Brain metastasis of hepatocellular carcinoma： A case report and review of the literature

INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most frequent malignancies in the world.     

Clinicopathological and prognostic implications of endoglin （CD105） expression in hepatocellular carcinoma and its adjacent non-tumorous liver

AIM: The expression pattern of endoglin (CD105) in hepatocellular carcinoma (HCC) has not been reported so far. We hypothesized that CD105 could differentially highlight a subset of microvessels in HCC, and intratumoral microvessel density (IMVD) by CD105 immunostaining (IMVD-CD105) could provide better prognostic information than IMVD by CD34 immunostaining (IMVD-CD34). METHODS: Paraffin blocks of tumor and adjacent non-tumorous liver tissues from 86 patients who underwent curative resection of HCC were used for this study. Serial sections were stained for CD105 and CD34, respectively, to highlight the microvessels. IMVD was counted according to a standard protocol. RESULTS: In the HCC tissues, CD105 was either negatively or positively stained only in a subset of microvessels. In contrast, CD34 showed positive and more extensive microvessel staining in all cases examined. However, in the adjacent non-tumorous liver sections, CD105 showed a diffuse pattern of microvessel staining in 20 of 86 cases, while CD34 showed negative or only focal staining of the sinusoids around portal area. Correlation with clinicopathological data demonstrated that lower scores of IMVD-CD105 were found in larger sized tumors [mean 41.4/0.74 mm2 (>5 cm tumor) vs 65.9/0.74 mm2 (≤5 cm tumor), P= 0.043] and more aggressive tumors, as indicated by venous infiltration [36.8/0.74 mm2 (present) vs 64.2/0.74 mm2 (absent), P = 0.020], microsatellite nodules [35.1/0.74 mm2 (present) vs 65.9/0.74 mm2 (absent), P= 0.012], and advanced TNM tumor stage [38.8/0.74 mm2 (stage 3 or 4) vs 68.3/0.74 mm2 (stage 1 or 2), P= 0.014]. No prognostic significance was observed when median values were used as cut-off points using either IMVD-CD105 or IMVD-CD34. However, the presence of the diffuse pattern of CD105 expression in the adjacent non-tumorous liver tissues predicted a poorer disease-free survival (median 8.6 vs 21.5 mo, P= 0.026). CONCLUSION: Our data demonstrate that a lower IMVD-CD105 is associated with larger and more aggressive tumors. In this study, IMVD-CD105 did not provide significant prognostic information. However, active angiogenesis as highlighted by diffuse CD105 staining of the microvessels in the adjacent non-tumorous liver tissues is predictive of early recurrence.     

Experimental study on ultrasound-guided intratumoral injection of“Star-99”in treatment of hepatocellular carcinoma of nude mice

AIM: To investigate the anti-cancer effect and the immunological mechanism of ultrasound-guided intratumoral injection of Chinese medicine "Star-99" in hepatocellular carcinoma (HCC) of nude mice.METHODS: Twenty-eight human hepatocellular carcinoma SMMC-7721 transplanted nude mice, 14 of hypodermically implanted and 14 of orthotopic liver transplanted, were randomly divided into three groups of which 14 mice with Star-99, and 7 with ethanol and saline respectively. Ten days after the transplantation the medicines were injected into the tumors of all the nude mice once every 5 days.After 4 injections the nude mice were killed. The diameters of three dimension of the tumors were measured by high frequency ultrasound before and after the treatment and the tumor growth indexes* (TGI) were calculated.Radioimmunoassay was used to detect the serum levels of interleukin-2 (IL-2) and tumor necrosis factor (TNF)-alpha.The tumor tissues were sent for flow cytometry (FCM) DNA analysis. Apoptotic cells were visualized by TUNEL assay.All the experiments were carried out by double blind method. zRESULTS: The TGI of Star-99 group (0.076±0.024) was markedly lower than that of the saline group (4.654±1.283)(P＜0.01). It also seemed to be lower than that of the ethanol group (0.082±0.028), but not significantly different (P＞0.05).Serum levels of IL-2 and TNF-α were markedly higher than those of ethanol group and saline groups (P＜0.05). The mean apoptotic index (AI: percentage of TUNEL signal positive cells)in Star-99 group (48.98±5.09 %) was significantly higher than that of the ethanol group (11.95±2.24 %) and the saline group (10.48±3.85 %) (P＜0.01). FCM DNA analysis showed that the appearance rate of the apoptosis peak in Srar-99group was 92.9 %, markedly higher than that of the ethanol group (14.3 %) and the saline group (0.0 %) (P＜0.01).Correlation (r=0.499, P＜0.05) was found between AI and serum level of TNF-α.CONCLUSION: Star-99 has an effect on the elevation of the serum levels of IL-2 and TNF-α. ft indicates that Star99 has the function of enhancing the cellular immunity and inducing cancer cell apoptosis. The correlation between AI and serum level of TNF-α indicates that the elevation of the serum of TNF-α induced by Star-99 may be an important factor in the promotion of the hepatic cancer cell apoptosis.Star-99 has strong effects on the inhibition and destruction of cancer cells. Its curative effect is as good as ethanol. Its major mechanisms can be as follows: (1) it increases the serum levels of IL-2 and TNF-α and triggers cellular immunity. (2) It can induce cancer cells apoptosis, the effective mechanism of the Star-99 is different from that of the ethanol. The mechanisms of triggering the immunologic function of the organism and inducing cell apoptosis are, of particular significance. This study will provide a new pathway of drug administration and an experimental basis for the treatment of HCC with Chinese herbal, and the study of Star-99 in the treatment of tumor is of profound significance with good prospects.     

Microarray-based method for detecting methylation changes of p16^Ink4a gene 5＇-CpG islands in gastric carcinomas

AIM: Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high-throughput approach for analyzing DNA methylation. The aim of this study was to describe a microarray-based method for detecting changes of DNA methylation in cancer. METHODS: This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Therefore, the amplified product might contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. Nine sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of p16 gene CpG islands in gastric carcinomas. The results were further validated by methylation-specific PCR (MSP). RESULTS: The experimental results showed that the microarray assay could successfully detect methylation changes of p16 gene in 18 gastric tumor samples. Moreover, it could also potentially increase the frequency of detecting p16 methylation from tumor samples than MSP. CONCLUSION: Microarray assay could be applied as a useful tool for mapping methylation changes in multiple CpG loci and for generating epigenetic profiles in cancer.     

瘦素启动子甲基化在骨关节炎发病中的作用

目的 探讨瘦素启动子甲基化在骨关节炎(OA)发病中的作用.方法 取OA组和对照组(创伤行截肢手术,除外OA等关节炎性疾病)患者膝骨关节标本,培养膝关节软骨细胞,采用不同浓度(5.0 μmol/L、10.0 μmol/L、20.0 μmol/L)不同时间(12 h、24 h、48 h、72 h、96h、120 h、168 h)5-氮杂-2'-脱氧胞苷(5-Aza-CdR)刺激软骨细胞,实时定量PCR检测膝关节软骨细胞瘦素mRNA表达,同时使用Epityper DNA甲基化分析技术枪测该基因启动子部分区域(-280～+79)的甲基化状态.结果 (1)10μmol/L 5-Aza-CdR刺激OA组软骨细胞,其瘦素mRNA表达水平增高,72 h最为显著.(2)OA组5-Aza-CdR刺激72 h后软骨细胞瘦素mRNA表达最高,对照组无5-Aza-CdR刺激软骨细胞瘦素mRNA表达最低.(3)使用非先导分层聚类分析法对瘦素启动子区域进行分析,2组甲基化模式存在差异,且 5-Aza-CdR刺激前后甲基化模式也不相同.结论 瘦素启动子部分位点的去甲基化可能是引起该基因异常表达导致OA发生的始动因素之一.

Abstract：

Objective To investigate the effects of 5-Aza-CdR( methylation transferase inhibitor) on the expression levels of leptin gene in chondrocytes and methylation states of leptin promoter region between osteoarthritis (OA) group and control. Methods The chondrocytes in osteoarthritis group were treated with 5-Aza-CdR with different doses and time-points, and the expression level of leptin was detected by real-time polymerase chain reaction for picking up the optimum dose and time-point. Next, the chondrocytes in 5 osteoarthritis patients and 5 control patients (amputation due to severe trauma) were treated with 5-Aza-CdR. Lastly, leptin mRNA expression levels in the four groups osteoarthritis and control chondrocytes treated with/without 5-Aza-CdR were measured by real-time PCR and the methylation state of promoter region ( - 280- + 79) was detected by epityper quantitative DNA methylation analysis. Results ( 1 ) After treating the chondrocytes in OA groups with 10 μmol/L 5-Aza-CdR for 72 h, the mRNA expression levels of leptin were increased significantly. ( 2 ) The mRNA expression levels of leptin were significantly different among the four groups ( P ＜ 0. 05 ), and the chondrocytes in osteoarthritis groups treated with 5-Aza-CdR showed a marked induction of leptin mRNA expression. (3) Analysis of quantitative methylation data using an unsupervised hierarchical clustering algorithm, showed that methylation patterns of leptin promoter was different between control and osteoarthritis chondrocyte treated with/without 5-Aza-CdR. Conclusion Demethylation of leptin promoter might up-regulate leptin gene expression level and it might contribute to osteoarthritis.     

Number of CpG islands and genes in human and mouse.

Estimation of gene number in mammals is difficult due to the high proportion of noncoding DNA within the nucleus. In this study, we provide a direct measurement of the number of genes in human and mouse. We have taken advantage of the fact that many mammalian genes are associated with CpG islands whose distinctive properties allow their physical separation from bulk DNA. Our results suggest that there are approximately 45,000 CpG islands per haploid genome in humans and 37,000 in the mouse. Sequence comparison confirms that about 20% of the human CpG islands are absent from the homologous mouse genes. Analysis of a selection of genes suggests that both human and mouse are losing CpG islands over evolutionary time due to de novo methylation in the germ line followed by CpG loss through mutation. This process appears to be more rapid in rodents. Combining the number of CpG islands with the proportion of island-associated genes, we estimate that the total number of genes per haploid genome is approximately 80,000 in both organisms.     

肝细胞肝癌谷胱甘肽硫转移酶P1甲基化研究

目的本研究探讨谷胱甘肽硫转移酶（GST）PI启动子CpG岛甲基化与肝细胞肝癌（HCC）的关系。方法用甲基化特异性PCR技术检测53例HCC肿瘤组织及其癌旁非肿瘤组织中，GSTP1基因启动子CpG岛甲基化状况。结果GSTP1基因在HCC肿瘤组织中的甲基化率显著高于癌旁非肿瘤组织（X^2=19．08，P〈0．001），在Ⅲ-Ⅳ期肿瘤中的甲基化率显著高于Ⅰ-Ⅱ期肿瘤（X^2=4．84，P=0．028），在不同大小的肿瘤之间、单个结节与多个结节的肿瘤之间以及包膜完整的肿瘤与包膜不完整的肿瘤之间甲基化率的差异均无显著性。结论GSTP1启动子CDG岛甲基化可能参与肝细胞的痛性转变。     

胃癌组织RASSF1基因启动子区甲基化的意义

目的:探讨抑癌基因RASSF1A启动子在胃癌组织及胃良性病变中甲基化的发生情况及与临床特征的关系.方法:采用甲基化特异性聚合酶链反应(MS- PCR)方法检测32例胃癌组织及32例相应的癌旁组织,以及46例胃良性病变中RASSF1A基因甲基化发生情况.结果:在32例胃癌DNA标本中,RASSF1A基因甲基化发生率为62.5%(20/32).而在32例癌旁组织中,只有1例存在甲基化,占3.1%(1/32),二者之间比较差异有统计学意义(P<0.05).在胃良性病变,浅表性胃炎和萎缩性胃炎中甲基化发生频率分别为3.3%和37.5%.RASSF1A基因甲基化发生率与胃癌分化程度、肿瘤大小及淋巴结转移之间无显著相关性.结论:RASSF1A基因启动子区甲基化在胃癌的发生发展中起重要作用.