Functional links between Snail-1 and Cx43 account for the recruitment of Cx43-positive cells into the invasive front of prostate cancer

Abstract

Suppressive function of connexin(Cx)43 in carcinogenesis was recently contested by reports that showed a multifaceted function of Cx43 in cancer progression. These studies did not attempt to model the dynamics of intratumoral heterogeneity involved in the metastatic cascade. An unorthodox look at the phenotypic heterogeneity of prostate cancer cells in vitro enabled us to identify links between Cx43 functions and Snail-1-regulated functional speciation of invasive cells. Incomplete Snail-1-dependent phenotypic shifts accounted for the formation of phenotypically stable subclones of AT-2 cells. These subclones showed diverse predilection for invasive behavior. High Snail-1 and Cx43 levels accompanied high motility and nanomechanical elasticity of the fibroblastoid AT-2_Fi2 subclone, which determined its considerable invasiveness. Transforming growth factor-β and ectopic Snail-1 overexpression induced invasiveness and Cx43 expression in epithelioid AT-2 subclones and DU-145 cells. Functional links between Snail-1 function and Cx43 expression were confirmed by Cx43 downregulation and phenotypic shifts in AT-2_Fi2, DU-145 and MAT-LyLu cells upon Snail-1 silencing. Corresponding morphological changes and Snail-1 downregulation were seen upon Cx43 silencing in AT-2_Fi2 cells. This indicates that feedback loops between both proteins regulate cell invasive behavior. We demonstrate that Cx43 may differentially predispose prostate cancer cells for invasion in a coupling-dependent and coupling-independent manner. When extrapolated to in vivo conditions, these data show the complexity of Cx43 functions during the metastatic cascade of prostate cancer. They may explain how Cx43 confers a selective advantage during cooperative invasion of clonally evolving, invasive prostate cancer cell subpopulations.

Fenofibrate enhances barrier function of endothelial continuum within the metastatic niche of prostate cancer cells

“…Where indicated, cancer cells were stained with CellTracker Orange CMRA (10 µM, Invitrogen). For gap junctional intercellular coupling (GJIC) analyses, calcein-loaded (Invitrogen) donor (DU-145) cells were plated on monolayers of acceptor (HUVEC) cells grown on coverslips in Petri dishes at the ratio of 1:50 [39] to evaluate intercellular calcein transfer and coupling index defined as the percentage of donor cells coupled with at least one acceptor cell. To determine the cytotoxic effect of FF, HUVECs incubated with various concentrations of FF for 6, 24 and 48 h were harvested, and the number of viable cells was determined by the fluorescence diacetate/ethidium bromide test.…”

“…Image acquisition was performed with a Leica DMI6000B microscope (DMI7000 version; Leica Microsystems, Wetzlar, Germany) equipped with the Total Internal Reflection Fluorescence, Nomarski Differential Interference Contrast (DIC) and Interference Modulation Contrast (IMC) modules. LAS-AF deconvolution software was used for image processing as described previously [39]. Three dimensional (3D) images were registered using a Leica TCS SP5 confocal microscope (Leica Microsystems, Mannheim, Germany) using 63Â HCX PL APO CS oil immersion (NA 1.4) objective lens.…”

9-AAA inhibits growth and induces apoptosis in human melanoma A375 and rat prostate adenocarcinoma AT-2 and Mat-LyLu cell lines but does not affect the growth and viability of normal fibroblasts

“…One can expect that, in heterogeneous cell populations such as populations of cancer cell lines (32,33), the cells that proliferate are present alongside cells that die by apoptosis or necrosis. The present study demonstrated that the inhibition of increased cell numbers in cancer cell cultures in the presence of 5-FU and 9-AAA resulted from an increased proportion of dying to proliferating cells over time.…”

“…1E and F; AT-2 P=0.016, P=0.01, P=0.01, P=0.01; MAT-LyLu P=0.018, P=0.01, P=0.01, P=0.01, respectively). At ii) In parallel, the samples from the same cell culture were analyzed with the modern method using FlowSight, which analyzes thousands of cells (33). The results obtained using the two methods combined yielded well-corresponding results (Fig.…”

“…The cells were analyzed with a FlowSight image flow cytometer and Ideas 5.0 software (Amnis Corporation, Seattle, WA, USA). For each sample, images of 10,000 single cells were analyzed at 488 and 785 nm light wavelength using three channels, as described previously (33). For the second method, the cells were fixed in 3.5% formaldehyde for 20 min.…”