A. Organotypic sections were stained for the proliferation marker Ki-67 (brown). B. Proliferation was defined as a percentage of Ki-67-positive cells among the total number of carcinoma cells per microscopic field (2006magnification; n = total number of fields analyzed, 3? fields per organotypic section). C. Apoptotic cells were detected by TUNEL assay (green) and caspase-3 staining (red). Apoptotic cell death was quantified in terms of TUNEL (D) and caspase-3-positive (F) cells as a percentage of total number of carcinoma cells per microscopic field (2006magnification; n = total number of fields analyzed, 3? fields per organotypic section). Mann-Whitney U-test, ***p,0.001, *p,0.05. G. 20 mg of total protein of lysed cell extracts was separated by SDS-PAGE and immunoblotted with antibodies against signaling molecules of the Bcl-family apoptosis pathway, anti-apoptotic Bcl-xL and pro-apoptotic Bax. b-actin was used as a loading control. H. The relative band intensities were quantified (n = 3 Western analyses from separate protein extractions; mean 6 SEM). Students t-test, *p,0.05.
epithelial characteristics in the MET process. Approximately a 2fold excess of E-cadherin in A431 human epidermoid carcinoma cells has been shown inhibit their invasion [41] which is in line with the degree of E-cadherin up-regulation induced by arresten in our experiments (Table S1). Our data therefore suggest that carcinoma cells undergo changes resembling MET in the presence of arresten. Arresten mediates its effects on endothelial cells through integrin receptors. Arresten is known to bind to a1b1 integrin and this ligation is shown to lead to the inhibition of focal adhesion kinase (FAK)/c-Raf/MEK1/2/p38/ERK1 mitogen-activated protein kinase pathway and suppression of endothelial cell migration, proliferation, and tube formation [16?9,44]. Integrin a1 is also required for the anti-survival effect of arresten in endothelial cells [18]. Using ECIS measurements we showed here that the high impedance of Arr-HSC cells was reduced upon treatment with the function-blocking a1 integrin antibody. These data suggest that a1b1 integrin mediates the promoting effect of arresten on HSC-3 cell-cell contacts and cell spreading that are disturbed upon antibody binding. Blocking of a2b1 integrin receptor had a strong inhibitory effect on both the Arr-HSC and the Ctrl-HSC cell attachment suggesting that this receptor mediates interactions that do not involve arresten. Thus, the upregulation of E-cadherin on cell-cell junctions and the concomitant less invasive behavior may be linked to modulation of integrin a1b1 signalling by arresten. The manipulation of b1 integrin and subsequent signaling pathways can lead to reversion of the malignant phenotype [45?6]. The ECM proteoglycan versican, known to interact and signal through b1-integrin [47], was recently shown to induce MET in MDA-MB-231 cells [48] further supporting the concept that alterations in the ECM can regulate epithelial plasticity. We also consider it possible that the excess of arresten disturbs the cell-matrix interactions in the collagen I-based 3D organotypic model resulting in induction of cell death.
ECM molecules, such as collagen I, for example, induce EMT by an integrin and FAK-mediated regulation of cadherins, both by disrupting E-cadherin adhesion complex and by upregulating Ncadherin expression [39,27,49]. A correctly assembled collagen IV network supports the differentiated epithelial cell phenotype, and disruption of this network by administration of the a1(IV)NC1 domain has been shown to facilitate EMT in mouse proximal tubular epithelial cells in vitro [50]. This observation differs from the epithelial morphology-promoting effect of arresten on oral carcinoma cells shown here, but these two phenomena represent distinct types of transitions [22] and diverse cells may respond in a different manner to stromal signals. Assadian et al. published recently a study which shows that p53 can induce an antiangiogenic program whereby expression of a1(IV) chain is upregulated, stabilized by prolyl-4-hydroxylase and efficiently processed by MMPs to an arresten-containing peptide [20]. This p53-dependent ECM remodeling was suggested to destabilize the vascular collagen IV network and thereby prevent endothelial cell adhesion and migration leading to reduced angiogenesis and tumor growth in vivo and in vitro. Our observations on the inhibition of tumor angiogenesis and growth by arresten are in line with these observations, but our data suggest that arresten also reduces proliferation, induces apoptosis and facilitates epithelial plasticity in tumor cells. As tumor cells respond to many biologically active molecules in biphasic manner [51?2], the effects of arresten may also vary depending on its level. To date, the systemic or local concentration of arresten is not known [20], although a pilot study by Ramazani et al. suggests that the normal circulatory level of collagen IV is around 100 ng/ml in healthy humans giving us some cues on the level or arresten [53]. We show here for the first time that arresten directly modulates the behavior of carcinoma cells, and propose that this occurs at least partially via binding to integrin a1b1.
Figure 6. Arr-HSC cell spreading is impaired in the presence of a function-blocking antibody to a1 integrin. A. The impedance, reflecting cell adhesion and spreading, was measured for Ctrl-HSC and Arr-HSC cells using electric cell-substrate impedance sensing (ECIS) (mean of duplicate wells of representative ECIS plates). The Arr-HSC cells showed markedly higher impedance at a low frequency than the control cells. B. Treatment of Arr-HSC cells with a specific function-blocking a1 antibody reduced the impedance when compared to the untreated Arr-HSC cells. Treatment of Arr-HSC cells with integrin a2 antibody almost completely abolished the cell spreading. C. Ctrl-HSC cells showed reduced spreading in the presence of integrin a2 antibody while a1 antibody had no effect on impedance. ongoing MET-like events, and subsequently became less motile and more apoptotic. However, the MET-like events may not always be beneficial for survival, as MET has also been reported during the establishment of metastases. Furthermore, some ECM molecules have been found to contribute to the formation of premetastatic niches [30,54]. In summary, since arresten is a potent inhibitor of angiogenesis, and also exerts strong anti-invasive effects on carcinoma cells, it could be considered a candidate for drug development efforts. However, the MET-inducing property of arresten and its role in primary tumors and metastases should be first characterized in detail.
purified recombinant arresten (0, 5, or 20 mg/ml), and same amount was also added to the lower chambers. The cells were allowed to migrate overnight, fixed in 10% TCA, washed and stained with 0.1% crystal violet. The cells that had migrated to the underside of the membrane were counted under a Leica DMRB microscope (Leica Microsystems). In the co-culture migration assays the inserts were equilibrated for 2 h with media collected from Arr-HSC cells for 24 h. The Ctrl-HSC cells were then suspended in the co-culture medium and plated on the Transwell wells as described above.
