Angiopoietin 2 induces glioma cell invasion by stimulating matrix metalloprotease 2 expression through the alphavbeta1 integrin and focal adhesion kinase signaling pathway

Accumulating evidence reveals a significant correlation between angiopoietin 2 (Ang2) expression and tumor invasion and metastasis in various human cancers, but the major focus of recent studies has been on the angiogenic effects of Ang2. We recently reported that Ang2-stimulated glioma cell invasion results from the up-regulation and activation of matrix metalloprotease 2 (MMP-2) in tumor cells. In this study, we identify a novel mechanism by which Ang2 stimulates MMP-2 expression leading to glioma cell invasion. We show that Ang2 interacts with alpha(v)beta(1) integrin in Tie2-deficient human glioma cells, activating focal adhesion kinase (FAK), p130(Cas), extracellular signal-regulated protein kinase (ERK) 1/2, and c-jun NH(2)-terminal kinase (JNK) and substantially enhancing MMP-2 expression and secretion. The Ang2/alpha(v)beta(1) integrin signaling pathway was attenuated by functional inhibition of beta(1) and alpha(v) integrins, FAK, p130(Cas), ERK1/2, and JNK. Furthermore, expression of a negative regulator of FAK, FAK-related nonkinase, by U87MG/Ang2-expressing glioma xenografts suppressed Ang2-induced MMP-2 expression and glioma cell infiltration in the murine brain. These data establish a functional link between Ang2 interaction with alpha(v)beta(1) integrin and glioma cell invasion through the FAK/p130(Cas)/ERK1/2 and JNK-mediated signaling pathway.     

Tumor cell invasiveness correlates with changes in integrin expression and localization

In nontumorigenic mammary epithelial cells (EpH4), transforming growth factor-beta (TGFbeta1) causes cell cycle arrest/apoptosis, but induces epitheliomesenchymal transition (EMT) in Ha-Ras-transformed EpH4 cells (EpRas). EMT is closely correlated with late-stage tumor progression and results in fibroblastic, migratory cells displaying a mesenchymal gene expression program (FibRas). EpRas and FibRas cells showed strongly increased cell substrate adhesion to fibronectin, collagens I/IV and laminin 1. Furthermore, Ras transformation caused enhanced or de-novo expression of the integrin subunits beta1, alpha2 and alpha3, or alpha5 and alpha6, respectively, the latter subunits being even more strongly expressed in FibRas cells. Importantly, polarized EpRas cells expressed integrin subunits beta1 and alpha6 at distinct (apical and lateral) membrane domains, while FibRas cells coexpressed these integrins and alpha5 at the entire plasma membrane. During EMT, EpRas cells formed an alpha5beta1 complex and deposited its ligand fibronectin into the extracellular matrix. Function-blocking alpha5 antibodies attenuated migration, and caused massive apoptosis in EpRas cells undergoing TGFbeta1-induced EMT in collagen gels, but failed to affect EpRas- or FibRas-derived structures. We conclude that functional alpha5beta1 integrin is centrally implicated in EMT induction. Importantly, FibRas cells also failed to deposit the alpha6beta4 ligand laminin 5, suggesting that alpha6beta4 is no longer functional after EMT and replaced by mesenchymal integrins such as alpha5beta1.     

Integrin beta1 is required for the invasive behaviour but not proliferation of squamous cell carcinoma cells in vivo

Integrin beta1 is both overexpressed and in an 'active' conformation in vulval squamous cell carcinomas (VSCCs) compared to matched normal skin. To investigate the significance of integrin beta1 deregulation we stably knocked-down integrin beta1 expression in the VSCC cell line A431. In vitro analysis revealed that integrin beta1 is required for cell adhesion, cell spreading and invasion. However, integrin beta1 is not required for cell growth or activation of FAK and ERK signalling in vitro or in vivo. Strikingly, while control tumours were able to invade the dermis, integrin beta1 knockdown tumours were significantly more encapsulated and less invasive.     

beta1-integrin mediates asbestos-induced phosphorylation of AKT and ERK1/2 in a rat pleural mesothelial cell line

Integrin-mediated signalling has been implicated in asbestos-induced carcinogenesis. In studies here, we examined signal transduction events associated with integrin-directed cell reactions triggered by crocidolite asbestos in the pleural mesothelial cell line 4/4 RM-4. Crocidolite fibres induced a significant time- and dose-dependent activation of the extracellular-signal-regulated kinases ERK1 and ERK2. ERK activation was specifically inhibited by integrin-blocking agents, that are integrin-binding peptides containing the sequence arginine-glycine-aspartic acid (RGD), and monoclonal antibodies against the integrin beta1-chain. Integrin-dependent activation of ERK1/2 in response to asbestos appeared to be independent of focal adhesion kinase pp125FAK (FAK) since FAK autophosphorylation remained unaffected in crocidolite-exposed mesothelial cells. Instead, we observed striking similarities in the kinetics of asbestos-induced ERK1/2 responses and phosphorylation of protein kinase B (AKT) at serine 473, a possible target residue for integrin-linked kinase. As with ERK activation, asbestos-induced AKT stimulation was significantly blocked by both the RGD-peptide and the beta1-integrin antibodies. These studies are the first to establish that in mesothelial cells ERK1/2 and AKT are simultaneously phosphorylated upon asbestos exposure in a beta1-integrin-dependent manner.     

Prostatic carcinoma cell migration via alpha(v)beta3 integrin is modulated by a focal adhesion kinase pathway

The highly invasive human prostate cancer PC3 cell line was found to express the alpha(v)beta3 integrin; in contrast, the noninvasive LNCaP prostate cancer cell line did not express alpha(v)beta3. PC3 cells adhered to and migrated on vitronectin (VN), an alpha(v)beta3 ligand expressed in mature bone where prostate cancer cells preferentially metastasize. In contrast, LNCaP cells did not adhere to or migrate on VN. Analysis of primary human prostate cancer cells isolated from 16 surgical specimens, showed that these cells expressed alpha(v)beta3, whereas normal prostate epithelial cells did not. In addition, only primary prostate cancer cells adhered to and migrated on VN. The role of alpha(v)beta3 in mediating prostate epithelial cell migration was confirmed using LNCaP cell transfectants expressing beta3 (beta3-LNCaP). Exogenous expression of alpha(v)beta3 induced LNCaP cells to adhere to and migrate on VN. In response to alpha(v)beta3 engagement, increased tyrosine phosphorylation of focal adhesion kinase (FAK), a signaling molecule activated by integrins and able to modulate cell migration, was detected. Transfection of FAK-related nonkinase, known to compete with FAK for its correct localization and phosphorylation, caused inhibition of beta3-LNCaP cell migration, specifically on VN. These data indicate that de novo expression of alpha(v)beta3 integrin in prostate cancer cells generates a migratory phenotype that is modulated by a FAK signaling pathway. This study points to alpha(v)beta3 as potential target in prostate cancer cell invasion and metastasis.     

Fibronectin-integrin (alpha5beta1) modulates migration and invasion of murine melanoma cell line B16F10 by involving MMP-9

Cell adhesion to extracellular matrix (ECM) initiates signaling cascade regulated by cell surface integrin receptors, which affects the proliferation and invasion of cells. Cells cultured in the presence of ECM ligand fibronectin (FN) stimulate secretion of matrix metalloproteinases (MMPs), facilitating cancer cell invasion and metastasis. Among all the members of the MMP family, MMP-9 is of crucial importance in tumor invasion and metastasis. The present study aims at studying the effects of integrin receptor alpha5beta1 and its ligand FN on expression of MMP-9 in murine melanoma cell line B16F10 and understanding the molecular mechanism(s) involved. The main experimental methods performed in the study were gelatin zymography, immunoblot, real-time RT-PCR, immunocytochemistry, enzyme linked immunosorbent assay (ELISA), transwell chamber assay, and in vivo metastasis assay in syngenic (C57BL6J) mice. The study reports that FN induces the activity, mRNA, and protein expression of MMP-9 and initiates its proteolytic activation in B16F10 cells. Blockage of the alpha5 receptor abrogated the FN-mediated stimulatory response on MMP-9 in B16F10 cells. Inhibitor studies and immunoblot analysis strongly suggest the involvement of focal adhesion kinase (FAK), extracellular regulated kinase (ERK), and phosphatidylinositol-3-kinase (PI-3K) in the FN-mediated responses. Immunocytochemical analysis showed the nuclear localization of nuclear factor-kappaB (NF-kappaB) might lead to activation of MMP-9 gene upon FN treatment. This study demonstrates that integrin receptor alpha5beta1 and FN interaction induces the invasive potential of B16F10 cells and MMP-9 induction is the downstream effectors in the process. This system serves as a novel model system to understand the molecular mechanism of melanoma growth and invasion.     

Downregulation of urokinase plasminogen activator receptor expression inhibits Erk signalling with concomitant suppression of invasiveness due to loss of uPAR-beta1 integrin complex in colon cancer cells

Cancer invasion is regulated by cell surface proteinases and adhesion molecules. Interaction between specific cell surface molecules such as urokinase plasminogen activator receptor (uPAR) and integrins is crucial for tumour invasion and metastasis. In this study, we examined whether uPAR and beta1 integrin form a functional complex to mediate signalling required for tumour invasion. We assessed the expression of uPAR/beta1 integrin complex, Erk signalling pathway, adhesion, uPA and matrix metalloproteinase (MMP) expression, migration/invasion and matrix degradation in a colon cancer cell line in which uPAR expression was modified. Antisense inhibition of the cell surface expression of uPAR by 50% in human colon carcinoma HCT116 cells (A/S) suppressed Erk-MAP kinase activity by two-fold. Urokinase plasminogen activator receptor antisense treatment of HCT116 cells was associated with a 1.3-fold inhibition of adhesion, approximately four-fold suppression of HMW-uPA secretion and inhibition of pro-MMP-9 secretion. At a functional level, uPAR antisense resulted in a four-fold decline in migration/invasion and abatement of plasmin-mediated matrix degradation. In empty vector-transfected cells (mock), uPA strongly elevated basal Erk activation. In contrast, in A/S cells, uPA induction of Erk activation was not observed. Urokinase plasminogen activator receptor associated with beta1 integrin in mock-transfected cells. Disruption of uPAR-beta1 integrin complex in mock-transfected cells with a specific peptide (P25) inhibited uPA-mediated Erk-MAP kinase pathway and inhibited migration/invasion and plasmin-dependent matrix degradation through suppression of pro-MMP-9/MMP-2 expression. This novel paradigm of uPAR-integrin signalling may afford opportunities for alternative therapeutic strategies for the treatment of cancer.     

Proteolysis of integrin alpha5 and beta1 subunits involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells

Our previous report demonstrated that all-trans-retinoic acid (ATRA) induces detachment and death under serum starvation in several human tumor cell lines. In this study, we examined the influence of cell-extracellular matrix interaction on the ability of ATRA to induce apoptosis. Plating of human hepatoma Hep3B cells onto poly-hydroxyethylmethacrylate-coated plates in the absence of serum resulted in the acceleration of ATRA-induced apoptosis. In contrast, ATRA-induced apoptosis was significantly suppressed by plating cells onto Matrigel-coated plates but not suppressed by culturing onto collagen-, laminin-, vitronectin-, or fibronectin-coated plates. Exogenously added soluble collagen, laminin, fibronectin, vitronectin or Matrigel failed to suppress ATRA-induced apoptosis. Results from the adhesion assay indicated that the cell attachment to fibronectin was significantly inhibited by ATRA. Treatment with perturbing antibody against integrin alpha5 or beta1 subunits resulted in promotion of ATRA-induced apoptosis. Moreover, the proteolytic cleavage of alpha5beta1 integrin and focal adhesion kinase (FAK) proteins is linked to the early phase of the ATRA-induced apoptotic process. Furthermore, ATRA-induced detachment, death, and cleavage of alpha5beta1 integrin and FAK were drastically suppressed by plating cells onto Matrigel-coated plates. These findings provide evidence that abrogation of cell adhesion, through proteolysis of alpha5beta1 integrin and FAK, is closely linked to ATRA-induced apoptosis in Hep3B cells.     

Activation of focal adhesion kinase enhances the adhesion and invasion of pancreatic cancer cells via extracellular signal-regulated kinase-1/2 signaling pathway activation

Background  

Interaction with integrin and focal adhesion kinase (FAK) regulates the cancer cell adhesion and invasion into extracellular matrix (ECM). In addition, phosphorylation of FAK correlates with the increase of cell motility and invasion. Adhesion and spreading of cancer cells on a variety of ECM proteins, including collagen type IV (Coll IV), leads to an increase in tyrosine phosphorylation and activation of FAK. In this study, we investigated the mechanism of activation of FAK and its downstream extracellular signal-regulated kinase (ERK)-1/2 signaling following stimulation by interleukin (IL)-1α and adhesion to ECM with subsequent enhancement of pancreatic cancer cell adhesion and invasion.     

Osteopontin increases lung cancer cells migration via activation of the alphavbeta3 integrin/FAK/Akt and NF-kappaB-dependent pathway

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which is abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and cell proliferation via interaction with its receptor, alphavbeta3 integrin. However, the effect of OPN on migration activity in human lung cancer cells is mostly unknown. Here we found that OPN increased the migration via activation of alphavbeta3 integrin in human lung cancer cells (A549 cells). Phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002), Akt inhibitor or ERK inhibitor (PD98059) inhibited the OPN-induced increase in the migration of lung cancer cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), p85 subunit of PI3K, serine 473 of Akt and ERK. In addition, treatment of A549 cells with NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) inhibited OPN-induced migration of lung cancer cells. Stimulation of A549 cells with OPN also induced IkappaB kinase alpha/beta (IKK alpha/beta) phosphorylation, IkappaBalpha phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. The OPN-mediated increases in IKK alpha/beta, IkappaBalpha and p65 Ser(536) phosphorylation were inhibited by Ly294002, Akt inhibitor and PD98059. Co-transfection with FAK, p85, Akt and ERK mutants also reduced the OPN-induced kappaB-luciferase activity. Taken together, these results suggest that OPN acts through alphavbeta3 integrin, which in turn activates the FAK, PI3K, Akt, ERK and NF-kappaB pathways, contributing to the migration of lung cancer cells.     

Alpha6beta1 integrin induces proteasome-mediated cleavage of erbB2 in breast cancer cells

ErbB2 and alpha6 integrin have been implicated in malignancy of breast cancer cells. Here we have determined the influence of alpha6beta1 integrin on erbB2 signaling in anchorage-independent growth, using MDA-MB435 breast cancer cells. Firstly, we transfected the cells with erbB2 cDNA, and isolated cells with high or low levels of alpha6beta1 integrin by cell sorting (alpha6H-ErbB and alpha6L-ErbB). We found that an erbB ligand, heregulin beta1, enhanced growth activity of alpha6L-ErbB cells, but not alpha6H-ErbB cells. Secondly, we established cells expressing a beta4 integrin deletion mutant (beta4-deltacyt), which selectively inhibited alpha6beta1 integrin expression and adhesion to laminin-1. Again, heregulin beta1 enhanced the growth of erbB2 cDNA-transfected beta4-deltacyt cells, but not mock cells. Western blot analysis revealed that heregulin beta1 stimulated phosphorylation of Akt and its downstream molecules, GSK3beta and p70S6kinase, and that the extent of phosphorylation was greater in ErbB2/beta4-deltacyt cells than ErbB2/mock cells. Furthermore, we found that the erbB2 cytoplasmic domain was truncated in ErbB2/mock cells, which was independent of ligand stimulation and adhesion, and was suppressed by proteasome inhibitors. These results suggest that alpha6beta1 integrin inhibits erbB2 signals by inducing proteasome-dependent proteolytic cleavage of the erbB2 cytoplasmic domain, and may thereby contribute to the regulation of tumor growth.     

The Walter Herbert Lecture. Control of cell motility and tumour invasion by extracellular matrix interactions.

Integrins are heterodimeric transmembrane proteins with large ectodomains and a short cytoplasmic tail inside the cell. They mediate cell adhesion to extracellular matrix proteins and to the surfaces of other cells. In many cases the sequence recognised by the integrins in the extracellular matrix proteins is the tripeptide Arg-Gly-Asp (RGD). Short synthetic peptides containing this sequence can inhibit invasion in vitro and tumour dissemination in vivo. Thus, the alpha 5 beta 1 fibronectin binding integrin appears to be the key integrin in the invasion of at least melanoma, osteosarcoma and glioblastoma cells. Modulation of the level and activities of this integrin can suppress invasion, whereas the alpha v beta 3 vitronectin binding integrin appears to be associated with increased invasiveness. There is increasing evidence that some of these effects are mediated through signals elicited by the binding of integrins to their target proteins. This possibility has generated a great deal of interest in the cytoplasmic molecules that might mediate the integrin-associated signalling.     

Nm23-H1 suppresses hepatocarcinoma cell adhesion and migration on fibronectin by modulating glycosylation of integrin beta1

Background

Nm23 gene was isolated as a metastatic suppressor gene. The antimetastatic effect of Nm23 has been an enigma for more than 10 years. Little is known about its molecular mechanisms. In this study we overexpressed Nm23-H1 in H7721 cells and observed reduction of cell adhesion, migration and extension of actin stress fibers in cells stimulated by fibronectin (Fn).

Methods

pcDNA3/Nm23-H1 was introduced into H7721 cells, and expression of Nm23-H1 was monitored by RT-PCR and western blot. Cell adhesion, actin extension and wound-induced migration assays were done on dishes coated with fibronectin. Phosphorylation of focal adhesion kinase (FAK) and total amount of integrin alpha5 and beta1 in Nm23-H1 transfected cells and control cells were measured by western blot. Flow cytometry was used to detect expression of surface alpha5 and beta1 integrin. N-glycosylation inhibitor tunicamycin was used to deglycosylate the integrin beta1 subunit.

Results

Overexpression of nm23-H1 in H7721 cells reduced cell adhesion, migration and extension of actin stress fibers on dishes coated with Fn. Phosphorylation of FAK in Nm23-H1 transfected cells was also attenuated. Integrin alpha5 and beta1 gene messages were unaltered in nm23-H1 overexpressed cells as detected by RT-PCR. However, while cell surface integrin alpha5 was unchanged, surface expression of beta1 integrin was downregulated. Western blot also showed that the total amounts of integrin alpha5 and beta1 were unaltered, but the level of mature integrin beta1 isoform was decreased significantly. Furthermore, partially glycosylated precursor beta1 was increased, which indicated that the impaired glycosylation of integrin beta1 precursor might contribute to the loss of cell surface integrin beta1 in nm23-H1 overexpressed cells.

Conclusion

These results suggest that by modulating glycosylation of integrin beta1, nm23-H1 down-regulates integrin beta1 subunit on cell surface and mediates intracellular signaling and subsequent suppression of the invasive process, including cell adhesion and migration.     

Changes in the levels of integrin and focal adhesion kinase (FAK) in human melanoma cells following 532 nm laser treatment.

Despite the increase in laser therapy, concern remains that sublethal treatment of pre-malignant lesions may adversely affect the biological behaviour of surviving cells. Integrin receptors mediate interaction of cells with the extracellular matrix and their occupation leads to focal adhesion kinase (FAK) activation. Using our previously established model we have now investigated subcellular changes and compared integrin and FAK concentrations, the degree of FAK phosphorylation and its association with the beta1 integrin in laser vs. non-laser treated cells. We treated cells with laser generated from a frequency doubled Q-switched (Nd:YAG) laser system (532 nm) at 0.4 J/cm2 twice per week for 4 weeks. Using cell lysates we performed Western immunoblotting 24 hr later to detect integrin subunits and FAK proteins and immunoprecipitation to investigate FAK phosphorylation and its association with beta1. Cell morphology was examined using electron microscopy. SK23 and G361 cells exhibited an 3.4- and 11.2-fold increase, respectively, in FAK protein following laser treatment. FAK phosphorylation in SK23 cells was increased by 82%, whereas FAK phosphorylation in G361 cells was reduced slightly (2%). Furthermore, both alpha3 and 4 integrins were up-regulated, by approximately 4-fold and 7- to 9-fold, respectively. In addition, the beta1 integrin was proteolysed in both cell lines and the levels of FAK associated with beta1 was increased (2.1- and 2.7-fold, respectively). Finally, laser treatment of SK23 cells caused an increased number of cell processes. Sublethal 532 nm laser light thus induces changes in integrin and FAK concentrations and subsequently influences cellular attachment and morphology.     

Dissecting the role of matrix metalloproteinases (MMP) and integrin alpha(v)beta3 in angiogenesis in vitro: absence of hemopexin C domain bioactivity, but membrane-Type 1-MMP and alpha(v)beta3 are critical

Matrix metalloproteinase (MMP)-2 and its hemopexin C domain autolytic fragment (also called PEX) have been proposed to be crucial for angiogenesis. Here, we have investigated the dependency of in vitro angiogenesis on MMP-mediated extracellular proteolysis and integrin alpha(v)beta3-mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic inhibitors BB94, CT1399, and CT1847 inhibited endothelial cell invasion, as did neutralizing anti-membrane-type 1-MMP (MT1-MMP) antibodies and tissue inhibitor of MMP (TIMP)-2 and TIMP-3 but not TIMP-1. This confirmed the pivotal importance of MT1-MMP over other MMPs in this model. Invasion was also inhibited by a nonpeptidic antagonist of integrin alpha(v)beta3, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin alpha(v)beta(3). Moreover, no specific binding of pro-MMP-2 to integrin alpha(v)beta3 was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin alpha(v)beta3 , and this in a PEX-independent manner. Likewise, integrin alpha(v)beta3 -expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MT1-MMP and alpha(v)beta3 - dependent but MMP-2 independent and does not support a role for PEX in alpha(v)beta3 integrin binding or in modulating angiogenesis in this system.     

Ascites induces modulation of alpha6beta1 integrin and urokinase plasminogen activator receptor expression and associated functions in ovarian carcinoma

Interactions between cancer cells and the surrounding medium are not fully understood. In this study, we demonstrate that ascites induces selective changes in the expression of integrins and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) in ovarian cancer cells. We hypothesise that this change of integrin and uPA/uPAR expression triggers signalling pathways responsible for modulating phenotype-dependent functional changes in ovarian cancer cells. Human ovarian surface epithelial (HOSE) cell lines and epithelial ovarian cancer cell lines were treated with ascites for 48 h. Ascites induced upregulation of alpha6 integrin, without any change in the expression of alphav, beta1 and beta4 integrin subunits. Out of the four ovarian cancer cell lines studied, ascites induced enhancement in the expression of uPA/uPAR in the more invasive OVCA 433 and HEY cell lines without any change in the noninvasive OVHS1 and moderately invasive PEO.36 cell lines. On the other hand, no change in the expression of alpha6 integrin or uPAR, in response to ascites, was observed in HOSE cells. In response to ascites, enhancement in proliferation and in adhesion was observed in all four ovarian cancer cell lines studied. In contrast, no significant increase in proliferation or adhesion by ascites was observed in HOSE cells. Ascites-induced expression of uPA/uPAR correlated with the increased invasiveness of HEY and OVCA 433 cell lines but was not seen in OVHS1, PEO.36 and HOSE cell lines. Upregulation of alpha6 integrin and uPA/uPAR correlated with the activation of Ras and downstream Erk pathways. Ascites-induced activation of Ras and downstream Erk can be inhibited by using inhibitory antibodies against alpha6 and beta1 integrin and uPAR, consistent with the inhibition of proliferation, adhesion and invasive functions of ovarian cancer cell lines. Based on these findings, we conclude that ascites can induce selective upregulation of integrin and uPA/uPAR in ovarian cancer cells and these changes may modulate the functions of ovarian carcinomas.     

Insulin-like growth factor-1 induces adhesion and migration in human multiple myeloma cells via activation of beta1-integrin and phosphatidylinositol 3'-kinase/AKT signaling

Insulin-like growth factor-1 (IGF-I) is a growth and survival factor in human multiple myeloma (MM) cells. Here we examine the effect of IGF-I on MM cell adhesion and migration, and define the role of beta1 integrin in these processes. IGF-I increases adhesion of MM.1S and OPM6 MM cells to fibronectin (FN) in a time- and dose-dependent manner, as a consequence of IGF-IR activation. Conversely, blocking anti-beta1 integrin monoclonal antibody, RGD peptide, and cytochalasin D inhibit IGF-I-induced cell adhesion to FN. IGF-I rapidly and transiently induces association of IGF-IR and beta1 integrin, with phosphorylation of IGF-IR, IRS-1, and p85(PI3-K). IGF-I also triggers phosphorylation of AKT and ERK significantly. Both IGF-IR and beta1 integrin colocalize to lipid rafts on the plasma membrane after IGF-I stimulation. In addition, IGF-I triggers polymerization of F-actin, induces phosphorylation of p125(FAK) and paxillin, and enhances beta1 integrin interaction with these focal adhesion proteins. Importantly, using pharmacological inhibitors of phosphatidylinositol 3'-kinase (PI3-K) (LY294002 and wortmannin) and extracellular signal-regulated kinase (PD98059), we demonstrate that IGF-I-induced MM cell adhesion to FN is achieved only when PI3-K/AKT is activated. IGF-I induces a 1.7-2.2 (MM.1S) and 2-2.5-fold (OPM6) increase in migration, whereas blocking anti-IGF-I and anti-beta1 integrin monoclonal antibodies, PI3-K inhibitors, as well as cytochalasin D abrogate IGF-I-induced MM cell transmigration. Finally, IGF-I induces adhesion of CD138+ patient MM cells. Therefore, these studies suggest a role for IGF-I in trafficking and localization of MM cells in the bone marrow microenvironment. Moreover, they define the functional association of IGF-IR and beta1 integrin in mediating MM cell homing, providing the preclinical rationale for novel treatment strategies targeting IGF-I/IGF-IR in MM.     

Studies on the expression of alpha(v)beta3 integrin receptors in non-malignant and malignant human cervical tumor tissues

Integrins are group of cell surface receptors, which mediate adhesion between cell-cell and cell-extracellular matrix proteins of the basement membrane. Integrins play an important role in cellular growth, development, morphology, signalling and also in tumor development. Among the integrin group of cell surface receptors one of the most important member is alpha(v)beta3 integrin receptor. Evidences say that the expression of this integrin receptor is regulated during tumor development. Large numbers of studies have been done to establish the role of alpha(v)beta3 integrin receptor in human melanoma systems. Expression of this receptor in metastatic but not benign melanomas suggests a role for this integrin in the regulation of tumor proliferation. Alpha(v)beta3 integrin receptor also plays a significant role in tumor angiogenesis, apoptosis and signal transduction process. Recent studies show that collagenase MMP-2 binds directly to integrin alpha(v)beta3 on the surface of invasive tumor cells and facilitates tumor cell invasion. In this present communication we studied the expression of alpha(v)beta3 integrin receptor in malignant and non-malignant cervical tumor tissues. Because receptor ligand interaction is a cell surface phenomenon the membrane fraction of tumor tissues was separated and the expression of alpha(v)beta3 integrin receptor was assayed by ELISA and immunoprecipitation from membrane extracted protein fraction. Comparative ELISA and immunoprecipitation clearly demonstrates much higher expression of alpha(v)beta3 vitronectin integrin receptor in the membrane fraction of malignant human cervical tumor tissues than nonmalignant tissue membrane fractions.     

整合素α5β1在IFNα-2b抑制白血病细胞系K562细胞增殖中的作用

背景与目的:慢性粒细胞白血病ph 细胞大量增殖与整合素β1介导的增殖抑制功能存在障碍密切相关。本研究通过观察整合素α5β1在干扰素(IFNα-2b)抑制慢性粒细胞白血病细胞K562增殖中的作用,探讨整合素β1在CML发病机制中的作用。方法:流式细胞仪检测K562细胞表面整合素α5β1的表达并检测纤维粘连蛋白(FN)与K562细胞的结合以及整合素α5β1单抗阻断两者结合的程度;MTT法测定整合素α5β1对IFNα-2b抑制K562细胞增殖的影响;RT-PCR法检测IFNα-2b作用后K562细胞中粘着斑激酶(FAK)表达量的改变。结果:整合素α5和β1在K562细胞表面高表达,分别为(97.59±1.04)%、(99.24±0.52)%,而健康人骨髓单核细胞为(64.05±2.38)%、(72.40±3.56)%;IFNα-2b不能改变其表达量,但可增强K562细胞整合素与FN的结合能力;K562细胞与FN的结合可被整合素α5和β1单抗阻断。与未处理组相比,IFNα-2b处理组的K562细胞发生增殖抑制(P<0.05),细胞内FAK基因表达水平增加2.21倍(P<0.05)。整合素α5和β1单抗可增强IFNα-2b引起的增殖抑制作用,并阻断IFNα-2b引起的FAK基因表达水平的增加。结论:IFNα-2b通过恢复整合素α5β1功能,增强FN与K562细胞表面整合素α5β1结合的能力而抑制K562细胞增殖并提高细胞内FAK表达量。     

Thiolutin, an inhibitor of HUVEC adhesion to vitronectin, reduces paxillin in HUVECs and suppresses tumor cell-induced angiogenesis.

Recent studies have shown that integrin alpha v beta 3, a receptor for vitronectin, plays an important role in tumor-induced angiogenesis and tumor growth and that antagonists of alpha v beta 3 inhibit angiogenic processes including endothelial cell adhesion and migration. On the other hand, most inhibitors of integrin alpha v beta 3 are peptide antagonists that include the Arg-Gly-Asp (RGD) motif. We therefore reasoned that non-peptide inhibitors of endothelial cell adhesion to vitronectin might be useful for inhibition of tumor angiogenesis in vivo. We screened for low-molecular-weight natural products able to inhibit adhesion of human umbilical vein endothelial cells (HUVECs) to vitronectin, and pyrrothine group compounds including aureothricin, thioaurin and thiolutin were isolated from microbial culture broths. Of these compounds, thiolutin inhibited adhesion of HUVECs to vitronectin the most effectively (IC(50), 0.83 microM). In vivo experiments showed that thiolutin significantly suppressed angiogenesis induced by tumor cells (S-180), a pathological form of neovascularization, in a mouse dorsal air sac assay system. To explore the mechanism of inhibition of HUVEC adhesion to vitronectin by thiolutin, we examined the effect of this agent on intracellular cell adhesion signaling. We found that the amount of paxillin in HUVECs was significantly reduced by thiolutin treatment, while those of other focal adhesion proteins including vinculin and focal adhesion kinase (FAK) were not. Metabolic labeling experiments showed that thiolutin enhanced degradation of paxillin in HUVECs. Protease inhibitors (MG115 and E64-D) decreased the rate of degradation of the paxillin induced by thiolutin and partially restored thiolutin-induced inhibition of HUVEC adhesion to vitronectin. Based on these findings, we concluded that thiolutin, an inhibitor of HUVEC adhesion to vitronectin, reduces the paxillin level in HUVECs and suppresses tumor cell-induced angiogenesis in vivo.