Importantly, such approaches bypass the drug-resistance of cancer cells without provoking the selection of malignant cancer cell lineages. total of 25 M FF exerted corresponding effects on Cx43-mediated gap junctional coupling, EGF production, and ERK1/2 activation in HUVEC/A549 co-cultures. It also directly augmented endothelial barrier function via the interference with focal adhesion kinase (FAK)/RhoA/Rac1-regulated endothelial cell adhesion/contractility/motility and prompted the selective transmigration of epithelioid A549 cells. N-acetyl-L-cysteine abrogated FF effects on HUVEC activation, suggesting the involvement of PPAR-independent mechanism(s) in its action. Our data identify a novel Cx43/EGF/ERK1/2/FAK/RhoA/Rac1-dependent signaling axis, which determines the efficiency of lung cancer cell diapedesis. FF interferes with its activity and reduces the susceptibility of endothelial cells to A549 stimuli. These findings provide the rationale for the implementation of FF in the therapy of malignant lung cancers. < 0.05 and ** < 0.01). Error bars represent SEM. All results are representative of CGS 21680 HCl at least three impartial experiments ( 3). Scale bar = 40 m. Note that the relatively efficient diapedesis of A549 cells is usually considerably inhibited by FF. 2.2. A549 Cells Impair Endothelial Barrier Function via Intercellular Cx43/EGF/ERK1/2-Dependent Signaling To identify the mechanisms underlying the attenuation of the endothelial barrier function by A549 cells, we further focused on the mediators of A549-induced HUVEC activation. Protein array analyses demonstrated the expression of numerous angioactive factors in A549 cells (such as FGF-2, Serpin E1, and uPA), and the up-regulation of EGF in A549/HUVEC co-cultures (Physique 2A). Concomitantly, HUVECs displayed increased motility in A549-conditioned medium (Physique 2B and Physique S1B), which suggests the role of paracrine, EGF-dependent signaling in HUVEC activation by A549 cells. Notably, we also observed a high functionality of gap junctions in HUVEC continua (Physique 2C and Physique S2A). This was accompanied by somewhat limited GJIC between A549 cells and HUVEC, as demonstrated by the relatively low value of a coupling index estimated for HUVEC/A549 co-cultures (Ci = 17.6%). A549-induced activation of HUVECs was correlated with an increased abundance of connexin(Cx)43+ plaques in HUVEC/A549 co-cultures Cx43 (Physique 2D). Moreover, the inhibition of Cx43-mediated GJIC by 18--glicyrrhetinic acid (AGA; 70 M, cf. Physique S2C in Supplementary data) and Cx43 down-regulation by siRNA (Physique S3) led to the distinct attenuation of HUVEC activation by A549 cells (Physique 2E and Physique S1C,D), in the absence of nonspecific effects of control siRNA (Physique S3). Thus, Cx43-mediated communication between A549 cells and HUVECs may up-regulate EGF, which further activates HUVECs in a para/autocrine manner. Actually, ectopic administration of EGF resulted in the activation of HUVECs, whereas chemical inhibition of the EGF receptor (by PD158780, 20 M) and of ERK1/2 (by UO126, 50 M) led to the attenuation of this process (Physique 2F; Figures S1E,F and S4). Collectively, these data indicate the involvement of the Cx43/EGF/ERK1/2 axis in A549-induced HUVEC activation. Open in a separate window Physique 2 A549 cells impair the endothelial barrier function via the activation of the Cx43/EGF/ERK1/2-dependent intercellular signaling axis. (A) A549 cells were seeded onto HUVEC monolayers as in Physique 1 and co-cultured for 24 h. Then, the expression CGS 21680 HCl of angioactive proteins was semi-quantitively estimated with an antibody array kit (see Materials and Methods). Plots show the densitometrically estimated dot intensities, illustrating the protein amounts in A549 CGS 21680 HCl cells (in a.u.; left) or in A549/HUVEC co-cultures relative to the HUVEC control. (B) A549-conditioned medium (3:5) was added to HUVECs and their motility was estimated with time-lapse videomicroscopy for 7 h. (C) Calcein-loaded HUVEC (left) or A549 cells (right) were seeded onto HUVEC monolayers and GJIC (coupling ratio-Ci) was estimated by a calcein transfer assay after 1 h. Concomitantly, Cx43 expression in HUVECs and in HUVEC/A549 co-cultures was estimated with immunofluorescence (D). (E) The effect of AGA (70 M) and Cx43 silencing by siRNA on HUVEC motility. (F) HUVECs were cultured in the presence of EGF or A549/HUVEC co-cultures were established as above and the effects of EGFR- or ERK1/2 inhibitor (PD158780 and UO126, respectively) on HUVEC motility were Rabbit polyclonal to ERCC5.Seven complementation groups (A-G) of xeroderma pigmentosum have been described. Thexeroderma pigmentosum group A protein, XPA, is a zinc metalloprotein which preferentially bindsto DNA damaged by ultraviolet (UV) radiation and chemical carcinogens. XPA is a DNA repairenzyme that has been shown to be required for the incision step of nucleotide excision repair. XPG(also designated ERCC5) is an endonuclease that makes the 3 incision in DNA nucleotide excisionrepair. Mammalian XPG is similar in sequence to yeast RAD2. Conserved residues in the catalyticcenter of XPG are important for nuclease activity and function in nucleotide excision repair estimated with time-lapse videomicroscopy. Error bars represent SEM. Scale bar = 40 m. The statistical significance of the differences was tested with one-way ANOVA followed by post-hoc Tukeys.