Acquired drug resistance interferes with the susceptibility of prostate cancer cells to metabolic stress.

BACKGROUND: Metformin is an inhibitor of oxidative phosphorylation that displays an array of anticancer activities. The interference of metformin with the activity of multi-drug resistance systems in cancer cells has been reported. However, the consequences of the acquired chemoresistance for the adaptative responses of cancer cells to metformin-induced stress and for their phenotypic evolution remain unaddressed. METHODS: Using a range of phenotypic and metabolic assays, we assessed the sensitivity of human prostate cancer PC-3 and DU145 cells, and their drug-resistant lineages (PC-3_DCX20 and DU145_DCX20), to combined docetaxel/metformin stress. Their adaptation responses have been assessed, in particular the shifts in their metabolic profile and invasiveness. RESULTS: Metformin increased the sensitivity of PC-3 wild-type (WT) cells to docetaxel, as illustrated by the attenuation of their motility, proliferation, and viability after the combined drug application. These effects correlated with the accumulation of energy carriers (NAD(P)H and ATP) and with the inactivation of ABC drug transporters in docetaxel/metformin-treated PC-3 WT cells. Both PC-3 WT and PC-3_DCX20 reacted to metformin with the Warburg effect; however, PC-3_DCX20 cells were considerably less susceptible to the cytostatic/misbalancing effects of metformin. Concomitantly, an epithelial-mesenchymal transition and Cx43 upregulation was seen in these cells, but not in other more docetaxel/metformin-sensitive DU145_DCX20 populations. Stronger cytostatic effects of the combined fenofibrate/docetaxel treatment confirmed that the fine-tuning of the balance between energy supply and expenditure determines cellular welfare under metabolic stress. CONCLUSIONS: Collectively, our data identify the mechanisms that underlie the limited potential of metformin for the chemotherapy of drug-resistant tumors. Metformin can enhance the sensitivity of cancer cells to chemotherapy by inducing their metabolic decoupling/imbalance. However, the acquired chemoresistance of cancer cells impairs this effect, facilitates cellular adaptation to metabolic stress, and prompts the invasive front formation.

CD44+ cells determine fenofibrate-induced microevolution of drug-resistance in prostate cancer cell populations.

Combinations of metabolic blockers (incl. fenofibrate) with chemotherapeutic drugs interfere with the drug-resistance of prostate cancer cells. However, their effect on cancer stem cells-dependent microevolution of prostate cancer malignancy remains unaddressed. Here, we hypothesize that the combined docetaxel/fenofibrate treatment prompts the selective expansion of cancer stem cells that affects the microevolution of their progenies. Accordingly, we adapted a combined in vitro/in vivo approach to identify biological and therapeutic consequences of this process. Minute subpopulations of docetaxel-resistant CD133high and/or CD44high cancer stem cell-like (SCL) cells were found in prostate cancer DU145 and PC3 cell populations. When pretreated with docetaxel, they readily differentiated into docetaxel-resistant CD44negative "bulk" cells, thus accounting for the microevolution of drug-resistant cell lineages. Combined docetaxel/fenofibrate treatment induced the generation of poly(morpho)nuclear giant cells and drug-resistant CD44high SCL cells. However, the CD44negative offspring of docetaxel- and docetaxel/fenofibrate-treated SCLs remained relatively sensitive to the combined treatment, while retaining enhanced resistance to docetaxel. Long-term propagation of drug-resistant SCL-derived lineages in the absence of docetaxel/fenofibrate resulted in their reverse microevolution toward the drug-sensitivity and invasive phenotype. Consequently, prostate tumors were able to recover from the combined docetaxel/fenofibrate stress after the initial arrest of their expansion in vivo. In conclusion, we have confirmed the potential of fenofibrate for the metronomic treatment of drug-resistant prostate tumors. However, docetaxel/fenofibrate-induced selective expansion of hyper-resistant CD44high SCL prostate cells and their "bulk" progenies prompts the microevolution of prostate tumor drug-resistance. This process can limit the implementation of metabolic chemotherapy in prostate cancer treatment.

Hydrolysis of Schiff bases with phenyl-ethynyl-phenyl system: The importance for biological and physicochemical studies.

A series of new Schiff bases containing the phenyl-ethynyl-phenyl system was synthesized and their thermal stability, photophysical and electrochemical properties were investigated. Moreover, DFT calculations were performed to obtain the optimized ground-state geometry and distribution of the HOMO and LUMO levels as well as IR spectra of the prepared compounds. It was found that, the photoluminescence of synthesized imines was negligible in all investigated organic solvents except for the PBS/ACN mixture. As was proved in further studies, this phenomenon was related to the partial hydrolysis of imines, which is the source of the fluorogenic aldehyde causing the aggreggacion incrased-emision effect. In further research, due to the susceptibility of the azomethines to partial hydrolysis, the biological activity of 2-{(E)-[4-(phenylethynyl) phenyl]imino}phenol (1b), substrate (2-aminophenol) and Cu(II)-1b complex was analyzed. The biological tests showed, that 1b (as example of imine resveratrol analogue), demonstrated its increased cytostatic activity in prostate cancer cellular system. It was proved that the non-hydrolyzed imine was crucial for the cytotoxic effect. This activity could be ascribed to its Cu(II) complexing capability as showed in our previous research.

Potentially Bioaccessible Phenolics from Mung Bean and Adzuki Bean Sprouts Enriched with Probiotic-Antioxidant Properties and Effect on the Motility and Survival of AGS Human Gastric Carcinoma Cells.

Gastric digests from mung (MBS) and adzuki (ABS) bean sprouts enriched with probiotic Lactobacillus plantarum 299v were tested for their antioxidant potential, as well as antiproliferative and antimotility properties, in human stomach cancer cells (AGS). The digest of ABS contained quercetin and kaempferol derivates, while kaempferol and apigenin derivates were dominant in MBS. Compared to the controls, the probiotic-rich sprouts had a higher antioxidant potential-by 13% and 9%, respectively. Adzuki bean sprouts decreased the viability of AGS already at low concentrations (25% motility inhibitions). MBS and ABS displayed dose-independent cytostatic effects. The ABS extracts decreased the proliferation of AGS more effectively than the MBS extracts-0.2‱ ABS exerted c.a. 70% of inhibitions. Moreover, the phytochemicals from the probiotic-rich sprouts considerably reduced this activity. The increased vinculin level, the apoptotic shape of cell nuclei, and the reduced cell motility and proliferation indicate that the extracts exhibited cytostatic and cytotoxic activity.

Cytoprotective Compounds Interfere with the Nutraceutical Potential of Bread Supplemented with Green Coffee Beans.

The proliferation and motile activity of prostate epithelial (Pnt2) and cancer cells (DU-145; PC-3) in the presence of bioavailable compounds from green coffee beans (GCB), wholemeal wheat bread (WMWB), and its GCB-fortified variant were analyzed. The considerable cytostatic and anti-invasive activity of GCB extracts was correlated with its phenolic contents. WMWB extract contained significantly lower levels of phenolics but still displayed relatively high cytostatic activity. However, the cytostatic properties of WMWB compounds were hardly augmented by 3% GCB flour supplementation. The cytoprotective activity of the WMWB compounds exerts a negative impact on the cytostatic activity of GCB compounds. These data confirm the relatively high chemopreventive potential of GCB. However, they also indicate that subtle interactions between bioavailable compounds in GCB and WMWB can negatively affect the nutraceutic potential of the fortified bread. Apparently, gastrointestinal processing differentially regulates the availability of individual compounds and affects the balance between the cytostatic and cytoprotective activity of the whole product. Our data show that comprehensive research is necessary before the fortification of a specific carrier with a specific supplement can be recommended.

Metastatic prostate cancer cells are highly sensitive to 3-bromopyruvic acid.

AIMS: 3-Bromopyruvate (3-BP), an alkylating agent and a glycolytic inhibitor, is a promising anticancer agent, which can be efficient also against multidrug-resistant cancer cells. The aim of this study was to examine how 3-BP affects the survival and mobility of rat (MAT-LyLu and AT-2) and human (DU-145 and PC-3) metastatic prostate cancer cell lines. MAIN METHODS: Cytotoxicity was estimated with Neutral Red. Cell mobility was analyzed by time-lapse microscopic monitoring of trajectories of individual cells at 5-min intervals for 6h. ATP was estimated with luciferin/luciferase and glutathione (GSH) with o-phthalaldehyde. Actin cytoskeleton was visualized with phalloidin conjugated with Atto-488. KEY FINDINGS: All metastatic prostate cell lines studied were very sensitive to 3-BP (IC50 of 4-26µM). 3-Bromopyruvate drastically reduced cell movement even at concentrations of 5-10µM after 1h treatment. This compound depleted also cellular ATP and GSH, and disrupted actin cytoskeleton. SIGNIFICANCE: The data obtained suggest that 3-BP can potentially be useful for treatment of metastatic prostate cancer and, especially, be efficient in limiting metastasis.

Fenofibrate Augments the Sensitivity of Drug-Resistant Prostate Cancer Cells to Docetaxel.

Metronomic agents reduce the effective doses and adverse effects of cytostatics in cancer chemotherapy. Therefore, they can enhance the treatment efficiency of drug-resistant cancers. Cytostatic and anti-angiogenic effects of fenofibrate (FF) suggest that it can be used for the metronomic chemotherapy of drug-resistant prostate tumors. To estimate the effect of FF on the drug-resistance of prostate cancer cells, we compared the reactions of naïve and drug-resistant cells to the combined treatment with docetaxel (DCX)/mitoxantrone (MTX) and FF. FF sensitized drug-resistant DU145 and PC3 cells to DCX and MTX, as illustrated by their reduced viability and invasive potential observed in the presence of DCX/MTX and FF. The synergy of the cytostatic activities of both agents was accompanied by the inactivation of P-gp-dependent efflux, dysfunction of the microtubular system, and induction of polyploidy in DCX-resistant cells. Chemical inhibition of PPARα- and reactive oxygen species (ROS)-dependent pathways by GW6471 and N-acetyl-L-cysteine, respectively, had no effect on cell sensitivity to combined DCX/FF treatment. Instead, we observed the signs of adenosine triphosphate (ATP) deficit and autophagy in DCX/FF-treated drug-resistant cells. Furthermore, the cells that had been permanently propagated under DCX- and DCX/FF-induced stress did not acquire DCX/FF-resistance. Instead, relatively slow proliferation of DCX-resistant cells was efficiently inhibited by FF. Collectively, our observations show that FF reduces the effective doses of DCX by interfering with the drug resistance and energy metabolism of prostate cancer cells. Concomitantly, it impairs the chemotherapy-induced microevolution and expansion of DCX/FF-resistant cells. Therefore, FF can be applied as a metronomic agent to enhance the efficiency of palliative chemotherapy of prostate cancer.

Fenofibrate Interferes with the Diapedesis of Lung Adenocarcinoma Cells through the Interference with Cx43/EGF-Dependent Intercellular Signaling.

Extravasation of circulating cancer cells is regulated by the intercellular/intracellular signaling pathways that locally impair the endothelial barrier function. Co-cultures of human umbilical vein endothelial cells (HUVECs) with lung adenocarcinoma A549 cells enabled us to identify these pathways and to quantify the effect of fenofibrate (FF) on their activity. A549 cells induced the disruption and local activation of endothelial continuum. These events were accompanied by epidermal growth factor (EGF) up-regulation in endothelial cells. Impaired A549 diapedesis and HUVEC activation were seen upon the chemical inhibition of connexin(Cx)43 functions, EGF/ERK1/2-dependent signaling, and RhoA/Rac1 activity. A total of 25 µM 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 PPARα-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.

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.

Connexin-dependent intercellular stress signaling in tissue homeostasis and tumor development.

Cellular stress responses determine tissue development, homeostasis and pathogenesis. Paracrine signaling, exchange of mechanical stimuli and intercellular transfer of small metabolites via connexin-built gap junctional channels are involved in the cellular stress detection and propagation of stress stimuli in multicellular networks. Cellular stress responses are also regulated through the activity of unpaired connexons (hemichannels) and via the intracellular interference of connexins with the cell cycle and pro-apoptotic machinery. Therefore, connexins are considered as multidirectional transmitters of the "outside-in" and "inside-out" stress signaling that are crucial for tissue homeostasis, regeneration and pathology. In particular, the disturbance of connexin function during the multi-stage process of tumor development leads to abnormal reactions of tumor cells to stress stimuli. In this review, we outline the current knowledge on the multidirectional role of connexins in the detection of stress signals. We also discuss the role of connexin-mediated intercellular transmittance of stress signals in tumour promotion, progression and metastatic cascade. HIGHLIGHTS: 1. Connexins and gap junctions protect cells from the microenvironmental stress and are involved in propagation and intracellular processing of stress signals. 2. The quality and quantity of stress stimuli, which may lead to cell adaptation or death by apoptosis, is determined by intrinsic properties of connexins and the cell phenotype. 3. Connexin deficiency increases the resistance of tumor cells to the "outside-in" stress signaling. 4. The connexin-mediated "inside-out" stress signaling participates in tumor cell invasion during the metastatic cascade.

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.

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

Pathologic accumulation of myofibroblasts in asthmatic bronchi is regulated by extrinsic stimuli and by the intrinsic susceptibility of bronchial fibroblasts to transforming growth factor-ß (TGF-ß). The specific function of gap junctions and connexins in this process has remained unknown. Here, we investigated the role of connexin43 (Cx43) in TGF-ß-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-ß1-induced myofibroblastic differentiation than were their nonasthmatic counterparts. TGF-ß1 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-ß1-induced Smad2 activation and fibroblast-myofibroblast transition. Transient Cx43 silencing in asthmatic (Cx43high) fibroblasts by Cx43 small interfering RNA attenuated the TGF-ß1-triggered Smad2 activation and myofibroblast formation. Direct interactions of Smad2 and Cx43 with ß-tubulin were demonstrated by co-immunoprecipitation assay, whereas the sensitivity of these interactions to TGF-ß1 signaling was confirmed by Förster Resonance Energy Transfer analyses. Furthermore, inhibition of the TGF-ß1/Smad pathway attenuated TGF-ß1-triggered Cx43 up-regulation and myofibroblast differentiation of asthmatic fibroblasts. Chemical inhibition of gap junctional intercellular communication with 18 α-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-ß1/Smad-dependent differentiation of bronchial fibroblasts. Thus, our observations point to Cx43 as a novel profibrotic factor in asthma progression.

Efficient and non-toxic gene delivery by anionic lipoplexes based on polyprenyl ammonium salts and their effects on cell physiology.

BACKGROUND: One of the major challenges limiting the development of gene therapy is an absence of efficient and safe gene carriers. Among the nonviral gene delivery methods, lipofection is considered as one of the most promising. In the present study, a set of cationic polyprenyl derivatives [trimethylpolyprenylammonium iodides (PTAI)] with different lengths of polyprenyl chains (from 7, 8 and 11 to 15 isoprene units) was suggested as a component of efficient DNA vehicles. METHODS: Optimization studies were conducted for PTAI in combination with co-lipid dioleoylphosphatidylethanolamine on DU145 human prostate cancer cells using: size and zeta potential measurements, confocal microscopy, the fluorescein diacetate/ethidium bromide test, cell counting, time-lapse monitoring of cell movement, gap junctional intercellular coupling analysis, antimicrobial activity assay and a red blood cell hemolysis test. RESULTS: The results obtained show that the lipofecting activity of PTAI allows effective transfection of plasmid DNA complexed in negatively-charged lipoplexes of 200-500 nm size into cells without significant side effects on cell physiology (viability, proliferation, morphology, migration and gap junctional intercellular coupling). Moreover, PTAI-based vehicles exhibit a potent bactericidal activity against Staphylococcus aureus and Escherichia coli. The developed anionic lipoplexes are safe towards human red blood cell membranes, which are not disrupted in their presence. CONCLUSIONS: The developed carriers constitute a group of promising lipofecting agents of a new type that can be utilized as effective lipofecting agents in vitro and they are also an encouraging basis for in vivo applications.

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

OBJECTIVE: Extravasation of circulating cancer cells is an important step of the metastatic cascade and a potential target for anti-cancer strategies based on vasoprotective drugs. Reports on anti-cancer effects of fenofibrate (FF) prompted us to analyze its influence on the endothelial barrier function during prostate cancer cell diapedesis. RESEARCH DESIGN AND METHODS: In vitro co-cultures of endothelial cells with cancer cells imitate the 'metastatic niche' in vivo. We qualitatively and quantitatively estimated the effect of 25 µM FF on the events which accompany prostate carcinoma cell diapedesis, with the special emphasis on endothelial cell mobilization. RESULTS: Fenofibrate attenuated cancer cell diapedesis via augmenting endothelial cell adhesion to the substratum rather than through the effect on intercellular communication networks within the metastatic niche. The inhibition of endothelial cell motility was accompanied by the activation of PPARα-dependent and PPARα-independent reactive oxygen species signaling, Akt and focal adhesion kinase (FAK) phosphorylation, in the absence of cytotoxic effects in endothelial cells. CONCLUSIONS: Fenofibrate reduces endothelial cell susceptibility to the paracrine signals received from prostate carcinoma cells, thus inhibiting endothelial cell mobilization and reducing paracellular permeability of endothelium in the metastatic niche. Our data provide a mechanistic rationale for extending the clinical use of FF and for the combination of this well tolerated vasoactive drug with the existing multidrug regimens used in prostate cancer therapy.