Methylation of tumor associated genes in tissue and plasma samples from liver disease patients

To investigate whether aberrant hypermethylation in plasma DNA could be used as diagnosis makers for hepatocellular carcinoma (HCC), we performed methylation-specific PCR (MSP) to check the methylation status of five tumor associated genes in 36 cases of

Pseudogenization of the tumor-growth promoter angiogenin in a leaf-eating monkey

Physiological functions of human genes may be studied by gene-knockout experiments in model organisms such as the mouse. This strategy relies on the existence of one-to-one gene orthology between the human and mouse. When lineage-specific gene duplication occurs and paralogous genes share a certain degree of functional redundancy, knockout mice may not provide accurate functional information on human genes. Angiogenin is a small protein that stimulates blood-vessel growth and promotes tumor development. Humans and related primates only have one angiogenin gene, while mice have three paralogous genes. This makes it difficult to generate angiogenin-knockout mice and even more difficult to interpret the genotype-phenotype relation from such animals should they be generated. We here show that in the douc langur (Pygathrix nemaeus), an Asian leaf-eating colobine monkey, the single-copy angiogenin gene has a one-nucleotide deletion in the sixth codon of the mature peptide, generating a premature stop codon. This nucleotide deletion is found in five unrelated individuals sequenced, and therefore is likely to have been fixed in the species. Five colobine species that are closely related to the douc langur have intact angiogenin genes, suggesting that the pseudogenization event was recent and unique to the douc langur lineage. This natural knockout experiment suggests that primate angiogenin is dispensable even in the wild. Further physiological studies of douc largurs may offer additional information on the role of this cancer-related gene in normal physiology of primates, including humans. Our findings also provide a strong case for the importance of evolutionary analysis in biomedical studies of gene functions. (C) 2003 Elsevier Science B.V. All rights reserved.

Somatic mutations of mitochondrial genome in early stage breast cancer

The complete mitochondrial genomes of the primary cancerous, matched paracancerous normal and distant normal tissues from 10 early-stage breast cancer patients were analyzed in this study, with special attempt (i) to investigate whether the reported high

Molecular identification and expression analysis of tumor necrosis factor receptor-associated factor 2 in grass carp Ctenopharyngodon idella

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial component of almost the entire tumor necrosis factor receptor superfamily signaling pathway. In the present study, a TRAF2 gene has been cloned from grass carp (Ctenopharyngodon idella) by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The full-length cDNA is 3162 bp, including a 60 bp 5' untranslated region (UTR), a 1611 bp open reading frame, and a 1491 bp 3' UTR. The polyadenylation signal (AATAAA) and the mRNA instability motifs (ATTTTA, ATTTA) were followed by a poly(A) tail in the 3' UTR. No signal peptide or transmembrane region has been found in the putative amino acids of grass carp TRAF2 (gcTRAF2). Phylogenetic tree analysis clearly showed that gcTRAF2 is nearest to the TRAF2 gene of goldfish. The identity of gcTRAF2 with its homologs in other vertebrates ranges from 56% to 97%. It is characterized by one RING-type signature at the N-terminus, one zinc finger in the middle part, and one conserved TRAF domain consisting of a C-proximal (TRAF-C) subdomain and a N-proximal (TRAF-N) subdomain. The identity of TRAF-C among all TRAF2 homologs in vertebrates varies from 78% to 97%, whereas the identity of TRAF-N ranges from 56% to 100%. The recombinant gcTRAF2 has been expressed in Escherichia coli using pET-32a expression vector. The rabbit anti-gcTRAF2 polyclonal antibody was obtained. The expression of gcTRAF2 in different organs was examined by real-time quantitative polymerase chain reaction and Western blot analysis. It was widely distributed in heart, head kidney, thymus, brain, gill, liver, spleen, and trunk kidney. This is the first report of a TRAF2 homolog molecule in fish.

Androgen receptor targets NFKB and TSPI to suppress prostate tumor growth in vivo

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti-tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR-expressing cells formed tumors in male mice at a much lower rate than the AR-negative controls. Moreover, the AR-expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti-angiogenic protein, thrombospondin-1. AR activation caused a decrease in RelA, a subunit of the pro-survival transcription factor NF kappa B, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti-apoptotic targets, Bcl-2 and IL-6. Increased apoptosis within AR-expressing tumors was likely due to the NF kappa B suppression, since it was restricted to the cells lacking nuclear (active) NF kappa B. Thus we for the first time identified combined decrease of NF kappa B and increased TSP1 as molecular events underlying the AR anti-tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early-stage prostate cancer. (C) 2007 Wiley-Liss, Inc.

Dioxin-like components in human breast milk collected from Hong Kong and Guangzhou

The H4IIE rat hepatoma cell line was employed as a cell model to screen 7-ethoxyresorufin O-deethylase (EROD)-TCDD equivalents (EROD-TEQ) of human breast milk samples collected from Hong Kong and Guangzhou, China. The screening methods employed a 96-well plate spectrofluorometer-EROD assay. For cell-line validation, our results demonstrated a dose-dependent increase in the Ah receptor-mediated response (i.e., CYP1A1 mRNA and EROD) of the cells upon exposure to a number of known Ah receptor agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzothiophene, benzo[a]pyrene, and beta-naphthaflavone. TCDD induced CYP1A1 mRNA and EROD was in a close positive correlation (r = 0.98). For the screening of dioxin-like compounds, breast milk samples collected during lactation weeks 3-5 were used. One hundred (from Hong Kong) and 48 (from Guangzhou) breast milk samples were assayed, of which 65% and 68% of the samples, respectively, showed detectable dioxin-like activities using the H4IIE cell EROD screening method. For sixty-five samples from Hong Kong the mean EROD-TEQ values ranged from 58.1 to 96.5 pg/g of milk fat for those aged 21-36 years while 32 samples from Guangzhou had mean values of 98.8-202.1 pg/g of milk fat. In comparisons of the EROD-TEQ values for different age groups from both cities, there were no significant differences (P < 0.05). However, the mean and median EROD-TEQ values of the Guangzhou population were in general higher than those of the Hong Kong population. The results of the present study indicate that it is feasible to use the H4IIE cell-line as a model for screening dioxin-like compounds in human breast milk. In addition, the method is rapid and cost-effective, particularly for a routine and high-throughput sample screening analysis, compared to the costly and time-intensive chemical analytical techniques. (C) 2003 Elsevier Inc. All rights reserved.

瓦山安息香中苯并呋喃促雌激素合成的机理研究

雌激素是人体内重要的激素之一，具有广泛的生理功能。雌激素缺乏与许多疾病相关，如卵巢功能低下，更年期综合征以及骨质疏松等；雌激素过剩也将导致某些疾病，如乳腺癌、卵巢癌、子宫内膜癌等。目前，如何降低肿瘤组织中的雌激素水平而达到治疗肿瘤的目的，已经得到广泛的研究，但促雌激素生成或调节卵巢功能药物或其相关研究则很少。 本实验室前期的研究发现，瓦山安息香属植物果实中的乙醇提取物具有促雌激素生成作用，通过活性追踪和结构鉴定，确认促E2 生成的主要成分为苯并呋喃类化合物。苯并呋喃类化合物的作用与芳香酶有关，但其确切的作用机理有待证实和深入研究。 为了探讨安息香苯并呋喃类化合物的促雌激素合成的作用机理，拟采用如下的实验方案： 1、细胞学方面，对小鼠3T3-L1 前脂肪细胞、人乳腺癌细胞MCF-7、MDA-MB-231 以及人卵巢癌细胞OVCAR-3、OVCAR-4、OVCAR-5、OVCAR-8、IGROV1 等细胞株，采用RT-PCR 和ELISA 方法研究芳香酶Aro基因的表达和雌二醇E2 的生成，芳香酶抑制剂Formestane 作为阳性对照，研究时效曲线和量效曲线，确定安息香苯并呋喃类化合物SP25 的有效浓度和作用时间。 2、RNAi 方面，设计合成了针对人芳香酶Aro基因的3 对RNAi 序列，转染入细胞，芳香酶促进剂Forskolin 和地塞米松、芳香酶抑制剂Formestane 作为阳性对照，采用实时定量PCR 技术，研究RNA 干扰后，安息香苯并呋喃类化合物SP25 对人芳香酶Aro基因表达水平瓦山安息香苯并呋喃促雌激素合成的机理研究的影响。 3、雌激素受体方面，设计一段ERE 的雌激素调控元件，构建重组荧光素酶报告基因载体，瞬时转染人乳腺癌细胞株MDA-MB-231，建立针对雌激素受体的报告基因筛选模型，观察安息香苯并呋喃类化合物SP25 对雌激素受体的选择性和亲和力，从受体水平考察安息香苯并呋喃类化合物SP25 促进雌激素生成的药理学机理。 实验结果显示： 1、分化后的小鼠3T3-L1 前脂肪细胞、人乳腺癌细胞MCF-7 、MDA-MB-231 以及人卵巢癌细胞OVCAR-3、OVCAR-4、OVCAR-8 等细胞株具有芳香酶基因的表达。睾酮向雌二醇的转化能够被芳香酶抑制剂Formestane 所阻断，其中OVCAR-3 最适合进行下一步的RNAi研究。 2、RNAi 实验结果显示，设计的3 对RNAi 序列中R2 的干扰效果最强，相应的阴性对照C2 与R2 的表达量相差118 倍（24 小时）和19 倍（48 小时），显示R2/C2 这组序列可用于进一步的RNAi 试验。以R2 干扰OVCAR-3 细胞株，药物作用24、48 小时后，芳香酶抑制剂Formestane 与R2 相对表达量相比分别为0.83 倍和0.04 倍；芳香酶促进剂Forskolin 与R2 相对表达量相比分别为3.61 和1.84 倍；芳香酶促进剂地塞米松与R2 相对表达量相比分别为5.76 倍和3.49倍；苯并呋喃类化合物SP25 与R2 相对表达量相比分别为8.13 倍和4.59 倍。实验证实安息香苯并呋喃类化合物SP25 能够促进因RNAi 而发生基因沉默的人芳香酶Aro表达水平的上调。 3、雌激素受体实验结果显示，构建成功重组pERE-pGL3-promoter 荧光素酶报告基因载体和基于报告基因系统的雌激素受体激动剂或拮抗剂的细胞筛选模型。实验结果表明安息香苯并呋喃类化合物SP25 与雌激素受体ERα和ERβ亲和力选择性之比约为3：1 ，SP25通过与雌激素受体ERα结合作用其受体，刺激芳香酶的表达。 本课题通过RNA 干扰、ELISA、荧光实时定量PCR、报告基因筛选模型等技术手段，从细胞水平、蛋白酶水平和基因表达水平、雌激素受体水平等方面系统地研究了从瓦山安息香属植物果实中提取的苯并呋喃SP25 促进促雌激素生成的机理研究。试验结果显示苯并呋喃类化合物SP25 促雌激素生成的主要作用机制是直接促进芳香酶基因表达水平，以及与雌激素受体a 结合，刺激芳香酶活性。 Estrogen is an important hormone that has versatile physiologicalfunctions. Lack of estrogen will lead to many diseases such as lower ovarianfunction, climacteric syndrome and osteoporosis. Excessive estrogen alsoinduces breast carcinoma, oophoroma and endometrial carcinoma and otherdiseases. To depress the estrogen level in tumor tissue to cure carcinomawas widely studied, but there is only few studies reported on the induction ofestrogen and on the regulation of ovary function. We found that the extracts from seeds of Styrax perkinsiae couldpromote the synthesis of estrogen. The active compounds benzofurans wereidentified. Effect of benzofurans may be related to aromatase, but the mechanism was not clear. To reveal the mechanism of these benzofurans to promote estrogensynthesis, the following protocols were adopted: 1 Cytology: 3T3-L1 preadipocytes,human ovary carcinoma celllines OVCAR-3,OVCAR-4,OVCAR-5,OVCAR-8,IGROV1 andbreast carcinoma cell lines MCF-7 and MDA-MB-231 were usedto determine Aro gene expression and estrogen production withRT-PCR AND ELISA methods. Formestane, an aromataseinhibitor, was used as positive control. And dose-curve,time-curve and the effective concentration of SP25 were also studied. 2 Designed 3 pairs of RNAi for human aromatase gene, andtransfected into cell. Aromatase inducer Forskolin andDexamethasone, and aromatase inhibitor Formestane were usedas positive controls. We studied the change of Aro expressionlevel with SP25 by using real-time PCR after RNA interfering. 3 Estrogen Receptor: We constructed the recombined Luciferasereport vector and establish a screening system for estrogenagonist and antagon. With this system, we studied the affinity ofSP25 and estrogen receptor. Results: 1 Differentiated 3T3-L1 preadipocytes¡¢human ovary carcinomacell lines:OVCAR-3, OVCAR-4, OVCAR-8 and breast carcinomacell lines MCF-7, MDA-MB-231 had detected aromatase geneexpression.And OVCAR-3 is more suitable for further aromatasegene function research. 2 In RNAi assay, R2 has a strong interfering effcet in OVCAR-3 cellline, and ratio of C2 (the negative control) to R2 were 118 times(24 hours) and 19 times (48 hours). This means sucessful inRNA interfering. After R2 acted on OVCAR-3 cell line, the ratiosof formestane to R2 were 0.83 and 0.04 times, 5.76 and 3.49times (Dex), 3.61 and 1.84 times (forskolin) and 8.13 and 4.59times (sp25) after drug treated 24 or 48 hours respectively.These results indicated that SP25 can directly induce aromatasegene up-regulation. 3 We had constructed pERE-pGL3-promoter recombined vectorand the Luciferase report gene screening system. Luciferasereport gene assay showed that sp25 had a higher affinity with strogen receptor alpha than estrogen receptor beta, this indicated that SP25 can act on estrogen receptor and induce aromatase. Our results revealed that the mechanisms of benzofuran to promoteestrogen were the upregulation aromatase gene expression and promotion ofaromatase activity and have partially elective affinity with estrogen receptoralpha.

齐墩果酸的微生物转化

齐墩果酸（OA）是一个分布广泛、含量丰富的天然三萜化合物，常以皂苷元的形式广泛存在于植物中,具有多种重要生物活性。但是OA许多活性较弱，且生物利用度低，限制了其在临床上的应用。一是OA水溶性差;二是抗癌活性仍与临床应用的抗癌药物相差比较大。 真菌在微生物转化中具有种类多、培养条件比较简单等特点，为了寻找到具有转化OA能力的菌株，采取一步发酵的方法，在18株实验室保藏真菌菌株中筛选到5株目的菌株,TLC分析显示有转化效果。 随后采用二步发酵的方法作为复筛，验证5株菌株转化能力，波谱分析结果表明5株菌株对OA确实有转化作用。 选择5株菌种中代号1F-2 2菌株作为放大实验菌株，分离转化产物，得到OA衍生物108（相对分子量414m/z）和1010（相对分子量340 m/z），分离出的产物用于活性检测。寻找到产物108的RP-HPLC分离条件，质谱得出二者相对分子质量。 为验证OA转化产物抗肿瘤活性，首次研究了OA对卵巢癌细胞株IGROV1和人乳腺癌细胞株MDA-MB-231作用，通过细胞增殖抑制实验、用MTT法检测细胞活性，结果表明齐墩果酸可降低卵巢癌细胞株IGROV1和乳腺癌细胞MDA-MB-231细胞增殖能力并呈剂量依赖性，对肿瘤细胞株的半数有效抑制浓度化IC50 分别为36.58μg/mL和38.8μg/mL (P<0．01)。OA能抑制肿瘤细胞活性，并且OA对卵巢癌细胞株IGROV1抑制活性高于乳腺癌细胞MDA-MB-231。 在此基础上，转化产物108和1010对卵巢癌细胞株IGROV1和人乳腺癌细胞株MDA-MB-231的抑制作用也进行研究，MTT实验结果表明，转化产物对两株癌细胞也有抑制活性（P<0．01）。 总之，本文工作为进一步开展齐墩果酸类化合物结构改造和抗肿瘤活性的研究奠定了基础。 Oleanolic acid (OA) is a triterpenoid widely distributed in the nature which possesses various important bioactivities. OA also serves as aglycon of many natural saponins. However, the relatively weak activities and poor bioavailability hinder its clinical use. Firstly, poor water-solubility results in worse bioavailability. Secondly, compared with clinical antitumor drug, the antitumor effect of OA has a great difference, it is worse. Many fungi have ability to transform nature products into a variety of derivatives, and transformation conditions of fungi are simple. Attempt to obtain fungi strains able to biotransform OA, we carried out the following experiments: To investigate the biotransformation 0f OA by strains supplied firstly, we used one-step fermentation method to screen the aimed strains from 18 fungus strains stored in our laboratory. On the basis of the initial screening experiments, we found 5 aimed strains. The TLC results showed that the 5 fungi strains could transform OA into other components derivatives. Then we used two-step fermentation method as secondly screening. We repeated the five strains to do the experiments, analytical data of the results proved the transformation indeed. In the followed experiments work, we chose 1F-2 2 strain as large-scale transformation fungus from the aimed fungi. We got two biotransformation products of OA by 1F-2 2, and named those derivatives 108 and 1010. We found RP-HPLC separation conditions of product 108. The two products were characterized by ESI-MS. To verify the anti-tumor activity of biotransformation products of OA, we studied the inhibition effect of oleanolic acid on the ovarian carcinomas IGROV1 and breast cancer cell line MDA-MB-231 firstly. With an assay based on a tetrazolium dye (MTT), the effects of various concentrations of oleanolic acid on ovarian carcinomas IGROV1 and breast cancer cell line MDA-MB-231 were studied. MTT method was used to measure the tumor cells viability. Compared with the control group, oleanolic acid can significantly inhibit the viability of the ovarian carcinoma cells IGROV1 and MDA-MB-231 breast cancer cell line (P<0．01), IC50 values were 36.58μg/mL or 38.8μg/mL. Oleanolic acid can inhibit the malignant tumor cells viability, and inhibitory activity of OA to ovarian carcinomas IGROV1 was higher than to breast cancer cell line MDA-MB-231. On this basis, we studied the anti-tumor activity of the two derivatives of OA [called 108 (414 m/z) and 1010(340 m/z)]. It came to the conclusion that the two derivatives also showed potent inhibitory effect on the growth of these tumor cells（P<0．01）. Therefore, the results of studies will benefit the further investigating on the relationships of structures and antitumor activities of OA.

Experimental verification of therapeutic doses for the superficially-placed tumor radiotherapy with heavy ions at HIRFL

Up to now, clinical trials of heavy-ion radiotherapy for superficially placed tumors have been carried out for six times and over 60 selected patients have been treated with 80—100 MeV/u carbon ions supplied by the Heavy Ion Research Facility in Lanzhou (HIRFL) at the Institute of Modern Physics, Chinese Academy of Sciences since November, 2006. A passive irradiation system and a dose optimization method for radiotherapy with carbon-ion beams have been developed. Experimental verification of longitudinally ...

Heavy-ion conformal irradiation in the shallow-seated tumor therapy terminal at HIRFL

Basic research related to heavy-ion cancer therapy has been done at the Institute of Modern Physics (IMP), Chinese Academy of Sciences since 1995. Now a plan of clinical trial with heavy ions has been launched at IMP. First, superficially placed tumor treatment with heavy ions is expected in the therapy terminal at the Heavy Ion Research Facility in Lanzhou (HIRFL), where carbon ion beams with energy up to 100 MeV/u can be supplied. The shallow-seated tumor therapy terminal at HIRFL is equipped with a passive beam delivery system including two orthogonal dipole magnets, which continuously scan pencil beams laterally and generate a broad and uniform irradiation field, a motor-driven energy degrader and a multi-leaf collimator. Two different types of range modulator, ripple filter and ridge filter with which Guassian-shaped physical dose and uniform biological effective dose Bragg peaks can be shaped for therapeutic ion beams respectively, have been designed and manufactured. Therefore, two-dimensional and three-dimensional conformal irradiations to tumors can be performed with the passive beam delivery system at the earlier therapy terminal. Both the conformal irradiation methods have been verified experimentally and carbon-ion conformal irradiations to patients with superficially placed tumors have been carried out at HIRFL since November 2006.

Capability verification of the beam delivery system in the superficially-placed tumor therapy terminal at HIRFL

The passive beam delivery system in the superficially-placed tumor therapy terminal at Heavy Ion Researc h Facility in Lanzhou (HIRFL), which includes two orthogonal dipole magnets as scanning system, a motor-driven energy degrader as range-shifter, series of ridge filters as range modulator and a multileaf collimator, is introduced in detail. The capacities of its important components and the whole system have been verified experimentally. The tests of the ridge filter for extending Bragg peak and the range shifter for energy adjustment show both work well. To examine the passive beam delivery system, a beam shaping experiment were carried out, simulating a three-dimensional (3D) conformal irradiation to a tumor. The encouraging experimental result confirms that 3D layer-stacking conformal irradiation can be performed by means of the passive system. The validation of the beam delivery system establishes a substantial basis for upcoming clinical trial for superficially-placed tumors with heavy ions in the therapy terminal at HIRFL.

Physical property measurement of therapeutic carbon ion beam in the shallow-seated tumor therapy terminal at HIRFL

For the first time the physical properties of therapeutic carbon-ion beam supplied by, the shallow-seated tumor therapy terminal at the Heavy Ion Research Facility in Lanzhou (HIRFL) are measured. For a 80.55MeV/u C-12 ion beam delivered to the therapy terminal, the homogeneity of irradiation fields is 73.48%, when the beam intensity varied in the range of 0.001-0.1nA (i.e. 1 X 10(6) - 1 X 10(8) particles per second). The stability of the beam intensity within a few minutes is estimated to be 80.87%. The depth-dose distribution of the beam at the isocenter of the therapy facility is measured, and the position of the high-dose Bragg peak is found to be located at the water-equivalent depth of 13.866mm. Based on the relationship between beam energy and Bragg peak position, the corresponding beam energy at the isocenter of the therapy terminal is evaluated to be 71.71MeV/u for the original 80.55MeV/u C-12 ion beam, which consisted basically with calculation. The readout of the previously-used air-free ionization chamber regarding absorbed dose is calibrated as well in this experiment. The results indicate that the performance of the therapy facility should be optimized further to meet the requirements of clinical trial.

Potentiality of phosphorylation of BRCA1 at Ser 1524 to activate p21 in response to X-ray irradiation

The breast and ovarian cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein, which functions as a tumor suppressor gene. Many studies suggested that multiple functions of BRCA1 may contribute to its tumor suppressor activity, including roles in cell cycle checkpoints, apoptosis and transcription. It is postulated that phosphorylation of BRCA1 is an important means by which its cellular functions are regulated. In this study, we employed phospho-Ser-specific antibody recognizing Ser-1524 to study BRCA1 phosphorylation under conditions of DNA damage and the effects of phosphorylation on BRCA1 functions. The results showed that 10 Gy X-ray treatment significantly induced phosphorylation of Ser-1524 but not total BRCA1 protein levels. The expression both of p53 and p21 increased after irradiation, but ionizing radiation (IR) -induced activation of p21 was prior to that of p53. The percentages of G0/G1 phase remarkably increased after IR. In addition, no detectable levels of 89 kDa fragment of PARP, a marker of apoptotic cells, were observed. Data implied that IR-induced phosphorylation of BRCA1 at Ser-1524 might activatep21 protein, by which BRCA1 regulated cell cycle, but play no role in apoptosis.