Focal adhesion kinase-related non-kinase ameliorates liver fibrosis by inhibiting aerobic glycolysis via the FAK/Ras/c-myc/ENO1 pathway

BACKGROUND Hepatic stellate cell(HSC)hyperactivation is a central link in liver fibrosis development.HSCs perform aerobic glycolysis to provide energy for their activation.Focal adhesion kinase(FAK)promotes aerobic glycolysis in cancer cells or fibroblasts,while FAK-related non-kinase(FRNK)inhibits FAK phosphorylation and biological functions.AIM To elucidate the effect of FRNK on liver fibrosis at the level of aerobic glycolytic metabolism in HSCs.METHODS Mouse liver fibrosis models were established by administering CCl4,and the effect of FRNK on the degree of liver fibrosis in the model was evaluated.Transforming growth factor-β1 was used to activate LX-2 cells.Tyrosine phosphorylation at position 397(pY397-FAK)was detected to identify activated FAK,and the expression of the glycolysis-related proteins monocarboxylate transporter 1(MCT-1)and enolase1(ENO1)was assessed.Bioinformatics analysis was performed to predict putative binding sites for c-myc in the ENO1 promoter region,which were validated with chromatin immunoprecipitation(ChIP)and dual luciferase reporter assays.RESULTS The pY397-FAK level was increased in human fibrotic liver tissue.FRNK knockout promoted liver fibrosis in mouse models.It also increased the activation,migration,proliferation and aerobic glycolysis of primary hepatic stellate cells(pHSCs)but inhibited pHSC apoptosis.Nevertheless,opposite trends for these phenomena were observed after exogenous FRNK treatment in LX-2 cells.Mechanistically,the FAK/Ras/c-myc/ENO1 pathway promoted aerobic glycolysis,which was inhibited by exogenous FRNK.CONCLUSION FRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-myc/ENO1 pathway,thereby improving liver fibrosis.FRNK might be a potential target for liver fibrosis treatment.

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.

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.

Focal adhesion kinase and Src phosphorylations in HGF-induced proliferation and invasion of human cholangiocarcinoma cell line, HuCCA-1

AIM: To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression.METHODS: Previously isolated HuCCA-1 cells were re-characterized by immunofluorescent staining and reverse transcriptase-polymerase chain reaction assay for the expression of cytokeratin 19, HGF and c-Met mRNA. Cultured HuCCA-1 cells were treated with HGF and determined for cell proliferation and invasion effects by MTT and invasion assays. Western blotting, immunoprecipitation, and co-immunoprecipitation were also performed to study the phosphorylation and interaction of FAK and Src. A novel Src inhibitor (AZM555130) was applied in cultures to investigate the effects on FAK phosphorylation inhibition and on cell proliferation and invasion.RESULTS: HGF enhanced HuCCA-1 cell proliferation and invasion by mediating FAK and Src phosphorylations.FAK-Src interaction occurred in a time-dependent manner that Src was proved to be an upstream signaling molecule to FAK. The inhibitor to Src decreased FAK phosphorylation level in correlation with the reduction of cell proliferation and invasion.CONCLUSION: FAK plays a significant role in signaling pathway of HGF-responsive cell line derived from cholangiocarcinoma. Autophosphorylated Src, induced by HGF, mediates Src kinase activation, which subsequently phosphorylates its substrate, FAK, and signals to cell proliferation and invasion.

Effect of focal adhesion kinase on cytoskeletal arrangement of HepG2 cells induced by hypoxia

Objective： To study focal adhesion kinase （FAK） expression in hypoxic HepG2 cells and the effect of FAK siRNA on cytoskeletal arrangement of HepG2 cells induced by hypoxia. Methods： HepG2 cells were cultured in 21% O2 and 1% O2. Morphological changes were observed after hypoxia treatment. Western blot was used to measure FAK expression. The siRNA expression vector pshRNA-FAK targeting the mRNA of FAK and vector pGensil-2 （as a control） were constructed, and then transfected into HepG2 cells. Western blot was used to detect FAK. The cytoskeletal arrangement of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was analyzed by phalloidin. The migratory ability of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was analyzed by cell migration assay. Results： Hypoxia-treated cells displayed a more elongated shape with a large degree of cell detachment. FAK expression increased in hypoxic HepG2 cells. FAK protein level was decreased by 75.64% ± 3.12% （P 〈 0.01） after the pshRNA-FAK transfection. Hypoxia induced cytoskeletal arrangement of HepG2 cells. However, cytoskeletal arrangement of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was inhibited in 1% O2. As cell migration assay showed, the migrating number of HepG cells transfected with pshRNA-FAK was significantly lower than that of control （P 〈 0.05）. Conclusion： The expression of FAK in hypoxic HCC might have a close relationship to the cytoskeletal arrangement of HepG2 cells induced by hypoxia. Up-regulation of FAK expression may be one of mechanisms of cytoskeletal arrangement and invasion of hepatocellular carcinoma induced by hypoxia.

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.

Inhibition of focal adhesion kinase enhances antitumor response of radiation therapy in pancreatic cancer through CD8+ T cells

Objective:Pancreatic ductal adenocarcinoma(PDAC)is a deadly malignancy,due in large part to its resistance to conventional therapies,including radiotherapy(RT).Despite RT exerting a modest antitumor response,it has also been shown to promote an immunosuppressive tumor microenvironment.Previous studies demonstrated that focal adhesion kinase inhibitors(FAKi)in clinical development inhibit the infiltration of suppressive myeloid cells and T regulatory(T regs)cells,and subsequently enhance effector T cell infiltration.FAK inhibitors in clinical development have not been investigated in combination with RT in preclinical murine models or clinical studies.Thus,we investigated the impact of FAK inhibition on RT,its potential as an RT sensitizer and immunomodulator in a murine model of PDAC.Methods:We used a syngeneic orthotopic murine model to study the effect of FAKi on hypofractionated RT.Results:In this study we showed that IN10018,a small molecular FAKi,enhanced antitumor response to RT.Antitumor activity of the combination of FAKi and RT is T cell dependent.FAKi in combination with RT enhanced CD8+T cell infiltration significantly in comparison to the radiation or FAKi treatment alone(P<0.05).FAKi in combination with radiation inhibited the infiltration of granulocytes but enhanced the infiltration of macrophages and T regs in comparison with the radiation or FAKi treatment alone(P<0.01).Conclusions:These results support the clinical development of FAKi as a radiosensitizer for PDAC and combining FAKi with RT to prime the tumor microenvironment of PDAC for immunotherapy.

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.

Focal adhesion kinase signaling is necessary for the hydrogen sulfide-enhanced proliferation,migration,and invasion of HTR8/SVneo human trophoblasts

Objective:Hydrogen sulfide(H_(2)S)has been elucidated that it promotes migration and invasion in human placenta trophoblasts.However,the signaling pathway underlying H_(2)S-based regulation of trophoblasts remains unknown.Hence,we investigated the potential effect of sodium hydrosulfide(NaHS),an exogenous H_(2)S donor,on extravillous trophoblasts.Methods:The Cell Counting Kit-8 was used to detect the proliferative activity of trophoblasts and to screen the optimal concentration of NaHS.The migration and invasion of HTR8/SVneo cells were measured by Transwell assays.Gene expression was determined by quantitative real-time PCR analysis.Protein expression was determined by western blot.Results:We found that NaHS could promote the proliferation,migration,and invasion of HTR8/SVneo cells.The phosphorylation of focal adhesion kinase(FAK),Src,and extracellular signal-regulated kinase(ERK)were activated by NaHS.Moreover,NaHS also upregulated the expression of matrix metalloproteinase-2(MMP-2)and MMP-9,downregulated the expression of E-cadherin in HTR8/SVneo cells.The application of NaHS could increase the expression of cystathionine-β-synthase.Conclusion:Both FAK-Src signaling and the upstream signaling cascade of ERK activation play a significant important role in NaHS-induced proliferation,migration,and invasion via upregulating activity of MMP-2,MMP-9,and downregulating E-cadherin in HTR8/SVneo cells.These novel findings may provide a strong foundation for the clinical application of H_(2)S donor drugs.

Expression, distribution and function of the focal adhesion kinase (pp125^(FAK)) during murine ectoplacental cone outgrowth in vitro

Mouse embryo implantation is a complex process that includes trophoblast cells derived from ectoplacental cone (EPC) adhesion to and migration through the extracellular matrix (ECM) of uterine endometrium and invasion into the decidua. At the time of implantation, fibronectin (FN) is abundant in the decidua and is distributed pericellularly around each individual stromal cell, and its receptor (integrin α-5β-1) expression on trophoblast populations is up-regulated. The focal adhesion kinase, a 125 ku protein tyrosine kinase (pp125 FAK), is tyrosine phosphorylated upon integrin engagement with its ECM ligand, and its tyrosine phosphorylation sites then serve as the binding sites which couple it with cellular proteins that contain Src SH2 or SH3 domains. Through these linkages, pp125 FAK may integrate multiple signals triggered by integrins. The model of EPC culture %in vitro% was used to study the expression, distribution and function of pp125 FAK during EPC outgrowth on FN. Results indicated that, pp125 FAK primarily expressed and distributed in cellular focal adhesions of the front edge of trophoblast outgrowth from EPC, and was localized in the peripheral region of the individual migrating trophblast cell; antibody or antisense oligodeoxynucleotide to pp125 FAK inhibited EPC attachment and outgrowth, as well as trophoblast cells spreading and migration. This experiment demonstrated that pp125 FAK as an integrin-mediated signaling molecule was involved in EPC outgrowth %in vitro%, and played an important role during trophoblast cells interaction with FN.

Specific shRNA targeting of FAK influenced collagen metabolism in rat hepatic stellate cells

AIM:To investigate the effects and mechanism of disruption of focal adhesion kinase(FAK) expression on collagen metabolism in rat hepatic stellate cells(HSC).METHODS:The plasmids expressing FAK short hairpin RNA(shRNA) were transfected into HSC-T6 cells,and the level of FAK expression was determined by both real-time quantitative polymerase chain reaction(QPCR) and Western blotting analysis.The production of type collagen and type collagen in FAK-disrupted cells was analyzed by real-time Q-PCR.The level of collagen metabolism proteins,including matrix metalloproteinases-13(MMP-13) and tissue inhibitors of metalloproteinases-1(TIMP-1) was also determined by both real-time Q-PCR and Western blotting analysis.RESULTS:The transfection of FAK shRNA plasmids into HSC resulted in disrupted FAK expression.Compared with the HK group,the levels of type collagen and type collagen mRNA transcripts in FAK shRNA plas-mid group were signif icantly decreased(0.69 ± 0.03 vs 1.96 ± 0.15,P = 0.000;0.59 ± 0.07 vs 1.62 ± 0.12,P = 0.020).The production of TIMP-1 in this cell type was also signif icantly reduced at both mRNA and protein levels(0.49 ± 0.02 vs 1.72 ± 0.10,P = 0.005;0.76 ± 0.08 vs 2.31 ± 0.24,P = 0.000).However,the expression of MMP-13 mRNA could be significantly up-regulated by the transfection of FAK shRNA plasmids into HSC(1.74 ± 0.20 vs 1.09 ± 0.09,P = 0.000).CONCLUSION:These data support the hypothesis that shRNA-mediated disruption of FAK expression could attenuate extracellular matrix(ECM) synthesis and promote ECM degradation,making FAK a potential target for novel anti-f ibrosis therapies.

The Expression of Integrinβ1 and FAK in Pituitary Adenomas and Their Correlation with Invasiveness

The expression and possible role of integrin-focal adhesion kinase signal pathway in invasive pituitary adenomas were explored. Forty-nine human pituitary adenomas were detected for the expression of integrinβ1 （INTβ1） and focal adhesion kinase （FAK） by immunohistochemistry, and their correlation with the invasiveness of pituitary adenomas as well as between themselves was ana- lyzed. The results showed that INTβ1 was expressed in 46 cases （93.9%） and FAK in 36 cases （73.5%）, respectively, and their expression levels were highly correlated with tumor invasiveness, but not with the tumor types. It was suggested that the integrin-focal adhesion kinase signal pathway plays a role in the invasiveness of pituitary adenomas.

Biglycan 及 FAK 信号通路对结肠癌细胞增殖能力的影响及分子机制

目的 研究Biglycan及FAK信号通路对结肠癌细胞增殖的调控作用及其分子机制. 方法 构建Biglycan过表达的人结肠癌HCT116细胞并采用FAK抑制剂处理. 分为5组：正常对照组、空载体对照组、空载体抑制剂处理组、Biglycan过表达组和Biglycan 过表达抑制剂处理组,处理时间为24h .通过Western blot 及MTT 检测的方法,观察各组HCT116 细胞的增殖能力以及FAK、p-FAK、PCNA、p53 的表达情况.结果 过表达Biglycan 能够显著促进HCT116 细胞的增殖以及FAK 的磷酸化（P ＜0.01）,并导致PCNA 表达水平的显著升高和p53 表达水平的显著抑制（P ＜0.01）;而FAK 抑制剂PF-562271 作用能够明显抑制细胞的增殖能力,Biglycan对p-FAK、PCNA、p53 蛋白表达水平的调控被抑制（P ＜0.01）.结论 Biglycan通过促进FAK 信号通路的活化调控结肠癌细胞的增殖.

Activated focal adhesion kinase involved in adhesion and migration of vascular smooth muscle cells stimulated by fibronectin

OBJECTIVE: To study the effects of focal adhesion kinase (FAK) phosphorylation on smooth muscle cells (SMCs) adhesion and migration stimulated by fibronectin. METHODS: Adhesion and migration of cultured SMCs were stimulated by different concentrations of fibronectin (FN), FAK and its phosphorylation were detected by immunoprecipitation and Western blot. FAK antisense oligodeoxynucleotides (ODNs) were transfected into SMCs by cationic lipid to investigate its modulatory effects on tyrosine phosphorylation. SMCs adhesion and migration were also measured by morphological enumeration and modified Boyden Chambers, respectively. RESULTS: FAK were expressed when SMCs adhesion and migration were successfully simulated by different concentrations of FN. FAK phosphorylation were detected only at 20 microg/ml FN or more. FAK antisense ODNs were transfected efficiently by cationic lipid and FAK phosphorylation was inhibited substantially. The SMCs migration rate in the 5 - 60 microg/ml FN groups was reduced by 17.89% - 27.67%. Cell migration stimulated by FN at 10, 20, 40 and 60 microg/ml were reduced by 23.26%, 21.63%, 19.31% and 17.88%, respectively (P

Activated focal adhesion kinase involved in adhesion and migration of vas cular smooth muscle cells stimulated by fibronectin

To study the effects of focal adhesion kinase (FAK) phosphorylation on smooth mu scle cells (SMCs) adhesion and migration stimulated by fibronectin Methods Adhesion and migration of cultured SMCs were stimulated by different concentrati ons of fibronectin (FN), FAK and its phosphorylation were detected by immunoprec ipitation and Western blot FAK antisense oligodeoxynucleotides (ODNs) were tra nsfected into SMCs by cationic lipid to investigate its modulatory effects on ty rosine phosphorylation SMCs adhesion and migration were also measured by morph ological enumeration and modified Boyden Chambers, respectively Results FAK were expressed when SMCs adhesion and migration were successfully simulated by different concentrations of FN FAK phosphorylation were detected only at 20 ?μg/ml FN or more FAK antisense ODNs were transfected efficiently by cationi c lipid and FAK phosphorylation was inhibited substantially The SMCs migration rate in the 5-60?μg/ml FN groups was reduced by 17 89%-27 67% Cell migrat ion stimulated by FN at 10, 20, 40 and 60?μg/ml were reduced by 23 26%, 21 6 3%, 19 31% and 17 88%, respectively ( P 【0 05) Conclusions FAK phosphorylation and FAK mediated signal transduction play important roles i n SMCs adhesion and migration stimulated by ECM The process can be inhibited e ffectively by FAK antisense ODNs

MicroRNA-7 regulates glioblastoma cell invasion via targeting focal adhesion kinase expression

Background Invasion growth is the most characteristic biological phenotype of glioblastoma, but the molecular mechanism in glioma cell invasion is poorly understood. Recent data have showed that microRNA plays an essential role in tumor invasion. Our study aimed to explore the mechanism of miR-7 involved in the control of glioblastoma cell invasion. Methods Glioma cell invasion was evaluated by transwell and scratch assays after up-regulation of miR-7 using miR-7 mimics in U87 and U251 cells. Luciferase reporter assay was used to determine focal adhesion kinase （FAK） as a target of miR-7. The levels of miR-7, matrix metalloproteinases （MMP）-2 and MMP-9 mRNA were detected by PCR assay, and the levels of FAK, MMP-2, MMP-9, total and phosphorylation serine/threonine kinase （AKT）, and extracellular signal-regulated kinase （ERK） 1/2 were measured by Western blotting analysis. Results Over-expression of miR-7 inhibited the invasion and migration activity of U87 and U251 cells. And up-regulation of miR-7 reduced FAK protein expression, Further, luciferase reporter assay showed that miR-7 modulated FAK expression directly by binding 3＇UTR of FAK mRNA. In addition, miR-7 repressed p-ERK1/2 and p-AKT level, MMP-2 and MMP-9 expression. Finally, the inverse relationship between FAK and miR-7 expression was certificated in human glioma tissues. Conclusion To our knowledge, these data indicate for the first time that miR-7 directly regulates cell invasion by targeting FAK in glioblastoma and that miR-7 could be a potential therapeutic target for glioblastoma intervention.

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

Safflower Yellow Inhibits Progression of Hepatocellular Carcinoma by Modulating Immunological Tolerance via FAK Pathway

Objective To explore the anti-tumor effect of safflower yellow(SY)against hepatocellular carcinoma(HCC)and the underlying potential mechanism.Methods An in vitro model was established by mixing Luc-Hepa1-6 cells and CD3^(+)CD8^(+)T cells,followed by adding programmed cell death protein 1(PD-1)antibody(Anti-mPD-1)with or without SY.The apoptosis was detected by flow cytometry and the level of inflammatory cytokines was determined by enzyme-linked immunosorbent assay.The protein levels of programmed cell death 1 ligand 1(PD-L1),chemokine ligand(CCL5),C-X-C motif chemokine ligand 10(CXCL10)were measured by Western blot.An in situ animal model was established in mice followed by treatment with anti-mPD-1 with or without SY.Bioluminescence imaging was monitored with an AniView 100 imaging system.To establish the FAK-overexpressed Luc-Hepa1-6 cells,cells were transfected with adenovirus containing pcDNA3.1-FAK for 48 h.Results The fluorescence intensity,apoptotic rate,release of inflammatory cytokines,and CCL5/CXCL10 secretion were dramatically facilitated by anti-mPD-1(P<0.01),accompanied by an inactivation of PD-1/PD-L1 axis,which were extremely further enhanced by SY(P<0.05 or P<0.01).Increased fluorescence intensity,elevated percentage of CD3+CD8+T cells,facilitated release of inflammatory cytokines,inactivated PD-1/PD-L1 axis,and increased CCL5/CXCL10 secretion were observed in Anti-mPD-1 treated mice(P<0.01),which were markedly enhanced by SY(P<0.05 or P<0.01).Furthermore,the enhanced effects of SY on inhibiting tumor cell growth,facilitating apoptosis and inflammatory cytokine releasing,suppressing the PD-1/PD-L1 axis,and inducing the CCL5/CXCL10 secretion in Anti-mPD-1 treated mixture of Luc-Hepa1-6 cells and CD3+CD8+T cells were abolished by FAK overexpression(P<0.01).Conclusion SY inhibited the progression of HCC by mediating immunological tolerance through inhibiting FAK.

Regulation of mouse blastocyst adhesion,outgrowth and matrix metalloproteinase-2 by focal adhesion kinase

The interaction of extracellular matrix-integrin markedly influences the adhesion, outgrowth, differentiation and expression of serine proteinases by the blastocyst, so it is regarded as a vital factor in blastocyst implantation. Al- though the mechanism of extracellular interactions between extracellular matrix and integrins has been well elucidated, the roles of the signaling molecules in the extracellular ma- trix-integrin signal transduction pathway in blastocyst im- plantation are unknown. This limits the understanding of blastocyst implantation and ECM-integrin signal transduc- tion pathway. In the present study, in vitro blastocyst culture and indirect immunocytochemistry, matrix metallopro- teinases (MMPs) zymography and antisense oligodeoxynu- cleotide (ODN) were used to investigate the expression of a fundamental molecule of integrin-dependent signal trans- duction pathways, focal adhesion kinase (FAK), in mouse blastocysts and its influence on mouse blastocyst adhesion, outgrowth and MMP-2. The results showed that mouse blas- tocysts expressed FAK. FAK protein was clustered in the peripheral migrating trophoblast cells and dispersed in the central area of blastocyst outgrowth. Fibronectin triggered pro-MMP-2 and 64 kD MMP-2 activities. The antisense ODN to FAK attenuated pro-MMP-2 and 64 kD MMP-2 activities which decreased abruptly and tended to disappear with increasing concentrations of the antisense ODN. Both mouse blastocyst adhesion and outgrowth on fibronectin were also influenced by the antisense ODN. Up to 20 mg/mL of the antisense ODN concentration, the adhesion and out- growth rates were decreased in a dose-dependent manner. The results indicated that FAK influenced mouse blastocyst adhesion, outgrowth and MMP-2 activity by intracellular signal transduction. In other words, FAK regulates mouse implantation in terms of blastocyst adhesive and invasive abilities.

Integrins and focal adhesion kinase in the malignant behavior of gliomas

Glioblastoma multiforme(GBM)is the most common type of glioma and is associated with a very poor prognosis.The standard treatment includes radiotherapy concurrent with temozolomide,however recently the Food and Drug Administration approved bevacizumab for use in patients with progressive glioblastoma following prior therapy.The limited number of treatment options points to the need for novel effective therapeutic approaches.A promising approach is the use of tyrosine kinase inhibitors(TKIs)in GBM treatment.However,the results from the majority of clinical trials using TKIs are not very encouraging.One growing area is the development of tumor-homing peptides that resemble the integrin recognition sequence RGD.In this article,the role of integrins and focal adhesion kinase in malignant glioma is reviewed,and an experimental study is proposed that will apply a strategy for peptide-mediated delivery of compounds deep into tumor parenchyma using tumor-homing peptides.