大肠癌中nm23－H_1表达的定量研究及临床意义

为研究肿瘤转移相关基因ｎｍ２３－Ｈ１ｍＲＮＡ在人大肠癌中的表达及其与临床病理的关系，应用ＲＴ－ＰＣＲ定量技术，对３６例大肠癌组织中ｎｍ２３－Ｈ１基因表达进行了定量研究。结果表明，大肠癌中ｎｍ２３－Ｈ１表达显著高于正常大肠粘腹；ＤｕｋａｓＤ期中其表达显著低于ＤｕｋｅｓＡ、Ｂ、Ｃ期；有远处转移者其表达亦显著低于无远处转移者，提示：ｎｍ２３－Ｈ１基因可能参与大肠癌的恶性进展，其表达程度与大肠癌远处或广泛转移呈负相关。     

散发性结肠癌中β-catenin mRNA表达的即时聚合酶链反应分析

目的探讨散发性结直肠癌(SCRC)组织中β-catenin mRNA 的含量与 SCRC 组织中的β-catenin 蛋白异常表达以及临床病理学指标间的关系。方法用 TaqMan 即时荧光定量聚合酶链反应(PCR)方法检测了81例 SCRC 组织和28例癌旁正常黏膜组织的β-catenin mRNA 含量(每个样本的β-catenin cDNA 拷贝数与 GAPDH cDNA 拷贝数的比值作为β-catenin mRNA 含量);以 EnVision 二步法检测了β-catenin 蛋白在 SCRC 组织和癌旁正常黏膜组织中的表达。结果在 SCRC 组织中β-catenin mRNA 含量(2.527±2.284)低于癌旁正常黏膜组织中的含量(5.003±3.326),差异有统计学意义。有淋巴结转移组的β-catenin mRNA 含量(1.827±1.288)低于无淋巴结转移组(3.359±2.881),P<0.05;溃疡型和浸润型生长组β-catenin mRNA 含量(2.202±2.035)低于隆起型生长组(3.108±2.610),P<0.05;在β-catenin 胞质或胞核异常表达组和无胞质或胞核异常表达组织之间,β-catenin mRNA 含量的差异无统计学意义。结论β-catenin mRNA 的低转录水平与 SCRC 的淋巴结转移及溃疡浸润型生长相关,而与β-catenin 蛋白的胞质胞核异常表达无明显关联。定量检测β-cateninmRNA 水平可能是一种判断 SCRC 生物学行为的有用方法。     

A clinicopathological analysis of KISS1 and KISS1R expression in colorectal cancer

Kisspeptins, the products of the KISS1 gene have tumor suppressing and antimetastatic properties. We aimed to study KISS1 and KISS1R expression in colorectal cancer. We analyzed KISS1 and KISS1R expression using immunohistochemistry and image analysis in normal and malignant tissue samples from 111 patients with colorectal adenocarcinoma. KISS1 expression was much higher in the normal than in the malignant colonic mucosa. Regarding malignant tissues, KISS1 levels were higher in larger tumors, in stage III and IV cancers, in cancers with lymph node metastasis and in tumors located in the distal part of the large intestine. Patients with greater KISS1 levels had worse prognosis. No KISS1R expression was detected in normal or malignant tissues or in liver metastases. KISS1 expression is reduced during the malignant transformation of the colonic mucosa. However, larger and advanced colorectal cancers express more KISS1, without reaching the former normal levels, and increased KISS1 levels are associated with worse prognosis. Finally, neither the normal nor the malignant colonic epithelial cells produce KISS1R.     

应用蛋白质组学分析结直肠癌促泌素和糖调节蛋白78蛋白变化及其意义

目的分析结直肠癌、配对正常黏膜组织差异表达蛋白质,寻找与结直肠癌发生、发展相关的分子标记物。方法固相pH梯度双向凝胶电泳分离结直肠癌患者配对癌及正常黏膜组织的总蛋白,液相色谱串联质谱鉴定差异表达蛋白质点。半定量逆转录聚合酶链反应（RT—PCR）、Western blot及免疫组织化学（EnVision法）方法检测促泌素（SCGN）及糖调节蛋白78（GRP78）两个差异表达蛋白质及组织定位,并检测54例神经内分泌肿瘤SCGN的表达。结果比较结直肠癌、配对正常黏膜组织蛋白质表达图谱,共获得5倍以上差异蛋白质点35个,癌组织中表达上调15个、下调20个。质谱分析鉴定14个蛋白质。RT-PCR及Western blot证实结直肠癌组织SCGN低表达,免疫组织化学染色发现正常结直肠黏膜神经内分泌细胞及98％（53／54）的神经内分泌肿瘤SCGN阳性。GRP78蛋白在结直肠癌组织中的表达明显高于正常黏膜组织,但在mRNA水平上癌组织和正常组织之间差异无统计学意义。结论双向凝胶电泳结合质谱分析是筛选肿瘤异常表达蛋白质的有效手段,所获得的35个差异表达候选蛋白质可能与结直肠癌的发生、发展有关。SCGN是一个候选神经内分泌标记物,GRP78蛋白表达增高可能涉及翻译后修饰。     

结直肠癌患者肠道UGT1A基因的多态性表达

目的：从mRNA水平及蛋白质水平分析结直肠粘膜UGT1A基因位点的多态性表达。 方法： 结直肠癌病例组40例，正常人肠道粘膜对照组20例。逆转录聚合酶链反应分析结直肠癌组、正常人群肠道粘膜UGT1A mRNA表达；免疫印迹法检测各组UGT1A蛋白的表达。 结果： (1)UGT1A mRNA量的差异表达：结直肠癌组织中UGT1A mRNA表达明显低于其周围正常粘膜，而后者低于正常人群肠粘膜组织的表达量，P<0.01。结直肠癌病例组、正常人群结直肠粘膜组织呈现个体差异表达。(2)结直肠粘膜组织UGT1A各同Ⅰ型的多态性表达：癌组织、癌周正常粘膜及在正常人群肠道粘膜中UGT1A各同Ⅰ型表达例数各不相同。UGT1A1、1A3、1A4、1A6、1A9 mRNA表达水平在癌组织表达低于周围正常粘膜，P<0.01；而UGT1A8、1A10在癌组织中mRNA表达量高于周围正常粘膜，P<0.01。(3)UGT1A蛋白的差异表达：其灰度值比值在癌组织显著低于周围正常组织，后者又显著低于正常人肠粘膜，P<0.01。各组蛋白表达的深浅度亦各不相同。 结论： (1)UGT1A基因位点的多态表达不仅存在于结直肠癌组织及周围的正常组织，亦存在于正常人群肠粘膜；(2)结直肠粘膜上皮UGT1A基因位点在转录水平及功能水平均存在着多态调节，不同个体对致癌物的易感性不同可能是这种差异表达的结果。     

人卵巢癌中三种MAGE家族基因表达和患者HLA表型的初步分析

分析卵巢癌中MAGE家族基因成员MAGE 1, 2 , 3基因mRNA表达和患者HLAI类分子表型 ,为卵巢癌的免疫治疗提供依据。应用RT PCR技术检测卵巢癌和正常组织中MAGE 1, 2 , 3基因mRNA表达 ,同时采用血清学方法检测卵巢癌患者HLAI类分子表型 ,并分析基因表达和HLAI类分子表型分布的相互关系。 31例卵巢癌中阳性表达率分别MAGE 1为7/31(2 3% ) ,MAGE 2为 2 0 /31(64% )和MAGE 3为 6/31(19% ) ,其中至少一种MAGE基因表达为 2 7/31(90 % ) ,而正常组织除睾丸外未见任何一种基因表达。患者的HLAI类分子表型多为HLA A2、A11、A19和B15、B35、B40。对卵巢癌中MAGE 1, 2 , 3基因特异性表达分析和HLAI类分子表型的鉴定 ,有助于肿瘤的分子诊断和免疫治疗     

Expression of IAP family proteins in esophageal cancer

Members of the inhibitor of apoptosis protein (IAP) family, including survivin, have been reported to be expressed in many tumors. However, their expression in esophageal cancer has not been clarified completely. We investigated the expression of mRNA for IAP family proteins in samples from esophageal cancers and their adjacent normal mucosa tissues by real-time quantitative RT-PCR. The survivin expression in esophageal cancer was significantly higher than that in normal mucosa (P < 0.05). Other IAP family proteins including cIAP1, cIAP2, NAIP and XIAP tended to show stronger expression in cancer tissue than normal mucosa, although the differences were not significant. As to the histological type of tumor, poorly differentiated squamous cell carcinomas exhibited significantly higher level of expression than well-differentiated carcinomas (P < 0.05). The proportion of apoptotic cells of cancer tissue inversely correlated with the intensity of survivin expression (P < 0.05). Immunohistochemical staining demonstrated cytoplasmic as well as nuclear expression of survivin in esophageal cancer, and further, in situ hybridization analysis demonstrated cytoplasmic expression of mRNA for survivin. The results suggest that the expression of IAP family proteins, especially survivin, may be associated with the biological character of esophageal cancer, such as apoptosis.     

Expression of ATP1AL1, a non-gastric proton pump,in human colorectum

The expression of mRNAs encoding a human nongastric proton pump (ATP1AL1) in the colorectum was investigated. The real-time PCR gave significant levels of signals not only in the distal part of human colon and rectum, but also in the proximal part of the colon. ATP1AL1 mRNA was overexpressed in 12 out of 20 human colorectal adenocarcinomas compared with the level in the accompanying normal mucosa. It is noted that astonishing levels of the mRNA overexpression were found in 4 carcinomas, which were detected even by Northern blot. The very high levels of ATP1AL1 mRNA expression in some cancer tissues may be connected to an unknown specific pathophysiological condition.     

Methylation status of T-lymphoma invasion and metastasis 1 promoter and its overexpression in colorectal cancer

T-lymphoma invasion and metastasis 1 has been implicated in tumor invasion and metastasis. However, the regulatory mechanisms underlying aberrant T-lymphoma invasion and metastasis 1 expression in human colorectal cancer have not been well defined. To investigate the relationship between methylation status and expression levels of T-lymphoma invasion and metastasis 1 gene, methylation-specific polymerase chain reaction, and immunohistochemistry staining were performed in 232 matched samples of human colorectal cancer tissue and normal colorectal mucosa. Results showed that T-lymphoma invasion and metastasis 1 protein was overexpressed in colorectal cancer, especially in metastatic cases (P < .001). The degree of T-lymphoma invasion and metastasis 1 promoter methylation was a little lower in cancer tissues than in matched normal mucosa (P < .05), and the expression level of T-lymphoma invasion and metastasis 1 was inversely related to the methylation status in cancer tissues (P < .001). Colon cancer cell lines HT29 and LS174T were treated with demethylating agent 5-aza-2'-deoxycytidine, resulting in promoter hypomethylation accompanied by reexpression of T-lymphoma invasion and metastasis 1 mRNA and protein. In contrast, colon cancer cell lines SW620 and LoVo were treated with hypermethylation agent S-adenosylmethionine, resulting in T-lymphoma invasion and metastasis 1 promoter hypermethylation, accompanied by suppression of T-lymphoma invasion and metastasis 1 expression and inhibition of cell growth, plate colony formation, and migration. The present study demonstrates that overexpression of T-lymphoma invasion and metastasis 1 is associated with hypomethylation status of T-lymphoma invasion and metastasis 1 promoter region in colorectal cancer tissues. It suggests that promotor hypomethylation of T-lymphoma invasion and metastasis 1 may play a role in the progression and metastasis of colorectal cancer. Pharmacologic reversal of T-lymphoma invasion and metastasis 1 promoter hypomethylation may inhibit cell proliferation and migration.     

Period 1 and estrogen receptor-beta are downregulated in Chinese colon cancers

To investigate whether Period 1 (PER1) and Estrogen receptor-beta (ER2) are associated with occurrence and development of Chinese colorectal cancers. By using RT-quantitative PCR, tissue microarray (TMA) and immunohistochemistry, we detected mRNA levels and protein levels of PER1 and ER2 in the cancerous tissues and paired normal adjacent tissues in patients with colorectal cancer. Survival analyses were performed by the Kaplan-Meier method utilizing log-rank test and univariate and multivariate Cox proportional modeling to measure 5-year disease-free survival (DFS) and overall survival (OS). Real-time PCR showed that, the delta Ct value (tumor tissue vs. normal mucosa) of PER1 or ER2 is 8.51±2.81 vs. 7.34±2.08 or 12.39±2.43 vs. 9.76±1.75, expression of PER1 and ER2 decreased significantly in tumor tissues compared with noncancerous mucosas of patients with or without metastasis (both of P values <0.001). Spearman test revealed that PER1 and ER2 were significantly down-regulated in cancerous tissues (r=0.283; P<0.001) which was also confirmed by immunohistochemistry of specimens from 203 colon cancer patients in a TMA format. The reduction of PER1 was associated with gender and distant metastasis (P=0.037 and P<0.001, respectively) whereas the decline of ER2 was associated with age (P=0.043) by analyzing the clinical data. However, we were not capable of detecting any association between PER1 level or ER2 level and overall survival (OS) or disease free survival (DFS). It is the first observation of correlated reduction of PER1 and ER2 in Chinese colon cancers, and they do play a certain role in colorectal cancer.     

Expression of Wnt ligands and Frizzled receptors in colonic mucosa and in colon carcinoma.

AIMS: Signalling through the Wnt pathway is integrally associated with colon carcinogenesis. Although activating mutations in the genes for adenomatous polyposis coli (APC) and beta-catenin are clearly associated with colon cancer, less is understood about the role of the upstream secreted ligands (Wnts) and their receptors (frizzled, Fz) in this process. In other systems, increased Wnt signalling has been shown to alter the expression of components of this pathway. This study was designed to test the hypothesis that colon cancer is characterised by aberrant expression of specific Wnt genes and Fz receptors. METHODS: The expression of Wnt genes was assessed by in situ, antisense RNA hybridisation in paraffin wax embedded samples of normal and malignant human colon tissues with probes specific for the individual Wnt genes. The expression of Fz1 and Fz2 was determined by immunoperoxidase based antibody staining on human tissues. RESULTS: Changes in the expression of some ligands and receptors were seen in colon cancer. For example, Wnt2 mRNA was detected in colon cancer but was undetectable in normal colonic mucosa. Differential expression of Wnt5a in normal mucosa was also noted, with increased expression at the base of the crypts compared with the luminal villi and slightly increased expression in colon cancer. Wnt7a exhibited minimal expression in both normal and malignant colon tissues, whereas other Wnt ligands including Wnts 1, 4, 5b, 6, 7b, and 10b were expressed equally and strongly in both normal and malignant colon tissues. In defining cellular responses and phenotype, the type and distribution of Fz receptors may be as important as the pattern of Wnt ligand expression. No expression of Fz receptor 1 and 2 was seen in normal colonic mucosa and in well differentiated tumours. However, poorly differentiated tumours exhibited a high degree of Fz receptor expression, especially at the margin of cellular invasion. CONCLUSIONS: These data indicate that the expression of members of the Wnt signal transduction pathway, distinct from APC and beta-catenin, is integrally associated with the process of colon carcinogenesis. Wnt2, and possibly Wnt5a, may be involved in the progression from normal mucosa to cancer and the expression of Fz1/2 receptors may be involved in processes associated with tumour invasion. Altered expression of these Wnts and Fz receptors may prove useful as prognostic or diagnostic markers for patients with colon cancer.     

Evaluation of cyclin D1 mRNA expression in gastric and colorectal cancers

Cyclin D1 is a G1 cyclin that controls the transition of the cell cycle from G1 phase to S phase, and its gene is located on chromosome 11q13. We evaluated the expression of cyclin D1 mRNA in surgically resected specimens of gastric and colorectal cancers using quantitative RT-PCR. In this method, cDNA derived from cyclin D1 mRNA was amplified in a tube together with an internal control. The expression of cyclin D1 mRNA was high in 8 of 36 gastric cancer tissues (22%) and 9 of 27 (33%) colorectal cancer tissues, compared to normal mucosal tissues. In gastric cancers, the rate of cyclin D1 mRNA expression (an index of the density of DNA bands) was significantly higher in patients with tumors invading beyond the submucosal layer, regional lymph nodes and lymphatic vessels (i.e., patients with stage III or IV). In colorectal cancers, the rate of cyclin D1 mRNA expression was significantly higher in patients with venous invasion. Moreover, in patients with colorectal cancer, the survival rate of high-expression group was significantly lower than in low-expression group. Our results suggested that overexpression of cyclin D1 mRNA reflected the severity of gastric cancer and poor prognosis of colorectal cancer.     

Dipeptidyl Peptidase 10, a Novel Prognostic Marker in Colorectal Cancer

Purpose

The dipeptidyl peptidase IV (DPPIV) gene family exhibits multiple functions and is involved in the pathogenesis of various diseases. It has attracted pharmaceutical interest in the areas of metabolic disorders as well as cancer. However, clinicopathologic significance of DPPIV family in colorectal cancer is not fully understood.

Materials and Methods

The clinical relevance of DPPIV and DPP10 expression was determined by immunohistochemical staining, and by assessing its clinicopathologic correlation in 383 colorectal cancer patients with known clinical outcomes.

Results

DPPIV was not expressed in normal colon mucosa, but it showed luminal expression in 52 of the 383 colorectal cancers (13.5%). DPPIV expression in tumors was associated with right-sided location of the colon (p=0.010) and more advanced tumor stage (p=0.045). DPP10 was expressed in normal colonic mucosa, but its expression varied in primary colorectal cancer tissues. Loss of DPP10 expression was found in 11 colorectal cancers (CRCs) (2.9%), and multivariate analysis showed that loss of DPP10 expression was an independent factor for poor patient prognosis (p=0.008).

Conclusion

DPP10 may play a role in disease progression of colorectal cancer and loss of DPP10 expression in primary CRC is significantly associated with poor survival outcomes.     

KISS1 expression in colorectal cancer

Kisspeptins, the products of the KISS1 gene, are involved in cancer invasion, migration, metastasis and angiogenesis, while they induce apoptosis in various cancers. Herein, we studied KISS1 expression in colorectal cancer. We analyzed KISS1 expression using immunohistochemistry and image analysis in normal and malignant tissue samples from 60 patients with colorectal adenocarcinoma. The results correlated with various clinicopathological parameters. The expression of KISS1 was much higher in normal than in malignant colonic mucosa. However, among malignant tissues, KISS1 expression was higher in larger tumors (>4 cm) than in smaller ones (≤4 cm) and in stages III and IV than in stages I and II. In addition, it was higher in patients with lymph node metastases. Moreover, KISS1 levels in the normal mucosa and their difference from those in the malignant mucosa were higher in the right part of the large intestine than in the left one. KISS1 expression is reduced during the malignant transformation of the colonic mucosa and there is a difference in the expression pattern between the right and the left part of the large intestine. However, larger and advanced colorectal tumors express higher KISS1 levels than smaller and localized ones.     

Overexpression of the obesity hormone leptin in human colorectal cancer

BACKGROUND: Leptin is an adipocyte-derived neurohormone, high levels of which are found in obese individuals. Leptin controls energy expenditure, acting in the brain, and regulates different processes in peripheral organs. Recent studies have suggested that leptin may be involved in cancer development and progression. AIMS: To analyse leptin expression in human colorectal cancer as well as in colorectal mucosa and colorectal adenomas. METHODS: Leptin expression was assessed by immunohistochemistry in 166 colorectal cancers, 101 samples of colorectal mucosa and 41 adenomas. Leptin concentration in colorectal cancer was correlated with selected clinicopathological features. RESULTS: Immunoreactivity for leptin was observed in 51.2% (85/166) of primary colorectal cancers. In adenomas leptin expression was observed in 14.6% (6/41) of studied cases. In normal mucosa, leptin was present at low levels, except in tumour bordering areas where its concentration appeared to reflect levels in the adjacent cancer tissue. Leptin expression in colorectal cancer significantly correlated with tumour G2 grade (p = 0.002) as well as with histological type (adenocarcinoma) of tumours (p = 0.044). CONCLUSIONS: Results indicate that leptin is overexpressed in human colorectal cancer, which suggests that the hormone might contribute to colorectal cancer development and progression.     

Expression of caveolin-1 is correlated with Akt-1 in colorectal cancer tissues

Caveolin is a major component of caveolae which is a plasma membrane microdomain. The emerging role of caveolin in tumorigenesis was based mainly on in vitro experiments with cancer cell lines. We performed semi-quantitative RT-PCR for caveolin, Akt and EGFR to understand the role of caveolins in colorectal tumor biology. Cancer tissue samples and the neighboring normal colon mucosa were obtained from 95 colorectal cancer patients who underwent operations at Ewha Womans University Mokdong Hospital. With these fresh tissues, semi-quantitative RT-PCR was performed by coamplification of the gene for caveolin-1, EGFR and Akt-1 with beta-actin. The average age was 60.21+/-13.33 years old, and sex ratio was 1.44:1. Caveolin-1 is more expressed in tumors than normal mucosa (P=0.025). The expression of caveolin-1 and Akt-1 had a definitive positive relationship (P=0.002). But, the expression of caveolin-1 and EGFR was not significantly related. We could not find correlations between caveolin-1 expression and clinical factors. In conclusion, caveolin-1 is more expressed in cancer tissues than normal colon and related with Akt-1, not with EGFR expression in colorectal cancer tissues, which suggests that signaling for caveolin-1 affects Akt-1 activation, but this reaction is not initiated by EGFR stimulation in colon cancer.