Focal Adhesion Kinase Signal Transduction Research Articles

Dragon’s Blood Protect Rat Blood-Brain Barrier Dysfunction Induced by Simulated Microgravity Effect

Dragon’s blood (DB) has shown a protective effect on neurological diseases. Microgravity (MG) or simulated MG (SMG) can induce blood–brain barrier (BBB) dysfunction, which is a characteristic feature of neurological disorders. This study’s purpose was to evaluate the effect of DB on SMG-induced BBB dysfunction and explore its signaling pathway. Both DB and vitamin C (Vc) were administered orally for tail-suspended rats within 3 weeks. DB and Vc solutions were added to human brain microvascular endothelial cells (HCMEC/D3) cells, which were then exposed to SMG for 24 h. The protective effect of DB was assessed by hematoxylin and eosin and Nissl staining, ultrastructure observation, and permeability in rats. Cell apoptosis and the distribution of tight junction (TJ) and adherens junction (AJ) proteins and filamentous actin (F-actin) were examined in HCMEC/D3. The oxidative stress and inflammation, and TJ and AJ protein expressions were determined in rat brain and HCMEC/D3. The focal adhesion kinase (FAK) signaling pathway proteins were determined. DB protected SMG-induced rat BBB disruption by improving neuronal apoptosis, repairing widened intercellular space, and decreasing BBB permeability. DB effectively relieved SMG-induced HCMEC/D3 damage by inhibiting cell apoptosis and restoring F-actin spindle distribution. High doses of DB upregulated TJ and AJ protein expressions and decreased oxidative stress and proinflammatory cytokine levels in rat brain and HCMEC/D3. DB enhanced the expressions of FAK signal transduction proteins and F-actin/globular actin (G-actin) ratio in rat brain and HCMEC/D3, suggesting that DB promotes actin cytoskeleton polymerization, benefits the endothelial cell–cell and cell–extracellular matrix adhesion, and, in consequence, contributes to BBB integrity.

Oxidative stress-induced FAK activation contributes to uterine serous carcinoma aggressiveness.

Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer (EC), characterized by its high propensity for metastases. In fact, while endometrioid endometrial carcinoma (EEC), which accounts for 85% of EC, presents a good prognosis, USC is the most frequently fatal. Herein, we used for the first time a peptide-based tyrosine-kinase-activity profiling approach to quantify the changes in tyrosine kinase activation between USC and EEC. Among the tyrosine kinases highly activated in USC, we identified focal adhesion kinase (FAK). We conducted mechanistic studies using cellular models. In a USC cell line, targeting FAK either by inhibitors PF-573228 and defactinib (VS-6063) or by gene silencing limits 3D cell growth and reduces cell migration. Moreover, results from our studies suggest that oxidative stress is increased in USC tumors compared to EEC ones. Reactive oxygen species (ROS) induce tyrosine phosphorylation of FAK and a concomitant tyrosine phosphorylation of paxillin, a mediator of FAK signal transduction. Mechanistically, by tracking hundreds of individual cells per condition, we show that ROS increased cell distance and migration velocity, highlighting the role of ROS-FAK-PAX signaling in cell migration. Both defactinib and ROS scavenger N-acetylcysteine (NAC) revert this effect, pointing toward ROS as potential culprits for the increase in USC cell motility. A proof of concept of the role of FAK in controlling cell growth was obtained in in vivo experiments using cancer-tissue-originated spheroids (CTOS) and a patient-derived orthotopic xenograft model (orthoxenograft/PDOX). Defactinib reduces cell proliferation and protein oxidation, supporting a pro-tumoral antioxidant role of FAK, whereas antioxidant NAC reverts FAK inhibitor effects. Overall, our data points to ROS-mediated FAK activation in USC as being responsible for the poor prognosis of this tumor type and emphasize the potential of FAK inhibition for USC treatment.

The involvement of TGF-β1 /FAK/α-SMA pathway in the antifibrotic impact of rice bran oil on thioacetamide-induced liver fibrosis in rats.

The objective of the current study is to investigate the effect of rice bran oil (RBO) on hepatic fibrosis as a characteristic response to persistent liver injuries. Rats were randomly allocated into five groups: the negative control group, thioacetamide (TAA) group (thioacetamide 100 mg/kg thrice weekly for two successive weeks, ip), RBO 0.2 and 0.4 groups (RBO 0.2mL and 0.4 mL/rat/day, po) and standard group (silymarin 100 mg/kg/day, po) for two weeks after TAA injection. Blood and liver tissue samples were collected for biochemical, molecular, and histological analyses. Liver functions, oxidative stress, inflammation, liver fibrosis markers were assessed. The obtained results showed that RBO reduced TAA-induced liver fibrosis and suppressed the extracellular matrix formation. Compared to the positive control group, RBO dramatically reduced total bilirubin, AST, and ALT blood levels. Furthermore, RBO reduced MDA and increased GSH contents in the liver. Simultaneously RBO downregulated the NF-κβ signaling pathway, which in turn inhibited the expression of some inflammatory mediators, including Cox-2, IL-1β, and TNF-α. RBO attenuated liver fibrosis by suppressing the biological effects of TGF-β1, α-SMA, collagen I, hydroxyproline, CTGF, and focal adhesion kinase (FAK). RBO reduced liver fibrosis by inhibiting hepatic stellate cell activation and modulating the interplay among the TGF-β1 and FAK signal transduction. The greater dosage of 0.4 mL/kg has a more substantial impact. Hence, this investigation presents RBO as a promising antifibrotic agent in the TAA model through inhibition of TGF-β1 /FAK/α-SMA.

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.

Progress in research on focal adhesion kinase signal transduction and cross-talk

Progress in research on focal adhesion kinase signal transduction and cross-talk

Activation of the focal adhesion kinase signal transduction pathway in cervical carcinoma cell lines and human genital epithelial cells immortalized with human papillomavirus type 18.

The inappropriate activation of protein-tyrosine kinases (PTKs) has been associated with initiation and progression of several types of human cancers. We therefore postulated that immortalization by DNA tumor viruses results in the induction of PTKs fundamental to these processes. An RT-PCR-based screen was thus used to identify PTKs that were abundantly expressed in HPV-18-immortalized epithelial cells and HPV-containing carcinoma cell lines. One of the genes isolated in this screen was the focal adhesion kinase (FAK; pp125FAK), a cytoplasmic protein kinase that is activated in v-src transformed cells or by stimulation with mitogenic polypeptides. FAK also becomes catalytically active upon integrin engagement with extracellular matrix proteins, such as fibronectin. We found that FAK expression and activity were significantly elevated in HPV-18 E6/E7-immortalized human genital epithelial cells relative to their primary cell counterparts. Protein expression and tyrosine phosphorylation of the putative FAK substrate, paxillin, were also notably increased upon HPV-18 immortalization of genital epithelial cells and in HPV-containing cervical carcinoma cell lines. Most significantly, these cells expressed markedly higher levels of both intracellular and extracellular fibronectin, thus providing a mechanism for activation of FAK and increased tyrosine phosphorylation of paxillin. These findings suggest a role for the integrin/FAK-mediated signaling pathway in cervical carcinogenesis and represent one of the first demonstrations of a tyrosine kinase whose activity is elevated following viral immortalization.

Actin Disruption Inhibits Bombesin Stimulation of Focal Adhesion Kinase (pp125FAK) in Prostate Carcinoma

Jasplakinolide is a member of a new class of antitumor agents targeting the actin cytoskeleton with activity against prostate cancer. Focal adhesion kinase (FAK) is an actin-associated mediator of mitogenic peptides. We hypothesized that the neuropeptide bombesin would activate FAK in prostate carcinoma, and that disruption of the actin network would block FAK activation and inhibit cell growth. Methods: PC-3 human prostate carcinoma cells were exposed to 50–200 nMjasplakinolide (Jas) or cytochalasin E (CyE) in cytotoxicity experiments. FAK phosphorylation was measured in cells stimulated with 0.01–10 nMbombesin; separate cells were pretreated 6 hr with 50–500 nMJas or CyE. Cell lysates and anti-FAK immunoprecipitates were subjected to SDS-PAGE, Western blotting, and detection with anti-actin or anti-phosphotyrosine. Depolymerized G-actin was separated from total actin by ultracentrifugation. Cytoskeletal changes were confirmed by fluorescence microscopy. Results: Jas (GI50= 47 ± 7 nM) and CyE (GI50= 61 ± 20 nM) potently inhibited PC-3 growth (P< 0.01 vs control). Bombesin rapidly stimulated tyrosine phosphorylation of FAK in a dose dependent manner. FAK phosphorylation was inhibited to near-basal levels (50% of bombesin stimulated) by 500 nMJas (63%) and 500 nMCyE (61%). Conclusions: Bombesin stimulated FAK in prostate carcinoma cells. Jasplakinolide, which induced over-polymerization of actin, and CyE, which depolymerizes actin, both inhibited bombesin-stimulated phosphorylation of FAK and inhibited PC-3 cell growth. Actin-disrupting agents block FAK signal transduction, which may be critical to their antitumor activity in prostate carcinoma.