Erucin, a natural isothiocyanate, exerts pro-angiogenic properties in cultured endothelial cells and reverts angiogenic impairment induced by high glucose.

Insufficient vessel maintenance adversely impacts patients in terms of tissue reperfusion following stroke or myocardial infarction, as well as during wound healing. Angiogenesis impairment is a feature typical of metabolic disorders acting at the cardiovascular level, such as diabetes. Therapeutic angiogenesis regulation offers promising clinical implications, and natural compounds as pro-angiogenic nutraceuticals hold valuable applications in regenerative medicine. By using cultured endothelial cells from human umbilical veins (HUVEC) we studied functional and molecular responses following exposure to erucin, a natural isothiocyanate derived from Brassicaceae plants and extracted from the seeds of rocket. Erucin (at nanomolar concentrations) promotes cell migration and tube formation, similar to vascular endothelial growth factor (VEGF), through mobilizing paxillin at endothelial edges. At the molecular level, erucin induces signaling pathways typical of angiogenesis activation, namely Ras, PI3K/AKT, and ERK1/2, leading to VEGF expression and triggering its autocrine production, as pharmacological inhibition of soluble VEGF and VEGFR2 dampens endothelial functions. Furthermore, erucin, alone and together with VEGF, preserves endothelial angiogenic functions under pathological conditions, such as those induced in HUVEC by high glucose (HG) exposure. Erucin emerges as a compelling candidate for therapeutic revascularization applications, showcasing promising prospects for natural compounds in regenerative medicine, particularly in addressing angiogenesis-related disorders.

Surface-induced phase separation of reconstituted nascent integrin clusters on lipid membranes.

Integrin adhesion complexes are essential membrane-associated cellular compartments for metazoan life. The formation of initial integrin adhesion complexes is a dynamic process involving focal adhesion proteins assembled at the integrin cytoplasmic tails and the inner leaflet of the plasma membrane. The weak multivalent protein interactions within the complex and with the plasma membrane suggest that liquid-liquid phase separation could play a role in the nascent adhesion assembly. Here, we report that solid-supported lipid membranes supplemented with phosphoinositides induce the phase separation of minimal integrin adhesion condensates composed of integrin ß1 tails, kindlin, talin, paxillin, and FAK at physiological ionic strengths and protein concentrations. We show that the presence of phosphoinositides is key to enriching kindlin and talin on the lipid membrane, which is necessary to further induce the phase separation of paxillin and FAK at the membrane. Our data demonstrate that lipid membrane surfaces set the local solvent conditions for steering the membrane-localized phase separation even in a regime where no condensate formation of proteins occurs in bulk solution.

[Lutein inhibits the adhesion, invasiveness and metastasis of human prostate cancer PC-3M cells].

OBJECTIVE: To explore the effects of lutein on the adhesion, invasiveness and metastasis of human prostate cancer PC-3M cells and its action mechanism. METHODS: We divided human prostate cancer PC-3M cells into a control, a low-dose lutein, a medium-dose lutein and a high-dose lutein group, and treated them with 0, 10, 20 and 40 µmol/L lutein, respectively. Then we examined the adhesion of the cells to matrix by cell adhesion assay and the changes in cell pseudopodia by Phalloidin staining, detected the expressions of paxillin, matrix metalloproteinase 2 (MMP-2), MMP-9, recombinant tissue inhibitors of metalloproteinase 1 (TIMP-1), E-cadherin, N-cadherin and vimentin by Western blot, determined the invasiveness and migration of the cells by scratch and Transwell assays, and observed their dynamic movement by high-intension imaging. RESULTS: Compared with the control, the lutein intervention groups showed significant reduction in the number of the cells adhered to matrix, the number of cell pseudopodia, the expressions of paxillin, MMP-2, MMP-9, N-cadherin and vimentin, the rates of migration, invasion and metastasis, and the distances of displacement and movement of the cells. However, the expressions of TIMP-1 and epithelial-mesenchymal transition-related E-cadherin were upregulated significantly. CONCLUSION: Lutein can inhibit cell adhesion, reduce the expressions of MMPs, and suppress cell invasion and migration by inhibiting the process of epithelial-mesenchymal transition.

Phyllanthus taxodiifolius Beille Disrupted N-cadherin, Vimentin, Paxillin and Actin Stress Fibers in Glioblastoma.

OBJECTIVE: Glioblastoma is the most aggressive and lethal brain tumor in adults with highly invasive properties. In this present study, we explored the effects of Phyllanthus taxodiifolius Beille extract on molecules known to be hallmarks of aggressive glioblastoma including N-cadherin and vimentin, mesenchymal markers, as well as paxillin, a major adaptor protein that regulates the linking of focal adhesions to the actin cytoskeleton. METHODS: P. taxodiifolius were air-dried, powdered and percolated with methanol, filtered, concentrated and lyophilized to yield a crude methanol extract. C6 glioblastoma cell line was used in this study. The expression of N-cadherin and vimentin, as well as the activation of paxillin was determined using Western blot analysis. The effect of the extract on focal adhesions and actin cytoskeleton were investigated using immunofluorescence staining and confocal imaging. RESULTS: In the presence of 40 µg/ml Phyllanthus taxodiifolius Beille extract, the expression of N-cadherin and vimentin were significantly decreased (p<0.001 and p<0.05, respectively). Activation of paxillin was also diminished as indicated by a reduction of phosphorylated-paxillin (p<0.01). Consequently, actin stress fibers in glioblastoma cells were abolished as evidenced by the decrease in focal adhesion (p<0.001) and stress fibers numbers (p<0.001). CONCLUSION: Our study demonstrates for the first time that P. taxodiifolius interferes with multiple key molecules related to pathological hallmarks of glioblastoma. These molecules are involved with cell contacts, focal adhesions, and the formation and stabilization of actin stress fibers, which are required for glioblastoma metastatic behavior. These results provide further evidence supporting the potential of P. taxodiifolius and its bioactive compounds as anti-cancer agents.

Hesperetin Inhibits TGF-ß1-Induced Migration and Invasion of Triple Negative Breast Cancer MDA-MB-231 Cells via Suppressing Fyn/Paxillin/RhoA Pathway.

Triple-negative breast cancer is an aggressive subtype of breast cancer with poor clinical outcomes and poor prognosis. Hesperetin is an active component extracted from Citrus fruits and Traditional Chinese Medicine has a wide range of pharmacological effects. Here, we assessed the anti-migration and anti-invasive effects and explored inhibitory mechanisms of hesperetin on metastasis of human triple negative breast cancer MDA-MB-231 cells. Cell viability experiments revealed that 200 µM hesperetin has a clear inhibitory effect on MDA-MB-231 cells. TGF-ß1 treatment induces apparent tumor progression in MDA-MB-231 cells including aberrant wound-healing and invasion ability, which is effectively suppressed by hesperetin co-treatment. Additionally, hesperetin inhibited the TGF-ß1-mediated actin stress fiber formation. Western blot results showed that hesperetin suppressed the TGF-ß1-mediated (i) activation of Fyn, (ii) phosphorylation of paxillin at Y31, Y88, and Y118 sites, (iii) the increased expression of RhoA, and (iv) activation of Rho-kinase. We demonstrated the increased interaction of Fyn with paxillin and RhoA protein in the TGF-ß1-induced metastasis of MDA-MB-231 cells. Small interfering RNA Fyn inhibited phosphorylation of paxillin (Y31) and activation of Rho-kinase induced by TGF-ß1. In conclusion, hesperetin has a significant inhibitory effect on migration and invasion of MDA-MB-231 cells induced by TGF-ß1, which might be attributed to inhibiting the Fyn/paxillin/RhoA pathway.

RHBDD2 overexpression promotes a chemoresistant and invasive phenotype to rectal cancer tumors via modulating UPR and focal adhesion genes.

The current standard of care for locally advanced rectal cancer (RC) is neoadjuvant radio-chemotherapy (NRC) with 5-fluorouracil (5Fu) as the main drug, followed by surgery and adjuvant chemotherapy. While a group of patients will achieve a pathological complete response, a significant percentage will not respond to the treatment. The Unfolding Protein Response (UPR) pathway is generally activated in tumors and results in resistance to radio-chemotherapy. We previously showed that RHBDD2 gene is overexpressed in the advanced stages of colorectal cancer (CRC) and that it could modulate the UPR pathway. Moreover, RHBDD2 expression is induced by 5Fu. In this study, we demonstrate that the overexpression of RHBDD2 in CACO2 cell line confers resistance to 5Fu, favors cell migration, adhesion and proliferation and has a profound impact on the expression of both, the UPR genes BiP, PERK and CHOP, and on the cell adhesion genes FAK and PXN. We also determined that RHBDD2 binds to BiP protein, the master UPR regulator. Finally, we confirmed that a high expression of RHBDD2 in RC tumors after NRC treatment is associated with the development of local or distant metastases. The collected evidence positions RHBDD2 as a promising prognostic biomarker to predict the response to neoadjuvant therapy in patients with RC.

Aspalathin-rich green Aspalathus linearis extract suppresses migration and invasion of human castration-resistant prostate cancer cells via inhibition of YAP signaling.

BACKGROUND: More than 80% of advanced prostate cancer (PCa) cases have bone metastasis, with a 5-year survival rate of 25%. Previously, we reported that GRT, a standardized, pharmaceutical-grade aspalathin-rich extract (12.78 g aspalathin/100 g extract), prepared from green rooibos produced from the leaves and fine stems of Aspalathus linearis, inhibits the proliferation of PCa cells, meriting this investigation to determine if GRT can suppress the migration and invasion of castration-resistant prostate cancer (CRPC) cells. PURPOSE: In the present study, we investigated whether GRT extract can interfere with the migration and invasion of human CRPC cells. METHODS: Transwell assays were used to explore the effects of GRT on the migration and invasion of CRPC cells. Micro-Western Array (MWA) and Western blot analysis were carried out to unravel the underlying molecular mechanism(s). RESULTS: Treatment with 25-100 µg/ml GRT suppressed the migration and invasion of LNCaP C4-2B and 22Rv1 CRPC cells. MWA and Western blot analysis indicated that GRT treatment suppressed the protein level of yes-associated protein (YAP), macrophage stimulating 1 protein (MST1), phospho-MST1/phospho-MST2 T183/T180, and paxillin, but increased the abundance of E-cadherin. Over-expression of YAP rescued the suppressive effects of GRT on migration and invasion of CRPC cells. Treatment with the major flavonoid of GRT - the C-glucosyl dihydrochalcone, aspalathin - at a concentration of 75-100 µg/ml also reduced the migration and invasion of CRPC cells, and the inhibition was partially rescued by YAP over-expression. CONCLUSIONS: GRT treatment suppresses the migration and invasion of CRPC cells via inhibition of YAP signaling and paxillin.

Cucurbitacin B suppresses metastasis mediated by reactive oxygen species (ROS) via focal adhesion kinase (FAK) in breast cancer MDA-MB-231 cells.

Metastasis is responsible for the majority of cancer-related deaths and prevention of metastasis remains a big challenge for cancer therapy. Cucurbitacin B (Cuc B) is a natural triterpenoid with potent anticancer activities while its effect on metastasis remains unclear. In the present study, the inhibitory effect and mechanisms of Cuc B on metastasis were investigated in MDA-MB-231 breast cancer cells. The cells were treated with or without Cuc B, and the cytotoxicity was determined by MTT assay. The effect of Cuc B on metastasis was evaluated with wound healing, transwell, and adhesion assays. Furthermore, the adhesion of cancer cells to endothelial cells was determined. The protein expression was determined by Western blotting. Cuc B (< 100 nmol·L-1) showed no obvious cytotoxicity to MDA-MB-231 cells, but significantly inhibited migration, invasion, and adhesion to Matrigel, fibronectin, type I collagen, and endothelial cells. Cuc B dramatically inhibited the phosphorylation of focal adhesion kinase (FAK) and paxillin in dose- and time-dependent manners. Furthermore, Cuc B induced intracellular reactive oxygen species (ROS) generation, which could be reduced by N-acetyl-l-cysteine (NAC). In addition, NAC pretreatment could reverse Cuc B-induced suppression of migration and adhesion, expression of FAK, but showed no effect on paxillin expression. In summary, Cuc B suppressed ROS-dependent metastasis through FAK pathway in breast cancer MDA-MB-231 cells, demonstrating novel mechanisms for the anticancer effects of Cuc B.

Furanodiene presents synergistic anti-proliferative activity with paclitaxel via altering cell cycle and integrin signaling in 95-D lung cancer cells.

Furanodiene (FUR) is a natural terpenoid isolated from Rhizoma Curcumae, a well-known Chinese medicinal herb that presents anti-proliferative activities in several cancer cell lines. Recently, we found that the combined treatment of FUR with paclitaxel (TAX) showed synergetic anti-proliferative activities in 95-D lung cancer cells. Herein, we showed that FUR reduced the cell numbers distributed in mitosis phase induced by TAX while increased those in G1 phase. The protein levels of cyclin D1, cyclin B1, CDK6 and c-Myc were all down-regulated in the group of combined treatment. The dramatically down-regulated expression of integrin ß4, focal adhesion kinase and paxillin might partially contribute to the synergic effect. Though FUR alone obviously induced endoplasmic reticulum stress, this signaling pathway may not contribute to the synergetic anti-proliferative effect as the protein expression of CHOP and BIP was similar in FUR alone and combined treatment group.

Ganoderiol A-enriched extract suppresses migration and adhesion of MDA-MB-231 cells by inhibiting FAK-SRC-paxillin cascade pathway.

Cell adhesion, migration and invasion are critical steps for carcinogenesis and cancer metastasis. Ganoderma lucidum, also called Lingzhi in China, is a traditional Chinese medicine, which exhibits anti-proliferation, anti-inflammation and anti-metastasis properties. Herein, GAEE, G. lucidum extract mainly contains ganoderiol A (GA), dihydrogenated GA and GA isomer, was shown to inhibit the abilities of adhesion and migration, while have a slight influence on that of invasion in highly metastatic breast cancer MDA-MB-231 cells at non-toxic doses. Further investigation revealed that GAEE decreased the active forms of focal adhesion kinase (FAK) and disrupted the interaction between FAK and SRC, which lead to deactivating of paxillin. Moreover, GAEE treatment downregulated the expressions of RhoA, Rac1, and Cdc42, and decreased the interaction between neural Wiskott-Aldrich Syndrome protein (N-WASP) and Cdc42, which impair cell migration and actin assembly. To our knowledge, this is the first report to show that G.lucidum triterpenoids could suppress cell migration and adhesion through FAK-SRC-paxillin signaling pathway. Our study also suggests that GAEE may be a potential agent for treatment of breast cancer.

Antitumour effects of Yangzheng Xiaoji in human osteosarcoma: the pivotal role of focal adhesion kinase signalling.

The present study examined, in vitro and in vivo, the potential antitumour effects of Yangzheng Xiaoji (YZXJ), a traditional Chinese medical formula used in cancer treatment, on osteosarcoma, a tumour type recently found to be sensitive to YZXJ. The human osteosarcoma cell line MG63 was used in cell-matrix adhesion and cell growth assays. The same cell line was used in an in vivo tumour model by establishing subcutaneous osteosarcoma xenografts. Oral and intraperitoneal routes were used to deliver the YZXJ extract. The effect of YZXJ on the activation of focal adhesion kinase (FAK) and paxillin was evaluated by immunofluorescence methods. It was found that YZXJ exhibited a significant inhibitory effect on cell-matrix adhesion as demonstrated by a cell-based assay and electric cell-substrate impedance sensing (ECIS) analysis. The effect was observed together with a reduction in phospho-FAK and phospho-paxillin in the cells when treated with YZXJ. In the in vivo tumour model, YZXJ was found to significantly inhibit the growth of osteosarcoma with a sustained effect observed when YZXJ was delivered intraperitoneally. YZXJ sensitized cells to the effect of FAK inhibitor in vitro and in vivo. It is concluded that Yangzheng Xiaoji plays a significant role in cell-matrix adhesion and tumour growth, likely by inhibiting the activation of the FAK pathway. The therapeutic role of Yangzheng Xiaoji in osteosarcoma warrants further investigation.

Citrullination of fibronectin modulates synovial fibroblast behavior.

INTRODUCTION: Rheumatoid arthritis is an autoimmune arthritis characterized by joint destruction. Anti-citrullinated protein antibodies are pathologic in rheumatoid arthritis, but the role of the citrullinated proteins themselves is much less clear. Citrullination is the conversion of the arginine residues of a protein to citrulline. In the inflamed rheumatoid joint there is increased protein citrullination. Several proteins are citrullinated in rheumatoid arthritis, including collagen type II, fibrinogen, and fibronectin. Fibronectin is thought to mediate the adhesion of joint-invading synovial fibroblasts to the rheumatoid cartilage in addition to regulating other synovial fibroblast functions. However, the effect of citrullinated fibronectin on synovial fibroblasts is unknown. METHODS: To investigate the effect of citrullinated fibronectin on synovial fibroblast behavior, we cultured normal murine, arthritic murine, and human rheumatoid synovial fibroblasts. We then compared several synovial fibroblast functions in the presence of fibronectin versus citrullinated fibronectin. We assessed adhesion with time-lapse microscopy, migration with transwell assays, focal adhesion kinase and paxillin phosphorylation by western blot, and focal matrix degradation by fluorescent gelatin degradation. RESULTS: Normal synovial fibroblasts have impaired adhesion, spreading, migration, and integrin-mediated phosphorylation of focal adhesion kinase and paxillin on citrullinated fibronectin. Murine arthritic and human rheumatoid synovial fibroblasts also have impaired adhesion and spreading on citrullinated fibronectin, but focal matrix degradation is unaffected by citrullinated fibronectin. CONCLUSION: Citrullination of fibronectin alters synovial fibroblast behavior and may affect how these cells adhere to and invade the joint and travel through the bloodstream. This work suggests an important role for the interaction of synovial fibroblasts with citrullinated matrix in the pathophysiology of rheumatoid arthritis.

NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy.

Muscular dystrophies are common, currently incurable diseases. A subset of dystrophies result from genetic disruptions in complexes that attach muscle fibers to their surrounding extracellular matrix microenvironment. Cell-matrix adhesions are exquisite sensors of physiological conditions and mediate responses that allow cells to adapt to changing conditions. Thus, one approach towards finding targets for future therapeutic applications is to identify cell adhesion pathways that mediate these dynamic, adaptive responses in vivo. We find that nicotinamide riboside kinase 2b-mediated NAD+ biosynthesis, which functions as a small molecule agonist of muscle fiber-extracellular matrix adhesion, corrects dystrophic phenotypes in zebrafish lacking either a primary component of the dystrophin-glycoprotein complex or integrin alpha7. Exogenous NAD+ or a vitamin precursor to NAD+ reduces muscle fiber degeneration and results in significantly faster escape responses in dystrophic embryos. Overexpression of paxillin, a cell adhesion protein downstream of NAD+ in this novel cell adhesion pathway, reduces muscle degeneration in zebrafish with intact integrin receptors but does not improve motility. Activation of this pathway significantly increases organization of laminin, a major component of the extracellular matrix basement membrane. Our results indicate that the primary protective effects of NAD+ result from changes to the basement membrane, as a wild-type basement membrane is sufficient to increase resilience of dystrophic muscle fibers to damage. The surprising result that NAD+ supplementation ameliorates dystrophy in dystrophin-glycoprotein complex- or integrin alpha7-deficient zebrafish suggests the existence of an additional laminin receptor complex that anchors muscle fibers to the basement membrane. We find that integrin alpha6 participates in this pathway, but either integrin alpha7 or the dystrophin-glycoprotein complex is required in conjunction with integrin alpha6 to reduce muscle degeneration. Taken together, these results define a novel cell adhesion pathway that may have future therapeutic relevance for a broad spectrum of muscular dystrophies.

Src kinases as therapeutic targets for cancer.

Src family kinases (SFKs) have a critical role in cell adhesion, invasion, proliferation, survival, and angiogenesis during tumor development. SFKs comprise nine family members that share similar structure and function. Overexpression or high activation of SFKs occurs frequently in tumor tissues and they are central mediators in multiple signaling pathways that are important in oncogenesis. SFKs can interact with tyrosine kinase receptors, such as EGFR and the VEGF receptor. SFKs can affect cell proliferation via the Ras/ERK/MAPK pathway and can regulate gene expression via transcription factors such as STAT molecules. SFKs can also affect cell adhesion and migration via interaction with integrins, actins, GTPase-activating proteins, scaffold proteins, such as p130(CAS) and paxillin, and kinases such as focal adhesion kinases. Furthermore, SFKs can regulate angiogenesis via gene expression of angiogenic growth factors, such as fibroblast growth factor, VEGF, and interleukin 8. On the basis of these important findings, small-molecule SFK inhibitors have been developed and are undergoing early phase clinical testing. In preclinical studies these agents can suppress tumor growth and metastases. The agents seem to be safe in humans and could add to the therapeutic arsenal against subsets of cancers.

Viscum album agglutinin-I induces degradation of cytoskeletal proteins in leukaemia PLB-985 cells differentiated toward neutrophils: cleavage of non-muscle myosin heavy chain-IIA by caspases.

The role of the anti-cancer agent Viscum album agglutinin-I (VAA-I) in leukaemia PLB-985 cells differentiated toward a neutrophil-like phenotype by dimethylsulphoxide (PLB-985D) has never been studied. This study investigated whether or not VAA-I can induce cytoskeletal breakdown in PLB-985D cells, as previously observed in undifferentiated PLB-985 cells. VAA-I was found to induce apoptosis in PLB-985D cells, as assessed by cytology and by degradation of gelsolin, an event known to occur via caspase-3 activation. VAA-I induced cytoskeletal breakdown based on the disruption of the F-actin network and cleavage of paxillin, vimentin and lamin B(1). In addition, we demonstrated, for the first time, that non-muscle myosin heavy chain IIA (NMHC-IIA) was cleaved by VAA-I treatment. Degradation of NMHC-IIA was reversed by the pan caspase inhibitor z-VAD-fmk in PLB-985D cells and neutrophils. However, unlike lamin B(1), no NMHC-IIA was detected on the cell surface of apoptotic neutrophils. In conclusion, PLB-985D cells responded in a similar manner to neutrophils regarding the degradation of the tested cytoskeletal. Therefore, PLB-985D cells may provide a suitable substitute for neutrophils in screening experiments, preventing extensive neutrophil cell isolation.

Focal adhesion kinase and paxillin: novel regulators of brain sexual differentiation?

Steroid-mediated sexual differentiation of the brain is a developmental process that permanently organizes the brain into a male or female phenotype. Previous studies in the rodent have examined the steroid-mediated mechanisms of male brain development. In an effort to identify molecules involved in female brain development, a high-throughput proteomics approach called PowerBlot was used to identify signaling proteins differentially regulated in the neonatal male and female rat hypothalamus during the critical period for brain sexual differentiation. Focal adhesion kinase (FAK) and paxillin, both members of the focal adhesion complex family of proteins, were significantly elevated in the newborn female compared with the male hypothalamus. Sex differences in these proteins were not detected in brain regions that are not subject to substantial organizational effects of steroids. Estrogens, the aromatized products of testosterone in the male, can both masculinize and defeminize the male brain. Daily estradiol administration to neonatal females significantly reduced FAK and paxillin in the hypothalamus, and aromatase inhibition increased paxillin in males to levels comparable with females. Androgens also appear to modulate paxillin levels in combination with estrogen action. Across development, hypothalamic levels of FAK were significantly elevated in females compared with males on postnatal d 6. Synaptic circuits in the hypothalamus develop sex differences perinatally. Estradiol treatment of cultured hypothalamic neurons significantly enhanced axon branching (P<0.01), consistent with the phenotype of FAK-deficient neurons. Together, these data implicate FAK and paxillin as regulators of sex differences in neuronal morphology.