & Canjing Zhang 2 & Xianxian Sui 3 & Shengxuan Cao 4 & Feng Tang 5 & Shuhui Sun 6 & Songmei Wang 3 &
Bobin Chen 1

Received: 21 July 2017 / Accepted: 12 December 2017

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Summary
We investigated the anti-tumour effects and the underlying molecular mechanisms of a new oral histone deacetylase inhibitor
(HDACi), chidamide, in NK/T cell lymphoma (NKTCL), a rare and highly aggressive non-Hodgkin lymphoma with poor
outcomes. SNT-8 and SNK-10 NKTCL cell lines were exposed to different concentrations of chidamide for the indicated time.
The treated cells were analysed for cell proliferation, cell cycle progression, and cell apoptosis. Proteins in the AKT/mTOR and
MAPK signalling pathways and the DNA damage response (DDR) cell cycle checkpoint pathway were measured by Western
blotting. Chidamide inhibited cell proliferation in a dose- and time-dependent manner, arrested cell cycle progression at the G0/
G1 phase, and induced apoptosis in the NKTCL cell lines. In addition, we found that chidamide suppressed the phosphorylation
levels of proteins in the AKT/mTOR and MAPK signalling pathways and activated the DDR cell cycle checkpoint pathway, that
is, the ATM-Chk2-p53-p21 pathway. Expression of EBV genes was also assessed by Real-Time PCR. Chidamide induced EBV
lytic-phase gene expression in EBV-positive NKTCL. Our results provide evidence that chidamide shows antitumour effects by
inhibiting the AKT/mTOR and MAPK signalling pathways and activating the ATM-Chk2-p53-p21 signalling pathway in vitro.

Keywords NK/T cell lymphoma . Chidamide . DNA damage response . Cell cycle checkpoint pathway

Electronic supplementary material The online version of this article

(https://doi.org/10.1007/s10637-017-0552-y) contains supplementary
Introduction
material, which is available to authorized users.
NK/T cell lymphoma (NKTCL), a peculiar form of lymphoma,
* Songmei Wang is an aggressive malignancy that occurs frequently in Asian
smwang2@fudan.edu.cn countries but rarely in Western countries [1–4]. EBV plays an
* Bobin Chen essential role in NKTCL carcinogenesis [5, 6]. The localized
bbchen@fudan.edu.cn form of NKTCL can be treated with radiotherapy with or with-
out chemotherapy, and its 5-year overall survival (OS) is ap-
1
Department of Hematology, Huashan Hospital, Shanghai Medical proximately 40–90% [7, 8]. Although the disseminated form is
College, Fudan University, 12 Urumqi Middle Road,
Shanghai 200040, China treated mainly with anthracycline-based chemotherapies, such
2 as CHOP (cyclophosphamide, doxorubicin, vincristine, and
Institutes of Biomedical Sciences, Shanghai Medical College, Fudan
University, Shanghai, China prednisone), it is resistant to chemotherapy and has a poor
3 prognosis because of frequent expression of P-glycoprotein.
Laboratory of Medical Molecular Biology, Experimental Teaching
Center, School of Basic Medical Sciences, Fudan University, Dongan L-asparaginase was introduced to treat stage III/IV patients in
Road, Shanghai 200032, China the early 2000s, but the disseminated form still has a poor
4
Shanghai Medical College, Fudan University, Shanghai, China prognosis with less than 30% of 5-year OS [9–11].
5 Many studies proposed a vast array of available treatment
Department of Pathology, Huashan Hospital, Shanghai Medical
College, Fudan University, Shanghai, China options for the disseminated form, such as P-GEMOX
6 (pegaspargase, gemcitabine and oxaliplatin) [12], MESA
Key Laboratory of Medical Molecular Virology, School of Basic
Medical Sciences, Fudan University, Shanghai, China (methotrexate, etoposide, dexamethasone and asparaginase)
 Invest New Drugs

[13], MIDLE (methotrexate, ifosfamide, dexamethasone, L- information prompted us to investigate the therapeutic poten-
asparaginase and etoposide) [14], autologous and allogeneic tial of chidamide for NKTCL treatment.
H S C T [1 5 ] , i m m u n o t h e r a p y ( a n t i - P D 1 a n t ib o d y The aim of this study was to investigate the effects of
pembrolizumab) [16], and epigenetic therapy (demethylation chidamide on the inhibition of NKTCL proliferation and to
drugs and deacetylation drugs) [17]. But according to clinical explore its potential molecular mechanisms. We found that
trials, the reliability and long-term effects of these treatment are chidamide inhibited cell growth and induced apoptosis in the
not good. Thus, much attention has been focused on identifying SNT-8 and SNK-10 NKTCL cell lines via the AKT/mTOR
new therapeutic targets for the development of novel drugs, and MAPK pathways. Furthermore, chidamide could induce
with the aim of improving NKTCL treatment outcomes. cell cycle arrest at the G0/G1 phase through a molecular
The role of histone deacetylases (HDACs) in cancer and mechanism dependent on ATM-Chk2-p53-p21 signal path-
the therapeutic potential of histone deacetylase inhibitors way activation. We also demonstrated that chidamide is an
(HDACis) as emerging drugs in cancer treatment have effective inducer of EBV lytic-phase gene expression in
attracted increasing amounts of attention. Some well- NKTCL cell lines. These findings suggest the potential clini-
recognized mechanisms of HDACi lethality include regula- cal application of chidamide as a novel therapeutic drug for
tion of the cell cycle, apoptosis, DNA-damage responses treating NKTCL.
(DDRs), metastasis, angiogenesis, autophagy, and other cellu-
lar processes [18]. HDACs play crucial roles in cancer by
modulating chromatin remodelling and maintaining a dynam- Results
ic acetylation balance among DNA damage-related proteins.
Consequently, the relevance of DDRs in HDACi-induced ap- Chidamide inhibited the viability of NKTCL cell lines
optosis is important [19].
Various chemotherapeutic agents and ionizing UV radia- SNT-8 and SNK-10 cell lines were cultured with various con-
tion can activate DDRs, including DNA repair and cell cycle centrations of chidamide for 24 h, 48 h and 72 h. The effects of
checkpoint pathways, which allow cells to address both en- chidamide on viability in the SNT-8 and SNK-10 cell lines
dogenous and exogenous sources of DNA damage [20, 21]. were measured using the CCK-8 assay. We found that
The cell cycle checkpoint pathways can allocate more time for chidamide decreased SNT-8 and SNK-10 cell viability in a
DNA repair to preserve genomic integrity. Failure to accurate- time and dose-dependent manner (Fig. 1).
ly repair DNA damage will lead to permanent cell cycle arrest
and apoptosis, resulting in the delay or prevention of Chidamide arrested NKTCL cell lines at the G0/G1
tumourigenesis [22]. In cell cycle checkpoint pathways, the phase
ataxia-telangiectasia mutated (ATM) kinase is activated
through the autophosphorylation of the Ser1981 residue, To determine whether cell cycle progression was arrested by
which subsequently activates the distal transducer kinase chidamide in the tested cell lines, we evaluated the effects of
checkpoint kinase 2 (Chk2), resulting in G0/G1 checkpoint chidamide on the cell cycle distribution of SNT-8 and SNK-10
activation [23, 24]. In addition, as a critical mediator of the cells by flow cytometry with PI staining. In SNK-10 cells,
G0/G1 checkpoint, wild type p53 can be phosphorylated by after incubation with different concentrations of chidamide
ATM kinase, then activates p21, which binds and further in- for 48 h, the proportion of G0/G1 cells was increased from
hibits cyclin A/Cdk2 and cyclin E/Cdk2 complexes, DNA 51.96% to 65.75% after treatment with 8 μM chidamide, but
repair proteins and, upon protracted checkpoint activation, this increasing was not statistically significant (Fig. 2a). In
apoptotic cell death promoters [25, 26]. SNT-8 cells, chidamide increased the proportion of G0/G1
Up to now, four HDACis (vorinostat, romidepsin, cells in a dose-dependent manner. At the same time, the per-
belinostat, and panobinostat) have been approved to treat can- centage of S phase cells was obviously decreased (Fig. 2b).
cer by the US Food and Drug Administration (FDA). The p21 and cyclin E are the key factors that mediate the tran-
China Food and Drug Administration (CFDA) approved sition of cells from G1 phase to S phase. To investigate wheth-
chidamide to treat relapsed/refractory peripheral T-cell lym- er chidamide could influence the expression levels of these two
phoma (PTCL). Chidamide (CS055/HBI-8000) (Fig. 1a), a factors, the protein levels of p21 and cyclin E were detected by
novel benzamide type of HDACi, has clear subtype selectivity Western blotting in SNT-8 and SNK-10 cells after treatment
and provides distinct regulation of gene expression. Previous with various concentrations of chidamide for 48 h or 4 μM
studies have shown that chidamide induces cell growth inhi- chidamide for different time. Our data showed that chidamide
bition and apoptosis in many cancers, including lung cancer up-regulated p21 protein expression, down-regulated cyclin E
[27], colon cancer [28], hepatocellular carcinoma [29], pan- protein expression in NKTCL cell lines (Fig. 2c).
creatic cancer [30] and haematologic malignancies, such as These results suggested that chidamide inhibited NKTCL
myelodysplastic syndromes [31] and leukaemia [32]. This cell proliferation through inducing cell cycle arrest at the G0/
Invest New Drugs

Fig. 1 Chidamide inhibited the

viability of NKTCL cell lines. a
The chemical structure of
chidamide. b SNT-8 cells were
treated for 24 h, 48 h or 72 h with
chidamide at the following
concentrations: 0 μM (Control),
0.5 μM, 2.5 μM, 5 μM, or
10 μM. c SNK-10 cells were
treated for 24 h, 48 h or 72 h with
chidamide at the following
concentrations: 0 μM (Control),
1 μM, 2 μM, 4 μM, or 8 μM.
Effects of chidamide on SNK-10
and SNT-8 cell viability were
measured by CCK-8 assay. Time-
and dose-response curves were
produced using GraphPad
software

G1 phase. The mechanism by which chidamide induces cell As shown in Fig. 4a, chidamide reduced p-AKT expression
cycle arrest in NKTCL cell lines may be associated with p21 in both cell lines without affecting the level of total AKT.
and cyclin E. There were no changes in the p-AKT levels in cells treated
with a low concentration of chidamide. However, treatment
with a high concentration of chidamide significantly inhibited
Chidamide induced NKTCL cell lines apoptosis
p-AKT protein expression in both SNT-8 and SNK-10 cell
through mitochondria-mediated caspase pathway
lines. In addition, minor changes were found in the expression
of p-mTOR after treatment with a low concentration of
Many chemotherapeutic agents promote cancer cell apoptosis.
chidamide, but an obvious decrease was observed in SNT-8
We investigated the effect of chidamide on apoptosis in
cells after treatment with a high dose of chidamide for a long
NKTCL cell lines using annexin V/PI dual staining and flow
incubation time.
cytometry. Our data showed that chidamide induced apoptosis
Chidamide inhibited the expression of p-JNK and p-Erk in
in both SNT-8 and SNK-10 cells (Fig. 3a and b).
SNT-8 and SNK-10 cells in a concentration- and time-
Next, Bcl-2, Bax, PARP and survivin were analysed by
dependent manner. However, the expression levels of p-P38
immunoblotting. The data showed that chidamide had no ob-
and total P38 were unchanged (Fig. 4b and c).
vious effects on the expression of Bax, while it down-
These results suggested that chidamide inhibited cell
regulated Bcl-2 (Fig. S1) and survivin, and induced the cleav-
growth and induced apoptosis in NKTCL cell lines through
age of PARP in the two cell lines (Fig. 3c). Collectively, these
regulation of the AKT/mTOR and MAPK pathways.
results suggest that the mechanism through which chidamide
induced apoptosis in NKTCL cell lines may be associated
with mitochondria-mediated caspase pathway.
Chidamide activated the cell cycle checkpoint
pathway in NKTCL cell lines
Chidamide inhibited MAPK and AKT signalling
pathways in NKTCL cell lines To elucidate the molecular mechanism underlying the
chidamide-induced cell cycle arrest and apoptosis in
In NKTCL, AKT and MAPK pathways are often over activat- NKTCL cell lines, we hypothesized that chidamide can acti-
ed. To further investigate the mechanisms through which vate the cell cycle checkpoint pathway. We examined the ef-
chidamide affects NKTCL cell lines, the expression levels of fects of chidamide on p-ATM, ATM, p-Chk2, and γH2A.X
AKT and MAPK signal pathways were analysed using protein expression in NKTCL cell lines by Western blotting
Western blotting. (Fig. 5).
 Invest New Drugs

Fig. 2 Chidamide arrested NKTCL cell lines at the G0/G1 phase. a cytometry. Representative flow cytometry results are shown. b Cell
SNT-8 cells were treated for 48 h with chidamide at the following distribution at the G0/G1, S, and G2/M phases of the cell cycle.
concentrations: 0 μM (Control), 0.5 μM, 2.5 μM, 5 μM, or 10 μM. *p < 0.05, **p < 0.01, ***p < 0.001 vs control. C. Western blotting
SNK-10 cells were treated for 48 h with chidamide at the following showed the protein levels of p21 and cyclin E after treatment with
concentrations: 0 μM (Control), 1 μM, 2 μM, 4 μM, or 8 μM. Cell different concentrations of chidamide for 48 h or 4 μM chidamide for
cycle distribution of SNT-8 and SNK-10 cells was analysed by flow different lengths of time

SNT-8 and SNK-10 cells were treated with 4 μM and 5 μM These results suggested that chidamide caused serious DNA
chidamide for 0 h, 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h, and damage and activated the cell cycle checkpoint pathway of the
the expression of p-ATM and ATM was detected by Western DNA damage response, the ATM-Chk2-p53-p21 pathway, to
blotting. The two cell lines were treated with various concen- induce G1 phase arrest and apoptosis.
trations of chidamide for 48 h, and the expression of p-ATM,
ATM, p-Chk2, pp53 and p53 was detected by Western blot- Chidamide induced EBV lytic-phase gene expression
ting. Next, the NKTCL cell lines were treated with 4 μM and in NKTCL cell lines
5 μM chidamide for 0 h, 1 h, 6 h, 12 h, 24 h and 48 h, and
Western blotting showed the expression of H2A.X phosphor- Since EBV plays an essential role in NKTCL etiology, we
ylated at Ser 139, which is a marker of DNA damage. investigated the mRNA expression of three EBV lytic-phase
As shown in Fig. 5, after cultured with chidamide at in- genes (BZLF1, BRLF1, and BMRF1) and one EBV latent-
creasing concentrations and time, NKTCL cells showed in- phase gene (EBNA1) in two NKTCL cell lines by Real-Time
creased expression of p-ATM, γH2A.X, p-Chk2 and pp53. PCR (Fig. S2). The results showed that chidamide had no
Invest New Drugs

Fig. 3 Chidamide induced NKTCL cell lines apoptosis through 0.01, ***p < 0.001 vs control. c Western blotting showed the protein
mitochondria-mediated caspase pathway. a Apoptosis in SNT-8 and levels of PARP, cleaved-PARP and survivin. After treatment with
SNK-10 cells was analysed by flow cytometry after 48 h of treatment different concentrations of chidamide for 48 h or 4 μM chidamide for
with chidamide. Percent apoptosis (including early and late apoptotic different lengths of time, cleavage of PARP was induced, and survivin
cells) was determined by flow cytometry. b Representative histograms was down-regulated in the two cell lines
of annexin V-FITC/PI double-staining flow cytometry. *p < 0.05, **p <

obvious effect on the expression of EBNA1, while it signifi- histone deacetylase (HDAC) inhibitor that is designed to
cantly increased the mRNA expression of the three lytic-phase block the catalytic pocket of class I HDACs [34] to selectively
genes in NKTCL cell lines. The results suggested that inhibit the activity of HDAC1, 2, 3 and 10 [35, 36]. Recently,
chidamide promoted EBV into lytic phase in EBV-positive Chidamide was reported to exert antitumour effects on various
NKTCL. cancer cell lines [27, 30, 32], but not include NKTCL cell
lines. In this study, we demonstrated that chidamide induced
apoptotic cell death and inhibited cell growth in the SNT-8 and
Discussion SNK-10 NKTCL cell lines.
Induction of apoptosis in cancer cells is one of the strate-
Natural killer/T-cell lymphoma (NKTCL) is a highly aggres- gies used in anti-cancer therapy. We found that chidamide
sive haematological malignancy. It is often resistant to con- induced apoptosis in NKTCL cell lines in a dose- and time-
ventional chemotherapy, and the development of new thera- dependent manner (Fig. 1). Furthermore, our results demon-
peutic regimens is urgently needed to improve prognosis [33]. strated that chidamide induced cell cycle arrest at the G0/G1
Chidamide is a novel benzamide type of subtype-selective phase in SNT-8 and SNK-10 cells (Fig. 2), which are similar
 Invest New Drugs

Fig. 5 Chidamide activated the cell cycle checkpoint pathway in

NKTCL cell lines. Protein expression levels of p-ATM, ATM, p-Chk2,
pp53, p53 and γH2A.X in NKTCL cell lines (SNK-8 and SNT-10) that
were untreated and treated with various concentrations of chidamide for
different lengths of time were shown by Western blotting

found that chidamide decreased the protein levels of PARP and

simultaneously up-regulated the pro-apoptotic protein cleaved-
PARP (Fig. 3c). Furthermore, chidamide had no obvious effect
on the expression of Bax (Fig. S1), while it down-regulated the
Fig. 4 Chidamide inhibited MAPK and AKT signalling pathways in
anti-apoptotic protein survivin and Bcl-2, up-regulated p21
NKTCL cell lines. Expression levels of AKT, p-AKT, mTOR, p-mTOR, and down-regulated cyclin E. These results suggest that
JNK, p-JNK, Erk, p-Erk, P38 and p-P38 in cells were determined by mitochondria-mediated caspase cascade, p21, and cyclin E
Western blotting. a Western blotting showed the protein levels of the may play vital roles in the chidamide-induced apoptosis and
AKT/mTOR pathway. b Western blotting showed the protein levels of
the MAPK pathway. Cells were treated with chidamide at different con-
cell cycle arrest in NKTCL cell lines.
centrations for 48 h. c Cells were treated with 4 μM chidamide for dif- The cell proliferation and survival AKT and MAPK
ferent lengths of time pathways are activated in NKTCL cell lines and in the
neoplastic cells of most NKTCL primary tumours.
to the previously reported effects of chidamide on MDS cells Phosphorylated AKT has often been detected in NKTCL
[31] and leukaemia cells [32]. [40, 41]. Treatment of the NK-92 NKTCL cell line with
Previous studies have revealed that chidamide achieves anti- the PI3K inhibitor LY294002 resulted in a reduction of p-
tumour effects through various mechanisms, including: 1) up- AKT and cell apoptosis [40]. Taken together, these studies
regulation of pro-apoptotic proteins (e.g., cleaved-PARP) and highlight the importance of the MAPK and AKT/mTOR
down-regulation of antiapoptotic proteins (e.g., survivin) [37]; pathways in NKTCL survival; thus, targeting MAPK and
2) induction of the endogenous cyclin-dependent kinase AKT might be an effective approach for treating NKTCL.
(CDK) inhibitor p21 [38]; 3) inhibition of the PI3K/AKT, In the present study, we found that chidamide reduced the
Jak/Stat, and MAPK/JNK signalling pathways [28, 37, 39]; expression levels of p-JNK, p-Erk and p-AKT in SNT-8
and 4) ROS generation and induction of DNA damage [36, and SNK-10 cells. The results show that the AKT/mTOR
37], as well as disruption of cell cycle checkpoints, which has and MAPK pathways play an essential role in chidamide-
been frequently reported in neoplastic cells. In this study, we induced cell death (Fig. 4).
Invest New Drugs

DNA damage causes genomic instability and, ultimately,

may lead to cancer. Induction of DNA damage, such as DNA
double-strand breaks (DSBs), has been shown to be an effec-
tive treatment for cancer. We proved that chidamide-induced
cell death involved DNA damage. This was confirmed by the
increased level of phosphorylated H2A.X (γH2A.X) detected
by Western blotting (Fig. 5c). γH2A.X is considered to be a
sensitive marker for DSBs and is essential for caspase-
activated DNase-mediated nucleosomal DNA fragmentation.
Deregulation of cell cycle progression is one of the
hallmarks of cancer. Hence, targeting the regulatory
components of the cell cycle is considered to be an
important strategy for cancer treatment. Cyclin-
dependent kinase (CDK) inhibitor p21 (also known as
p21WAF1/Cip1) is one of these components that promotes Fig. 6 A simplified scheme of chidamide inhibiting NKTCL cell lines.
cell cycle arrest in response to many stimuli [42]. p21 Chidamide inhibits NKTCL cell proliferation and induces G0/G1 phase
arrest and apoptosis through down-regulating the AKT/mTOR and
inhibits cyclin E/CDK2, which results in the inhibition
MAPK signalling pathways. Down-regulated p-AKT causes cell
of CDK2-dependent phosphorylation of RB and the se- apoptosis by activating PARP and inhibiting survivin. On the other
questration of E2F1, thus inhibiting E2F1-dependent hand, down-regulated p-AKT causes p53 activation. Chidamide induces
gene transcription and progression into and through the DNA damage by activating ATM, which phosphorylates H2A.X, Chk2
and p53 to induce cell cycle arrest at the G0/G1 phase via p21 activation
S phase [43]. Depending on the extent of DNA-damage,
and cyclin E down-regulation. Chidamide-mediated anticancer activity
the induction of p21 by p53 inhibits cyclin E/CDK2 and may occur through molecular mechanisms dependent on ATM-Chk2-
thereby inhibits the G1/S transition. We showed that p53-p21 signal pathway activation
chidamide up-regulated p21 protein expression, down-
regulated cyclin E protein expression and, consequently, NKTCL effects. Chidamide, EBV-targeted agents, has poten-
arrested the cell cycle at the G0/G1 phase (Fig. 2). The tial for treating NKTCL patients, especially for those who
stability of the p21 protein, which is the regulator of the have weak cell cycle checkpoint activity.
G1/S transition, is largely regulated by the DNA dam-
age response kinases ATM, Chk2 and p53 [44]. The
G1/S checkpoint is the first line of defence in cancer
cells. At this checkpoint, Chk2 is activated by ATM to Materials and methods
phosphorylate p53, thus preventing the activation of cy-
clin E/CDK2 [18]. We showed that chidamide induced Chidamide
the rapid activation of ATM-Chk2-p53-p21 kinases (Fig.
5). Thus, chidamide has potential use in NKTCL treat- Chidamide was provided by Shenzhen Chipscreen
ment through activating the DDR cell cycle checkpoint Biosciences Ltd. (Shenzhen, China) and was dissolved in
pathway. dimethylsulfoxide (DMSO) at a concentration of 50 mM.
It is well known that EBV is related to NKTCL carcinogen- The stock solution was stored at −80 °C until use. The final
esis [5, 6]. Since the latent-lytic EBV switch was been investi- concentration of DMSO in the treatment medium was main-
gated as a potential therapy for NKTCL [45, 46], we examined tained below 0.1%.
whether chidamide had influence on EBV lytic cycle. As shown
in our results, chidamide induced the mRNA expression of EBV
lytic genes, without influencing on the latent gene. Collectively, Cell lines and cell culture
these results suggested that chidamide activates EBV lytic rep-
lication in NKTCL cells. The NKTCL cell lines, SNT-8 and SNK-10, which were pro-
In summary, it demonstrated that chidamide inhibited cell vided by Dr. Norio Shimizu at Tokyo Medical and Dental
proliferation and induced cell apoptosis and cell cycle arrest at University, were cultured in RPMI-1640 (HyClone, Utah,
the G0/G1 phase in NKTCL cell lines (Fig. 6). We propose USA) media supplemented with 10% heat-inactivated human
that chidamide inhibits NKTCL growth by two mechanisms: plasma, and 700 U/ml recombinant human interleukin-2 (IL-2)
induction of apoptosis and inhibition of cell cycle progression, (Peprotech, Rock Hill, NJ, USA). SNK-10 was established from
which are possibly mediated by the inhibition of the MAPK the peripheral blood of a NKTCL patient with a chronic active
and AKT/mTOR pathways. In addition, chidamide activates EBV infection [47]. SNT-8 is an EBV-positive NKTCL cell line
the DDR cell cycle checkpoint pathway to achieve its anti- derived from the primary lesions of a Japanese patient [48].
 Invest New Drugs

Cell viability Antibodies

Cell viability was measured using the CCK-8 assay (Dojindo Antibodies against cyclin E, PARP, p21, phospho-p53 (Ser15),
China CO., Ltd) following the manufacturer’s instructions. phospho-AKT (Ser473), phospho-mTOR (Ser2448), mTOR,
The cell viability ratio was calculated as follows: Cell viability phospho-JNK (Thr183/Tyr185), JNK, phospho-Erk (Thr202/
ratio (%) = [OD (Chidamide) – OD (Blank)]/ [OD (Control) – Tyr204), Erk, phospho-p38 (Thr180/Tyr182), and phospho-
OD(Blank)] × 100. Each experiment was carried out using six Chk2 (Thr68) were purchased from Cell Signaling
replicates, and the results were calculated from three indepen- Technology (Boston, MA, USA). The primary antibodies an-
dent experiments. ti-survivin, anti-AKT (Ser473), anti-p38, anti-phospho ATM
(Ser1981), anti-ATM, and anti-γH2A.X (Ser139), anti-Bax,
anti-Bcl-2 were purchased from Abcam® (Cambridge, MA,
Cell cycle analysis USA). Anti-p53 antibody was purchased from Santa Cruz
Biotechology (Texas, USA). Anti-GAPDH and anti-β-actin
Cells were treated with vehicle (0.1% DMSO) or different were obtained from Wuhan Goodbio Technology Co., Ltd.
concentrations of chidamide. After a 48 h incubation, cells
were harvested and fixed by incubation in 75% ethanol Real-time PCR
(500 μl) at 4 °C for 2 h. Cells were then collected by centri-
fugation at 1000 rpm for 5 min and washed twice with PBS. Total RNA was extracted using Trizol Reagent (life technolo-
Subsequently, cells were incubated with 500 μl of PI/RNase gies, Carlsbad, USA) following manufacturer’s instructions.
Staining Buffer (BD Pharmingen Biosciences, San Diego, cDNA was synthesized using ReverTra Ace qPCR RT Kit
CA, USA) at 4 °C for 30 min in the dark. Stained cells were (Toyobo, Osaka, Japan) and subjected to Real-Time PCR
analysed using a FACSCalibur flow cytometer (Becton using a SYBR® Green qPCR Master Mix (Bio-Rad,
Dickinson, San Jose, CA, USA), and the data were analysed Hercules, USA) on a CFX96 Touch™ Real-Time PCR
using the Modfit LT software (Verity Software House, Detection System. All reactions were performed in duplicate.
Topsham, ME, USA). Primers used for Real-Time PCR were as follows:
β-actin-Forword:5’-ACTGGAACGGTGAAGGTGAC
AG-3’,Reverse:5’-GGTGGCTTTTAGGATGGCAAG-
Cell apoptosis analysis 3’;BZLF1-Forword:5’-TACAAGAATCGGGTGGCTTC-
3’,Reverse:5’-GCACATCTGCTTCAACAGGA-3’;BMRF1-
Cells were seeded in six-well plates and incubated with vari- F o r w o r d : 5 ’ - A C C T G C C G T T G G AT C T TA G T G -
ous concentrations of chidamide for 48 h. Cell apoptosis was 3’,Reverse:5’-GGCGTTGTTGGAGTCCTGTG-3’;BRLF1-
determined using an annexin V-FITC apoptosis kit (BD Forword:5’-GAAGCCCGGTGCCCAAAG-3’,Reverse:5’-
Pharmingen Biosciences, San Diego, CA, USA) according GTGTCACTGTTGCCCGAGTC-3’;EBNA1-Forword:5’-
to the manufacturer’s instructions. Cells were analysed on a C G T T T G G G A G A G C T G AT T C T- 3 ’ , R e v e r s e : 5 ’ -
FACScan flow cytometer (BD Biosciences) and using the CCCCTCGTCAGACATGATTC-3′. The relative expression
FlowJo software (Tree Star, Ashland, OR, USA). of RNA was calculated as below:
The relative expression = 2 -(ΔΔCT), where ΔCT = (cycle
threshold (CT) detected gene) - (CT β-actin) and
Western blot analysis ΔΔCT = ΔCT (chidamide) - ΔCT (Control).

Cells were seeded in six-well plates at a density of 3 × 105 Statistical analysis

cells per well and were exposed to different doses of
chidamide for different lengths of time. Then, cells were har- All data were expressed as the mean± standard deviation (SD)
vested and lysed in RIPA buffer (Beyotime, Nantong, China) and were analysed by one-way ANOVA using STATA13.0.
in the presence of the protease inhibitor PMSF (Beyotime, The results were calculated with Graphpad software (version
Nantong, China) and a phosphatase inhibitor (Roche, Basel, 6.0) (Graphpad Software, San Diego, CA). P < 0.05 was con-
Switzerland). The concentration of protein in the cell lysates sidered to be statistically significant.
was measured using a BCA assay (Beyotime, Nantong,
China). Equal amounts of protein were boiled in SDS sample Acknowledgements We thank Dr. Dongju Li (Laboratory of Medical
buffer, separated by SDS-PAGE and transferred onto PVDF Molecular Biology, Experimental Teaching Center, School of Basic
Medical Sciences, Fudan University), and Dr. Yu Zhang (First
membranes (Millipore, Billerica, MA, USA). After blocking
Department of Internal Medicine, Tokyo Medical University, Japan) for
with 5% skim milk for 1 h, the PVDF membranes were probed technical support. We thank Dr. Norio Shimizu for providing the SNT-8
with the corresponding antibodies. and SNK-10 cell lines.
Invest New Drugs

Funding The study was supported by the Chinese Medicine Scientific asparaginase and postchemotherapeutic radiotherapy. Ann
Research Foundation of the Shanghai Municipal Commission of Health Hematol 94(7):1175–1184
and Family Planning (Grant No. 2014JP003A), the Science and 12. Wang J-H, Wang L, Liu C-C, Xia Z-J, Huang H-Q, Lin T-Y, Jiang
Technology Development Fund of Bao Shan District (Grant No. 13-E- W-Q, Lu Y (2016) Efficacy of combined gemcitabine, oxaliplatin
34) and the National Natural Science Foundation of China (Grant No. and pegaspargase (P-gemox regimen) in patients with newly diag-
81470352 and 81770208). nosed advanced-stage or relapsed/refractory extranodal NK/T-cell
lymphoma. Oncotarget 7(20):29092
Compliance with ethical standards 13. Liang R, Gao GX, Chen JP, Wang JS, Wang XM, Zeng Y, Bai QX,
Zhang T, Yang L, Dong BX, Gu HT, Shu MM, Hao CX, Wang JH,
Zhang N, Chen XQ (2016) A phase 2 study of methotrexate,
Conflict of interest The authors declare that they have no conflict of
etoposide, dexamethasone, and pegaspargase chemotherapy for
interest.
newly diagnosed, relapsed, or refractory extranodal natural killer/
T-cell lymphoma, nasal type: a multicenter trial in Northwest China.
Ethical approval This article does not contain any studies with human Hematol Oncol. https://doi.org/10.1002/hon.2325
participants or animals performed by any of the authors. 14. Yoon DH, Kim SJ, Jeong SH, Shin D-Y, Bae SH, Hong J, Park SK,
Yhim H-Y, Yang D-H, Lee H (2016) Phase II trial of concurrent
chemoradiotherapy with L-asparaginase and MIDLE chemothera-
py for newly diagnosed stage I/II extranodal NK/T-cell lymphoma,
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