Activating Transcription Factor 4 Confers a Multidrug Resistance Phenotype to Gastric Cancer Cells through Transactivation of SIRT1 Expression
et al. (2012) Activating Transcription Factor 4 Confers a Multidrug Resistance Phenotype to Gastric Cancer Cells
through Transactivation of SIRT1 Expression. PLoS ONE 7(2): e31431. doi:10.1371/journal.pone.0031431
Activating Transcription Factor 4 Confers a Multidrug Resistance Phenotype to Gastric Cancer Cells through Transactivation of SIRT1 Expression
Hongwu Zhu. 0
Limin Xia. 0
Yongguo Zhang. 0
Honghong Wang 0
Wenjing Xu 0
Hao Hu 0
Jing Wang 0
Yi Gang 0
Sumei Sha 0
Bin Xu 0
Daiming Fan 0
Yongzhan Nie 0
Kaichun Wu 0
Wael El-Rifai, Vanderbilt University Medical Center, United States of America
0 Department of Gastroenterology and State Key Laboratory of Cancer Biology, Xijing Hospital, Fourth Military Medical University , Xi'an , People's Republic of China
Background: Multidrug resistance (MDR) in gastric cancer remains a major challenge to clinical treatment. Activating transcription factor 4 (ATF4) is a stress response gene involved in homeostasis and cellular protection. However, the expression and function of ATF4 in gastric cancer MDR remains unknown. In this study, we investigate whether ATF4 play a role in gastric cancer MDR and its potential mechanisms. Methodology/Principal Findings: We demonstrated that ATF4 overexpression confered the MDR phenotype to gastric cancer cells, while knockdown of ATF4 in the MDR variants induced re-sensitization. In this study we also showed that the NAD+-dependent histone deacetylase SIRT1 was required for ATF4-induced MDR effect in gastric cancer cells. We demonstrated that ATF4 facilitated MDR in gastric cancer cells through direct binding to the SIRT1 promoter, resulting in SIRT1 up-regulation. Significantly, inhibition of SIRT1 by small interfering RNA (siRNA) or a specific inhibitor (EX-527) reintroduced therapeutic sensitivity. Also, an increased Bcl-2/Bax ratio and MDR1 expression level were found in ATF4overexpressing cells. Conclusions/Significance: We showed that ATF4 had a key role in the regulation of MDR in gastric cancer cells in response to chemotherapy and these findings suggest that targeting ATF4 could relieve therapeutic resistance in gastric cancer.
Funding: This study was supported by combined grants from the National Natural Science Foundation of China (NO.81090270, NO.81090273, and NO.81000864),
the Chinese Postdoctoral Science Foundation (NO.20100471776), the National Key and Basic Research Development Program of China (NO. 2010CB529302), and
the National Municipal Science and Technology Project (2009ZX09103-667 and 2009ZX09301-009-RC06). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Multidrug resistance is usually the main cause for failure of
chemotherapy against malignant tumors, including gastric cancer
.The term multidrug resistance is classically used to define a
resistance phenotype where cells become resistant simultaneously to
different drugs with no obvious structural resemblance and with
different cellular targets . MDR occurs more frequently with novel
drugs that have more significant effectiveness after their first application
in cancer treatment. The clinical usefulness of multiple drugs is limited
by both natural and acquired tumor cell resistance, which almost
always is multifactorial in nature . The factors that may affect
drug sensitivity include: accelerated drug efflux, drug activation and
inactivation, alterations in the drug target, DNA methylation,
processing of drug-induced damage, and evasion of apoptosis .
Gastric cancer is relatively insensitive to chemotherapeutics.
The MDR mechanisms in gastric cancer cells have been broadly
investigated in our laboratory and elsewhere [1,4,5], yet they have
not been fully elucidated, indicating that other unknown molecules
or pathways may be involved in the development of MDR.
In mammalian cells, eukaryotic translation initiation factor 2 a
subunit (eIF2a) is phosphorylated by different eIF2a kinases in
response to different stress signals, including anoxia/hypoxia,
endoplasmic reticulum stress, amino acid deprivation, and
oxidative stress. This phosphorylation event leads to a rapid decrease
in global protein biosynthesis concurrent with induction of
translational expression of genes, including ATF4 that function to
alleviate cellular damage from stress [6,7]. Although ATF4 may
play a pro-apoptotic role under conditions of severe or prolonged
stress, ATF4 is a potent stress-responsive gene thought to play a
protective role by regulating cellular adaptation to adverse
circumstances in the integrated stress response (ISR) [8,9,10].
Recently, overexpression of ATF4 was reported to be prominent
in a wide variety of tumors and to protect tumor cells against
multiple stresses, as well as a range of cancer therapeutic agents
[11,12,13,14,15,16,17]. The potential mechanisms responsible for
this protection include autophagy induction, promotion of DNA
damage repair, and up-regulation of intracellular glutathione
[12,13,14,17]. However, the expression and function of ATF4 in
gastric cancer MDR remains unknown.
In this study, we reported that ATF4 was significantly
upregulated in the MDR response of gastric cancer cells compared
with parental control cells. Knockdown of ATF4 by siRNA
significantly sensitized cells with MDR to a variety of
chemotherapeutic agents, whereas up-regulation of ATF4 in SGC7901 and
AGS cells rendered them multidrug resistant. We also showed that
ATF4 promoted gastric cancer MDR partly through up-regulating
expression of SIRT1. And SIRT1 inhibition could partly reverse
the gastric cancer MDR phenotype mediated by ATF4. These
data suggest that targeting ATF4 may provide a novel therapeutic
option for reversing clinical gastric cancer MDR.
ATF4 modulates the MDR phenotype of gastric cancer
To determine whether ATF4 is involved in the development of
MDR in gastric cancer cells, ATF4 levels were detected by
Western blot and qPCR in the SGC7901 cell line and its MDR
variants, SGC7901/VCR and SGC7901/ADR. Both protein and
mRNA levels of ATF4 were much higher in the resistant cell lines
than in parental cells (Fig. 1A).
To investigate whether ATF4 overexpression is sufficient to
induce a MDR phenotype in gastric cancer cells, ATF4 expression
cDNA was stably transfected into SGC7901 and AGS cells. First,
CDDP sensitivity was tested using a colony formation assay. As
shown by the quantification of the colony formation assay, ATF4
overexpression resulted in a nearly 3-fold increase in colony
numbers compared with empty vector-expressing cells (Fig. 1B).
MTT assays also indicated that the IC50 values of
SGC7901ATF4 for ADR, VCR, CDDP, and 5-FU were significantly
increased as compared to empty vector transfected cells(Fig. 1D).
As ATF4 levels are elevated in MDR gastric cancer cells, we
further wanted to determine whether targeting ATF4 could
resensitize the MDR cell lines. Knockdown of ATF4 by siRNA in the
SGC7901/ADR and SGC7901/VCR cells led to a 2- to 3-fold
reduction in cell number when used in combination with CDDP
(Fig. 1C). Data in Fig. 1E also suggest that down-regulation of
ATF4 significantly reverses the resistance of SGC7901/ADR cells
in response to chemotherapy.
As inhibition of apoptosis is one of important mechanisms of
MDR, we also investigated the capacity of the SGC7901/ADR
cells transfected with the specific ATF4 siRNA to undergo
CDDPinduced apoptosis by Hoechst staining and DNA fragmentation
assays. Treatment of SGC7901-ATF4 and SGC7901/ADR-SCR
cells with the indicated concentrations of CDDP for 36 hours did
not induce any apoptosis, as assessed by Hoechst nuclear staining
(Fig. 1F) and DNA fragmentation assays (Fig. 1G). In contrast,
SGC7901-Vector and SGC7901/ADR-siATF4 cells displayed
significant apoptosis, with the more frequent appearance of
condensed and fragmented nuclei and DNA ladder formation.
Moreover, more obvious cleavage of procaspase-3 was observed
after treatment with CDDP in SGC7901-Vector and SGC7901/
ADR-siATF4 cells as compared to SGC7901-ATF4 and
SGC7901/ADR-SCR cells, respectively (Fig. 1H).
Taken together, these results indicate that ATF4 confers a
MDR phenotype to gastric cancer cells and that targeting ATF4
provides a method of sensitizing resistant cells to chemical
mechanisms [18,19,20,21]. We were curious to determine whether
SIRT1, which is a stress-related gene critical to MDR
development, could be the downstream target of ATF4 responsible for
mediating ATF4-induced MDR in gastric cancer cells.
SIRT1 levels in LV-Vector and LV-ATF4 stably transfected
SGC7901 cells were assayed by qPCR and Western blot. The
overexpression of ATF4 was associated with increased SIRT1
expression at both the transcriptional (Fig. 2A, left) and translational
levels (Fig. 2B, left). In contrast, siRNA knockdown of ATF4 in
SGC7901/ADR cells resulted in a significant reduction of
endogenous SIRT1 expression (Fig. 2A and 2B, right). These results suggest
that ATF4 up-regulates SIRT1 expression in gastric cancer cells.
To further investigate the molecular mechanisms involved in
ATF4-related MDR of gastric cancer, we also examined MDR1,
MRP, Bcl-2, and Bax expression levels in the gastric cancer cells
used above. As shown in Fig. 2B, ATF4-proficient cells expressed
more MDR1 as compared to the control cells. Meanwhile, no
obvious difference in MRP expression was found in any of these
cell lines. Interestingly, both Bcl-2 and Bax expression levels were
up-regulated in ATF4-proficient cells, compared with the control
cell lines, while the expression of Bax showed only slight changes,
indicating that an up-regulation of the Bcl-2 to Bax ratio might
suppress the drug-induced apoptosis in ATF4-overexpressing
gastric cancer cells.
These results indicate that ATF4 promotes MDR ability of
gastric cancer cells through multiple mechanisms.
ATF4 transactivates SIRT1 promoter activity and directly
binds to the SIRT1 promoter
To determine whether ATF4 mediates SIRT1 gene
transcription, 293T cells were co-transfected with the 1.2 kb SIRT1
promoter reporter plasmid and the ATF4 expression plasmid. The
luciferase reporter assay showed that the SIRT1 promoter activity
was markedly activated by ATF4 in a dose-dependent manner
In an attempt to gain specific insight into the mechanisms of
SIRT1 induction, we examined the possible induction pathways
from ATF4. By analyzing the 59-flanking sequence of the SIRT1
gene with bioinformatics softwares (Tfsitescan service, TESS, and
Genomatix), two ATF4 putative binding sites were identified
within the 2950 to 2600 bp region of the SIRT1 promoter
To determine whether SIRT1 is a direct target of ATF4, ChIP
with the ATF4 antibody using SGC7901-ATF4 cells showed
enrichment of both binding sites within the SIRT1 promoter
region, indicating that the RNA and subsequent protein level
increases of SIRT1 in ATF4-expressing cell lines are likely due to a
direct interaction of ATF4 with the SIRT1 gene promoter (Fig. 3C).
To investigate the role of the two ATF4 binding sites in
regulating SIRT1 transactivation, site-directed mutagenesis was
used to mutate these sites. Luciferase reporter assay showed that
either mutating the binding site 1 or binding site 2 reduced the
SIRT1 promoter activity induced by ATF4. Furthermore,
mutation of both binding sites abolished the SIRT1 promoter
activity. These results suggested that both ATF4 binding sites are
involved in the transactivation of SIRT1 promoter (Fig. S1).
Taken together, these results indicate that SIRT1 is a direct
transcriptional target of ATF4.
ATF4 up-regulates the expression of SIRT1, MDR1, Bcl-2,
and Bax in gastric cancer cells
Previous studies have reported that cells overexpressing SIRT1
displayed decreased sensitivity to chemotherapy by multiple
SIRT1 inhibition by siRNA partly reverses the MDR
phenotype of ATF4-overexpressing gastric cancer cells
The identification of ATF4-mediated SIRT1 expression level
increases in gastric cancer cells, prompted us to analyze the role of
this pathway in gastric cancer MDR. To address this, we
compared the in vitro drug sensitivity in ATF4 stably transfected
gastric cancer cells after transfection of SIRT1 siRNA or scrambled
siRNA by colony formation and MTT assays. Knockdown of
SIRT1 by siRNA in SGC7901-ATF4 cells led to a .40%
reduction in colony number when used in combination with
CDDP (Fig. 4A, upper). This effect was also observed in
AGSATF4 cells (Fig. 4A, lower). Moreover, data from the MTT assay
also indicated that knockdown of SIRT1 could re-sensitize
SGC7901-ATF4 cells to chemical drugs, but this did not occur
in scrambled siRNA transfected cells (Fig. 4B).
To determine whether SIRT1 protects the cells from
CDDPinduced apoptosis, SGC7901-ATF4 cells transfected with SIRT1
siRNA or scrambled siRNA were treated with CDDP and labeled
with Annexin V and PI. The apoptotic cells were identified by
Annexin V labeling. The apoptotic percentage of SIRT1 siRNA
transfected SGC7901-ATF4 cells was significantly higher than
that of the control cells (Fig. 5A, 72.8% vs. 25.9%). The
appearance of condensed and fragmented nuclei was also
increased in SIRT1 siRNA transfected cells compared to the
control cells (Fig. 5B). Furthermore, cleavage of procaspase-3 was
observed as early as 12 h after treatment with 10 mg/ml CDDP in
SIRT1 siRNA transfected SGC7901-ATF4 cells, but not in
scrambled siRNA treated cells, even after 24 h of CDDP
treatment (Fig. 5C). These results suggest that SIRT1
overexpression suppresses CDDP-induced apoptosis.
To study the effect of down-regulation of SIRT1 by siRNA on
MDR associated molecules, we examined MDR1, MRP, Bcl-2,
and Bax expression levels in SGC7901-ATF4 cells following
transfection with SIRT1 siRNA or scrambled siRNA.
Downregulation of MDR1 was observed in the SIRT1 siRNA-treated
cells (Fig. 5D) compared to the control cells. In contrast, no
obvious difference of MRP, Bcl-2, and Bax expression levels were
found between the samples.
These observations indicate that SIRT1 mediates the
ATF4induced MDR effect in gastric cancer cells.
Inhibition of SIRT1 activity re-sensitizes ATF4 transfected
cells to DNA-damaging agents
To provide evidence that SIRT1 catalytic activity is also
responsible for the ATF4-induced MDR, SGC7901-ATF4 cells
were pretreated with EX-527, a novel, potent and specific
smallmolecule inhibitor of SIRT1, and followed by treatment with
different chemical drugs. First, we determined the basal
cytotoxicity of EX-527 in LV-Vector and LV-ATF4 stably transfected
SGC7901 cells. The MTT assay revealed that EX-527 at
concentrations up to 10 mM did not inhibit, but rather slightly
increased, the viability of both cell lines (Fig. 6A). Next, we
examined SIRT1, ATF4, MDR1, MRP, Bcl-2, and Bax
expression levels after 24 hours incubation with or without the
indicated doses of EX-527 in the gastric cancer cells used above.
Only the expression of MDR1 were down-regulated by EX-527 in
a concentration-dependent manner (Fig. 6B). Then we
preincubated SGC7901-ATF4 cells with vehicle or EX-527 (0.5, 1, 2, 4,
and 10 mM) for 24 h, and then CDDP- and 5-FU-mediated cell
death was monitored. As shown in Fig. 6C, EX-527 significantly
enhanced the cytotoxicity of both drugs in a dose-dependent
manner. We also determined the possible synergistic effect of
EX527 on different doses of CDDP- and 5-FU-mediated inhibition of
cell proliferation in SGC7901-ATF4 cells. As expected, 10 mM
EX-527 is sufficient to potentiate the cytotoxicity of both drugs
These results suggest that SIRT1 activity also plays a critical
role in the ATF4-induced gastric cancer MDR and this role might
be mediated partly through MDR1 expression.
MDR poses significant clinical challenges to the effective
chemotherapy of many human malignancies. The mechanisms
by which cells acquire resistance are multiple and complex, so
more extensive understanding of them, as well as identification of
novel mechanisms for chemoresistance, will be particularly helpful
in providing better therapeutic options. This study is the first
report that high levels of ATF4, commonly seen in tumor cells
under stressful circumstances, confers gastric cancer cells with a
MDR phenotype, and it identifies that this effect is mediated partly
by transactivation of SIRT1 expression.
ATF4 and SIRT1 are evolutionarily conserved stress response
genes involved in a broad spectrum of biological processes, many
of which are salutary for homeostasis and cellular protection
[22,23,24]. Both of these genes are induced in response to a variety
of stresses, including oxygen deprivation (hypoxia/anoxia),
oxidative stress, DNA damage, nutritional deprivation, and
chemotoxic stress. Levenson VV et al. first reported that changes
in expression of ATF4 could play a role in the pleiotropic
resistance to different classes of DNA-targeting drugs . In
recent years, several studies had found that ATF4 was involved
directly or indirectly in the development of drug resistance
through autophagy, the glutathione-dependent redox system, and
DNA damage repair [11,12,13,14,15,16,17,25,26]. Here we show
that the protective ability of ATF4 indeed mediates a MDR
phenotype in ATF4-overexpressing gastric cancer cell lines in
response to chemotherapy. Our findings clearly show that
overexpression of ATF4 in gastric cancer cells was associated
Figure 2. ATF4 up-regulates SIRT1 expression in gastric cancer cells. (A) mRNA levels of SIRT1 in LV-Vector and LV-ATF4 stably transfected
SGC7901 cell lines (left) and LV-SCR and LV-siATF4 stably transfected SGC7901/ADR cells (right) were subjected to qPCR. GAPDH were used as an
internal control. Data represent the means 6 S.D. of three independent experiments. (B) Cell lysates from cells in section A and their respective
nontreated counterparts(NC) were blotted with the indicated antibodies. b-actin was used as an internal control.
with more resistance, while knockdown of ATF4 induced
resensitization. These data suggest that ATF4 is probably an
important downstream mediator of resistance caused by multiple
mechanisms and is therefore a valuable therapeutic target. Yet, as
one of the most important transcriptional mediators of the ISR
which activates a variety of target genes that promote restoration
of homeostasis, ATF4 may also mediate resistance by other
mechanisms. In our study, SIRT1 was found to be up-regulated in
ATF4-overexpressing cells compared to vector transfected cells. In
contrast, knockdown of ATF4 with ATF4 specific siRNA led to a
down-regulation of SIRT1 in MDR gastric cancer cells. Our
results suggest that SIRT1 might be a downstream mediator of
ATF4-induced gastric cancer MDR.
As a member of the ATF subfamily of the basic-region leucine
zipper (bZIP) transcription factors , ATF4 has the potential to
act as either a transcriptional activator or a transcriptional
repressor via ATF or cAMP responsive element (CRE) binding
sites . The consensus binding site for ATF was defined as
TGACGT (C/A) (G/A) , which is a sequence identical to the
CRE consensus element (TGACGTCA) . Also, the highly
conserved core motif ACGT in most CREs  can bind to
different bZIP factors, depending on the flanking bases of the core
motif [22,30,31]. In our study two putative ATF-CRE binding
sites were found in the 1.2 kb SIRT1 promoter region, and ATF4
directly activated SIRT1 transcription via binding to both binding
elements. However, how the two binding sites play their roles
Figure 4. SIRT1 inhibition by siRNA suppressed the ATF4-induced gastric cancer MDR phonotype. (A) SGC7901-ATF4 and AGS-ATF4 cells
were transfected with scrambled siRNA (SCR) or SIRT1 siRNA (siSIRT1). Seventy-two hours later, Cell lines were treated continuously with either 0 or
0.25 mg/ml cisplatin for 14 d; media was changed every 3 d. Cells were plated in triplicate, and the experiment was repeated three times.
Representative wells are shown. Graphs provide average quantification as a percentage of the nontreated cells. Inset, relative SIRT1 protein
expression by Western blot. (B) SGC7901-ATF4 cells were transfected with scrambled siRNA (SCR) or SIRT1 siRNA (siSIRT1). Seventy-two hours later,
both cell lines were treated with the indicated doses of different drugs for additional 72 h. In vitro drug sensitivity was tested by MTT assay. Data
represent the means 6 S.D. of three independent experiments.
under detailed stress circumstances remains unknown and requires
Mammalian SIRT1 is the closest homologue of the yeast Sir2
and the most extensively studied SIRT family member. It is
heavily implicated in the regulation of cellular processes that
determine longevity, including anti-apoptosis, neuronal protection,
and cellular senescence or ageing . Recently, an increasing
number of studies have implicated increased expression of SIRT1
with resistance to chemotherapy and ionizing radiation
[18,19,20,33,34,35,36]. For example, SIRT1 overexpression has
been found in drug-resistant neuroblastoma, osteosarcoma,
mammary, ovarian, prostate, colon, and lung cancer cell lines
compared with their drug-sensitive counterparts. All these drug
resistant effects of SIRT1 could possibly be due to its
antiapoptotic effect [37,38] and silencing of tumor suppressor genes
. Finally, we might predict that, if SIRT1 is involved in the
ATF4-induced MDR, inhibition of SIRT1 should affect the
sensitivity of ATF4-overexpressing cells in response to
chemotherapy. As expected, both siRNA and pharmacological inhibition of
SIRT1 could re-sensitize ATF4-overexpressing cells to chemical
drugs. In addition, our study indicates that SIRT1 protects cells
from death partly through an anti-apoptotic effect.
It has been reported that MDR1 was up-regulated in cells with
increased SIRT1 expression [18,36]. In this study, we also
demonstrated that MDR1 is up-regulated in ATF4-overexpressing
cells, and knockdown of SIRT1 with SIRT1 specific siRNA or
inhibiting its activity with EX-527 could lead to down-regulation
of MDR1, which is consistent with a drug-resistant role by SIRT1.
Figure 5. Effect of down-regulation of SIRT1 by siRNA on apoptosis and MDR related molecules. (A) SGC7901-ATF4 cells were
transfected with scrambled siRNA (SCR) or SIRT1 siRNA (siSIRT1). Seventy-two hours later, cells were incubated for additional 36 h in fresh medium in
the absence or presence of cisplatin at 5 mg/ml. After drug treatment, the cells were labeled with Annexin V and PI. The distribution pattern of live
and apoptotic cells was determined by FACS analysis. (B) SGC7901-ATF4 cells were transfected by the same way in section A and then treated with
5 mg/ml of cisplatin for 36 h. Then Hoechst 33258 nuclear staining was performed to detect apoptotic cells. (C) SGC7901-ATF4 cells were transfected
by the same way in section A and were incubated for additional 624 h in fresh medium with 10 mg/ml of cisplatin. At the time indicated, protein
extracts were collected and subjected to immunoblot analysis for caspase-3 (uncleaved and cleaved forms). b-actin was used as an internal control.
(D) SGC7901-ATF4 cells were transfected by the same way in section A. Seventy-two hours later, cell lysates were blotted with the indicated
antibodies. b-actin was used as an internal control.
Figure 6. Inhibition of SIRT1 activity reintroduce sensitivity in ATF4-overexpressing cell lines. (A) LV-Vector and LV-ATF4 stably
transfected SGC7901 cell lines were incubated with or without the indicated doses of EX-527. Ninety-six hours later, cell viabilities were determined
by MTT assay. (B) Stably transfected SGC7901 cell lines in section A were incubated with or without EX-527 (110 mM) for 24 h, and total cell lysates
were subjected to immunoblotting with the indicated antibodies. b-actin was used as an internal control. (C) SGC7901-ATF4 cells were preincubated
with the indicated doses of EX-527 for 24 h. Then SGC7901-Vector and SGC7901-ATF4 cells were exposed to cisplatin (1 mg/ml) or 5-fluorouracil
(1.25 mg/ml) for additional 72 h. Cell viabilities were determined by MTT assay. (D) SGC7901-ATF4 cells were preincubated with or without EX-527
(10 mM) for 24 h. Then the cells were exposed to the indicated doses of cisplatin or 5-fluorouracil for additional 72 h. Cell viabilities were determined
by MTT assay. All data represent the means 6 S.D. of three independent experiments. Graphs provide average quantification as a percentage of the
However, the Bcl-2/Bax ratio, which was up-regulated in the
ATF4-overexpressing cells, was SIRT1-independent, suggesting
that SIRT1-independent mechanisms also play a role in the
ATF4-induced MDR in gastric cancer cells.
In summary, we demonstrate that ATF4 confers a MDR
phenotype to gastric cancer cells, and this effect is partly mediated
by transactivation of SIRT1 overexpression. Moreover, ATF4 is a
valid target in drug-resistant gastric tumors, and developing
effective inhibitors of ATF4 should be taken into consideration in
the future. These findings provide novel insights into the role of
ATF4 in controlling SIRT1 expression and into its
stressresistance features in tumorigenesis and chemotherapy. This is
especially important for clinical consideration, as ATF4 can be
upregulated by oxygen deprivation, oxidative stress, nutritional
deprivation and almost all the adverse stressors in a tumor
microenvironment, which could be hijacked by cancer cells to
evade proliferation inhibition and cell death in response to
chemotherapy. Therefore, interventions predicated on disrupting
stress-induced ATF4 expression in cancer cells may be effective in
circumventing or reversing drug resistance in gastric cancer.
Materials and Methods
For detailed methods, please see Text S1.
Cell culture and reagents
The human gastric adenocarcinoma cell lines SGC7901
(obtained from the Academy of Military Medical Science, Beijing,
China) and the MDR variants, SGC7901/ADR and SGC7901/
VCR (established and maintained in our laboratory), and AGS
(obtained from the cell bank of Chinese Academy of Sciences,
Shanghai, China) were cultured in RPMI-1640 medium
supplemented with 10% fetal bovine serum (Hyclone) and penicillin/
streptomycin. 293T cells (also obtained from the cell bank of
Chinese Academy of Sciences) were cultured in DMEM
supplemented with 10% fetal bovine serum. To maintain the
MDR phenotype, adriamycin (with a final concentration of
0.5 mg/ml) and vincristine (with a final concentration of 1 mg/
ml) were added to the culture media for SGC7901/ADR and
SGC7901/VCR cells, respectively. EX-527 (Sigma) was dissolved
in DMSO at the indicated concentrations. Adriamycin (ADR),
vincristine (VCR), cisplatin (CDDP), and 5-fluorouracil (5-FU)
were dissolved in normal saline at indicated concentrations.
Cell transfection and stable cell lines
The human ATF4 expression plasmid (pCMV5-ATF4) was
kindly provided by Professor Amy S. Lee . Lentiviral vector
encoding siRNA specific to ATF4 and control siRNA were
generated with the use of PLKO.1-TRC (Addgene) and were
designated as LV-siATF4 and LV-SCR control, respectively.
Lentiviral vector encoding human ATF4 gene were constructed in
FUW-teto (Addgene), designated as LV-ATF4. The empty vector
was used as negative control, designated as LV-Vector. Stable cell
lines were generated by transfection of indicated lentiviral
constructs followed by selection in puromycin or zeocin
(Invitrogen), respectively. Cell transfection and generation of stable cell
lines were performed using standard procedures. The sequences of
the siRNA constructs can be found in Text S1.
The collection of protein extracts and immunoblotting analysis
were performed using standard procedures. For antibody sources,
please see Text S1.
Colony formation assay
The colony formation assay was performed, as previously
described , with slight modifications (Text S1).
Annexin V staining and FACS analysis
Annexin V staining and FACS analysis were performed using
standard procedures. Cells negative for both PI and Annexin V
staining were classified as live cells, cells that stained positive for
Annexin V only were classified as early apoptotic cells, and PI
positive and Annexin V positive cells were cells undergoing late
stages of apoptosis.
DNA fragmentation assay
DNA fragments were extracted with the DNA Ladder
Extraction Kit with Spin Column (C0008, Beyotime Co., Beijing,
China) according to the manufacturers protocol. The DNA
fragments were separated using gel electrophoresis on a 1%
agarose gel containing 0.1 mg/ml ethidium bromide.
Hoechst Staining was performed according to manufacturers
protocol (C0003, Beyotime Co.). Cells were visualized with a
DP70 invert Immunofluorescence microscope (Olympus). Cells
with condensed and fragmented nuclei were judged to be
In vitro drug sensitivity assay
ADR, VCR, CDDP, and 5-FU were all freshly prepared before
each experiment. Drug sensitivity was measured using a
3-(4,5dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT)
assay according to the standard protocol (Text S1).
Quantitative real-time PCR (qPCR)
Quantitative real-time PCR was performed using a LightCycler
480 II system (Roche) and SYBR Green detection (TaKaRa).
Sequences of the primers can be found in Text S1.
Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed according to the manufacturers
protocol (P2078, Beyotime Co.) with slight modifications.
Chromatin solutions were sonicated and incubated with anti-ATF4 or
with control IgG, and rotated overnight at 4uC. DNAprotein
cross-links were reversed and chromatin DNA was purified and
subjected to PCR analysis. The primers 59-ACC CCT CGT TTT
ACA TCT-39 and 59-TTT GGA GTC CTT CCT TTC-39 were
used to amplify the SIRT1 distal promoter sequence (A1,
nucleotides 2974 to 2843), and the primers 59-ACC CAA
CAA ACC CAT TCT-39 and 59-CCT CCT GGG AAG ACC
TTT-39 were used to amplify the SIRT1 proximal promoter
sequence (A2, nucleotides 2781 to 2647). The primers for
GAPDH, 59-TAC TAG CGG TTT TAC GGG CG-39 and
59TCG AAC AGG AGG AGC AGA GAG CGA-39, were used as a
negative control. As a positive control for the ATF4-DNA
interaction, the primers 59-TGG TTG GTC CTC GCA GGC
AT-39 and 59-CGC TTA TAC CGA CCT GGC TCC T-39,
which were designed to amplify the asparagine synthetase (ASNS)
promoter region that contains at least two sites reported to bind
ATF4 , were also used. After amplification, PCR products
were resolved on a 1.5% agarose gel and visualized by ethidium
Reporter gene assay
The 1.2 kb human SIRT1 promoter sequence (21100 to
+100 bp) was synthesized and cloned into the XhoI and HindIII
sites of the pGL3-Basic vector. The resulting construct was
confirmed by DNA sequencing. 293T cells were then
co-transfected with the SIRT1 promoter reporter plasmid, the pRL-TK
plasmid (Promega, USA), and the pCMV5-ATF4 plasmid by
using Lipofectamine2000 (Invitrogen). Forty eight hours after
transfection, cells were washed three times with cold
phosphatebuffered saline (PBS). Then, the cells were lysed in 100 ml of
Passive Lysis Buffer (Promega) and shaken for 15 minutes. Firefly
luciferase and Renilla luciferase activities were measured using the
Dual-Luciferase Reporter Assay System (Promega) with a
Varioskan Flash microplate reader (Thermo Scientific). Relative
activity was defined as the ratio of firefly luciferase activity to
Renilla luciferase activity and was calculated by dividing the
luminescence intensity obtained with the assay for firefly luciferase
by that of the Renilla luciferase. All measurements were performed
in triplicate, and the assays were repeated three times in 293 T
Each experiment was repeated at least three times. All data
were presented as mean value 6 S.D. The difference between the
means was analyzed with Students t test. All statistical analyses
were performed using SPSS16.0 software (Chicago, IL).
Significance was set at the 5% level.
Figure S1 Effect of mutated ATF4 binding sites on the
activity of the SIRT1 promoter. 293T cells were
cotransfected with pCMV-ATF4 and wild type SIRT1,
MUT1, SIRT1-MUT2, or MUT1+MUT2 reporter, and the
relative luciferase activity was determined. The luciferase activity
of the mock pCMV-Taq group was designated as 1.00. The results
are the mean 6 S.D. of three experiments performed in duplicate.
*, P,0.05. The left side is a schematic representation of the
reporter gene constructs. The bar graphs on the right side
represent the relative levels of luciferase activity in each of the
Supplementary Material and Methods.
We thank Professor Amy S. Lee for kindly providing us with the
pCMV5ATF4 plasmid. We also thank Mr. Taidong Qiao and Ms. Zheng Chen for
their excellent technical assistance.
Conceived and designed the experiments: KW YN DF HZ. Performed the
experiments: HZ YZ LX HW WX HH JW JX YG SS BX. Analyzed the
data: KW HZ LX. Wrote the paper: KW HZ LX.
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