Mitochondrial activity and metabolic reprogramming influence the phenotype of cancer cells and resistance to targeted therapy. We previously established that an insulin-like growth factor 1 ...(IGF-1)-inducible mitochondrial UTP carrier (PNC1/SLC25A33) promotes cell growth. This prompted us to investigate whether IGF signaling is essential for mitochondrial maintenance in cancer cells and whether this contributes to therapy resistance. Here we show that IGF-1 stimulates mitochondrial biogenesis in a range of cell lines. In MCF-7 and ZR75.1 breast cancer cells, IGF-1 induces peroxisome proliferator–activated receptor γ coactivator 1β (PGC-1β) and PGC-1α–related coactivator (PRC). Suppression of PGC-1β and PRC with siRNA reverses the effects of IGF-1 and disrupts mitochondrial morphology and membrane potential. IGF-1 also induced expression of the redox regulator nuclear factor-erythroid-derived 2-like 2 (NFE2L2 alias NRF-2). Of note, MCF-7 cells with acquired resistance to an IGF-1 receptor (IGF-1R) tyrosine kinase inhibitor exhibited reduced expression of PGC-1β, PRC, and mitochondrial biogenesis. Interestingly, these cells exhibited mitochondrial dysfunction, indicated by reactive oxygen species expression, reduced expression of the mitophagy mediators BNIP3 and BNIP3L, and impaired mitophagy. In agreement with this, IGF-1 robustly induced BNIP3 accumulation in mitochondria. Other active receptor tyrosine kinases could not compensate for reduced IGF-1R activity in mitochondrial protection, and MCF-7 cells with suppressed IGF-1R activity became highly dependent on glycolysis for survival. We conclude that IGF-1 signaling is essential for sustaining cancer cell viability by stimulating both mitochondrial biogenesis and turnover through BNIP3 induction. This core mitochondrial protective signal is likely to strongly influence responses to therapy and the phenotypic evolution of cancer.
A new intracellular O(2) (icO(2)) sensing probe is presented, which comprises a nanoparticle (NP) formulation of a cationic polymer Eudragit RL-100 and a hydrophobic phosphorescent dye ...Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP). Using the time-resolved fluorescence (TR-F) plate reader set-up, cell loading was investigated in detail, particularly the effects of probe concentration, loading time, serum content in the medium, cell type, density, etc. The use of a fluorescent analogue of the probe in conjunction with confocal microscopy and flow cytometry analysis, revealed that cellular uptake of the NPs is driven by nonspecific energy-dependent endocytosis and that the probe localizes inside the cell close to the nucleus. Probe calibration in biological environment was performed, which allowed conversion of measured phosphorescence lifetime signals into icO(2) concentration (μM). Its analytical performance in icO(2) sensing experiments was demonstrated by monitoring metabolic responses of mouse embryonic fibroblast cells under ambient and hypoxic macroenvironment. The NP probe was seen to generate stable and reproducible signals in different types of mammalian cells and robust responses to their metabolic stimulation, thus allowing accurate quantitative analysis. High brightness and photostability allow its use in screening experiments with cell populations on a commercial TR-F reader, and for single cell analysis on a fluorescent microscope.
The gastrointestinal microbiota is emerging as a unique and inexhaustible source for metabolites with potential to modulate G‐protein coupled receptors (GPCRs). The ghrelin receptor growth hormone ...secretagogue receptor (GHSR)‐1a is a GPCR expressed throughout both the gut and the brain and plays a crucial role in maintaining energy balance, metabolism, and the central modulation of food intake, motivation, reward, and mood. To date, few studies have investigated the potential of the gastrointestinal microbiota and its metabolites to modulate GPCR signaling. Here we investigate the ability of short‐chain fatty acids (SCFAs), lactate, and different bacterial strains, including Bifidobacterium and Lactobacillus genera, to modulate GHSR‐1a signaling. We identify, for what is to our knowledge the first time, a potent effect of microbiota‐derived metabolites on GHSR‐1a signaling with potential significant consequences for host metabolism and physiology. We show that SCFAs, lactate, and bacterial supernatants are able to attenuate ghrelin‐mediated signaling through the GHSR‐1a. We suggest a novel route of communication between the gut microbiota and the host via modulation of GHSR‐1a receptor signaling. Together, this highlights the emerging therapeutic potential in the exploration of the microbiota metabolome in the specific targeting of key GPCRs, with pleiotropic actions that span both the CNS and periphery.—Torres‐Fuentes, C., Golubeva, A. V., Zhdanov, A. V., Wallace, S., Arboleya, S., Papkovsky, D. B., El Aidy, S., Ross, P., Roy, B. L., Stanton, C., Dinan, T. G., Cryan, J. F., Schellekens, H. Short‐chain fatty acids and microbiota metabolites attenuate ghrelin receptor signaling. FASEB J. 33, 13546‐13559 (2019). www.fasebj.org
•Oxygen respirometry is used to quantify the effects of antimicrobials on bacteria.•The effects of Lauroyl Arginate Ethyl Ester on different samples are investigated.•The assay allows simple ...quantification of toxicity and calculation of EC50.•The platform offers rapid, automated and parallel analysis of multiple samples.•It supersedes traditional testing methods.
Microbial spoilage and foodborne diseases cause significant economic and productivity losses. There is a need for novel approaches and antimicrobial treatments to extend shelf life of products, improve quality and microbial safety, and reduce spoilage and waste, and new assessment methods. Traditional assays for testing the toxicity of antimicrobials are time consuming, labour intensive, give crude estimations of toxicity, and cannot analyse complex samples such as crude food homogenates. Using a model antimicrobial compound Lauroyl Arginate Ethyl Ester (LAE), we describe a new analytical methodology based on optical oxygen sensing and respirometry to investigate the effects of various antimicrobial treatments on pure bacterial cultures, meat microbiota and packaged meat samples. By measuring and analysing the time profiles of O2 probe signal (phosphorescence lifetime) in incubating test samples, we were able to visualise the toxic effects of LAE on the different bacterial specie, generate time and dose response curves, calculate EC50 and generation times of test organisms. The new multi-parametric toxicity testing platform allows for rapid, automated and parallel analysis of multiple samples under a range of antimicrobial concentrations and conditions.
Abstract
Monitoring of tissue O
2
is essential for cancer development and treatment, as hypoxic tumour regions develop resistance to radio- and chemotherapy. We describe a minimally invasive ...technique for the monitoring of tissue oxygenation in developing grafted tumours, which uses the new phosphorescence lifetime based Tpx3Cam imager. CT26 cells stained with a near-infrared emitting nanoparticulate O
2
probe NanO2-IR were injected into mice to produce grafted tumours with characteristic phosphorescence. The tumours were allowed to develop for 3, 7, 10 and 17 days, with O
2
imaging experiments performed on live and euthanised animals at different time points. Despite a marked trend towards decreased O
2
in dead animals, their tumour areas produced phosphorescence lifetime values between 44 and 47 µs, which corresponded to hypoxic tissue with 5–20 μM O
2
. After the O
2
imaging in animals, confocal Phosphorescence Lifetime Imaging Microscopy was conducted to examine the distribution of NanO2-IR probe in the tumours, which were excised, fixed and sliced for the purpose. The probe remained visible as bright and discrete ‘islands’ embedded in the tumour tissue until day 17 of tumour growth. Overall, this O
2
macro-imaging method using NanO2-IR holds promise for long-term studies with grafted tumours in live animal models, providing quantitative 2D mapping of tissue O
2
.
Optochemical sensors are actively used in cell analysis, however existing systems have limitations with respect to their robustness and analytical performance. We have developed advanced multimodal ...and multi-parametric solid-state pH sensors for cell analysis based on hydrophobic protonable metal-free porphyrins, such as octaethylporphine (OEP), and octaethylporphine-ketone (OEPK), as fluorescent pH indicators. The internally referenced ratiometric intensity and nanosecond lifetime-based versions of the pH sensors were developed and also multiplexed with the O2 sensors based on phosphorescent PtOEP dye. We optimised the key parameters of the pH sensor, including: dye encapsulation matrix, the type and concentration of proton transfer reagent, measurement pH range and pKa, dye concentrations and cross-talk with the O2 sensor. Subsequently, sensor coatings were deposited on common substrates used in cell analysis (96-well plates), fine-tuned for their operational performance, dual O2/pH sensing functionally and the ability to measure Extracellular Acidification (ECAR) and Oxygen Consumption (OCR) rates in biological samples containing cells. The optimised sensors with stable internally referenced calibrations, convenient spectral characteristics and low cytotoxicity, were demonstrated with cultured cells and 3D spheroid structures, measuring their ECAR, OCR and responses to stimulation. These pH and dual pH/O2 sensors are well-suited for detailed metabolic studies of biological samples on widely available laboratory equipment.
•New high performance optochemical pH sensors and dual pH/O2 sensors have been developed.•Fluorescent porphyrin pH indicator dye and phosphorescence Pt-poprhyrin O2 indicator dye embedded in plasticised PVC matrix produced sensor coatings .•The sensors operate in internally referenced ratiometric intensity, fluorescence and phosphorescence lifetime based sensing modes.•Sensor characteristics were optimised for cell analysis applications, and demonstrated in ECA and OCR measurements with mammalian cells.
Cell analysis by optochemical sensing represents large and important niche in life and biomedical sciences. We present advanced multi-modal, multi-analyte sensing platform and dedicated materials for ...cell analysis based on the substituted phosphorescent Pt(II)- or Pd(II)-porphyrin indicator dyes bearing dual O2 and pH sensing functionality (MePor-SB). The study includes screening of the different host matrices for the sensor, synthesis and evaluation of new MePor-SB derivatives with altered protonation behaviour, development of the ratiometric version of the pH sensor, assessment of photoluminescent signal enhancement options, deposition of sensor coatings on common cell analysis substrates and their demonstration of sensor performance in Oxygen Consumption Rate (OCR) and Extracellular Acidification (ECA) measurements with relevant cell models. The main outcomes include the elaboration of the structure-function relationships for this biosensor system, development of the self-referenced tandem O2/pH and OCR/ECA sensing system which enables calibration-free operation, demonstration of the sensor operation on common plastic substrates in cell analysis, benchmarking against the existing platforms and real-life experiments with cells. This new sensing platform shows potential for wide practical use.
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•An advanced multi-modal, multi-analyte optochemical sensing platform for cell analysis is described.•It uses dedicated phosphorescent porphyrin dyes and sensor coatings with dual O2 and pH sensing functionality.•The sensors provide self-referencing, quantitative readout of O2 concentration and pH.•It allows simultaneous high-throughput analysis of oxygen consumption and extracellular acidification rates in cultured cells.
Abnormal accumulation of oncometabolite fumarate and succinate is associated with inhibition of mitochondrial function and carcinogenesis. By competing with α-ketoglutarate, oncometabolites also ...activate hypoxia inducible factors (HIFs), which makes oncometabolite mimetics broadly utilised in hypoxia research. We found that dimethyloxalylglycine (DMOG), a synthetic analogue of α-ketoglutarate, commonly used to induce HIF signalling, inhibits O2 consumption in cancer cell lines HCT116 and PC12, well before activation of HIF pathways. A rapid suppression of cellular respiration was accompanied by a decrease in histone H4 lysine 16 acetylation and not abolished by double knockdown of HIF-1α and HIF-2α. In agreement with this, production of NADH and state 3 respiration in isolated mitochondria were down-regulated by the de-esterified DMOG derivative, N-oxalylglycine. Exploring the roles of DMOG as a putative inhibitor of glutamine/α-ketoglutarate metabolic axis, we found that the observed suppression of OxPhos and compensatory activation of glycolytic ATP flux make cancer cells vulnerable to combined treatment with DMOG and inhibitors of glycolysis. On the other hand, DMOG treatment impairs deep cell deoxygenation in 3D tissue culture models, demonstrating a potential to relieve functional stress imposed by deep hypoxia on tumour, ischemic or inflamed tissues. Indeed, using a murine model of colitis, we found that O2 availability in the inflamed colon tissue rapidly increased after application of DMOG, which could contribute to the known therapeutic effect of this compound. Overall, our results provide new insights into the relationship between mitochondrial function, O2 availability, metabolic reprogramming and associated diseases.
•DMOG treatment rapidly decreases NADH levels and inhibits respiration in cancer cells.•NOG, the de-esterified DMOG, inhibits state 3 respiration in isolated mitochondria.•Decrease in respiration precedes and does not depend on activation of HIF signalling.•Combined treatment with DMOG and inhibitors of glycolysis depletes cellular ATP.•DMOG increases oxygenation of 3D cell culture and inflamed mouse colon tissue.
Oxygenation condition at the cellular level is a critical factor in tissue physiology and common pathophysiological states including cancer, metabolic disorders, ischemia-reperfusion injury and ...inflammation. O
and ROS signalling and hypoxia research are rapidly growing areas spanning life and biomedical sciences, but still many current cell and tissue models and experimental set ups lack physiological relevance, particularly precise control of cellular O
. Quenched-phosphorescence O
sensing enables implementation of such in situ control of cellular O
and the creation of physiological conditions in respiring samples analysed in vitro. The advantages of optical O
sensing are the non-invasive, contactless, real-time, quantitative monitoring of O
concentration, which can be performed in the gas or liquid phase, macroscopically or microscopically, by point measurement or in imaging mode, with sub-cellular spatial resolution, in a flexible manner and with various cell and tissue models. Significantly, this same technology can also be used to probe the metabolism of cells and tissue under specific oxygenation conditions and their responses to changing conditions. Here we describe the range of available O
sensing systems and tools, their analytical capabilities, uses in cell/tissue physiology and hypoxia research, and strategies for integration in routine experimental procedures.
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