透明质酸对人结肠癌细胞SW480增殖、粘附和侵袭能力的影响

背景与目的:透明质酸广泛存在于结肠癌间质中,本研究旨在探讨透明质酸(hyaluronan,HA)对体外培养的人结肠癌SW480细胞增殖、粘附和侵袭能力的影响.材料与方法:体外培养的SW480细胞被随机分为3组:对照组(无血清培养基培养)、HA1(HA为10 μg/ml)和HA2(HA为20 μg/ml)组,经培养不同时间后,以MTT实验和软琼脂细胞集落形成实验(soft agar clone formation assay)比较SW480细胞增殖能力,用平板粘附模型和Boyden chamber小室模型比较SW480细胞粘附和侵袭能力.结果:HA1和HA2组与对照组相比,细胞增殖数量、软琼脂细胞集落、粘附于平板和穿过Boyden chamber隔膜的细胞数皆显著增加(P＜0.05),且呈剂量依赖性.结论:HA能增强SW480细胞体外增殖、粘附和侵袭能力.     

CCL21/CCR7轴促进人肺癌A549细胞的趋化与侵袭

目的：研究CCL21/CCR7轴对肺癌A549细胞定向趋化与侵袭活性的影响.方法：RT-PCR法从临床肺腺癌标本中扩增出CCR7编码区序列,定向克隆至载体pEGFP-N1中,稳定转染A549细胞,Boyden小室法检测转染前后A549细胞对CCL21的趋化和侵袭活性的改变.结果：CCL21作用下多聚碳酸酯膜背面的转染后A549细胞数明显多于转染前的A549细胞数.结论：CCL21/CCR7轴能够促进A549细胞的趋化与侵袭,其可能参与了肺癌淋巴结转移的过程.进一步研究CCL21/CCR7在肺癌中的作用将有助于阐明肺癌淋巴结转移的机制.     

肝细胞生长因子对结肠癌细胞SW620增殖、侵袭的影响

摘要：目的探讨肝细胞生长因子（Hepatocyte growth factor,HGF）对人结肠癌细胞株SW620的增殖和侵袭能力的影响。方法应用MTT法分析HGF对SW620细胞增殖的影响,确定最佳促增殖浓度;应用基底膜重建实验和电镜观察HGF对细胞侵袭能力的影响;应用PCR技术检测HGF对MMP-2 mRNA表达的影响。结果当HGF浓度≥10ng/ml时促进SW620增殖且呈质量浓度依赖性; HGF明显促进SW620 移行且呈质量浓度依赖关系;加入 HGF 后,SW620细胞内的微丝明显增多,MMP-2表达水平升高。结论HGF可以在体外促进人结肠癌细胞SW620的增殖和移行。     

CCL21在人结肠癌SW480细胞侵袭过程中的作用

背景与目的：既往研究显示趋化冈子受体CCR7在胃肠道肿瘤中表达上调,并与淋巴管浸润和淋巴结转移相关。本研究旨在探讨趋化因子CCL21及其受体CCR7在人结肠癌SW480细胞体外侵袭中的作用。方法：在趋化因子CCL21作用的条件下,用划痕实验和Transwel!小室检测SW480细胞侵袭能力．免疫印迹检测SW480细胞中金属基质蛋白酶-9（matrix metalloproteinase-9．MMP-9）表达;CCL21预处理的SW480细胞在含足叶乙甙（VP—16）的体系中培养,MTr法检测细胞增殖活力,流式细胞术和Hoechst33258染色检测细胞凋亡。结果：CCL21（100ng／mL）组中SW480细胞向划痕处爬行的速度明显快于阴性对照组：阴性对照组与CCL21（100ng／mL）组中穿膜细胞数分别为（48±4）个和（113±7）个,差异有统计学意义（P〈0．05）;CCL21（100ng／mL）组中MMP-9的相对表达水平为0．83±0．02,高于阴性对照组的0．38±0．0l（P〈0．05）。CCL21单独作用不能促进SW480细胞增殖;VP-16组中SW480细胞的增殖抑制率为68．3％,CCL21预处理增强SW480细胞活力,CCL21（100ng／mL）中抑制率降至47．4％：VP-16组与CCL21（100ng／mL）组中凋亡率分别为（65．2±5．2）％和（48．7±3．1）％,差异有统计学意义（P〈0．05）。结论：CCL21／CCR7可以促进结肠癌SW480细胞的体外侵袭力,并增强其在微环境中的生存能力。     

凝血因子Ⅶa促进SW620细胞增殖与迁移的机制探讨

目的 体外探讨凝血因子Ⅶa促进结肠癌细胞株SW620增殖与迁移的作用机制.方法 采用蛋白酶激活受体2激动剂(PAR2-AP)、凝血因子Ⅶa等处理SW620细胞,以实时定量PCR检测SW620细胞中白细胞介素8(IL-8)、组织因子(TF)及半胱氨酸蛋白酶7(caspase-7)mRNA的表达水平;以酶联免疫吸附试验(ELISA)检测细胞上清IL-8蛋白的含量;以Xa生成法检测细胞TF活性;以Western blot法检测细胞磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)水平.结果 PAR2-AP、凝血因子Ⅶa能够增加SW620细胞中IL-8基因和蛋白的表达,上调TF mRNA水平及活性,下调caspase-7基因表达和p-p38 MAPK水平,单克隆抗TF及抗PAR2抗体均可抑制凝血因子Ⅶa的作用.结论 凝血因子Ⅶa与细胞表面TF形成复合物,通过活化PAR2,上调结肠癌细胞株SW620中IL-8、TF的表达,下调细胞caspase-7表达,从而促进细胞增殖与迁移能力,p38 IVIAPK在此过程中起负性调节作用.     

CD44v3短发夹RNA对透明质酸诱导人结肠癌SW480细胞体外粘附和侵袭的抑制作用

目的:研究CD44v3的短发夹RNA(short hairpin RNA,shRNA)对体外培养的人结肠癌SW480细胞CD44v3表达及透明质酸诱导的粘附侵袭行为的抑制作用。方法:设计和构建带有U6启动子的CD44v3基因特异性的shRNA干扰表达质粒,转染至SW480细胞,以RT-PCR和Western blot检测SW480细胞转染前后CD44v3表达,以平板粘附模型和Boyden小室模型检测SW480细胞转染前后粘附和侵袭能力。结果:和转染前相比,转染了RNA干扰表达质粒的SW480细胞CD44v3mRNA和蛋白表达、以及受透明质酸诱导而粘附于平板和穿过Boyden小室隔膜的细胞数皆显著减少(P<0.01)。结论:针对CD44v3的短发夹RNA能显著抑制透明质酸诱导人结肠癌SW480细胞体外粘附和侵袭行为。     

槲皮素对人结肠癌SW480细胞体外侵袭能力的影响

目的：研究槲皮素对结肠癌SW480细胞的体外侵袭能力的影响,并探讨其可能的作用机制。方法：结肠癌SW480细胞经槲皮素处理后用Transwell小室的方法检测其体外黏附、侵袭和运动能力。明胶酶谱分析法检测SW480细胞分泌的基质金属蛋白酶活性。结果：结肠癌SW480细胞经槲皮素处理后,体外侵袭、运动能力呈剂量依赖性下降,SW480细胞MMP-2及MMP-9的分泌下降（P〈0.05）。结论：槲皮素在体外剂量依赖性地抑制结肠癌SW480细胞的转移能力,这可能与槲皮素抑制MMP-2及MMP-9的分泌有关。     

nm23-H1对肝癌细胞株SMMC7721转移特性的抑制

[目的]研究转移抑制基因nm23-H1与肝癌细胞转移行为的关系.[方法]体外构建真核细胞表达重组体DNA pcDNA 3.1(-)-nm23-H1;采用Lipofectamine介导将目的基因nm23-H1体外转染肝癌细胞株SMMC7721,通过软琼脂克隆实验、内皮细胞异质粘附实验和Boyden小室侵袭实验对转染细胞的生物学活性进行检测.[结果]粘附实验中,实验组肿瘤细胞株与内皮细胞粘附计数为(50～60)/mm2,少于对照组的(200～300)/mm2,实验组细胞株粘附性降低,t检验P＜0.05;软琼脂克隆实验对照组形成多个细胞克隆(47%～62%),而实验组无克隆形成,克隆能力明显降低;Boyden小室侵袭实验中,每视野穿透Matrigel膜的细胞数分别为1.34～3.26和27.46～32.94,实验组细胞株侵袭能力明显低于对照组细胞株,t检验P＜0.01.[结论]nm23-H1能够降低肿瘤细胞株SMMC7721在体外的转移潜能.     

miR-34a对结肠癌细胞SW480增殖、迁移和侵袭能力的影响及机制探讨

目的 探讨miR-34a对结肠癌细胞株SW480增殖、侵袭和迁移能力的影响及可能的作用机制。方法将miR-34a过表达慢病毒、空病毒载体转染SW480细胞,未做处理细胞作为空白对照组。Real-time PCR检测各组细胞内miR-34a的表达;CCK8法检测细胞增殖能力;划痕实验、Transwell小室实验检测细胞迁移和侵袭能力;Western blot实验检测细胞内E-cadherin和Vimentin蛋白表达。结果 与空病毒载体组及空白对照组相比,转染组细胞中miR-34a的表达增高,且细胞增殖效率、侵袭和迁移能力降低(P＜0.05),miR-34a使E-cadherin蛋白表达增加、Vimentin蛋白表达降低。结论 miR-34a可抑制结肠癌细胞SW480增殖、侵袭和迁移能力,并能影响E-cadherin和Vimentin的表达,miR-34a有望成为干预结肠癌转移和复发的分子靶点。     

奥曲肽对人结肠癌细胞抑制及凋亡的影响

目的观察奥曲肽抑制人结肠癌SW620细胞增殖并诱导其凋亡的作用。方法奥曲肽作用于体外培养的SW620细胞后,通过MTT比色法测定细胞增殖抑制作用;采用AO/EB染色荧光显微镜观察奥曲肽诱导SW620细胞凋亡后的形态学改变;应用流式细胞术(FCM)分析细胞凋亡率。结果奥曲肽作用SW620细胞后,MTT检测显示细胞增殖活性受到明显抑制,且呈一定浓度依赖性;AO/EB染色可见典型凋亡小体,PI染色流式法显示细胞凋亡率明显上升。结论奥曲肽具有抑制体外培养人结肠癌SW620细胞增殖,并有效诱导其凋亡作用。     

A critical role of CCR7 in invasiveness and metastasis of SW620 colon cancer cell in vitro and in vivo

Tumor cell migration and metastasis are critically regulated by chemokines and their receptors. CC Chemokine Receptor 7 (CCR7) plays a critical role in mediating chemotactic and invasive responses in cancers. However, whether or not CCR7 is a desired target of cancer therapy needs further investigation in terms of its biodegradation and availability in vivo. In this study, we employed RNA interference technology to detect the in vitro effects of anti-CCR7 siRNAs on proliferation and invasiveness of SW620 cells. We also evaluated the ability of these siRNAs to inhibit the lymphogenesis and the lymph node metastasis in xenografted SW620 tumor mouse. The chemotaxis and invasion assay in the animal model showed that blocking CCR7 expression at the mRNA level by a siRNA impaired invasion of colon cancer cells and inhibited lymph node metastasis of colon cancer and lymphogenesis. Therefore, CCR7 might be a desired target for cancer therapy and novel drug development.     

Expression of chemokine receptor CCR7 is associated with cervical lymph node metastasis of oral squamous cell carcinoma

Tumor cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. The present study was designed to examine the expression of chemokine receptor CCR7 in oral squamous cell carcinoma (OSCC), and to investigate the possible role of CCR7/CCL21 interaction in neck lymph node metastasis of OSCC. By using immunohistochemistry, RT-PCR and Western Blot, expression of CCR7 was examined in 85 cases of oral squamous cell carcinoma, and Tca8113 and ACC cell lines. CCL21-mediated cell migration was assayed in Matrigel-coated chemotaxis chamber. In vitro adhesion assay was shown for banding of tumor cell lines to submandibular lymph nodes with or without anti-CCR7 antibody treatment. Immunohistochemical staining showed 65.9% (56/85) of positive CCR7 expression in OSCC tissues. CCR7 expression was significantly higher in patients with lymph node metastasis compared with those without lymph node metastasis (P=0.015) and was also associated with tumor size (P=0.014), and clinical stage (P=0.009). RT-PCR and Western Blot also confirmed positive CCR7 expression in oral squamous cell carcinoma and Tca8113 cell line, and negative CCR7 expression in normal oral mucosa and ACC cell line. CCL21 stimulation increased the ability of CCR7-positive Tca8113 cells passing through the Matrigel membrane. CCR7-positive Tca8113 cells also showed stronger adhesion to lymph nodes, which could be partly blocked by anti-CCR7 antibody incubation. These results indicated that the chemotactic CCR7/CCL21 interaction may be a possible mechanism for induction of directional lymph node metastasis by oral squamous cell carcinoma.     

Tumor necrosis factor‐α induces prostate cancer cell migration in lymphatic metastasis through CCR7 upregulation

Understanding the mechanism of lymph node metastasis, a poor prognostic sign for prostate cancer, and the further dissemination of the disease is important to develop novel treatment strategies. Recent studies have reported that C‐C chemokine receptor 7 (CCR7), whose ligand is CCL21, is abundantly expressed in lymph node metastasis and promotes cancer progression. Tumor necrosis factor‐α (TNF‐α) is chronically produced at low levels within the tumor microenvironment. The aim of this study was to determine whether TNF‐α promotes prostate cancer dissemination from metastatic lymph nodes through activation of the CCL21/CCR7 axis. First, human prostate cancer cells were determined to express both TNF‐α and CCR7. Second, low concentrations of TNF‐α were confirmed to induce CCR7 in prostate cancer cells through phosphorylation of ERK. Finally, CCL21 was found to promote the migration of prostate cancer cells through phosphorylation of the protein kinase p38. Our results suggest that TNF‐α leads to the induction of CCR7 expression and that the CCL21/CCR7 axis might increase the metastatic potential of prostate cancer cells in lymph node metastasis.     

CXCL12 promotes CCR7 ligand–mediated breast cancer cell invasion and migration toward lymphatic vessels

Chemokines are a family of cytokines that mediate leukocyte trafficking and are involved in tumor cell migration, growth, and progression. Although there is emerging evidence that multiple chemokines are expressed in tumor tissues and that each chemokine induces receptor‐mediated signaling, their collaboration to regulate tumor invasion and lymph node metastasis has not been fully elucidated. In this study, we examined the effect of CXCL12 on the CCR7‐dependent signaling in MDA‐MB‐231 human breast cancer cells to determine the role of CXCL12 and CCR7 ligand chemokines in breast cancer metastasis to lymph nodes. CXCL12 enhanced the CCR7‐dependent in vitro chemotaxis and cell invasion into collagen gels at suboptimal concentrations of CCL21. CXCL12 promoted CCR7 homodimer formation, ligand binding, CCR7 accumulation into membrane ruffles, and cell response at lower concentrations of CCL19. Immunohistochemistry of MDA‐MB‐231–derived xenograft tumors revealed that CXCL12 is primarily located in the pericellular matrix surrounding tumor cells, whereas the CCR7 ligand, CCL21, mainly associates with LYVE‐1⁺ intratumoral and peritumoral lymphatic vessels. In the three‐dimensional tumor invasion model with lymph networks, CXCL12 stimulation facilitates breast cancer cell migration to CCL21‐reconstituted lymphatic networks. These results indicate that CXCL12/CXCR4 signaling promotes breast cancer cell migration and invasion toward CCR7 ligand–expressing intratumoral lymphatic vessels and supports CCR7 signaling associated with lymph node metastasis.     

Association between the expression of chemokine receptors CCR7 and CXCR3, and lymph node metastatic potential in lung adenocarcinoma

Chemokines and their receptors are essential for leukocyte trafficking, and are also involved in cancer metastasis to specific organs. Although the migration of tumor cells into the lymph nodes is an important aspect of cancer, the processes involved are poorly understood. Chemokine receptors CCR7 and CXCR3 have been shown to play an important role in tumor cell migration and lymph node metastasis. Therefore, the assessment of chemokine receptor expression on lung adenocarcinomas may improve the prediction of the spread of this carcinoma to the lymph nodes. In this study, we examined the expression and function of these two chemokine receptors (CCR7 and CXCR3) in lung adenocarcinoma. By using flow cytometry, they were detected in all of the lung adenocarcinoma cell lines examined. In the chemotaxis assays, A549 cells exhibited CCL21-induced migration, which was significantly suppressed by neutralizing anti-CCR7 antibody. The CXCL10-induced migration of A549 cells was also significantly suppressed by neutralizing anti-CXCR3 antibody. In clinical lung adenocarcinoma samples, we found the expression of CCR7 and CXCR3 in 65 and 90% cases, respectively, most of which had lymph node metastasis. Importantly, the expression of CCR7 was significantly associated with lymph node metastasis, although the expression of CXCR3 was not. These results suggest that the activation of CCR7 and CXCR3 with their ligands preferentially stimulates lung adenocarcinoma metastasis to the draining lymph nodes.     

Expression of chemokine receptor CCR7 is associated with lymph node metastasis of gastric carcinoma

The interactions of chemokine receptor CCR7 and its ligands are essential for migration of lymphocytes and dendritic cells to lymph nodes. In this study, we found that 4 of 6 (67%) gastric carcinoma cell lines tested expressed functional CCR7 for the chemokine CCL21/6Ckine, as demonstrated by calcium mobilization and actin polymerization assays. Moreover, we also showed that signaling through CCR7 induced chemotactic and invasive responses in CCR7-positive gastric carcinoma cells. In clinical samples, immunohistochemical assay showed that CCR7-positive carcinoma cells were detected in 42 of 64 (66%) cases and a significant difference in both lymph node metastasis (P < 0.001) and lymphatic invasion (P < 0.001) between CCR7-positive and -negative cases. Patients with CCR7-positive tumors had a significantly poorer prognosis than those with CCR7-negative tumors (P < 0.05). Stepwise regression analysis revealed that the most important factor related to lymph node metastasis was the expression of CCR7. These results indicated that CCR7 and its ligands interaction is associated with preferential lymph node metastasis of gastric carcinoma.