B2基因结构及其在结直肠癌中的表达

目的　明确B2基因与胰岛素样生长因子结合蛋白 相关蛋白 1(insulin likegrowthfactorbindingproteinrelatedprotein 1,IGFBP rP1)基因在结构上的关系以及其在结直肠肿瘤中的表达改变。方法　用 5′RACE(rapidamplificationofcDNAend)技术获得B2全长 ,用半定量逆转录 聚合酶链反应(RT PCR)法和免疫组织化学SP法检测 76例结直肠癌组织和配对的正常黏膜以及 6个结肠癌细胞系(SW480 ,SW1116,SW62 0 ,HCT8,CoLo2 0 5和LoVo)中B2的表达水平。结果　获得的B2基因全长 112 5bp ,与IGFBP rP1一致性为 112 2 / 112 5。mRNA水平上 ,B2 /IGFBP rP1的表达水平从正常 癌旁 腺瘤 腺癌逐步递增 (P <0 0 5 )。除SW480外 ,其余 5个细胞系中均无该基因的表达。蛋白水平上 ,腺癌与正常黏膜之间B2 /IGFBP rP1的表达阳性率差异有显著意义 (P <0 0 1)。B2 /IGFBP rP1在侵袭前沿肿瘤细胞中高表达的病例 ,发生淋巴结转移概率较高 ,肿瘤的浸润深度较深 ,B2 /IGFBP rP1的阳性程度也较高 (P <0 0 5 )。结论　B2与IGFBP rP1是同一个基因 ;B2 /IGFBP rP1的表达改变可能与结直肠肿瘤的发生、发展有关 ;高表达B2 /IGFBP rP1的肿瘤细胞可能具有高的侵袭潜能     

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.     

A critical role for Rac1 in tumor progression of human colorectal adenocarcinoma cells

Colorectal adenocarcinoma is the second cause of cancer mortality in developed countries. Rac1 is a member of the family of Rho GTPases that regulates many intracellular signaling pathways, including those involved in tumorigenesis, invasion, and metastasis. We have investigated the role of Rac1 in colorectal tumor progression by genetic modification of the human colorectal adenocarcinoma cell line SW620 to either overexpress Rac1 or lack Rac1 expression. Tumor behavior was studied by orthotopic injection of stably modified cell lines into the cecal wall of athymic nude mice, a model that replicates the histopathological appearance and clinical behavior of human colorectal adenocarcinoma in humans. While overexpression of Rac1 resulted in an accelerated tumorigenic process, inducing a faster mortality rate, inhibition of Rac1 completely suppressed tumor formation. These results suggest that Rac1 plays a major role in colorectal adenocarcinoma progression. Finally, interference with Rac1 function may provide an important tool to block the malignant phenotype of colorectal adenocarcinoma cells.     

Expression of KITENIN in human colorectal cancer and its relation to tumor behavior and progression

KAI1 COOH-terminal interacting tetraspanin (KITENIN) contributes to tumor invasion and metastasis in various cancers. The aim of current study was to evaluate whether KITENIN affects tumor cell invasion and prognosis in human colorectal cancers. We investigated the biologic role of KITENIN on tumor cell invasion by using small interfering RNA in Caco2, DLD1, and SW480. We evaluated the expression of KITENIN and activator protein-1 (AP-1) target genes in human colorectal cancer tissues. The tumor cell invasion was decreased by knockdown of KITENIN in three tested cell lines. The mRNA expression of cyclin D1 and COX-2 was decreased in KITENIN knockdown Caco2 and the mRNA expression of MMP-3 and COX-2 was decreased in KITENIN knockdown DLD1 and SW480. The extracellular-signal protein kinase 1/2 (ERK1/2) phosphorylation was decreased in KITENIN knockdown in three tested cell lines. Expression of KITENIN and AP-1 target genes was significantly increased in human colorectal cancer tissues. The ERK1/2, c-Jun N-terminal kinase (JNK) and p38 phosphorylations were increased in human colorectal cancer tissues. Expression of KITENIN was significantly associated with lymphovascular invasion, depth of invasion, lymph node metastasis, tumor stage and poor survival. These results indicate that KITENIN is associated with human colorectal cancer progression including invasion and metastasis.     

CHRDL1 调节结直肠癌增殖、 迁移和侵袭

目的 探讨腱蛋白样蛋白 1 (chordin-like 1, CHRDL1) 对结直肠癌 (colorectal cancer, CRC) 进 展的调节作用。 方法 数据库分析 CHRDL1 与 CRC 进展、 预后关系, 构建 CHRDL1 过表达 SW480、 HT29 细胞系并检测细胞增殖、 迁移和侵袭, 体外移植瘤实验检测肿瘤生长, 分析 CHRDL1 过表达对 CRC 细胞迁 移和侵袭作用机制。 结果 CHRDL1 在 CRC 进展中呈高表达且可影响预后。 CHRDL1 过表达可促进细胞增 殖、 迁移、 侵袭和移植瘤生长, 促进 Vimentin、 N-Cadherin、 snail 表达, 抑制 E-Cadherin 表达, CHRDL1 与 snail 存在互作关系, 干扰 snail 表达可逆转上述指标趋势。 结论 CHRDL1 可促进 CRC 细胞增殖, 还可通 过 Snail 促进 CRC 细胞上皮间质转化来发挥促进细胞迁移、 侵袭作用。     

MicroRNA-145 suppresses cell migration and invasion by targeting paxillin in human colorectal cancer cells

A number of cancers show increased expression of paxillin which plays a central role in tumor progression, including colorectal cancer. However, the mechanisms causing paxillin upregulation remains unclear. In our study, bioinformatics analyses suggested that paxillin is predicted to be a direct target of miR-145. We firstly identified paxillin as a new target of miR-145 and demonstrated that miR-145 inhibits paxillin expression by binding to the paxillin mRNA 3’UTR. Therefore, we assume overexpression of paxillin induced by suppression of miR-145 may promote cell migration and invasion. We detected the expression of paxillin and miR-145 in human colorectal cancer tissues by real-time quantitative PCR. Higher expression of paxillin and lower expression of miR-145 was observed in colorectal cancer tissues than corresponding paracancerous tissue. Moreover, the expression of paxillin was negatively correlated with miR-145 expression. A dual-luciferase reporter assay was used to confirm that paxillin was a direct target of miR-145. In CRC cell lines, overexpression of miR-145 could downregulate paxillin protein expression levels, and ectopic overexpression of miR-145 mimics or inhibitor could inhibit or promote cell migration, invasion, proliferation and clone formation in vitro. Taken together, these data suggested that miR-145 plays a pivotal role in colon cancer through inhibiting cell proliferation migration and invasion, and miR-145 may serve as a tumor suppressor by targeting paxillin gene.     

RhoGDI2在结直肠癌中的表达及临床意义

目的：探讨RhoGDI2在结直肠癌中表达及临床意义。方法：通过免疫组化检测 RhoGDI2 在结直肠癌组织、癌旁组织及转移淋巴结中表达,分析其与CRC患者临床因素及生存期之间的关系。结果：RhoGDI2在癌旁组织、肿瘤原发灶及转移淋巴结中表达逐渐升高,差异有统计学意义（P＜0.001）; RhoGDI2表达与淋巴结转移、远处转移关系密切（P＜0.05）;高表达RhoGDI2者总生存期及无病生存期短。结论：RhoGDI2在结直肠癌组织中高表达,且与结直肠癌淋巴结转移、远处转移明显相关,高表达RhoGDI2者总生存期及无病生存期短。     

CXCR4/CXCL12 expression profile is associated with tumor microenvironment and clinical outcome of liver metastases of colorectal cancer

Interaction between CXCR4 and CXCL12 plays a role in tumor progression. The present study examined CXCR4, CXCL12 and CD133 expression in liver metastases of colorectal cancer (CLM) and determined whether the expression profiles affect the tumor microenvironment and thus progression, and whether they could serve as a prognostic marker for survival. Liver metastases of colorectal cancer collected from 92 patients were evaluated by CXCR4, CXCL12 and CD133 immunohistochemistry and clinicopathological data were analyzed. The expression profile of CXCR4 was determined in the colorectal cancer cell line, SW48. The expression of cytoplasmic CXCR4 was higher in 36 (39%) patients than that indicated by CXCR4 staining intensity of hepatocytes. High levels of nuclear CXCR4 expression in 23 (25%) patients significantly correlated with CXCL12 expression in hepatocytes. Nuclear CXCR4 expression was increased in the cancer cells after exposure to CXCL12. Univariate and multivariate analyses demonstrated that the high levels of nuclear CXCR4 and CXCL12 expression in hepatocytes were significantly better prognostic factors for overall and hepatic disease-free survival in patients with CLM. The expression of CXCR4 and CXCL12 in CLM may have an interactive effect that could alter the tumor microenvironment. CXCR4 expression in metastatic liver tumors together with the upregulation of CXCL12 in hepatocytes may help to predict the clinical outcomes of patients with CLM after hepatectomy.     

MiR-873-5p inhibits cell migration,invasion and epithelial-mesenchymal transition in colorectal cancer via targeting ZEB1

Recent studies have demonstrated that dysregulation of mircoRNAs (miRNAs) greatly affected biological processes of human cancers, including colorectal cancer. As a member of miRNAs family, miR-873-5p has been proved to be a tumor suppressor in some human cancers. Here, we aim to investigate the effects of miR-873-5p on the migration, invasion and epithelial-mesenchymal transition (EMT) of colorectal cancer cells. The low expression of miR-873-5p in colorectal cancer cells was identified by conducting qRT-PCR analysis. Gain of function assays were designed and conducted to demonstrate the specific function of miR-873-5p overexpression in colorectal cancer progression. Transwell assay and western blot assay were conducted and revealed that miR-873-5p inhibited cell migration, invasion and EMT formation. To find the downstream molecular mechanism of miR-873-5p, mechanism assays were designed and performed to find the downstream target of miR-873-5p. ZEB1 (Zinc finger E-box-binding homeobox 1) was certified to be the target of miR-873-5p through bioinformatics analysis, luciferase activity assay and pull-down assay. Finally, rescue assays were carried out to demonstrate the effects of miR-873-5p-ZEB1 axis on the migration, invasion and EMT process of colorectal cancer cells. In conclusion, we confirmed that miR-873-5p suppressed cell migration, invasion and EMT in colorectal cancer via targeting ZEB1.     

eEF1A2 promotes cell migration, invasion and metastasis in pancreatic cancer by upregulating MMP-9 expression through Akt activation

eEF1A2 is a protein translation factor involved in protein synthesis that is overexpressed in various cancers, with important functions in tumor genesis and progression. We have previously showed that the ectopic expression of eEF1A2 is correlated with lymph node metastasis and perineural invasion in pancreatic cancer. In this study, we investigated the functional role of eEF1A2 in the regulation of cell migration, invasion, and metastasis in pancreatic cancer. Furthermore, we investigated the potential molecular mechanisms involved. By evaluating the invasive ability of a panel of pancreatic cancer cell lines with different metastatic potentials, eEF1A2 expression in cells was positively associated with their invasive ability. The knockdown of eEF1A2 by siRNA decreased the migration and invasion of PANC-1 cells. By contrast, the ectopic expression of exogenous eEF1A2 significantly promoted the migration and invasion of SW1990 cells. Stable eEF1A2 overexpression in a nude mouse model of peritoneal metastasis likewise dramatically enhanced the intraperitoneal metastatic ability of SW1990 cells. In addition, eEF1A2 overexpression could upregulate MMP-9 expression and activity. A significant positive correlation between the overexpression of both eEF1A2 and MMP-9 was observed in pancreatic cancer tissues. The inhibition of MMP-9 activity reduced the promoting effect of eEF1A2 on cell migration and invasion. Furthermore, eEF1A2-mediated cell migration and invasion, as well as MMP-9 expression and upregulation, were largely dependent on the eEF1A2-induced Akt activation. The findings suggested the potentially important role of eEF1A2 in pancreatic cancer migration, invasion, and metastasis. Thus, the results provide evidence of eEF1A2 as a potential therapeutic target in the treatment of aggressive pancreatic cancer.     

Expression of the adult intestinal stem cell marker Lgr5 in the metastatic cascade of colorectal cancer

The recently advanced cancer stem cell model postulates that progression and metastasis of cancer are mainly driven by tumor cells with stem cell properties. Intestinal cancer stem cells are difficult to study due to the lack of reliable markers, but expression of the Wnt target gene Lgr5 is promising to define at least stem cell like cells in intestinal and colorectal cancer. The aim of this study was to find a possible link of stem cell like cancer cells to the metastatic process of colorectal cancer. To this end, we evaluated immunohistochemical Lgr5 expression in 31 distant metastases and in primary tumor compartments with relevance for metastasizing, comprising 89 colorectal carcinomas. Lgr5 expression was seen in 51.6% of distant metastases. 12.9%, 14.8% and 26.7% of primary tumors with histologically confirmed tumor buds, angioinvasion and perineural infiltrates, respectively, showed evidence of Lgr5 expression in these tumor compartments. However, distant metastases, which were derived from carcinomas with such Lgr5 positive tumor compartments, showed 6- to 11.5-fold higher median value of Lgr5 expression compared to those metastases derived from tumors without Lgr5 expressing cells in these compartments. These differences between the metastases were statistically significant, if being related to tumor buds (all tumors; p = 0.047) and to vascular infiltrates (stage IV tumors; p = 0.007). In conclusion, our results point to rare evidence of Lgr5 positive stem cell like cells in the metastatic cascade of colorectal cancer, but these few cells might be biologically powerful in the metastatic process of cancer subsets. Clonal analysis is necessary to proof this hypothesis.     

ST14/MT-SP1影响MT2-MMP和TIMP-2的表达而增强结直肠癌细胞的侵袭力

目的： 膜型丝氨酸蛋白酶（ST14 / MT-SP1）和它的同源物在细胞迁移和肿瘤转移中起重要作用。本研究目的是评估ST14/MT-SP1过度表达如何影响结直肠癌细胞的侵袭能力。方法: 全长人ST14/MT-SP1基因被瞬时转染到结直肠癌细胞系RKO。表达产物由Ni2+-亲和层析柱纯化并通过明胶酶谱法分析蛋白的明胶酶活性。用ECM体外侵润试验确定细胞的体外侵袭力。用cDNA微阵列法测定ST14/MT-SP1转染细胞中MMPs和TIMPs表达变化情况。用实时定量PCR 来验证这些基因表达的变化。结果: 人全长ST14/MT-SP1基因被转染到结直肠癌细胞系RKO后，纯化表达的蛋白具有明胶酶的活力。RKO细胞过度表达ST14/MT-SP1 后其体外侵润转移能力显著增强(P<0.01),而ST14/MT-SP1蛋白被阻断后使SW480和SW620细胞的侵袭能力降低(P<0.01)。进一步发现，ST14/MT-SP1 过度表达使RKO 细胞的 MT2-MMP(MMP-15)表达显著上调（约2.5倍） 和TIMP2表达下调（约0.35倍）。结论: ST14/MT-SP1 过度表达导致了结直肠癌细胞侵袭力增强，这种能力的增强是由于ST14/MT-SP1自身具有明胶酶的活性和ST14/MT-SP1 能上调 MT2-MMP与下调TIMP-2的表达。因此,ST14/MT-SP1过度表达可能增强结直肠癌细胞的侵袭能力。     

具有相同遗传背景的人结肠癌细胞系生物学特性的鉴定

目的 对三种取自不同转移部位的结肠癌细胞系进行生物学特性的鉴定.方法 从体内成瘤性、原位移植后的自发性转移能力,以及体外生长、克隆形成率、粘附、运动、侵袭能力等方面,探讨具有相同遗传背景的SW480、SW620以及SW480/M5三种细胞系生物学特性的差异.结果 体内实验证明SW480具有多器官转移的潜能,SW620只具有淋巴结转移的能力,SW480/M5只具有肝脏转移的能力,SW480/M5的皮下瘤生长速度最快,其次是SW620细胞;体外实验证明W48/M5和SW620的体外生长、克隆形成能力均强于SW480细胞,而两者对纤维黏连蛋白的粘附能力较SW480细胞弱,SW480/M5的运动及侵袭能力强于SW480及SW620细胞.结论 与SW480细胞相比,SW480/M5和SW620细胞具有一定的器官亲和力,三者的遗传背景一致,是研究结肠癌晚期演进的遗传改变的重要资源.     

不同人结直肠肿瘤细胞系对常用抗肿瘤药物体外化疗敏感性的比较

 目的：观察5种常用抗肿瘤药物对这些人结直肠肿瘤细胞系的生长抑制作用,探讨5种常用抗肿瘤药物对11株人结直肠肿瘤细胞系的作用强度以及比较其体外敏感性,研究不同抗肿瘤药物对人结直肠癌细胞系HCT116和SW480热休克蛋白27（HSP27）和HSP70表达水平的影响。方法：采用CCK-8（Cell Counting Kit-8）法检测5种常用抗肿瘤药物分别对11株人结直肠肿瘤细胞系的生长抑制效应,计算50%抑制浓度（50% inhibitory concentration, IC50）及敏感指数,并比较不同人结直肠肿瘤细胞系对5种抗肿瘤药物的敏感性,Western blotting检测HSP27和HSP70蛋白表达水平。结果：11株人结直肠肿瘤细胞系对5-氟尿嘧啶(5-FU)和奥沙利铂(OHP)均比较敏感,没有明显耐药性;5株人结直肠肿瘤细胞系对丝裂霉素(MMC)敏感,6株中度敏感;除SW1116 外的10株人结直肠肿瘤细胞系都对多西紫杉醇(DXL)敏感,而SW1116细胞对DXL表现出明显耐药性;除LS174T和SW1116外的9株人结直肠肿瘤细胞系都对伊立替康(IFL)表现出中度敏感,LS174T细胞对IFL表现敏感,而SW1116细胞对IFL表现出明显耐药性。抗肿瘤药物作用于人结直肠癌细胞系HCT116和SW480使HSP27的表达上调,但HSP70的表达水平变化不明显。结论：LS174T是多药敏感细胞株,SW1116是多药耐药细胞株,5-FU和OHP为广谱抗结直肠肿瘤药物;化疗药物的敏感性及HSP27表达量检测对临床选择化疗药物具有一定的提示意义。     

Fas/Fas ligand interaction in human colorectal hepatic metastases: A mechanism of hepatocyte destruction to facilitate local tumor invasion

This study demonstrates a novel role for the Fas pathway in the promotion of local tumor growth by inducing apoptotic cell death in normal hepatocytes at the tumor margin in colorectal hepatic metastases. Our results show that >85% of lymphocytes infiltrating colorectal liver cancer express high levels of Fas-ligand (Fas-L) by flow cytometry. Using immunohistochemistry of tumor tissue we showed strong Fas expression in noninvolved hepatocytes, whereas Fas-L expression was restricted to tumor cells and infiltrating lymphocytes at the tumor margin. Apoptosis was observed in 45 +/- 13% of the Fas(high) hepatocytes at the tumor margin whereas only 7 +/- 3% tumor cells were apoptotic (n = 10). In vitro, primary human hepatocytes expressed Fas receptor and crosslinking with anti-Fas antibody induced apoptosis in 44 +/- 5% of the cells compared with 4. 6 +/- 1.0% in untreated controls (P = 0.004). Both tumor-infiltrating lymphocytes (TIL) and human metastatic colon cancer cells cells are able to induce Fas-mediated apoptosis of primary human hepatocytes in coculture cytotoxic assays. TIL induced apoptosis in 47 +/- 9% hepatocytes compared with control 4.3 +/- 1. 0% (P = 0.009) and this effect was reduced by anti-human Fas-L mAb (18.7 +/- 1.3%, P = 0.009). SW620 cells induced apoptosis in 26 +/- 2% hepatocytes compared with control 5.6 +/- 1.7% (P = 0.004) and this was reduced to 11.2 +/- 1.8% (P = 0.004) in the presence of anti-human Fas-L mAb. These data suggest that the inflammatory response at the margin of colorectal liver metastases induces Fas expression in surrounding hepatocytes, allowing them to be killed by Fas-L-bearing TIL or tumor cells and facilitating the invasion of the tumor into surrounding liver tissue.     

Prognostic significance of expression of matrix metalloproteinase in colorectal adenocarcinomas and their metastases

Expression of matrix metalloproteinases 2 and 9 was studied in primary tumors and their metastases in patients with colorectal cancer. The correlation of immunoreactivity with clinical morphological signs, prognosis of the disease, and development of metastases in the liver was analyzed. The level of expression and distribution of markers in patients with colorectal cancer with metastases in the liver differed from that in control patients without metastases. Enhanced expression of matrix metalloproteinases 2 and 9 was detected in colorectal cancer patients with distant metastases. Increased expression of matrix metalloproteinase 9 was associated significantly with low histological differentiation of the tumor, deeper tumor invasion, and was more often observed in tumors of colorectal cancer patients with unfavorable prognosis. Thus, matrix metalloproteinase 9 is a valuable marker for clinical observation and prognosis in patients with this location of the tumor process. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 4, pp. 434–437, April, 2007     

Overexpression of EIF-5A2 is associated with metastasis of human colorectal carcinoma

Our previous study has suggested an oncogenic role of eIF-5A2 in ovarian tumorigenesis. Abnormalities of eIF-5A2, however, in colorectal carcinoma are unclear. In this study, amplification and overexpression of eIF-5A2 in colorectal carcinoma were studied by fluorescence in situ hybridization and immunohistochemistry using colorectal carcinoma tissue microarrays, including 139 primary colorectal carcinomas and their adjacent normal mucosa, 22 paired premalignant adenomas, and 42 metastatic tumors. The immunohistochemistry results showed that overexpression of EIF-5A2 was detected in none of normal epithelial mucosa, 35.3% of colorectal adenomas, 53.2% of primary colorectal carcinomas, and 67.6% of metastases. Amplification of eIF-5A2 was detected in 15.8% (16/101) of informative colorectal carcinomas, and most of them showed overexpression of EIF-5A2. In primary colorectal carcinomas, the frequency of EIF-5A2 overexpression was significantly higher in colorectal carcinomas with lymphovascular invasion (61.2%) than that in colorectal carcinomas without lymphovascular invasion (36.6%, P < .05). In addition, significant positive associations were found between EIF-5A2 overexpression and the tumors' later pN and pM stages, as well as increased tumor cell proliferation (P < .05). These findings suggest that overexpression of EIF-5A2 in colorectal carcinomas may be important in the acquisition of a metastatic phenotype and plays an important role in colorectal carcinoma development and progression.