Damian Ryszawy

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

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.

Anticancer and antioxidant activity of bread enriched with broccoli sprouts.

This study is focused on antioxidant and anticancer capacity of bread enriched with broccoli sprouts (BS) in the light of their potential bioaccessibility and bioavailability. Generally, bread supplementation elevated antioxidant potential of product (both nonenzymatic and enzymatic antioxidant capacities); however, the increase was not correlated with the percent of BS. A replacement up to 2% of BS gives satisfactory overall consumers acceptability and desirable elevation of antioxidant potential. High activity was especially found for extracts obtained after simulated digestion, which allows assuming their protective effect for upper gastrointestinal tract; thus, the anticancer activity against human stomach cancer cells (AGS) was evaluated. A prominent cytostatic response paralleled by the inhibition of AGS motility in the presence of potentially mastication-extractable phytochemicals indicates that phenolic compounds of BS retain their biological activity in bread. Importantly, the efficient phenolics concentration was about 12 μM for buffer extract, 13 μM for extracts after digestion in vitro, and 7 μM for extract after absorption in vitro. Our data confirm chemopreventive potential of bread enriched with BS and indicate that BS comprise valuable food supplement for stomach cancer chemoprevention.

Triterpene saponosides from Lysimachia ciliata differentially attenuate invasive potential of prostate cancer cells.

Neither androgen ablation nor chemotherapeutic agents are effective in reducing the risk of prostate cancer progression. On the other hand, multifaceted effects of phytochemicals, such as triterpene saponins, on cancer cells have been suggested. A promising safety and tolerability profile indicate their possible application in the treatment of advanced prostate cancers. We analyzed the specificity, selectivity and versatility of desglucoanagalloside B effects on human prostate cancer cells derived from prostate cancer metastases to brain (DU-145 cells) and bone (PC-3 cells). Prominent growth arrest and apoptotic response of both cell types was observed in the presence of sub-micromolar desglucoanagalloside B concentrations. This was accompanied by cytochrome c release and caspase 3/7 activation. A relatively low cytostatic and pro-apoptotic response of cancer cells to a desglucoanagalloside B analog, anagallosaponin IV, illustrated the specificity of the effects of desglucoanagalloside B, whereas the low sensitivity of normal prostate PNT2 cells to desglucoanagalloside B showed the selectivity of its action. Inhibition of cancer cell motility was observed in the presence of both saponins, however only desglucoanagalloside B attenuated cancer cell invasive potential, predominantly through an effect on cell elastic properties. These data demonstrate the versatility of its effects on prostate cancer cells. In contrast to PNT2 cells, cancer cells tested in this study were relatively resistant to mitoxantrone. The multifaceted action of desglucoanagalloside B on basic cellular traits, crucial for prostate cancer progression, opens perspectives for elaboration of combined palliative therapies and new prostate cancer prophylaxis regimens.

Connexin43 Controls the Myofibroblastic Differentiation of Bronchial Fibroblasts from Patients with Asthma

&NA; Pathologic accumulation of myofibroblasts in asthmatic bronchi is regulated by extrinsic stimuli and by the intrinsic susceptibility of bronchial fibroblasts to transforming growth factor‐&bgr; (TGF‐&bgr;). The specific function of gap junctions and connexins in this process has remained unknown. Here, we investigated the role of connexin43 (Cx43) in TGF‐&bgr;‐induced myofibroblastic differentiation of fibroblasts derived from bronchoscopic biopsy specimens of patients with asthma and donors without asthma. Asthmatic fibroblasts expressed considerably higher levels of Cx43 and were more susceptible to TGF‐&bgr;1‐induced myofibroblastic differentiation than were their nonasthmatic counterparts. TGF‐&bgr;1 efficiently up‐regulated Cx43 levels and activated the canonical Smad pathway in asthmatic cells. Ectopic Cx43 expression in nonasthmatic (Cx43low) fibroblasts increased their predilection to TGF‐&bgr;1‐induced Smad2 activation and fibroblast‐myofibroblast transition. Transient Cx43 silencing in asthmatic (Cx43high) fibroblasts by Cx43 small interfering RNA attenuated the TGF‐&bgr;1‐triggered Smad2 activation and myofibroblast formation. Direct interactions of Smad2 and Cx43 with &bgr;‐tubulin were demonstrated by co‐immunoprecipitation assay, whereas the sensitivity of these interactions to TGF‐&bgr;1 signaling was confirmed by Förster Resonance Energy Transfer analyses. Furthermore, inhibition of the TGF‐&bgr;1/Smad pathway attenuated TGF‐&bgr;1‐triggered Cx43 up‐regulation and myofibroblast differentiation of asthmatic fibroblasts. Chemical inhibition of gap junctional intercellular communication with 18 &agr;‐glycyrrhetinic acid did not affect the initiation of fibroblast‐myofibroblast transition in asthmatic fibroblasts but interfered with the maintenance of their myofibroblastic phenotype. Collectively, our data identified Cx43 as a new player in the feedback mechanism regulating TGF‐&bgr;1/Smad‐dependent differentiation of bronchial fibroblasts. Thus, our observations point to Cx43 as a novel profibrotic factor in asthma progression.

Proteomic and bioinformatic analysis of a nuclear intrinsically disordered proteome

UNLABELLED Intrinsically disordered proteins (IDPs) are biologically active and crucial for cell function although they do not possess defined three-dimensional architecture. IDPs are especially prevalent in eukaryotic proteomes, and large-scale experiments have shown that many IDPs are nuclear proteins. Bioinformatic analyses have also demonstrated that the vast majority of transcription factors contain extended regions of intrinsic disorder. In the current study, we isolated and functionally analyzed IDPs expressed in the nuclei of HEK293 human cells. According to the results of MS analysis followed by subsequent analysis with the bioinformatic tools IUPred and RAPID (regression-based accurate predictor of intrinsic disorder), a heat-treatment method was able to enrich the nuclear lysate in IDPs. For approximately 85% of the proteins obtained, IUPred predicted a sequence of 30 or more consecutive disordered residues (DRs), and for approximately 83% of the proteins RAPID reported a content of at least 25% DRs (compared to ~66% and 49%, respectively, for the nuclear lysate). Gene Ontology analysis in terms of molecular function revealed that the obtained fraction was generally enriched in proteins involved in the process of transcription and especially in transcription factors. We also showed experimentally that IDPs are overrepresented in the cell nucleus. SIGNIFICANCE Intrinsically disordered proteins (IDPs) are crucial cellular molecules and are especially numerous in eukaryotes. In particular, IDPs act as signaling and regulatory proteins, and impairment in their functioning may lead to serious diseases. Large-scale bioinformatic studies of IDPs have provided essential knowledge about this group of proteins. However, experimental data reflect the actual situation in living cells. Our study is the first large-scale proteomic analysis of nuclear IDPs. We showed experimentally that IDPs are overrepresented in the nucleus in comparison to the whole cell. Analysis of molecular function indicated that the nuclear intrinsically disordered proteome (IDP-ome) is enriched in proteins involved in transcription regulation and especially in transcription factors. The IDP isolation method from human cell nuclei presented in this article could be further applied in differential proteomic studies.

Morpho-physiological heterogeneity of cells within two rat prostate carcinoma cell lines AT-2 and MAT-LyLu differing in the degree of malignancy observed by cell cloning and the effects of caffeine, theophylline and papaverine upon a proportion of the clones

Analysis of the heterogeneity of clones of single cells from two established lines of the Dunning series of rat prostate carcinoma differing in the degree of malignancy, AT-2 (moderately malignant) and MAT-LyLu (highly malignant), was conducted. The results showed that not only the original tumors and primary cell cultures were heterogeneous but also the established cell lines of tumor origin. In the MAT-LyLu cell line, the clones of cells with morpho-physiological features characteristic of malignant cells dominated, whereas diverse types of clones were present in the AT-2 cell line. Differences in cell morphology (EMT), multi-layering and release from contact inhibition followed by active migration were observed and found to be correlated with increased expression of proteins involved in cancer cell invasiveness: connexin 43 and transcription factor Snail. Caffeine, theophylline and papaverine, reported to show anticancer activity in vivo, were found to decrease the proportion of clones displaying malignant cell features in the AT-2 cell line. At the tested concentrations, these compounds reversibly retarded cell growth but did not inhibit it. The results showed that the heterogeneity of cell populations within the cell lines should be taken into account in experiments carried out in vitro on established model cancer cell lines.

Connexin43high prostate cancer cells induce endothelial connexin43 up-regulation through the activation of intercellular ERK1/2-dependent signaling axis

Connexin(Cx)43 regulates the invasive potential of prostate cancer cells and participates in their extravasation. To address the role of endothelial Cx43 in this process, we analyzed Cx43 regulation in human umbilical vein endothelial cells in the proximity of Cx43high (DU-145 and MAT-LyLu) and Cx43low prostate cancer cells (PC-3 and AT-2). Endothelial Cx43 up-regulation was observed during the diapedesis of DU-145 and MAT-LyLu cells. This process was attenuated by transient Cx43 silencing in cancer cells and by chemical inhibition of ERK1/2-dependent signaling in endothelial cells. Cx43 expression in endothelial cells was insensitive to the inhibition of gap junctional intercellular coupling between Cx43high prostate cancer and endothelial cells by 18α-glycyrrhetinic acid. Instead, endothelial Cx43 up-regulation was correlated with the local contraction of endothelial cells and with their activation in the proximity of Cx43high DU-145 and MAT-LyLu cells. It was also sensitive to pro-inflammatory factors secreted by peripheral blood monocytes, such as TNFα. In contrast to Cx43low AT-2 cells, Cx43low PC-3 cells produced angioactive factors that locally activated the endothelial cells in the absence of endothelial Cx43 up-regulation. Collectively, these data show that Cx43low and Cx43high prostate cancer cells can adapt discrete, Cx43-independent and Cx43-dependent strategies of diapedesis. Our observations identify a novel strategy of prostate cancer cell diapedesis, which depends on the activation of intercellular Cx43/ERK1/2/Cx43 signaling axis at the interfaces between Cx43high prostate cancer and endothelial cells.

Invasive Cx43high sub-line of human prostate DU145 cells displays increased nanomechanical deformability

Connexin(Cx)43high cells are preferentially recruited to the invasive front of prostate cancer in vitro and in vivo. To address the involvement of Cx43 in the regulation of human prostate cancer DU145 cell invasiveness, we have analysed the nanoelasticity of invasive Cx43high sub-sets of DU145 cells by atomic force microscopy (AFM). The Cx43high DU145 cells displayed considerably higher susceptibility to mechanical distortions than the wild type DU145 cells. Transient Cx43 silencing had no effect on their elastic properties. Our data confirm the relationship between the invasive potential, Cx43 expression and nanoelasticity of the DU145 cells. However, they also show that Cx43 is not directly involved in the maintenance of DU145 invasive phenotype.

Invasive bronchial fibroblasts derived from asthmatic patients activate lung cancer A549 cells in vitro

Epidemiological data suggests that there are functional links between bronchial asthma and lung carcinogenesis. Bronchial fibroblasts serve a prominent role in the asthmatic process; however, their involvement in lung cancer progression remains unaddressed. To estimate the effect of the asthmatic microenvironment on the invasiveness of lung cancer cells, the present study compared the behavior of human non-small cell lung cancer A549 cells exposed to the signals from human bronchial fibroblasts (HBFs) derived from non-asthmatic donors (NA HBFs) and from asthmatic patients (AS HBFs). NA HBFs did not significantly affect A549 motility, whereas AS HBFs and the media conditioned with AS HBF/A549 co-cultures increased Snail-1/connexin43 expression and motility of A549 cells. In contrast to NA HBFs, which formed A549-impenetrable lateral barriers, α-SMA+ AS HBFs actively infiltrated A549 monolayers and secreted chemotactic factors that arrested A549 cells within AS HBF/A549 contact zone. However, small sub-populations of A549 cells could release from this arrest and colonize distant regions of AS HBF monolayers. These data indicated that the interactions between lung cancer cells and HBFs in asthmatic bronchi may facilitate the colonization of lung tumors by fibroblasts. It further stabilizes the tumor microenvironment and potentially facilitates collective colonization of novel bronchial loci by cancer cells. Potential mechanistic links between the asthmatic process and lung cancer progression suggest that bronchial asthma should be included in the list of potential prognostic markers for lung cancer therapy.

PO-235 Fenofibrate overcomes the drug-resistance of human prostate cancer cells

Introduction Microevolution of drug-resistant cancer cell populations is a serious obstacle for currently available cancer therapies. Reports on the inhibitory effects of fenofibrate (FF) on the growth, survival and invasiveness of prostate cancer cells suggest its potential for metronomic strategies of prostate cancer therapy. Therefore, we assessed the interference of FF with the drug-resistance of prostate cancer cells. Material and methods Additive effects of DCX/FF on the propagation, invasiveness and drug-resistance of native prostate cancer cells and of their invasive DCX-resistant variants (DU145_DCX20 and DU145_DCX50) were estimated with time-lapse and fluorescence microscopy. Flow-cytometric and cytofluorimetric tests were performed to assess the interference of FF with ATP production, pro-apoptotic and pro-autophagic pathways; and with the activity of ABC transporters. Results and discussions When administered alone, 2.5 nM DCX significantly attenuated the proliferation of native DU145 cells, but exerted no effect on the viability of DU145_DCX20 and DU145_DCX50 cells. FF (25 mM) sensitised these cells to DCX through PPARa/ROS-independent interference with intracellular ATP production and P-gp activity, as demonstrated by control assays with elacridar. Concomitantly, DCX/FF treatment considerably reduced neoplastic and invasive potential of drug-resistant DU145 cells via the activation of mTOR-sensitive suicidal autophagy signalling(s). Conclusion Our observations suggest that FF can be applied to reduce the effective doses of chemotherapeutic drugs, to attenuate their adverse effects and to inhibit the microevolution of drug-resistant cells induced by chemotherapy. Thus, it can be considered as an metronomic agent that can enhance the efficiency of long-term palliative prostate cancer treatment.