ABSTRACT
Aberrant expression of glycans, i.e., oligosaccharide moiety covalently attached to proteins or lipids, is characteristic of various cancers, including urothelial ones. The binding of lectins to glycans is classified as molecular recognition, which makes lectins a strong tool for understanding their role in developing diseases. Here, we present a quantitative approach to tracing glycan-lectin interactions in cells, from the initial to the steady phase of adhesion. The cell adhesion was measured between urothelial cell lines (non-malignant HCV29 and carcinoma HT1376 and T24 cells) and lectin-coated surfaces. Depending on the timescale, single-cell force spectroscopy, and adhesion assays conducted in static and flow conditions were applied. The obtained results reveal that the adhesion of urothelial cells to two specific lectins, i.e., phytohemagglutinin-L and wheat germ agglutinin, was specific and selective. Thus, these lectins can be applied to selectively capture, identify, and differentiate between cancer types in a label-free manner. These results open up the possibility of designing lectin-based biosensors for diagnostic or prognostic purposes and developing strategies for drug delivery that could target cancer-associated glycans.
Subject(s)
Lectins , Urinary Bladder Neoplasms , Humans , Lectins/metabolism , Urinary Bladder Neoplasms/metabolism , Phytohemagglutinins/pharmacology , Wheat Germ Agglutinins , Polysaccharides/metabolismABSTRACT
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.
Subject(s)
Antineoplastic Agents , Cytostatic Agents , Metformin , Prostatic Neoplasms , Humans , Male , Docetaxel/pharmacology , Docetaxel/therapeutic use , Taxoids/pharmacology , Taxoids/therapeutic use , Cytostatic Agents/pharmacology , Cytostatic Agents/therapeutic use , Drug Resistance, Neoplasm , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Prostatic Neoplasms/metabolism , Metformin/pharmacology , Metformin/therapeutic use , Stress, PhysiologicalABSTRACT
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.
ABSTRACT
Recruiting circulating cells based on interactions between surface receptors and corresponding ligands holds promise for capturing cells with specific adhesive properties. Our study investigates the adhesion of skin cells to specific lectins, particularly focusing on advancements in lectin-based biosensors with diagnostic potential. We explore whether we can successfully capture normal skin (melanocytes and keratinocytes) and melanoma (WM35, WM115, WM266-4) cells in a low-shear flow environment by coating surfaces with lectins. Specifically, we coated surfaces with Dolichos biflorus (DBA) and Maackia Amurensis (MAL) lectins, which were used to detect and capture specific skin cells from the flow of cell mixture. Alterations in glycan expression (confirmed by fluorescent microscopy) demonstrated that DBA binds predominantly to normal skin cells, while MAL interacts strongly with melanoma cells. Assessing adhesion under static and dynamic low-shear stress conditions (up to 30 mPa) underscores the reliability of DBA and MAL as markers for discriminating specific cell type. Melanocytes and keratinocytes adhere to DBA-coated surfaces, while melanoma cells prefer MAL-coated surfaces. A comprehensive analysis encompassing cell shape, cytoskeleton, and focal adhesions shows the independence of our approach from the inherent characteristics of cells, thus demonstrating its robustness. Our results carry practical implications for lectin-biosensor designs, emphasizing the significance of glycan-based discrimination of pathologically altered cells. Combined with microfluidics, it demonstrates the value of cell adhesion as a discriminant of cancer-related changes, with potential applications spanning diagnostics, therapeutic interventions, and advanced biomedical technologies.
Subject(s)
Biosensing Techniques , Cell Adhesion , Skin Neoplasms , Humans , Biosensing Techniques/methods , Biosensing Techniques/instrumentation , Glycosylation , Skin Neoplasms/pathology , Melanoma/pathology , Melanoma/diagnosis , Keratinocytes/cytology , Skin/pathology , Skin/chemistry , Lectins/chemistry , Lectins/metabolism , Cell Line, Tumor , Melanocytes/cytology , Melanocytes/metabolism , Microfluidics/methods , Microfluidic Analytical Techniques/instrumentationABSTRACT
The present study investigates silicone transfer occurring during microcontact printing (µCP) of lectins with polydimethylsiloxane (PDMS) stamps and its impact on the adhesion of cells. Static adhesion assays and single-cell force spectroscopy (SCFS) are used to compare adhesion of nonmalignant (HCV29) and cancer (HT1376) bladder cells, respectively, to high-affinity lectin layers (PHA-L and WGA, respectively) prepared by physical adsorption and µCP. The chemical composition of the µCP lectin patterns was monitored by time-of-flight secondary ion mass spectrometry (ToF-SIMS). We show that the amount of transferred silicone in the µCP process depends on the preprocessing of the PDMS stamps. It is revealed that silicone contamination within the patterned lectin layers inhibits the adhesion of bladder cells, and the work of adhesion is lower for µCP lectins than for drop-cast lectins. The binding capacity of microcontact printed lectins was larger when the PDMS stamps were treated with UV ozone plasma as compared to sonication in ethanol and deionized water. ToF-SIMS data show that ozone-based treatment of PDMS stamps used for µCP of lectin reduces the silicone contamination in the imprinting protocol regardless of stamp geometry (flat vs microstructured). The role of other possible contributors, such as the lectin conformation and organization of lectin layers, is also discussed.
ABSTRACT
Aqueous microgels are distinct entities of soft matter with mechanical signatures that can be different from their macroscopic counterparts due to confinement effects in the preparation, inherently made to consist of more than one domain (Janus particles) or further processing by coating and change in the extent of crosslinking of the core. Motivated by the importance of the mechanical properties of such microgels from a fundamental point, but also related to numerous applications, we provide a perspective on the experimental strategies currently available and emerging tools being explored. Albeit all techniques in principle exploit enforcing stress and observing strain, the realization differs from directly, as, e.g., by atomic force microscope, to less evident in a fluid field combined with imaging by a high-speed camera in high-throughput strategies. Moreover, the accompanying analysis strategies also reflect such differences, and the level of detail that would be preferred for a comprehensive understanding of the microgel mechanical properties are not always implemented. Overall, the perspective is that current technologies have the capacity to provide detailed, nanoscopic mechanical characterization of microgels over an extended size range, to the high-throughput approaches providing distributions over the mechanical signatures, a feature not readily accessible by atomic force microscopy and micropipette aspiration.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Connexins/metabolism , Neoplasms/etiology , Stress, Physiological/physiology , Animals , Cell Communication , Cell Transformation, Neoplastic/metabolism , Gap Junctions/metabolism , Homeostasis , Humans , Neoplasms/metabolism , Neoplasms/pathology , Signal TransductionABSTRACT
This study examines the nutraceutical (phenolics content, antioxidant activity, biological activity) and nutritional potential (starch and protein digestibility) of wheat pasta supplemented with 1-4% of powdered parsley leaves. Compared to the control, the potentially bioaccessible fraction of pasta fortified with 4% parsley leaves was characterized by 67% increased phenolics content, a 146% higher antiradical ability and 220% additional reducing power. Elevation of these parameters in fortified pasta was accompanied by an augmentation of its antiproliferative effect on carcinoma cells, which confirms their biological relevance. Supplementation of pasta had no significant effect on starch digestibility, while negatively affecting protein digestibility (a reduction by about 20% for pasta with a 4% supplement). Electrophoretic and chromatographic analyses indicated the presence of phenolic interactions with proteins and/or digestive enzymes. Fortification improved the nutraceutical and nutritional potential of the studied pasta; however, the final effect is made by many factors, including phenolics-food matrix interactions.
Subject(s)
Food, Fortified , Nutritive Value , Petroselinum , Triticum , Dietary Supplements , Humans , Petroselinum/chemistry , Phenols/analysis , Plant Leaves/chemistry , Triticum/chemistryABSTRACT
Numerous adverse effects limit the applicability of mitoxantrone for the treatment of drug-resistant tumors, including carcinosarcoma. Here, we estimated the additive effects of mitoxantrone and curcumin, a plant-derived biomolecule isolated from Curcuma longa, on the neoplastic and invasive potential of carcinosarcoma cells in vitro. Curcumin augmented the cytostatic, cytotoxic and anti-invasive effects of mitoxantrone on the Walker-256 cells. It also strengthened the inhibitory effects of mitoxantrone on the motility of drug-resistant Walker-256 cells that had retained viability after a long-term mitoxantrone/curcumin treatment. Thus, curcumin reduces the effective doses of mitoxantrone and augments its interference with the invasive potential of drug-resistant carcinosarcoma cells.