flavin-adenine-dinucleotide and Prostatic-Neoplasms

Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture autofluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. Among these intrinsic fluorophores, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD)-essential coenzymes in cellular respiration-have been used as intrinsic fluorescent biomarkers for metabolic states in cancer and other pathologies. Traditional FLIM imaging for NAD(P)H, FAD, and in particular fluorescence lifetime redox ratio (FLIRR) requires a sequential multiwavelength excitation to avoid spectral bleed-through (SBT). This sequential imaging complicates image acquisition, may introduce motion artifacts, and reduce temporal resolution. Testing several two-photon excitation wavelengths in combination with optimized emission filters, we have proved a FLIM imaging protocol, allowing simultaneous image acquisition with a single 800-nm wavelength excitation for NADH and FAD with negligible SBT. As a first step, standard NADH and FAD single and mixed solutions were tested that mimic biological sample conditions. After these optimization steps, the assay was applied to two prostate cancer live cell lines: African-American (AA) and Caucasian-American (LNCaP), used in our previous publications. FLIRR result shows that, in cells, the 800-nm two-photon excitation wavelength is suitable for NADH and FAD FLIM imaging with negligible SBT. While NAD(P)H signals are decreased, sufficient photons are present for accurate lifetime fitting and FAD signals are measurably increased at lower laser power, compared with the common 890-nm excitation conditions. This single wavelength excitation allows a simplification of NADH and FAD FLIM imaging data analysis, decreasing the total imaging time. It also avoids motion artifacts and increases temporal resolution. This simplified assay will also make it more suitable to be applied in a clinical setting.

Topics: Antibiotics, Antineoplastic; Doxorubicin; Flavin-Adenine Dinucleotide; Fluorescence; Humans; Male; Microscopy, Fluorescence, Multiphoton; NADP; Photons; Prostatic Neoplasms; Tumor Cells, Cultured

In prostate cancer, bone is a frequent site of metastasis; however, the molecular mechanisms of this tumor tropism remain unclear. Here, we integrate a microfluidic coculture platform with multi-photon imaging based techniques to assess both phenotypic cell behavior and FAD fluorescence intensity and fluorescence lifetime in the same cell. This platform combines two independent assays normally performed with two different cell populations into a single device, allowing us to simultaneously assess both phenotypic cell behavior and enzyme activity. We observed that the osteotropic prostate cancer cell line (C4-2B), when in a coculture with bone marrow stromal cells (MC3T3-E1), has increased protrusive phenotype and increased total and protein-bound FAD compared to its parent cell line (LNCaP). We hypothesized that an increase in ROS-generating APAO activity may be responsible for these effects, and found that the effects were decreased in the presence of the antioxidant N-Acetyl Cysteine (NAC). This suggests that an ROS-related signaling mechanism at the bone metastatic site may be correlated with and play a role in increased invasion of metastasizing prostate cancer cells. The studies performed using this combined platform will lead to new insights into the mechanisms that drive prostate cancer metastasis.

Topics: Acetylcysteine; Cell Line, Tumor; Coculture Techniques; Flavin-Adenine Dinucleotide; Humans; Male; Mesenchymal Stem Cells; Microfluidics; Microscopy, Fluorescence, Multiphoton; Prostatic Neoplasms; Reactive Oxygen Species

Stokes shift spectroscopy (S3) is an emerging approach toward cancer detection. The goal of this paper is to evaluate the diagnostic potential of the S3 technique for the detection and characterization of normal and cancerous prostate tissues. Pairs of cancerous and normal prostate tissue samples were taken from each of eight patients. Stokes shift spectra were measured by simultaneously scanning both the excitation and emission wavelengths while keeping a fixed wavelength interval Δλ=20  nm between them. The salient features of this technique are the highly resolved emission peaks and significant spectral differences between the normal and cancerous prostate tissues, as observed in the wavelength region of 250 to 600 nm. The Stokes shift spectra of cancerous and normal prostate tissues revealed distinct peaks around 300, 345, 440, and 510 nm, which are attributed to tryptophan, collagen, NADH, and flavin, respectively. To quantify the spectral differences between the normal and cancerous prostate tissues, two spectral ratios were computed. The findings revealed that both ratio parameters R1=I297/I345 and R2=I307/I345 were excellent diagnostic ratio parameters giving 100% specificity and 100% sensitivity for distinguishing cancerous tissue from the normal tissue. Our results demonstrate that S3 is a sensitive and specific technique for detecting cancerous prostate tissue.

Topics: Collagen; Flavin-Adenine Dinucleotide; Humans; Male; NAD; Pilot Projects; Prostatic Neoplasms; Sensitivity and Specificity; Spectrometry, Fluorescence; Tryptophan

Riboflavin (Rf) and its metabolic analogs flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential for normal cellular growth and function. Their intracellular transport is regulated by the riboflavin carrier protein (RCP), which has been shown to be over-expressed by metabolically active cancer cells. Therefore, FAD-decorated ultrasmall superparamagnetic iron oxide nanoparticles (FAD USPIO) were developed as the first carrier-protein-targeted molecular MR agents for visualizing tumor metabolism. FAD USPIO were synthesized using an adsorptive, fluorescent and non-polymeric coating method, and their physicochemical properties were characterized using TEM, SEM, FTIR, MRI and fluorescence spectroscopy. In vitro analyses showed the biocompatibility of FAD USPIO, and confirmed that they were strongly and specifically taken up by cancer (LnCap) and endothelial (HUVEC) cells. In vivo molecular MRI together with subsequent histological validation finally demonstrated that FAD USPIO efficiently accumulate in tumors and tumor blood vessels, indicating that RCP-targeted diagnostic nanoparticles are interesting new materials for the assessment of vascular metabolism in tumors.

Topics: Animals; Cell Line; Cell Line, Tumor; Dextrans; Flavin-Adenine Dinucleotide; Fluorescent Dyes; Humans; Magnetic Resonance Imaging; Magnetite Nanoparticles; Male; Membrane Transport Proteins; Mice; Mice, Nude; Molecular Imaging; Nanoparticles; Prostate; Prostatic Neoplasms

Stokes Shift (SS) Spectroscopy (SSS) of normal and abnormal breast and prostate tissues were studied. SS spectra is measured by simultaneously scanning both the excitation and emission wavelengths while keeping a fixed wavelength interval of Δλ = 20 nm. Characteristic, highly resolved peaks and significant spectral differences between normal and different pathological tissues of breast and prostate tissues were observed. The SS spectra of normal and different pathological breast and prostate tissues show the distinct peaks around 300, 350, 450, 500 and 600 nm may be attributed to tryptophan, collagen, NADH, flavin and porphyrin, respectively. Results of the current study demonstrate that the SS spectral changes due to tryptophan, collagen, hemoglobin, NADH, FAD and porphyrin have good diagnostic potential; therefore can be targeted as native tumor markers.

Topics: Adult; Breast Neoplasms; Carcinoma, Ductal, Breast; Female; Fibroadenoma; Flavin-Adenine Dinucleotide; Humans; Male; Middle Aged; NAD; Pilot Projects; Porphyrins; Prostatic Hyperplasia; Prostatic Neoplasms; Spectrometry, Fluorescence; Spectrophotometry; Young Adult