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Keywords:

  • deacetylase;
  • epigenetic;
  • gastric cancer;
  • mammalian target of rapamycin;
  • p21

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements and funding
  9. References

Deregulation of the mammalian target of rapamycin pathway (mTOR pathway) is associated with human cancer. The relationship between mTOR pathway and histone acetylation is still unclear in gastric cancer (GC). Immunohistochemistry was used to examine the phosphorylation of mTOR and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in GC tissues. MKN45 and SGC7901 cells were treated with the mTOR inhibitor rapamycin (RAPA) alone or in combination with the phosphatidylinositol 3-kinase inhibitor LY294002 and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Small interfering RNA (siRNA) technology was also used to knockdown mTOR. Phosphorylated mTOR and phosphorylated 4E-BP1 were expressed in 71.1% and 68.4% of the human GC tissues tested, respectively; significantly higher than the levels in para-cancerous tissues (50% and 57.9%) and normal tissues (44.6% and 29%). RAPA markedly inhibited cell proliferation, induced G1 cell cycle arrest, and reduced phosphorylation of p70 S6 protein kinase (p70S6K) and 4E-BP1 in GC cells, particularly when used in combination with LY294002 or TSA. The mRNA expression of the tumour suppressor gene p21WAF1 increased significantly in GC cells treated with both RAPA and TSA. Histone acetylation also increased after RAPA and TSA treatment or siRNA knockdown of mTOR. Our findings suggest that the mTOR pathway is activated in GC, and also that inhibition of mTOR enhances the ability of TSA to suppress cell proliferation and lead to cell cycle arrest via increasing histone acetylation and p21WAF1 transcription in human MKN45 and SGC7901 GC cells.


Abbreviations
mTOR

mammalian target of rapamycin

4E-BP1

eukaryotic translation initiation factor 4E-binding protein 1

GC

gastric cancer

RAPA

rapamycin

HDAC

histone deacetylase

TSA

trichostatin A

siRNA

small interfering RNA

p70S6K

p70 S6 protein kinase

PI3K

phosphatidylinositol 3-kinase

CIP/KIP

CDK-interacting protein/kinase inhibition protein

ERK

extracellular signal-regulated kinase

MAPK

mitogen-activated protein kinase

Dnmt

DNA-methyltransferase

CDKI

cyclin-dependent kinase inhibitor

TSC2

tuberous sclerosis 2

FLT3

FMS-like tyrosine kinase 3

ITD

internal tandem duplication

SIRT1

sirtuin 1

ChIP

chromatin immunoprecipitation

FCM

flow cytometry

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements and funding
  9. References

Despite improvements in surgical resection techniques and the efficacy of multimodality therapy regimens, gastric cancer (GC) still has the second highest mortality rate of all cancers worldwide (Jansen et al., 2005). In clinical studies, molecular-targeted therapies have significantly improved the outcome of patients with advanced solid malignancies (Cunningham et al., 2004; Hurwitz et al., 2004; Piccart-Gebhart et al., 2005).

The phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signalling pathway (PI3K/Akt/mTOR pathway) is important for the survival and growth of cells, and is an attractive target for antitumour therapy (Arkenau, 2009; Zagouri et al., 2011). An important downstream target of PI3K/Akt is mTOR, which mediates phosphorylation of the p70S6K and 4E-BP1 (Abraham, 2002); these proteins are responsible for the translation and expression of a large number of proteins, including D-type cyclins and c-myc (Troussard et al., 2000; Sola and Troussard, 2003). Activation of mTOR in response to mitogens leads to cell-cycle progression from the G1 to the S phase. It is postulated that dysregulation of the mTOR signalling pathway plays a role in tumourigenesis. The PI3K/Akt/mTOR pathway is thought to be activated in 30–50% of prostate cancers, 30–60% of malignant gliomas, 30–50% of endometrial carcinomas, >50% of melanomas, >30% of renal cell carcinomas and approximately 10% of breast cancers (Vignot et al., 2005). Recently, activation of the mTOR pathway has also been identified in gastric carcinoma (Al-Batran et al., 2012). Therefore, the mTOR pathway is an attractive target for antitumour therapy. Several mTOR inhibitors have been developed, including rapamycin and its analogues CCI-779, RAD001(everolimus) and AP23573. After showing promising results in phase I trials, the potential clinical significance of these drugs is currently under evaluation in several phase II and II trials on patients with solid tumours or some hematological malignancies. Recently, the efficacy of everolimus in patients with previously treated metastatic GC was supported by preliminary results from a phase II study conducted in Korea (Al-Batran et al., 2012). However, studies have revealed that RAPA can induce activation of Akt, which can attenuate the potential antitumour activity of RAPA. It has been suggested that mTOR and PI3K inhibitors (such as LY294002) might be promising options for combination therapy (Dowling et al., 2010; Lang et al., 2010; Wander et al., 2011).

However, aberrant gene transcription resulting from epigenetic changes is a frequent event during the molecular pathogenesis of malignant transformation. Histone deacetylation is critical for maintaining a closed chromatin structure, and altered histone deacetylation is related to aberrant gene transcription for a variety of genes, such as p21WAF1, in a number of malignancies (Zhu and Otterson, 2003). P21WAF1 is a member of the CDK-interacting protein/kinase inhibition protein (CIP/KIP) family, which inhibit the late G1/S checkpoint kinases. Increased expression of p21WAF1 can induce growth arrest in transformed cells (Polyak et al., 1994). Histone acetylation is major mechanism of regulation of the p21WAF1 gene in some cell lines (Fang and Lu, 2002). In contrast to genetic defects, the reversible nature of the epigenetic aberrations observed in cancer cells constitute an attractive therapeutic target; hence, a number of HDAC inhibitors are undergoing preclinical testing in combination therapy for cancer at present (Giannini et al., 2012).

Important relationships exist between the activation of some signalling pathways and epigenetic changes. Regarding the relationship between the extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) signalling pathway and DNA methylation, Richardson and colleagues suggested that the expression of DNA-methyltransferase (Dnmt) could be decreased by inhibiting signalling through the ras-MAPK pathway in human T cells (Deng et al., 1998). We had found that inhibition of the ERK/MAPK pathway could decrease DNA methylation in colon cancer cells (Lu et al., 2007). However, it is not clear if the PI3K/Akt/mTOR pathway and the expression of genes related to the cell cycle regulate cell proliferation as independent factors, or if their roles are interrelated. Furthermore, it is unknown whether epigenetic modifications are involved in the process by which inhibition of the mTOR pathway influences the cell cycle and suppresses cell proliferation. Our group has been interested in TSA (a HDAC inhibitor), which has been used in epigenetic therapy for cancer, specifically in gastric carcinoma (Fang et al., 2004; Lu et al., 2007). As mentioned above, RAPA can induce Akt activation which attenuates the potential antitumour activity of RAPA in some cancer cell lines.

This study investigates the activation of mTOR and 4E-BP1 in human GC, and their relationship with the clinical characteristics of GC. We have also focused on the effects of inhibiting the mTOR signalling pathway using RAPA and/or LY294002 on the suppression of cell proliferation and epigenetic modifications induced by RAPA and TSA in GC cell lines.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements and funding
  9. References

Immunohistochemical analysis of phosphorylated mTOR and 4E-BP1 in human gastric cancer specimens

To determine the expression of activated mTOR in human GC specimens, tissue arrays were constructed from formalin fixed, paraffin-embedded tissues, and stained using antibodies against phosphorylated mTOR (Ser2448) and phosphorylated 4E-BP1 (Thr70; Cell Signaling Technologies, MA, USA). All of the specimens were obtained from 38 GC patients who had undergone surgery at Shanghai Renji Hospital between July 2009 and January 2010. The protocol was approved by the ethics committee of Shanghai Jiaotong University School of Medicine, Renji Hospital, and the research was performed in accordance with the provisions of the Declaration of Helsinki of 1975. We obtained one or two pieces of tissue each from the cancerous mucosa, para-cancerous mucosa (within 5 cm of the cancerous areas), and non-cancerous mucosa (further than 5 cm from the cancerous areas) for every patient. The tissue pieces were frozen in liquid nitrogen, and 5 µm-thick sections were fixed in formalin and embedded in paraffin. None of the patients received preoperative treatments such as radiotherapy or chemotherapy. The array comprised both intestinal-type (n = 16) and diffuse-type (n = 22) gastric adenocarcinomas of various tumour stages (Table 1). The slides were deparaffinised in xylene, exposed to a graded series of alcohols [100%, 95%, 80% ethanol/ddH2O (v/v)], rehydrated in PBS (pH 7.5), and endogenous peroxidases were blocked using H2O2. The slides were incubated with the primary antibodies (1:50 dilution) at 4°C overnight. After washing with PBS, biotinylated universal antibody (1:50 dilution; Vectastain Universal Elite ABC Kit, Vector Laboratories, Burlingame, CA, USA) was added to the tissue sections. Phosphorylated-mTOR and phosphorylated-4E-BP1 were visualised using Vectastain Elite ABC reagent (Vector Laboratories), followed by incubation with diaminobenzidine. The nuclei were counterstained with hematoxylin. Negative controls were performed by omitting the primary antibody. Tissues from human prostate adenocarcinoma served as a positive control (Sahin et al., 2004). Immunoreactivity for mTOR and 4E-BP1 were independently rated by two pathologists and the staining intensity was scored as absent (−), mild (+) or strong (++), in accordance with a recently published scoring system for mTOR expression (Zhou et al., 2004).

Table 1. Pathological features of the 38 gastric carcinomas
ParameterCategoryNumber (total = 38)
Lauren's classificationIntestinal16 (42%)
 Diffuse22 (58%)
T-StageT12 (5%)
 T223 (61%)
 T35 (13%)
 T48 (21%)
GradeG115 (39%)
 G211 (29%)
 G312 (32%)
Node018 (47%)
 >020 (53%)

Cell culture and reagents

MKN45 and SGC7901 cells were maintained in RPMI-1640 medium (Gibco/BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) in a 5% CO2 humidified air atmosphere at 37°C.

TSA and LY294002 were purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in dimethyl sulfoxide (DMSO, as the control vehicle) and used at a final concentration of 1 and 10 µM. RAPA was purchased from Cell Signaling Technology, dissolved in DMSO and used at a final concentration of 10 nM.

Cell viability assay and proliferation studies

Cell viability was assessed using MTT [3-(4,5-dimethylthiazol-2-l)-2,5- diphenyltetrazolium bromide] (Sigma). Briefly, 5 × 103 cells were seeded per well in 96-well flat-bottom plates, cultured for 24 h, serum-starved for 18 h and treated with appropriate concentrations of LY294002, RAPA or TSA in the presence of 10% FBS for 48 or 72 h. After treatment, 20 µL of MTT (5 mg/mL in PBS) was added to each well, incubated for 3 h at 37°C, the cells were lysed by the addition of 0.1 N HCl in isopropanol, and the absorption values were measured at 570 nm. The percentage of cell survival was determined as follows: Aexp group/Acontrol × 100%.

To assay proliferation, MKN45 or SGC7901 cells (50,000) were plated in normal growth medium, cultured overnight, and treated with DMSO, TSA, RAPA, LY294002 or different combination of these reagents. The cells were trypsinised and counted using a Coulter counter (CASY®, Roche, CH).

Flow cytometric analysis of cell cycle progression

Cell cycle analysis was carried out by flow cytometry (FCM) (Bender et al., 1999). Approximately 1 × 108 cells were removed from the treated and mock-treated cultures after 24 h, washed twice with PBS, fixed in ice-cold ethanol for 1 h, centrifuged, and resuspended and incubated in 1% (v/v) Triton X-100 (Sigma) and 0.01% RNase (Sigma) for 10 min at 37°C. DNA was stained with 0.05% propidium iodide for 20 min at 4°C in the dark. Cell cycle distributions were determined using a flow cytometer (FACSCalibur; B.D., USA). The data obtained from 10,000 cells was analysed using the MultiCycle software package (Phoenix, San Diego, CA, USA).

RNAi and transient transfections

Expression of mTOR was inhibited using the commercial SignalSilence™ mTOR siRNA kit (Cell Signaling Technology, Danvers, MA, USA) as previously described (Lu et al., 2007). The kit included a human mTOR siRNA, control siRNA (fluorescein conjugate), and a siRNA transfection reagent. Selective silencing of mTOR was confirmed by Western blotting using phospho-p70- and phospho-4E-BP1-specific antibodies (Cell Signaling Technologies, MA, USA).

Real-time RT-PCR

Expression of p21WAF1 was quantified using real-time RT-PCR. Total RNA was isolated using Trizol® reagent (Invitrogen-Gibco BRL, Carlsbad, CA, USA). Reverse transcription reactions using 5 µg of total RNA in a final reaction volume of 20 µL were done using SuperScript II reverse transcriptase (Invitrogen Life Technologies). Relative quantification data was obtained using the comparative Ct method using the ABI PRISM 7700 Sequence Detection System (software version 1.6; Applied Biosystems, Foster City, CA). The primers were manufactured by Shengong Company (Shanghai, China). The sequences of the p21WAF1 primers were as follows: sense 5′-CTG GAG ACT CTC AGG GTC GAA-3′ and antisense 5′-GGA TTA GGG CTT CCT CTT GGA-3′. Real-time PCR was also performed using primers for β-actin (sense 5′-CTG GCA CCC AGC ACA ATG-3′ and antisense 5′-GGA CAG CGA GGC CAG GAT-3′). Results were expressed as a ratio of the number of copies of the target gene to the number of copies of β-actin. The Ct values were measured and the average Ct of triplicate samples was calculated. Significant alterations in mRNA expression were defined as a threefold difference in the expression level after treatment relative to control cells (Scanlan et al., 2002).

Western blotting analysis

Whole-cell extracts were prepared from both treated, mock-treated, transfected and mock-transfected MKN45 and SGC7901 cells as previously described (Fang et al., 2001). Primary antibodies against Akt/phospho-Akt (Ser473), p70S6K/phospho- p70S6K (Thr421/Ser424), and 4E-BP1/phospho-4E-BP1 (Thr70) were purchased from Cell Signaling Technology. Antibodies against Histone H4/Ace-H4 and Histone H3/Ace-H3 were obtained from Upstate Biotechnology (Lake Placid, NY, USA). An antibody against GAPDH (Sigma) was used as a control for protein input.

Chromatin immunoprecipitation (ChIP) assay

ChIP analysis used chromatin from vehicle- or drug-treated cells. Chromatin was fixed and immunoprecipitated with a ChIP assay kit (Upstate Biotechnology, Lake Placid). Briefly, the cells were cross-linked with 1% formaldehyde, washed with ice-cold phosphate-buffered saline containing a protease inhibitor mixture (Sigma) and 1 mM phenylmethylsulfonyl fluoride, and lysed in SDS lysis buffer. Nuclei were sonicated for a total of 40 s to shear the DNA, the lysates were pelleted, and the supernatants diluted. The average size of the sonicated DNA fragments subjected to immunoprecipitation was 500 bp, as determined by ethidium bromide gel electrophoresis. Diluted lysates were pre-cleared with salmon sperm DNA/protein A-agarose 50% slurry for 30 min at 4°C prior to immunoprecipitation with the specified antibodies or rabbit non-immune IgG at 4°C overnight. After pelleting, the protein A precipitates were washed sequentially for 5 min each at 4°C with a low-salt immune complex wash buffer, high-salt immune complex wash buffer, LiCl immune complex wash buffer, and finally TE buffer (10 mM Tris–Cl, pH 7.5, 1 mM EDTA). The antigen/antibody complexes were extracted twice in 250 µL of elution buffer (1% SDS, 0.1 M NaHCO3), and 20 µL of 5 M NaCl solution was added to the combined eluates and incubated at 65°C for 4 h. The samples were treated with 10 µL of 0.5 M EDTA, 20 µL of 1 M Tris–HCl, pH 6.5 and 1 µL of 20 µg/mL proteinase K at 45°C for 1 h. The recovered DNA was purified with a DNA Clean-up kit (Qiagen, Hilden, Germany); samples of input DNA were prepared in the same way. The presence of the selected DNA sequence was assessed by PCR using primers flanking the p21WAF1 promoter region of interest (Table 2). PCR for the input was performed using genomic DNA. The input fraction corresponded to 1% of the chromatin solution before immunoprecipitation. The PCR products were analysed on 2% agarose gels.

Table 2. Sequences of primers and reaction condition for ChIPs-PCR
GenePrimer (forward) (5′ [RIGHTWARDS ARROW] 3′)Primer (reverse) (5′ [RIGHTWARDS ARROW] 3′)Reaction condition
  • *

    P1, nucleotides −576 to −293; P2, nucleotides −51 to +77.

p21WAF1(P1)*CGT GGT GGT GGT GAG CTA GACTG TCT GCA CCT TCG CTC CT296 bp, 95°C 5 min; 95°C 1 min, 58°C 1 min, 72°C 1 min, 35cycles; 72°C 5 min
p21WAF1(P2)*GGT TGT ATA TCA GGG CCGCTC TCA CCT CCT CTG AGT GC128 bp, 95°C 5 min; 95°C 1 min, 58°C 1 min, 72°C 1 min, 35cycles; 72°C 5 min
γ-actinGGA CCT GGC TGG CCG GGA CCGTG GCC ATC TCC TGC TCG AA153 bp, 95°C 5 min; 95°C 1 min, 56°C 1 min, 72°C 1 min, 35cycles; 72°C 5 min

Data analysis

Each growth inhibition or flow cytometry experiment was repeated 5–6 times and the mean ± SD is presented. For growth inhibition studies, the values were expressed as a percentage of untreated control cells. Comparisons between treated groups were done with one-way ANOVA. Relationships between different clinicopathological variables were analysed using the Kruskal–Wallis test using SAS 6.12 software; P < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements and funding
  9. References

Phosphorylation of mTOR/4E-BP1 increases during gastric tumourigenesis and progression

To investigate the potential role of activation of the mTOR pathway in GC tumourigenesis and progression, we examined the phosphorylation of mTOR and 4E-BP1 in cancerous, para-cancerous, and non-cancerous tissues from human gastric mucosa. mTOR and 4E-BP1 were phosphorylated in 71.1% and 68.4%, respectively, of the GC tissues tested; significantly higher than the levels observed in the para-cancerous tissues (50% and 57.9%) and normal tissues (44.6% and 29%, P < 0.05; Table 3). p-mTOR immunoreactivity was well-distributed within the cytoplasm of the tumour glands or cancerous cells (Figure 1); however, p-4E-BP1 occurred in both the cytoplasm and nuclei of the tumour cells or glands in GC slides (Figure 1). We examined whether p-mTOR/p-4E-BP1 was associated with the clinicopathological features of GC, including the Lauren classification, stage, grade, lymph node metastasis, patient age, and tumour size. p-4E-BP1 was positively correlated with the tumour size (P < 0.05), with a mean tumour size of 6.26 ± 2.63 for p-4E-BP1-positive cases versus 4.21 ± 1.27 cm in p-4E-BP1-negative cases. However, p-mTOR was only positively correlated with tumour size in p-mTOR-positive cases; however, p-mTOR-negative cases also had a large tumour size. Thus, additional studies with a larger sample size will be needed to clarify this phenomenon. However, there was no strong evidence of any association between phosphorylation of mTOR or 4E-BP1 and other clinicopathological feature in GC (Table 4).

Table 3. Phosphorylation of mTOR and 4E-BP1 in different gastric tissues
 p-mTOR n (%)p-4E-BP1 n (%)
(−)(+)(++)(−)(+)(++)
  1. Ca, cancerous tissues; P, para-cancerous tissues; N, non-cancerous tissues.

Ca11 (28.9)22 (57.9)5 (13.2)12 (31.6)17 (44.7)9 (23.7)
P19 (50)18 (47.4)1 (2.6)16 (42.1)13 (34.2)9 (23.7)
N21 (55.3)17 (44.7)0 (0)27 (71)9 (23.7)2 (5.3)
P <0.05 <0.01  
image

Figure 1. Activation of mTOR and 4E-BP1 in human gastric adenocarcinoma specimens. Sections of resected intestinal-type gastric cancer, diffuse-type gastric cancer, para-cancerous and normal tissues were stained using antibodies for phospho-mTOR and phospho-4E-BP1.

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Table 4. Association of p-mTOR and p-4E-BP1 with patient characteristics
 p-mTORp-4E-BP1
 (−)(+)(++)P-value(−)(+)(++)P-value
  • a

    Median (range).

Lauren's classification   0.57   0.06
Diffuse5 (22.7)14 (63.6)3 (13.6) 10 (45.5)9 (40.9)3 (13.6) 
Intestinal6 (37.5)8 (50)2 (12.5) 2 (12.5)8 (50)6 (37.5) 
T-Stage   0.67   0.15
T10 (0)2 (100)0 (0) 1 (50)0 (0)1 (50) 
T27 (30.4)12 (52.2)4 (17.4) 6 (26.1)12 (52.2)5 (21.7) 
T32 (40)2 (40)1 (20) 0 (0)4 (80)1 (20) 
T42 (25)6 (75)0 (0) 5 (62.5)1 (12.5)2 (25) 
Grade   0.66   0.26
G14 (26.7)9 (60)2 (13.3) 2 (13.3)8 (53.3)5 (33.3) 
G25 (45.5)5 (45.5)1 (9.1) 4 (36.3)4 (36.3)3 (27.3) 
G32 (16.7)8 (66.7)2 (16.7) 6 (50)5 (41.7)1 (8.3) 
Node   0.29   0.82
03 (16.7)12 (66.7)3 (16.7) 5 (27.8)9 (50)4 (22.2) 
>08 (40)10 (50)2 (10) 7 (35)8 (40)5 (25) 
Age (y)74 (41–91)a57 (31–79)67 (41–76)0.161 (33–78)a67 (43–91)53 (31–76)0.5
Tumour size (cm)7.05 ± 2.584.56 ± 1.867.6 ± 2.3304.21 ± 1.275.91 ± 2.546.92 ± 2.810

RAPA increases the inhibition of cell growth by epigenetic drugs in MKN45 and SGC7901 gastric cancer cells

The response of MKN45 and SGC7901 cells to growth inhibition by TSA, RAPA and LY294002 were examined. Dose-dependent studies using MTT tests showed that treatment of these cells with RAPA at 10 nmol/L significantly inhibited cell viability (Figure 2A). However, more significant growth inhibition occurred when the cell lines were treated in combination with RAPA and TSA or RAPA and LY294002 and TSA, particularly at 72 h (Figures 2B and 2C).

image

Figure 2. Inhibition of the mTOR pathway increases the effects of TSA on growth-inhibition and the cell cycle. (A) Cells were cultured in serum-free medium for 24 h, then incubated with various concentrations of RAPA for 48 h. Growth was assessed using the MTT assay; data is presented as a percentage of control vehicle-treated cells cultured in the same plate at 48 h from an average of six replicate experiments. (B and C) mTOR pathway inhibition and HDAC inhibition suppresses cell growth in MKN45 and SGC7901 cells. The experiments were performed in triplicate. Cells were treated (alone or in combination) for 48 or 72 h with 1 µM TSA, 10 nM RAPA or 10 µM LY294002; P < 0.01. L, LY294002; LR, LY294002 + RAPA; LRT: LY294002 + RAPA + TSA.

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Inhibition of the mTOR pathway enhances cell cycle arrest by TSA

To investigate the influence of mTOR inhibition on cell cycle arrest induced by TSA, the effects of RAPA, LY294002 and TSA on p21WAF1-mediated G1-phase arrest were evaluated. Treatment of MKN45 and SGC7901 cells with RAPA alone blocked the cell cycle in the G1 phase (P < 0.05; Figure 3). Cells treated with a combination of RAPA and TSA or LY294002, RAPA and TSA blocked the cell cycle in the G1 phase more significantly than treatment with TSA alone (P < 0.01).

image

Figure 3. Cell cycle arrest after treatment of drugs in gastric cancer cell lines. Cells were treated (alone or in combination) for 24 h with 1 µM TSA, 10 nM RAPA or 10 µM LY294002. T, TSA; R, RAPA; L, LY294002; LR, LY294002 + RAPA (P < 0.05).

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PI3K/Akt/mTOR signalling inhibitors downregulate the phosphorylation of Akt, p70S6K and 4E-BP1, whereas HDAC inhibitors enhance this effect

GC cells were incubated with PI3K/Akt/mTOR signalling inhibitors, and the cell lysates were analysed for Akt, p70S6K and 4E-BP1 phosphorylation by Western blot analysis. The protein levels of total Akt, p70S6K and 4E-BP1 did not decrease in the RAPA- and LY294002-treated lysates compared with the vehicle controls. Phosphorylation of Akt did not increase after RAPA treatment (Figure 4), but treatment with RAPA and LY294002 decreased the phosphorylation of p70S6K and 4E-BP1 in MKN45 and SGC7901 cells compared to cells treated with RAPA alone (Figure 5). Similar effects were also observed in mTOR siRNA-transfected cells (Figure 6). 4E-BP1 was phosphorylated to a lesser extent in cells treated with PI3K/Akt/mTOR signalling inhibitors and TSA compared to cells treated with RAPA or LY294002 alone. Similar results were also obtained for phospho-p70S6K. Interestingly, RAPA alone or in combination with LY294002 or TSA did downregulate the total levels of 4E-BP1 protein expression.

image

Figure 4. Inhibition of Akt downregulates the phosphorylation of Akt; however, RAPA does not induce Akt phosphorylation. Cells were treated (alone or in combination) for 6 h with 1 µM TSA, 10 nM RAPA or 10 µM LY294002. L, LY294002; LR, LY294002 + RAPA. Equal amounts of protein were analysed by Western blotting using antibodies against total Akt and phospho-Akt. The data is representative of three replicate experiments.

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image

Figure 5. PI3K/Akt/mTOR inhibitors downregulate the phosphorylation of p70S6K and 4E-BP1. Cells were treated (alone or in combination) for 6 h with 1 µM TSA, 10 nM RAPA or 10 µM LY294002. L, LY294002; LR, LY294002 + RAPA; LRT: LY294002 + RAPA + TSA. Equal amounts of protein were analysed by Western blotting using antibodies against total p70S6K, 4E-BP1, phospho-p70S6K or phospho-4E-BP1. The data is representative of three replicate experiments.

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image

Figure 6. SiRNA-mediated inhibition of mTOR downregulates the phosphorylation of p70S6K and 4E-BP1, and increases acetylation of histone H3 in SGC7901 cells. Cells were transiently transfected with siRNA, and protein lysates were prepared at the indicated times after transfection. Quantification of the target protein bands relative to GAPDH is shown below the blots. M, Untransfected MKN45 cells; Mi-0, MKN45 cells transfected with scrambled siRNA (control); Mi, MKN45 cells transfected with SignalSilenceTM mTOR siRNA. G, Untransfected SGC7901 cells; Gi-0, SGC7901 cells transfected with scrambled siRNA (control); Gi, SGC7901 cells transfected with SignalSilenceTM mTOR siRNA.

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RAPA increases the upregulation of p21WAF1 by TSA

Inhibition of the mTOR pathway induced cell cycle arrest (Figure 3). However, members of the cyclin-dependent kinase inhibitor (CDKI) family, including p21WAF1, inhibit a wide range of cyclin CDK complexes involved in G1- and S-phase progression. Thus, real-time PCR was used to ascertain whether inhibition or blocking of the mTOR pathway had an effect on the expression of GC-related tumour suppressor genes. This assay was also used to clarify whether epigenetic changes and mTOR signalling can interact with these tumour-related genes. TSA increased the transcription of p21WAF1, the effect being more significant in the presence of RAPA (Figure 7A).

image

Figure 7. TSA upregulates p21WAF1 and increases acetylation of histones H3 and H4, which can be enhanced by PI3K/Akt/mTOR inhibitors. Cells were treated (alone or in combination) for 6 h with 1 µM TSA, 10 nM RAPA or 10 µM LY294002. L, LY294002; LR, LY294002 + RAPA; LRT: LY294002 + RAPA + TSA. (A) Realtime PCR analysis of p21WAF1 are expressed in arbitrary units relative to the mock control cells; data is representative of three replicate real-time RT-PCR experiments. (B) Cells were treated for 24 h with different drugs as indicated. Equal amounts of protein were analysed by Western blotting using antibodies against ace-H3 or ace-H4; data are representative of three replicate experiments.

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PI3K/Akt/mTOR signalling pathway inhibitors enhance the effect of a HDAC inhibitor on histones H3 and H4 acetylation

Deacetylation of chromatin by histone deacetylase (HDAC) complexes has been implicated in the repression of gene expression (Eberharter and Becker, 2002). The association between cell cycle arrest and p21WAF1 expression prompted us to investigate the relationship between HDAC inhibitors and mTOR signalling in histone acetylation. RAPA and LY294002 enhanced the ability of TSA to induce increased acetylation of histones H3 and H4 (Figure 7B), which is consistent with the effects of knocking down mTOR using a siRNA (Figure 6).

Inhibition of mTOR augments the ability of TSA to promote the binding of chromatin-associated acetylated histones H4 and H3 to the p21WAF1 promoter

Having demonstrated that transcription of p21WAF1 (Figure 7A) and acetylation of histones H3 and H4 (Figure 7B) increased more significantly in GC cells treated with a combination of RAPA and TSA, ChIP assays were used to determine whether histone acetylation is involved in regulation of the p21WAF1 gene. TSA combined with RAPA greatly increased chromatin-associated acetylated histones H4 and H3 at the regions from −576 to-293 and −51 to +77 of the p21WAF1 promoter (Figure 8). Treatment with TSA alone or TSA combined with LY294002 and RAPA also increase it slightly in the association of acetylated histones H4 and H3 to the p21WAF1 promoter, whereas cells treated with RAPA alone, LY294002 alone, or a combination of RAPA and LY294002 did not increase in the binding of acetylated histones H3 and H4 to the p21WAF1 promoter.

image

Figure 8. RAPA and LY294002 accentuate the ability of TSA to augment chromatin-associated acetylation of histones H4 and H3 at the p21WAF1 promoter. (A) Schematic representation of the p21WAF1 promoter. Primer sets are indicated as P1 and P2. (B) ChIP assays were performed using MKN45 and SGC7901 cells that had been treated as indicated using antibodies against acetylated histones H4 and H3 to immunoprecipitate and PCR amplify of the regions from −576 to −293 and −51 to +77 of the p21WAF1 promoter. 1–7: input, 8–14: H4, 15–21: H3, 22–28: control (no antibody); 1, 8, 15, 22: DMSO (vehicle control); 2, 9, 16, 23: RAPA (R); 3, 10, 17, 24: LY294002 (L); 4, 11, 18, 25: TSA (T); 5, 12, 19, 26: RAPA + TSA (RT); 6, 13, 20, 27: LY294002 + RAPA (LR); 7, 14, 21, 28: LY294002 + RAPA + TSA (LRT).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements and funding
  9. References

The kinase mTOR is responsible for mitogen-induced cell proliferation/survival signalling. By preventing phosphorylation of 4E-BP1, mTOR inhibitors can downregulate the expression of such genes and induce G1 cell cycle arrest (Huang et al., 2003). In our patient cohort, the levels of phosphorylated mTOR and phosphorylated 4E-BP1 were significantly higher in GC tissues than the para-cancerous tissues and normal tissues (P < 0.05). The levels of phosphorylated mTOR and phosphorylated 4E-BP1 correlated positively with tumour size, although a larger number of samples are required to confirm this observation. This finding led us to investigate the effects of RAPA, an inhibitor of mTOR, on the proliferation of human GC cells. We confirmed that RAPA could inhibit mTOR and prevent further phosphorylation of p70S6K and 4E-BP1, which reduces the expression of proteins involved in transcription, translation and cell cycle control. Even high concentrations of RAPA (100 nM, 48 h) failed to inhibit GC cell proliferation by more than 50%. In an in vitro study, inhibition of mTOR using RAD001 increased the levels of p-Akt in breast and prostate cancer cells (O'Reilly et al., 2006). Interestingly, mTOR inhibition failed to induce Akt activation in MKN45 and SGC7901 cells. We hypothesise that high concentrations of RAPA might promote the phosphorylation of Akt in some, but not, all cell lines (Han et al., 2007). However, LY294002 could enhance the effects of RAPA in GC cells. The data from this study is consistent with the results of another study, which also demonstrated that LY294002 enhanced the ability of RAPA to inhibit the proliferation of T-cells (Breslin et al., 2005).

We found that inhibition of the PI3K/Akt/mTOR signalling pathway using LY294002 or RAPA could induce GC cell cycle arrest at the G1 phase; this inhibition was more significant when the cells were treated in combination with TSA. Additionally, the induction of cell cycle arrest was accordance with the expression of p21WAF1. Deacetylation of chromatin by HDAC complexes has been implicated in the repression of genes such as p21WAF1. The link between cell cycle arrest and p21WAF1 expression prompted us to investigate the relationship between HDAC inhibitors and PI3K/Akt/mTOR signalling in histone acetylation.

Alterations to epigenetic mechanisms are known to be associated with the biology of cancerous lesions and their clinical outcome (Ziech et al., 2010; Chik et al., 2011; Brennan and Flanagan, 2012); such mechanisms include DNA methylation, chromatin remodelling, histone replacement, and alterations to histone tails and non-coding RNA (Dawson and Kouzarides, 2012; Gigek et al., 2012). Imbalances in histone acetylation/deacetylation within promoter regions contribute to the deregulation of gene expression and have been associated with carcinogenesis and cancer progression (Sawan and Herceg, 2010). In mammalian cells, it has been proposed that the ability of the tuberous sclerosis 2 (TSC2) tumour suppressor gene to regulate vascular endothelial growth factor involves regulation by HDACs (Brugarolas et al., 2003). Thus, there appears to be a precedent for the involvement of mTOR in histone acetylation. FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD)-transformed cells also require concurrent Akt and mTOR blockage to undergo apoptosis after HDAC inhibitor treatment in leukemia cells (Cai et al., 2006). In the current study, treatment with RAPA or LY294002 alone failed to alter histone H3 or H4 acetylation under basal conditions, which led us to conclude that the PI3K/Akt/mTOR pathway has an indirect effect on histone acetylation. TSA-sensitive and TSA-insensitive mechanisms may be involved in the regulation of histone acetylation. For example, PI3K inhibitors limit the expression of sirtuin 1 (SIRT1) – a NAD-dependent and TSA-insensitive deacetylase in a human melanoma cell line (Csiszar et al., 2005; Wang et al., 2005), and to alter p300 histone acetyltransferase activity (Chen et al., 2004). Inhibition of the EGFR/PI3K/Akt cell survival pathway also promotes the ability of TSA to induce cell death and inhibit migration in human ovarian cancer cells (Zhou et al., 2006). Suppression of PI3K/Akt/mTOR signalling is a determinant of the sensitivity of lung adenocarcinoma cells to the novel HDAC inhibitor FK228 (Kodani et al., 2005). Further elucidation of the mechanisms by which TSA increases p21WAF1 expression, and the crosstalk between the PI3K/Akt/mTOR pathway and histone acetylation in human GC cells may provide novel clues for the treatment of GC.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements and funding
  9. References

High levels of mTOR phosphorylation and 4E-BP1 phosphorylation in GCs indicate that activation of the mTOR pathway occurs during GC tumourigenesis and progression. By influencing histone acetylation and transcription of p21WAF1, inhibition of the PI3K/Akt/mTOR pathway enhances the effect of TSA that suppress cell proliferation and lead to cell cycle arrest in human GC cell lines (summarised in Figure 9); the exact mechanisms responsible for this process remain unclear. However, in light of the present data and consideration of previous findings, we can hypothesise that a close relationship exists between the PI3K/Akt/mTOR signalling pathway and altered histone acetylation in GC, which may represent a potential target for tumour therapy.

image

Figure 9. Proposed mechanism by which RAPA enhances the ability of TSA to suppress cell proliferation and arrest the cell cycle via epigenetic modifications in human gastric cancer cell lines. The bulky solid arrow represents the findings of the present study, the bulky dotted arrow represents uncertain findings from the current study, the other arrows indicate findings from previous studies.

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Acknowledgements and funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements and funding
  9. References

Thanks are given to Hong-yin Zhu, En-lin Li and Wei-qi Gu for performing the real-time PCR, Ms. Guanfeng Shen for performing the FCM.

This work was supported by the National Natural Science Foundation for Distinguished Young Scholars [grant number 30625034] and the National Natural Science Foundation [grant number 81001070].

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  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements and funding
  9. References
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