ABSTRACT
Pancreatic ductal adenocarcinoma (PDA) is an extremely metastatic and lethal disease. Here, in both murine and human PDA, we demonstrate that extracellular matrix architecture regulates cell extrusion and subsequent invasion from intact ductal structures through tumor-associated collagen signatures (TACS). This results in early dissemination from histologically premalignant lesions and continual invasion from well-differentiated disease, and it suggests TACS as a biomarker to aid in the pathologic assessment of early disease. Furthermore, we show that pancreatitis results in invasion-conducive architectures, thus priming the stroma prior to malignant disease. Analysis in potentially novel microfluidic-derived microtissues and in vivo demonstrates decreased extrusion and invasion following focal adhesion kinase (FAK) inhibition, consistent with decreased metastasis. Thus, data suggest that targeting FAK or strategies to reengineer and normalize tumor microenvironments may have roles not only in very early disease, but also for limiting continued dissemination from unresectable disease. Likewise, it may be beneficial to employ stroma-targeting strategies to resolve precursor diseases such as pancreatitis in order to remove stromal architectures that increase risk for early dissemination.
Subject(s)
Carcinoma, Pancreatic Ductal/genetics , Focal Adhesion Kinase 1/genetics , Gene Expression Regulation, Neoplastic , Neoplasms, Experimental , Pancreatic Neoplasms/genetics , RNA, Small Interfering/genetics , Tumor Microenvironment/genetics , Animals , Apoptosis , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/therapy , Cell Line, Tumor , Cell Movement , Focal Adhesion Kinase 1/antagonists & inhibitors , Focal Adhesion Kinase 1/biosynthesis , Humans , Mice , Mice, Transgenic , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/therapyABSTRACT
Tenon's capsule fibroblasts are the main cellular components of filtration tract scar that limit the success rate of glaucoma filtration surgery. Scar formation results from infiltration and proliferation of fibroblasts into damaged areas, meanwhile synthesis of extracellular matrix glycoproteins. Integrins are cell surface receptors for extracellular molecules that mediate cell adhesion, spreading, migration, and invasion. They bind their ligands often through recognition of short amino-acid sequences-arginine-glycine-aspartic acid (RGD). Peptides that contain RGD sequence can compete with RGD containing insoluble matrix proteins for binding to the integrin receptor and thus prevent the downstream signaling pathway. Increasing evidence supports that ß1-integrin/focal adhesion kinase (FAK)/Akt signal pathway plays an important role in fibrogenesis and scar formation in different tissues. In consideration of advantages of peptide hydrogel, that is well biocompatibility, gel state, degradability, good drug loading, we designed and fabricated an RGD peptide hydrogel, and hypothesized that it could inhibit the expression of ß1-integrin, FAK, and Akt in Tenon's capsule fibroblasts. Rheology results showed that 1% wt Fmoc-FFGGRGD peptide solution could self-assemble into hydrogel. Western blot analysis revealed that there were statistical differences between control group and 1% wt group in ß1-integrin/ß-actin, FAK/ß-actin, Akt/ß-actin respectively (*p < .05). The relative mRNA expression of ß1-integrin, FAK, Akt in control group and 1% wt group were also statistically different respectively (*p < .05). We proved that 1% wt Fmoc-FFGGRGD self-assembly peptide hydrogel could inhibit the expression of ß1-integrin, FAK and Akt in Tenon's capsule fibroblasts. It is a promising way to solve scar formation of glaucoma filter channel.
Subject(s)
Fibroblasts/metabolism , Focal Adhesion Kinase 1/biosynthesis , Gene Expression Regulation/drug effects , Hydrogels , Integrin beta1/biosynthesis , Oligopeptides , Proto-Oncogene Proteins c-akt/biosynthesis , Tendons/metabolism , Animals , Hydrogels/chemistry , Hydrogels/pharmacology , Oligopeptides/chemistry , Oligopeptides/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
The continuous rise in relapse rate and mortality for multiple myeloma (MM) demands an effective treatment option. The microRNAs are emerging nowadays for their promising therapeutic potential. Earlier, we reported involvement of Versican (VCAN) in myeloma pathogenesis which could be inhibited by miR-144 and miR-199 in stroma. However, there is dearth of literature showcasing the direct effect of these miRs in association with VCAN in MM. Expression of miR-144 and miR-199 was determined in myeloma cell lines (RPMI8226 & U266). These miRs were inhibited by small oligos to elucidate changes in expression of VCAN along with variation in parameters such as proliferation, apoptosis, migration and invasion in vitro. Moreover, effect on certain downstream signaling cascades was also evaluated. Lastly, interaction of miRs with VCAN was assessed by reporter luciferase assay. microRNAs expression were found significantly elevated in myeloma cells in comparison to stromal levels reported previously. The antagomirs-mediated inhibition of miR-144 and miR-199 significantly induced VCAN expression in myeloma cells along with alteration in myeloma-associated parameters in favor of myeloma pathogenesis with downstream activation of FAK/STAT3 signaling. Interestingly, miR-144 found to have direct binding with VCAN 3' UTR while miR-199 possess different mechanism. The inhibition of miR-144 and miR-199 contributed in myeloma progression via upregulation of VCAN in vitro affirming the translational significance of VCAN and associated microRNAs in MM. These miRs, hence might be employed for targeting VCAN and might emerge as an effective therapy for the better outcome of MM in clinical settings in future.
Subject(s)
Focal Adhesion Kinase 1/biosynthesis , Gene Expression Regulation, Neoplastic , MicroRNAs/metabolism , Multiple Myeloma/metabolism , Neoplasm Proteins/biosynthesis , RNA, Neoplasm/biosynthesis , STAT3 Transcription Factor/biosynthesis , Signal Transduction , Up-Regulation , Versicans/biosynthesis , Cell Line, Tumor , Focal Adhesion Kinase 1/genetics , Humans , MicroRNAs/genetics , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Neoplasm Proteins/genetics , RNA, Neoplasm/genetics , STAT3 Transcription Factor/genetics , Versicans/geneticsABSTRACT
Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that mediates multiple cellular functions such as survival, invasion, and migration. FAK has been found to be over-expressed in various human cancers, including melanoma. FAK molecule has several tyrosine, serine, and threonine phosphorylation sites which have an important regulatory role. Tyrosine phosphorylation of FAK has been extensively studied, however little is known about the role of serine phosphorylation. We sought to examine the frequency and extent of serine-910 phosphorylated FAK (P-FAKSer910) expression in a spectrum of melanocytic proliferations as well as it's correlation with other histopathologic predictors and its effect on patient's survival. Clinicopathologic features and immunohistochemical expression of P-FAKSer910 were evaluated in 147 melanocytic proliferations: 73 primary melanoma (PM), 19 metastatic melanoma (MetM), 2 melanoma in situ, and 53 melanocytic nevi (MN). Higher cytoplasmic intensity predicted better overall survival (OS) in PM (χ=5.69; P=0.034) and was associated with 48% decrease in death risk (HR, 0.52; 95% CI, 0.28-0.95; P=0.036). In contrast, increased nuclear intensity was significantly associated with better disease-free survival (DFS) when stratified by tumor stage Log-rank test, trend of survival (χ=5.83, P=0.015) and independently on multivariate analysis when subcategorized into 3-tier categories (HR, 0.43; 95% CI, 0.18-0.98; P=0.045). Also, Clark's level and tumor-infiltrating lymphocytes (TILS) were independent predictors of DFS. Cytoplasmic intensity correlated inversely with American Joint Commission on Cancer stage in primary melanoma cases as well with vascularity in both primary and metastatic melanoma. Nuclear intensity independently correlated negatively with angioinvasion and TILS when subcategorized to 3 tier system. We found American Joint Commission on Cancer tumor stage, Clark's level, depth, ulceration, TILS, mitosis, angioinvasion, and tumor vascularity predictors of both DFS and OS. There was no significant difference in cytoplasmic or nuclear expression among the major categories of melanocytic proliferation. In this pilot immunohistochemistry-based study, we found P-FAKSer910 expression in melanoma by cytoplasmic intensity to correlate with better OS while nuclear intensity correlated with better DFS.
Subject(s)
Focal Adhesion Kinase 1/biosynthesis , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Melanoma , Skin Neoplasms , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Cohort Studies , Disease-Free Survival , Female , Focal Adhesion Kinase 1/genetics , Follow-Up Studies , Humans , Male , Melanoma/enzymology , Melanoma/genetics , Melanoma/mortality , Melanoma/pathology , Middle Aged , Phosphorylation , Serine/genetics , Serine/metabolism , Skin Neoplasms/enzymology , Skin Neoplasms/genetics , Skin Neoplasms/mortality , Skin Neoplasms/pathology , Survival RateABSTRACT
It is well recognized that the interaction between stem cells and their physical microenvironment plays a fundamental role in controlling cell behaviors and directing lineage commitment, which eventually determines cell fate. Any change in the physical characteristics of the extracellular matrix in terms of topography, geometry, and stiffness has a strong effect on this interaction. Nevertheless, the precise biomechanism that regulates the responses of stem cells to the biophysical properties of substrates is not fully understood. In this study, we generated a series of polydimethylsiloxane (PDMS) substrates with different stiffness properties and explored the whole process involved in the determination of osteogenic lineage in stem cells from the human apical papilla (hSCAPs) in response to substrate stiffness. We first found that the hSCAPs responded to different substrate stiffnesses by changing their cell morphologies and cytoskeletons (via changes in α-tubulin and ß-tubulin in microtubules and F-actin in microfilaments). We then found that the hSCAPs secreted more fibronectin in response to the stiffer substrates. We next found that fibronectin interacted with focal adhesion kinase (FAK) and paxillin in the FA plaques, and moreover, the expressions of FAK and paxillin were enhanced as the substrate stiffness increased. We further found that FAK and paxillin directly interacted with ß-catenin. Furthermore, the accumulation of ß-catenin in the nuclear region was strengthened as the substrate stiffness increased. We finally detected the changes of Lef-1 and TCF-1 in osteogenic-induced hSCAPs and found that their expressions were enhanced as the substrate stiffness increased. Lef-1 and TCF-1, as the transcriptional factors in the nucleus, potentially bound to the promoter region of Runx2 and might ultimately determine the osteogenic lineage in hSCAPs. These results indicate the important effect of stiffness in the microenvironment on the osteogenic lineage of hSCAPs and increase the understanding of the biomechanisms involved in the molecular signal cascade during mechanosensing, mechanotransduction, and stem cell differentiation, which will be useful in the biological fields of cell-matrix/cell-cell interactions and tissue engineering/regenerative medicine.
Subject(s)
Cell Differentiation , Extracellular Matrix/chemistry , Mechanotransduction, Cellular , Osteogenesis , Stem Cells/metabolism , Cytoskeleton/metabolism , Fibronectins/biosynthesis , Focal Adhesion Kinase 1/biosynthesis , Humans , Stem Cells/cytologyABSTRACT
Objective- Increasing evidence shows that resveratrol has antiatherogenic effects, but its underlying mechanisms are unknown. Thus, we evaluated the molecular mechanisms underlying the antiatherogenic effect of resveratrol. Approach and Results- Using the previously established mouse atherosclerosis model of partial ligation of the left carotid artery, we evaluated the role of resveratrol in antiatherosclerosis. We attempted to determine the mechanisms associated with focal adhesions using vascular endothelial cells. The results showed that resveratrol stimulated focal adhesion kinase cleavage via resveratrol-increased expression of lactoferrin in endothelial cells. Furthermore, we found that an N-terminal focal adhesion kinase fragment cleaved by resveratrol contained the FERM (band 4.1, ezrin, radixin, and moesin)-kinase domain. Furthermore, resveratrol inhibited lipopolysaccharide-stimulated adhesion of THP-1 human monocytes by decreased expression of ICAM-1 (intercellular adhesion molecule-1). A decreased ICAM-1 level was also observed in the left carotid artery of mice treated with resveratrol. To understand the relationship between resveratrol-induced antiinflammation and focal adhesion disruption, endothelial cells were transfected with FERM-kinase. Ectopically expressed FERM-kinase, the resveratrol-cleaved focal adhesion kinase fragment, was found in the nuclear fraction and inhibited the transcription level of icam-1 via the Nrf2 (nuclear factor erythroid 2-related factor 2)-antioxidant response element complex. Finally, ectopically expressed FERM-kinase blocked tumor necrosis factor-α- or IL- (interleukin) stimulated monocytic binding to endothelial cells. Conclusions- Our results show that resveratrol inhibits the expression of ICAM-1 via transcriptional regulation of the FERM-kinase and Nrf2 interaction, thereby blocking monocyte adhesion. These suppressive effects on the inflammatory mechanism suggest that resveratrol delayed the onset of atherosclerosis.
Subject(s)
Atherosclerosis/prevention & control , Cell Adhesion/drug effects , Monocytes/drug effects , Resveratrol/pharmacology , Active Transport, Cell Nucleus , Animals , Atherosclerosis/metabolism , Atherosclerosis/pathology , Carotid Arteries/drug effects , Carotid Arteries/metabolism , Carotid Stenosis , Disease Models, Animal , Down-Regulation/drug effects , Endothelium, Vascular/metabolism , Enzyme Induction , Focal Adhesion Kinase 1/biosynthesis , Focal Adhesion Kinase 1/metabolism , Inflammation , Lactoferrin/metabolism , Ligation , Mice , Mice, Knockout, ApoE , Monocytes/metabolism , NF-E2-Related Factor 2/metabolism , Random Allocation , Transcription, GeneticABSTRACT
BACKGROUND: Increasing evidence supports the association of microRNA with tumor occurrence and development. However, the expression of miR-6875-3p and its role in cell proliferation, invasion and metastasis in hepatocellular carcinoma (HCC) remains elusive. METHODS: The expression of miR-6875-3p and BTG2 in HCC tissues and cell lines was detected by using in situ hybridization, immunohistochemistry and qRT-PCR, respectively. A western blot assay, qRT-PCR and Luciferase reporter assay were employed to study the interaction between miR-6875-3p and BTG2. Cell proliferation invasion and metastasis were measured by MTT, transwell and matrigel analyses in vitro. In vivo, tumorigenicity and metastasis assays were performed in nude mice. RESULTS: We found that miR-6875-3p were elevated expressed in HCC tissues and cell lines, and negatively correlated with BTG2 expression, while positively correlated with tumor staging, size, degree of differentiation, and vascular invasion of HCC. Moreover, in vitro and in vivo assays showed that miR-6875-3p regulates EMT and improve the proliferation, metastasis and stem cell-like properties of HCC cells. BTG2 was identified as a direct and functional target of miR-6875-3p via the 3'-UTR of BTG2. We also confirmed that miR-6875-3p plays its biological functions via the BTG2/FAK/Akt pathway. CONCLUSION: Our study provides evidence that high expression of miR-6875-3p can promote tumorigenesis of HCC in vitro and in vivo, so as to function as a novel oncogene in HCC. In mechanism, we found that miR-6875-3p plays its biological functions via the BTG2/FAK/Akt pathway.
Subject(s)
Carcinoma, Hepatocellular/genetics , Focal Adhesion Kinase 1/metabolism , Immediate-Early Proteins/metabolism , Liver Neoplasms/genetics , MicroRNAs/biosynthesis , Proto-Oncogene Proteins c-akt/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Proliferation/physiology , Down-Regulation , Focal Adhesion Kinase 1/biosynthesis , Focal Adhesion Kinase 1/genetics , Hep G2 Cells , Heterografts , Humans , Immediate-Early Proteins/biosynthesis , Immediate-Early Proteins/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/genetics , MicroRNAs/metabolism , Middle Aged , Neoplasm Invasiveness , Neoplasm Metastasis , Signal Transduction , Transfection , Tumor Suppressor Proteins/biosynthesis , Tumor Suppressor Proteins/geneticsABSTRACT
Titanium (>99.6% purity) and its anodically oxidized modifications, with and without deposited silver nanodendrites regarding its biocompatibility were evaluated. In human gingival fibroblasts and osteoblast cell lines grown on tested samples, the level of expression of genes encoding αV (ITGAV) and ß1 (ITGB1) integrin subunits also genes encoding focal adhesion (FAK) and extracellular-signal regulated (ERK) kinases was assessed. For this purpose, the qualitative and quantitative PCR technique was used. The expression of studied genes was dependent on the origin of cell lines and the type of evaluated material. The high expression of PBGD and ITGAV genes in fibroblasts grown on the surface of anodically modified titanium with deposited silver nanodendrites indicates potentially high biocompatibility of these samples for soft tissue cells. The high expression of the ITGB1 and ERK1 genes and the enhanced expression of the FAK gene in osteoblasts cells grown on the tested material was also observed. Summarizing, the nanocrystalline Ti modified with silver deposits showed higher biocompatibility in comparison with the conventional pure Ti samples.
Subject(s)
Coated Materials, Biocompatible , Dendrimers , Fibroblasts/metabolism , Materials Testing , Metal Nanoparticles/chemistry , Osteoblasts/metabolism , Silver , Titanium , Cell Line, Tumor , Coated Materials, Biocompatible/chemistry , Coated Materials, Biocompatible/pharmacology , Dendrimers/chemistry , Dendrimers/pharmacology , Fibroblasts/cytology , Focal Adhesion Kinase 1/biosynthesis , Gene Expression Regulation/drug effects , Humans , Integrin alpha5/biosynthesis , Integrin beta1/biosynthesis , Mitogen-Activated Protein Kinase 3/biosynthesis , Osteoblasts/cytology , Silver/chemistry , Silver/pharmacology , Titanium/chemistry , Titanium/pharmacologyABSTRACT
OBJECTIVE: SCCRO/DCUN1D1/DCN1 (squamous cell carcinoma-related oncogene/defective in cullin neddylation 1 domain containing 1/defective in cullin neddylation) is considered as an oncogene, but its role in the prostate cancer (PC) is still not clear. The current study aims to investigate the expression of SCCRO in PC tumor tissues, further its clinical significance, and proliferation inhibiting effect on PC cells in vitro. PATIENTS AND METHODS: RT-PCR was used to detect the expression of SCCRO in PC tissue and corresponding adjacent normal tissues from 160 cases, and its relationship with clinical pathological characteristics was analyzed. Small interfering RNA (siRNA) expression plasmid targeting SCCRO gene was constructed and transferred into PC cell line Lncap. The effect on proliferation was observed by CCK8 assay, and its influence on invasion and migration of Lncap cells was studied by Transwell Matrigel assay after SCCRO gene was silenced. The expression of focal adhesion kinase (FAK) and matrix metalloproteinase-2 (MMP-2) influenced by SCCRO silencing were detected by Western blot. RESULTS: mRNA expression of SCCRO protein increased significantly in cancer tissues compared to adjacent normal tissue, especially for T3+T4, N+, and III+IV patients (p<0.05). SCCRO expression was an independent prognostic factor (p<0.05). After SCCRO gene was knocked down by siRNA, the SCCRO protein level decreased 78.4% in the siRNA-3 group. By CCK8 assay, knocking down SCCRO in Lncap significantly reduced the cell proliferation, as well as its migration and invasion capability compared to siRNA-control group (p<0.01) by transwell invasion and migration assay. The expression of FAK and MMP-2 also reduced in siRNA-3 group compared to siRNA control group (p<0.01). CONCLUSIONS: SCCRO is associated with progression and prognosis of PC. After SCCRO gene was transferred, the growth of Lncap cells was inhibited, and ability of invasion and migration decreased by reducing the expression of FAK and MMP-2. SCCRO has potential to become a new target for the treatment of PC.
Subject(s)
Cell Proliferation , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins/genetics , Cell Line, Tumor , Focal Adhesion Kinase 1/biosynthesis , Focal Adhesion Kinase 1/genetics , Gene Silencing , Humans , Intracellular Signaling Peptides and Proteins , Male , Matrix Metalloproteinase 2/biosynthesis , Matrix Metalloproteinase 2/genetics , Proteins , RNA, Small InterferingABSTRACT
BACKGROUND: Neuroblastoma (NB) often presents with metastatic disease and poor survival. The need for new prognostic markers remains invaluable. The FAK-Src-Paxillin protein system is associated with aggressive phenotype in adult malignancies but is largely unexplored in pediatric NB. OBJECTIVE: To assess FAK-Src-Paxillin protein expression in human NB cell lines and clinical cytology material and to delineate its association with survival. DESIGN/METHODS: Western blot and immunohistochemistry were applied for FAK-Src-Paxillin expression in NB cell lines and 23 human cytology specimens, respectively. Protein expression in human clinical samples was correlated with clinicopathological parameters, MYCN amplification and survival. RESULTS: FAK, Src and Paxillin proteins are expressed in human NB cells lines, and can be detected in clinical cytology specimens from NB patients, (59%, 32% and 33% respectively). Simultaneous FAK-Src-Paxillin expression was noted in 30% of NB patients. Children with concomitant positivity FAK, Src, and Paxillin tumors, as well as MYCN amplification, had increased mortality compared to those without. CONCLUSIONS: FAK-Src-Paxillin system is a marker of unfavorable prognosis for human NB patients but also a promising therapeutic target.
Subject(s)
Biomarkers, Tumor/biosynthesis , Focal Adhesion Kinase 1/biosynthesis , Gene Expression Regulation, Neoplastic , Neuroblastoma , Paxillin/biosynthesis , Proto-Oncogene Proteins pp60(c-src)/biosynthesis , Animals , Child , Child, Preschool , Disease-Free Survival , Female , Humans , Infant , K562 Cells , Male , Mice , N-Myc Proto-Oncogene Protein/biosynthesis , NIH 3T3 Cells , Neuroblastoma/metabolism , Neuroblastoma/mortality , Neuroblastoma/pathology , Survival RateABSTRACT
The study was conducted to evaluate the effects of platelet-rich plasma (PRP), supernatant of PRP (SPRP) obtained by centrifugation, and supernatant of activated PRP (SActi-PRP) obtained by Ca2+ solution-treated PRP on collagen biosynthesis, prolidase activity, and ß1-integrin signaling in cultured human skin fibroblasts. Incubation of fibroblasts with 5% PRP for 24 h contributed to ~5-fold increase in collagen biosynthesis compared to the control. In the cells treated with 5% of SPRP or SActi-PRP, collagen biosynthesis showed a 3-fold increase of the control. PRP, SPRP, and SActi-PRP stimulated prolidase activity similar to collagen biosynthesis. Collagen biosynthesis and prolidase activity are regulated by ß1-integrin receptor signaling. Incubation of fibroblasts with PRP for 24 h contributed to a dose-dependent increase in the expression of ß1-integrin receptor, while SActi-PRP increased the process to a much lower extent. SPRP had no effect on the ß1-integrin receptor expression. All the studied fractions of blood increased the expression of FAK as well as the expression of phosphorylated MAP-kinases. However, PRP was found to be the most effective stimulator of expression of these particular kinases. These studies suggest that a complex of factors, including growth factors, adhesion molecules, and prolidase contained in PRP, all evoke growth and collagen-promoting activities in human dermal fibroblasts.
Subject(s)
Collagen/biosynthesis , Dipeptidases/metabolism , Fibroblasts/metabolism , Integrin beta1/drug effects , Platelet-Rich Plasma/chemistry , Skin/metabolism , Cells, Cultured , DNA/biosynthesis , Dose-Response Relationship, Drug , Fibroblasts/drug effects , Fibroblasts/enzymology , Focal Adhesion Kinase 1/biosynthesis , Focal Adhesion Kinase 1/genetics , Humans , Mitogen-Activated Protein Kinases/metabolism , Signal Transduction/drug effects , Skin/cytology , Skin/drug effectsABSTRACT
An improved expression protocol is proposed for amino acid type-specific [13C], [15N]-isotope labeling of proteins in baculovirus-infected (BV) insect cell cultures. This new protocol modifies the methods published by Gossert et al. (J Biomol NMR 51(4):449-456, 2011) and provides efficient incorporation of isotopically labeled amino acids, with similar yields per L versus unlabeled expression in rich media. Gossert et al. identified the presence of unlabeled amino acids in the yeastolate of the growth medium as a major limitation in isotope labeling using BV-infected insect cells. By reducing the amount of yeastolate in the growth medium ten-fold, a significant improvement in labeling efficiency was demonstrated, while maintaining good protein expression yield. We report an alternate approach to improve isotope labeling efficiency using BV-infected insect cells namely by replacing the yeast extracts in the medium with dialyzed yeast extracts to reduce the amount of low molecular weight peptides and amino acids. We report the residual levels of amino acids in various media formulations and the amino acid consumption during fermentation, as determined by NMR. While direct replacement of yeastolate with dialyzed yeastolate delivered moderately lower isotope labeling efficiencies compared to the use of ten-fold diluted undialized yeastolate, we show that the use of dialyzed yeastolate combined with a ten-fold dilution delivered enhanced isotope labeling efficiency and at least a comparable level of protein expression yield, all at a scale which economizes use of these costly reagents.
Subject(s)
Isotope Labeling/methods , Amino Acids/analysis , Amino Acids/chemistry , Animals , Baculoviridae , CD4 Antigens/biosynthesis , CD4 Antigens/chemistry , CD4 Antigens/isolation & purification , Carbon Isotopes , Culture Media/analysis , Culture Media/chemistry , Focal Adhesion Kinase 1/biosynthesis , Focal Adhesion Kinase 1/chemistry , Focal Adhesion Kinase 1/isolation & purification , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular , Protein Biosynthesis , Sf9 Cells , SpodopteraABSTRACT
Furanodiene is one of the major bioactive components isolated from the natural product of the plant, Curcuma wenyujin Y.H. Chen et C. Ling. Furanodiene has been found to exert anticancer effects in various types of cancer cell lines, as well as exhibit antimetastatic activities. However, the antimetastatic capacity of furanodiene in combination with the common chemotherapy drug doxorubicin has not been investigated. We found that doxorubicin at a non-toxic concentration induced cell migration and cell invasion in highly metastatic breast cancer cells. Combinational treatments with furanodiene and doxorubicin blocked the invasion and migration of MDA-MB-231 breast cancer cells in vitro. We also clarified the effects of the combination on the signaling pathways involved in migration, invasion, and cytoskeletal organization. When combined with doxorubicin, furanodiene downregulated the expression of integrin αV and ß-catenin and inhibited the phosphorylation of paxillin, Src, focal adhesion kinase (FAK), p85, and Akt. Moreover, combinational treatments also resulted in a decrease in matrix metalloproteinase-9 (MMP-9). Further study demonstrated that the co-treatments with furanodiene did not significantly alter the effects of doxorubicin on the tubulin cytoskeleton, represented by no influence on the expression levels of RhoA, Cdc42, N-WASP, and α/ß tubulin. These observations indicate that furanodiene is a potential agent that may be utilized to improve the anticancer efficacy of doxorubicin and overcome the risk of chemotherapy in highly metastatic breast cancer.
Subject(s)
Breast Neoplasms/drug therapy , Curcuma/chemistry , Doxorubicin/administration & dosage , Furans/administration & dosage , Heterocyclic Compounds, 2-Ring/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Female , Focal Adhesion Kinase 1/biosynthesis , Furans/chemistry , Gene Expression Regulation, Neoplastic/drug effects , Heterocyclic Compounds, 2-Ring/chemistry , Humans , Matrix Metalloproteinase 9/biosynthesis , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Neoplasm Proteins/biosynthesis , Plant Extracts/chemistry , Signal Transduction/drug effectsABSTRACT
PURPOSE: Gemcitabine-based chemotherapy remains one of the standards in management of metastatic breast cancer. However, intrinsic and acquired resistance to gemcitabine inevitably occurs. The aims of this study were to assess the efficacy of the combination of src inhibition and gemcitabine in gemcitabine-resistant breast cancer cells. METHODS AND RESULTS: By using colony formation, sphere forming, flow cytometry, cell counting kit-8 and transwell assays, 231/GEM-res (gemcitabine-resistant) cell line, which was 10 times more resistant, was shown to have elevated drug tolerance, enhanced proliferative and self-renewal abilities, compared with its parental cells. Inhibition of src by both saracatinib (AZD0530) and siRNA could partially reverse gemcitabine resistance and attenuate resistance-associated anti-apoptosis, migration and stem cell capacities. In addition, the combination of src inhibition and gemcitabine had synergistic antitumor effects. Western blot analysis revealed up-regulation of pro-apoptotic protein BAX, along with the down-regulation of anti-apoptotic proteins (BCL-XL, Survivin), migration associated proteins (p-FAK, MMP-3) and cancer stem cell (CSC) markers (CD44, Oct-4), which was probably mediated by AKT/c-Jun pathway. CONCLUSION: In highly gemcitabine-resistant 231 cells, src inhibition can synergize with gemcitabine, reverse drug resistance, inhibit tumor growth/metastasis/stemness of cancer stem cells, possibly via the AKT/c-Jun pathway.
Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Benzodioxoles/pharmacology , Deoxycytidine/analogs & derivatives , Drug Resistance, Neoplasm/genetics , Enzyme Inhibitors/pharmacology , JNK Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Quinazolines/pharmacology , Triple Negative Breast Neoplasms/drug therapy , src-Family Kinases/antagonists & inhibitors , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Deoxycytidine/pharmacology , Drug Synergism , Female , Focal Adhesion Kinase 1/biosynthesis , Gene Expression Regulation, Neoplastic , Humans , Hyaluronan Receptors/biosynthesis , Inhibitor of Apoptosis Proteins/biosynthesis , Matrix Metalloproteinase 3/biosynthesis , Octamer Transcription Factor-3/biosynthesis , RNA Interference , RNA, Small Interfering/genetics , Survivin , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology , Tumor Cells, Cultured , bcl-2-Associated X Protein/biosynthesis , bcl-X Protein/biosynthesis , src-Family Kinases/genetics , GemcitabineABSTRACT
UNLABELLED: There are limited therapy options for advanced thyroid cancer, including papillary and anaplastic thyroid cancer (PTC and ATC). Focal adhesion kinase (FAK) regulates cell signaling by functioning as a scaffold and kinase. Previously, we demonstrated that FAK is overexpressed and activated in thyroid cancer cells and human PTC clinical specimens. However, it remains unclear whether patients with advanced thyroid cancer will benefit from FAK inhibition. Therefore, the dual functions of FAK in mediating protumorigenic processes and thyroid tumorigenesis were investigated. Evidence here shows that FAK expression predominantly regulates thyroid cancer cell growth, viability, and anchorage-independent growth. FAK inhibition, with PF-562,271 treatment, modestly reduced tumor volumes, while FAK depletion, through shRNA knockdown, significantly reduced tumor volumes in vivo A role for FAK expression in tumor establishment was demonstrated in a model of PTC, where FAK knockdown tumors did not develop. FAK depletion also led to a significant decrease in overall metastatic burden. Interestingly, pretreatment with a FAK inhibitor resulted in a paradoxical increase in metastasis in a model of ATC, but decreased metastasis in a model of PTC. These data provide the first evidence that FAK expression is critical for the regulation of thyroid tumorigenic functions. IMPLICATIONS: This study demonstrates that FAK expression, but not kinase activity alone, predominantly mediates thyroid tumor growth and metastasis, indicating that targeting the scaffolding function(s) of FAK may be an important therapeutic strategy for advanced thyroid cancer, as well as other FAK-dependent tumors. Mol Cancer Res; 14(9); 869-82. ©2016 AACR.
Subject(s)
Focal Adhesion Kinase 1/biosynthesis , Thyroid Neoplasms/enzymology , Thyroid Neoplasms/pathology , Animals , Carcinogenesis/metabolism , Carcinogenesis/pathology , Cell Growth Processes/physiology , Cell Line, Tumor , Female , Focal Adhesion Kinase 1/antagonists & inhibitors , Focal Adhesion Kinase 1/metabolism , Heterografts , Humans , Mice , Mice, Nude , Neoplasm Metastasis , Signal Transduction , Thyroid Neoplasms/geneticsABSTRACT
Gastric carcinoma is the third leading cause of lethal cancer worldwide. Previous studies showed that Notch1 receptor intracellular domain (N1IC), the activated form of Notch1 receptor, promotes gastric cancer progression. It has been demonstrated that a significant cross-talk interplays between Notch pathways and microRNAs (miRNAs) in controlling tumorigenesis. This study identified an intronic microRNA-151 (miR-151), which consists of two mature miRNAs, miR-151-3p and miR-151-5p, as a Notch1 receptor-induced miRNA in gastric cancer cells. Activation of Notch1 pathway enhanced expressions of miR-151 and its host gene, focal adhesion kinase (FAK), in gastric cancer cells. The levels of miR-151 in gastric cancer samples were higher than those of adjacent non-tumor samples. Activated Notch1 pathway induced CBF1-dependent FAK promoter activity. The ectopic expression of miR-151 promoted growth and progression of SC-M1 gastric cancer cells including cell viability and colony formation, migration, and invasion abilities. Activated Notch1 pathway could augment progression of gastric cancer cells through miR-151-5p and FAK. The mRNA levels of pluripotency genes, Nanog and SOX-2, tumorsphere formation ability, tumor growth, and lung metastasis of SC-M1 cells were elevated by activated Notch1 pathway through miR-151-5p. Furthermore, miR-151-5p could target 3'-untranslated region (3'-UTR) of p53 mRNA and down-regulate p53 level in SC-M1 cells. Mechanistically, Notch1/miR-151-5p axis contributed to progression of SC-M1 cells through down-regulation of p53 which in turn repressed FAK promoter activity. Taken together, these results suggest that Notch1 pathway and miR-151-5p interplay with p53 in a reciprocal regulation loop in controlling gastric carcinogenesis.
Subject(s)
MicroRNAs/metabolism , Receptor, Notch1/metabolism , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Animals , Cell Line, Tumor , Disease Progression , Focal Adhesion Kinase 1/biosynthesis , Focal Adhesion Kinase 1/genetics , HEK293 Cells , Heterografts , Humans , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , K562 Cells , Mice , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/genetics , Promoter Regions, Genetic , Receptor, Notch1/genetics , Stomach Neoplasms/pathology , Transfection , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
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.
Subject(s)
Colonic Neoplasms/genetics , Focal Adhesion Kinase 1/biosynthesis , Matrix Metalloproteinase 2/biosynthesis , Matrix Metalloproteinase 9/biosynthesis , RNA, Long Noncoding/genetics , Caco-2 Cells , Cell Proliferation/genetics , Cell Survival/genetics , Colonic Neoplasms/pathology , Focal Adhesion Kinase 1/genetics , Gene Expression Regulation, Neoplastic , Humans , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/genetics , Neoplasm Invasiveness/geneticsABSTRACT
Directed cell migration is a crucial orchestrated process in embryonic development, wound healing, and immune response. The underlying substrate can provide physical and/or chemical cues that promote directed cell migration. Here, using electrospinning we developed substrates of aligned poly(lactic-co-glycolic acid) nanofibres to study the influence of glial cells on endothelial cells (ECs) in a 3-dimensional (3D) co-culture model. ECs build blood vessels and regulate their plasticity in coordination with neurons. Likewise, neurons construct nerves and regulate their circuits in coordination with ECs. In our model, the neuro-vascular cross-talk was assessed using a direct co-culture model of human umbilical vein endothelial cells (HUVECs) and rat Schwann cells (rSCs). The effect of rSCs on ECs behavior was demonstrated by earlier and higher velocity values and genetic expression profiles different of those of HUVECs when seeded alone. We observed 2 different gene expression trends in the co-culture models: (i) a later gene expression of angiogenic factors, such as interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), and (ii) an higher gene expression of genes involved in actin filaments rearrangement, such as focal adhesion kinase (FAK), Mitogen-activated protein kinase-activated protein kinase 13 (MAPKAPK13), Vinculin (VCL), and Profilin (PROF). These results suggested that the higher ECs migration is mainly due to proteins involved in the actin filaments rearrangement and in the directed cell migration rather than the effect of angiogenic factors. This co-culture model provides an approach to enlighten the neurovascular interactions, with particular focus on endothelial cell migration.
Subject(s)
Cell Movement/genetics , Endothelial Cells/metabolism , Nanofibers/chemistry , Schwann Cells/metabolism , Animals , Coculture Techniques , Endothelial Cells/cytology , Focal Adhesion Kinase 1/biosynthesis , Focal Adhesion Protein-Tyrosine Kinases/biosynthesis , Gene Expression Regulation , Human Umbilical Vein Endothelial Cells , Humans , Interleukin-8/biosynthesis , Lactic Acid/chemistry , Lactic Acid/pharmacology , Paxillin/biosynthesis , Polyglycolic Acid/chemistry , Polyglycolic Acid/pharmacology , Polylactic Acid-Polyglycolic Acid Copolymer , Rats , Schwann Cells/cytology , Vascular Endothelial Growth Factor A/biosynthesis , Vinculin/biosynthesisABSTRACT
Skeletal muscle has a remarkable ability to respond to different physical stresses. Loading muscle through exercise, either anaerobic or aerobic, can lead to increases in muscle size and function while, conversely, the absence of muscle loading stimulates rapid decreases in size and function. A principal mediator of this load-induced change is focal adhesion kinase (FAK), a downstream non-receptor tyrosine kinase that translates the cytoskeletal stress and strain signals transmitted across the cytoplasmic membrane by integrins to activate multiple anti-apoptotic and cell growth pathways. Changes in FAK expression and phosphorylation have been found to correlate to specific developmental states in myoblast differentiation, muscle fiber formation and muscle size in response to loading and unloading. With the capability to regulate costamere formation, hypertrophy and glucose metabolism, FAK is a molecule with diverse functions that are important in regulating muscle cell health.
Subject(s)
Cell Differentiation/physiology , Focal Adhesion Kinase 1/biosynthesis , Gene Expression Regulation, Enzymologic/physiology , Muscle, Skeletal/enzymology , Signal Transduction/physiology , Stress, Physiological/physiology , Animals , Humans , Muscle, Skeletal/cytology , Myoblasts, Skeletal/cytology , Myoblasts, Skeletal/enzymologyABSTRACT
Colorectal cancer (CRC) metastasis dissemination to secondary sites represents the critical point for the patient's survival. The microenvironment is crucial to cancer progression, influencing tumour cell behaviour by modulating the expression and activation of molecules such as integrins, the cell-extracellular matrix interacting proteins participating in different steps of the tumour metastatic process. In this work, we investigated the role of α5ß1 integrin and how the microenvironment influences this adhesion molecule, in a model of colon cancer progression to the liver. The culture medium conditioned by the IHH hepatic cell line, and the extracellular matrix (ECM) proteins, modulate the activation of α5ß1 integrin in the colon cancer cell line HCT-116, and drives FAK phosphorylation during the process of cell adhesion to fibronectin, one of the main components of liver ECM. In these conditions, α5ß1 modulates the expression/activity of another integrin, α2ß1, involved in the cell adhesion to collagen I. These results suggest that α5ß1 integrin holds a leading role in HCT-116 colorectal cancer cells adhesion to the ECM through the modulation of the intracellular focal adhesion kinase FAK and the α2ß1 integrin activity. The driving role of the tumour microenvironment on CRC dissemination, here detected, and described, strengthens and adds new value to the concept that α5ß1 integrin can be an appropriate and relevant therapeutic target for the control of CRC metastases.