Association of NDRG1 Gene Promoter Methylation with Reduced NDRG1 Expression in Gastric Cancer Cells and Tissue Specimens

NDRG1 (N-myc downstream-regulated gene 1) plays a role in cell differentiation and suppression of tumor metastasis. This study aims to determine the expression of NDRG1 mRNA and protein in gastric cancer cell lines and tissue specimens and then assess the possible cause of its aberrant expression. Six gastric cancer cell lines and 20 pairs of normal and gastric cancer tissue samples were used to assess NDRG1 expression using Real-time PCR and Western blot. High-resolution melting analysis (HRM) and methylation-specific PCR (MSP) were performed to detect gene mutation and methylation, respectively, in cell lines and tissues samples. Expression of NDRG1 mRNA and protein was downregulated in gastric cancer cell lines and tissues. Specifically, expression of NDRG1 mRNA and protein was lower in all six gastric cancer cell lines than that of normal gastric cells, while 15 out of 20 cases of gastric cancer tissues had the reduced levels of NDRG1 mRNA and protein. HRM data showed that there was no mutation in NDRG1 gene, but MSP data showed high levels of NDRG1 gene promoter methylation in the CpG islands in both cell lines and tissue samples. Moreover, treatment with the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine upregulated NDRG1 expression in gastric cancer HGC27 cells, but not in the histone deacetylase inhibitor trichostatin A-treated HGC27 cells. In conclusion, this study has shown that expression of NDRG1 mRNA and protein was reduced in gastric cancer cell lines and tissues, which is due to methylation of NDRG1 gene promoter. Further study will unearth the clinical significance of the reduced NDRG1 protein in gastric cancer.     

Mapping and Expression Analysis of Chicken NDRG1 and NDRG3 Genes

N-myc downstream-regulated genes 1 and 3 (NDRG1 and NDRG3) are members of the alpha/beta hydrolase superfamily. Phylogenetic analysis of the family demonstrated that human NDRG1 and 3 belong to a subfamily. The mapping and gene expression patterns of these genes represent one step toward further investigation into their possible roles in the chicken (Gallus gallus). To map these genes in the chicken chromosome, a 6000 rads chicken-hamster radiation hybrid panel (ChickRH6) was used. Primers were designed according to the published human sequences for amplification of those two genes. We compared the corresponding human mRNA sequences with the predicted coding sequences of the chicken NDRG1 and 3 genes and found that the assembled contigs shared a high percentage of similarity with the human genes. PCR of samples from ChickRH6 revealed that the locations of NDRG1 and 3 are linked to the markers MYC (58 cRs away, LOD score 4.52) and SEQ0265 (10 cRs away, LOD score 17.81), respectively. This result adds two new markers to the chicken RH map, and it reinforces that the RH technique is indeed a powerful tool for mapping genes due to its rapidity, precision, convenience, and reproducibility. In addition, we detected the gene expression and distribution of chicken NDRG1 and 3 in seven tissues, including heart, liver, spleen, lung, muscle, brain, and thymus, by RT-PCR, and found that NDRG1 is relatively ubiquitously expressed in all the tested tissues and highly expressed in heart and liver, whereas NDRG3 is high in heart, muscle, and brain.     

NDRG1对人肠癌细胞失巢凋亡的影响

目的:探讨NDRG1对体外培养的人肠癌细胞系失巣凋亡的影响。方法:采用慢病毒系统将NDRG1表达单元转入人肠癌细胞系SW620、HCT8中,建立相应的过表达稳定细胞系;通过siRNA的方法干扰HCT116和LOVO细胞系中NDRG1的表达,分别在非贴壁培养的情况下培养48小时,采用流式细胞术和TUNEL染色检测细胞的凋亡情况。结果:在贴壁培养条件下,NDRG1过表达并没有显著影响肠癌细胞的生长及增殖,而NDRG1特异性siRNA干扰HCT116细胞中NDRG1的表达后,其凋亡率无明显变化(P0.05)。在悬浮培养条件下,NDRG1过表达的肠癌细胞的失巢凋亡率显著低于正常对照组(P0.05),而用三种不同的siRNA干扰HCT116及LOVO细胞中NDRG1的表达后,其失巢凋亡率均显著高于正常对照组(P0.05)。结论:NDRG1在体外可抑制人肠癌细胞的失巢凋亡。     

Prostate Tissue Metal Levels and Prostate Cancer Recurrence in Smokers

Although smoking is not associated with prostate cancer risk overall, smoking is associated with prostate cancer recurrence and mortality. Increased cadmium (Cd) exposure from smoking may play a role in progression of the disease. In this study, inductively coupled plasma mass spectrometry was used to determine Cd, arsenic (As), lead (Pb), and zinc (Zn) levels in formalin-fixed paraffin embedded tumor and tumor-adjacent non-neoplastic tissue of never- and ever-smokers with prostate cancer. In smokers, metal levels were also evaluated with regard to biochemical and distant recurrence of disease. Smokers (N?=?25) had significantly higher Cd (median ppb, p?=?0.03) and lower Zn (p?=?0.002) in non-neoplastic tissue than never-smokers (N?=?21). Metal levels were not significantly different in tumor tissue of smokers and non-smokers. Among smokers, Cd level did not differ by recurrence status. However, the ratio of Cd ppb to Pb ppb was significantly higher in both tumor and adjacent tissue of cases with distant recurrence when compared with cases without distant recurrence (tumor tissue Cd/Pb, 6.36 vs. 1.19, p?=?0.009, adjacent non-neoplastic tissue Cd/Pb, 6.36 vs. 1.02, p?=?0.038). Tissue Zn levels were also higher in smokers with distant recurrence (tumor, p?=?0.039 and adjacent non-neoplastic, p?=?0.028). These initial findings suggest that prostate tissue metal levels may differ in smokers with and without recurrence. If these findings are confirmed in larger studies, additional work will be needed to determine whether variations in metal levels are drivers of disease progression or are simply passengers of the disease process.     

骨形态发生蛋白7 在前列腺癌中的表达

目的:探讨骨形态发生蛋白( BMP-7)在前列腺癌组织中的表达及其与临床分期之间的关系.方法:应用免疫印迹法检测30例前列腺癌患者及30例前列腺良性增生患者前列腺组织中BMP-7的表达情况.结果:前列腺癌组织中BMP-7的表达显著高于前列腺良性增生组织,且BMP-7的表达随前列腺癌的临床分期、Gleason分级增高而增加.结论:BMP-7在前列腺癌中的表达明显增高,其表达量与临床分期相关,前列腺癌组织中BMP-7的表达增高提示预后不佳.     

Overexpressed MicroRNA-182 Promotes Proliferation and Invasion in Prostate Cancer PC-3 Cells by Down-Regulating N-myc Downstream Regulated Gene 1 (NDRG1)

MicroRNAs, non-coding 20–22 nucleotide single-stranded RNAs, result in translational repression or degradation and gene silencing of their target genes, and significantly contribute to the regulation of gene expression. In the current study, we report that miR-182 expression was significantly upregulated in prostate cancer tissues and four cell lines, compared to benign prostatic hyperplasia tissues and normal prostatic epithelial (RWPE-1) cells. Ectopic overexpression of miR-182 significantly promotes the proliferation, increases the invasion, promotes the G1/S cell cycle transition and reduces early apotosis of PC-3 cells, while suppression of miR-182 decreased the proliferation and invasion, inhibits the G1/S cell cycle transition and increase early apotosis of PC-3 cells. Additionally, we demonstrated that miR-182 could downregulate expression of NDRG1 by directly targeting the NDRG1 3′-untranslated region. In conclusion, our results suggest that miR-182 plays an important role in the proliferation of human prostate cancer cells by directly suppressing the tumor supressor gene NDRG1. We uncovered a new epigenetic regulation of NDRG1.     

一个新的前列腺癌细胞系分泌蛋白磷酸甘油酸酯激酶的鉴定与分析

目的：建立前列腺癌细胞系C4-2B分泌蛋白双向电泳图谱,并对感兴趣的蛋白进行质谱鉴定,对其表达调控进行初步分析。方法：收集前列腺癌细胞系C4-2B的分泌蛋白,利用双向电泳结合质谱的方法对感兴趣的蛋白进行鉴定,而后利用Western blot等方法对结果进行验证和分析。结果：得到较为稳定的前列腺癌细胞系分泌蛋白C4-2B双向电泳图谱,鉴定了一个新的前列腺癌细胞系分泌蛋白磷酸甘油酸酯激酶,并发现1nmol/L人工合成雄激素R1881可上调其表达。结论：建立了简便的前列腺癌细胞系分泌蛋白双向电泳及质谱分析的研究方法,初步证明磷酸甘油酸酯激酶是前列腺癌细胞系C4-2B新的分泌蛋白,且人工合成雄激素R1881可诱导其表达上调。     

人NDRG3 cDNA的克隆与表达

目的：为了获得NDRG3(N-Myc downstream regulated gene3)的cDNA,以成人脑组织为模板。方法：由特异性的引物通过RT—PCR方法扩增人NDRG3的编码基因,大小约为1．1 kb。将此基因插入PMD 18-T载体中,并进行酶切鉴定和序列测定。然后,将测序正确的片段亚克隆人原核表达载体pPROEX-HTb表达载体中,转化DH5α感受态细胞,并在LB培养基中获得高效表达。结果：这表明成功地进行了人NDRG3的克隆和表达,为进一步研究NDRG3的功能奠定了良好的基础。     

Methylation of APC and GSTP1 in Non-Neoplastic Tissue Adjacent to Prostate Tumour and Mortality from Prostate Cancer

Background

Markers that can discriminate between indolent and aggressive prostate tumours are needed. We studied gene methylation in non-neoplastic tissue adjacent to prostate tumour (NTAT) in association with prostate cancer mortality.

Methods

From two cohorts of consecutive prostate cancer patients diagnosed at one pathology ward in Turin, Italy, we selected 157 patients with available NTAT and followed them up for more than 14 years. We obtained DNA from NTAT in paraffin-embedded prostate tumour tissues and used probe real-time PCR to analyse methylation of the glutathione S-transferase (GSTP1) and adenomatous polyposis coli (APC) gene promoters.

Results

Prevalence of APC and GSTP1 methylation in the NTAT was between 40 and 45%. It was associated with methylation in prostate tumour tissue for the same two genes as well as with a high Gleason score. The hazard ratio (HR) of prostate cancer mortality was 2.38 (95% confidence interval: 1.23–4.61) for APC methylation, and 2.92 (1.49–5.74) for GSTP1 methylation in NTAT. It changed to 1.91 (1.03–3.56) and 1.60 (0.80–3.19) after adjusting for Gleason score and methylation in prostate tumour tissue. Comparison of 2 vs. 0 methylated genes in NTAT revealed a HR of 4.30 (2.00–9.22), which decreased to 2.40 (1.15–5.01) after adjustment. Results were stronger in the first 5 years of follow-up (adjusted HR: 3.29, 95% CI: 1.27–8.52).

Conclusions

Changes in gene methylation are an early event in prostate carcinogenesis and may play a role in cancer progression. Gene methylation in NTAT is a possible prognostic marker to be evaluated in clinical studies.     

人NDRG1的融合表达、纯化及抗体制备

NDRG1是在N myc缺陷的小鼠胚胎组织中发现的一异常高表达的新基因 .在研究高同型半胱氨酸血症引起动脉粥样硬化的机制时 ,在培养的人脐静脉内皮细胞 (HUVEC)中发现了人NDRG1 .为了研究人NDRG1的功能以及结构与功能之间的关系 ,用RT PCR技术 ,从人脑总RNA中克隆NDRG1cDNA ,进行序列测定后 ,将NDRG1插入pPROEXHTb表达载体中 ,转化E .coliDH5α感受态细胞 ,并在LB培养基中获得高效可溶表达 .表达的 6His NDRG1融合蛋白经Ni2 + NTA偶联的琼脂糖珠纯化 ,通过圆二色性分析其二级结构 ,结果为 :α螺旋 :2 3 6 ,β片层 :1 8 6 ,转角 :2 5 7,无规卷曲 :32 0 .用此融合表达的蛋白免疫家兔 ,制备得到高效价的抗体 ,利用固定于硝酸纤维素膜上的NDRG1抗原亲和吸附纯化抗血清提高了抗体的特异性 ,为进一步研究NDRG1的功能打下了良好的基础     

Androgen-Regulated Expression of Arginase 1, Arginase 2 and Interleukin-8 in Human Prostate Cancer

Background

Prostate cancer (PCa) is the most frequently diagnosed cancer in North American men. Androgen-deprivation therapy (ADT) accentuates the infiltration of immune cells within the prostate. However, the immunosuppressive pathways regulated by androgens in PCa are not well characterized. Arginase 2 (ARG2) expression by PCa cells leads to a reduced activation of tumor-specific T cells. Our hypothesis was that androgens could regulate the expression of ARG2 by PCa cells.

Methodology/Principal Findings

In this report, we demonstrate that both ARG1 and ARG2 are expressed by hormone-sensitive (HS) and hormone-refractory (HR) PCa cell lines, with the LNCaP cells having the highest arginase activity. In prostate tissue samples, ARG2 was more expressed in normal and non-malignant prostatic tissues compared to tumor tissues. Following androgen stimulation of LNCaP cells with 10 nM R1881, both ARG1 and ARG2 were overexpressed. The regulation of arginase expression following androgen stimulation was dependent on the androgen receptor (AR), as a siRNA treatment targeting the AR inhibited both ARG1 and ARG2 overexpression. This observation was correlated in vivo in patients by immunohistochemistry. Patients treated by ADT prior to surgery had lower ARG2 expression in both non-malignant and malignant tissues. Furthermore, ARG1 and ARG2 were enzymatically active and their decreased expression by siRNA resulted in reduced overall arginase activity and l-arginine metabolism. The decreased ARG1 and ARG2 expression also translated with diminished LNCaP cells cell growth and increased PBMC activation following exposure to LNCaP cells conditioned media. Finally, we found that interleukin-8 (IL-8) was also upregulated following androgen stimulation and that it directly increased the expression of ARG1 and ARG2 in the absence of androgens.

Conclusion/Significance

Our data provides the first detailed in vitro and in vivo account of an androgen-regulated immunosuppressive pathway in human PCa through the expression of ARG1, ARG2 and IL-8.     

RNA结合蛋白QKI-5在前列腺癌中的表达分析

目的:检测前列腺癌及癌旁正常前列腺组织中RNA结合蛋白QKI-5的表达情况并分析其临床意义。方法:收集前列腺癌及与之匹配的癌旁组织124例,通过免疫组化染色、Western blot、实时PCR方法检测其QKI-5的表达水平,并分析QKI-5的表达与前列腺癌临床病理特征的关系。结果:前列腺癌组织中QKI-5蛋白及m RNA表达水平均明显低于癌旁正常前列腺组织,并且随着Gleason评分的增高而降低(P0.05)。前列腺癌组织中QKI-5的表达与其Gleason评分(r=-0.939,P0.05)、TNM分期(r=-0.913,P0.05)、血清PSA值(r=-0.743,P0.05)均密切相关。结论:QKI-5在前列腺癌的发生发展过程中可能起抑癌基因的作用,并可能作为前列腺癌的诊断、病情分析和预后评估的参考指标。     

The Metastasis Suppressor,N-myc Downregulated Gene 1 (NDRG1), Is a Prognostic Biomarker for Human Colorectal Cancer

Metastasis remains to be one of the most prevalent causes leading to poor long-term survival of colorectal cancer (CRC) patients. The clinical significances of tumor metastatic suppressor, N-myc downregulated gene 1 (NDRG1), have been inconsistently reported in a variety of cancerous diseases. In this study with 240 CRC clinical specimens, we showed that NDRG1 expression was significantly decreased in most of CRC tissues compared to the paired non-tumor counterparts. Statistical analysis revealed a significant inverse correlation of NDRG1 expression with tumor stage, differentiation status and metastasis. Compared with NDRG1-negative group, NDRG1-positve group had better disease-free/overall survival (p = 0.000) over 5 years’ follow-up. Furthermore, NDRG1 was considered to be an independent prognostic factor for overall survival (p = 0.001) and recurrence (p = 0.003). Our study concludes that NDRG1 is a novel favorable predictor for the prognosis in CRC patients.     

Pterostilbene Acts through Metastasis-Associated Protein 1 to Inhibit Tumor Growth,Progression and Metastasis in Prostate Cancer

The development of natural product agents with targeted strategies holds promise for enhanced anticancer therapy with reduced drug-associated side effects. Resveratrol found in red wine, has anticancer activity in various tumor types. We reported earlier on a new molecular target of resveratrol, the metastasis-associated protein 1 (MTA1), which is a part of nucleosome remodeling and deacetylation (NuRD) co-repressor complex that mediates gene silencing. We identified resveratrol as a regulator of MTA1/NuRD complex and re-activator of p53 acetylation in prostate cancer (PCa). In the current study, we addressed whether resveratrol analogues also possess the ability to inhibit MTA1 and to reverse p53 deacetylation. We demonstrated that pterostilbene (PTER), found in blueberries, had greater increase in MTA1-mediated p53 acetylation, confirming superior potency over resveratrol as dietary epigenetic agent. In orthotopic PCa xenografts, resveratrol and PTER significantly inhibited tumor growth, progression, local invasion and spontaneous metastasis. Furthermore, MTA1-knockdown sensitized cells to these agents resulting in additional reduction of tumor progression and metastasis. The reduction was dependent on MTA1 signaling showing increased p53 acetylation, higher apoptotic index and less angiogenesis in vivo in all xenografts treated with the compounds, and particularly with PTER. Altogether, our results indicate MTA1 as a major contributor in prostate tumor malignant progression, and support the use of strategies targeting MTA1. Our strong pre-clinical data indicate PTER as a potent, selective and pharmacologically safe natural product that may be tested in advanced PCa.