血管抑制素基因真核表达载体的构建及转染研究

目的：构建血管抑制素（angiostatin,AG)基因真核细胞表达载体并进行转染研究。方法：根据已发表的血管抑制基因的核苷酸序列,设计并合成一对引物、以鼠的纤溶酶原核酸为模板进行PCR扩增,将其基因克隆到真核表达载体pRC/CMV中,进行序列分析和酶切鉴定后,运用 质体将重组质粒pRC/CMV-AG转入胰腺癌SW1990细胞中,观察其表达情况,以及其对血管内皮细胞生长的作用。结果：PCR扩增出一长1．4kb的片断,酶切和序列分析证明含完整的AG基因序列,与已知的血管抑制素基因的同源性很高（＞96％）。Western blot证实重组体pRC/CMV-AG有较高的表达,对血管内皮细胞的生长有一定的抑制作用。结论：重组体pRC/CMV-AC是一种高效真核表达载体;克隆的AG基因能抑制血管内皮细胞的生长。     

Tiam1蛋白表达介导PI3K/Akt/mTOR信号通路对老年胰腺癌细胞转移、侵袭的影响

目的 探讨T淋巴瘤侵袭转移诱导基因(Tiam1)蛋白表达介导PI3K/Akt/mTOR信号通路对老年胰腺癌细胞转移、侵袭的影响。方法 选择行手术确诊的老年胰腺癌患者106例，收集其癌组织标本及配对癌旁正常组织，Western Blot法检测胰腺组织Tiam1蛋白表达；选择人胰腺癌细胞株SW1990,采用浓度为50 ng/mL的HGF溶液和生理盐水处理并作为HGF组和空白组，转染Tiam1 minic作为Tiam1组，CCK8法、Transwell小室侵袭实验、划痕实验检测转染后胰腺癌细胞增殖、侵袭和转移能力；Western Blot法检测胰腺组织PI3K/Akt/mTOR相关蛋白表达，明确Tiam1蛋白表达与PI3K/Akt/mTOR信号通路的关系。结果 胰腺癌组织中Tiam1表达量高于癌旁组织中Tiam1表达量(P<0.05)。各组在转染0 h、12 h时细胞增殖率比较无明显差异(P>0.05),转染24 h、48 h、60 h时，与空白组比较，Tiam1组和HGF组细胞增殖率升高，且Tiam1组高于HGF组(P<0.05);与空白组比较，Tiam1组和HGF组侵袭...     

人 MEG3基因质粒载体的构建及其对人胰腺癌SW1990细胞增殖的影响

目的：构建人类母源性印记基因3（MEG3）的真核表达载体，转染得到过表达的人胰腺癌SW1990细胞株，分析 MEG3过表达对 SW1990细胞增殖的影响。方法根据 GenBank 中 MEG3的基因序列，通过化学合成的方法进行全基因合成得到人 MEG3基因全长，将其构建入 pcDNA3.0真核表达载体中，得到 pcDNA3.0-MEG3表达载体，转染胰腺癌细胞 SW1990。采用 RT-PCR 及荧光定量 PCR 技术检测转染细胞中 MEG3基因的表达量。采用 MTT 试剂盒对转染后 SW1990细胞增殖能力的变化进行检测。实验中设转染 pcDNA3.0的 SW1990细胞为阴性对照组，普通 SW1990细胞为空白对照组。结果成功构建了 MEG3真核表达质粒pcDNA3.0-MEG3，并成功转染 SW1990细胞。与两对照组相比，转染后细胞的 MEG3基因表达量显著提高，提高约895倍（F ＝73.592，P ﹤0.01）。MTT 检测结果显示，MEG3基因明显抑制 SW1990细胞的增殖，实验组72 h 时吸光度值为0.81±0.06，与阴性对照组（1.17±0.07）和空白对照组（1.08±0.03）相比，3组差异有统计学意义（ F ＝33.489，P ﹤0.01）。结论本研究成功构建了MEG3真核表达质粒 pcDNA3.0-MEG3，并证实 MEG3基因及其产物对胰腺癌 SW1990细胞增殖有明显的抑制作用。     

MYEOV对人胰腺癌细胞增殖和迁移的影响

目的 探讨骨髓瘤过表达基因MYEOV在人胰腺癌细胞中的表达情况及其下调对胰腺癌SW1990细胞增殖和迁移能力的影响。方法 构建慢病毒载体GV248，转染胰腺癌细胞株SW1990获得SW1990-sh1和SW1990-sh2两个实验组，以空白质粒转染作为阴性对照组，未干预细胞为空白对照组。qPCR和Western blot检测转染前后MYEOV mRNA与蛋白的表达水平；CCK-8法测定细胞增殖能力；划痕实验分析细胞迁移能力。qPCR检测TGF-β/SMAD通路中相关SMADs mRNA表达水平。结果 与正常胰腺导管上皮细胞系HPDE6相比，MYEOV mRNA在6株胰腺癌细胞中表达水平明显增加（P<0.01）；但仅在PANC-1和SW1990细胞系中检测到低水平MYEOV蛋白表达。实验组中MYEOV mRNA和蛋白表达均明显下调。与对照组相比，SW1990细胞的增殖和迁移能力明显下降（P<0.001）。下调MYEOV能降低TGF-β通路中SMAD1、SMAD4、SMAD5和SMAD9 mRNA表达（P<0.01），而SMAD2、SMAD3和SMAD7 mRNA仅在SW1990-sh2组中表达下调（P<0.01）。结论 MYEOV在胰腺癌细胞系中转录水平高，MYEOV可通过TGF-β/SMAD通路促进胰腺癌细胞的增殖和迁移能力。     

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。     

miR-144对胰腺癌SW1990细胞增殖、迁移、侵袭及PI3K通路的影响

探讨miR-144对胰腺癌SW1990细胞增殖、迁移、侵袭及磷脂酰肌醇-3激酶（PI3K）通路的影响。方法 采用实时荧光定量PCR（RT-qPCR）检测人正常胰腺上皮细胞系HPDE与胰腺癌SW1990细胞中miR-144表达水平。将miR-144阴性对照质粒、miR-144 mimic质粒分别转染至SW1990细胞,分别命名为阴性转染组、miR-144过表达组,未经处理的细胞命名为空白对照组。采用RT-qPCR法检测各组miR-144表达水平;CCK-8法检测细胞增殖情况;平板细胞克隆形成实验检测菌落形成;Transwell检测细胞迁移及侵袭能力;Western blot法检测PI3K通路中PI3K及其磷酸化蛋白（p-PI3K）、丝氨酸-苏氨酸蛋白激酶（Akt）及其磷酸化蛋白（p-Akt）的表达水平。结果 SW1990细胞中miR-144的表达水平显著低于HPDE细胞（P<0.05）;miR-144过表达组中miR-144表达水平显著高于空白对照组和阴性转染组（P<0.05）、细胞增殖率、菌落形成、迁移及侵袭数量和p-PI3K、p-Akt蛋白表达水平均显著低于空白对照组、阴性转染组（均P<0.05）。结论 miR-144在胰腺癌细胞SW1990中呈低表达,上调miR-144表达水平有效抑制胰腺癌细胞增殖、迁移及侵袭,其机制可能与PI3K通路抑制有关。     

NK4基因联合奥沙利铂对人结肠癌细胞株HCT116凋亡和侵袭的影响

目的 探讨NK4基因联合奥沙利铂（oxaliplatin, L-OHP）在人结肠癌细胞HCT116凋亡和侵袭中的作用。方法 构建NK4质粒表达载体,将NK4基因转染至HCT116细胞,采用流式细胞术分析NK4基因联合奥沙利铂对HCT116细胞凋亡的影响,Transwell小室法检测NK4基因联合奥沙利铂对该细胞侵袭的影响。采用金正均Q值法判断NK4基因联合奥沙利铂是否有协同作用;Western blot法检测不同药物作用下相关机制蛋白p-Akt的变化情况。结果 在HCT116细胞中成功构建NK4质粒表达载体,NK4组 mRNA表达水平高于阴性对照组（NC组）和空白组,差异有统计学意义（P<0.01）。与NC组相比,NK4基因联合奥沙利铂促细胞凋亡作用最显著（16.55% vs 46.86%,P<0.05）;抑制细胞侵袭能力最强（0.9% vs 77.4%,P<0.05）;金正均Q值法结果显示NK4基因联合奥沙利铂具有协同相加作用（0.85≤Q<1.15）,且细胞凋亡和侵袭相关蛋白p-Akt的表达在联合用药组中显著下调,亦显示协同作用（P<0.01）。结论 NK4基因协同奥沙利铂可能通过Akt相关信号通路诱导HCT116结肠癌细胞凋亡并抑制该细胞侵袭。     

沉默高尔基体α-甘露糖苷酶Ⅱ表达对人胃癌BGC-823细胞侵袭和迁移能力的影响

目的:探讨RNA干扰沉默高尔基体α-甘露糖苷酶Ⅱ(Golgi a-mannosidase Ⅱ,GM Ⅱ)基因表达对人胃癌BGC-823细胞侵袭和迁移的影响.方法:为沉默GM Ⅱ基因,构建了4个分别针对不同靶位点的短发夹RNA (short hairpin RNA,shRNA)质粒表达载体,同时合成阴性对照质粒表达载体.应用LipofectAMINE2000将质粒转染入BGC-823细胞.分别采用RT-PCR和蛋白质印迹法检测转染后GM Ⅱ mRNA和蛋白表达的变化,选出沉默效果最佳的质粒.应用Transwell侵袭实验和划痕损伤实验分别检测GM Ⅱ基因沉默对人胃癌细胞BGC-823侵袭和迁移能力的影响.结果:质粒载体pGPU6/GFP/Nco-1303沉默GM Ⅱ基因的效果最好.转染pGPU6/GFP/Neo-1303组穿透小室基质的细胞数明显少于空白对照组和转染阴性对照组(P＜0.05).无血清培养液培养24 h后,转染pGPU6/GFP/Neo-1303组细胞的迁移能力较空白对照组和转染阴性对照组明显降低(P＜0.05).结论:RNA干扰技术可沉默人胃癌BGC-823细胞中GMⅡmRNA和蛋白的表达,从而抑制BGC-823细胞的侵袭和迁移能力.GM Ⅱ可能成为胃癌治疗的新靶点之一.     

增强型绿色荧光蛋白基因稳定转染胰腺癌细胞

目的：建立稳定高表达增强型绿色荧光蛋白（EGFP）并能连续传代的胰腺癌细胞株。方法：将携带EGFP基因的pEGFP—C1质粒体外转染人胰腺癌细胞株SW1990，G418筛选稳定表达绿色荧光蛋白的细胞克隆并扩大培养，用荧光显微镜和流式细胞仪检测癌细胞荧光蛋白表达。结果：转染EGFP的胰腺癌细胞用G418筛选18天后，挑选克隆扩大培养，癌细胞几乎均见荧光蛋白表达并可连续稳定传至20代以上。结论：胰腺癌细胞株SW1990-EGFP的建立有助于构建理想的动物模型来研究肿瘤侵袭和转移的机制。     

MT1-MMP反义核酸抑制高转移人卵巢癌SW626细胞的侵袭效应

背景与目的:MT1-MMP(MMP-14)是新发现的一种膜型基质金属蛋白酶,研究表明MT1-MMP在肿瘤转移中发挥关键性作用。本研究应用反义核酸技术,观察了MT1-MMP反义核酸对高转移人卵巢癌SW626细胞体外生长和侵袭的抑制效应。方法:应用自行设计的引物,借助RT-PCR技术获得两端含不同限制酶位点的MT1-MMPcDNA片段,反向插入pcDNA3.1中,并应用脂质体介导的基因转染技术,将重组质粒导入SW626细胞中,应用MTT、Westernblot、明胶酶谱和体外侵袭实验等方法观察了SW626细胞转染前后,细胞生长、MT1-MMP蛋白表达、MMP-2和MMP-9酶活性及细胞体外侵袭能力等指标的变化。结果:成功构建了反义MT1-MMP真核表达载体(pMMP14as),将其转染入SW626细胞后,与空质粒转染组相比,MT1-MMP反义核酸转染组细胞MT1-MMP表达显著降低,抑制率为65.8%;MMP-2酶原的活化受到明显抑制;pMMP14as转染组的穿膜细胞百分数为(63.3±5.8)%,明显低于空质粒转染组(97.6±7.5)(P<0.05)%。结论:MT1-MMP反义核酸可明显抑制高转移SW626细胞体外生长和侵袭效应,提示MT1-MMP可作为人卵巢癌抗侵袭治疗的分子靶点。     

NK4 (HGF-antagonist/angiogenesis inhibitor) in cancer biology and therapeutics

Invasion and subsequent establishment of metastasis are devastating events for patients with cancer, but past therapeutic approaches have paid relatively little attention to these important issues. Hepatocyte growth factor (HGF) and its receptor, the c-Met tyrosine kinase, play roles in cancer invasion and metastasis in a wide variety of tumor cells. Activation of the c-Met receptor integrates multiple signal transduction pathways involved in cell-cell and cell-matrix interactions, cellular migration, and breakdown of the extracellular scaffold. Paracrine activation of the c-Met receptor by stromal-derived HGF mediates tumor-stromal interactions that facilitate invasion and metastasis. Likewise, aberrant expression of the c-Met receptor and autocrine or mutational activation of c-Met receptor tyrosine kinase are closely associated with the progression of malignant tumors. Based on this background, NK4, a competitive antagonist of HGF-c-Met association was prepared so as to block cancer invasion and metastasis. NK4, an internal fragment of HGF, binds to but does not activate the c-Met receptor, thereby competitively antagonizing the biological activities of HGF. Unexpectedly, NK4 was subsequently shown to be an angiogenesis inhibitor as well, and this angioinhibitory activity is independent of its action as an HGF-antagonist. Importantly, NK4 protein or NK4 gene therapy have been shown to inhibit tumor invasion, metastasis and angiogenesis, effectively converting malignant tumors into benign tumors. Targeting tumor invasion-metastasis and angiogenesis with NK4 seems to have considerable therapeutic potential for cancer patients. (Cancer Sci 2003; 94: 321–327)     

NK4基因转染对裸鼠胰腺癌移植瘤生长的影响

背景与目的:NK4不仅是肝细胞生长因子的拮抗剂,而且是血管形成的抑制剂。研究已经证实NK4可以抑制肿瘤的生长和转移,但有关其在胰腺癌中的作用,目前少见文献报道。为此,本研究旨在探讨NK4基因在裸鼠体内的抗胰腺癌作用及其可能的机制。方法:建立裸鼠胰腺癌皮下移植瘤模型,构建NK4基因真核细胞表达载体并转染入瘤体内,转染前后称其体重、瘤重和测肿瘤体积,采用免疫组织化学和脱氧核糖核酸末端转移酶介导的缺口末端标记技术对裸鼠胰腺癌组织中的凋亡细胞、增殖抗原和微血管密度进行观察和比较。结果:4周后,NK4转染组裸鼠移植瘤体积为(1.39±0.33)cm3,明显小于对照组和空载体组[(2.06±0.55)cm3和(1.90±0.36)cm3,P<0.01];其瘤重为(1.30±0.81)g,也显著低于对照组和空载体组[(3.45±1.88)g和(3.14±1.51)g,P<0.01],抑瘤率为62.29%。NK4转染组肿瘤细胞的凋亡指数为9.34±0.91,显著高于对照组和空载体组(4.13±0.79和3.94±1.03,P<0.001);而NK4转染组肿瘤细胞的增殖指数为53.88±4.30,与对照组间和空载体组比较无显著性差异(56.24±4.03和54.33±5.41,P>0.05);NK4转染组肿瘤组织的微血管密度为(12.24±4.63),明显低于对照组和空载体组(20.13±7.00和19.70±6.15,P<0.05)。结论:转染NK4基因可显著抑制裸鼠胰腺癌移植瘤的生长,其作用机制可能是通过抑制肿瘤新生血管的形成,促进肿瘤细胞凋亡。     

Radiation stimulates HGF receptor/c-Met expression that leads to amplifying cellular response to HGF stimulation via upregulated receptor tyrosine phosphorylation and MAP kinase activity in pancreatic cancer cells

Hepatocyte growth factor (HGF) is a stromal-derived cytokine that plays a crucial role in invasion and metastasis of tumor cells through the interaction with HGF receptor, c-Met, which is frequently overexpressed in pancreatic cancer. The present study was designed to investigate the change in HGF receptor and HGF-mediated signaling after irradiation in pancreatic cancer cells. Six cell lines from human pancreatic cancer were included in the study. Gamma-radiation was used for irradiation treatment. The changes in expression levels of c-Met were evaluated by immunoblot and confirmed morphologically by indirect immunofluorescence staining. Whether the resultant alteration in c-Met would cascade as biologically usable signals upon HGF ligation was traced by receptor tyrosine phosphorylation analysis and mitogen activated protein kinase (MAP kinase or MAPK) activity assay. The various biological responses to HGF (including cell proliferation, cell scattering, migration and invasion) were evaluated as well. We also used a 4-kringle antagonist of HGF, NK4, to block the HGF/c-Met signaling pathway. Both immunoblot and immunofluorescent analysis showed moderate increased expression of c-Met in 3 of 6 pancreatic cancer cell lines after irradiation. The actions seemed to be dose-responsible, which began at 3 hr and reached its peak value at 24 hr following irradiation. The radiation-increased expression of c-Met could transform into magnifying receptor tyrosine phosphorylation reaction and MAP kinase activity once the ligand was added, fairly corresponding with alteration in the receptor. Sequentially, the cellular responses to HGF, including scattering and invasion but not proliferation, were enhanced. Also, in the presence of HGF, the elevated receptor could help to recover the radiation-compromised cell migration. A recombinant HGF antagonist, NK4 could effectively block these aberrant effects activated by irradiation both in molecular and cellular levels, thus suggesting the deep involvement of the c-Met/HGF pathway in the enhanced malignant potential after irradiation. These results suggest that radiation may promote HGF-induced malignant biological behaviors of certain pancreatic cancer cells through the up-regulated HGF/c-Met signal pathway. Selectively targeted blockade of the HGF/c-Met pathway could help to abolish the enforced malignant behavior of tumor cells by irradiation and therefore may improve the efficacy of radiotherapy for pancreatic cancer.     

NK4, a four-kringle antagonist of HGF, inhibits spreading and invasion of human pancreatic cancer cells

Because of the highly aggressive behaviour, i.e. invasive, disseminative and metastatic properties, the outcome for patients with pancreatic cancer is morbid. A better understanding and interference with the malignant behaviour of pancreatic cancer may provide new directions for treatment. We report here the induction of highly motile and invasive properties in human pancreatic cancer cells by hepatocyte growth factor (HGF) and blockage of these properties by NK4, a newly identified antagonist for HGF. In all of eight human pancreatic cancer cell lines we used (AsPC-1, BxPC-3, H-48N, KP-1N, KP-2, KP-3, MIA PaCa-2 and SUIT-2 cells), the c-Met/HGF receptor was expressed at varying levels. Although weak mitogenic activity of HGF was seen only in SUIT-2 and KP-3 cells, HGF strongly stimulated migration and invasion of these pancreatic cancer cells, except for BxPC-3 and MIA PaCa-2 cells. In contrast, migration and invasion potently induced by HGF in KP-1N, KP-3 and SUIT-2 cells were inhibited by NK4. The invasion of SUIT-2 cells was also potently stimulated with the influence of cocultured pancreatic fibroblasts and by ascitic fluid obtained after pancreatic cancer resection, however, invasiveness of the cancer cells in such conditions was practically abolished by NK4. Consistently, the ascitic fluid in patients who had undergone pancreatic cancer surgery contained high levels of HGF. These findings mean that HGF is probably involved in invasion, dissemination, and metastasis of pancreatic cancer, particularly through tumour-stromal interaction and after resection of the pancreatic cancer. NK4, an effective antagonist of HGF, may prove to have the potential for anti-invasion/metastasis.     

Plasminogen activator inhibitor-1 (PAI-1) gene transfection inhibits the liver metastasis of pancreatic cancer by preventing angiogenesis

Plasminogen activator inhibitor-1 (PAI-1) is a unique type of serine protease inhibitor and one of the key regulators of tumor invasion and metastasis. The purpose of this study was to elucidate the effect of PAI-1 gene transfection on liver metastasis and its mechanism by using the human high liver metastasis pancreatic cancer cell line, SW1990. PAI-1-transfected SW1990 (SW/PAI-1) produced a significantly higher level of PAI-1 in supernatant than parental cells. While no difference was observed for the production of u-PA and u-PA activity in the supernatant, cell proliferation of SW/PAI-1 was slightly suppressed on the 7th day of incubation compared to parental cells. Cellular invasion, in vivo tumorigenesis in xenograft and liver metastasis were significantly suppressed in SW/PAI-1 cells compared to parental cells. The angiogenesis of xenograft by detecting microvascular density and the production of metastasis-related factors, such as VEGF and TGF-beta1, were also decreased in SW/PAI-1 cells. These findings suggested that PAI-1 gene transfection might have the ability to prevent the liver metastasis of pancreatic cancer by modulating angiogenesis.     

Radiation to stromal fibroblasts increases invasiveness of pancreatic cancer cells through tumor-stromal interactions

Radiotherapy represents a major treatment option for patients with pancreatic cancer, but recent evidence suggests that radiation can promote invasion and metastasis of cancer cells. Interactions between cancer cells and surrounding stromal cells may play an important role in aggressive tumor progression. In the present study, we investigated the invasive phenotype of pancreatic cancer cells in response to coculture with irradiated fibroblasts. Using in vitro invasion assay, we demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of pancreatic cancer cells and, surprisingly, the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts. The hepatocyte growth factor (HGF) secretion from fibroblasts remained unchanged after irradiation, whereas exposure of pancreatic cancer cells to supernatant from irradiated fibroblasts resulted in increased phosphorylation of c-Met (HGF receptor) and mitogen-activated protein kinase activity, possibly or partially via increased expression of c-Met. We also demonstrated that scattering of pancreatic cancer cells was accelerated by the supernatant from irradiated fibroblasts. The enhanced invasiveness of pancreatic cancer cells induced by coculture with irradiated fibroblasts was completely blocked by NK4, a specific antagonist of HGF. These data suggest that invasive potential of certain pancreatic cancer cells is enhanced by soluble mediator(s) released from irradiated fibroblasts possibly through up-regulation of c-Met expression/phosphorylation and mitogen-activated protein kinase activity in pancreatic cancer cells. Our present findings further support the potential use of NK4 during radiotherapy for patients with pancreatic cancer.     

Hepatic gene expression of NK4, an HGF-antagonist/angiogenesis inhibitor, suppresses liver metastasis and invasive growth of colon cancer in mice

Hepatocyte growth factor (HGF) is involved in malignant behavior of cancer cells by enhancing invasion and metastasis. We earlier found that NK4, a four-kringle fragment of HGF, functions as both an HGF antagonist and an angiogenesis inhibitor. We have now carried out studies to determine if hydrodynamics-based delivery and expression of the NK4 gene would inhibit liver metastasis and invasive growth of colon carcinoma cells in mice. When the naked plasmid for NK4 was introduced into mice by hydrodynamics-based gene delivery, a high level of expression of NK4 was predominant in the liver. After intrasplenic inoculation of MC-38 murine colon carcinoma cells, the cells formed numerous metastatic nodules in the liver and showed invasive growth behavior. On the other hand, when mice were given the NK4 plasmid, hepatic gene expression of NK4 inhibited the liver metastasis and subsequent growth associated with a decrease in microvessel density. Likewise, intrahepatic invasion of cancer cells was inhibited by NK4 gene expression, and this anti-invasive effect was associated with in situ inhibition of c-Met receptor tyrosine phosphorylation. Moreover, NK4 gene expression prolonged survival of these mice. Taken together with the knowledge that the majority of deaths from colon cancer are due to liver metastasis, the potential therapeutic use of hepatic gene expression of NK4 for metastatic colon cancer treatment can be given consideration.     

Inhibition of growth, invasion, and metastasis of human pancreatic carcinoma cells by NK4 in an orthotopic mouse model

Hepatocyte growth factor (HGF) is involved in malignant behavior of cancers as a mediator in tumor-stromal interactions through enhancing tumor invasion and metastasis. We found recently that NK4, a four-kringle fragment of HGF, functions as both an HGF-antagonist and an angiogenesis inhibitor. We have now determined whether blockade of the HGF-c-Met/HGF receptor pathway and tumor angiogenesis by administration of recombinant NK4 would inhibit growth, invasion, and metastasis of human pancreatic carcinoma implanted into the pancreas of nude mice. When treatment with NK4 or anti-HGF neutralizing antibody was initiated from the third day after orthotopic injection of SUIT-2 human pancreatic cancer cells, both NK4 and anti-HGF antibody suppressed the conversion of orthotopic pancreatic tumors from carcinoma in situ to aberrantly invading cancers during days 3-14. On the other hand, when the treatment was begun on day 10, a time when cancer cells were already invading surrounding tissues, NK4 but not anti-HGF antibody inhibited tumor growth, peritoneal dissemination, and ascites accumulation at 4 weeks after the inoculation. Antitumor effects of NK4 correlated with decreased microvessel density in pancreatic tumors thereby indicating that the antiangiogenic activity of NK4 may have mainly contributed to its antitumor effects. Moreover, although NK4-treatment was initiated from the end stage (day 24 after tumor inoculation), NK4 prolonged survival time of mice, and the suppression of peritoneal dissemination, ascites accumulation, and invasion of metastasized cancer cells into the peritoneal wall were remarkable. We propose that simultaneous targeting of both tumor angiogenesis and the HGF-mediated invasion-metastasis may prove to be a new approach to treating patients with pancreatic cancer.     

Induction of hepatocyte growth factor activator gene expression under hypoxia activates the hepatocyte growth factor/c-Met system via hypoxia inducible factor-1 in pancreatic cancer

Hepatocyte growth facor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. Our previous study demonstrated that tumor–stromal interaction under hypoxia augments the aggressive invasive features of pancreatic cancer line PK8 through activated HGF/c-Met signaling. The present study investigated whether or not hypoxia increases HGFA expression in PK8 cells and promotes the processing of HGF, and leads to c-Met activation. Moreover, HGFA promoter assays were performed to define whether hypoxia inducible factor-1 alpha (HIF-1α) directly activates the HGFA promoter in a hypoxia-dependent fashion. As a result, hypoxia induced the HGFA mRNA and protein expression in PK8 and the elevation under hypoxia was inhibited by the transfection of HIF-1α siRNA, thus indicating HIF-1α-dependent induction of HGFA. The transfection of siRNA against HGFA to PK8 cells suppressed the conversion to the active HGF, which is secreted from fibroblast MRC5. Furthermore, the phosphorylation of c-Met and cancer invasion of PK8 cells were decreased by the transfection of HGFA siRNA under hypoxia. Using the luciferase reporter system, HIF-1α was shown to transactivate the HGFA promoter under hypoxia. These experiments demonstrated for the first time that HGFA is a novel HIF-1 target gene. Under hypoxia, HGFA might be overexpressed and secreted from pancreatic cancer cells, which contributes to accelerate processing of HGF from fibroblast, resulting in the activation of the c-Met pathway. HGF/HGFA/c-Met recruited between cancer-stromal fibroblasts is activated under hypoxic conditions and therefore might play a central role in the aggressive invasion of pancreatic cancer. ( Cancer Sci 2008; 99: 1341–1347)     

Intraperitoneal injection of adenovirus-mediated NK4 gene suppresses peritoneal dissemination of pancreatic cancer cell line AsPC-1 in nude mice

NK4, composed of the N-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts not only as a competitive antagonist for HGF but also as a potent angiogenesis inhibitor. This study was designed to assess a therapeutic potential of adenovirus-mediated NK4 gene transfer for disseminated pancreatic cancer cells in the peritoneal lavage of nude mice. We constructed a recombinant adenovirus NK4 (Ad-NK4), which encodes a secretable form of human NK4. In vitro migration of AsPC-1 (human pancreatic cancer cell line) was stimulated by HGF, and it was completely inhibited by Ad-NK4 transfection. Weekly intraperitoneal injections of Ad-NK4 could suppress the development of tumor nodules in a nude mouse peritoneal dissemination model. NK4 expression was detected in the disseminated nodules, liver, pancreas, spleen, and mesenterium. Immunohistochemical study of the disseminated tumors showed a remarkable decrease in microvessel density and an increase in number of apoptotic tumor cells in the Ad-NK4-treated mice. Survival of the Ad-NK4-treated mice was significantly improved. This study indicates that the intraperitoneal transduction of adenovirus-mediated NK4 gene may be a useful therapeutic modality to prevent the development of peritoneal dissemination of pancreatic cancer.