A small molecule induces integrin ß4 nuclear translocation and apoptosis selectively in cancer cells with high expression of integrin ß4.

Increased integrin ß4 (ITGB4) level is accompanied by malignant progression of multiple carcinomas. However, selective therapeutic strategies against cancer cells expressing a high level of ITGB4 have not been reported. Here, for the first time, we report that a chiral small molecule, SEC, selectively promotes apoptosis in cancer cells expressing a high level of ITGB4 by inducing ITGB4 nuclear translocation. Nuclear ITGB4 can bind to the ATF3 promoter region and activate the expression of ATF3, then upregulate the downstream pro-apoptosis genes. Furthermore, SEC promoted the binding of annexin A7 (ANXA7) to ITGB4 and increased ANXA7 GTPase activity. Activated ANXA7 promoted ITGB4 nuclear translocation by triggering ITGB4 phosphorylation at Y1494. SEC also inhibited the growth of xenograft tumors in the avian embryo model. We identified a small molecule, SEC, with selective pro-apoptosis effects on cancer cells with high expression of ITGB4, both in vitro and in vivo, by triggering the binding of ITGB4 and ANXA7, ITGB4 nuclear trafficking, and pro-apoptosis gene expression.

Expression and function of laminin and integrins on adhesion/migration of primary culture cells derived from rat oral epithelium.

BACKGROUND AND OBJECTIVE: It remains controversial whether or not the junctional epithelium cells that are directly attached to teeth migrate on the enamel surface, as those cells are able to adhere firmly to the enamel. The aim of this study was to investigate the expression patterns of laminin gamma(2), integrin beta(4) and integrin alpha(3), and to examine their potential function in cell migration. MATERIAL AND METHODS: Oral epithelium cells obtained from Sprague-Dawley rats were established in primary culture. We employed a wound-healing assay to characterize the direction of cell extension at the start of cell migration, and observed different localizations of laminin and integrins using immunofluorescence. For functional analyses of integrins, we employed a phosphatidylinositol-3-kinase (PI3K) activator to promote integrin beta(4) function and used P1B5 to inhibit integrin alpha(3) function, and we analyzed the percentage of re-epithelialization as the migration function. RESULTS: Marked accumulation of laminin gamma(2) was detected in the peripheral cytoplasm of cells adjacent to the wound area, as shown by the results of the migration assay. Integrin beta(4) was detected in the distal cell processes of actively migrating cells, while integrin alpha(3) was found in cell membranes of cells adjacent to the wound area. In the functional analyses, the percentage of re-epithelialization was significantly lower in the PI3K-activator group and in the P1B5-treated group (2.5% and 7.2%, respectively) than in the control group (39.0%) (p < 0.01). CONCLUSION: The results suggest that laminin gamma(2) is secreted as a foothold for cell migration, that integrin beta(4) participates in cell adhesion and that integrin alpha(3) is involved in cell migration in the primary culture cells.

Effect of the dental adhesive, 4-META/MMA-TBB resin, on adhesion and keratinization of regenerating oral epithelium.

BACKGROUND AND OBJECTIVE: The 4-META/MMA-TBB [4-(2-methacryloxyethyl)trimellitic anhydride/methyl methacrylate-tributylborane] resin is widely used as a dental adhesive. It has also been applied in the dressing of gingival wound surfaces following periodontal surgery. However, its effect on the regeneration and/or cell attachment of the oral epithelium remains to be clarified. To evaluate the effect of the resin applied as a wound dressing, we investigated expression of laminin 5, integrin beta(4) and cytokeratin 14 in regenerating oral epithelium treated with this resin following gingivectomy from the viewpoint of cell attachment and differentiation. MATERIAL AND METHODS: The resin was applied to the entire wound surface in rats after gingival surgery, and regenerating epithelium was examined immediately and at 1, 3, 5, 7 and 14 days later. The resin was removed 2 weeks after application in some animals and tissue further examined at 1, 3, 5 and 7 days later. RESULTS: Regenerating epithelium under the resin was not keratinized, but became keratinized immediately after removal of the resin. Laminin 5 and integrin beta(4) were immunolocalized in the basal lamina, the internal basal lamina, in marginal cells of the regenerating epithelium and at the resin-regenerating epithelium interface. Cytokeratin 14 localized in the regenerating epithelium underneath the resin, as well as in healthy and regenerated junctional epithelial cells. CONCLUSION: These results suggest that this resin covers the wound surface and that the regenerating epithelium biologically adheres to the resin during the initial process of its regeneration.

Synthesis and discovery of a novel pyrazole derivative as an inhibitor of apoptosis through modulating integrin beta4, ROS, and p53 levels in vascular endothelial cells.

Recently, pyrazole derivatives as high affinity and selective A2A adenosine receptor antagonists have been reported. But, so far, there are no reports about the inhibitory effects of multi-substituted pyrazole derivatives on apoptosis of vascular endothelial cells (VECs). In this study, we synthesized six pyrazole derivatives and characterized the structures of the compounds by IR, (1)H NMR, mass spectroscopy, and element analysis. The biology assay showed that a novel pyrazole derivative, ethyl 3-(o-chlorophenyl)-5-methyl-1-phenyl-1H-pyrazole-4-carboxylate (MPD) at low concentration (25muM) increased VECs viability and inhibited VECs apoptosis induced by deprivation of serum and FGF-2. During this process, the levels of integrin beta4, reactive oxygen species (ROS), and p53 were depressed obviously. The data suggested that MPD was a potential inhibitor of apoptosis associated with the signal pathway mediated by integrin beta4, ROS, and p53 in VECs.

The effect of butyric acid on adhesion molecule expression by human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Short-chain fatty acids, such as butyric acid, are detected in periodontal pockets and are thought to be involved in the initiation and progression of periodontal disease. In the present study, we examined the effects of butyric acid on adhesion molecule expression by human gingival epithelial cells. MATERIAL AND METHODS: The human gingival carcinoma cell line, Ca9-22, was cultured in media that contained different concentrations of butyric acid. RESULTS: Cell numbers were significantly decreased in a dose-dependent manner by butyric acid at concentrations of > or = 0.2 mM. The expression of intercellular adhesion molecule-1 mRNA was significantly increased 6 h after stimulation. By contrast, the expression levels of integrins alpha 6 and beta 4 were decreased. Similar results were obtained by flow cytometry. CONCLUSION: The results of the present study indicate that butyric acid alters the expression of adhesion molecules by Ca9-22 cells. The elucidation of the mechanism of action of butyric acid on the periodontium may help to clarify several aspects of the onset and progression of periodontal disease.

Targeting integrin beta4 for cancer and anti-angiogenic therapy.

The integrins play key roles in the signaling networks that drive pathological angiogenesis and tumor progression. Integrin beta4 is a laminin receptor upregulated in tumor cells and angiogenic endothelial cells. Biochemical studies have indicated that beta4 combines with and enhances the signaling function of multiple receptor tyrosine kinases, including ErbB2, EGF-R and Met. Genetic studies have revealed that beta4 signaling promotes both angiogenesis and tumorigenesis. Here, I discuss the hypothesis that beta4 promotes both processes by amplifying receptor-tyrosine-kinase signaling. Therefore, I propose that a simultaneous blockade of beta4 and receptor-tyrosine-kinase signaling represents a rational approach to cancer and anti-angiogenic therapy.

Loss of beta4 integrin subunit reduces the tumorigenicity of MCF7 mammary cells and causes apoptosis upon hormone deprivation.

PURPOSE: The alpha6beta4 integrin, a laminin receptor, has been implicated from many studies in tumor progression and invasion. We showed that the beta4 integrin subunit associates with the ErbB-2 tyrosine kinase in human mammary carcinoma cell lines and that its overexpression in NIH3T3/ErbB-2-transformed cells causes a constitutive activation of phosphatidylinositol 3-kinase (PI3K), inducing a strong increase of their invasive capacity. In this study, we investigated the biological consequences of interference with the endogenous beta4 integrin subunit expression. EXPERIMENTAL DESIGN: In vitro and in vivo tumor growth and the biochemical consequences of beta4 integrin inactivation were studied in mammary tumor cells by using short hairpin RNA approach. RESULTS: Our data show that tumor growth of mammary tumor cells strictly depends on beta4 expression, confirming the relevance of beta4 protein in these cells. Moreover, interference with beta4 expression significantly reduces endogenous PI3K activity and AKT and mammalian target of rapamycin phosphorylation. Accordingly, with these results and considering that PI3K activity in mammary tumor plays a relevant role in hormone resistance, we asked whether beta4 expression might be relevant for hormone responsiveness in these cells. Data reported indicate that the interference with endogenous beta4 expression, upon hormone deprivation, induces caspase-9 and cytochrome c-mediated apoptosis, which is enhanced upon tamoxifen treatment. On the other hand, the expression of myr-AKT in MCF7 beta4-short hairpin RNA cells rescues the cells from apoptosis in the absence of hormones and upon tamoxifen treatment. CONCLUSIONS: Overall, these results confirm the relevance of beta4 expression in mammary tumors and indicate this integrin as a relevant target for tumor therapy.

Safrole oxide induces apoptosis by up-regulating Fas and FasL instead of integrin beta4 in A549 human lung cancer cells.

Previously, we found that 3,4-(methylenedioxy)-1-(2',3'-epoxypropyl)-benzene (safrole oxide) induced a typical apoptosis in A549 human lung cancer cells by activating caspase-3, -8, and -9. In this study, we further investigated which upstream pathways were activated by safrole oxide during the apoptosis. Immunofluorescence assay combined with laser scanning confocal microscopy revealed that both Fas and Fas ligand (FasL) were up-regulated by the small molecule. In addition, Fas protein distribution was altered, showing a clustering distribution instead of a homogeneous one. Subsequently, Western blot analysis confirmed the up-regulations of Fas and its membrane-binding form of FasL (m-FasL), as well as P53 protein. Conversely, safrole oxide hardly affected integrin beta4 subunit expression or distribution, which was reflected from the data obtained by immunofluorescence assay combined with laser scanning confocal microscopy. The results suggested that Fas/FasL pathway might be involved in safrole oxide-induced apoptosis of A549 cells, while integrin beta4 might be irrelevant to the apoptosis. Nevertheless, we first found the strong expression of integrin beta4 in A549 cells. The study first suggested that safrole oxide might be used as a small molecular promoter of Fas/FasL pathway to elicit apoptosis in A549 cells, which would lay the foundation for us to insight into the new strategies for lung cancer therapy.

Beta4 integrin is involved in statin-induced endothelial cell death.

HMG-CoA reductase inhibitors (statins) have been shown to inhibit angiogenesis. The molecular mechanism mediating the anti-endothelial activities of statins remains unclear. The present study demonstrated that the antiangiogenic effect of atorvastatin (ATV) was associated with endothelial death. Molecular profiling data identified a 29-fold upregulation of beta4 integrin mRNA. Western blot and flow cytometry confirmed robust increases of total and cell-surface beta4 integrin. Blockage of beta4 integrin activity by antagonizing antibody abrogated ATV-induced endothelial death. The endothelial death and beta4 integrin upregulation by ATV could be reversed by intermediate metabilites of the HMG-CoA reductase pathway mevalonate or GGPP, but not by FPP, suggesting that these effects were results of specific inhibition of the pathway. These data indicate that the HMG-CoA reductase might represent an important survival pathway in angiogenic endothelial cells and thus, a potential target for antiangiogenic therapy.

Rattlesnake venom induces apoptosis by stimulating PC-PLC and upregulating the expression of integrin beta4, P53 in vascular endothelial cells.

In the previous studies, we found that phosphatidylcholine-specific phospholipase C (PC-PLC) was implicated in apoptosis induced by rattlesnake venom in vascular endothelial cells (VEC) [Biochem. Biophys. Res. Commun. (1997b) 223, 182]. In order to find out other signal elements in this pathway and the mechanisms by which PC-PLC mediates apoptosis induced by rattlesnake venom in VEC, the expression of integrin beta4 and P53 was evaluated when the activity of PC-PLC was suppressed by D609 (tricyclodecan-9-yl-xanthogenate), a specific inhibitor of this enzyme. The increase of integrin beta4 and P53 expression induced by the venom was markedly suppressed when apoptosis of VEC was inhibited by D609. The data indicated that integrin beta4 and P53 play important roles in signal transduction of apoptosis induced by rattlesnake venom, and that PC-PLC might regulate apoptosis by up-regulating the expression of integrin beta4 and P53 in VEC.