Focal Adhesion Kinase Protein Expression Research Articles

Pyk2/FAK Signaling Is Upregulated in Recurrent Glioblastoma Tumors in a C57BL/6/GL261 Glioma Implantation Model.

The majority of glioblastomas (GBMs) recur shortly after tumor resection and recurrent tumors differ significantly from newly diagnosed GBMs, phenotypically and genetically. In this study, using a Gl261-C57Bl/6 mouse glioma implantation model, we identified significant upregulation of proline-rich tyrosine kinase Pyk2 and focal adhesion kinase (FAK) phosphorylation levels-pPyk2 (579/580) and pFAK (925)-without significant modifications in total Pyk2 and FAK protein expression in tumors regrown after surgical resection, compared with primary implanted tumors. Previously, we demonstrated that Pyk2 and FAK are involved in the regulation of tumor cell invasion and proliferation and are associated with reduced overall survival. We hypothesized that the use of inhibitors of Pyk2/FAK in the postsurgical period may reduce the growth of recurrent tumors. Using Western blot analysis and confocal immunofluorescence approaches, we demonstrated upregulation of Cyclin D1 and the Ki67 proliferation index in tumors regrown after resection, compared with primary implanted tumors. Treatment with Pyk2/FAK inhibitor PF-562271, administered through oral gavage at 50 mg/kg daily for two weeks beginning 2 days before tumor resection, reversed Pyk2/FAK signaling upregulation in recurrent tumors, reduced tumor volume, and increased animal survival. In conclusion, the use of Pyk2/FAK inhibitors can contribute to a delay in GBM tumor regrowth after surgical resection.

RIOK3 promotes pancreatic ductal adenocarcinoma cell invasion and metastasis by stabilizing FAK

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive cancer, characterized by a high metastatic burden. RIO Kinase 3 (RIOK3) has been shown to promote invasion and metastasis of PDAC by cytoskeleton remodeling, but the exact mechanism is still unknown. In this study, we analyzed transcriptome sequencing data from RIOK3 stable knockdown PANC-1 cells and TCGA-PDAC data and discovered that RIOK3 was substantially related to focal adhesion signaling in PDAC. Additionally, silencing RIOK3 dramatically decreased Focal Adhesion Kinase (FAK) protein expression and phosphorylation (Tyr397 and Tyr925 sites). Immunoprecipitation assay verified the interaction of RIOK3 and FAK. Furthermore, RIOK3 considerably increased the protein stability of FAK protein but not FAK-Y925F protein. The biological function of RIOK3 in increasing PDAC cell invasion and migration was shown to be dependent on FAK activation. Moreover, we discovered that RIOK3 mutations were mainly characterized by amplification. RIOK3 mRNA was found to be significantly elevated in PDAC tissues and was associated with a poor prognosis. Furthermore, RIOK3 mRNA was significantly upregulated in later T-stage, pre-existing lymph node metastases, and later pathological stage samples. Overall, our study found that RIOK3 promotes PDAC cell invasion and metastasis by stabilizing FAK protein expression and upregulating its phosphorylation. This also provides a new target for therapeutic modalities targeting FAK.

MiR-15p-5p Mediates the Coordination of ICAM-1 and FAK to Promote Endothelial Cell Proliferation and Migration.

Intercellular adhesion molecule-1 (ICAM-1) in endothelial cells is critical for neutrophil adhesion and transmigration across the endothelium. Focal adhesion kinase (FAK), which controls the turnover of focal adhesion to regulate cell adhesion and migration, plays a role in the resolution of inflammation. However, the coordinated involvement of ICAM-1 and FAK during endothelial inflammation has yet to be elucidated. This study reports that, as part of an inflammatory response, ICAM-1 controls FAK expression in endothelial cells via the microRNA miR-15b-5p. Induction of lung injury by lipopolysaccharide (LPS) resulted in higher levels of FAK expression in inflammatory tissues, while in ICAM-1 knockout mice, FAK expression was reduced in the lungs. FAK expression was also reduced in endothelial cells following ICAM-1 siRNA downregulation. Furthermore, ICAM-1 inhibited miR-15b-5p expression while increasing FAK mRNA and protein expression via binding of miR-15b-5p to the 3' untranslated region (UTR) of FAK. ICAM-1 inhibited miR-15b-5p promoter activity and hence reduced miR-15b-5p expression. FAK increased endothelial cell proliferation and migration, whereas miR-15b-5p inhibited cell proliferation and migration. These findings indicate that the inflammatory molecule ICAM-1 regulates FAK expression via miR-15b-5p levels, which in turn controls endothelial cell proliferation and migration.

Focal Adhesion Kinase Drives Resistance to Therapy in HPV-Negative Head and Neck Squamous Cell Carcinoma in a p53-Dependent Manner

Our group has previously demonstrated focal adhesion kinase (FAK) expression is associated with radioresistance and worse outcome in human papillomavirus negative (HPV-) head and neck squamous cell carcinoma (HNC). However, the relative importance of FAK in driving therapeutic resistance is unclear. FAK function can be inhibited by wild type (wt) p53, but p53 is mutated in over 80% of HPV(-) HNC. Based on these observations, we tested the hypothesis that mutant (mut) p53 permits or even drives FAK-mediated radioresistance.Two cohorts of HPV(-) HNC tumors treated with radiation (RT), single institution (n = 81) and TCGA (n = 324), were stratified by TP53 mutational status (wt versus mut) and FAK copy number/mRNA expression. In vivo shRNA screening in 5 human HNSCC xenograft flank models was performed using libraries focused on known "druggable" proteins and DNA damage repair combined with RT or carboplatin. Correlation between FAK protein expression and sensitivity to cisplatin was evaluated in 20 TP53 mut HNC cell lines. The relationship between p53, FAK and radioresponse was examined in vitro using UMSCC (p53 null) HNC cells forced to express wt or mut TP53. FAK function was modulated in these cells using shRNA or chemical inhibition (defactinib) and response to radiation or carboplatin was evaluated via immunoblot for DNA damage repair proteins and clonogenic assay. FAK and p53 interactions were evaluated via immunoprecipitation. An in vivo immunodeficient flank model using HN31 cells expressing FAK shRNA was used to assess in vivo radioresponse.High levels of FAK copy number and gene expression were associated with worse disease-free survival in TP53 mut HPV(-) HNC, but not TP53 wt tumors. In vivo shRNA screening identified FAK as an excellent target for either radio- or chemosensitization. FAK protein expression was correlated with cisplatin IC50 (P = 0.0083, r = 0.572) in our HNC cell line panel. Inhibition of FAK via shRNA in the HN31(TP53 mut) cell line led to radiosensitization both in vitro and in a xenograft flank model. Defactinib treatment led to increased sensitivity to radiation or carboplatin in TP53 mut (HN31) but not TP53 wt (HN30) tumor cell lines. In UMSCC1 cells pretreated with defactinib, TP53 mut expression resulted in significant radiosensitization, but TP53 wt expression did not. To examine the physical interaction between p53 and FAK, different TP53 constructs were expressed in UMSCC1 cells. Interestingly, missense mutation does not impair the ability of p53 to bind to FAK. However, FAK phosphorylation was higher in cells expressing mut p53 compared to wt. Moreover, FAK inhibition combined with radiation led to higher levels of ɣ-H2AX in mut p53 expressing cells.FAK is a potentially important target for therapeutic sensitization, primarily in TP53 mutated HNSCC.

Effects of fluid shear stress on expression of focal adhesion kinase in MG-63 human osteoblast-like cells on different surface modification of titanium

ABSTRACT This study aimed to investigate the effect of fluid shear stress (FSS) on cell proliferation and expression of focal adhesion kinase (FAK) in MG-63 cells on different modified titanium surfaces. MG63 cells were cultured on three different surfaces: glass slide, polished treatment (PT) titanium surface and sandblasted/acid-etched surfaces (SLA) titanium surface. The surface topography and roughness were evaluated by scanning electron microscopy (SEM) and atomic force microscopy (AFM), respectively. The cells were subjected to FSS, and the cell appearance before and after the stress was evaluated. MTT assay was applied to estimate cell proliferation. The mRNA and protein levels of FAK were determined by qRT-PCR and western blotting. Titanium plates demonstrated different surface microtopography. Parameter Ra values of SLA group were around 3.4 µm, which was higher than PT group. Exposure to the FSS of 12 dynes/cm2 significantly induced positive upregulation of cellular proliferation and the expression of FAK, which were directly correlated with the duration of exposure and surface. Cells in SLA group were able to endurance the longtime of FSS, especially under the FSS of 16 dynes/cm2. SLA surface had a positive influence on the expression of FAK. Different surface modifications created different microtopography of titanium plates. Cell proliferation and the mRNA and protein expression of FAK were stimulated by FSS and regulated by a marked synergistic effect of surface topography and the level and duration of FSS.

Focal Adhesion Kinase (FAK) Over-Expression and Prognostic Implication in Pediatric Hepatocellular Carcinoma.

Focal adhesion kinase (FAK) is over-expressed and is correlated with aggressiveness in adult hepatocellular carcinoma (HCC). Inhibition of FAK decreases HCC invasiveness by down-regulating Enhancer of Zeste homolog 2 (EZH2), an epigenetic controller, that acts in transcriptional repression of a large number of genes via histone 3 methylation of lysine 27 (H3K27me3). Here, we investigated the hepatic expression of total FAK, EZH2, H3K27me3, and proliferating cell nuclear antigen (PCNA) in 17 pediatric HCCs and 8 healthy livers (CTRL). Quantitative imaging analysis showed that FAK gene/protein expression is up-regulated in HCCs compared to CTRL and, among tumor samples the levels of this gene/protein are significantly higher in cirrhotic HCCs than in a healthy milieu. Accordingly, the protein levels of EZH2 were also significantly increased in HCCs from a cirrhotic background. Intriguingly, the protein expression of FAK, EZH2, and PCNA significantly inversely correlated with tumor size. Finally, in HCC samples, mainly in cirrhotic background, the up-regulation of FAK gene positively correlated with that observed in β-Catenin gene. Conclusion: FAK gene/protein is over-expressed in pediatric HCCs concomitantly to EZH2 protein and β-Catenin gene, with a more significant up-regulation in a cirrhotic background. This triad of interactors deserves further investigations for translational application.

A combined FAK, c-MET, and MST1R three-protein panel risk-stratifies colorectal cancer patients.

Focal adhesion kinase (FAK) is a key tyrosine kinase downstream of c-MET (or hepatocyte growth factor receptor, HGFR) and MST1R (macrophage-stimulating protein receptor or recepteur d'origine Nantais, RON) membrane receptors. The pathway plays an important role in cancer survival and invasion. In this study, we examined the protein expression of FAK, c-MET, and MST1R levels in a well-annotated cohort of 330 colorectal cancer patients. We found FAK to be overexpressed in colorectal adenocarcinomas (p = 0.0002), and FAK levels correlated positively with phospho-FAK levels (R2 = 0.81). In comparison, MST1R levels were not significantly different, and c-MET levels were slightly higher in the normal samples. We then developed a combined 3-protein panel of FAK, c-MET, and MST1R expression signatures that can robustly risk-stratify colorectal cancer across all stages into three clusters that differ in progression-free survival. The colorectal cancer subgroup with high FAK, low c-MET, and low MST1R protein levels showed the worst progression-free survival with particularly early progression of disease (p = 0.0053). Combined FAK, c-MET, and MST1R were independently prognostic for progression-free survival in stage II colorectal cancers in a multivariate model. The 3-protein panel provides a potentially clinically attractive method for risk-stratification and adjuvant therapy guidance, especially in stage II disease.

Prognostic and clinical significance of focal adhesion kinase expression in breast cancer: A systematic review and meta-analysis.

BackgroundThe prognostic significance of focal adhesion kinase (FAK) in breast cancer remains controversial. Here, we conducted a meta-analysis to explore the prognostic value of FAK expression in breast cancer.Materials and methodsPossible prognostic significance of protein or mRNA expression of FAK in breast cancer was investigated with searches of electronic databases for relevant publications. Pooled hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were extracted from eligible studies.ResultsA total of eight eligible studies which included 2604 participants were analyzed in this meta-analysis. Increased expression of FAK protein was found to significantly correlate with shorter overall survival (OS) (HR = 1.43, 95% CI: 1.12–1.83; P = 0.004), and not with disease-free survival (HR = 1.31, 95% CI: 0.92–1.85; P = 0.14). Elevated FAK protein expression was also associated with negative estrogen receptor (ER) expression (OR, 1.34; 95% CI, 1.06–1.68; P = 0.01), negative progesterone receptor (PR) expression (OR, 1.54; 95% CI, 1.22–1.93; P < 0.001), positive human epidermal growth factor receptor 2 (HER2) expression (OR, 1.64; 95% CI, 1.28–2.09; P < 0.001), triple-negative breast cancer (TNBC) (OR, 1.57; 95% CI, 1.14–2.17; P = 0.006), high nuclear grade (OR, 1.70; 95% CI, 1.05–2.78; P = 0.03), high Ki-67 expression level (OR, 2.87; 95% CI, 1.94–4.24; P < 0.001), and positive p53 status (OR, 2.28; 95% CI, 1.58–3.29; P < 0.001).ConclusionOur meta-analysis identifies an association between increased FAK protein expression and worse OS among breast cancer patients. Moreover, enhanced FAK expression is associated with negative ER expression, negative PR expression, positive HER2 expression, TNBC, high nuclear grade, high Ki-67 expression level, and positive p53 status in breast carcinoma.

Simvastatin ameliorates altered mechanotransduction in uterine leiomyoma cells

Uterine leiomyomas, the most common tumors of the female reproductive system, are characterized by excessive deposition of disordered stiff extracellular matrix and fundamental alteration in the mechanical signaling pathways. Specifically, these alterations affect the normal dynamic state of responsiveness to mechanical cues in the extracellular environment. These mechanical cues are converted through integrins, cell membrane receptors, to biochemical signals including cytoskeletal signaling pathways to maintain mechanical homeostasis. Leiomyoma cells overexpress β1 integrin and other downstream mechanical signaling proteins. We previously reported that simvastatin, an antihyperlipidemic drug, has antileiomyoma effects through cellular, animal model, and epidemiologic studies. This study aimed to examine the hypothesis that simvastatin might influence altered mechanotransduction in leiomyoma cells. This is a laboratory-based experimental study. Primary leiomyoma cells were isolated from 5 patients who underwent hysterectomy at the Department of Gynecology and Obstetrics of the Johns Hopkins University Hospital. Primary and immortalized human uterine leiomyoma cells were treated with simvastatin at increasing concentrations (0.001, 0.01, 0.1, and 1 μM, or control) for 48 hours. Protein and mRNA levels of β1 integrin and extracellular matrix components involved in mechanical signaling were quantified by quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence. In addition, we examined the effect of simvastatin on the activity of Ras homolog family member A using pull-down assay and gel contraction. We found that simvastatin significantly reduced the protein expression of β1 integrin by 44% and type I collagen by 60% compared with untreated leiomyoma cells. Simvastatin-treated cells reduced phosphorylation of focal adhesion kinase down to 26%-60% of control, whereas it increased total focal adhesion kinase protein expression. Using a Ras homolog family member A pull-down activation assay, we observed reduced levels of active Ras homolog family member A in simvastatin-treated cells by 45%-85% compared with control. Consistent with impaired Ras homolog family member A activation, simvastatin treatment reduced tumor gel contraction where gel area was 122%-153% larger than control. Furthermore, simvastatin treatment led to reduced levels of mechanical signaling proteins involved in β1 integrin downstream signaling, such as A-kinase anchor protein 13, Rho-associated protein kinase 1, myosin light-chain kinase, and cyclin D1. The results of this study suggest a possible therapeutic role of simvastatin in restoring the altered state of mechanotransduction signaling in leiomyoma. Collectively, these findings are aligned with previous epidemiologic studies and other reports and support the need for clinical trials.

Effect of celecoxib on protein expression of FAK and Cx43 in DMBA induced rat tongue carcinoma cells.

The pathogenesis of tongue cancer (TA) has not been fully illustrated. Cyclooxygenase-2 (COX-2) is correlated with the precancerous lesion of oral cavity mucosa and malignant transformation. The focal adhesion kinase (FAK) and gap junction protein connexin 43 (Cx43) are involved in the occurrence and progression of tumors. This study aimed to investigate the effect of celecoxib on the proliferation, malignant transformation, and expression of FAK and Cx43 proteins. Healthy male Sprague-Dawley (SD) rats (4 months old) were divided into control, model and celecoxib group. 7,12-dimethylbenzanthracene (DMBA) was used to generate tongue mucosal carcinoma, coupled with celecoxib intervention. At 8, 12, 16, and 20 weeks after induction, the rat survival status, the tumor formation rate and the tongue tissue morphology were observed. Meanwhile, the expression of FAK and Cx43 was also evaluated by using immunohistochemistry (IHC). Tumor occurrence rates after induction were 0, 26.67%, 66.67%, and 80% at 8, 12, 16, and 20 weeks, respectively. The celecoxib treatment decreased such rats to 0, 0, 0, and 13.33%, respectively (p<0.05 compared to model group). No significant change was observed in control group, whilst model group had mild to severe hyperplasia and squamous carcinoma with elongated time. Celecoxib treatment significantly improved the tissue morphology (p<0.05). The model group also had elevated FAK and depressed Cx43 protein expression (p<0.05). With elongated time, the FAK expression was further increased whilst Cx43 protein was depressed (p<0.05 compared to model group). The focal application of celecoxib effectively inhibited the DMBA-induced rat TA, possibly via regulating FAK and CX43 protein expression, and inhibiting oral epidermal hyperplasia.

The Clinical Implication of PTEN and FAK Expression in Gastric Cancer Patients

Objective: The tumor suppressor gene phosphatase and tensin homolog (PTEN) was reported to inhibit the growth and invasion of gastric cancer (GC) via the downregulation of focal adhesion kinase (FAK). To date, the clinical implication of PTEN and FAK expression in GC has not been well addressed. Methods: A total of 200 GC patients receiving curative surgery were enrolled. The clinicopathologic features according to the expression of PTEN and FAK protein using immunohistochemical staining were compared among patients. Results: Patients with high PTEN expression were more likely to have smaller tumor size, more well- and moderately differentiated tumors, a more superficial gross appearance, less scirrhous stromal reactions, more likely to have high FAK expression, and have less advanced pathologic tumor (T) category, node (N) category, and tumor, node, metastasis (TNM) stage and more distant metastases than patients with low PTEN expression. Multivariate analysis showed that PTEN/FAK expression status is an independent prognostic factor affecting overall survival (OS) and disease-free survival (DFS). Patients with PTEN(high)/FAK(low) had better OS and DFS, followed by those with PTEN(high)/FAK(high), those with PTEN(low)/FAK(low), and those with PTEN(low)/FAK(high) (OS: 83.3% versus 58.0% versus 46.2% versus 26.5%, respectively, P &amp;lt; 0.001; DFS: 83.3% versus 55.8% versus 30.8% versus 24.4%, respectively, P &amp;lt; 0.001). Conclusions: GC patients with high PTEN expression were more likely to have fewer tumor recurrences and a better prognosis than those with low PTEN expression. PTEN and FAK may have opposing effects on GC patient survival. Our results may have clinical impact on treatment of GC patients.

The Role of FAK in the Secretion of MMP9 after CD147 Stimulation in Macrophages.

To investigate whether focal adhesion kinase (FAK) can participate in the secretion of matrix metalloproteinase 9 (MMP9) after CD147 stimulation in THP-1 induced macrophages; thus, to explore the potential treatment perspectives for acute coronary syndrome (ACS).Phorbol-12-myristate-13-acetate (PMA) was used to induce THP-1 cells to differentiate into macrophages. To confirm the peak mRNA and protein expression of FAK and MMP9 after the stimulation of CD147, the macrophages were divided into 5 groups (0, 3, 6, 9, and 12 hours), with 0 hours group as control group. To investigate the role of FAK in the secretion of MMP9, with stimulation of CD147 for 9 hours, FAK inhibitor 14 was used to inhibit FAK Y397 phosphorylation. The mRNA and protein expressions were quantified by qRT-PCR and western blotting, respectively. (1) Relative mRNA expression of FAK and MMP9 were both significantly up-regulated (all P < 0.05) after stimulation of CD147, FAK peaked at 9 hours (3.908 ± 0.106 versus 1, P < 0.05), whereas MMP9 peaked at 6 hours (2.522 ± 0.062 versus 1, P < 0.05). (2) Relative protein expression of FAK, pFAK, and MMP9 were all significantly increased after CD147 stimulation (all P < 0.05), FAK (1.930 ± 0.024 versus 1, P < 0.05) and pFAK (1.737 ± 0.021 versus 1, P < 0.05) peaked at 9 hours, whereas MMP9 peaked at 6 hours (1.527 ± 0.033 versus 1, P < 0.05). (3) CD147 up-regulates FAK, pFAK, and MMP9 mRNA and protein expressions in a dose-dependent manner. (4) FAK inhibitor 14 significantly reduced the relative protein expression level of pFAK (0.077 ± 0.012 versus 1, P < 0.05) and MMP9 (0.133 ± 0.012) at 9 hours after CD147 stimulation.The results demonstrated that FAK Y397 phosphorylation was involved in the secretion of MMP9 after CD147 stimulation in macrophages and may play a role in the regulation of ACS.

Therapeutic effects of tyroservatide on metastasis of lung cancer and its mechanism affecting integrin-focal adhesion kinase signal transduction.

Tyroservatide (YSV) can inhibit the growth and metastasis of mouse lung cancer significantly. This study investigated the therapeutic effects of tripeptide YSV on metastasis of human lung cancer cells and explored its possible mechanism that affects integrin–focal adhesion kinase (FAK) signal transduction in tumor cells. YSV significantly inhibited the adhesion and the invasion of highly metastatic human lung cancer cell lines 95D, A549, and NCI-H1299. In addition, YSV significantly inhibited phosphorylation of FAK Tyr397 and FAK Tyr576/577 in the 95D, A549, and NCI-H1299 human lung cancer cells in vitro. And the mRNA level and protein expression of FAK in these human lung cancer cells decreased at the same time. YSV also significantly inhibited mRNA and protein levels of integrin β1 and integrin β3 in the 95D, A549, and NCI-H1299 human lung cancer cells. Our research showed that YSV inhibited adhesion and invasion of human lung cancer cells and exhibited therapeutic effects on metastasis of lung cancer.

RLj-RGD3, a Novel Three-RGD-Motif-Containing Recombinant Protein from Lampetra japonica, Protects PC12 Cells from Injury Induced by Oxygen-Glucose Deprivation and Reperfusion.

rLj-RGD3 is a 14.5 kDa recombinant protein with 3 RGD (Arg-Gly-Asp) motifs from the salivary gland secretions of Lampetra japonica, which is a histidine-rich and arginine-rich protein. Previous reports indicated that rLj-RGD3 has typical functions of RGD-toxin protein, such as platelet aggregation suppression tumour metastasis and angiogenesis inhibition. Because histidine and arginine have cerebral ischemia-reperfusion and neuroprotective functions, we investigated whether rLj-RGD3 has such activities and studied the mechanism. The effects of rLj-RGD3 on neuroprotection and antiapoptosis were determined. The expression level of focal adhesion kinase (FAK), p-FAK, Caspase-3, and Bcl-2 after oxygen-glucose deprivation and reperfusion (OGD-R) was examined. The viability of PC12 cells incubated with rLj-RGD3 at high concentrations (16 μmol/L) increased significantly due to its ability to protect the cells from apoptosis after OGD-R-induced injury. Furthermore, rLj-RGD3 attenuated the damage due to OGD-R. Most of the PC12 cells were apoptotic after OGD-R. In contrast, the number of apoptotic PC12 cells was significantly decreased in the group treated with a high-dose of rLj-RGD3. In addition, rLj-RGD3 activated FAK and p-FAK protein. rLj-RGD3 inhibited Caspase-3 and upregulated Bcl-2 protein expression in PC12 cells after OGD-R. The study provides the first evidence for neuroprotective effects of rLj-RGD3 in ischemic injury that may be partly mediated through inhibition of Caspase-3 and upregulation of Bcl-2, FAK, and p-FAK protein expression.

Hepatoprotective Effects of Traditional Chinese Medicine on Liver Fibrosis from Ethanol Administration following Partial Hepatectomy.

The aim of this study was to establish the effective hepatoprotective properties of traditional Chinese medicines (TCMs) in fibrotic rat liver regeneration after partial hepatectomy (PHx). Fibrosis was induced in rats by ethanol (EtOH, 5 ml/kg) administration for 6, 24, 72, and 168 h. The rats were then fed four TCMs (1 g/kg/day, Codonopsis pilosula (CP), Salvia miltorrhiza Bunge (SMB), Bupleurum kasi (BK), and Elephantopus scaber L (ESL)) to Spraque-Dawley rats for 6, 24, 72 and 168 h, respectively. Surgical 70% cirrhotic fibrosis PHx was then conducted at 6, 24, 72, and 168 h. The effects on liver regeneration were examined to estimate and measure hepatocyte growth factor (HGF), focal adhesion kinase (FAK), Cyclin D1, Cyclin E, and retinoblastoma protein (pRb) protein expression using Western blotting analysis. Cyclin D1, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2 and TIMP-3 mRNA by Reverse transcription polymerase chain reaction (RT-PCR) were analyzed in cirrhotic fibrosis rats. Transforming growth factor-β1 (TGF-β1), Cyclin D1, Cyclin E, pRb and E2F mRNA expression levels were determined in fibrotic rats following PHx using RT-PCR. We found elevated glutamyl oxaloacetic transaminase (GOT), glutamyl pyrubic transaminase (GPT), alkaline phosphatase (ALP), gammaglutamyl transpeptidase (γ-GT), glutathione (GSH), nonprotein sulfhydryl (NPSH) and total bilirubin in serum after 6 h EtOH administration. These levels were progressively decreased over 168 h. Total protein and albumin were reduced in serum after 6 h administration and then progressively increased. In contrast, tissues disorder histology and morphology were determined in liver sections. After rats were fed TCMs we found that SMB extraction not only induced HGF, FAK, Cyclin D1, and pRb protein expression and Cyclin D1 mRNA increases, but also reduced MMP-2 and MMP-9 after 24 and 72 h post injury. In the cell cycle S phase the Cyclin E protein expression was increased by ESL. CP induced TIMP-1, TIMP-2 and TIMP-3 mRNA increases in fibrotic rats. We detected liver regeneration in fibrotic rats. We also found that the liver regeneration index increased from 6 to 168 h post PHx. After 168 h fibrotic liver regeneration rats exhibited reduced TGF-β1 mRNA expression and enhanced Cyclin D1, Cyclin E, pRb and E2F mRNA expression. TCMs play a crucial role in the early mediating process in fibrotic rat liver regeneration after PHx.

MicroRNA‑7 suppresses human colon cancer invasion and proliferation by targeting the expression of focal adhesion kinase.

Previous studies have demonstrated that microRNA (miRNA) are essential in tumor development and invasion. The close association between focal adhesion kinase (FAK) and colon cancer (CC) has been previously reported. miRNA-7 (miR-7) inhibits the translation of FAK protein. Therefore, the present study aimed to assess the underlying molecular mechanism of miR-7 in human CC cell lines, to provide a novel therapeutic biomarker of CC in the future. The present study detected the expression of miR-7 in 60 CC tissues by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The association between the expression of miR-7 and clinical pathological factors was analyzed. Overexpression/underexpression of miR-7 were established by transfecting miR-7mimics/inhibitors into HCT-8 and Caco-2 cells. The transfected CC cell lines were used in cell viability and scratching assays. The regulation of FAK by miR-7 was analyzed by western blotting and RT-qPCR. It was demonstrated that the expression of miR-7 negatively correlated with lymph node metastasis and tumor node metastasis staging in CC (P<0.05). Inhibition of miR-7 led to an accelerated ability of proliferation and migration in CC cell lines. Additionally, overexpression of miR-7 inhibited the proliferation and migration of CC cells. In addition, it was also observed that miR-7 regulated the proliferation and migration of CC by regulating the protein expression of FAK, therefore, regulating the expression of matrix metalloproteinase (MMP)-2 and MMP-9. miR-7 inhibited the proliferation and migration of CC cells by regulating FAK. These findings suggested that miR-7 may be a novel therapeutic target for CC.

Expression of focal adhesion kinase in the eutopic endometrium of women with adenomyosis varies with dysmenorrhea and pelvic pain.

The aim of the present study was to examine whether the expression of focal adhesion kinase (FAK) is altered in the eutopic endometrium of female patients with adenomyosis, as compared with that of females without adenomyosis. The expression of FAK was assessed by immunohistochemical, western blot and reverse transcription-quantitative polymerase chain reaction analyses. An elevated expression of FAK mRNA and protein was identified in the eutopic endometrium of patients with adenomyosis compared with patients without adenomyosis (P<0.05). In addition, a positive correlation was detected between FAK protein expression and dysmenorrhea and pelvic pain in females with adenomyosis (P<0.05). The significant increase of FAK expression identified in the eutopic endometrium of females with adenomyosis, as well as the association of FAK protein expression with dysmenorrhea and pelvic pain, suggested that FAK may play a role in the pathogenesis of adenomyosis.

Doxycycline inhibits leukemic cell migration via inhibition of matrix metalloproteinases and phosphorylation of focal adhesion kinase.

Doxycycline, a tetracycline-based antibiotic, has been reported to attenuate melanoma cell migration through inhibiting the focal adhesion kinase (FAK) signaling pathway. However, it remains to be elucidated whether doxycycline exerts this effect on leukemia cell migration. The present study aimed to examine the role of doxycycline in leukemia cell migration. The invasion capacities of the human leukemia cell lines KG1a (acute myelogenous leukemia) and K562 (chronic myelogenous leukemia) were evaluated using Matrigel® matrix-coated Transwell® chamber assays; leukemic cell lines treated with doxycycline (1 µg/ml) or anti-β1-integrin antibodies were added to the upper chamber, while untreated cells were included as controls. Reverse transcription quantitative polymerase chain reaction was performed in order to further understand the influence of doxycycline treatment on the expression of FAK and gelatinases in the KG1a and K562 leukemic cell lines. In addition, FAK protein expression and phosphorylation were determined using western blot analysis in order to investigate the mechanism by which doxycycline inhibited leukemic cell migration. The results revealed that doxycycline treatment significantly attenuated the migration of KG1a and K562 cells, which was demonstrated to be associated with inhibition of the expression and phosphorylation of FAK. In addition, doxycycline treatment inhibited matrix metalloproteinase (MMP)-2 and MMP-9 expression. Furthermore, incubation with blocking anti-β1-integrin antibodies had an analogous inhibitory effect on leukemic cell migration to that of doxycycline. In conclusion, the results of the present study suggested that doxycycline attenuated leukemic cell migration through inhibiting the FAK signaling pathway. Therefore, doxycycline may have potential for use as a novel strategy for the treatment of leukemia.

The effects of focal adhesion kinase on the motility, proliferation and apoptosis of Caco2 and SMMC-7721 cells.

Focal adhesion kinase (FAK) plays important roles in cancer development. However, the significance of FAK expression in colorectal carcinoma and hepatocellular carcinoma has not been clarified. This study aims to explore the roles FAK played in the progression of colorectal carcinoma and hepatocellular carcinoma. RNAi method was used to inhibit the expression of FAK in Caco2 and SMMC-7721 cells. Reverse transcriptase polymerase chain reaction analysis and Western blot analysis were used to examine mRNA and protein expression of FAK. Then, the proliferation, motility and apoptosis of both types of cells were detected using MTT assay, wound healing/transwell assay and nuclear staining assay. The microstructure changes (F-actin, β-tubulin and lamin B1) of SMMC-7721 cells were visualized by immunofluorescence. FAK was overexpressed in both cell lines and down-regulation of FAK resulted in suppression of cell proliferation, inhibition of cell migration and invasion. The apoptosis of cells was increased significantly following the FAK expression inhibition. Moreover, actin polymerization, β-tubulin and lamin B1 expression of cells were significantly decreased. The results highlight the role of FAK in the progression of cancers. These findings suggest FAK serve as a potential therapeutic target for cancer therapy.

Insulin-like growth factor 1 increases apical fibronectin in blastocysts to increase blastocyst attachment to endometrial epithelial cells in vitro.

Does insulin-like growth factor 1 (IGF1) increase adhesion competency of blastocysts to increase attachment to uterine epithelial cells in vitro? IGF1 increases apical fibronectin on blastocysts to increase attachment and invasion in an in vitro model of implantation. Fibronectin integrin interactions are important in attachment of blastocysts to uterine epithelial cells at implantation. Mouse blastocysts (hatched or near completion of hatching) were cultured in serum starved (SS) medium with varying treatments for 24, 48 or 72 h. Treatments included 10 ng/ml IGF1 in the presence or absence of the PI3 kinase inhibitor LY294002, an IGF1 receptor (IGF1R) neutralizing antibody or fibronectin. Effects of treatments on blastocysts were measured by attachment of blastocysts to Ishikawa cells, blastocyst outgrowth and fibronectin and focal adhesion kinase (FAK) localization and expression. Blastocysts were randomly allocated into control and treatment groups and experiments were repeated a minimum of three times with varying numbers of blastocysts used in each experiment. FAK and integrin protein expression on Ishikawa cells was quantified in the presence or absence of IGF1. Fibronectin expression and localization in blastocysts was studied using immunofluorescence and confocal microscopy. Global surface expression of integrin αvβ3, β3 and β1 was measured in Ishikawa cells using flow cytometry. Expression levels of phosphorylated FAK and total FAK were measured in Ishikawa cells and blastocysts by western blot and image J analysis. Blastocyst outgrowth was quantified using image J analysis. The presence of IGF1 significantly increased mouse blastocyst attachment to Ishikawa cells compared with SS conditions (P < 0.01). IGF1 treatment resulted in distinct apical fibronectin staining on blastocysts, which was reduced by the PI3 kinase inhibitor LY294002. This coincided with a significant increase in blastocyst outgrowth in the presence of IGF1 (P < 0.01) or fibronectin (P < 0.001), which was abolished by LY294002 (P < 0.001). Apical expression of integrin αvβ3, β3 and β1 in Ishikawa cells was unaltered by IGF1. However, IGF1 increased phosphorylated FAK (P < 0.05) and total FAK expression in Ishikawa cells. FAK signalling is linked to integrin activation and can affect the integrins' ability to bind and recognize extracellular matrix proteins such as fibronectin. Treatment of blastocysts with IGF1 before co-culture with Ishikawa cells increased their attachment (P < 0.05). This effect was abolished in the presence of LY294002 (P < 0.001) or an IGF1R neutralizing antibody (P < 0.05). This study uses an in vitro model of attachment that uses mouse blastocysts and human endometrial cells. This involves a species crossover and although this use has been well documented as a model for attachment (as human embryo numbers are limited) the results should be interpreted carefully. This study presents mechanisms by which IGF1 improves attachment of blastocysts to Ishikawa cells and documents for the first time how IGF1 can increase adhesion competency in blastocysts. Failure of the blastocyst to implant is the major cause of human assisted reproductive technology (ART) failure. As growth factors are absent during embryo culture, their addition to embryo culture medium is a potential avenue to improve IVF success. In particular, IGF1 could prove to be a potential treatment for blastocysts before transfer to the uterus in an ART setting.