Inhibiting focal adhesion kinase:A potential target for enhancing therapeutic efficacy in colorectal cancer therapy

Focal adhesion kinase(FAK) is a major integrin- dep-endent tyrosine phosphorylated protein, recently, FAK association with colorectal cancer(CRC) has gained at-tention. The various cancer-promoting mechanisms that associated with FAK can be implicated in the progression of CRC. The interactions between structural features of FAK and various kinases could be closely related to growth, survival, and metastasis in CRC cells. These interactions include human epithelial growth factor re-ceptor, c-Met, platelet-derived growth factor receptor, vascular endothelial growth factor receptor, and Src. Such interactions can trigger the survival signaling of CRC cells and are also involved signaling downstream of phosphatidylinositol 3-kinase, AKT, and the extracellular regulated kinase. Based on this scientific background, many pharmaceutical companies are taking efforts to develop FAK inhibitors to treat solid cancer including CRC. Although the anti-cancer efficacies have been noted in many studies, the commercial drugs have not been deve-loped yet. Therefore, the FAK research on CRC is expec-ted to gain momentum and be highly appreciated as a potential field for developing the new drugs. Therefore, the studies on FAK that effect on the progression of human CRC s would be possible to suggest various app-roaches to CRC treatment, and FAK could be a potential target as an anticancer candidate for CRC therapies.

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 over-expressed 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.

The Overexpressed FAK （Focal Adhesion Kinase） in Higher Grade Human Urothelial Tumors

Malignant transformation of normal cells involves important structural and functional changes, particularly in cell adhesion. In this study, we wanted to assess whether changes in the expression of FAK, a tyrosine kinase, which is recruited to focal adhesions and plays a key role in cell migration, proliferation and survival, could reflect the invasive capacity of bladder carcinomas. The aim of this study was to evaluate the FAK expression in cancer ceils as an important prognostic factor of the evolution of bladder carcinomas. Tumor and paired peritumoral biopsies were obtained during transurethral endoscopic resection or cystectomy of bladder tumors in 280 patients at the Urology Unit of the Mustapha Hospital of Algiers and the Hospital of Tizi-Ouzou （Algeria）. The authors studied FAK expression in samples from bladder carcinomas at different stages of malignant transformation by western blot analysis using a specific anti-FAK antibody. Western blot is one of the most common laboratory techniques; it is used to detect the presence of a specific protein in a complex mixture extracted from cells. A weak increase in FAK expression was observed in tumors of grade 1 and 2 （1.65; 2.99） as compared to healthy tissues; it became particularly important in grade 3 tumors; the authors show that FAK levels significantly increased gradually according to the tumor stage.

Degradation of FAK-targeting by proteolytic targeting chimera technology to inhibit the metastasis of hepatocellular carcinoma

Liver cancer is a prevalent malignant cancer,ranking third in terms of mortality rate.Metastasis and recurrence primarily contribute to the high mortality rate of liver cancer.Hepatocellular carcinoma(HCC)has low expression of focal adhesion kinase(FAK),which increases the risk of metastasis and recurrence.Nevertheless,the efficacy of FAK phosphorylation inhibitors is currently limited.Thus,investigating the mechanisms by which FAK affects HCC metastasis to develop targeted therapies for FAK may present a novel strategy to inhibit HCC metastasis.This study examined the correlation between FAK expression and the prognosis of HCC.Additionally,we explored the impact of FAK degradation on HCC metastasis through wound healing experiments,transwell invasion experiments,and a xenograft tumor model.The expression of proteins related to epithelial-mesenchymal transition(EMT)was measured to elucidate the underlying mechanisms.The results showed that FAK PROTAC can degrade FAK,inhibit the migration and invasion of HCC cells in vitro,and notably decrease the lung metastasis of HCC in vivo.Increased expression of E-cadherin and decreased expression of vimentin indicated that EMT was inhibited.Consequently,degradation of FAK through FAK PROTAC effectively suppressed liver cancer metastasis,holding significant clinical implications for treating liver cancer and developing innovative anti-neoplastic drugs.

Overexpression of MiR-633 Suppresses the Tumorigenicity of Gastric Cancer Cells and Induces Apoptosis by Targeting MAPK1

Objective MicroRNA(miRNA/miR)-633 is dysregulated in several types of cancers and is involved in tumorigenesis.However,the function and role of this miRNA in gastric cancer(GC)are not fully understood.The aim of the present study was to evaluate miR-633 expression in GC cell lines and in GC tissue vs.adjacent normal tissue,and to determine its association with clinicopathological data.This work was extended to investigate the effects of miR-633 overexpression on tumor cells in vitro.Methods Reverse transcription-quantitative PCR(RT-qPCR)was used to detect and compare the expression level of miR-633 in GC cells,as well as in GC and normal adjacent tissue samples.The clinical significance of miR-633 was also analyzed.MiR-633 lentivirus(LV-miR-633)and negative control lentivirus(LV-NC)were generated and used to transduce SGC-7901 and HGC-27 GC cells in order to analyze the effect of miR-633 on their phenotype.The effects of miR-633 overexpression on GC cell proliferation,apoptosis,migration and invasion were investigated.The target gene of miR-633 was predicted,then confirmed using a dual luciferase reporter gene assay,RT-qPCR and Western blotting.Results MiR-633 was significantly downregulated in GC cell lines,as well as in GC tissue compared with adjacent normal tissue.Moreover,miR-633 expression was associated with the tumor/node/metastasis(TNM)stage,invasion depth,Borrmann classification and lymph node metastasis(P<0.05).Compared with the LV-NC group,transduction with LV-miR-633 reduced the proliferation,the number of clones,the wound healing rate,the number of invading cells and the number of cells in the G1 phase of the cell cycle(P<0.01).LV-miR-633 also increased the apoptosis rate(P<0.01).The expression level of mitogen-activated protein kinase(MAPK)1,high-mobility group box 3(HMGB3),claudin 1(CLDN1)and MAPK13 were downregulated in LV-miR-633-transduced cells(P<0.01).The dual luciferase reporter assay confirmed that the 3′-untranslated region of MAPK1 was the target site of miR-633(P<0.01).Conclusion MiR-633 acts as a tumor suppressor in GC,and its expression level is associated with TNM stage,invasion depth,Borrmann type and lymph node metastasis.Overexpression of miR-633 inhibits the proliferation and migration of GC cells and induces apoptosis and cell cycle arrest at the in G1 phase.In addition,miR-633 negatively regulates the expression of MAPK1,HMGB3,CLDN1 and MAPK13 and directly targets MAPK1.

Key Role of CD151-integrin Complex in Lung Cancer Metastasis and Mechanisms Involved

Tetraspanin CD151 was found to be upregulated in malignant cell types and has been identified as a tumor metastasis promoter.In this study,we aimed to examine the role of the CD151-integrin complex in lung cancer metastasis and the underlying mechanisms.CD151 QRD194–196→AAA194–196 mutant was generated and used to transfect A549 human lung adenocarcinoma cells.We found that there was no significant difference in CD151 protein expression between CD151 and CD151-AAA mutant groups.In vitro,CD151-AAA mutant delivery abrogated the migration and invasion of A549 cells,which was promoted by CD151 gene transfer.Furthermore,CD151-AAA delivery failed to activate FAK and p130Cas signaling pathways.Western blot and immunohistochemical staining showed strong CD151 expression in lung cancerous tissues but not in adjacent normal tissues.Increased level of CD151 protein was observed in 20 of the patients and the positive rate of CD151 protein in specimens was 62.5%(20/32).In addition,CD151 was co-localized withα3 integrin at the cell-cell contact site in carcinoma tissues.These results suggested that the disruption of the CD151-α3 integrin complex may impair the metastasis-promoting effects and signaling events induced by CD151 in lung cancer.Our findings identified a key role for CD151-α3 integrin complex as a promoter in the lung cancer.

通关藤对胃癌小鼠Src/FAK信号通路及Th1/Th2相关因子水平的影响

目的探讨通关藤对胃癌小鼠类固醇受体共激活因子(Steroid receptor coactivator,Src)/黏着斑激酶(Focaladhesion kinase,FAK)信号通路及Th1/Th2相关因子水平的影响。方法将SPF级成年雌雄各半C57BL/6小鼠60只,根据随机数字表法分为空白对照组、模型组(注射生理盐水)、环磷酰胺组(注射环磷酰胺)、通关藤低、中、高剂量组(分别注射0.1 ml、0.2 ml、0.4 ml通关藤注射液),每组各10只。干预1次/2 d,于干预第21天时处死所有小鼠,颈部取血。测量小鼠体质量以及胸腺、脾、肿瘤质量。HE染色观察小鼠瘤体病理形态变化。流式细胞术检测小鼠CD4^(+)、CD8^(+)以及CD4^(+)/CD8^(+)水平,ELISA法检测白细胞介素-2(Interleukin-2,IL-2)、白细胞介素-4(Interleukin-4,IL-4)、白细胞介素-10(Interleukin-10,IL-10)、干扰素-γ(Interferon-γ,INF-γ)水平,Western blot法检测Src、磷酸化Src(p-Src)、FAK、磷酸化FAK(p-FAK)蛋白表达水平。结果与模型组比较,通关藤各干预组脾指数、胸腺指数水平升高,且随通关藤剂量增加,脾指数、胸腺指数水平、抑瘤率均升高,差异有统计学意义(P<0.05)。环磷酰胺组脾指数和胸腺指数明显低于各通关藤剂量组,差异有统计学意义(P<0.05);环磷酰胺组和通关藤高剂量组抑瘤率明显高于通关藤低、中剂量组,差异有统计学意义(P<0.05)。HE染色结果显示,模型组肿瘤细胞排列整齐且密集,而环磷酰胺组和通关藤各剂量组肿瘤细胞密度有所减少,且分布不均,并呈不同程度的肿瘤细胞坏死灶。与模型组比较,通关藤各干预组IL-2、INF-γ水平升高,IL-4、IL-10水平降低,差异有统计学意义(P<0.05);相较于环磷酰胺组,通关藤低、中、高剂量组IL-2、INF-γ水平升高,IL-4、IL-10水平降低,差异有统计学意义(P<0.05),且通关藤干预剂量越高,IL-2、INF-γ水平升高越明显,IL-4、IL-10水平降低越明显(P<0.05)。与模型组比较,环磷酰胺组和通关藤各干预组CD4^(+)、CD4^(+)/CD8^(+)水平升高,CD8^(+)水平降低,差异有统计学意义(P<0.05);与环磷酰胺组比较,通关藤低、中、高剂量组CD4^(+)、CD4^(+)/CD8^(+)水平升高,CD8^(+)水平降低,差异有统计学意义(P<0.05),且通关藤干预剂量越高,CD4^(+)、CD4^(+)/CD8^(+)水平升高越明显,CD8^(+)水平降低越明显(P<0.05)。与模型组比较,环磷酰胺组和通关藤各干预组p-Src/Src、p-FAK/FAK蛋白水平降低,差异有统计学意义(P<0.05);与环磷酰胺组比较,通关藤低、中、高剂量组p-Src/Src、p-FAK/FAK蛋白水平升高,差异有统计学意义(P<0.05),且通关藤干预剂量越高,p-Src/Src、p-FAK/FAK蛋白水平降低越明显。结论通关藤可调节胃癌小鼠Src/FAK信号通路及Th1/Th2相关因子水平,从而发挥抑癌作用,且剂量越高,效果越显著。

周期蛋白依赖性激酶5对去势抵抗性前列腺癌细胞生长及侵袭转移的影响

目的研究周期蛋白依赖性激酶5(CDK5)对去势抵抗性前列腺癌(CRPC)细胞DU145生长及侵袭转移的影响。方法将CDK5小分子抑制剂20-223添加到细胞中,根据其浓度分为对照组(20-223浓度为0.00μmol/L)、2.00μmol/L组(20-223浓度为2.00μmol/L)、4.00μmol/L组(20-223浓度为4.00μmol/L)以及8.00μmol/L组(20-223浓度为8.00μmol/L),每组100株。采用CDK5小干扰RNA(siRNA)干扰CDK5在DU145细胞内的表达,CDK5抑制剂20-223抑制CDK5在DU145细胞内的活性;采用噻唑蓝(MTT)检测细胞生长活力,单克隆形成实验测定细胞增殖能力,采用膜联蛋白V-异硫氰酸荧光素(AnnexinV-FITC)和碘化丙啶(PI)双染法测定细胞凋亡,划痕实验和Transwell实验测定细胞的迁移能力,Western Blot测定Myc促癌基因(c-Myc)、SRY-box转录因子4(SOX4)及侵袭转移相关蛋白的表达情况。对比分析CDK5抑制剂20-223对CRPC细胞DU145生长、凋亡、侵袭转移能力,以及四组细胞c-Myc、SOX4、上皮间质转化(EMT)相关蛋白的表达量。结果在用药后24、48、72 h的细胞生长抑制率方面,2.00μmol/L组、4.00μmol/L组、8.00μmol/L组均高于对照组,差异有统计学意义(P<0.01)。CRPC细胞DU145生长活力随着CDK5抑制剂的浓度升高而降低,其生长受到显著抑制。2.00μmol/L组、4.00μmol/L组、8.00μmol/L组用药后48 h Q1象限、Q2象限、Q3象限、Q4象限的细胞凋亡率均高于对照组,差异有统计学意义(P<0.01)。4.00μmol/L组在用药后48 h的迁移率(2.28%)低于对照组(2.37%),差异有统计学意义(P<0.01)。细胞经过2 d的处理后,2.00μmol/L组、4.00μmol/L组、8.00μmol/L组c-Myc、SOX4、Vimentin、Zeb1蛋白的表达量均低于对照组,而E-cadherin的表达量高于对照组。CDK5基因沉默组CDK5、c-Myc、SOX4蛋白的表达明显低于对照组和NC组。结论CDK5在CRPC中的表达及活性下降,在一定程度上抑制了DU145细胞的恶性增殖及浸润,并且对其凋亡起到了促进作用。CDK5的潜力很强,属于去势抵抗药物的一种,当对其的活性进行抑制后,c-Myc、SOX4等蛋白水平明显下降,进而上调E-cadherin、Vimentin、Zeb1等蛋白水平,从而减弱了去势抵抗药物敏感性。

Focal adhesion kinase antisense oligodeoxynucleotides inhibit human pulmonary artery smooth muscle cells proliferation and promote human pulmonary artery smooth muscle cells apoptosis

Background Pulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in pulmonary vessel structural remodelling At present, the mechanisms related to proliferation of PASMCs are not clear Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein tyrosine kinase Recent research indicates that FAK is implicated in signalling pathways which regulate cytoskeletal organization, adhesion, migration, survival and proliferation of cells Furthermore, there are no reports about the role of FAK in human pulmonary artery smooth muscle cells (HPASMCs) We investigated whether FAK takes part in the intracellular signalling pathway involved in HPASMCs proliferation and apoptosis, by using antisense oligodeoxynucleotides (ODNs) to selectively suppress the expression of FAK protein Methods Cultured HPASMCs stimulated by fibronectin (40 μg/ml) were passively transfected with ODNs, sense FAK, mismatch sense and antisense FAK respectively Expression of FAK, Jun NH2 terminal kinase (JNK), cyclin dependent kinase 2 (CDK 2) and caspase 3 proteins were detected by immunoprecipitation and Western blots Cell cycle and cell apoptosis were analysed by flow cytometry In addition, cytoplasmic FAK expression was detected by immunocytochemical staining Results When compared with mismatch sense group, the protein expressions of FAK, JNK and CDK 2 in HPASMCs decreased in antisense FAK ODNs group and increased in sense FAK ODNs group significantly Caspase 3 expression upregulated in HPASMCs when treated with antisense ODNs and downregulated when treated with sense ODNs When compared with mismatch sense ODNs group, the proportion of cells at G 1 phase decreased significantly in sense ODNs group, while the proportion of cells at S phase increased significantly In contrast, compared with mismatch sense ODNs group, the proportion of cells at G 1 phase was increased significantly in antisense FAK ODNs group The level of cell apoptosis in antisense FAK group was higher than in the mismatch sense group and the latter was higher than sense FAK group In addition, the sense FAK ODNs group was strongly stained by immunocytochemistry, whereas the antisense FAK ODNs group was weakly stained Conclusions The results suggest that FAK relates to the proliferation of HPASMCs Antisense FAK ODNs inhibit HPASMCs proliferation and facilitate their apoptosis It is possible that FAK via JNK, CDK 2 signalling pathways enhances HPASMCs proliferation and via caspase 3 inhibits HPASMCs apoptosis

GPER1 promotes estrogen receptor negative breast cancer cell migration and invasion via non-genomic activation of c-Src/NF-κB/focal adhesion kinase cascade

Breast cancer metastasis is the root cause of deaths from breast cancer.Currently,endocrine therapy resistance in estrogen receptor(ER)-positive(ER^(+))breast cancer remains a major clinical issue.Moreover,ER-negative(ER^(-))breast cancer is often associated with distant recurrence and death.G-protein-coupled ER(GPER1)participates in endocrine therapy resistance and is involved in the malignant progression of breast cancer.However,the underlying detailed mechanisms remain obscure.Here we investigated the role and mechanism of GPER1 in the activation of focal adhesion kinase(FAK)using ER^(+)or ER
breast cancer cell lines.In SK-Br-3 cells(ERa^(-)/β/GPER1^(+)),both 17b-estradiol(E2)and the GPER1 agonist G1 resulted in rapid FAK phosphorylation.This action is due to GPER1 interaction with the non-receptor tyrosine kinase c-Src and subsequent activation of nuclear factor kappa B(NF-kB)signaling.Silencing of GPER1,c-Src or the nuclear factor kappa B p65 subunit blocked E2-or G1-induced SK-Br-3 cell migration and invasion.In MCF-7 cells(ERa^(+)/β(+)/GPER1^(+)),silencing of GPER1,but not ERa or ERb,abolished FAK phosphorylation induced by E2 or G1.In MDA-MB-231 cells(ERa^(-)/β^(+)/GPER1^(-)),E2 or G1 was also unable to stimulate E2-induced FAK phosphorylation.However,E2 and G1 regained the ability to induce FAK phosphorylation under conditions of overexpression of GPER1.In conclusion,we demonstrated that GPER1,but not ERa or ERb,mediates FAK phosphorylation induced by E2 via the c-Src/p65 signaling pathway,which enhances cell migration and invasion.These findings may shed light on novel therapeutic strategies based on GPER1/FAK signaling pathways in suppression of breast cancer metastasis.

基于网络药理学和实验验证探究槲皮素治疗乳腺癌转移的作用机制

[目的]利用网络药理学和实验验证探究槲皮素(quercetin)抑制乳腺癌转移(breast cancer metastasis,BCM)的作用机制。[方法]通过中药系统药理学数据库与分析平台(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,TCMSP)和Swiss Target Prediction平台预测槲皮素潜在靶点,利用人类基因数据库(Human Gene Database,GeneCards)和在线人类孟德尔遗传(Online Mendelian Inheritance in Man,OMIM)数据库检索BCM相关的靶点,运用Cytoscape 3.7.0软件构建槲皮素和BCM交集靶点的蛋白互作(protein-protein interaction,PPI)网络,使用R软件和Bioconductor安装包完成基因本体(gene oncology,GO)分析及京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路分析,结合分子对接探究槲皮素和通路关键靶蛋白的结合作用。运用噻唑蓝(methyl thiazolyl tetrazolium,MTT)实验、细胞划痕实验、Transwell实验和免疫印迹法探究槲皮素抑制BCM的相关机制。[结果]槲皮素与BCM共有112个交集靶点,KEGG富集分析提示局灶性黏附(focal adhesion,FAs)是槲皮素治疗BCM的主要通路,分子对接结果表明槲皮素与FAs通路的关键靶点具有较强的结合作用。MTT、划痕实验和Transwell实验提示,槲皮素能明显抑制BCAP-37和4T1细胞的增殖、迁移及侵袭能力(P<0.05)。免疫印迹实验结果表明,槲皮素抑制FAs通路中肉瘤基因(sarcoma gene,SRC)、局灶黏附激酶(focal adhesion kinase,FAK)和蛋白激酶B (protein kinase B,Akt)的活化程度,降低磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)蛋白表达(P<0.05)。[结论]槲皮素通过降低SRC/FAK/PI3K信号通路活性,达到抑制BCM的目的。

原发性肝癌组织中层粘连蛋白3、黏着斑激酶蛋白表达及其与患者临床病理特征和预后关系研究

目的:探讨层粘连蛋白3(LAMA3)、黏着斑激酶(FAK)蛋白在原发性肝癌组织中的表达情况,分析它们与患者临床病理特征和预后的关系。方法:选取接受手术治疗的原发性肝癌患者75例,收集癌组织和癌旁组织标本。采用免疫组化染色检测癌组织和癌旁组织LAMA3、FAK蛋白表达。术后随访肝癌患者3年,统计生存情况。分析肝癌组织LAMA3、FAK蛋白表达与患者临床病理特征的关系。采用Kaplan-Meier生存曲线分析LAMA3、FAK蛋白表达与原发性肝癌患者预后的关系。采用Logistic回归分析原发性肝癌患者预后的影响因素。结果:肝癌组织LAMA3和FAK蛋白阳性表达率高于癌旁组织LAMA3和FAK蛋白阳性表达率(均P<0.05)。有淋巴结转移和血管侵犯的患者肝癌组织LAMA3、FAK蛋白阳性表达率高于无淋巴结转移和无血管侵犯的患者(均P<0.05)。术后随访3年,生存患者35例,病死患者40例。Kaplan-Meier生存曲线分析显示,肝癌组织LAMA3、FAK蛋白表达阳性患者3年累积生存率低于LAMA3、FAK蛋白表达阴性患者(均P<0.05)。Logistic回归分析发现,有淋巴结转移、有血管侵犯、LAMA3蛋白阳性和FAK蛋白阳性是原发性肝癌患者术后3年预后的独立危险因素(均P<0.05)。结论:原发性肝癌组织中LAMA3、FAK蛋白呈高表达,且两者阳性表达与患者淋巴结转移、血管侵犯及预后有关,可能作为原发性肝癌潜在的预后标志物。

金雀异黄酮抑制OPN-FAK信号通路对非小细胞肺癌迁移和侵袭的影响

目的:探讨金雀异黄酮(GS)抑制骨桥蛋白(OPN)/黏着斑激酶(FAK)信号通路对非小细胞肺癌(NSCLC)迁移和侵袭的影响。方法:qRT-PCR检测四种NSCLC细胞系(H596、H520、A549、H1703)及正常人肺上皮细胞(BEAS-2B)中OPN、FAK mRNA表达水平;以A549细胞为研究目标,分别设置对照组、GS低浓度(GS-L)组(10μmoL/L)、GS中浓度(GS-M)组(20μmoL/L)、GS高浓度(GS-H)组(40μmoL/L)、重组骨桥蛋白(rOPN)组(15ng/mL)、GS-H+rOPN组(40μmoL/L GS+15ng/mL rOPN)。分别利用MTT法、流式细胞仪检测上述各组细胞增殖率、凋亡率;划痕实验及Transwell实验检测细胞迁移、侵袭变化;Western blot及qRT-PCR检测OPN/FAK信号通路相关基因和蛋白的表达。结果:与BEAS-2B细胞相比,A549细胞中OPN、FAK mRNA表达水平变化最为显著(P<0.05)。与对照组相比,GS-L组、GS-M组、GS-H组A549细胞增殖率、迁移率、侵袭及迁移数、OPN/FAK信号通路相关基因和蛋白表达显著降低,凋亡率显著增加(P<0.05);rOPN组A549细胞增殖率、迁移率、侵袭及迁移数、OPN/FAK信号通路相关基因和蛋白表达显著增加,凋亡率显著降低(P<0.05)。与GS-H组相比,GS-H+rOPN组A549细胞增殖率、迁移率、侵袭及迁移数、OPN/FAK信号通路相关基因和蛋白表达显著增加,凋亡率显著降低(P<0.05)。与rOPN组相比,GS-H+rOPN组A549细胞增殖率、迁移率、侵袭及迁移数、OPN/FAK信号通路相关基因和蛋白表达显著降低,凋亡率显著增加(P<0.05)。结论:GS可以抑制NSCLC-A549细胞的迁移、侵袭及增殖,促进其凋亡,可能与抑制OPN/FAK信号通路有关。

基于FAK/ERK途径探讨NRP-1对结直肠癌细胞的影响

目的探究神经纤毛蛋白-1(neuropilin-1,NRP-1)对结直肠癌(colorectal cancer,CRC)细胞行为的作用,及其对黏着斑激酶(focal adhesion kinase,FAK)/胞外信号调节激酶(extracellular signal-regulated kinases,ERK)信号途径的影响。方法将CRC细胞系DLD-1随机分为对照组、CT-707组、sh-NRP-1组、sh-NRP-1+CT-707组,按分组使用sh-NRP-1慢病毒液感染细胞与FAK抑制剂CT-707处理细胞,通过荧光显微镜观察感染慢病毒后细胞内增强型绿色荧光蛋白表达情况,实时荧光定量PCR和Western blot法检测细胞内NRP-1mRNA与蛋白的表达变化。CCK-8法和EdU染色检测细胞增殖能力,细胞划痕实验观察细胞划痕愈合率,Transwell实验检测细胞迁移与侵袭,Western blot法测定细胞内FAK/ERK相关蛋白表达情况。结果感染sh-NRP-1重组慢病毒的DLD-1细胞中可见明显绿色荧光,细胞内NRP-1mRNA相对表达量和蛋白相对表达量均显著下调(P<0.05),表明转染成功。与对照组比较,CT-707组和sh-NRP-1组的细胞增殖活性显著下降,EdU阳性百分比显著降低,细胞划痕愈合率显著降低,细胞迁移数目与侵袭数目均显著减少,FAK蛋白磷酸化水平和ERK蛋白磷酸化水平均显著下调(P<0.05);与sh-NRP-1组比较,sh-NRP-1+CT-707组细胞增殖活性显著下降,EdU阳性百分比和细胞划痕愈合率均显著降低,细胞迁移数目与侵袭数目也均显著减少,FAK蛋白磷酸化水平和ERK蛋白磷酸化水平均显著下调(P<0.05)。结论下调NRP-1表达能够发挥抑制CRC细胞增殖、迁移及侵袭的作用,该机制可能与调控FAK/ERK信号转导途径有关。

吉马酮调节FAK/YAP信号通路对非小细胞肺癌细胞顺铂耐药性的影响

目的 探讨吉马酮(GMC)调节局部黏着斑激酶(FAK)/Yes相关蛋白(YAP)通路对非小细胞肺癌(NSCLC)细胞顺铂(DDP)耐药性的影响。方法 将A549/DDP细胞分为CK组、低剂量GMC组(GMC-L组,5μmol/L)、中剂量GMC组(GMC-M组,10μmol/L)、高剂量GMC组(GMC-H组,20μmol/L)、GMC-H+pcDNA组(20μmol/L+转染pcDNA)、GMC-H+pcDNA-FAK组(20μmol/L+转染pcDNA-FAK)。检测各组细胞增殖、凋亡、迁移、侵袭情况;检测多药耐药基因1(MDR1)、增殖细胞核抗原(PCNA)、B淋巴细胞瘤-2(Bcl-2)、基质金属蛋白酶(MMP)-2、MMP-9、磷酸化的FAK(p-FAK)、YAP蛋白表达情况。结果 与CK组比较,GMC-L组、GMCM组、GMC-H组A549/DDP细胞OD450值、克隆形成率、划痕愈合率、侵袭细胞数、MDR1、PCNA、Bcl-2、MMP-2、MMP-9、p-FAK蛋白及核YAP蛋白表达降低,细胞凋亡率升高,且呈浓度依赖性(P<0.05);与GMC-H组、GMC-H+pcDNA组比较,GMC-H+pcDNA-FAK组A549/DDP细胞OD_(450)值、克隆形成率、划痕愈合率、侵袭细胞数、MDR1、PCNA、Bcl-2、MMP-2、MMP-9、p-FAK蛋白及核YAP蛋白表达升高,细胞凋亡率降低(P<0.05)。结论 GMC可能通过抑制FAK/YAP信号通路减弱NSCLC细胞DDP耐药性。

芦荟大黄素对高转移乳腺癌细胞MDA-MB-231体外转移潜能的影响

目的研究芦荟大黄素(Aloe emodin,AE)对人高转移乳腺癌细胞MDA-MB-231体外转移潜能的影响及其作用机制。方法 MTT法检测AE对MDA-MB-231细胞增殖的抑制作用;Transwell chamber法检测AE对MDA-MB-231细胞侵袭重组基底膜能力和趋化性运动能力的影响;RT-PCR、Western blot法检测AE对MDA-MB-231细胞黏着斑激酶(FAK)mRNA和蛋白表达的影响。明胶酶谱法检测MDA-MB-231细胞分泌的基质金属蛋白酶-9(MMP-9)活性。结果 80μmol·L-1 AE抑制MDA-MB-231细胞体外侵袭重组基底膜能力、趋化性运动能力,其抑制率分别为(52.98±5.46)%,(45.88±8.51)%。作用于MDA-MB-231细胞24 h后,AE下调FAK mRNA和蛋白表达,下调MDA-MB-231细胞分泌MMP-9。结论 AE抑制MDA-MB-231细胞体外侵袭能力、趋化性运动能力,其作用机制与其下调FAK表达和MMP-9的分泌有关。

黄芪、当归对血管平滑肌细胞粘着斑激酶表达和细胞凋亡的影响

目的:探讨黄芪、当归对体外培养的血管平滑肌细胞(VSMC)粘着斑激酶(FAK)表达和细胞凋亡的影响。方法:应用RT-PCR、Western blot检测黄芪、当归对FAK表达的影响;应用 Greiss试剂检测 VSMC培养液中NO_2^-含量;应用流式细胞仪检测细胞凋亡及bcl-2和caspase-3表达活性。结果:黄芪、当归注射液可以显著抑制由碱性成纤维细胞生长因子所诱导的FAK表达,增加细胞培养液中的NO_2^-含量,诱导cas-pase-3表达。下调bcl-2表达,促进体外培养的VSMC凋亡。结论:黄芪、当归诱导VSMC凋亡与其促进NO合成、抑制FAK表达、诱导促凋亡基因caspase-3表达及下调抗凋亡基因bcl-2表达有关。

苦杏仁苷抑制人肺癌NCI-H1299细胞体外侵袭的机制

探讨天然产物苦杏仁苷对人非小细胞肺癌NCI-H1299细胞体外侵袭与转移的抑制作用及其机制。采用MTS法检测苦杏仁苷对肿瘤细胞生长的影响;采用Transwell小室和划痕试验检测苦杏仁苷对细胞侵袭和迁移能力的影响;采用Western blot和RT-PCR试验检测苦杏仁苷对MMP-2/9、integrinβ1、integrinβ4、整合素连接激酶(ILK)、黏附斑激酶(FAK)、p-FAK和β-catenin的表达水平,以及Akt和RICTOR的磷酸化水平的影响。结果表明:苦杏仁苷可显著抑制H1299细胞体外增殖,0.5和1 mg/m L苦杏仁苷作用H1299细胞48 h后,肿瘤细胞侵袭、迁移能力显著下降,MMP-2/9、integrinβ1/4、ILK、FAK、β-catenin蛋白和mRNA表达,MMP-2/9活性以及FAK、Akt和RICTOR磷酸化水平显著下调(P均<0.05)。

FAK基因沉默诱导白血病细胞凋亡

目的:本研究靶向黏着斑激酶(FAK)基因,构建FAK-shRNA慢病毒载体,并研究FAK基因沉默对白血病细胞生长的影响。方法:化学合成人FAK基因的shRNA序列,应用分子生物学方法将该FAK shRNA序列插入含荧光素GFP慢病毒质粒。该FAK shRNA慢病毒载体在体外包装、浓缩,然后转染至人白血病细胞株。应用逆转录聚合酶链反应及蛋白质印迹方法检测FAK基因表达水平,予Annexin V标记检测细胞凋亡的情况。结果:经核苷酸序列分析提示FAK shRNA正确插入慢病毒质粒,该病毒载体在白血病细胞株的转染率为10%-25%。与对照组相比(无FAK shRNA的慢病毒载体),FAK shRNA慢病毒载体在细胞FAK mRNA及蛋白水平的抑制率分别为40%和70%。凋亡实验表明,对照组与FAK shRNA慢病毒载体组的Annexin V阳性率分别为(4.19±0.36)%和(8.48±0.58)%,两者差异明显(P<0.05)。结论:我们成功构建FAK shRNA慢病毒载体,该载体能抑制FAK基因表达并诱导白血病细胞凋亡。