Here we show that 1,25-dihydroxyvitamin D3 VD3, the active form of vitamin D significantly promoted apoptosis in the undifferentiated gastric cancer cell line HGC-27, and this was accompa
Trang 1synergy with trichostatin A ⁄sodium butyrate-induced and 5-aza-2¢-deoxycytidine-induced PTEN upregulation
Lina Pan1,*, Ammar F Matloob1,*, Juan Du2, Hong Pan1, Zhixiong Dong2, Jing Zhao2, Yu Feng1, Yun Zhong1, Baiqu Huang2and Jun Lu1
1 The Institute of Genetics and Cytology, Northeast Normal University, Changchun, China
2 The Key Laboratory of Molecular Epigenetics of Ministry of Education (MOE), Northeast Normal University, Changchun, China
Introduction
Gastric cancer is the fourth commonest malignancy
worldwide, especially in East Asian countries such as
Japan and China [1] The 5-year survival rate after
diagnosis of gastric cancer is 10–21% [2] Surgery and
chemotherapy are the major therapeutic methods for
gastric cancer, but the rate of recurrence after curative
resection is high Also, resistance to chemotherapeutic
agents and toxicity of drugs to normal tissues are the main problems in gastric cancer therapies [3] There-fore, there is an ongoing search for new therapeutic targets for gastric cancer
The physiologically active form of vitamin D, 1,25-dihydroxyvitamin D3(VD3), belongs to the secos-teroid hormone family, which controls calcium and
Keywords
1,25-dihydroxyvitamin D3;
5-aza-2¢-deoxycytidine; gastric cancer; PTEN;
trichostatin A ⁄ sodium butyrate
Correspondence
J Lu, Institute of Genetics and Cytology,
Northeast Normal University, 5268 Renmin
Street, Changchun 130024, China
Fax: +86 431 85099768
Tel: +86 431 85098729
E-mail: luj809@nenu.edu.cn
*These authors contributed equally to this
work
(Received 8 June 2009, revised 22 October
2009, accepted 8 December 2009)
doi:10.1111/j.1742-4658.2009.07542.x
Previous studies have shown an anticancer effect of vitamin D, but the mechanisms underlying this action have not been fully explored Here we show that 1,25-dihydroxyvitamin D3 (VD3, the active form of vitamin D) significantly promoted apoptosis in the undifferentiated gastric cancer cell line HGC-27, and this was accompanied by a concurrent increase in phos-phatase and tensin homolog deleted on chromosome 10 (PTEN) expression
on VD3 treatment In contrast, knockdown of PTEN expression by stable transfection of PTEN small interfering RNA greatly decreased the apo-ptosis rate We further demonstrated that VD3 induced PTEN expression through vitamin D receptor In addition, our evidence showed that vita-min D receptor, Egr-1 and p300 induced PTEN expression in a synergistic fashion Furthermore, we found that the histone deacetylase inhibitors trichostatin A and sodium butyrate and the methylation inhibitor 5-aza-2¢-deoxycytidine played important roles in vitamin D-induced apopto-sis through PTEN upregulation The data presented in this article suggest potential benefits of vitamin D in gastric cancer therapies in association with the use of trichostatin A⁄ sodium butyrate and 5-aza-2¢-deoxycytidine
Structured digital abstract
l MINT-7306489, MINT-7306501, MINT-7306512: P300 (uniprotkb:Q09472) physically inter-acts (MI:0914) with VDR (uniprotkb:P11473) and EGR1 (uniprotkb:P18146) by anti bait co-immunoprecipitation (MI:0006)
Abbreviations
5-Aza, 5-aza-2¢-deoxycytidine; ChIP, chromatin immunoprecipitation; CoIP, coimmunoprecipitation; Egr-1, early growth response gene 1; FITC, fluorescein isothiocyanate; HDAC, histone deacetylase; NaBu, sodium butyrate; PI, propidium iodide; PTEN, phosphatase and tensin homolog deleted on chromosome 10; siRNA, small interfering RNA; TSA, trichostatin A; VD3, 1,25-dihydroxyvitamin D 3; VDR, vitamin D receptor.
Trang 2phosphorus metabolism in normal development
Ear-lier studies revealed that VD3 could induce
differentia-tion and cell cycle arrest in a number of malignant
melanoma cells, including those in myeloid leukemia,
and breast, prostate, colon, skin and brain cancer [4–
8]
Phosphatase and tensin homolog deleted on
chromo-some 10 (PTEN) was originally discovered in 1997
[9–11] PTEN is a negative regulator of the ubiquitous
phosphatidylinositol 3-kinase pathway, and it is
fre-quently subject to loss of heterozygosity in various
human tumors, including brain, bladder, prostate and
endometrial cancers [9,10] Expression of PTEN is
reg-ulated by transcription factors such as p53 [12],
peroxi-some proliferator-activated receptor c [13], early
growth response gene 1 (Egr-1) [14], nuclear factor-jB
[15,16], and transforming growth factor-b [11]
More-over, functional vitamin D receptor (VDR) elements
have been identified in the promoter of PTEN,
sug-gesting that vitamin D may play a role in the
regula-tion of PTEN expression [17]
Epigenetic changes such as DNA methylation and
histone modifications play important roles in
silenc-ing tumor suppressor genes Dursilenc-ing the development
and progression of gastric cancer, a number of
tumor-related genes, including p53 [18], E-cadherin
[19,20], p14 [21], p15 [22,23], p16 [24], PTEN [25],
and RASSF1A [25], exhibit genetic and epigenetic
alterations Some of these genes can be reactivated
by the DNA methylation inhibitor
5-aza-2¢-deoxycyti-dine (5-Aza) [26] Earlier studies showed that
treat-ment with 5-Aza reduced DNA methylation and
subsequently upregulated PTEN expression in acute
lymphoblastic leukemia, melanoma and ovarian
can-cer cells [27–29] In gastric cancan-cer cells, treatment
with 5-Aza can greatly enhance PTEN expression
[30] It was also reported that use of 5-Aza in
com-bination with histone deacetylase (HDAC) inhibitors
may be an effective chemotherapeutic regimen for
patients with acute myeloid leukemia that is resistant
to conventional chemotherapy [31] Additionally,
there has been evidence that HDAC inhibitors can
improve the sensitivity of actinotherapy and
chemo-therapy [32,33]
The main aim of this study was, on the basis of the
above information, to determine whether vitamin D
had any effects on gastric cancer cells, and to
investi-gate the possible molecular processes involved in this
action As there has been evidence that the promoter
of PTEN is hypermethylated in gastric cancer [34], we
wanted to determine whether epigenetic modifiers such
as 5-Aza and trichostatin A (TSA)⁄ sodium butyrate
(NaBu) could change the expression of PTEN in
gas-tric cancer cells Moreover, we also intended to clarify the functional relationship between VDR and Egr-1, which is a major transcription factor of PTEN, in PTEN regulation and in PTEN-mediated cellular alter-ations in gastric cancer cells Overall, data arising from this study have provided the basis for the further investigations into the potential application of vita-min D as a novel molecular target in gastric cancer therapies in association with the use of TSA⁄ NaBu and 5-Aza
Results
Vitamin D induced apoptosis in gastric cancer cells through PTEN upregulation
Earlier studies revealed that vitamin D was able to induce cell differentiation and cell cycle arrest, in addition to its normal function of controlling cal-cium and phosphorus metabolism [4–8] To confirm this effect of vitamin D in gastric cancer, we treated undifferentiated HGC-27 adenocarcinoma cells with VD3, and estimated the apoptosis rate As shown in Fig 1A, the early apoptosis rate (Bd) was increased from 1.03% to 5.87%, and the late apopto-sis⁄ necrotic ratio (Bb) was increased from 1.46% to 5.55%, confirming that vitamin D could induce apoptosis in gastric cancer cells As functional VDREs were identified in the PTEN promoter [17], and the expression of PTEN declined in gastric can-cer [35], we speculated that PTEN might participate
in the VD3-induced apoptosis in gastric cancer cells
To investigate this, undifferentiated HGC-27 adenocar-cinoma cells were treated with VD3, and the expression
of PTEN was examined The results in Fig 1B show that PTEN expression was upregulated by VD3 at both the mRNA (upper) and protein (lower) levels To fur-ther establish the functional role of PTEN in VD3-induced apoptosis, we constructed a stably transfected cell line in which PTEN was knocked down by specific small interfering RNA (siRNA) The inhibitory effi-ciency of PTEN siRNA was first examined by using real-time RT-PCR (data not shown), and two clones with the highest RNA inhibitory efficiency were selected and further confirmed by using western blotting (Fig 1C) Eventually, clone 2 was used in the following experiments We then showed that the early apoptosis rate (Bd, Fig 1) was significantly reduced from 3.94%
to 1.31% in cells transfected with PTEN siRNA, as compared with cells transfected with control siRNA (Fig 1D) Meanwhile, the early apoptosis induced by VD3 was reduced from 6.06% to 2.12% in PTEN
siR-NA cells (Fig 1D) These results suggested that PTEN
Trang 3participated in the VD3-induced apoptosis in gastric
cancer cells
Vitamin D upregulated PTEN through VDR
To further study the mechanism by which vitamin D
induces PTEN expression, we transiently transfected
HGC-27 cells with the expression plasmid of VDR
for luciferase reporter assay The results showed that
overexpression of VDR stimulated the activity of the
PTEN promoter (Fig 2A) Similarly, exposure of
HGC-27 cells to VD3 after VDR transfection
upreg-ulated PTEN mRNA expression as much as 13-fold
(Fig 2B) As can be seen in Fig 2C, the PTEN
pro-tein level was also enhanced by VDR overexpression
Furthermore, we designed three primer pairs on the
PTEN promoter for chromatin immunoprecipitation
(ChIP) assays with antibody against VDR in
HGC-27 cells (Fig 2D) The ChIP data clearly
demon-strated the binding of VDR to the PTEN promoter,
especially on the RE B region (Fig 2E) These data
indicated that vitamin D induced PTEN expression through binding of VDR to the PTEN promoter
VDR synergistically activated PTEN with Egr-1 and p300
In a previous study, we showed that histone acetyl-transferase p300 activated PTEN expression in synergy with the transcriptional factor Egr-1 [36] To clarify the relationships among VDR, Egr-1 and p300 in regulat-ing PTEN expression, we transiently transfected 293T cells with VDR, Egr-1 and p300, either alone or in combination, before the expression of PTEN was esti-mated As shown in Fig 3A, overexpression of VDR
or p300 alone increased PTEN promoter activity by three-fold or four-fold Meanwhile, cotransfection with VDR⁄ 1, VDR ⁄ p300, 1 ⁄ p300 and VDR ⁄
Egr-1⁄ p300 resulted in the enhancement of PTEN promoter activity by 28-fold, 68-fold, 51-fold and over 700-fold, respectively (Fig 3A) Similar results were obtained by using quantitative real-time PCR (Fig 3B) and western
A
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Fig 1 Vitamin D induced apoptosis in gastric cancer cells through PTEN HGC-27 gastric cancer cells were treated with VD3 (10)7M ) for
24 h, and the apoptosis was evaluated by flow cytometry Apoptosis rates were calculated on the basis of 30 000 cells (A) HGC-27 cells were cultured in six-well plates, and after exposure to VD3 for 24 h, cells were collected for quantitative real-time PCR and western blotting (B) HGC-27 cells were transfected with siRNA targeting PTEN to construct stably transfected cell lines After being cultured with G418 for 15–22 days, individual colonies were isolated and expanded, and the expression of PTEN was examined by western blotting (C) Colony 2 was treated with VD3, and apoptosis was evaluated (D) *P < 0.05, **P < 0.01, (n = 3).
Trang 4blotting (Fig 3C) These experimental data clearly
indicated that VDR, Egr-1 and p300 were able to
work in synergy to significantly upregulate PTEN
expression
Next, we sought to explore the physical and
func-tional interactions among VDR, Egr-1 and p300 in
their synergistic action We performed
coimmunopre-cipitation (CoIP) assays after VDR, Egr-1 and p300
transfection We precipitated cell lysates with
anti-bodies against VDR, Egr-1 and p300, respectively The precipitates were then detected using immunoblotting with the above-mentioned antibodies The CoIP data revealed that VDR, Egr-1 and p300 were present in the same complex, supporting their cooperative effect
on PTEN regulation (Fig 3D) Collectively, these results showed that, as a nuclear receptor, VDR acted together with transcription factor Egr-1 and histone acetyltransferase p300 to upregulate PTEN expression
pc3.1 VDR pc3.1 VDR Control VD3
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Fig 2 Vitamin D upregulated PTEN through its receptor VDR HGC-27 cells were tran-siently transfected for 30 h with the expres-sion plasmid of VDR, and PTEN promoter reporter activity was measured (A) HGC-27 cells were transiently transfected with the pc3.1–VDR or pc3.1 vector before VD3 treatment, and the expression of PTEN was estimated by quantitative PCR (B) and wes-tern blotting (C) The diagram of PTEN pro-moter and the regions amplified by PCR in ChIP assays are shown (D) HGC-27 cells were collected and precipitated with anti-bodies against VDR after VD3 treatment, and ChIP assays were performed to exam-ine the binding of VDR on PTEN promoter (E) IP, immunoprecipitation *P < 0.05,
**P < 0.01, (n = 3).
WB: VDR WB: Egr-1 WB: p300
IgG VDR IgG Egr-1 IgG p300
α-PTEN α-actin
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Fig 3 VDR synergistically activated PTEN with Egr-1 and p300 Expression plasmids of VDR, Egr-1 and p300 were transiently transfected either alone or in combination, as indicated, and the expression of PTEN was examined by reporter activity assay (A), quantitative PCR (B), and western blotting (C) CoIP was used to detect the binding of VDR, Egr-1 and p300 after transient transfection with Egr-1, VDR and p300 (D) IP, immuno precipitation; WB, western blotting *P < 0.05, **P < 0.01, (n = 3).
Trang 5Vitamin D enhanced the apoptosis induced by
TSA⁄ NaBu
We showed above that histone acetyltransferase p300
increased PTEN expression synergistically with VDR
and Egr-1 (Fig 3), implying that histone acetylation
modification may participate in this process HDAC
inhibitors have emerged as accessory therapeutic
agents for multiple human cancers, because they can
restore the expression of certain tumor suppressor
genes [37] Moreover, our experimental data revealed
that VD3 was able to increase the apoptosis rates
induced by TSA and NaBu (Fig S1) We then tested
the effects of HDAC inhibitors on the expression of
PTEN, with emphasis on their synergistic action with
VD3 We treated HGC-27 cells with TSA and NaBu,
and assessed the expression level of PTEN As
expected, PTEN mRNA expression was significantly
enhanced by NaBu (over 300-fold) and VD3 (over
30-fold), and a synergistic effect of the combined use
of NaBu and VD3 was seen (over 700-fold) (Fig 4B)
A similar synergistic effect on the upregulation of PTEN, by 109-fold, was detected with combined treatment with TSA and VD3 (Fig 4A) To further investigate the function of PTEN and the cooperative effect of HDAC inhibitors and vitamin D on apopto-sis in gastric cancer cells, cell lines stably transfected with PTEN siRNA were treated with TSA⁄ NaBu, either alone or with VD3, and it was found that the cells transfected with PTEN siRNA exhibited a decline in early apoptosis rate from 8.84% to 2.65% with TSA treatment (Fig 4C), and from 15.65% to 5.59% with NaBu treatment (Fig 4D) Meanwhile, the early apoptosis rate was greatly reduced, from 25.09% to 1.55% with both TSA and VD3 (Fig 4C), and from 24.12% to 5.84% with both NaBu and VD3 (Fig 4D) These results suggested that PTEN participated in the apoptosis induced by HDAC inhibitors, and that VD3 could promote the apoptosis induced by TSA⁄ NaBu
Control T + VD3 TS A
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Fig 4 Vitamin D enhanced the apoptosis induced by TSA ⁄ NaBu HGC-27 gastric cancer cells were treated with VD3 and 200 n M TSA (A),
or VD3 and 2 m M NaBu (B), and western blotting was used to evaluate PTEN expression Cell lines stably transfected with PTEN siRNA or control siRNA were treated with VD3 and TSA ⁄ NaBu, respectively, and apoptosis was evaluated by flow cytometry The apoptosis rates were calculated on the basis of 30 000 cells (C) *P < 0.05, **P < 0.01, (n = 3).
Trang 6TSA⁄ NaBu increased the histone acetylation
level at the PTEN promoter
Next, we wanted to investigate whether TSA⁄ NaBu
truly altered the histone acetylation level at the PTEN
promoter We performed ChIP assays with antibodies
against acetylated histones after TSA⁄ NaBu
treat-ments The results showed that TSA was able to
enhance the acetylation level of histone H3 at all three
regions of the PTEN promoter (Fig 5A, left), whereas
it enhanced acetylation of histone H4 only at the RE
C region (Fig 5A, right) Meanwhile, NaBu induced
hyperacetylation of both histone H3 (Fig 5B, left) and
histone H4 (Fig 5B, right) These results indicated that
TSA⁄ NaBu induced PTEN expression by enhancing
the histone acetylation levels at the gene’s promoter
TSA⁄ NaBu upregulated Egr-1 and promoted its
binding to the PTEN promoter
Histone hyperacetylation is believed to favor an open
chromatin structure to facilitate the binding of major
transcription factors on the promoter We were therefore interested in determining whether TSA⁄ -NaBu had any effects on promoting the binding of Egr-1, a major transcription factor of PTEN To do this, we first examined the influences of TSA and NaBu on the expression of Egr-1, and we found that both TSA and NaBu upregulated Egr-1 in HGC-27 cells (Fig 5C) Consistent with this, ChIP assays with antibodies against Egr-1 demonstrated that TSA could markedly potentiate the binding of Egr-1 to the PTEN promoter (Fig 5D, left) The data pre-sented above had revealed that the histone acetyla-tion level of the PTEN promoter (Fig 5A), as well the binding ability of Egr-1, were increased by TSA (Fig 5D, left) Apparently, TSA treatment facilitated the binding of Egr-1 by enhancing the acetylation level of the PTEN promoter In contrast, NaBu had little effect on the binding of Egr-1 (Fig 5D, right) Taken together, these findings show that epige-netic mechanisms such as acetylation modification play an important role in the regulation of PTEN expression
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Fig 5 TSA ⁄ NaBu increased the histone acetylation level on the PTEN promoter HGC-27 cells were treated with TSA or NaBu for 24 h, and ChIP assays were performed with antibodies against Ac-H3 or Ac-H4 after TSA (A) and NaBu (B) stimulation HGC-27 cells were treated with TSA for 24 h, the mRNA level of Egr-1 was determined by quantitative PCR (C, left upper), and the protein level was estimated by western blotting (C, left lower) The expression level of Egr-1 was evaluated by quantitative PCR and western blotting after NaBu treatment (C, right) The change in Egr-1 binding to the PTEN promoter after stimulation was examined by ChIP assays (D) GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, immuno precipitation *P < 0.05, **P < 0.01, (n = 3).
Trang 7Vitamin D promoted the apoptosis induced by
5-Aza
We then investigated the effect of PTEN on the
apop-tosis induced by DNA methylation and vitamin D in
gastric cancer cells We treated HGC-27 cells with
5-Aza and VD3, either alone or in combination, and
we found that the expression of PTEN was
upregulat-ed by over 100-fold after combinupregulat-ed treatment with
5-Aza and VD3 (Fig 6A) Meanwhile, the early
apop-tosis rate dropped sharply from 26.45% to 2.44%, and
the late apoptosis⁄ necrotic rate declined from 17.74%
to 7.59%, in PTEN siRNA-transfected cells treated
with 5-Aza and VD3 (Fig 6B) Clearly, 5-Aza was
able to promote the apoptosis induced by vitamin D
through PTEN
5-Aza induced Egr-1 expression and enhanced its
binding to the PTEN promoter
It has been shown that 5-Aza is able to stimulate PTEN
expression and to induce apoptosis in acute
lymphoblas-tic leukemia cells [27] To evaluate the contribution of
5-Aza to the regulation of PTEN in gastric cancer cells,
we treated HGC-27 cells with 5-Aza; as a result, both
Egr-1 mRNA and protein levels were increased six-fold
(Fig 6C) Furthermore, ChIP assays with antibody
against Egr-1 showed that the binding of Egr-1 to the
PTEN promoter was increased after treatment with 5-Aza (Fig 6D) Thus, we concluded that PTEN partici-pated in vitamin D-induced apoptosis, and epigenetic modifications such as acetylation and DNA methylation were involved in the regulation of PTEN, which subse-quently affected the apoptosis of gastric cancer cells
Discussion
Recent studies have revealed that vitamin D is involved
in the control of various cellular processes, including cel-lular growth, differentiation, and apoptosis, in addition
to its known functions in calcium and phosphorus metabolism [4–8] In the present study, we have shown that VD3 can induce apoptosis in gastric cancer cells (Fig 1A), suggesting its potential use in cancer therapy However, it has been noted that vitamin D may possibly
be toxic when used in large amounts (i.e greater than
50 000 IUÆday)1), because it can cause abnormally high serum calcium levels (hypercalcemia) [38] Moreover, there is increasing evidence that HDAC inhibitors (e.g TSA and NaBu) and methylation inhibitors (e.g 5-Aza) can reactivate the expression of silenced genes, and hence restore normal cellular functions; this has inspired
a great deal of research interest in potential uses of these modifiers in tumor therapy [39,40] In this study, we explored the effects, as well as some underlying mecha-nisms, of the combined use of VD3 and TSA⁄ NaBu or
Control siRNA B1 B2
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11.70% 4.88%
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0.57% 17.74%
5 + VD3 5 + VD3 26.45% 2.44%
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ol 5-Aza VD3 5-Aza + VD3
Fig 6 Vitamin D promoted the apoptosis
induced by 5-Aza HGC-27 cells were
trea-ted with VD3 and 5-Aza (5 l M ), and western
blotting was used to assess PTEN
expres-sion (A) Cell lines stably transfected with
PTEN siRNA or control siRNA were treated
with VD3 and 5-Aza, and apoptosis was
evaluated by flow cytometry The apoptosis
rates were calculated on the basis of
30 000 cells (B) HGC-27 cells were cultured
in six-well plates before 5-Aza treatment,
and the expression of Egr-1 was examined
by quantitative PCR (C, upper) and western
blotting (C, lower) After exposure to 5-Aza
for 24 h, HGC-27 cells were collected and
precipitated with the antibody against Egr-1,
and the binding of Egr-1 was detected by
ChIP assays (D) IP, immunoprecipitation.
*P < 0.05, **P < 0.01, (n = 3).
Trang 85-Aza, on gastric cancer cells Our results indicated that
both TSA and NaBu could significantly promote the
apoptosis induced by VD3 (Fig 4C,D) Moreover, our
study showed that VDR acted as a transcription factor
of PTEN (Fig 2) Significantly, our study also revealed
that both TSA⁄ NaBu and 5-Aza promoted the
forma-tion of an open chromatin structure that potentiated the
binding of VDR to the PTEN promoter, and that VDR
was in turn activated by its ligand VD3 Overall, these
data may help in the development of a novel option for
accessory cancer therapy involving the combined use of
vitamin D and epigenetic modifiers This strategy also
has the advantage of the use of low concentrations of
vitamin D and HDAC inhibitors to minimize their
tox-icities, owing to their remarkable synergistic effects in
activating target genes
An interesting finding of this study is the correlation
between VD3 and the tumor suppressor gene PTEN
We showed that VD3 could induce the expression of
PTEN through VDR (Fig 2) Furthermore, the data
in Fig 3 also show correlations between VDR and
Egr-1, as well as between VDR and histone
acetyl-transferase p300 Further investigations revealed that
both TSA⁄ NaBu and 5-Aza were able to significantly
enhance the apoptosis induced by VD3 through PTEN
(Figs 4 and 6), and that the epigenetic modifications
played important roles in monitoring the expression of
PTEN (Figs 5 and 6) Thus, the results presented in
this article suggest a working model in which a low
concentration of VD3 exerts its stimulating effects on
the induction of apoptosis in gastric cancer cells, and
this process is tightly associated with the action of
epi-genetic modification agents such as TSA⁄ NaBu and
5-Aza, which either directly, or through transcription
factor Egr-1, influence the expression of PTEN
Poten-tially, our work may be useful as a clue for the
devel-opment of an optional therapeutic strategy for gastric
cancer involving the administration of vitamin D and
epigenetic modifiers
Experimental procedures
Plasmids
The luciferase reporter construct of the PTEN promoter was
obtained by subcloning the 1978 bp genomic DNA region
upstream of the human PTEN gene into the pGL-3basic-luc
vector [14] The human VDR expression plasmid was a gift
from Y C Li (Committee on Molecular Metabolism and
Nutrition, Biological Science Division, The University of
Chicago, IL, USA) [41] The expression plasmid containing
the wild-type p300 was generously provided by J Boyes
(Institute of Cancer Research, UK) The siRNA targeting
the PTEN gene (5¢-GACTTGAAGGCGTATACAG-3¢) [42] was synthesized and cloned into the BamHI⁄ HindIII sites in the pSliencer 4.1–CMV neo vector (Ambion, Austin, TX, USA), according to the published data
Cell culture Undifferentiated HGC-27 human gastric carcinoma and 293T human embryonic kidney epithelial cell lines were pur-chased from the Institute of Cell Biology, Shanghai, China Cells were cultured in IMDM supplemented with 10% fetal bovine serum, 100 UÆmL)1penicillin and 100 lgÆmL)1 strep-tomycin, and kept in a humidified atmosphere of 5% CO2 Stably transfected cell lines were maintained in IMDM sup-plemented with 10% fetal bovine serum in the presence of G418 (1000 lgÆmL)1)
Assessment of apoptosis Cells were seeded in 24-well plates and cultured for 18 h Following transient transfection or treatments with VD3 (Sigma), 5-Aza (Sigma), or TSA⁄ NaBu (Sigma, St Louis,
MO, USA), cells were cultured for 24 h The apoptosis assay was performed by using the annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit (Nanjingkaiji, Nanjing, China), and analysis was by flow cytometry (exita-tion at 488 nm; emission at 530 nm) with an FITC signal detector, and prodium iodide (PI) staining with a phycoery-thrin emission signal detector The apoptosis rates were calculated on the basis of 30 000 cells
Transient transfection and luciferase reporter assay
For transient transfection, cells were seeded in 24-well plates and cultured for 18 h, before being transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) After transfection, cells were cultured for 30 h before har-vest, washed with NaCl⁄ Pi, and lysed in 30 lL of lysis buf-fer Reporter gene expression was measured and quantified using a dual Luciferase Reporter Assay System (Promega, Madison, WI, USA) Relative luciferase activity was ana-lyzed by using a Turner Designs TD20⁄ 20 Luminometer (Sunnyvale, CA , USA) Firefly luciferase activity was nor-malized to the activity of the Renilla luciferase control Extracts from at least three independent transfection experi-ments were assayed in triplicate The results are shown as means ± standard deviations
RNA isolation and quantitative real-time PCR Total RNA was extracted following the TaKaRa RNAiso Reagent manual, and reverse transcribed into cDNA using the RT system supplied by Promega The resultant cDNA
Trang 9was diluted five-fold with RNase-free water Quantitative
real-time PCR was performed on an ABI Prism 7000
Sequence Detection System (Applied Biosystems, Foster
City, CA, USA), using SYBR Green (Toyobo, Japan) as a
dsDNA-specific fluorescent dye b-Actin was used for
standardizing PTEN and Egr-1 mRNA Amplification
prim-ers were 5¢-ACCAGTGGCACTGTTGTTTCAC-3¢ and
5¢-TTCCTCTGGTCCTGGTATGAAG-3¢ for the PTEN
gene [43], and 5¢-AGCCCTACGAGCACCTG-3¢ and
5¢-CGGTGGGTTGGTCATG-3¢ for the Egr-1 gene [36]
Data were analyzed by using the 2)DDCt method [44] All
results represent means ± standard deviations of three
independent experiments
Western blot and CoIP
Cells were treated with VD3 (10)7m), TSA (200 nm), NaBu
(2 mm), or 5-Aza (5 lm), incubated for 24 h, and then lysed
in lysis buffer (50 mm Tris⁄ HCl, 1% Nonidet P-40, 150 mm
NaCl, 1 mm EDTA, and 1 mm phenylmethanesulfonyl
fluo-ride) Cell lysates were separated by SDS⁄ PAGE in 15%
gels, then transferred to poly(vinylidene difluoride)
mem-branes (Millipore, Bedford, MA, USA), and subjected to
western blot analysis with rabbit polyclonal antibodies
against PTEN (Abcam, Cambridge, MA, USA) or Egr-1
(Santa Cruz Biotechnology, Santa Cruz, CA, USA), and
mouse monoclonal antibody against b-actin (Sigma) The
signals were visualized by using the chemiluminescent
sub-strate method with the SuperSignal West Pico kit provided
by Pierce Co (Rockford, IL, USA)
Coprecipitation of VDR with Egr-1 and p300 was
per-formed in 293T cells, using a protocol described previously
[45] The antibodies against Egr-1, VDR and p300 were
used for immunoprecipitation and western blotting
Construction of stably transfected cell lines
HGC-27 cells were transfected with pSliencer 4.1–CMV
neo-siPTEN or pSliencer 4.1–CMV neo vector, using the
Lipofectamine 2000 (Invitrogen) protocol Cells were
main-tained in IMDM supplemented with 10% fetal bovine
serum, 100 mgÆmL)1 penicillin, 100 mgÆmL)1 streptomycin,
and 1000 lgÆmL)1 G418 After 15–22 days, individual
clones were isolated and expanded The expression of PTEN
was knocked down in the cell lines with pSliencer 4.1–CMV
neo-siPTEN
ChIP
ChIP assays were carried out using a kit supplied by
Upstate, following the manufacturer’s protocol Cells were
plated at a density of 1· 105⁄ mL in six-well plates and
cul-tured for 24 h After treatments with TSA⁄ NaBu or 5-Aza,
cells were crosslinked in 2% formaldehyde for 10 min at
37C, and then lysed in SDS lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris, pH 8.1) with protease inhibitors The sonicated lysates were processed using a ChIP assay kit, essentially as described by the manufacturer (Upstate Bio-technology, Lake Placid, NY, USA) Antibodies against Ac-H3 and Ac-H4 were purchased from Upstate Antibody against Egr-1 was purchased from Santa Cruz Immunopre-cipitated chromatin was analyzed by quantitative PCR (ABI Prism 7000 Sequence Detection System Instrument, Applied Biosystems), using SYBR green dye with primers specific to sequences at the PTEN promoter
Statistical analysis Student’s test was used to calculate the statistical signifi-cance of the experimental data The level of signifisignifi-cance was set as *P < 0.05 and **P < 0.01
Acknowledgements
We thank E Adamson (Burnham Institute, USA) and
Y Chun Li (Committee on Molecular Metabolism and Nutrition, Biological Science Division, The University
of Chicago, Chicago, IL, USA) for providing plasmids This work was supported by grants from The National Basic Research Program of China (2005CB522404 and 2006CB910506), The Program for Changjiang Scholars and Innovative Research Team (PCSIRT) in Universi-ties (IRT0519), and The National Natural Science Foundation of China (30771232 and 30671184)
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