An updated overview of some factors that influence the biological effects of nanoparticles.

Nanoparticles (NPs) can be extremely effective in the early diagnosis and treatment of cancer due to their properties. The nanotechnology industry is developing rapidly. The number of multifunctional NPs has increased in the market and hundreds of NPs are in various stages of preclinical and clinical development. Thus, the mechanism underlying the effects of NPs on biological systems has received much attention. After NPs enter the body, they interact with plasma proteins, tumour cell receptors, and small biological molecules. This interaction is closely related to the size, shape, chemical composition and surface modification properties of NPs. In this review, the effects of the size, shape, chemical composition and surface modification of NPs on the biological effects of NPs were summarised, including the mechanism through which NPs enter cells, the resulting oxidative stress response, and the interaction with proteins. This review of the biological effects of NPs can not only provide theoretical support for the preparation of safer and more efficient NPs but also lay the foundation for their clinical application.

Molecular and clinical characteristics of IDH mutations in Chinese NSCLC patients and potential treatment strategies.

BACKGROUND: Isocitrate dehydrogenase (IDH) is an appealing target for anticancer therapy, and IDH (IDH1/2) inhibitors have been approved for targeted therapy of acute myeloid leukemia (AML) and Cholangiocarcinoma. The therapeutic potential of IDH inhibitors for non-small-cell lung cancer (NSCLC) patients is under active clinical investigation. Thus, it would be necessary and meaningful to study the molecular and clinical characteristics of IDH mutation in NSCLC patients, especially in the Chinese population. METHODS: A total of 17,978 Chinese patients with NSCLC who underwent next -generation sequencing (NGS) testing were retrospectively reviewed. RESULTS: We identified 161 unique IDH mutations in 361 of 17,978 patients (2.01%). Common active-site mutations, including IDH1R100 , IDH1R132 , IDH2R140 , and IDH2R172 , were detected in 154 patients (0.86%) and were associated with male sex (p = 0.004) and older age (p = 0.02). The IDH mutation spectra observed in NSCLC were quite different from those in glioma or AML. Patients with IDH active-site mutations exhibited significantly higher coalterations in KRAS (p. G12/13/61, 22.1% vs. 8.2%, p < 0.001) or BRAF (p. V600E, 6.5% vs. 1.0%, p < 0.001), but significantly lower coalterations in activating EGFR (e18-e20, 22.7 vs. 37.9%, p < 0.001) than IDH wild-type patients. Furthermore, we found that active-site IDH mutations were correlated with a short PFS (2-5.6 months) and short OS (2-9.5 months), which may arise as a resistance mechanism against common targeted drugs. In vitro, we experimentally observed that the combination of an IDH inhibitor and EGFR TKI could better inhibit lung cancer cell proliferation than an EGFR TKI alone. CONCLUSIONS: Taken together, this study reveals the molecular and clinical characteristics of IDH mutations in Chinese NSCLC patients and provides a theoretical basis for IDH-directed treatment. The potential of IDH mutations as response markers for targeted therapy warrants further investigation.

[Corrigendum] The Pleckstrin and Sec7 domain-containing gene as a novel epigenetic modification marker in human gastric cancer and its clinical significance.

Subsequently to the publication of the above article, an interested reader drew to the authors' attention that a pair of panels in Fig. 6C appeared to contain overlapping data, such that the data, which were intended to show the results from experiments performed under different experimental conditions, may have been derived from the same original source. The authors have re-examined their original data, and have realized that this figure was assembled incorrectly; essentially, the data panel showing the results of the SGC7901-Si-NC experiment in Fig. 6C was incorporated incorrectly during the process of assembling this figure. The corrected version of Fig. 6, showing all the correct data for Fig. 6C, is shown opposite. The authors confirm that this inadvertent error did not have any major impact on the conclusions reported in their paper, and are grateful to the Editor of International Journal of Oncology for allowing them this opportunity to publish a Corrigendum. Furthermore, the authors apologize to the readership for any inconvenience caused. [the original article was published in International Journal of Oncology 46: 195-204, 2015; DOI: 10.3892/ijo.2014.2736].

SAMD14 promoter methylation is strongly associated with gene expression and poor prognosis in gastric cancer.

BACKGROUND: Gastric cancer (GC) is the fifth most common malignancy worldwide and the third leading cause of cancer-related mortality. In recent years, SAMD14 has been studied in various malignant cancers; however, little is known about the exact mechanisms of SAMD14 involvement in carcinogenesis and malignant progression. METHODS: 60 paired GC-normal gastric tissues were evaluated for their SAMD14 mRNA expression in relation to SAMD14 gene promoter methylation. GC patient survival was assessed by Kaplan-Meier analyses and a Cox's proportional hazard model was employed for multivariate analyses. RESULTS: SAMD14 expression was significantly inversely correlated with the Borrmann type (P = 0.017), lymph node metastasis (P = 0.006) and tumor-node-metastasis (TNM) stage (P = 0.033). Methylation-specific PCR (MSP) revealed hyper-methylation of the SAMD14 promoter in 56.7% (34/60) of the primary GC tissues tested and in 10% (6/60) of matched non-malignant tissues. The SAMD14 promoter methylation status was also related to pathological differentiation, Borrmann type, TNM stage and lymph node metastasis. The results showed SAMD14 expression was significantly downregulated in Borrmann type, lymph node metastasis and TNM stage, which showed significantly higher methylation. SAMD14 promoter hyper-methylation was significantly associated with a poor prognosis and could serve as an independent marker for survival using multivariate Cox regression analysis. CONCLUSIONS: Our results indicated that promoter methylation was a key mechanism contributing to the downregulation of SAMD14 in GC. SAMD14 may be an epigenetically silenced tumor suppressor gene, and hyper-methylation of the SAMD14 promoter may serve as a biomarker to predict the clinical outcome of GC.

Osthole induces cell cycle arrest and apoptosis in head and neck squamous cell carcinoma by suppressing the PI3K/AKT signaling pathway.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most common lethal tumors with a high recurrence rate and low survival rate. Therefore, an urgent need exists for novel and effective treatment strategies for HNSCC patients. METHODS: Osthole, a natural ingredient extracted from Cnidium monnieri (L.) 'Cusson', has multiple pharmacological effects including antineoplastic activity. Regrettably, the antineoplastic effect of osthole in HNSCC cells remains undefined. We utilize in vitro assays to assess the anti-proliferative effects of osthole in HNSCC cells and tumorigenesis assays using FaDu cells in murine HNSCC models to assess in vivo function. Moreover, the possible molecular mechanisms of Osthole on HNSCC cells was also investigated. RESULTS: Our findings show that the anti-proliferation effect of osthole might function through induction of cell cycle arrest (G2/M phase) and apoptosis in HNSCC. Osthole could also down-regulating the protein level of cell cycle and apoptosis related proteins, such as Bcl-2, PARP1, Survivin, CyclinB1 and Cdc2, while up-regulating expression of Cleaved Caspase3/9, Cleaved PARP1 and Bax. Similarly, osthole suppressed the in vivo growth of FaDu cells in a subcutaneous tumor model. In terms of mechanism, our data show that osthole can suppress the PI3K/AKT pathway. CONCLUSIONS: In the current study, our in vitro and in vivo assay showed the suppressive effect of Osthole on HNSCC cells through induce cell cycle arrest (G2/M phase) and apoptosis. Moreover, the action mechanisms of Osthole on proliferation related signaling pathways was disclosed. Our present study suggests that osthole might be used as an effective therapeutic agent for patients with HNSCC.

Downregulation of A disintegrin and metallopeptidase with thrombospondin motif type 1 by DNA hypermethylation in human gastric cancer.

A disintegrin and metallopeptidase with thrombospondin motif type 1 (ADAMTS1) is a metalloproteinase with antiangiogenic activity. It was previously observed that the mRNA and protein levels of ADAMTS1 are downregulated in primary gastric tumors. The aim of the present study was to examine whether the reduction in the expression of ADAMTS1 is due to aberrant methylation of the gene in primary gastric tumor tissues and gastric cancer cell lines. In addition, the association between ADAMTS1 methylation and clinicopathological features in were investigated in patients with primary gastric cancer. The results revealed that the frequency of ADAMTS1 methylation in primary gastric tumor tissues was significantly higher, compared with the corresponding normal gastric tissues. The relative mRNA expression levels of ADAMTS1 were significantly lower in the methylated primary gastric tumor tissues, compared with the unmethylated primary gastric tumor tissues. A significant association was observed between the ADAMTS1 methylation status and the depth of tumor invasion and tumor, node, metastasis stage in primary gastric cancer. The mRNA expression of ADAMTS1 was significantly lower in 60% (3 of 5) of the gastric cancer cell lines. The relative mRNA expression levels of ADAMTS1 were significantly lower in the methylated gastric cancer cell lines, compared with the unmethylated gastric cancer cell lines. Furthermore, the expression of ADAMTS1 was significantly restored following treatment with the 5-Aza-2'-deoxycytidine demethylating agent in the MGC-803, HGC-27 and AGS gastric cancer cell lines, and the demethylation of the MGC-803 cell line inhibited cell invasion. Together, these results suggested for the first time, to the best of our knowledge, ADAMTS1 as a novel antitumor protease, and this function was lost following epigenetic silencing in the gastric cancer cells and gastric tumor tissues. Therefore, the aberrant methylation of ADAMTS1 may be involved in the development and progression of gastric cancer.

Aberrant SOX11 promoter methylation is associated with poor prognosis in gastric cancer.

BACKGROUND: Gastric cancer (GC) is the second most common cause of cancer mortality world-wide. In recent years, aberrant SOX11 expression has been observed in various solid and hematopoietic malignancies, including GC. In addition, it has been reported that SOX11 expression may serve as an independent prognostic factor for the survival of GC patients. Here, we assessed the SOX11 gene promoter methylation status in various GC cell lines and primary GC tissues, and evaluated its clinical significance. METHODS: Five GC cell lines were used to assess SOX11 expression by qRT-PCR. The effect of SOX11 expression restoration after 5-aza-2'-deoxycytidine (5-Aza-dC) treatment on GC growth was evaluated in GC cell line MKN45. Subsequently, 89 paired GC-normal gastric tissues were evaluated for their SOX11 gene promoter methylation status using methylation-specific PCR (MSP), and 20 paired GC-normal gastric tissues were evaluated for their SOX11 expression in relation to SOX11 gene promoter methylation. GC patient survival was assessed by Kaplan-Meier analyses and a Cox proportional hazard model was employed for multivariate analyses. RESULTS: Down-regulation of SOX11 mRNA expression was observed in both GC cell lines and primary GC tissues. MSP revealed hyper-methylation of the SOX11 gene promoter in 55.1% (49/89) of the primary GC tissues tested and in 7.9% (7/89) of its corresponding non-malignant tissues. The SOX11 gene promoter methylation status was found to be related to the depth of GC tumor invasion, Borrmann classification and GC differentiation status. Upon 5-Aza-dC treatment, SOX11 expression was found to be up-regulated in MKN45 cells, in conjunction with proliferation inhibition. SOX11 gene promoter hyper-methylation was found to be significantly associated with a poor prognosis and to serve as an independent marker for survival using multivariate Cox regression analysis. CONCLUSIONS: Our results indicate that aberrant SOX11 gene promoter methylation may underlie its down-regulation in GC. SOX11 gene promoter hyper-methylation may serve as a biomarker to predict the clinical outcome of GC.

Genome-wide analysis of histone modifications by ChIP-chip to identify silenced genes in gastric cancer.

The present study aimed to identify novel histone modification markers in gastric cancer (GC) by chromatin immunoprecipitation microarray (ChIP-chip) analysis and to determine whether these markers were able to discriminate between normal and GC cells. We also tested for correlations with DNA methylation. We probed a human CpG island microarray with DNA from a GC cell line (MKN45) by chromatin immunoprecipitation (ChIP). ChIP-reverse-transcriptase quantitative polymerase chain reaction PCR (RT-qPCR) was used to validate the microarray results. Additionally, mRNA expression levels and the DNA methylation of potential target genes were evaluated by RT-qPCR and methylation-specific PCR (MSP). The moults showed that 134 genes exhibited the highest signal-to-noise ratio of H3-K9 trimethylation over acetylation and 46 genes exhibited the highest signal-to-noise ratio of H3-K9 trimethylation over H3-K4 trimethylation in MKN45 cells. The ChIP-qPCR results agreed with those obtained from the ChIP-chip analysis. Aberrant DNA methylation status and mRNA expression levels were also identified for selected genes (PSD, SMARCC1 and Vps37A) in the GC cell lines. The results suggest that CpG island microarray coupled with ChIP (ChIP-chip) can identify novel targets of gene silencing in GC. Additionally, ChIP-chip is the best approach for assessing the genome-wide status of epigenetic regulation, which may allow for a broader genomic understanding compared to the knowledge that has been accumulated from single-gene studies.

The Pleckstrin and Sec7 domain-containing gene as a novel epigenetic modification marker in human gastric cancer and its clinical significance.

The Pleckstrin and Sec7 domain-containing (PSD) gene has been recently found to participate in the progression of several types of cancer. In the present study, we identified PSD as a candidate tumor suppressor gene silenced through epigenetic modification in gastric cancer (GC). PSD mRNA expression and DNA methylation were evaluated by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and methylation-specific PCR in GC cell lines and tissue samples. Involvement of histone modification in GC cell lines was examined by chromatin immunoprecipitation assay. We also used an siRNA-mediated approach to knock down the PSD gene in SGC7901 cells, which was utilized to detect the role of PSD in GC progression, followed by analysis of cell apoptosis and invasion. PSD gene expression was reduced in all GC cell lines compared with GES1 (an immortalized normal gastric cell line). In addition, PSD expression was markedly downregulated in gastric carcinoma tissues when compared to adjacent non-tumor tissues. Our data also indicated that PSD mRNA and protein levels were associated with tumor differentiation and lymph node metastasis. Aberrant DNA methylation status and histone modification were also found in GC cell lines. Enhanced gene expression was detected when the HGC27, AGS and BGC823 GC cell lines were treated with the DNA-demethylating agent 5-aza-2'-deoxycytidine. However, treatment with trichostatin A, a histone deacetylase inhibitor, had no effect on PSD expression in any of the GC cell lines. Suppression of PSD by siRNA led to enhanced SGC7901 cell invasion. The depletion of PSD expression inhibited cell proliferation and decreased apoptosis in SGC7901 cell lines. Knockdown of the PSD expression decreased caspase-3 and -7 protein levels in SGC7901 cells. PSD gene may function as a tumor suppressor in GC suggesting a vital role for DNA methylation and histone modification in PSD silencing. PSD expression might be a useful biomarker for epigenetic-based GC early diagnosis and may lead to the identification of new targets for pharmacological intervention.

Novel tumor-suppressor gene epidermal growth factor-containing fibulin-like extracellular matrix protein 1 is epigenetically silenced and associated with invasion and metastasis in human gastric cancer.

The present study aimed to investigate the role of histone modification and DNA methylation in epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) silencing in gastric cancer (GC). In the present study, four GC cell lines, and 45 paired normal and GC tissue samples were used to assess EFEMP1 expression using quantitative polymerase chain reaction (PCR), and EFEMP1 gene methylation status was evaluated by methylation-specific PCR. The involvement of histone modification in GC cell lines was examined by a chromatin immunoprecipitation (ChIP) assay. The results demonstrated that EFEMP1 mRNA level and methylation status in the EFEMP1 promoter region was associated with tumor differentiation, depth of tumor invasion and lymph node metastasis. DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) rapidly reduced DNA methylation and histone H3-K9 trimethylation at the silenced loci and reactivated EFEMP1 expression. By contrast, the histone deacetylase inhibitor trichostatin A markedly increased histone H3-K9 acetylation. However, it had no effect on DNA methylation, histone H3-K9 trimethylation or gene expression. In conclusion, the results suggested that EFEMP1 may function as a tumor suppressor in GC. Aberrant DNA methylation and histone H3-K9 trimethylation of EFEMP1 may be responsible for its downregulation in GC, and thus have an important role in tumor invasion and metastasis.

DNA promoter and histone H3 methylation downregulate NGX6 in gastric cancer cells.

Nasopharyngeal carcinoma-associated gene 6 (NGX6) is a novel candidate tumor metastasis suppressor gene. Our study was to determine whether DNA hypermethylation and histone modification at the NGX6 gene promoter play important roles in silencing NGX6 expression in gastric cancer. NGX6 expression was downregulated in all gastric cancer cells and 76.19 % tissues. In three GC cell lines, hypermethylated NGX6 loci were characterized by histone H3-K9 hypoacetylation and hypermethylation. Trichostatin A treatment could moderately increase H3-K9 acetylation at the silenced loci; however, it had no effect on DNA and H3-K9 methylation and minimal effects on NGX6 expression. In contrast, 5'aza-2'-deoxycytidine treatment could rapidly decrease DNA and H3-K9 methylation at the silenced loci, leading to the reexpression of NGX6. Combined treatment with 5'aza-2'-deoxycytidine and trichostatin A had synergistic effects on the reexpression of NGX6 at the hypermethylation loci. Our current study shows that NGX6 expression is downregulated in GC cancer cells and tissues due to NGX6 promoter methylation and H3-K9 methylation, but not H3-K9 acetylation. Our findings indicate that the downregulation of NGX6 expression contributes to the development and progression of gastric cancer. More studies are needed to determine the precise mechanism of NGX6 in the progression of gastric cancer.

Role of histone modifications and DNA methylation in the regulation of O6-methylguanine-DNA methyltransferase gene expression in human stomach cancer cells.

To determine a possible function of histone modifications in stomach carcinogenesis, we analyzed global and MGMT-promoter levels of di-methyl-H3-K9, di-methyl-H3-K4 and acetyl-H3-K9, as well as MGMT DNA methylation and mRNA expression following treatment with 5-aza-2' -deoxycytidine and/or Trichostatin A. We found that histone H3-K9 di-methylation, H3-K4 di-methylation, H3-K9 acetylation and DNA methylation work in combination to silence MGMT. The results indicate that histone modifications as well as DNA methylation may be involved in stomach carcinogenesis. In addition to its effect on DNA methylation, 5-aza-2' -deoxycytidine can act at histone modification level to reactivate MGMT expression in a region-specific and DNA methylation-dependent manner.

Promoter histone H3 lysine 9 di-methylation is associated with DNA methylation and aberrant expression of p16 in gastric cancer cells.

In the course of gastric cancer development, gene silencing by DNA hypermethylation is an important mechanism. While DNA methylation often co-exists with histone modifications to regulate gene expression, the function of histone modifications in gene silencing in gastric cancer has not been evaluated in detail. p16, a well-known tumor suppressor gene, is frequently silenced in DNA hypermethylation manner in gastric cancer. Accordingly, we chose p16 to clarify whether there is a correlation among histone H3 lysine 9 (H3-K9) di-methylation, H3-K9 acetylation, DNA methylation and p16 expression in human gastric cancer. Three gastric cancer cells, MKN-45, SGC-7901 and BGC-823, were treated with 5-aza-2'-deoxycytidine (5-Aza-dC) and/or trichostatin A (TSA). We investigated p16 promoter DNA methylation status, p16 mRNA levels, regional and global levels of di-methyl-H3-K9 and acetyl-H3-K9 in four groups: i) 5-Aza-dC, ii) TSA, iii) the combination of 5-Aza-dC and TSA and iv) control group with no treatments. p16 silencing is characterized by DNA hypermethylation, H3-K9 hypoacetylation and H3-K9 hypermethylation at the promoter region. Treatment with TSA, increased H3-K9 acetylation at the hypermethylated promoter, but did not affect H3-K9 di-methylation or p16 expression. By contrast, treatment with 5-Aza-dC, reduced H3-K9 di-methylation, increased H3-K9 acetylation at the hypermethylated promoter and reactivated the expression of p16. Combined treatment restored the expression of p16 synergistically. In addition, 5-Aza-dC and the combined treatment did not result in global alteration of H3-K9 di-methylation. These results suggest that H3-K9 di-methylation, H3-K9 acetylation and DNA methylation work in combination to silence p16 in gastric cancer. The decreased H3-K9 di-methylation correlates with DNA demethylation and reactivation of p16. H3-K9 di-methylation as well as DNA methylation related to p16 silencing is limited to the promoter region. In addition to its effect on DNA methylation, 5-Aza-dC can act at histone modification levels to reactivate p16 expression in region-specific and DNA methylation-dependent manner.

[Effects of 5-Aza-2'-deoxycytidine and trichostatin A on P16, hMLH1 and MGMT genes and DNA methylation in human gastric cancer cells].

OBJECTIVE: To investigate the effects of 5-Aza-2'-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) on DNA methylation and expression of P16, hMLH1 and MGMT genes in the human gastric cancer cell line MGC-803, and to explore the mechanism of P16, hMLH1 and MGMT gene silencing in human gastric cancer cells. METHODS: MGC-803 cells were cultured in RPMI-1640 medium and were treated with 5-Aza-dC or TSA. Methylation-specific polymerase chain reaction (MS-PCR) was used to detect the promoter methylation status of P16, hMLH1 and MGMT genes. RT-PCR was used to detect the mRNA expressions of P16, hMLH1 and MGMT. RESULTS: Promoter hypermethylation of P16, hMLH1 and MGMT genes were detected in MGC-803 cells, and mRNA expressions of P16, hMLH1 and MGMT were absent before treatment. After treatment with 5-Aza-dC, the promoter region of the P16, hMLH1 and MGMT gene exhibited a demethylation status, and their mRNA expressions were increased. The treatment with TSA had no effects on DNA demethylation or restoration of P16 or hMLH1 expression. P16, hMLH1 and MGMT mRNA relative expression levels after treatment with a combination of 5-Aza-dC and TSA were 0.412+/-0.030, 0.397+/-0.024 and 0.553+/-0.043 respectively, which were higher than those after 5-Aza-dC treatment alone (0.221+/-0.022, 0.214+/-0.018 and 0.156+/-0.017, all P<0.05). CONCLUSIONS: Promoter hypermethylation is a major mechanism of P16, hMLH1 and MGMT gene silencing in human gastric cancer cells. Treatment with 5-Aza-dC alone or the combination of 5-Aza-dC and TSA can reactivate the expressions of these genes.

[Histone H3 lysine 9 methylation is associated with the expression of hMLH1 and DNA methylation in gastric cancer cells].

OBJECTIVE: To identify the association of his tone H3 lysine 9 (H3-K9) methylation with DNA methylation and the expression of the mismatch repair gene hMLH1 in human gastric cancer cells. METHODS: Gastric cancer cells of the lines BGC-823 and MGC-803 were cultured and treated with 5-Aza-2'-deoxycytidine (5-Aza-dC), a demethylation agent, for 72 hour. Chromatin immunoprecipitation (ChIP) assay was used to assess the status of histone H3 lysine 9 methylation in the promoter regions of hMLH1 gene. Methylation-specific PCR (MSP) was used to evaluate the effect of 5-Aza-dC on DNA methylation status. RT-PCR was used to examine the hMLH1 gene expression. RESULTS: In the MGC-803 cells, silenced hMLH1 gene was characterized by DNA methylation and histone H3-K9 hypermethylation; 5-Aza-dC demethylated the DNA and reduced the histone H3-K9 methylation at silenced loci and resulted in reactivation of hMLH1 gene therein. Contrary to the MGC-803 cells, BGC-823 cells expressed hMLH1 gene with DNA demethylation and histone H3-K9 hypomethylation; and 5-Aza-dC had no effects on the gene expression, DNA methylation, and histone H3-K9 methylation therein. CONCLUSION: Hypermethylation of DNA in the promoter region is related to transcriptional silencing of hMLH1 gene. Histone H3-K9 methylation in different regions of the promoter studied correlates with DNA methylation status of hMLH1 gene in gastric cancer cells. Alteration of DNA methylation affects histone H3-K9 methylation. 5-Aza-dC can control hMLH1 expression, DNA methylation, and histone H3-K9 methylation in the promoter.

Re-expression of methylation-induced tumor suppressor gene silencing is associated with the state of histone modification in gastric cancer cell lines.

AIM: To identify the relationship between DNA hyper-methylation and histone modification at a hyperme-thylated, silenced tumor suppressor gene promoter in human gastric cancer cell lines and to elucidate whether alteration of DNA methylation could affect histone modification. METHODS: We used chromatin immunoprecipitation (ChIP) assay to assess the status of histone acetylation and methylation in promoter regions of the p16 and mutL homolog 1 (MLH1) genes in 2 gastric cancer cell lines, SGC-7901 and MGC-803. We used methylation-specific PCR (MSP) to evaluate the effect of 5-Aza-2'-deoxycytidine (5-Aza-dC), trichostatin A (TSA) or their combination treatment on DNA methylation status. We used RT-PCR to determine whether alterations of histone modification status after 5-Aza-dC and TSA treatment are reflected in gene expression. RESULTS: For the p16 and MLH1 genes in two cell lines, silenced loci associated with DNA hypermethylation were characterized by histone H3-K9 hypoacetylation and hypermethylation and histone H3-K4 hypomethylation. Treatment with TSA resulted in moderately increased histone H3-K9 acetylation at the silenced loci with no effect on histone H3-K9 methylation and minimal effects on gene expression. In contrast, treatment with 5-Aza-dC rapidly reduced histone H3-K9 methylation at the silenced loci and resulted in reactivation of the two genes. Combined treatment with 5-Aza-dC and TSA was synergistic in reactivating gene expression at the loci showing DNA hypermethylation. Similarly, histone H3-K4 methylation was not affected after TSA treatment, and increased moderately at the silenced loci after 5-Aza-dC treatment. CONCLUSION: Hypermethylation of DNA in promoter CpG islands is related to transcriptional silencing of tumor suppressor genes. Histone H3-K9 methylation in different regions of the promoters studied correlates with DNA methylation status of each gene in gastric cancer cells. However, histone H3-K9 acetylation and H3-K4 methylation inversely correlate with DNA methylation status of each gene in gastric cancer cells. Alteration of DNA methylation affects histone modification.