FAK mRNA Research Articles

Effect of TiO2 Nanotubes on Biological Activity of Osteoblasts and Focal Adhesion Kinase/Osteopontin Level

Osteoblasts are important cells for bone formation and play a major part in bone diseases and bone defects. Clinically, we usually adopt bone implants for related diseases. Also, nanotechnology is important in bones and joints. This study assessed the effects of TiO2 nanotubes of different diameters on osteoblast activity, FAK and OPN levels, aiming to provide an experimental foundation for selection of clinical bone implant materials. The morphology of MG-63 human osteosarcoma cells changed with expansion of TiO2 nanotubes’ diameter. From the biological activity, the cell proliferation and adhesion were enhanced as the diameter of the TiO2 nanotube was increased and its proliferation and adhesion were highest in the 100 nm TiO2 nanotube, which is related to increased ALP activity, FAK and OPN protein and mRNA expression. ELISA detected ALP activity and found that MG-63 cells cultured with 70 nm nanotube had strongest activity. Immune blotting and PCR results showed that, FAK and OPN activities were highest in 70 nm TiO2 nanotube cells. In summary, TiO2 nanotubes increased cell proliferation and adhesion by up-regulating the activities of FAK and OPN in a concentration-dependent relationship.

Upregulated lnc-HZ02 and miR-hz02 inhibited migration and invasion by downregulating the FAK/SRC/PI3K/AKT pathway in BPDE-treated trophoblast cells.

BPDE (benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide), a metabolite of environmental carcinogenic BaP, weakens the migration and invasion of human villous trophoblast cells and may further induce miscarriage. However, the underlying mechanisms remain largely unknown. In this study, we identified thatin trophoblast Swan 71 and HTR-8/SVneo cells, miR-hz02 upregulates the level of lnc-HZ02, which inhibits the expression of an RNA-binding protein HuR. HuR could interact with FAK mRNA and promoteits mRNA stability, thus upregulatingthe FAK level and the FAK/SRC/PI3K/AKT pathway, and finally maintainingthe normal migration and invasion of trophoblast cells. If trophoblast cells are exposed to BPDE, both miR-hz02 and lnc-HZ02 are upregulated, which reduce the level of HuR, weaken the interactions of HuR with FAK mRNA, downregulate FAK level and the FAK/SRC/PI3K/AKT pathway, and finally inhibit cell migration and invasion. This study provides a novel scientific understanding ofthe dysfunctions of human trophoblast cells.

Fak silencing impairs osteogenic differentiation of bone mesenchymal stem cells induced by uniaxial mechanical stretch

Background/purposeMechanical stretch plays a key role in promoting proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) in distraction osteogenesis (DO). A better understanding of how the extracellular biomechanical stimulation is transferred to intracellular signal expression will benefit DO. Focal adhesion kinase (FAK) is a key factor in integrin signaling pathway. However, little is known about the effect of integrin-FAK signaling during the process of stretch induced osteogenic differentiation of BMSCs. Materials and methodsA specific short hairpin RNAs (shRNAs) lentiviral expression vector was used to silence Fak gene and a well-established in vitro uniaxial dynamic stretching device was applied to stimulate DO. Fak silencing was confirmed by fluorescence microscopy and the detection of Fak mRNA and FAK, p-FAK protein expression. Alkaline phosphatase (ALP) activity, expression of osteogenic differentiation markers - runt-related transcription factor 2 (RUNX2/Runx2) and alkaline phosphatase (Alp) together with integrin upstream signal transduction molecules integrin beta-1 (ITGB1/Itgb1) and downstream signal transduction molecules integrin-linked kinase (ILK) were detected after the stretch. ResultsThe results showed that mechanical stretch in control groups significantly induced the osteogenic differentiation of BMSCs with increased ALP activity, expression of RUNX2/Runx2 and Alp, together with upregulated ITGB1/Itgb1 and ILK, which all vanished in Fak silencing group. ConclusionSilencing of the Fak gene inhibited the osteogenic differentiation of rat BMSCs induced by in vitro mechanical stretch through integrin signaling pathway.

WT1 associated protein promotes metastasis and chemo-resistance to gemcitabine by stabilizing Fak mRNA in pancreatic cancer

WT1 associated protein (WTAP), playing an important role in several malignancies owing to its complex function in transcriptional and post-transcriptional regulation, is an independent prognostic indicator for pancreatic cancer (PC). However, its specific role and underlying mechanism in PC remain unclear. In the present study, we found that WTAP could promote migration/invasion and suppress chemo-sensitivity to gemcitabine in PC. Further mechanical investigation revealed that WTAP could bind to and stabilize Fak mRNA which in turn activated the Fak-PI3K-AKT and Fak-Src-GRB2-Erk1/2 signaling pathways. In addition, GSK2256098, a specific Fak inhibitor, could reverse WTAP-mediated chemo-resistance to gemcitabine and metastasis in PC. Taken together, Fak inhibitor might be a promising therapeutic option for PC patients with WTAP overexpression.

AXL and CDCP1: A roadmap of innate resistance in EGFR mutant NSCLC.

e24003Background: Several non receptor tyrosine kinases (RTKs) activated by EGFR tyrosine kinase inhibitors (TKI) have been identified. Src family kinases (SFKs), FAK and STAT3 protein and mRNA lev...

MicroRNA-433 inhibits oral squamous cell carcinoma cells by targeting FAK.

We investigated the involvement of microRNA-433 (miR-433) in the proliferation, migration, and invasiveness of oral squamous cell carcinoma (OSCC). Totally 108 OSCC tissues and adjacent normal tissues from patients with OSCC were collected. Also, transplanted tumor formation experiment in nude mice was conducted to verify the effect of miR-433 and FAK on subcutaneous transplanted tumor. The CD44+ stem cell from SCC-9 were collected and assigned into the blank, miR-433 mimics, mimics control, miR-433 inhibitors, inhibitors control, siFAK and miR-433 inhibitors + siFAK groups. The qRT-PCR and western blotting were used to detect miR-433, FAK, ERK, MEK, pERK and pMEK after transfection. Flow cytometry, MTT assay, scratch test and Transwell assay were performed to determine the cell proportion, growth, migration and invasion of SCC-9 cells. In cell line SCC-9, expression of CD133, Oct-4, and BIM-1 was greater in CD44+ cells than CD44- cells, indicating that CD44+ cells had characteristics of tumor stem cells. Expression of FAK, ERK, MEK, p-ERK and p-MEK was decreased in tumor tissues from the CD44-, miR-433, and siFAK groups. Expression of MiR-433 mRNA was elevated, while levels of FAK, ERK, MEK, p-ERK, and p-MEK mRNA were all decreased in the miR-433 mimics group. In the miR-433 mimics and siFAK groups, cell proliferation, migration, and invasion were all decreased, while the opposite trends were seen in the miR-433 inhibitor group. These results indicate that miR-433 downregulates FAK through the ERK/MAPK signaling pathway to inhibit the proliferation, migration, and invasiveness of SCC-9 OSCC cells.

EditingFAKto be more metastatic

Cancer Drugs that inhibit cell migration pathways, such as that mediated by the kinase FAK, may prevent metastasis in cancer patients. Amin et al. found that the RNA-editing enzyme ADAR underpins FAK activity. In lung adenocarcinoma cells, ADAR bound to and edited FAK mRNA, improving its stability, increasing FAK protein levels, and enhancing the migration of cells. High levels of ADAR expression correlate with poor prognosis in patients. Thus, inhibiting FAK may improve therapeutic outcomes for patients with ADAR-positive lung adenocarcinoma. Sci. Signal. 10 , eaah3941 (2017).

Abstract 4095: c-Met activation mediates resistance to polo-like kinase 1 inhibitor-induced apoptosis in non-small cell lung cancer

Abstract Background: Inhibiting polo-like kinase 1 PLK1 may be an effective treatment for non-small cell lung cancer (NSCLC). PLK1 is a key regulator of mitosis and DNA damage checkpoints. PLK1 inhibitors are well tolerated, but only a few unselected patients with NSCLC respond to single-agent therapy. However, predictive biomarkers have not been used to select patients who are likely to experience a response to PLK1 inhibitors, and the mechanisms of resistance to PLK1 inhibitors have not been elucidated, making these unknowns a major gap in knowledge. To address this gap, we compared basal gene and protein expression in 63 NSCLC cell lines and discovered that mesenchymal NSCLC cell lines were more sensitive to PLK1 inhibitors than epithelial cell lines in vitro and in vivo. The induction of apoptosis in some NSCLC cell lines at very low drug concentrations and the need to find better therapy for mesenchymal NSCLC motivated us to further study PLK1 inhibition. Methods: To identify the pathways involved in PLK1 inhibitor-induced apoptosis, we used 3 pairs of isogenic NSCLC cell lines in which we had induced a mesenchymal phenotype using TGF-β. These isogenic lines were treated with the PLK1 inhibitor (volasertib) for 24 hours and levels of 301 proteins and phosphoproteins were simultaneously measured before and after treatment with volasertib using reverse phase protein array (RPPA). Results: The induction of a mesenchymal phenotype using TGF-β increased PLK1 inhibition-induced DNA damage and apoptosis in 3 NSCLC cell lines. To further elucidate mechanisms of resistance to PLK1 inhibition, we compared gene and protein expression in these isogenic cell lines, before and after PLK1 inhibition. There were 35, 12 and 43 proteins differentially regulated following PLK1 inhibition in epithelial vs. mesenchymal lines in HCC366, H1975, and HCC4006 cell lines, respectively at False Discovery rate 0.1. Phosphorylated FAK (Y397) and c-Met (Y1234/1235) were consistently inhibited following PLK1 inhibition in the mesenchymal lines but activated in the epithelial lines. These changes were confirmed by Western blotting. Total FAK and c-Met protein and mRNA levels were not affected, demonstrating post-translational changes. The inhibition of c-Met using EMD 1214063 led to FAK inhibition but FAK inhibition did not affect c-Met activation. The combination of c-Met inhibitor and volasertib increases sensitivity in NSCLC cell lines tested. The combinations led to more apoptosis than the single-agent inhibitors. Conclusions: NSCLC cell lines have diverse sensitivities to PLK1 inhibition, which is consistent with the results of clinical trials of PLK1 inhibitors in solid tumors, but no studies to date explain these diverse responses to PLK1 inhibition. We have identified c-Met activation as a previously unknown pathway of resistance to PLK1 inhibition in epithelial NSCLC. Citation Format: Ratnakar Singh, Li Shen, Pan Tong, Jing Wang, Faye M. Johnson. c-Met activation mediates resistance to polo-like kinase 1 inhibitor-induced apoptosis in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4095. doi:10.1158/1538-7445.AM2017-4095

MicroRNA-7 inhibits cell proliferation, migration and invasion in human non-small cell lung cancer cells by targeting FAK through ERK/MAPK signaling pathway.

ObjectiveTo investigate the effects of microRNA-7 (miR-7) on the proliferation, migration and invasion of non-small cell lung cancer NSCLC) cells by targeting FAK through ERK/MAPK signaling pathway.MethodsNSCLC tissues and adjacent normal tissues were obtained from 160 NSCLC patients after operation. NSCLC cell lines (A549, H1299 and H1355) and a normal human fetal lung fibroblast cell line (MRC-5) were obtained. NSCLC cells were assigned into miR-7 inhibitors, miR-7 mimics, blank, miR-7 mimics control, miR-7 inhibitors control, FAK siRNA and miR-7 inhibitors + FAK siRNA groups. The expressions of miR-7 and FAK mRNA in tissues and cell lines were detected by qRT-PCR and Western-Blotting. Cell proliferation, migration and invasion were detected by MTT assay, wound scratch assay and Transwell assay.ResultsCompared with adjacent normal tissues, miR-7 expression was down-regulated, but the mRNA and protein expressions of FAK, ERK and MAPK were up-regulated. Compared with the blank and mimics control groups, miR-7 significantly increased but FAK, ERK and MAPK expressions decreased in miR-7 mimics and FAK siRNA groups. Cell proliferation, migration and invasion were inhibited in the miR-7 mimics and FAK siRNA groups, while opposite regarding miR-7 inhibitors group.ConclusionThe miR-7 can inhibit the activation of ERK/MAPK signaling pathway by down-regulating FAK expression, thereby suppressing the proliferation, migration and invasion of NSCLC cells. The miR-7 and its target gene FAK may be novel targets for the diagnosis and treatment of NSCLC.

Bit1 knockdown contributes to growth suppression as well as the decreases of migration and invasion abilities in esophageal squamous cell carcinoma via suppressing FAK-paxillin pathway.

BackgroundThere is growing evidence that Bit1 exerts different roles in the development and progression of human cancers. Although Bit1 was highly exhibited in ESCC tissues in our previous study, its roles and molecular mechanisms implicated in development and progression of ESCC remain unknown.MethodsBit1 protein expression in ESCC cell lines and normal esophageal epithelial cell was detected by Western blotting. Bit1 protein expression mediated by Bit1 shRNA was investigated by Western blotting. MTT, migration assay, invasion experiment, ELISA and Flow cytometry were utilized to determine the effects of Bit1 knockdown on cell proliferation, migration, invasion and apoptosis, respectively. A xenograft model was used to examine in vivo tumourigenicity, and immunohistochemistry and TUNEL were utilized to evaluate the related protein expression and apoptosis. Gene microarray was determined by Agilent SurePrint G3 Human GE 8 × 60 K Microarray, the interaction of Bit1 and FAK proteins were detected by Immunoprecipitation and the key protein expressions of FAK-paxillin pathway were detected by Western blotting.ResultsWe found Bit1 expression in all human ESCC cell lines tested was significantly higher than that in normal esophageal epithelial cell Het-1A (P < 0.05), in which EC9706 presented the highest Bit1 level. Bit1 protein level was significantly downregulated at day 1 after transfection with specific shRNA against Bit1 (P < 0.05). At days 2 and 3, Bit1 level reached the lowest value after transfection with Bit1 shRNA. Moreover, Bit1 depletion contributed to growth inhibition in vitro and in vivo, reduced cell migration and invasion abilities, and induced cell apoptosis in EC9706 and TE1 cells. More importantly, Bit1 downregulation significantly lowered Bcl-2 and MMP-2 levels in EC9706 xenografted tumor tissues, meanwhile triggered apoptosis after treatment with different doses of Bit1 shRNA. Further gene microarray revealed that 23 genes in Bit1-RNAi group were markedly downregulated, whereas 16 genes were obviously upregulated. Notably, Bit1 intrinsically interacted with FAK protein in EC9706 cells. Moreover, paxillin was downregulated at mRNA and protein levels in Bit1 shRNA group, coupled with the decreases of FAK mRNA and protein expressions.ConclusionBit1 may be an important regulator in cell growth, apoptosis, migration and invasion of ESCC via targeting FAK-paxillin pathway, and thereby combinative manipulation of Bit1 and FAK-paxillin pathway may be the novel and promising therapeutic targets for the patients with ESCC.

GMP-grade platelet lysate enhances proliferation and migration of tenon fibroblasts.

Tenon's fibroblasts (TFs), widely employed as in vitro model for many ophthalmological studies, are routinely cultured with FBS. Platelet Lysate (PL), a hemoderivate enriched with growth factors and cytokines has been largely tested in several clinical applications and as substitute of FBS in culture. Here, we investigate whether PL can exert biological effects on TF populations similarly to other cell types. Results show that PL significantly enhances cell proliferation and migration vs. FBS, without influencing cell size/granularity. Upregulation of EGF, VEGF, KDR, MMP2-9, FAK mRNA levels also occurs and phosphorylation of AKT but not of ERK1/2 is significantly enhanced. The inhibition of the PI3kinase/AKT pathway with the specific inhibitor wortmannin, decreases PL-induced cell migration but not proliferation. Condition supernatants containing PL show increased bioavailability of Nitric Oxide and reduced levels of 8-Iso-PGF2-alpha, correlating with cell proliferation and migration. Pro-angiogenic/inflammatory soluble factors (GRO, Angiogenin, EGF, I-309, PARC) are exclusively or greater expressed in media containing PL than FBS. GMP-grade PL preparations positively influence in vitro biological effects of TFs representing a suitable and safer alternative to FBS.

Evidence for presence of Focal adhesion kinase and enhanced activity in epithelial and stromal cells of mice cervix during pregnancy

We recently provided the first evidence of a presence of a cluster of mechano‐transducing molecules in the cervix using proteomics analysis, including caveolin‐1 (CAV‐1), talin‐1, vinculin, pro‐filin and others. Here, we use confocal immunofluorescence, Western blot and real time PCR analysis to examine whether a key mechano‐transduction molecule, phosphorylated focal adhesion kinase protein and mRNA (pFAK and FAK mRNA) are expressed in the cervix during pregnancy (days 0, days 14, 15, 16), as well as identify its (pFAK) cellular distribution in mice. pFAK was localized in the epithelial and stromal (fibroblast) cells of the cervix of pregnant mice and its expression and activity increased with advancement in pregnancy but decreased postpartum. The expression was found to be confined to the cell membrane. We conclude that the growing fetus likely induces mechano‐transduction in the cervix and may influence cervical remodeling and most likely the timing of birth. Funding: Office of Student Research, Appalachian State University.

FAK Inhibition disrupts a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth.

Ovarian cancer ascites fluid contains matrix proteins that can impact tumor growth via integrin receptor binding. In human ovarian tumor tissue arrays, we find that activation of the cytoplasmic focal adhesion (FAK) tyrosine kinase parallels increased tumor stage, β5 integrin, and osteopontin matrix staining. Elevated osteopontin, β5 integrin, and FAK mRNA levels are associated with decreased serous ovarian cancer patient survival. FAK remains active within ovarian cancer cells grown as spheroids, and anchorage-independent growth analyses of seven ovarian carcinoma cell lines identified sensitive (HEY, OVCAR8) and resistant (SKOV3-IP, OVCAR10) cells to 0.1 μmol/L FAK inhibitor (VS-4718, formerly PND-1186) treatment. VS-4718 promoted HEY and OVCAR8 G0-G1 cell-cycle arrest followed by cell death, whereas growth of SKOV3-IP and OVCAR10 cells was resistant to 1.0 μmol/L VS-4718. In HEY cells, genetic or pharmacological FAK inhibition prevented tumor growth in mice with corresponding reductions in β5 integrin and osteopontin expression. β5 knockdown reduced HEY cell growth in soft agar, tumor growth in mice, and both FAK Y397 phosphorylation and osteopontin expression in spheroids. FAK inhibitor-resistant (SKOV3-IP, OVCAR10) cells exhibited anchorage-independent Akt S473 phosphorylation, and expression of membrane-targeted and active Akt in sensitive cells (HEY, OVCAR8) increased growth but did not create a FAK inhibitor-resistant phenotype. These results link osteopontin, β5 integrin, and FAK in promoting ovarian tumor progression. β5 integrin expression may serve as a biomarker for serous ovarian carcinoma cells that possess active FAK signaling.

MiR-138 and MiR-135 Directly Target Focal Adhesion Kinase, Inhibit Cell Invasion, and Increase Sensitivity to Chemotherapy in Cancer Cells

Focal Adhesion Kinase is a 125 kDa non-receptor kinase and overexpressed in many types of tumors. Recently, short noncoding RNAs, called microRNAs have been discovered as regulators of gene expression mainly through binding to the untranslated region (UTR) of mRNA. In this report we show that MiR-138 and MiR-135 down-regulated FAK expression in cancer cells. MiR-138 and MiR-135 inhibited FAK protein expression in different cancer cell lines. The computer analysis of 3'FAK-untranslated region (FAKUTR) identified one conserved MiR-138 binding site (CACCAGCA) at positions 3514-3521 and one conserved MiR-135 (AAGCCAU) binding site at positions 4278-4284 in the FAK-UTR. By a dual-luciferase assay we demonstrate that MiR-138 and MiR-135 directly bound the FAK untranslated region using FAK-UTR-Target (FAK-UTR) luciferase plasmid and inhibited its luciferase activity. The sitedirected mutagenesis of the MiR-138 and MiR-135 binding sites in the FAK-UTR abrogated MiR-138 and MiR-135-directed inhibition of FAK-UTR. Real-time PCR demonstrated that cells transfected with MiR-138 and MiR-135 expressed decreased FAK mRNA levels. Moreover, stable expression of MiR-138 and MiR-135 in 293 and HeLa cells decreased cell invasion and increased sensitivity to 5- fluorouracil (5-FU), FAK inhibitor, Y15, and doxorubicin. In addition, MiR-138 significantly decreased 293 xenograft tumor growth in vivo. This is the first report on regulation of FAK expression by MiR-135 and MiR138 that affected invasion, drug sensitivity, and tumor growth in cancer cells, which is important to the development of FAK-targeted therapeutics and understanding their novel regulations and functions.

Sphingosine kinase 1 enhances the proliferation and invasion of human colon cancer LoVo cells through up-regulating FAK pathway and the expression of ICAM-1 and VCAM-1

To investigate the effects of sphingosine kinase 1 (SphK1) on the proliferation, migration and invasion of human colon cancer LoVo cells, and to explore the related mechanisms. Human colon cancer LoVo cells were divided into three groups: phorbol 12-myristate 13-acetate (PMA) was used to induce the activation of SphK1 in the PMA group, N,N-dimethylsphingosine (DMS) used to suppress the activity of SphK1 in DMS group, and the cells treated with equal amount of 0.9 % NaCl instead of drugs served as the control group. The activity of SphK1 was assayed by autoradiography, the cell proliferation was assessed by MTT assay, cell migration and invasion were examined by Boyden chamber assay, concentrations of sICAM-1 and sVCAM-1 were assayed by ELISA, and RT-PCR and Western blot were used to evaluate the mRNA and protein expression in the cells. The activity of SphK1 was efficiently induced by PMA and significantly suppressed by DMS. PMA induced cell proliferation in a time- and dose-dependent manner. On the contrast, DMS suppressed cell proliferation in a time- and dose-dependent manner. After treating with PMA, the number of migrating and invasing cells were increased to 143.36 ± 8.73 and 118.46 ± 6.25, significantly higher than those of the control group (75.48 ± 6.12 and 64.19 ± 5.36). After treating with DMS, the number of migrating and invasing cells were decreased to 38.57 ± 3.24 and 32.48 ± 4.27, significantly lower than those of the control group (P < 0.01). The relative expression levels of FAK, ICAM-1 and VCAM-1 mRNA in the PMA group were 0.82 ± 0.06, 0.74 ± 0.05 and 0.89 ± 0.09, and those in the DMS group were 0.23 ± 0.02, 0.26 ± 0.03 and 0.37 ± 0.04, with significant differences between the PMA, DMS and control groups (P < 0.01). Compared with the control group, the relative expression levels of FAK and p-FAK proteins in the PMA group (0.52 ± 0.06 and 0.51 ± 0.06) were significantly elevated, and those of the DMS group (0.20 ± 0.03 and 0.09 ± 0.02) were significantly decreased. In addition, the concentrations of sICAM-1 and sVCAM-1 were significantly elevated with the activation of SphK1. On the contrary, those of the DMS group were significantly reduced with the suppression of SphK1 (Both P < 0.01). SphK1 may enhance the proliferation, migration and invasion of colon cancer LoVo cells through activating FAK pathway and up-regulating the expression of ICAM-1 and VCAM-1.

Effect of small interfering RNA transfection on FAK and DLC1 mRNA expression in OVCAR-3

RNA interference is an evolutionarily conserved cellular defense mechanism that protects cells from hostile genes and regulates the function of normal genes during growth and development. In this study, we established GFP-siFAK-DLC1 vector and transfect the vector into OVCAR-3 cells. RT-PCR and western blot analyses were performed for FAK, DLC1 mRNA, and protein expression in OVCAR-3 cells. ELISA method was used for caspase-3 and caspase-9 activities. These studies demonstrate that both recombinant pGFP-siFAK-DLC1 vector and pGFP-siCon-DLC1 vector may effectively promote DLC1 mRNA transcription and didn't affect siRNA effect. Recombinant vector (pGFP-siFAK-DLC1) may promote DLC1 gene expression, and effectively silence FAK gene expression. Silencing FAK mRNA expression and DLC1 mRNA expression may markedly enhance caspase-3 and caspase-9 activities in OVCAR-3 cells. These results showed that in ovarian cancer OVCAR-3 cell silencing FAK gene expression or / and increasing DLC-1 gene expression, could improve Caspase-3 and Caspase-9 protease activities. In the expression of DLC-1 and silence FAK expression group (double action group) effect was more significant as compared with the silence FAK gene group or expression of DLC-1 gene alone, difference was significant (p < 0.05).

Indole compounds inhibits migration of breast cancer cell by suppressing MMPs via reduction of FAK expression

Indole‐3‐carbinol (I3C) and indole[3,2‐b] carazole (ICZ) are major bioactive food components in cruciferous vegetables. Although previous studies have shown anticancer activity of I3C and ICZ in various cancer cells, it has not been determined how indole compounds regulates migration and related signaling pathways. In this study, we investigated the effects of I3C and ICZ on migration using breast cancer cells (MCF‐7 and MDA‐MB231). The growth inhibition of breast cancer cells by I3C and ICZ was measured by MTT assay, and 10 ¥ìM was chosen as maximum dose for migration assay because at this concentration, no significant interference in the cell growth or morphology was observed. Our results showed that I3C and ICZ were dramatically inhibited migration of breast cancer cells in a dose‐dependent manner, as observed in monolayer scratch assay. The MMP‐2 and ‐9 activity were also reduced by I3C. In addition, pretreatment of I3C was decreased FAK mRNA expression. Taken together, indole compounds inhibits migration of breast cancer cells by reduction of MMPs activity and FAK expression.

Effect of DLC-1 gene on tumour formation in nude mice inoculated with OVCAR-3

The aim of the present study was to examine effect of DLC-1 gene transfect OVCAR-3 cells in vivo on tumour growth in nude mice. Results showed that tumor development rate in OVCAR-3/DLC-1 group, OVCAR-3/pEGFP-C3 group and OVCAR-3 group were 87, 87 and 100%, respectively. After 40 days, tumour cells were not detected in peripheral blood of mice in every group. Low expression of P-FAK, P- P130 cas and P-JNK proteins was detected in group OVCAR-3/DLC-1, while high expression was found in group OVCAR-3/pEGFP-C3 and OVCAR-3. the brightness of the bands of P130cas mRNA, FAK mRNA and JNK mRNA in group OVCAR-3/DLC-1 became weaker, while the brightness of the bands become stronger in group OVCAR-3/pEGFP-C3 and group OVCAR-3. DLC-1, as a anti-tumor related target gene, possesses a promising future. Key words: DLC-1, ovarian cancer, nude mice, transfection.

Primary Cilia-Mediated Osteogenic Response to Fluid Flow Occurs via Increases in Focal Adhesion and Akt Signaling Pathway in MC3T3-E1 Osteoblastic Cells

Fluid flow is an important anabolic mechanical signal that results in the upregulation of osteogenic responses in bone cells. Primary cilium may be responsible for the sensing of mechanical signals and converting these into biochemical signals to maintain bone homeostasis through an unknown mechanism. Focal adhesions, sites of mechanical linkage between the cytoskeleton and extracellular matrix, play a role as mechanoreceptors that are involved in fluid flow-induced signaling. In this study, we first verified whether focal adhesions are required for primary cilia to upregulate fluid flow-induced bone forming activities in osteoblasts. Specifically, mRNA and protein levels of cyclooxygenase-2 (COX-2) and release of prostaglandin E2 (PGE2), which are important molecules that regulate bone formation, were quantified in MC3T3-E1 osteoblastic cells with or without primary cilia after exposure to 1 Pa of fluid flow-induced shear stress. Then, this experiment was continued on Arg-Gly-Asp-Ser (RGDS) peptide-treated cells after determining the formation of focal adhesions were inhibited. Second, signaling pathways, including Ras, ERK, and Akt, whereby primary cilia transmit fluid flow to cellular responses in osteoblastic cells were studied. Removal of primary cilia resulted in a significant decrease in COX-2 mRNA and protein and PGE2 release with a similar decrease in FAK mRNA and vinculin-positive focal adhesion sites in response to fluid flow. Furthermore, the removal of primary cilia suppressed the activation of p-Akt, but not Ras and p-ERK in response to fluid flow. Our results suggest that primary cilia lead to fluid flow-induced COX-2 induction and PGE2 release via increases in focal adhesions and their responses are activated by Akt phosphorylation in osteoblastic cells.

P53-Dependent repression of focal adhesion kinase in response to estradiol in breast cancer cell-lines

Mutations in the TP53 suppressor gene are frequent in breast cancers. These mutations are associated with poor prognosis, thought to be due to proliferative advantage and poor response to chemotherapy associated with loss of p53 function. The focal adhesion kinase ( FAK/ PTK2), a tyrosine kinase, is over-expressed in a variety of human tumors including breast cancers. FAK is a critical regulator of adhesion and motility and its over-expression is associated with increased metastatic potential. Recently, FAK promoter has been shown to contain p53 responsive elements and to be down-regulated by DNA-damage in a p53-dependent manner. Here, we have used five estrogen-dependent breast cancer cells lines with different p53 status, including an isogenic model, to show that FAK expression was regulated in a p53-dependent manner in response to estradiol. FAK protein and mRNA expression were down-regulated by estradiol in wild-type but not mutant p53 cells. Moreover, silencing wild-type p53 increased FAK expression, while over expressing p53 repressed FAK expression. ChIP experiment showed that p53 bound to FAK promoter in the presence of estradiol in p53 wild-type but not in mutant p53 cells, suggesting a direct role of p53 in down regulating FAK mRNA expression. FAK mRNA expression was also found to correlate with TP53 mutation status in a series of breast tumors. Finally, loss of FAK down-regulation in p53 mutant cells was correlated with increased proliferation and invasion upon estradiol stimulation, while FAK silencing reduced invasion. These results suggest that p53 is an important down regulator of FAK and that loss of p53 function in breast cancer may contribute to the metastatic potential of estrogen-responsive tumors through uncontrolled FAK expression upon estrogens stimulation.