Frequent inactivation of SPARC by promoter hypermethylation in colon cancers

Epigenetic modification of gene expression plays an important role in the development of human cancers. The inactivation of SPARC through CpG island methylation was studied in colon cancers using oligonucleotide microarray analysis and methylation specific PCR (MSP). Gene expression of 7 colon cancer cell lines was evaluated before and after treatment with the demethylating agent 5-aza-2'-deoxycytidine (5Aza-dC) by oligonucleotide microarray analysis. Expression of SPARC was further examined in colon cancer cell lines and primary colorectal cancers, and the methylation status of the SPARC promoter was determined by MSP. SPARC expression was undetectable in 5 of 7 (71%) colorectal cancer cell lines. Induction of SPARC was demonstrated after treatment with the demethylating agent 5Aza-dC in 5 of the 7 cell lines. We examined the methylation status of the CpG island of SPARC in 7 colon cancer cell lines and in 20 test set of colon cancer tissues. MSP demonstrated hypermethylation of the CpG island of SPARC in 6 of 7 cell lines and in all 20 primary colon cancers, when compared with only 3 of 20 normal colon mucosa. Immunohistochemical analysis showed that SPARC expression was downregulated or absent in 17 of 20 colon cancers. A survival analysis of 292 validation set of colorectal carcinoma patients revealed a poorer prognosis for patients lacking SPARC expression than for patients with normal SPARC expression (56.79% vs. 75.83% 5-year survival rate, p = 0.0014). The results indicate that epigenetic gene silencing of SPARC is frequent in colon cancers, and that inactivation of SPARC is related to rapid progression of colon cancers.     

5-Aza-2'-deoxycytidine induces p21WAF expression by demethylation of p73 leading to p53-independent apoptosis in myeloid leukemia

The DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) has significant therapeutic value for the treatment of patients with myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The demethylating effect of 5-Aza-CdR has been well characterized. In contrast, less is known about the molecular events downstream of the methylation inhibition. Here, 5-Aza-CdR induced apoptosis in AML cells (both p53 mutant and wild-type) but not in epithelial or normal PBMCs. Cell death was accompanied by activation of the mitochondrial apoptosis pathway, as shown by release of cytochrome c and AIF and loss of mitochondrial membrane potential (DeltaPsim). Activation of caspase-3 (but not -6 and -8) was detectable using Western blot analysis and measurement of caspase enzymatic activity. 5-Aza-CdR treatment resulted in the induction of p21, which correlated with the arrest of AML cells in the G1 cell cycle phase. Induction of p21 expression was independent of its promoter methylation status but mediated by 5-Aza-CdR-induced reexpression of the tumor-suppressor p73, a known upstream regulator of p21. The p73 promoter was hypermethylated in AML cell lines and in primary AML cells but not in epithelial cells, which were resistant toward 5-Aza-CdR. Therefore, 5-Aza-CdR-mediated specific killing of myeloid cells might be dependent on its ability to revert p73 promoter methylation and to reexpress p73 mRNA. In addition, exogenous expression of p73 rendered epithelial cells sensitive to apoptosis induced by 5-Aza-CdR or other cytostatic drugs. We therefore conclude that p73 is a relevant target for methylation-dependent efficacy of 5-Aza-CdR in AML cells.     

胃和结直肠腺癌患者血清RASSF1A基因启动子异常甲基化检测及其临床意义

目的 研究胃和结直肠腺癌患者血清RASSF1A基因启动子区域的甲基化状态及其临床意义.方法采用甲基化特异性聚合酶链反应(MSP)技术,检测47例胃腺癌患者、45例结直肠腺癌患者、60例胃肠道良性病变患者及30例健康志愿者血清RASSF1A基因启动子区域的甲基化状态,并同时检测25例进行手术的胃腺癌、结直肠腺癌患者的肿瘤...     

Cancer testis antigens expression in mesothelioma: role of DNA methylation and bioimmunotherapeutic implications

Recent evidences suggest that malignant mesothelioma may be sensitive to immunotherapy; however, little is known about malignant mesothelioma-associated tumour antigens. Focusing on cancer/testis antigens, the expression of well-characterised immunogenic tumour-associated antigens was investigated in malignant mesothelioma cells. At variance with MAGE-4 and NY-ESO-1, malignant mesothelioma cells frequently expressed MAGE-1, -2 and -3, GAGE 1-2, GAGE 1-6, SSX-2 and SSX 1-5, and distinct malignant mesothelioma cells concomitantly expressed at least four cancer/testis antigens. Additionally, the tumour-associated antigens RAGE-1 was expressed at high levels in both benign and malignant mesothelial cells. Lastly, treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine induced and up-regulated the expression of the cancer/testis antigen examined in malignant mesothelioma cells. Overall, these findings strongly suggest that cancer/testis antigens-based immunotherapy may represent a suitable therapeutic approach to malignant mesothelioma, and foresee the clinical use of 5-aza-2'-deoxycytidine to design new chemo-immunotherapeutic strategies in malignant mesothelioma patients.     

The prognostic value of CDKN2A hypermethylation in colorectal cancer: a meta-analysis

Background:

The prognostic value of CDKN2A promoter hypermethylation in colorectal cancer remains controversial. We systematically reviewed the evidence for assessment of CDKN2A methylation in colorectal cancer to elucidate this issue.

Methods:

Pubmed, Embase and ISI web of knowledge were searched to identify eligible studies to evaluate the association of CDKN2A hypermethylation and overall survival and clinicopathological features of colorectal cancer patients. Combined hazard ratios (HRs) or odds ratios (ORs) with 95% confidence interval (95% CI) were pooled using a random-effects model.

Results:

A total of 11 studies encompassing 3440 patients were included in the meta-analysis. CDKN2A hypermethylation had an unfavourable impact on OS of patients with colorectal cancer (HR 1.65, 95% CI 1.29–2.11). Subgroup analysis indicated that CDKN2A hypermethylation was significantly correlated with OS in Europe (HR 1.49; 95% CI 1.28–1.74) and Asia (HR 3.30; 95% CI 1.68–6.46). Furthermore, there was a significant association between CDKN2A hypermethylation and lymphovascular invasion (OR 1.68, 95% CI 1.15–2.47), lymph node metastasis (OR 1.68, 95% CI 1.09–2.59) and proximal tumour location (OR 2.09, 95% CI 1.34–3.26) of colorectal cancer.

Conclusion:

This meta-analysis indicated that CDKN2A hypermethylation might be a predictive factor for unfavourable prognosis of colorectal cancer patients.     

Increased cytotoxicity and bystander effect of 5-fluorouracil and 5-deoxy-5-fluorouridine in human colorectal cancer cells transfected with thymidine phosphorylase.

5-Fluorouracil (5-FU) and 5'-deoxy-5-fluorouridine (5'-DFUR), a prodrug of 5-FU, are anticancer agents activated by thymidine phosphorylase (TP). Transfecting the human TP cDNA into cancer cells in order to sensitize them to these pyrimidine antimetabolites may be an important approach in human cancer gene therapy research. In this study, an expression vector containing the human TP cDNA (pcTP5) was transfected into LS174T human colon carcinoma cells. Eight stable transfectants were randomly selected and analysed. The cytotoxic effects of 5-FU and 5'-DFUR were higher in TP-transfected cells as compared to wild-type cells. The maximal decreases in the IC50 were 80-fold for 5-FU and 40-fold for 5'-DFUR. The increase in sensitivity to these pyrimidines of TP-transfected cells significantly correlated with the increase in both TP activity and TP expression. Transfected clone LS174T-c2 but not wild-type cells exhibited formation of [3H]FdUMP from [3H]5-FU. In addition the LS174T-c2 clone enhanced the cytotoxic effect of 5'-DFUR, but also that of 5-FU, towards co-cultured parental cells. For both anti-cancer agents, this bystander effect did not require cell-cell contact. These results show that both 5-FU or 5'-DFUR could be used together with a TP-suicide vector in cancer gene therapy.     

METH-2 silencing and promoter hypermethylation in NSCLC

The antiangiogenic factor METH-2 (ADAMTS-8) was identified in a previous dual-channel cDNA microarray analysis to be at least two-fold under-represented in 85% (28 out of 33) of primary non-small-cell lung carcinomas (NSCLCs). This observation has been validated in an independent series of NSCLCs and adjacent normal tissues by comparative multiplex RT-PCR, and METH-2 mRNA expression was dramatically reduced in all 23 tumour samples analysed. Immunohistochemical analysis of the same sample set demonstrated that METH-2 was strongly expressed in 14 out of 19 normal epithelial sites examined but only one out of 20 NSCLCs. DNA methylation analysis of the proximal promoter region of this gene revealed abnormal hypermethylation in 67% of the adenocarcinomas and 50% of squamous cell carcinomas, indicating that epigenetic mechanisms are involved in silencing this gene in NSCLC. No homozygous deletions of METH-2 were found in lung cancer cell lines. Allelic imbalance in METH-2 was assessed by an intronic single nucleotide polymorphism (SNP) assay and observed in 44% of informative primary samples. In conclusion, the downregulation of METH-2 expression in primary NSCLC, often associated with promoter hypermethylation, is a frequent event, which may be related to the development of the disease.     

Targeted radiosensitisation by pegylated liposome-encapsulated 3', 5'-O-dipalmitoyl 5-iodo-2'-deoxyuridine in a head and neck cancer xenograft model

5-Iodo-2'-deoxyuridine (IUdR) is an effective radiosensitiser but its clinical development has been limited by toxicity. Prolonged intravenous infusions of IUdR are necessary for optimal tumour uptake but cause dose-limiting myelosuppression. The lack of selective tumour uptake can lead to radiosensitisation of adjacent normal tissues and enhanced local radiation toxicity. Liposomal IUdR delivery offers selective targeting of tumour tissues and avoidance of local and systemic toxicity. In these studies, we report the development of a pegylated liposome containing a lipophilic IUdR derivative (3', 5'-O-dipalmitoyl-5-iodo-2'-deoxyuridine) for use in a head and neck cancer xenograft model. Initial studies confirmed the ability of IUdR to sensitise two head and neck cancer cell lines to single fractions of radiotherapy (SFRT) and this effect was seen to correlate with the thymidine replacement index in KB cells. In vivo delivery of single doses of either unencapsulated IUdR or pegylated liposomal IUdR (PLIUdR) to nude mice bearing KB xenograft tumours did not enhance the effect of SFRT delivered 16 h later. When PLIUdR was delivered by a protracted administration schedule to a dose of 48 mg kg(-1) over 7 days, it enhanced the effect of both 4.5 Gy SFRT and fractionated radiotherapy. PLIUdR was at least as effective as unencapsulated IUdR delivered by multiple intravenous injections or continuous subcutaneous infusion. Immunohistochemistry with a specific anti-IUdR monoclonal antibody confirmed greater levels of tumour staining in tumours from animals treated with PLIUdR compared with those treated with unencapsulated IUdR.     

Van-Gogh-like 2 antagonises the canonical WNT pathway and is methylated in colorectal cancers

Background:

Aberrant activation of the canonical WNT signaling is a feature of colorectal cancer (CRC). Van-Gogh-like 2 (VANGL2) belongs to the non-canonical WNT pathway whose activation inhibits canonical WNT signaling. In this study, we investigated the role of VANGL2 and its epigenetic regulation in CRC.

Methods:

Van-Gogh-like 2 expression and promoter methylation after 5-aza-2′-deoxycytidine (5-aza) treatment were evaluated in CRC cells. DNA samples from 418 sporadic CRCs were tested for VANGL2 promoter methylation and microsatellite instability (MSI). Proliferation, colony formation and activation of the WNT pathway were tested in cells after VANGL2 overexpression.

Results:

Van-Gogh-like 2 mRNA was significantly higher in 5-aza-treated RKO, LOVO and SW48, whereas no differences were found in SW480. Van-Gogh-like 2 was fully methylated in RKO, SW48, HCT116, DLD1 and Caco2; partially methylated in LOVO, LS174T and SW837; and unmethylated in SW480, SW620 and HT29. Higher expression of VANGL2 mRNA was found in the unmethylated cell lines. In CRC specimens (8.93% MSI), methylated VANGL2 was associated with MSI, higher grade, proximal colon location and BRAF mutation. Van-Gogh-like 2 overexpression in SW480 significantly decreased proliferation, colony formation and β-catenin levels.

Conclusion:

Van-Gogh-like 2 is frequently methylated in MSI-CRCs with BRAF mutation and may act as a tumour suppressor gene, counteracting WNT/β-catenin signaling.     

Correlation between hypermethylation of the RASSF2A promoter and K-ras/BRAF mutations in microsatellite-stable colorectal cancers

Recently, RASSF2A was identified as a potential tumor suppressor epigenetically inactivated in human cancers. Here, we evaluated the methylation status of RASSF2A in colorectal cancer (CRC) and analyzed its correlation with K-ras/BRAF mutations, microsatellite instability status and other clinicopathological features. Using methylation-specific PCR and bisulfite sequencing, we analyzed the methylation status in primary CRC, adenomas and corresponding normal tissues and then compared it with the presence of K-ras and BRAF mutations. We also examined the expression and methylation status of RASSF2A in CRC cell lines. We found that aberrant methylation of RASSF2A promoter regions is associated with gene silencing in CRC cell lines. In primary CRC, the frequency of RASSF2A methylation was 72.6%, and it was found in 16 of 16 (100%) adenomas. In addition, there was a positive correlation between K-ras/BRAF mutations and RASSF2A methylation in primary CRC. Furthermore, a significant positive correlation between K-ras/BRAF mutations and RASSF2A methylation was also observed in microsatellite-stable (p = 0.033) and distal CRC (p = 0.025). These results show that RASSF2A methylation is a frequent event in colorectal tumorigenesis and positively correlates with K-ras/BRAF mutation in microsatellite-stable or distal CRC.     

Role of hMLH1 promoter hypermethylation in drug resistance to 5-fluorouracil in colorectal cancer cell lines

Loss of DNA mismatch repair (MMR) occurs in 10-15% of sporadic colorectal cancer, is usually caused by hMLH1 hypermethylation, and has been shown to confer resistance to various chemotherapeutic reagents, including 5-fluorouracil (5-FU). We tested the hypothesis that demethylation of the hMLH1 promoter in hypermethylated colorectal cancer cells would restore MMR proficiency and drug sensitivity to 5-FU. We used the MMR-deficient cell lines SW48, HCT116, HCT116+chr2 and the -proficient cell line HCT116+chr3. After treatment with the demethylating agent 5-Aza-2'-deoxycytidine (5 aza-dC), hMLH1 mRNA and protein expression were determined by RT-PCR and immunoblots. The methylation status for hMLH1 was investigated by methylation-specific PCR. Cells were subsequently treated with 5-FU and the growth characteristics ascertained by clonogenic assays. hMLH1 hypermethylation was reverted in SW48 cells 24 hr after treatment with 5 aza-dC and was accompanied by hMLH1 mRNA and protein reexpression. While 5 aza-dC alone did not affect the growth of SW48 cells, all other cell lines responded with a pronounced growth inhibition. 5-FU treatment strongly reduced the colony formation of HCT116+chr3 cells. These effects were significantly less in the MMR-deficient cells. Combined treatment of SW48 cells resulted in a similar growth pattern as seen in 5-FU only treated HCT116+chr3 cells. We demonstrate that in vitro resistance to 5-FU can be overcome by reexpression of hMLH1 protein through 5 aza-dC-induced demethylation in hypermethylated cell lines. Induction of the expression of methylated tumor suppressor or MMR genes could have a significant impact on the development of future chemotherapy strategies.     

5-氮杂-2’-脱氧胞苷对人前列腺癌裸鼠移植瘤生长及雄激素受体表达影响的研究

目的探讨5-氮杂-2’-脱氧胞苷(5-Aza-2’deoxycytidine,5-Aza-CdR)对LNCaP荷瘤裸鼠的肿瘤生长及雄激素受体(androgen receptor,AR)表达的影响。方法裸鼠皮下接种LNCaP细胞,建立人前列腺癌裸鼠移植瘤模型,待肿瘤长至300 mm3大小时,荷瘤裸鼠腹腔内注射5-Aza-CdR,0.25 mg/kg,隔天1次。对照组给予生理盐水注射。每3天测量肿瘤体积1次,共观察24天。实验结束时处死裸鼠,取出肿块称重后,肿瘤组织抽提RNA和蛋白质,分别应用实时定量PCR和western blot方法,检测AR在mRNA和蛋白质水平上的表达。结果 LNCaP荷瘤裸鼠经5-Aza-CdR治疗后,与对照组相比,肿瘤生长缓慢。第24天观察结束时,5-Aza-CdR处理组肿瘤体积[(982±83)mm3]显著低于对照组[(1 867±195)mm3](P<0.01)。移植瘤重量与对照组相比明显下降[(796±178)mg vs(1 267±252)mg],抑瘤率为37.2%,差异有统计学意义(P<0.01)。5-Aza-CdR组游离PSA(FPSA)浓度相对对照组显著下降,分别为(40.25±13.3)ng/mL和(79.7±13.2)ng/mL,差异有统计学意义(P<0.01)。荷瘤裸鼠经5-Aza-CdR处理后,AR在mRNA水平上的表达下降为对照组的0.5倍,在蛋白质水平上的表达也显著降低(P<0.01)。结论 5-Aza-CdR能够有效抑制LN-CaP荷瘤裸鼠移植瘤的生长,并显著降低AR基因表达,为前列腺癌的去甲基化治疗提供了理论依据。     

Effects of Promoter Region 5＇CpG Island Demethylation on the Biological Behavior of Human Colorectal Cancer RKO Cells in Vitro

OBJECTIVE To explore the relationship between the methylation status of the promoter 5＇CpG island region and the biological behavior of human colorectal cancer RKO cells in vitro. METHODS RKO cells were treated with a selective DNA methyltransferase inhibitor-5-aza-2＇-deoxycytidine （5-aza-CdR） for 72 h. Methylationspecific PCR （MSP）, T-A cloning and DNA sequence analysis were used to determinate the 5＇CpG island methylation status of the p16/CDKN2 tumor suppressor gene. Cell growth, morphological changes and apoptosis were analyzed by the MTT assay, flow cytometry, fluorescence staining and electron microscopy. RESULTS The 5＇CpG island of the p16/CDKN2 tumor suppressor gene in RKO cells was a typically hypermethylated. The DNA methyltransferase inhibitor （5-Aza-CdR） effectively reversed the hypermethylation status of the promoter region. With demethylation, RKO cell growth was suppressed, the cells doubling times were prolonged （P〈0.01） and apoptosis was induced, which showed a relationship. CONCLUSION A selective DNA methyltransferase （DNMT） inhibitor can inhibit proliferation by demethylation in 5＇CpG islands, and may be a potential new therapy target for colorectal cancer.     

Nuclear beta-catenin expression is closely related to ulcerative growth of colorectal carcinoma

Although most colorectal cancer develops based on the adenoma-adenocarcinoma sequence, morphologically, colorectal cancer is not a homogeneous disease entity. Generally, there are two distinct morphological types: polypoid and ulcerative colorectal tumours. Previous studies have demonstrated that K-ras codon 12 mutations are preferentially associated with polypoid growth of colorectal cancer; however, little is known about the molecular mechanism that determines ulcerative growth of colorectal cancer. beta-catenin complex plays a critical role both in tumorigenesis and morphogenesis. We examined the differential expression of beta-catenin and its related factors among different types of colorectal cancer in order to determine any relationship with gross tumour morphology. Immunohistochemical staining of beta-catenin, E-cadherin and MMP-7 was performed on 51 tumours, including 26 polypoid tumours and 25 ulcerative tumours. Protein truncation tests and single-strand conformational polymorphism for mutation of the adenomatous polyposis coli tumour suppressor gene, as well as single-strand conformational polymorphism for the mutation of beta-catenin exon 3 were also done. Nuclear expression of beta-catenin was observed in 18 out of 25 (72%) cases of ulcerative colorectal cancer and seven out of 26 (26.9%) cases of polypoid colorectal cancer. A significant relationship of nuclear beta-catenin expression with ulcerative colorectal cancer was found (P<0.001). However, this finding was independent of adenomatous polyposis coli tumour suppressor gene mutation and E-cadherin expression. Together with previous data, we propose that different combinations of genetic alterations may underlie different morphological types of colorectal cancer. These findings should be taken into consideration whenever developing a new genetic diagnosis or therapy for colorectal cancer.     

MicroRNA-9-5p increases the sensitivity of colorectal cancer cells to 5-fluorouracil by downregulating high mobility group A2 expression

Chemotherapy drug 5-fluorouracil (5-FU) is the first-line treatment for colorectal cancer (CRC); however, 5-FU resistance decreases CRC therapeutic efficiency. A previous study revealed that microRNA (miR)-9-5p serves an antitumor effect in CRC. However, the effect of miR-9-5p in CRC chemoresistance remains unknown. In the present study, two CRC cell lines, including HT-29 and HCT-116 cells, were used to investigate the impact of miR-9-5p in overcoming 5-FU resistance. The results revealed that treatment with 5-FU decreased CRC cell viability and upregulated miR-9-5p expression in both CRC cells. Knockdown of miR-9-5p decreased HCT-116 cell sensitivity to 5-FU and inhibited apoptosis. By contrast, miR-9-5p overexpression enhanced the sensitivity of HT-29 cells to 5-FU and induced apoptosis. Additionally, it was confirmed that miR-9-5p directly targeted high mobility group A2 (HMGA2). HMGA2 overexpression reversed miR-9-5p-induced HT-29 apoptosis. The present study indicated that miR-9-5p enhanced the sensitivity of CRC cells to 5-FU via downregulating HMGA2 expression.     

Candidate tumor suppressor gene SLC5A8 is frequently down-regulated by promoter hypermethylation in prostate tumor

Background

The prostate gland is the most common site of cancer and the third leading cause of cancer mortality in men. Solute carrier family 5 (iodide transporter), member 8 (SLC5A8) was proposed as a potential tumor suppressor gene which is silenced by epigenetic changes in various tumors. The aim of this study was to investigate the significance of DNA methylation in SLC5A8 expression in prostate tumors.

Methods

DNA methylation status of the promoter region and expression of SLC5A8 were evaluated in prostate cancer cell lines, tumor and adjacent non-tumor prostate tissues from same prostate cancer patients, by using bisulphite-modified sequencing, RT-PCR and quantitative methylation-specific PCR (QMSP) analysis.

Results

The reduced or lost expression of SLC5A8 was observed in 70% of the tumor tissues. The bisulphite-modified sequencing analysis on the prostate cancer cell lines which do not express SLC5A8 detected the densely methylated SLC5A8 promoter region. SLC5A8 was reactivated by treatment with DNA methyl transferase inhibitor, 5-azacytidine but not by trichostatin A (TSA). Higher methylation at the promoter region of SLC5A8 in primary prostate tumor tissues was detected as compared with those in adjacent non-tumor tissues (7/10, 70%).

Conclusions

These data suggested that DNA methylation in the SLC5A8 promoter region suppressed the expression of SLC5A8 in prostate tumor.