Investigation of Mitochondrial Metabolic Response to Doxorubicin in Prostate Cancer Cells: An NADH, FAD and Tryptophan FLIM Assay

Alam, Shagufta Rehman; Wallrabe, Horst; Švindrych, Zdeněk; Chaudhary, Ajay Kumar; Christopher, Kathryn G.; Chandra, Dhyan; Periasamy, Ammasi

doi:10.1038/s41598-017-10856-3

Cited by 85 publications

(95 citation statements)

References 47 publications

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“…It is becoming increasingly apparent that organelles “talk” to each other to regulate key cellular functions (Murley & Nunnari, ; Quirós et al , ; Valm et al , ), but the mechanisms involved and potential roles in disease pathogenesis remain largely unexplored. Using a 2P‐FLIM assay for label‐free imaging of mitochondrial activity in live cells (Lakowicz, ; Alam et al , ; Wallrabe et al , ) (Fig A and B), we describe here a previously unknown form of inter‐organelle communication, from lysosomes to mitochondria, and reveal details of its function and mechanism of action, and how its dysregulation may represent a seminal process in AD pathogenesis and a defining molecular defect in tuberous sclerosis.…”

Section: Discussionmentioning

confidence: 94%

A novel lysosome‐to‐mitochondria signaling pathway disrupted by amyloid‐β oligomers

et al. 2018

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The mechanisms of mitochondrial dysfunction in Alzheimer's disease are incompletely understood. Using two‐photon fluorescence lifetime microscopy of the coenzymes, NADH and NADPH, and tracking brain oxygen metabolism with multi‐parametric photoacoustic microscopy, we show that activation of lysosomal mechanistic target of rapamycin complex 1 (mTORC1) by insulin or amino acids stimulates mitochondrial activity and regulates mitochondrial DNA synthesis in neurons. Amyloid‐β oligomers, which are precursors of amyloid plaques in Alzheimer's disease brain and stimulate mTORC1 protein kinase activity at the plasma membrane but not at lysosomes, block this Nutrient‐induced Mitochondrial Activity (NiMA) by a mechanism dependent on tau, which forms neurofibrillary tangles in Alzheimer's disease brain. NiMA was also disrupted in fibroblasts derived from two patients with tuberous sclerosis complex, a genetic disorder that causes dysregulation of lysosomal mTORC1. Thus, lysosomal mTORC1 couples nutrient availability to mitochondrial activity and links mitochondrial dysfunction to Alzheimer's disease by a mechanism dependent on the soluble building blocks of the poorly soluble plaques and tangles.

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Section: Discussionmentioning

confidence: 94%

A novel lysosome‐to‐mitochondria signaling pathway disrupted by amyloid‐β oligomers

et al. 2018

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“…The non-phosphorylated form of NADH acts as an electron donor in the mitochondrial electron transport chain. Typically, in cancer cells with a positive response to chemotherapy an increase of the relative contribution of protein-bound NAD(P)H and of the mean fluorescence lifetime of NAD(P)H is observed, which could be a consequence of glycolysis inhibition or the upregulation of mitochondrial respiration, or both (4,6,9). The fluorescence lifetime of NADH depends significantly on the state of the cofactor (whether "free" or "protein-bound") (3).…”

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confidence: 99%

“…Previous studies have demonstrated that fluorescence lifetime imaging (FLIM) of NAD(P)H is capable of detecting early cellular responses to anticancer treatments (4)(5)(6)(7)(8). Typically, in cancer cells with a positive response to chemotherapy an increase of the relative contribution of protein-bound NAD(P)H and of the mean fluorescence lifetime of NAD(P)H is observed, which could be a consequence of glycolysis inhibition or the upregulation of mitochondrial respiration, or both (4,6,9). The majority of these studies were carried out on in vitro models-monolayer cells or 3D tumor spheroids.…”

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In vivo metabolic and SHG imaging for monitoring of tumor response to chemotherapy

et al. 2018

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Although chemotherapy remains one of the main types of treatment for cancer, treatment failure is a frequent occurrence, emphasizing the need for new approaches to the early assessment of tumor response. The aim of this study was to search for indicators based on optical imaging of cellular metabolism and of collagen in tumors in vivo that enable evaluation of their response to chemotherapy. The study was performed on a mouse colorectal cancer model with the use of cisplatin, paclitaxel, and irinotecan. The metabolic activity of the tumor cells was assessed using fluorescence lifetime imaging of the metabolic cofactor reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H. Second harmonic generation (SHG) imaging was used to analyze the extent and properties of collagen within the tumors. We detected an early decrease in the free/bound NAD(P)H ratio in all treated tumors, indicating a shift toward a more oxidative metabolism. Monitoring of collagen showed an early increase in the amount of collagen followed by an increase in the extent of its orientation in tumors treated with cisplatin and paclitaxel, and decrease in collagen content in the case of irinotecan. Our study suggests that changes in cellular metabolism and fibrotic stroma organization precede morphological alterations and tumor size reduction, and that this indicates that NAD(P)H and collagen can be considered as intrinsic indicators of the response to treatment. This is the first time that these parameters have been investigated in tumors in vivo in the course of chemotherapy with drugs having different mechanisms of action.COLORECTAL cancer is one of the most common cancers in the world. The current standard treatments for colorectal cancer include surgery, radiation therapy, and chemotherapy. Treatment options and recommendations depend on several factors, such as the type and stage of the cancer, possible side effects, and the patient's preferences and overall health.In standard clinical practice neoadjuvant chemotherapy is used for patients with stage II and III сolorectal cancer to facilitate resection of those tumors that were once considered unresectable. Adjuvant chemotherapy is recommended for Stage III colorectal cancer after resection to prevent tumor recurrence and the development of metastases (1). When resection of all known tumor sites is no longer possible or advisable, palliative care is offered. Current guidelines on the chemotherapy of сolorectal cancer prescribe the use of platinum drugs (oxaliplatin, cisplatin, or similar), or irinotecan in various combinations with a fluoropyrimidine (capecitabine or 5-fluorouracil). Additionally, paclitaxel can be used for chemotherapy of colorectal cancer in combination with 5-fluorouracil.

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“…FAD and NAD(P)H cycle from reduced to oxidized forms, thus facilitating redox reactions during energy production (e.g., oxidative phosphorylation and glycolysis). Examples of this include diagnostic imaging to reveal boundaries of demarcation between normal and cancerous tissues or measurement of optical redox ratios (29,32,(34)(35)(36)(37)(38)(39)(40)(41). The signals that result vary depending on the NAD(P)H reduced or oxidized form (20,22), location inside of the cell (e.g., mitochondria, cytosol) (23), and binding status with enzymes (e.g., malate, lactate, isocitrate, and succinate dehydrogenases) (24,25).…”

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confidence: 99%

“…Yet for many studies, the bound enzyme might depend on the variation in the metabolism and is of lesser importance compared to the purpose for detecting changes in the NAD(P)H autofluorescence. Examples of this include diagnostic imaging to reveal boundaries of demarcation between normal and cancerous tissues or measurement of optical redox ratios (29,32,(34)(35)(36)(37)(38)(39)(40)(41).…”

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confidence: 99%

Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time‐resolved flow cytometry

et al. 2018

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Autofluorescence from the intracellular metabolite, NAD(P)H, is a biomarker that is widely used and known to reliably screen and report metabolic activity as well as metabolic fluctuations within cells. As a ubiquitous endogenous fluorophore, NAD(P)H has a unique rate of fluorescence decay that is altered when bound to coenzymes. In this work we measure the shift in the fluorescence decay, or average fluorescence lifetime (1–3 ns), of NAD(P)H and correlate this shift to changes in metabolism that cells undergo during apoptosis. Our measurements are made with a flow cytometer designed specifically for fluorescence lifetime acquisition within the ultraviolet to violet spectrum. Our methods involved culture, treatment, and preparation of cells for cytometry and microscopy measurements. The evaluation we performed included observations and quantification of the changes in endogenous emission owing to the induction of apoptosis as well as changes in the decay kinetics of the emission measured by flow cytometry. Shifts in NAD(P)H fluorescence lifetime were observed as early as 15 min post‐treatment with an apoptosis inducing agent. Results also include a phasor analysis to evaluate free to bound ratios of NAD(P)H at different time points. We defined the free to bound ratios as the ratio of ‘short‐to‐long’ (S/L) fluorescence lifetime, where S/L was found to consistently decrease with an increase in apoptosis. With a quantitative framework such as phasor analysis, the short and long lifetime components of NAD(P)H can be used to map the cycling of free and bound NAD(P)H during the early‐to‐late stages of apoptosis. The combination of lifetime screening and phasor analyses provides the first step in high throughput metabolic profiling of single cells and can be leveraged for screening and sorting for a range of applications in biomedicine. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

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Investigation of Mitochondrial Metabolic Response to Doxorubicin in Prostate Cancer Cells: An NADH, FAD and Tryptophan FLIM Assay

Cited by 85 publications

References 47 publications

A novel lysosome‐to‐mitochondria signaling pathway disrupted by amyloid‐β oligomers

A novel lysosome‐to‐mitochondria signaling pathway disrupted by amyloid‐β oligomers

In vivo metabolic and SHG imaging for monitoring of tumor response to chemotherapy

Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time‐resolved flow cytometry

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