Vitamin D stimulates apoptosis in gastric cancer cells in synergy 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 Huang2 and Jun Lu1 The Institute of Genetics and Cytology, Northeast Normal University, Changchun, China The Key Laboratory of Molecular Epigenetics of Ministry of Education (MOE), Northeast Normal University, Changchun, China 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 June 2009, revised 22 October 2009, accepted 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 phosphatase 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 apoptosis rate We further demonstrated that VD3 induced PTEN expression through vitamin D receptor In addition, our evidence showed that vitamin 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 apoptosis 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 interacts (MI:0914) with VDR (uniprotkb:P11473) and EGR1 (uniprotkb:P18146) by anti bait coimmunoprecipitation (MI:0006) 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] Therefore, 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 secosteroid hormone family, which controls calcium and 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 D3; VDR, vitamin D receptor FEBS Journal 277 (2010) 989–999 ª 2010 The Authors Journal compilation ª 2010 FEBS 989 Vitamin D induces apoptosis through PTEN L Pan et al phosphorus metabolism in normal development Earlier studies revealed that VD3 could induce differentiation 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 chromosome 10 (PTEN) was originally discovered in 1997 [9–11] PTEN is a negative regulator of the ubiquitous phosphatidylinositol 3-kinase pathway, and it is frequently subject to loss of heterozygosity in various human tumors, including brain, bladder, prostate and endometrial cancers [9,10] Expression of PTEN is regulated by transcription factors such as p53 [12], peroxisome proliferator-activated receptor c [13], early growth response gene (Egr-1) [14], nuclear factor-jB [15,16], and transforming growth factor-b [11] Moreover, functional vitamin D receptor (VDR) elements have been identified in the promoter of PTEN, suggesting that vitamin D may play a role in the regulation of PTEN expression [17] Epigenetic changes such as DNA methylation and histone modifications play important roles in silencing tumor suppressor genes During 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¢-deoxycytidine (5-Aza) [26] Earlier studies showed that treatment with 5-Aza reduced DNA methylation and subsequently upregulated PTEN expression in acute lymphoblastic leukemia, melanoma and ovarian cancer cells [27–29] In gastric cancer cells, treatment with 5-Aza can greatly enhance PTEN expression [30] It was also reported that use of 5-Aza in combination 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 chemotherapy [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 investigate 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 gas990 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 alterations in gastric cancer cells Overall, data arising from this study have provided the basis for the further investigations into the potential application of vitamin 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 calcium 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 apoptosis ⁄ 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 cancer [35], we speculated that PTEN might participate in the VD3-induced apoptosis in gastric cancer cells To investigate this, undifferentiated HGC-27 adenocarcinoma 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 further establish the functional role of PTEN in VD3induced apoptosis, we constructed a stably transfected cell line in which PTEN was knocked down by specific small interfering RNA (siRNA) The inhibitory efficiency 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 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 siRNA cells (Fig 1D) These results suggested that PTEN FEBS Journal 277 (2010) 989–999 ª 2010 The Authors Journal compilation ª 2010 FEBS L Pan et al Vitamin D induces apoptosis through PTEN 10 B1 102 1.46% PI 10 Control 10 B3 B4 10 10 10 Annexin V-FITC 1.68% 10 10 10 102 Annexin V-FITC ** 20 103 Control siRNA 103 B1 10 30 10 PTEN siRNA 10 0.68% 3.05% B4 10 Control 10 VD3 B1 3.94% 10 10 10 Annexin V-FITC B2 1.8 α-PTEN α-actin Control VD3 101 10 10 2.09% Control B4 10 3.26% 0.94% PI B2 1.11% 100 B3 B1 101 10 B3 3 10 B2 Control α-PTEN α-actin 5.87% PI Relative mRNA level B4 D 40 0.5 0.3 Control #1 #2 VD3 B 5.55% 10 B3 1.03% B2 102 B1 1.31% 10 10 10 Annexin V-FITC B2 103 3.16% 0.53% PI PI 102 0.73% 10 C 103 B1 B2 PI A3 101 VD3 B3 10 B4 6.06% 100 101 102 Annexin V-FITC 103 VD3 B3 B4 2.12% 102 10 10 Annexin V-FITC 10 Fig Vitamin D induced apoptosis in gastric cancer cells through PTEN HGC-27 gastric cancer cells were treated with VD3 (10)7 M) 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 was treated with VD3, and apoptosis was evaluated (D) *P < 0.05, **P < 0.01, (n = 3) participated in the VD3-induced apoptosis in gastric cancer cells indicated that vitamin D induced PTEN expression through binding of VDR to the PTEN promoter Vitamin D upregulated PTEN through VDR VDR synergistically activated PTEN with Egr-1 and p300 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 upregulated PTEN mRNA expression as much as 13-fold (Fig 2B) As can be seen in Fig 2C, the PTEN protein 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 HGC27 cells (Fig 2D) The ChIP data clearly demonstrated the binding of VDR to the PTEN promoter, especially on the RE B region (Fig 2E) These data In a previous study, we showed that histone acetyltransferase p300 activated PTEN expression in synergy with the transcriptional factor Egr-1 [36] To clarify the relationships among VDR, Egr-1 and p300 in regulating PTEN expression, we transiently transfected 293T cells with VDR, Egr-1 and p300, either alone or in combination, before the expression of PTEN was estimated 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 ⁄ Egr-1, VDR ⁄ p300, Egr-1 ⁄ p300 and VDR ⁄ Egr1 ⁄ 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 FEBS Journal 277 (2010) 989–999 ª 2010 The Authors Journal compilation ª 2010 FEBS 991 Vitamin D induces apoptosis through PTEN A B C 16 24 14 Relative mRNA level 28 Relative luc assay L Pan et al 20 16 12 pc3.1 VDR ** 1.1 1.6 1.3 12 α-PTEN 10 α-VDR α-actin pc3.1 VDR pc3.1 VDR Control VD3 Fig Vitamin D upregulated PTEN through its receptor VDR HGC-27 cells were transiently transfected for 30 h with the expression 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 western blotting (C) The diagram of PTEN promoter and the regions amplified by PCR in ChIP assays are shown (D) HGC-27 cells were collected and precipitated with antibodies against VDR after VD3 treatment, and ChIP assays were performed to examine the binding of VDR on PTEN promoter (E) IP, immunoprecipitation *P < 0.05, **P < 0.01, (n = 3) VDR pc3.1 Control VD3 D ATG RE A –1703 E RE B –1477 RE C –920 –784 –413 –156 Control Vitamin D IP/input RE A RE B α-VDR RE C blotting (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 functional interactions among VDR, Egr-1 and p300 in their synergistic action We performed coimmunoprecipitation (CoIP) assays after VDR, Egr-1 and p300 transfection We precipitated cell lysates with anti- A 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 B pc3.1 VDR Egr-1 Egr-1 p300 p300 * VDR + Egr-1 VDR + Egr-1 ** VDR + p300 VDR + p300 ** Egr-1 + p300 Egr-1 + p300 * VDR + Egr-1 + p300 ** VDR + Egr-1 + p300 20 40 60 80 600 700 800 Relative luc assay C pc3.1 VDR * 2.5 1.1 1.3 1.7 1.9 1.9 2.0 VDR – + – – + + + – Egr-1 – + + – – + – + p300 – + – + – – + ** 10 15 20 25 30 Relative mRNA level 35 D α-PTEN α-actin IP: IgG VDR IgG Egr-1 IgG p300 input WB: Egr-1 WB: VDR WB: p300 + Fig 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) 992 FEBS Journal 277 (2010) 989–999 ª 2010 The Authors Journal compilation ª 2010 FEBS L Pan et al Vitamin D induces apoptosis through PTEN B ** 120 Relative mRNA level Relative mRNA level A 80 40 Control TSA 1.1 800 ** 600 ** 400 90 60 30 VD3 TSA + VD3 Control NaBu 1.3 1.1 1.4 VD3 NaBu + VD3 1.1 1.6 α-PTEN α-actin 6.58% 102 100 B3 B4 8.84% 100 101 102 Annexin V-FITC 103 B1 103 16.31% 10 103 103 6.35% 4.71% 10 101 B4 100 B3 25.09% 100 101 102 Annexin V-FITC 103 VD 7.09% 1.53% 101 NaBu 100 15.65% 102 B2 3.51% 6.63% 101 100 B3 24.12% 102 101 100 Annexin V-FITC 103 N + VD3 N + VD3 103 5.59% 100 101 102 Annexin V-FITC 103 B1 101 B4 B4 B3 103 34.20% 1.91% 100 B3 1.55% 101 102 100 Annexin V-FITC 102 B2 B1 T + VD3 B4 VD ntr ol B4 100 101 102 Annexin V-FITC B2 T + VD3 100 B3 101 100 B3 PI 101 7.79% 0.86% B2 NaBu 2.65% 100 101 102 Annexin V-FITC 103 B1 B2 0.60% B4 PTEN siRNA 103 B1 B2 TSA PI 100 B3 Co Bu 102 101 10 TSA PI Na VD 4.39% 1.91% PI 102 Control siRNA 103 B1 B2 PI 0.79% D PTEN siRNA PI 103 B1 B2 PI PI 102 Control siRNA N+ C 103 B1 TS A T+ Co n VD tro l α-PTEN α-actin 103 B4 5.84% 102 100 101 Annexin V-FITC 103 Fig Vitamin D enhanced the apoptosis induced by TSA ⁄ NaBu HGC-27 gastric cancer cells were treated with VD3 and 200 nM TSA (A), or VD3 and mM 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) Vitamin 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 apoptosis 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 FEBS Journal 277 (2010) 989–999 ª 2010 The Authors Journal compilation ª 2010 FEBS 993 Vitamin D induces apoptosis through PTEN Control TSA * 10 C * PTEN/GAPDH PTEN/GAPDH 20 Control TSA 15 10 * * RE A RE B Ac-H3 RE C 2.0 * 1.6 1.2 0.8 0.4 0.0 RE A RE B Ac-H4 16 12 Control TSA Egr-1 RE C 2.7 20 15 10 ** RE A RE B Ac-H3 RE C ** 2.5 * ** RE C 1.0 0.5 RE B Ac-H4 1.5 * ** RE A Control NaBu 2.0 Control TSA Control NaBu Control NaBu IP/input * PTEN/GAPDH PTEN/GAPDH 25 10 IP/input Control NaBu 1.5 α-Egr-1 α-actin Control TSA D ** NaBu Control Egr-1 α-Egr-1 α-actin B 30 * 20 Relative mRNA level 25 Relative mRNA level A L Pan et al RE A RE B α-Egr-1 RE C 0.0 RE A RE B α-Egr-1 RE C Fig 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) TSA ⁄ 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 treatments 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 994 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 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 presented above had revealed that the histone acetylation 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 epigenetic mechanisms such as acetylation modification play an important role in the regulation of PTEN expression FEBS Journal 277 (2010) 989–999 ª 2010 The Authors Journal compilation ª 2010 FEBS L Pan et al Vitamin D induces apoptosis through PTEN B103 ** 10 10 10 B2 Control siRNA B1 ** PI 10 5-Aza B3 ** 10 VD3 5-Aza + VD3 Control 5-Aza 103 B1 PTEN 1.1 102 1.9 5- 10 10 102 Annexin V-FITC 17.74% 10 10 B3 10 B4 1.72% B1 10 100 102 Annexin V-FITC B2 3.94% 10 7.59% B3 B4 2.44% 100 10 102 Annexin V-FITC 10 Control 5-Aza * 35 5-Aza B3 + VD3 26.45% 102 10 Annexin V-FITC 4.88% 10 B4 PTEN siRNA B2 + VD3 40 B2 D * 10 10 0.57% 100 C 30 IP/input 0 B1 11.60% 10 A VD za + VD A za 5- C on tro l α-PTEN α-actin PI 1.3 10 B4 102 11.70% 10.93% 1.87% PI 40 30 20 10 10 120 Relative mRNA level Fig Vitamin D promoted the apoptosis induced by 5-Aza HGC-27 cells were treated with VD3 and 5-Aza (5 lM), and western blotting was used to assess PTEN expression (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) 160 PI Relative mRNA level A α-Egr-1 α-actin Control 5-Aza We then investigated the effect of PTEN on the apoptosis 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 upregulated by over 100-fold after combined treatment with 5-Aza and VD3 (Fig 6A) Meanwhile, the early apoptosis 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 lymphoblastic leukemia cells [27] To evaluate the contribution of 5Aza 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 15 * 1.6 Vitamin D promoted the apoptosis induced by 5-Aza 20 10 Control 5-Aza Egr-1 25 * RE A RE B α-Egr-1 RE C PTEN promoter was increased after treatment with 5Aza (Fig 6D) Thus, we concluded that PTEN participated in vitamin D-induced apoptosis, and epigenetic modifications such as acetylation and DNA methylation were involved in the regulation of PTEN, which subsequently 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 cellular 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 Iday)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 mechanisms, of the combined use of VD3 and TSA ⁄ NaBu or FEBS Journal 277 (2010) 989–999 ª 2010 The Authors Journal compilation ª 2010 FEBS 995 Vitamin D induces apoptosis through PTEN L Pan et al 5-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 formation 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 toxicities, 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 also show correlations between VDR and Egr-1, as well as between VDR and histone acetyltransferase p300 Further investigations revealed that both TSA ⁄ NaBu and 5-Aza were able to significantly enhance the apoptosis induced by VD3 through PTEN (Figs and 6), and that the epigenetic modifications played important roles in monitoring the expression of PTEN (Figs 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 epigenetic modification agents such as TSA ⁄ NaBu and 5Aza, which either directly, or through transcription factor Egr-1, influence the expression of PTEN Potentially, our work may be useful as a clue for the development 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 996 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 purchased from the Institute of Cell Biology, Shanghai, China Cells were cultured in IMDM supplemented with 10% fetal bovine serum, 100 mL)1 penicillin and 100 lgỈmL)1 streptomycin, and kept in a humidified atmosphere of 5% CO2 Stably transfected cell lines were maintained in IMDM supplemented with 10% fetal bovine serum in the presence of G418 (1000 lgỈmL)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 (exitation at 488 nm; emission at 530 nm) with an FITC signal detector, and prodium iodide (PI) staining with a phycoerythrin 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 harvest, washed with NaCl ⁄ Pi, and lysed in 30 lL of lysis buffer Reporter gene expression was measured and quantified using a dual Luciferase Reporter Assay System (Promega, Madison, WI, USA) Relative luciferase activity was analyzed by using a Turner Designs TD20 ⁄ 20 Luminometer (Sunnyvale, CA , USA) Firefly luciferase activity was normalized to the activity of the Renilla luciferase control Extracts from at least three independent transfection experiments 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 FEBS Journal 277 (2010) 989–999 ª 2010 The Authors Journal compilation ª 2010 FEBS L Pan et al was 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 primers 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)7 m), 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, mm EDTA, and mm phenylmethanesulfonyl fluoride) Cell lysates were separated by SDS ⁄ PAGE in 15% gels, then transferred to poly(vinylidene difluoride) membranes (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 substrate method with the SuperSignal West Pico kit provided by Pierce Co (Rockford, IL, USA) Coprecipitation of VDR with Egr-1 and p300 was performed 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 maintained in IMDM supplemented with 10% fetal bovine serum, 100 mgỈmL)1 penicillin, 100 mgỈmL)1 streptomycin, and 1000 lgỈmL)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 · 105 ⁄ mL in six-well plates and cultured for 24 h After treatments with TSA ⁄ NaBu or 5-Aza, cells were crosslinked in 2% formaldehyde for 10 at Vitamin D induces apoptosis through PTEN 37 °C, 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 Biotechnology, Lake Placid, NY, USA) Antibodies against Ac-H3 and Ac-H4 were purchased from Upstate Antibody against Egr-1 was purchased from Santa Cruz Immunoprecipitated 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 significance of the experimental data The level of significance 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 Universities (IRT0519), and The National Natural Science Foundation of China (30771232 and 30671184) References Peek RM Jr & Blaser MJ (2002) Helicobacter pylori and 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Simmen RC (2005) The soy isoflavone genistein promotes apoptosis in mammary epithelial cells by inducing the tumor suppressor PTEN Carcinogenesis 26, 1793–1803 43 Tell G, Pines A, Arturi F, Cesaratto L, Adamson E, Puppin C, Presta I, Russo D, Filetti S & Damante G (2004) Control of phosphatase and tensin homolog (PTEN) gene expression in normal and neoplastic zthyroid cells Endocrinology 145, 4660–4666 44 Livak KJ & Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method Methods 25, 402– 408 45 Han S, Lu J, Zhang Y, Cheng C, Han L, Wang X, Li L, Liu C & Huang B (2006) Recruitment of histone deacetylase by transcription factors represses interleukin-5 transcription Biochem J 400, 439–448 Supporting information The following supplementary material is available: Fig S1 Vitamin D enhanced the apoptosis induced by the histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate (NaBu), and the methylation inhibitor 5-aza-2¢-deoxycytidine (5-Aza) Fig S2 Vitamin D induced apoptosis in gastric cancer cells Fig S3 Dose-dependent experiments with different agents This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 989–999 ª 2010 The Authors Journal compilation ª 2010 FEBS 999 ... was treated with VD3, and apoptosis was evaluated (D) *P < 0.05, **P < 0.01, (n = 3) participated in the VD3-induced apoptosis in gastric cancer cells indicated that vitamin D induced PTEN expression... deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate (NaBu), and the methylation inhibitor 5-aza-2¢-deoxycytidine (5-Aza) Fig S2 Vitamin D induced apoptosis in gastric cancer cells. .. α-actin Control 5-Aza We then investigated the effect of PTEN on the apoptosis induced by DNA methylation and vitamin D in gastric cancer cells We treated HGC-27 cells with 5-Aza and VD3, either alone