flavin-adenine-dinucleotide and Neoplasms

Small molecules targeting the flavin adenine dinucleotide (FAD)-dependent histone lysine demethylase LSD family have displayed therapeutic promise against various diseases. Nine clinical candidates targeting the classic demethylase-dependent functions of the LSD family are currently being investigated for treating cancers, neurodegenerative diseases, etc. Moreover, targeting noncatalytic functions of LSDs also represents an emerging strategy for treating human diseases. In this Perspective, we provide full structural and functional landscape of the LSD family and action modes of different types of LSD inhibitors including natural products, peptides, and synthetic compounds, aiming to reveal new druggable space for the design of new LSD inhibitors. Particularly, we first classify these inhibitors into three types based on their unique binding modes. Additionally, the strategies targeting the demethylase-independent functions of LSDs are also briefly discussed. This Perspective may benefit the discovery of new LSD inhibitors for probing LSD biology and/or treating human diseases.

Topics: Drug Discovery; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Histone Demethylases; Humans; Neoplasms; Peptides

Optical imaging using the endogenous fluorescence of metabolic cofactors has enabled nondestructive examination of dynamic changes in cell and tissue function both in vitro and in vivo. Quantifying NAD(P)H and FAD fluorescence through an optical redox ratio and fluorescence lifetime imaging (FLIM) provides sensitivity to the relative balance between oxidative phosphorylation and glucose catabolism. Since its introduction decades ago, the use of NAD(P)H imaging has expanded to include applications involving almost every major tissue type and a variety of pathologies. Recent Advances: This review focuses on the use of two-photon excited fluorescence and NAD(P)H fluorescence lifetime techniques in cancer, neuroscience, tissue engineering, and other biomedical applications over the last 5 years. In a variety of cancer models, NAD(P)H fluorescence intensity and lifetime measurements demonstrate a sensitivity to the Warburg effect, suggesting potential for early detection or high-throughput drug screening. The sensitivity to the biosynthetic demands of stem cell differentiation and tissue repair processes indicates the range of applications for this imaging technology may be broad.. As the number of applications for these fluorescence imaging techniques expand, identifying and characterizing additional intrinsic fluorophores and chromophores present in vivo will be vital to accurately measure and interpret metabolic outcomes. Understanding the full capabilities and limitations of FLIM will also be key to future advances.. Future work is needed to evaluate whether a combination of different biochemical and structural outcomes using these imaging techniques can provide complementary information regarding the utilization of specific metabolic pathways.

Topics: Animals; Cells; Flavin-Adenine Dinucleotide; Humans; Molecular Imaging; NAD; Neoplasms; Optical Imaging; Signal Transduction

NAD(P)H quinone oxidoreductase 1 (NQO1) catalyses the two electron reduction of quinones and a wide range of other organic compounds. Its physiological role is believed to be partly the reduction of free radical load in cells and the detoxification of xenobiotics. It also has non-enzymatic functions stabilising a number of cellular regulators including p53. Functionally, NQO1 is a homodimer with two active sites formed from residues from both polypeptide chains. Catalysis proceeds via a substituted enzyme mechanism involving a tightly bound FAD cofactor. Dicoumarol and some structurally related compounds act as competitive inhibitors of NQO1. There is some evidence for negative cooperativity in quinine oxidoreductases which is most likely to be mediated at least in part by alterations to the mobility of the protein. Human NQO1 is implicated in cancer. It is often over-expressed in cancer cells and as such is considered as a possible drug target. Interestingly, a common polymorphic form of human NQO1, p.P187S, is associated with an increased risk of several forms of cancer. This variant has much lower activity than the wild-type, primarily due to its substantially reduced affinity for FAD which results from lower stability. This lower stability results from inappropriate mobility of key parts of the protein. Thus, NQO1 relies on correct mobility for normal function, but inappropriate mobility results in dysfunction and may cause disease.

Topics: Catalytic Domain; Dicumarol; Enzyme Inhibitors; Enzyme Stability; Flavin-Adenine Dinucleotide; Gene Expression; Humans; Models, Molecular; Mutation; NAD(P)H Dehydrogenase (Quinone); Neoplasms; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Multimerization

Traditional antitumor drugs inhibit the proliferation and metastasis of tumour cells by restraining the replication and expression of DNA. These drugs are usually highly cytotoxic. They kill tumour cells while also cause damage to normal cells at the same time, especially the hematopoietic cells that divide vigorously. Patients are exposed to other serious situations such as a severe infection caused by a decrease in the number of white blood cells. Energy metabolism is an essential process for the survival of all cells, but differs greatly between normal cells and tumour cells in metabolic pathways and metabolic intermediates. Whether this difference could be used as new therapeutic target while reducing damage to normal tissues is the topic of this paper. In this paper, we introduce five major metabolic intermediates in detail, including acetyl-CoA, SAM, FAD, NAD

Topics: Acetyl Coenzyme A; Antineoplastic Agents; Carcinogenesis; Cell Proliferation; Disease Progression; Flavin-Adenine Dinucleotide; Humans; NAD; Neoplasm Invasiveness; Neoplasms; S-Adenosylmethionine; Tetrahydrofolates

Succinate dehydrogenase (SDH) also known as complex II or succinate:quinone oxidoreductase is an enzyme involved in both oxidative phosphorylation and tricarboxylic acid cycle; the processes that generate energy. SDH is a multi-subunit enzyme which requires a series of proteins for its proper assembly at several steps. This enzyme has medical significance as there is a broad range of human diseases from cancers to neurodegeneration related to SDH malfunction. Some of these disorders have recently been linked to defective assembly factors, reinvigorating further research in this area. Apart from that this enzyme has agricultural importance as many fungicides have been/will be designed targeting specifically this enzyme in plant fungal pathogens. In addition, we speculate it might be possible to design novel fungicides specifically targeting fungal assembly factors. Considering the medical and agricultural implications of SDH, the aim of this review is an overview of the SDH assembly factors and critical analysis of controversial issues around them.

Topics: Animals; Citric Acid Cycle; Coenzymes; Flavin-Adenine Dinucleotide; Fungal Proteins; Fungi; Fungicides, Industrial; Gene Expression; Humans; Membrane Transport Proteins; Mitochondria; Molecular Chaperones; Neoplasms; Neurodegenerative Diseases; Oxidative Phosphorylation; Plants; Protein Isoforms; Protein Subunits; Proteins; Saccharomyces cerevisiae Proteins; Succinate Dehydrogenase

Lysine-specific demethylase 1 (LSD1), the first identified histone demethylase, was belonged to the superfamily of the flavin adenine dinucleotide (FAD)-dependent amine oxidases. LSD1 specifically demethylates mono- or dimethylated dimethylated histone H3 lysine4 (H3K4) and H3 lysine 9 (H3K9) via a redox process. Recently evidences showed that LSD1 played an important role in a broad spectrum of biological processes, including cell proliferation, adipogenesis, spermatogenesis, chromosome segregation and embryonic development. Furthermore, LSD1 also could promote progress of tumor by inhibiting the tumor suppressor activity of p53. To date, as a potential drug for discovering anti-tumor drugs, the medical significance of LSD1 inhibitors have been greatly appreciated. Here, we reviewed the remarkable progress being made in understanding of LSD1, mainly on its structure, basic function and medical application in tumor therapy.

Topics: Adipogenesis; Animals; Cell Proliferation; Chromosome Segregation; Drug Discovery; E2F1 Transcription Factor; Embryonic Stem Cells; Epithelial-Mesenchymal Transition; Flavin-Adenine Dinucleotide; Histone Demethylases; Histones; Humans; Mice; Molecular Targeted Therapy; Neoplasms; Phosphorylation; Retinoblastoma Protein; Tumor Suppressor Protein p53

Statistical studies have demonstrated that various agents may reduce the risk of cancer's development. One of them is activity of flavin-dependent enzymes such as flavin-containing monooxygenase (FMO)(GS-OX1), FAD-dependent 5,10-methylenetetrahydrofolate reductase and flavin-dependent monoamine oxidase. In the last decade, many papers concerning their structure, reaction mechanism and role in the cancer prevention were published. In our work, we provide a more in-depth analysis of flavin-dependent enzymes and their contribution to the cancer prevention. We present the actual knowledge about the glucosinolate synthesized by flavin-containing monooxygenase (FMO)(GS-OX1) and its role in cancer prevention, discuss the influence of mutations in FAD-dependent 5,10-methylenetetrahydrofolate reductase on the cancer risk, and describe FAD as an important cofactor for the demethylation of histons. We also present our views on the role of riboflavin supplements in the prevention against cancer.

Topics: 5,10-Methylenetetrahydrofolate Reductase (FADH2); Animals; Flavin-Adenine Dinucleotide; Humans; Monoamine Oxidase; Neoplasm Proteins; Neoplasms; Riboflavin; Risk Factors; Vitamin B Complex

In recent years fluorescence microscopy has become a widely used tool for tissue imaging and spectroscopy. Optical techniques, based on both linear and non-linear excitation, have been broadly applied to imaging and characterization of biological tissues. Among fluorescence techniques used in tissue imaging applications, in recent years two and three-photon excited fluorescence have gained increased importance because of their high-resolution deep tissue imaging capability inside optically turbid samples. The main limitation of steady-state fluorescence imaging techniques consists in providing only morphological information; functional information is not detectable without technical improvements. A spectroscopic approach, based on lifetime measurement of tissue fluorescence, can provide functional information about tissue conditions, including its environment, red-ox state, and pH, and hence physiological characterization of the tissue under investigation. Measurement of the fluorescence lifetime is a very important issue for characterizing a biological tissue. Deviation of this property from a control value can be taken as an indicator of disorder and/or malignancy in diseased tissues. Even if much work on this topic has still to be done, including the interpretation of fluorescence lifetime data, we believe that this methodology will gain increasing importance in the field of biophotonics. In this paper, we review methodologies, potentials and results obtained by using fluorescence lifetime imaging microscopy for the investigation of biological tissues.

Topics: Animals; Flavin-Adenine Dinucleotide; Humans; Microscopy, Fluorescence; NAD; Neoplasms; Spectrometry, Fluorescence

Topics: Adenosine Triphosphate; Animals; Azo Compounds; Carcinogens; Carcinoma, Hepatocellular; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Humans; Liver Neoplasms; Male; Neoplasm Transplantation; Neoplasms; Neoplasms, Experimental; Nucleotidyltransferases; Rats; Riboflavin; Riboflavin Deficiency

Topics: Adult; Animals; Child; Congenital Abnormalities; Female; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Homeostasis; Humans; Intestinal Absorption; Liver; Neoplasms; Neoplasms, Experimental; Pregnancy; Pregnancy Complications; Rats; Riboflavin; Riboflavin Deficiency; Thyroid Hormones

This work aims to develop a clinically compatible system that can perform breast tissue analysis in a more time efficient process than conventional histopathological assessment. The potential for such a system to be used in vivo in the operating room or surgical suite to improve patient outcome is investigated.. In this work, 80 matched pairs of invasive ductal carcinoma and adjacent normal breast tissue were measured in a combined time-resolved fluorescence and diffuse reflectance (DA) system. Following measurement, the fluorescence intensity of collagen and flavin adenine dinucleotide (FAD); the fluorescence lifetime of collagen, nicotinamide adenine dinucleotide (NADH), and FAD; the DA; absorption coefficient; and reduced scattering coefficient were extracted. Samples then underwent histological processing and H&E staining to classify composition as tumor, fibroglandular, and/or adipose tissue.. Statistically significant differences in the collagen and FAD fluorescence intensity, collagen and FAD fluorescence lifetime, DA, and scattering coefficient were found between each tissue group. The NADH fluorescence lifetime and absorption coefficient were statistically different between the tumor and fibroglandular groups, and the tumor and adipose groups. While many breast tissue analysis studies label fibroglandular and adipose together as "normal" breast tissue, this work indicates that some differences between tumor and fibroglandular tissue are not the same as differences between tumor and adipose tissue. Observations of the reduced scatter coefficient may also indicate further classification to include fibro-adipose may be necessary. Future work would benefit from the additional tissue classification.. With observable differences in optical parameters between the three tissue types, this system shows promise as a breast analysis tool in a clinical setting. With further work involving samples of mixed composition, this combined system could potentially be used intraoperatively for rapid margin assessment.

Topics: Breast; Breast Neoplasms; Female; Flavin-Adenine Dinucleotide; Humans; NAD; Neoplasms; Spectrometry, Fluorescence

Topics: Biomimetic Materials; Biomimetics; Energy Metabolism; Flavin-Adenine Dinucleotide; Humans; Hypothermia; NAD; Neoplasms; Tumor Microenvironment

Protein phosphorylation is a common phenomenon in human flavoproteins although the functional consequences of this site-specific modification are largely unknown. Here, we evaluated the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects as well as in the structural stability of the antioxidant and disease-associated human flavoprotein NQO1 using biophysical and biochemical methods. In vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by H/D exchange in different ligation states provided structural insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. We show that site-specific phosphorylation of human NQO1 may cause pleiotropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease. Our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale.

Topics: Antioxidants; Flavin-Adenine Dinucleotide; Flavoproteins; Humans; Mutation; NAD(P)H Dehydrogenase (Quinone); Neoplasms; Phosphorylation; Proteasome Endopeptidase Complex; Protein Binding

Accessible tools are needed for rapid, non-destructive imaging of patient-derived cancer organoid (PCO) treatment response to accelerate drug discovery and streamline treatment planning for individual patients.. To segment and track individual PCOs with wide-field one-photon redox imaging to extract morphological and metabolic variables of treatment response.. Redox imaging of the endogenous fluorophores, nicotinamide dinucleotide (NADH), nicotinamide dinucleotide phosphate (NADPH), and flavin adenine dinucleotide (FAD), was used to monitor the metabolic state and morphology of PCOs. Redox imaging was performed on a wide-field one-photon epifluorescence microscope to evaluate drug response in two colorectal PCO lines. An automated image analysis framework was developed to track PCOs across multiple time points over 48 h. Variables quantified for each PCO captured metabolic and morphological response to drug treatment, including the optical redox ratio (ORR) and organoid area.. The ORR (NAD(P)H/(FAD + NAD(P)H)) was independent of PCO morphology pretreatment. Drugs that induced cell death decreased the ORR and growth rate compared to control. Multivariate analysis of redox and morphology variables identified distinct PCO subpopulations. Single-organoid tracking improved sensitivity to drug treatment compared to pooled organoid analysis.. Wide-field one-photon redox imaging can monitor metabolic and morphological changes on a single organoid-level, providing an accessible, non-destructive tool to screen drugs in patient-matched samples.

Topics: Flavin-Adenine Dinucleotide; Humans; Microscopy, Fluorescence, Multiphoton; NAD; Neoplasms; Optical Imaging; Organoids; Oxidation-Reduction

Over a quarter million of protein phosphorylation sites have been identified so far, although the effects of site-specific phosphorylation on protein function and stability, as well as their possible impact in the phenotypic manifestation in genetic diseases are vastly unknown. We investigated here the effects of phosphorylating S82 in human NADP(H):quinone oxidoreductase 1, a representative example of disease-associated flavoprotein in which protein stability is coupled to the intracellular flavin levels. Additionally, the cancer-associated P187S polymorphism causes inactivation and destabilization of the enzyme. By using extensive in vitro and in silico characterization of phosphomimetic S82D mutations, we showed that S82D locally affected the flavin binding site of the wild-type (WT) and P187S proteins thus altering flavin binding affinity, conformational stability and aggregation propensity. Consequently, the phosphomimetic S82D may destabilize the WT protein intracellularly by promoting the formation of the degradation-prone apo-protein. Noteworthy, WT and P187S proteins respond differently to the phosphomimetic mutation in terms of intracellular stability, further supporting differences in molecular recognition of these two variants by the proteasomal degradation pathway. We propose that phosphorylation could have critical consequences on stability and function of human flavoproteins, important for our understanding of genotype-phenotype relationships in their related genetic diseases.

Topics: Amino Acid Sequence; Binding Sites; Cell Line; Computational Biology; Flavin-Adenine Dinucleotide; Humans; Kinetics; Molecular Dynamics Simulation; Mutation; NAD(P)H Dehydrogenase (Quinone); Neoplasms; Phosphorylation; Protein Binding; Protein Denaturation; Protein Stability; Proteome; Proteomics

Multiphoton FLIM microscopy offers many opportunities to investigate processes in live cells, tissue and animal model systems. For redox measurements, FLIM data is mostly published by cell mean values and intensity-based redox ratios. Our method is based entirely on FLIM parameters generated by 3-detector time domain microscopy capturing autofluorescent signals of NAD(P)H, FAD and novel FLIM-FRET application of Tryptophan and NAD(P)H-a2%/FAD-a1% redox ratio. Furthermore, image data is analyzed in segmented cells thresholded by 2 × 2 pixel Regions of Interest (ROIs) to separate mitochondrial oxidative phosphorylation from cytosolic glycolysis in a prostate cancer cell line. Hundreds of data points allow demonstration of heterogeneity in response to intervention, identity of cell responders to treatment, creating thereby different sub-populations. Histograms and bar charts visualize differences between cells, analyzing whole cell versus mitochondrial morphology data, all based on discrete ROIs. This assay method allows to detect subtle differences in cellular and tissue responses, suggesting an advancement over means-based analyses.

Topics: Animals; Cytosol; Disease Models, Animal; Doxorubicin; Flavin-Adenine Dinucleotide; Glucose; Heterografts; Humans; Microscopy, Fluorescence; Mitochondria; Molecular Imaging; NAD; NADP; Neoplasms; Oxidation-Reduction; Oxidative Phosphorylation

Paclitaxel, a widely used antimicrotubular agent, predominantly eliminates rapidly proliferating cancer cells, while slowly proliferating and quiescent cells can survive the treatment, which is one of the main reasons for tumor recurrence and non-responsiveness to the drug. To improve the efficacy of chemotherapy, biomarkers need to be developed to enable monitoring of tumor responses. In this study we considered the auto-fluorescent metabolic cofactors NAD(P)H and FAD as possible indicators of cancer cell response to therapy with paclitaxel. It was found that, among the tested parameters (the fluorescence intensity-based redox ratio FAD/NAD(P)H, and the fluorescence lifetimes of NAD(P)H and FAD), the fluorescence lifetime of NAD(P)H is the most sensitive in tracking the drug response, and is capable of indicating heterogeneous cellular responses both in cell monolayers and in multicellular tumor spheroids. We observed that metabolic reorganization to a more oxidative state preceded the morphological manifestation of cell death and developed faster in cells that were more responsive to the drug. Our results suggest that noninvasive, label-free monitoring of the drug-induced metabolic changes by noting the NAD(P)H fluorescence lifetime is a valuable approach to characterize the responses of cancer cells to anti-cancer treatments and, therefore, to predict the effectiveness of chemotherapy.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Biomarkers; Flavin-Adenine Dinucleotide; Humans; Microscopy, Fluorescence, Multiphoton; NADP; Neoplasms; Oxidation-Reduction; Paclitaxel; Tumor Cells, Cultured

Once whole-genome sequencing has reached the clinical practice, a main challenge ahead is the high-throughput and accurate prediction of the pathogenicity of genetic variants. However, current prediction tools do not consider explicitly a well-known property of disease-causing mutations: their ability to affect multiple functional sites distant in the protein structure. Here we carried out an extensive biophysical characterization of fourteen mutant variants at two cancer-associated sites of the enzyme NQO1, a paradigm of multi-functional protein. We showed that the magnitude of destabilizing effects, their molecular origins (structural vs. dynamic) and their efficient propagation through the protein structure gradually led to functional perturbations at different sites. Modulation of these structural perturbations also led to switches between molecular phenotypes. Our work supports that experimental and computational perturbation analyses would improve our understanding of the molecular basis of many loss-of-function genetic diseases as well as our ability to accurately predict the pathogenicity of genetic variants in a high-throughput fashion.

Topics: Flavin-Adenine Dinucleotide; Humans; Models, Molecular; Mutation; NAD(P)H Dehydrogenase (Quinone); Neoplasms; Phenotype; Protein Conformation; Protein Folding

Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73α oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73α, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention.

Topics: Allosteric Regulation; Flavin-Adenine Dinucleotide; Genetic Predisposition to Disease; Humans; Mutation; NAD(P)H Dehydrogenase (Quinone); Neoplasms; Polymorphism, Single Nucleotide; Proteasome Endopeptidase Complex; Protein Binding; Protein Conformation; Protein Domains; Protein Interaction Domains and Motifs; Tumor Protein p73; Tumor Suppressor Protein p53

Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S, and to develop new pharmacological therapies to rescue this function.

Topics: Binding Sites; Caco-2 Cells; Crystallography, X-Ray; Dicumarol; Enzyme Stability; Flavin-Adenine Dinucleotide; HCT116 Cells; HeLa Cells; Humans; NAD(P)H Dehydrogenase (Quinone); Neoplasms; Polymorphism, Single Nucleotide; Protein Binding; Protein Conformation; Protein Multimerization

Alteration in the cellular energy metabolism is a principal feature of tumors. An important role in modifying cancer cell metabolism belongs to the cancer-associated fibroblasts. However, the regulation of their interaction has been poorly studied to date. In this study we monitored the metabolic status of both cell types by using the optical redox ratio and the fluorescence lifetimes of the metabolic co-factors NAD(P)H and FAD, in addition to the intracellular pH and the hydrogen peroxide levels in the cancer cells, using genetically encoded sensors. In the co-culture of human cervical carcinoma cells HeLa and human fibroblasts we observed a metabolic shift from oxidative phosphorylation toward glycolysis in cancer cells, and from glycolysis toward OXPHOS in fibroblasts, starting from Day 2 of co-culturing. The metabolic switch was accompanied by hydrogen peroxide production and slight acidification of the cytosol in the cancer cells in comparison with that of the corresponding monoculture. Therefore, our HeLa-huFb system demonstrated metabolic behavior similar to Warburg type tumors. To our knowledge, this is the first time that these 3 parameters have been investigated together in a model of tumor-stroma co-evolution. We propose that determination of the start-point of the metabolic alterations and understanding of the mechanisms of their realization can open a new ways for cancer treatment.

Topics: Cell Communication; Coculture Techniques; Fibroblasts; Flavin-Adenine Dinucleotide; Fluorescence; HeLa Cells; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Intracellular Space; NADP; Neoplasms; Oxidation-Reduction

Subpopulations of cells that escape anti-cancer treatment can cause relapse in cancer patients. Therefore, measurements of cellular-level tumor heterogeneity could enable improved anti-cancer treatment regimens. Cancer exhibits altered cellular metabolism, which affects the autofluorescence of metabolic cofactors NAD(P)H and FAD. The optical redox ratio (fluorescence intensity of NAD(P)H divided by FAD) reflects global cellular metabolism. The fluorescence lifetime (amount of time a fluorophore is in the excited state) is sensitive to microenvironment, particularly protein-binding. High-resolution imaging of the optical redox ratio and fluorescence lifetimes of NAD(P)H and FAD (optical metabolic imaging) enables single-cell analyses. In this study, mice with FaDu tumors were treated with the antibody therapy cetuximab or the chemotherapy cisplatin and imaged in vivo two days after treatment. Results indicate that fluorescence lifetimes of NAD(P)H and FAD are sensitive to early response (two days post-treatment, P<.05), compared with decreases in tumor size (nine days post-treatment, P<.05). Frequency histogram analysis of individual optical metabolic imaging parameters identifies subpopulations of cells, and a new heterogeneity index enables quantitative comparisons of cellular heterogeneity across treatment groups for individual variables. Additionally, a dimensionality reduction technique (viSNE) enables holistic visualization of multivariate optical measures of cellular heterogeneity. These analyses indicate increased heterogeneity in the cetuximab and cisplatin treatment groups compared with the control group. Overall, the combination of optical metabolic imaging and cellular-level analyses provide novel, quantitative insights into tumor heterogeneity.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cetuximab; Cisplatin; Flavin-Adenine Dinucleotide; Humans; Microscopy, Fluorescence, Multiphoton; NADP; Neoplasms; Oxidation-Reduction; Single-Cell Analysis; Time Factors; Tumor Burden; Xenograft Model Antitumor Assays

There were two primary objectives of this study: (1) to determine whether treatment of a tumour site with systemically administered thermally sensitive liposomes and local hyperthermia (HT) for triggered release would have dual anti-tumour effect on the primary heated tumour as well as an unheated secondary tumour in a distant site, and (2) to determine the ability of non-invasive optical spectroscopy to predict treatment outcome. The optical end points studied included drug levels, metabolic markers flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide phosphate (NAD(P)H), and physiological markers (total haemoglobin (Hb) and Hb oxygen saturation) before and after treatment.. Mice were inoculated with SKOV3 human ovarian carcinoma in both hind legs. One tumour was selected for local hyperthermia and subsequent systemic treatment. There were four treatment groups: control, DOXIL (non-thermally sensitive liposomes containing doxorubicin), and two different thermally sensitive liposome formulations containing doxorubicin. Optical spectroscopy was performed prior to therapy, immediately after treatment, and 6, 12, and 24 h post therapy.. Tumour growth delay was seen with DOXIL and the thermally sensitive liposomes in the tumours that were heated, similar to previous studies. Tumour growth delay was also seen in the opposing tumour in the thermally sensitive liposome-treated groups. Optical spectroscopy demonstrated correlation between growth delay, doxorubicin (DOX) levels, and changes of NAD(P)H from baseline levels. Hb and Hb saturation were not correlated with growth delay.. The study demonstrated that thermally sensitive liposomes affect the primary heated tumour as well as systemic efficacy. Non-invasive optical spectroscopy methods were shown to be useful in predicting efficacy at early time points post-treatment.

Topics: Animals; Antibiotics, Antineoplastic; Cell Line, Tumor; Combined Modality Therapy; Doxorubicin; Female; Flavin-Adenine Dinucleotide; Hemoglobins; Humans; Hyperthermia, Induced; Mice; NADP; Neoplasms; Oxygen; Polyethylene Glycols; Spectrum Analysis; Treatment Outcome; Tumor Burden

Correlation coefficient mapping has been applied to intrinsic fluorescence spectra of colonic tissue for the purpose of cancer diagnosis. Fluorescence emission spectra were collected of 57 colonic tissue sites in a range of 4 physiological conditions: normal (29), hyperplastic (2), adenomatous (5), and cancerous tissues (21). The sample-sample correlation was used to examine the ability of correlation coefficient mapping to determine tissue disease state. The correlation coefficient map indicates two main categories of samples. These categories were found to relate to disease states of the tissue. Sensitivity, selectivity, predictive value positive, and predictive value negative for differentiation between normal tissue and all other categories were all above 92%. This was found to be similar to, or higher than, tissue classification using existing methods of data reduction. Wavelength-wavelength correlation among the samples highlights areas of importance for tissue classification. The two-dimensional correlation map reveals absorption by NADH and hemoglobin in the samples as negative correlation, an effect not obvious from the one-dimensional fluorescence spectra alone. The integrity of tissue was examined in a time series of spectra of a single tissue sample taken after tissue resection. The wavelength-wavelength correlation coefficient map shows the areas of significance for each fluorophore and their relation to each other. NADH displays negative correlation to collagen and FAD, from the absorption of emission or fluorescence resonance energy transfer. The wavelength-wavelength correlation map for the decay set also clearly shows that there are only three fluorophores of importance in the samples, by the well-defined pattern of the map. The sample-sample correlation coefficient map reveals the changes over time and their impact on tissue classification. Correlation coefficient mapping proves to be an effective method for sample classification and cancer detection.

Topics: Adenomatous Polyposis Coli; Algorithms; Collagen; Colon; Colonic Neoplasms; Data Interpretation, Statistical; Flavin-Adenine Dinucleotide; Hemoglobins; Humans; Hyperplasia; NAD; Neoplasms; Sensitivity and Specificity; Spectrometry, Fluorescence; Time Factors

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C in tumor cells. Thus, surprisingly, the same enzymatic reaction that protects normal cells activates cytotoxic drugs used in cancer chemotherapy. The 2.1-A crystal structure of rat liver quinone reductase reveals that the folding of a portion of each monomer is similar to that of flavodoxin, a bacterial FMN-containing protein. Two additional portions of the polypeptide chains are involved in dimerization and in formation of the two identical catalytic sites to which both monomers contribute. The crystallographic structures of two FAD-containing enzyme complexes (one containing NADP+, the other containing duroquinone) suggest that direct hydride transfers from NAD(P)H to FAD and from FADH2 to the quinone [which occupies the site vacated by NAD(P)H] provide a simple rationale for the obligatory two-electron reductions involving a ping-pong mechanism.

Topics: Amino Acid Sequence; Animals; Benzoquinones; Binding Sites; Crystallography, X-Ray; Flavin-Adenine Dinucleotide; Flavoproteins; Liver; Models, Chemical; Models, Molecular; Molecular Conformation; Molecular Sequence Data; NAD(P)H Dehydrogenase (Quinone); NADP; Neoplasms; Oxidation-Reduction; Prodrugs; Rats; Triazines

Topics: Adenine; Azo Compounds; Carbon Dioxide; Flavin-Adenine Dinucleotide; Liver; Neoplasms; p-Dimethylaminoazobenzene