hFRNK基因对胃泌素诱导的人结肠癌细胞侵袭力的影响

目的 观察腺病毒介导hFRNK基因对胃泌素所诱导的人结肠癌Colo320WT细胞侵袭力的影响.方法 试验分为胃泌素组、hFRNK组和对照组,胃泌素组用100 μmol/L胃泌素诱导结肠癌Col0320WT细胞12 h;hFRNK组,首先用脂质体瞬时转染腺病毒受体pCR3.1-CAR于Col0320WT细胞48 h,然后用100 μmol/L胃泌素干预结肠癌Colo320WT细胞12 h,再用重组腺病毒(pAdhFRNK)感染细胞;对照组为未经处理的Colo320WT细胞.用免疫印迹检测hFRNK基因黏着斑激酶(FAK)397位酪氨酸(FAKTyr397)的磷酸化表达,激光共聚焦显微镜观察FAKTyr397在细胞板状伪足的表达情况,免疫共沉淀检测hFRNK基因对四联信号复合物FAK-Src-Doek180一p130Cas形成的影响,Pull-down法检测hFRNK对Rac蛋白活性的影响.结果 胃泌素诱导后,磷酸化FAKTyrr397明显增强;与胃泌素组相比,hFRNK组中FAKTyr397表达下降,FAKTyr397定位到细胞板状伪足的量明显减少,FAK、Src、Dockl80和p130Cas 四联信号复合物没有形成,Rac的活性降低.结论 hFRNK基因可阻断胃泌素引起的FAK的磷酸化,阻断FAKTyr397摹积到细胞的板状伪足,阻止四联信号复合物FAK-Src-Dock180-p130Cas的形成以及Rac的活化,为hFRNK基因防治肿瘤的侵袭和转移提供理论依据.     

人结肠癌细胞胃泌素-黏着斑激酶通路的研究

目的研究胃泌素对人结肠癌细胞信号分子黏着斑激酶(FAK)酪氨酸磷酸化和蛋白质表达的影响。方法使用胆囊收缩素2受体(CCK2R)的真核表达载体pCR3.1/CCK2R,转染人结肠癌细胞株Colo320,上调胃泌素作用;使用胃泌素拮抗剂下调胃泌素作用。使用胃泌素按浓度和时间梯度刺激细胞,以免疫沉淀和蛋白质印迹法检测FAK酪氨酸磷酸化和蛋白质表达情况。结果胃泌素能够引起FAK酪氨酸磷酸化,具有时间和剂量依赖性;CCK2R表达上调可以增强此作用;胃泌素拮抗剂具有相反作用。结论FAK是胃泌素发挥效应的下游信号分子,以酪氨酸磷酸化的形式发挥作用。胃泌素CCKRFAK信号通路在胃泌素引起的肿瘤细胞增殖过程中发挥重要作用。     

胃泌素对结肠癌细胞中粘着斑激酶通路下游E-钙粘蛋白/β-连环蛋白复合物的影响

目的探讨胃泌素对结肠癌细胞CoLo320WT中粘着斑激酶（FAK）通路下游E-钙粘蛋白／β-连环蛋白（E-cadherin／β-catenin）复合物分布的影响；方法脂质体转染表达胃泌索受体CCK-2R的pCR3．1／OR质粒于结肠癌细胞CoLo320中。G418筛选出稳定表达CCK-2R的阳性克隆，RT-PCR鉴定，转染成功命为CoLo320WT。应用10^-8mmol／L 胃泌素（G17）以时间梯度（0h、1h、6h、12h、24h、48h）干预CoLo320WT细胞，同时应用10^-6mmol／L胃泌素受体拮抗剂L365，260干预CoLo320WT细胞30min，再予10^-8mmol／L胃泌素干预。采用免疫印迹法检测磷酸化的FAK Tyr397和总FAK的表达。采用免疫共沉淀和免疫印迹法检测CoLo320WT中TX-100溶解和未溶部分中的E-钙粘蛋白和β-连环蛋白的表达。用免疫细胞化学法观察E-钙粘蛋白和β-连环蛋白的在胞膜、胞质和胞核的分布。结果随着胃泌素干预时间的延长，细胞中磷酸化的FAK Tyr397的表达量呈增加趋势，12h达最大值。胃泌素受体拮抗剂L365，260阻断后磷酸化的FAK Tyr397表达减少。而胃泌素对总FAK没有明显影响。TX-100可溶性部分中E-钙粘蛋白和酽连环蛋白的量在胃泌素干预后表达减少，拮抗剂L365，260阻断后又增加。而TX-100不溶解部分中表达却相反。免疫细胞化学观察到在胃泌素干预下CoLo320WT细胞中E-钙粘蛋白和β-连环蛋白的分布发生胞质和胞核转移。结论胃泌素与其受体CCK-2受体结合，磷酸化的FAK Tyr397、激活FAK通路进而影响结肠癌细胞中E-钙粘蛋白和β-连环蛋白的分布，促进结肠癌细胞侵袭和转移。     

粘着斑激酶反义寡核苷酸与胃癌细胞生长及凋亡的关系

根据FAKcDNA序列设计合成与FAKmRNA碱基序列互补的FAK反义寡核苷酸,导入BGC－823胃癌细胞,通过MTT比色法、细胞免疫化学染色法、逆转录－聚合酶链反应（RT－PCR）、细胞周期及电镜等方法,检测FAK反义寡核苷酸对人胃癌细胞的影响。结果示：①胃癌细胞生长明显受到抑制,抑制效应呈浓度和时间依赖性。②细胞内P125蛋白和FAKmRNA表达均明显下降。③经FAK反义寡核苷酸处理后BGC－823胃癌细胞发生凋亡,FCM普上可见明显的凋亡峰,电镜观察呈现凋亡早期形态改变,认为FAK反义寡核甘酸导入BGC－823胃癌细胞后,使BGC－823胃癌细胞FAKmPNA及P125蛋白表达水平下降,抑制PGC－823胃癌细胞增列,同时诱导BGC－823胃癌细胞发生凋亡。     

黏着斑激酶在结肠癌组织中的表达及意义

目的探讨黏着斑激酶（FAK）在结肠癌发生、发展中的作用。方法采用RT-PCR法检测30例新鲜结肠癌及与之相对应的癌旁组织的FAK mRNA表达；同时用Western印迹法检测20例新鲜结肠癌及相对应的癌旁组织FAK蛋白表达水平，调平每对组织FAK蛋白含量后再行FAK Tyr397磷酸化位点蛋白检测。结果30例结肠癌组织FAK mRNA阳性率为90．0％，其表达值为0．745±0．530，对应癌旁组织阳性率为43．3％，其表达值为0．241±0．131（P〈0．01）；20例结肠癌组织FAK蛋白阳性率为95．0％，表达值为0．482±0．150；对应癌旁组织阳性率为60．0％，表达值为0．269±0．015；P均〈0．01。20例结肠癌组织中FAK Tyr397磷酸化蛋白阳性率为90．0％（18／20），表达值为0．385±0．021；而对应癌旁组织阳性率为20％（4／20），表达值为0．110±0．005，P均〈0．01。结论FAK特别是FAK Tyr397磷酸化蛋白表达增加在结肠癌的发生、发展中可能起重要作用。     

粘着斑激酶相关非激酶质粒转染抑制肝星状细胞周期进程及机制研究

粘着斑激酶（focal adhesion kinase，FAK）磷酸化后，对多种类型细胞的生物学行为具有重要影响，如促进增殖，正性调控细胞周期，增强粘附、迁移，控制凋亡等。粘着斑激酶相关非激酶（FAK—related non-kinase，FRNK）是FAK的内源性抑制剂。本研究应用体外细胞培养技术，以纤维连接蛋白（fibronectin，FN）刺激肝星状细胞（hepatic stellate cell，HSC）增殖，在脂质体介导下瞬时转染FRNK质粒，探讨选择性阻断FAK磷酸化对HSC增殖周期及细胞周期相关蛋白的影响。     

肝细胞生长因子对肝细胞癌细胞系SMMC-7721黏着斑激酶的影响

目的:探讨肝细胞生长因子(HGF)对黏着斑激酶(FAK)、Src表达及活性的影响以及PI3K在该过程中的作用.方法:LY294002预处理阻断PI3K的活性、HGF处理SMMC-7721后检测FAK和Src的表达、磷酸化及在细胞内的分布. 应用免疫印迹技术检测FAK、Src的表达及磷酸化, 免疫荧光技术检测FAK在SMMC-7721细胞中的分布.结果:HGF(50 μg/L)可以促进FAK Y397位点的磷酸化, 对FAK的表达没有影响. 应用PI3K抑制物LY294002处理后, FAK Y397的磷酸化水平显著降低. HGF处理后, FAK主要位于细胞边缘, 呈簇状分布, 应用PI3K抑制物, FAK在细胞内弥散分布. HGF处理后, Src激酶和SrcY416的磷酸化水平明显增加, 而经LY294002处理后, Src激酶与Src Y416的磷酸化水平明显降低.结论:肝细胞癌中, HGF以PI3K依赖性的方式促进FAK和Src的活化.     

黄白抑瘤方对人直肠癌Colo320细胞凋亡与Caspase3及聚ADP核糖聚合酶表达的影响

[目的]探讨黄白抑瘤方对Colo320细胞凋亡与凋亡相关蛋白Caspase3及聚ADP核糖聚合酶（PARP）表达的影响,以明确其治疗结直肠癌的生物学基础。[方法]使用不同浓度黄白抑瘤方体外干预Colo320细胞,检测不同浓度黄白抑瘤方对Colo320细胞凋亡的影响,检测其处理后Colo320细胞内凋亡相关蛋白Caspase3、PARP的表达。[结果]黄白抑瘤方作用Colo320细胞48h后,Colo320细胞出现了不同程度的亚二倍体凋亡峰,相较对照组细胞的早凋率明显上升（P〈0．01）;凋亡相关蛋白Caspase3和PARP表达差异有统计学意义（P〈0．01）。[结论]黄白抑瘤方能参与人直肠癌Colo320细胞的胞内凋亡通路,诱导细胞凋亡,从而达到抗肿瘤的作用。     

结缔组织生长因子反义寡核苷酸抑制血管紧张素Ⅱ诱导人近曲肾小管上皮细胞肥大米

目的：进一步探讨结缔组织生长因子（connective tissue grnwth factor，CTGF）反义寡核苷酸对血管紧张素Ⅱ（AngII）诱导的肾小管细胞肥大的影响。方法：采用体外培养的人肾近端小管上皮细胞株（HK2），分别采用考马斯亮蓝法、流式细胞分析技术及扫描电镜和透射电镜，观察CTGF反义寡核苷酸对AngII诱导的细胞内总蛋白含量、细胞周期分布改变及细胞直径和超微结构改变的影响。结果：AngⅡ干预HK2细胞48h后，细胞内总蛋白含量显著增加（P＜0．01）；这种作用可被CTGF反义寡核苷酸显著抑制，且呈时间和浓度依赖性。AngⅡ干预HK2细胞后，细胞大部分阻滞在G0-G1期（P＜0.01），而CTGF反义寡核苷酸可逆转这种周期阻滞现象（P＜0．05）。经AngⅡ干预的HK2细胞，细胞平均直径显著增加，细胞超微结构显示表面微绒毛减少、细胞内粗面内质网增多、线粒体减少、高尔基体增加，这些作用均可被CTGF反义寡核苷酸所抑制。结论：CTGF反义寡核苷酸对AngⅡ诱导HK2细胞肥大具有显著的抑制作用，提示CTGF可能介导了AngⅡ诱导的肾小管上皮细胞肥大。     

黏着斑激酶在结肠癌细胞中的表达及对其迁移的影响

[目的]观察局部黏着斑激酶（FAK）在结肠癌细胞中的表达及对结肠癌细胞侵袭动力和生长的影响。[方法]应用RT-PCR、Western blot方法测定结肠癌细胞株（HT-29）和正常肠上皮细胞中FAK的表达；在血小板源性生长因子（PDGF）刺激肿瘤细胞后，应用细胞黏附分析和体外侵袭试验方法，测定不同时间点细胞的体外黏附能力变化及侵袭能力，同时行FAK表达测定。[结果]结肠癌细胞较正常上皮细胞存在较高水平的FAK表达。PDGF干预组与非干预组比较，能促进HT-29的黏附功能（90．0％）和侵袭能力（54．5％），在该过程中，FAK的表达和活性升高（P〈0．05）。[结论]FAK对结肠癌细胞的体外黏附与迁移发挥重要的作用。     

Reduced expression of focal adhesion kinase disrupts insulin action in skeletal muscle cells

Integrins mediate interactions between cells and extracellular matrix proteins that modulate growth factor signaling. Focal adhesion kinase (FAK) is a key multifunctional integrin pathway protein. We recently reported that disruption of FAK impairs insulin-mediated glycogen synthesis in hepatocytes. To test the hypothesis that FAK regulates skeletal muscle insulin action, we reduced FAK expression in L6 myotubes using FAK antisense. In untransfected myotubes, insulin stimulated both FAK tyrosine phosphorylation and kinase activity. Cells treated with antisense FAK showed 78 and 53% reductions in FAK mRNA and FAK protein, respectively, whereas insulin receptor substrate 1/2 and paxillin abundance were unaffected. Insulin-stimulated U-(14)C-glucose incorporation into glycogen was abolished by FAK antisense, and 2-deoxy-glucose uptake and glucose transporter 4 (GLUT4) translocation were both markedly attenuated. Antisense FAK did not alter GLUT1 or GLUT3 protein abundance. Immunofluorescence staining showed decreased FAK Tyr(397) phosphorylation and reduced actin stress fibers. Thus, in skeletal myotubes, FAK regulates the insulin-mediated cytoskeletal rearrangement essential for normal glucose transport and glycogen synthesis. Integrin signaling may play an important regulatory role in muscle insulin action.     

粘着斑激酶及磷酸化粘着斑激酶在结肠癌中的表达及其临床意义

目的研究粘着斑激酶(FAK)和磷酸化粘着斑激酶(phospho-FAK,Tyr397)在结肠癌中的表达水平及其与侵袭和转移的关系.方法用免疫组织化学S-P法检测45例结肠癌和对应癌旁组织的FAK和磷酸化FAK(Tyr397)的表达.结果癌与癌旁组织FAK表达率分别为82.22%(37/45)和28.89%(13/45),磷酸化FAK(Tyr397)表达率分别为66.78%(30/45)和26.78%(12/45),两者比较差异有显著性(P值均＜0.01).癌组织中FAK表达率在分化程度(P＜0.05)、浸润深度(P＜0.01)和淋巴结转移(P＜0.05)中有显著性差异,磷酸化FAK(Tyr397)在分化程度(P＜0.01)、浸润深度(P＜0.05)和淋巴结转移(P＜0.01)中也有显著性差异,FAK和磷酸化FAK(Tyr397)在年龄(P>0.05)、性别(P>0.05)及远处转移(P>0.05)中差异无显著性.结论结肠癌中FAK和磷酸化FAK(Tyr397)的表达水平明显升高,与分化程度、浸润、淋巴结转移相关.     

Progress in researches about focal adhesion kinase in gastrointestinal tract

Focal adhesion kinase (FAK) is a 125-kDa non-receptor protein tyrosine. Growth factors or the clustering of integrins facilitate the rapid phosphorylation of FAK at Tyr-397 and this in turn recruits Src-family protein tyrosine kinases, resulting in the phosphorylation of Tyr-576 and Tyr-577 in the FAK activation loop and full catalytic FAK activation. FAK plays a critical role in the biological processes of normal and cancer cells including the gastrointestinal tract. FAK also plays an important role in the restitution, cell survival and apoptosis and carcinogenesis of the gastrointestinal tract. FAK is overexpressed in cancer cells and its over-expression and elevated activities are associated with motility and invasion of cancer cells. FAK has been proposed as a potential target in cancer therapy. Small molecule inhibitors effectively inhibit the kinase activity of FAK and show a potent inhibitory effect for the proliferation and migration of tumor cells, indicating a high potential for application in cancer therapy.     

Pressure activates colon cancer cell adhesion by inside-out focal adhesion complex and actin cytoskeletal signaling

BACKGROUND AND AIMS: Few circulating tumor cells implant or cause metastasis. We hypothesized that venous or lymphatic pressure or iatrogenic pressure during resection activates signals governing malignant colonocyte adhesion. METHODS: We studied the effect of 15 mm Hg increased pressure for 30 minutes on adhesion of primary human colon cancer cells and SW620 colonocytes to collagen and endothelial cells. We modulated integrin affinity with extracellular cations. We assessed binding affinity by detachment assay; integrin surface expression by flow cytometry; and focal adhesion kinase (FAK), Src, and extracellular signal-regulated kinase (ERK) activation by Western analysis and Src in vitro kinase assay. We inhibited Src (PP2), FAK (small RNA interference, SiRNA, or FRNK transfection), MEK (PD98059), PKC (calphostin C), and actin destabilization (phalloidin). RESULTS: Pressure and manganese stimulated primary and SW620 colonocyte adhesion to collagen. Pressure also stimulated SW620 adhesion to endothelial monolayers. Pressure strengthened SW620 binding force to matrix without changing integrin surface expression. Pressure activated SW620 FAK and Src, but not ERK. Manganese did not. Calcium-inhibited adhesion but stimulated FAK (but not Src). PP2 prevented pressure activation of Src, Src phosphorylation of FAK576, and pressure-stimulated adhesion but not FAK397 autophosphorylation. FRNK transfection or FAK SiRNA also prevented pressure-stimulated adhesion. FAK SiRNA ablated pressure-activated FAK397, Src, and FAK576 phosphorylation. Neither Src nor FAK inhibition blocked cation effects. Phalloidin prevented pressure-stimulated adhesion. PD98059 or calphostin C did not. CONCLUSIONS: In contrast to divalent cations, extracellular pressure may increase integrin affinity and promote colon cancer adhesion via actin dependent inside-out FAK and Src signals. This mechanotransduced pathway may regulate metastasizing tumor cell adhesion.     

Ethanol Enhances the Interaction of Breast Cancer Cells Over‐Expressing ErbB2 With Fibronectin

Background: Ethanol is a tumor promoter and may enhance the metastasis of breast cancer. However, the underlying cellular/molecular mechanisms remain unknown. Amplification of ErbB2 or HER2, a receptor tyrosine kinase of the ErbB family, is found in 20 to 30% of patients with breast cancer. We have previously demonstrated that the effect of ethanol on the migration/invasion of breast cancer cells positively correlated with the expression levels of ErbB2. Adhesion to the extracellular matrix (ECM) is an important initial step for cancer cell invasion and metastasis. In this study, we investigated the effects of ethanol on the adhesion of MCF7 breast cancer cells over‐expressing ErbB2 (MCF7^ErbB2) to human plasma fibronectin. Methods: To test the hypothesis that ethanol may enhance the attachment of human breast cancer cells to fibronectin, an important component of the ECM, we evaluated the effect of ethanol on the expression of focal adhesions, cell attachment, and ErbB2 signaling in cultured MCF7^ErbB2 cells. Results: Exposure to ethanol drastically enhanced the adhesion of MCF^ErbB2 cells to fibronectin and increased the expression of focal adhesions. Ethanol induced phosphorylation of ErbB2 at Tyr1248, FAK at Tyr861, and cSrc at Try216. Ethanol promoted the interaction among ErbB2, FAK, and cSrc, and the formation of a focal complex. AG825, a selective ErbB2 inhibitor, attenuated the ethanol‐induced phosphorylation of ErbB2 and its association with FAK. Furthermore, AG825 blocked ethanol‐promoted cell/fibronectin adhesion as well as the expression of focal adhesions. Conclusions: Our results suggest that ethanol enhances the adhesion of breast cancer cells to fibronectin in an ErbB2‐dependent manner, and the FAK pathway plays an important role in ethanol‐induced formation of a focal complex.     

CrkII regulates focal adhesion kinase activation by making a complex with Crk-associated substrate, p130Cas

CrkII is an adaptor protein possessing oncogenic potential despite the lack of an enzymatic domain. We investigated here the physiological functions of CrkII by studying its ability to induce anchorage-independent cell growth. We found that inhibition or null mutation of focal adhesion kinase (FAK) blocked the anchorage-independent growth induced by CrkII overexpression, indicating that FAK is a critical determinant of the transforming activity of CrkII. CrkII overexpression enhanced the autophosphorylation of FAK at Tyr-397 and tyrosine phosphorylation of p130(Cas) (Crk-associated substrate, Cas) upon stimulation of integrin by fibronectin. Moreover, the constitutive phosphorylation of FAK and Cas was observed in CrkII-overexpressing cells, even when they were in the suspended condition, consistent with the ability of CrkII to induce anchorage-independent growth. Using Cas-deficient cells, we showed Cas function to be essential for both the CrkII-induced phosphorylation of FAK (Tyr-397) and anchorage-independent cell growth. The CrkII-induced FAK autophosphorylation depended upon CrkII-Cas complex formation. Furthermore, we showed that CrkII knockdown resulted in defects in integrin-mediated events, such as cell spreading, haptotactic migration, and FAK autophosphorylation. The integrin-mediated FAK autophosphorylation was also reduced in Cas-deficient cells. These results suggest that the CrkII-Cas complex functions in integrin-mediated FAK activation signaling. Our findings show the importance of CrkII in integrin-mediated events, acting upstream of FAK to affect the activation of this kinase, which appears to have a central role in this pathway.     

Gastrin suppresses growth of CCK2 receptor expressing colon cancer cells by inducing apoptosis in vitro and in vivo

BACKGROUND & AIMS: The role of amidated gastrin17 (G17) and the gastrin/CCKB/CCK2 receptor in colorectal carcinogenesis is still a controversial issue. Here, we investigated the effect of G17 on proliferation and apoptosis of CCK2 receptor-expressing human colon cancer cell lines in vitro and in vivo. METHODS: Proliferation was determined by cell counting and cell cycle analysis. Apoptosis was analyzed by annexin V staining, TUNEL staining, caspase-3/7 assay, and JC1 (delta psi) assay. Signal-transduction pathways were analyzed by Western blotting and gel-shift and luciferase assays. An in vivo tumor model with subcutaneously inoculated colon cancer cells in SCID mice was used, and systemic hypergastrinemia was induced by omeprazole. RESULTS: In Colo320 cells stably transfected with the wild-type CCK2 receptor (Colo320wt) or in Lovo cells endogenously expressing CCK2 receptors, G17 treatment inhibited proliferation along with a G2/M cell cycle arrest. Furthermore, the administration of G17 significantly augmented apoptosis of CCK2 receptor-expressing cells. In contrast, G17 had no effect on proliferation and apoptosis in Colo320 cells stably transfected with a tumor-derived CCK2 receptor mutant (Colo320mut) or in cells lacking CCK2 receptor expression. Systemic hypergastrinemia in severe combined immunodeficiency (SCID) mice suppressed the growth of Colo320wt tumors accompanied by enhanced apoptosis as compared with untreated tumors. In contrast, omeprazole did not affect Colo320mut tumors reflecting a loss-of-function state of the CCK2(mut) receptor. This is supported by the observation that, in Colo320wt cells, but not in Colo320mut cells, G17 treatment induced the MAPK/ERK/AP-1 pathway and inhibited the activity of NF-kappaB. CONCLUSIONS: G17 exerts an antiproliferative and proapoptotic effect on human colon cancer cells expressing the wild-type CCK2 receptor. This supports the view that amidated gastrin prevents rather than promotes colorectal carcinogenesis.     

ERK1／2抑制剂PD98059对大肠癌细胞侵袭和转移的影响

目的观察细胞外信号调节蛋白激酶（ERK1／2）抑制剂PD98059对大肠癌细胞侵袭和转移的影响,探讨其可能机制。方法PD98059不同浓度（0、10、20、40μmo1／L）干预大肠癌细胞24h。用Boyden小室检测其体外运动和侵袭能力,Western blot法检测ERK1／2磷酸化蛋白和ERK1／2蛋白表达情况。结果随着PD98059剂量增大,大肠癌细胞穿膜数逐渐减少（P〈0．05）,磷酸化ERK1／2蛋白水平逐渐降低（P〈0．05）,ERK1／2蛋白水平无明显变化（P〉0．05）。结论PD98059能抑制大肠癌细胞的运动和侵袭力;其机制与PD98059抑制磷酸化ERK1／2有关。