Metabolic study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone to the enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in vitro in human bronchial epithelial cells using chiral capillary electrophoresis

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) with one chiral center at the carbinol is a major metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). As tobacco specific N-nitrosamines (TSNAs), NNK and NNAL are the most pulmonary carcinogens in tobacco products and smoke. In this paper, a chiral CE method modified with highly sulfated β-cyclodextrin (S-β-CD) was developed to investigate the stereoselective formation of NNAL from NNK in vitro in normal human bronchial epithelial (NHBE) cells. Combined with solid phase extraction (SPE) of the cell samples, NNK and NNAL enantiomers were baseline separated under the proposed CE conditions, with satisfactory recoveries (72.5-113% for NNK and (±)-NNAL) and low limits of detection (LOD, 2.5-3 μg/mL for NNK and (±)-NNAL). The cytotoxicity of NNK in NHBE cells was investigated through the cell counting kit (CCK) assay and proved to be highly dependent on the NNK's concentration. The metabolic results obtained from CE analysis demonstrated that NNK was preferentially metabolized to (+)-NNAL through carbonyl reduction. Meanwhile, the ratio of [(+)-NNAL]/[(-)-NNAL] was independent of NHBE cells' incubation time with NNK, but could be changed according to the original incubation concentration of NNK. This chiral CE method could be useful for the study on toxicology and metabolic transformations of related TSNAs.     

5-Aminolevulinic acid-induced fluorescence in bronchial tumours: dependency on the patterns of tumour invasion

5-ALA-induced protoporphyrin IX (PPIX) fluorescence kinetics was quantified by fluorescence microscopy in three-dimensional organ co-cultures of human bronchial epithelium, which were infiltrated by four different lung tumour cell lines (EPLC-M31, LCLC-103H, NCI-H125 and NCI-H841). Corresponding fluorescence measurements were performed in monolayer cultures of these tumour cell lines and BEAS-2B cells as a model for normal bronchial epithelium by flow cytometry. Significant differences of fluorescence intensities (FI) between the tumours were detected in organ co-cultures as well as in single cell measurements. Relative FI values in organ co-cultures (FI(EPLC-32M1)>FI(LCLC-H103)>FI(NCI-H125)>FI(NCI-H841)) did not correspond to the measurements in single cells (FI(LCLC-H103)>FI(NCI-H125)>FI(NCI-H841)>FI(EPLC-32M1)). Histology of organ co-cultures revealed different patterns of invasion and tumour cell densities depending on the tumour type. After correction of FI in the co-cultures to tumour cell density the correlation coefficient for fluorescence values between both models increased considerably. Thus, additionally to distinctive features of 5-ALA metabolism, patterns of tumour invasion may be a factor determining 5-ALA-induced fluorescence. Considering these results, a pronounced heterogeneity of 5-ALA-induced fluorescence might be expected in different bronchial tumours in vivo. This could interfere with the diagnostic reliability of 5-ALA-induced fluorescence for early tumour detection.     

Validation of a Fast and Simple HPLC-UV Method for the Quantification of Adenosine Phosphates in Human Bronchial Epithelial Cells

A new HPLC method for the simultaneous quantitative analysis of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) was developed and validated. ATP, ADP, and AMP were extracted from human bronchial epithelial cells with a rapid extraction procedure and separated with a C18 column (3 × 150 mm, 2.7 µm) using isocratic elution with a mobile phase consisting of 50 mM of potassium hydrogen phosphate (pH 6.80). The absorbance was monitored at 254 nm. The calibration curves were linear in 0.2 to 10 µM, selective, precise, and accurate. This method allowed us to quantify the nucleotides from two cell models: differentiated NHBE primary cells grown at the air–liquid interface (ALI) and BEAS-2B cell line. Our study highlighted the development of a sensitive, simple, and green analytical method that is faster and less expensive than other existing methods to measure ATP, ADP, and AMP and can be carried out on 2D and 3D cell models.     

Safety Evaluation of Far-UV-C Irradiation to Epithelial Basal Cells in the Corneal Limbus

Basal cells in the corneal limbus play an important role in the turnover cycle because they are the source of all cells that constitute the corneal epithelium. We examined the penetration depth of ultraviolet (UV) light in the corneal limbus and assessed the safety of Far-UV-C on stem cells in the basal area of the corneal limbus. Rats were irradiated with UV at peaks of 207, 222, 235, 254 and 311 nm while under anesthesia. The UV penetration depth in the rat corneal limbal epithelium was wavelength dependent: 311 nm UV-B and 254 nm UV-C reached the basal cells of the epithelium, and 235 nm radiation reached the middle area; however, 207 and 222 nm UV-C reached only the superficial layer of the epithelium. Porcine cornea, which is similar to the human eye in size and structure, were irradiated with 222 and 254 nm UV-C. As in rats, 222 nm UV-C reached only the superficial layer of the porcine corneal limbal epithelium. These results indicate that Far-UV-C, such as radiation of wavelengths of 207 and 222 nm, could not reach corneal epithelial stem cells, i.e. the cells remained intact. It is unlikely that the turnover of the corneal epithelium is obstructed or disrupted by exposure to Far-UV-C.     

Reconstruction of Rabbit Corneal Layer Composed of Corneal Fibroblasts and Corneal Epithelium on the Lyophilized Amniotic Membrane

Introduction Thehumancornea,animportantopticalcompo nentthatfocusesthelightontheiris,isatransparent avasculartissuethatalsoplaysaprotectiveroleforthe eye.Therefore,anyinjury,diseaseorcelldamagethat canleadtoopacificationofthecornea,canimpairthe vision,and…     

Bioevaluation of 99mTc(CO)3–Guanine in vitro and in vivo

The aim of this study is to examine biological behaviour of radiolabeled guanine with [Tc(CO)₃]⁺ core in vitro and in vivo. In vitro biological behavior of ^99mTc(CO)₃–Gua was evaluated on Lung (A-549), Breast (MCF-7), Colonic (Caco) carcinoma cell lines and normal human bronchial epithelial (NHBE). ^99mTc(CO)₃–Gua compound showed high uptake on A-549 cell line when compared to NHBE cell line. Biodistribution characteristics of ^99mTc(CO)₃–Gua was evaluated using New Zeland Rabbits. Scintigraphic results showed that a high level of radioactivity was observed in the lungs and liver shortly after administration of the ^99mTc(CO)₃–Gua and excretion takes place via both renal and hepatobiliary route. It was concluded that ^99mTc(CO)₃–Gua could be used as a nucleotide radiopharmaceutical for imaging purposes.     

The distribution of beta-adrenergic receptors in guinea pig lungs and their changes in experimental allergic asthma

In this paper, the distribution of pulmonary beta-adrenergic receptors (beta-receptors) in normal and experimental allergic asthmatic guinea pigs was determined using autoradiographic method. The results showed that the distribution of beta-receptors in the lung sections was widespread. There was a significant decrease of beta-receptor density in several tissue structures of asthmatic guinea pig lungs compared with the controls, a 23.73% decrease in beta-receptor binding sites in bronchiolar smooth muscles and a 18.65% decrease in bronchial smooth muscles; a 23.53%, a 14.23% and a 17.16% decrease in bronchiolar epithelium, in bronchial epithelium and in alveolar epithelium respectively. The results indicated that the beta-receptor decrease in smooth muscles in experimental asthmatic guinea pig lungs might be one of the important factors which made the tension of smooth muscles increase. The relationship between the beta-receptor decrease of other sites and bronchial asthma needs to be studied further.     

Studies of chemical fixation effects in human cell lines using Raman microspectroscopy

The in vitro study of cellular species using Raman spectroscopy has proven a powerful non-invasive modality for the analysis of cell constituents and processes. This work uses micro-Raman spectroscopy to study the chemical fixation mechanism in three human cell lines (normal skin, normal bronchial epithelium, and lung adenocarcinoma) employing fixatives that preferentially preserve proteins (formalin), and nucleic acids (Carnoy’s fixative and methanol–acetic acid). Spectral differences between the mean live cell spectra and fixed cell spectra together with principal components analysis (PCA), and clustering techniques were used to analyse and interpret the spectral changes. The results indicate that fixation in formalin produces spectral content that is closest to that in the live cell and by extension, best preserves the cellular integrity. Nucleic acid degradation, protein denaturation, and lipid leaching were observed with all fixatives and for all cell lines, but to varying degrees. The results presented here suggest that the mechanism of fixation for short fixation times is complex and dependent on both the cell line and fixative employed. Moreover, important spectral changes occur with all fixatives that have consequences for the interpretation of biochemical processes within fixed cells. The study further demonstrates the potential of vibrational spectroscopy in the characterization of complex biochemical processes in cells at a molecular level.     

Microanatomical and biochemical origins of normal and precancerous cervical autofluorescence using laser-scanning fluorescence confocal microscopy

Clinical studies have shown that in vivo fluorescence spectroscopy can improve the diagnosis of cervical precancer. Recent work suggests that epithelial fluorescence increases, whereas stromal fluorescence decreases, with precancer. However, the microanatomic and biochemical sources of fluorescence in living cervical tissue have not yet been established. This study aims to characterize the origins of living normal and precancerous cervical fluorescence at microscopic levels using laser-scanning fluorescence confocal microscopy. Ten pairs of colposcopically normal and abnormal biopsies were obtained; transverse, 200 microm thick, short-term tissue cultures were prepared and imaged when viable with UV (351-364 nm) and 488 nm excitation before and after addition of the vital dye, Mitotracker Orange. In normal epithelium basal epithelial cells showed cytoplasmic fluorescence; parabasal, intermediate and superficial cells showed fluorescence only at the periphery of the cell. In low-grade precancers cytoplasmic fluorescence was visible in the bottom one-third of the epithelium; in high-grade precancers cytoplasmic fluorescence was visible throughout the lower two-thirds of the epithelium. Cytoplasmic fluorescence was colocalized with the MitoTracker probe and is attributed to mitochondrial reduced form of nicotinamide adenine dinucleotide at UV excitation and mitochondrial flavin adenine dinucleotide at 488 nm excitation. Stromal fluorescence originated from matrix fibers; with the development of precancer the density and fluorescence intensity of matrix fibers decrease. Autofluorescence properties of precancerous cervix reflect an increased number of metabolically active mitochondria in epithelial cells and a reduced stromal fluorescence, which can be an indicator for altered communication between precancerous epithelium and stroma. These changes can explain differences in in vivo fluorescence spectra of normal and precancerous cervical tissue.     

Intraspinal endodermal cyst: ultrastructural study of abnormal cilia.

Abnormal cilia were demonstrated in the lining epithelial cells of three cases of intraspinal endodermal (bronchogenic) cyst. The changes comprised a wide spectrum of ultrastructural abnormalities, including (a). cilia with abnormal axonemal microtubules, (b). swollen cilia, (c). compound cilia with or without excessive ciliary matrix, (d). naked cilia without limiting membrane, and (e). intracytoplasmic cilia and aggregates of microtubules. Of these, compound cilia and swollen cilia were most common. Cilia with dynein arm deficiency were not observed. Ciliary abnormalities found in the present study were very similar to those described in the bronchial epithelium of various diseases. The present findings suggest that the lining epithelium of intraspinal endodermal cyst shares similar ciliogenesis and susceptibility to abnormal ciliary formation as that of the bronchial epithelium.     

Direct analysis of laser capture microdissected cells by MALDI mass spectrometry

Laser capture microdissection (LCM) has become an important tool in biological research, permitting isolation of specific cell populations from frozen tissue samples containing a mixture of cell types. Cells obtained by LCM can be directly analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). We report here methodology for the preparation and analysis of LCM captured cells with MALDI MS, giving high sensitivity and mass resolution. Comparison of the spectra obtained from cell populations of interest can identify unique disease or function-related protein markers. Using this approach, mass spectra obtained from human breast tissue containing invasive mammary carcinoma and normal breast epithelium using LCM were compared. Over 40 peaks were identified that significantly differed in intensity between invasive mammary carcinoma and normal breast epithelium. In addition, mass spectra are presented that show protein patterns from mouse liver and mouse colon crypts. The reported tissue preparation procedure and subsequent analysis by MALDI MS provide a new methodology for protein discovery involving LCM captured cells.     

Discrimination of cancerous and non-cancerous cell lines by headspace-analysis with PTR-MS

Proton transfer reaction mass spectrometry (PTR-MS) has been used to analyze the volatile organic compounds (VOCs) emitted by in-vitro cultured human cells. For this purpose, two pairs of cancerous and non-cancerous human cell lines were selected:1. lung epithelium cells A-549 and retinal pigment epithelium cells hTERT-RPE1, cultured in different growth media; and 2. squamous lung carcinoma cells EPLC and immortalized human bronchial epithelial cells BEAS2B, cultured in identical growth medium. The VOCs in the headspace of the cell cultures were sampled: 1. online by drawing off the gas directly from the culture flask; and 2. by accumulation of the VOCs in PTFE bags connected to the flask for at least 12 h. The pure media were analyzed in the same way as the corresponding cells in order to provide a reference. Direct comparison of headspace VOCs from flasks with cells plus medium and from flasks with pure medium enabled the characterization of cell-line-specific production or consumption of VOCs. Among all identified VOCs in this respect, the most outstanding compound was m/z = 45 (acetaldehyde) revealing significant consumption by the cancerous cell lines but not by the non-cancerous cells. By applying multivariate statistical analysis using 42 selected marker VOCs, it was possible to clearly separate the cancerous and non-cancerous cell lines from each other.     

Photodynamic therapy with endogenous protoporphyrin IX: basic principles and present clinical experience

5-Aminolaevulinic acid (ALA) is a precursor of protoporphyrin IX (Pp IX) in the biosynthetic pathway for haem. Certain types of cells have a large capacity to synthesize Pp IX when exposed to an adequate concentration of exogenous ALA. Since the conversion of Pp IX into haem is relatively slow, such cells tend to accumulate photosensitizing concentrations of Pp IX. Pp IX photosensitization can be induced in cells of the epidermis and its appendages, but not in the dermis. Moreover, since ALA in aqueous solution passes readily through abnormal keratin, but not through normal keratin, the topical application of ALA in aqueous solution to actinic keratoses or superficial basal cell or squamous cell carcinomas induces Pp IX photosensitization that is restricted primarily to the abnormal epithelium. Subsequent exposure to photoactivating light selectively destroys such lesions. In our ongoing clinical trial of ALA-induced Pp IX photodynamic therapy, the response rate for basal cell carcinomas following a single treatment has been 90% complete response and 7.5% partial response for the first 80 lesions treated. The cosmetic results have been excellent, and patient acceptance has been very good.     

Ursolic Acid inhibits cigarette smoke extract-induced human bronchial epithelial cell injury and prevents development of lung cancer

Cigarette smoking is the main cause of chronic obstructive pulmonary disease and lung cancer. The present study was aimed to explore the chemopreventive effect of ursolic acid (UA) on these diseases. In the CSE treated normal human bronchial epithelial cell model, UA alleviated cytotoxicity caused by CSE, recovered the intracellular redox balance, and relieved the stimulation of external deleterious factors as well. UA mitigated CSE-induced DNA damage through the Nrf2 (nuclear factor erythroid 2-related factor 2) pathway. Moreover, UA inhibited lung cancer development in the model established by A549 cells in nude mice in vivo. For the first time, our results indicate that UA could be developed as a potential lung cancer chemopreventive agent.     

Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow

Development of an in vitro living cell-based model of the intestine that mimics the mechanical, structural, absorptive, transport and pathophysiological properties of the human gut along with its crucial microbial symbionts could accelerate pharmaceutical development, and potentially replace animal testing. Here, we describe a biomimetic 'human gut-on-a-chip' microdevice composed of two microfluidic channels separated by a porous flexible membrane coated with extracellular matrix (ECM) and lined by human intestinal epithelial (Caco-2) cells that mimics the complex structure and physiology of living intestine. The gut microenvironment is recreated by flowing fluid at a low rate (30 μL h(-1)) producing low shear stress (0.02 dyne cm(-2)) over the microchannels, and by exerting cyclic strain (10%; 0.15 Hz) that mimics physiological peristaltic motions. Under these conditions, a columnar epithelium develops that polarizes rapidly, spontaneously grows into folds that recapitulate the structure of intestinal villi, and forms a high integrity barrier to small molecules that better mimics whole intestine than cells in cultured in static Transwell models. In addition, a normal intestinal microbe (Lactobacillus rhamnosus GG) can be successfully co-cultured for extended periods (>1 week) on the luminal surface of the cultured epithelium without compromising epithelial cell viability, and this actually improves barrier function as previously observed in humans. Thus, this gut-on-a-chip recapitulates multiple dynamic physical and functional features of human intestine that are critical for its function within a controlled microfluidic environment that is amenable for transport, absorption, and toxicity studies, and hence it should have great value for drug testing as well as development of novel intestinal disease models.     

Extreme dark cytotoxicity of Nile Blue A in normal human fibroblasts.

Early reports using mouse models indicated that Nile Blue A (NBA) is taken up more efficiently by tumor cells than normal tissue and retards tumor growth. NBA also shows both dark toxicity and phototoxicity of human tumor cells in vitro. However, studies on the dark toxicity of NBA and the effects of NBA-mediated photodynamic treatment in normal human cells are lacking. In the current study we have examined the cytotoxicity of NBA in normal human fibroblasts, spontaneously immortalized Li-Fraumeni Syndrome (LFS) cells and three different human tumor cell lines. The normal human fibroblasts showed extreme sensitivity to NBA compared with LFS cells and the human tumor cell lines. Treatment with 0.1 microgram/mL of NBA for 1 h reduced the colony formation of normal human fibroblasts by greater than 95%, but had no significant effect on the colony formation of LFS cells. No significant numbers of apoptotic cells were detected in either normal human fibroblasts or LFS cells following this drug concentration. Thus, unlike photodynamic therapy with some other photosensitizers, the dark toxicity of NBA was not caused by apoptosis. Although the drug uptake was higher in normal human fibroblasts compared with LFS cells, the difference in sensitivity between normal human fibroblasts and LFS cells could not be accounted for by the difference in drug uptake alone. In addition, we could not detect any significant photocytotoxic effect of NBA in either normal human fibroblasts or LFS cells for a drug concentration of 0.05 microgram/mL at light exposures of up to 6.7 J/cm2. These data indicate an extreme sensitivity of normal human fibroblasts to NBA and an inability to produce a significant photocytotoxic effect on human cells using NBA concentrations that have relatively low toxicity for normal human fibroblasts.     

Pharmacokinetics and pharmacodynamics of tetra(m-hydroxyphenyl)chlorin in the hamster cheek pouch tumor model: comparison with clinical measurements

The pharmacokinetics (PK) of the photosensitizer tetra(m-hydroxyphenyl)chlorin (mTHPC) was measured by optical fiber-based light-induced fluorescence spectroscopy (LIFS) in the normal and tumoral cheek pouch mucosa of 29 Golden Syrian hamsters with chemically induced squamous cell carcinoma. Similar measurements were carried out on the normal oral cavity mucosa of five patients up to 30 days after injection. The drug doses were between 0.15 and 0.3 mg per kg of body weight (mg/kg), and the mTHPC fluorescence in the tissue was excited at 420 nm. The PK in both human and hamster exhibited similar behavior although the PK in the hamster mucosa was slightly delayed in comparison with that of its human counterpart. The mTHPC fluorescence signal of the hamster mucosa was smaller than that of the human mucosa by a factor of about 3 for the same injected drug dose. A linear correlation was found between the fluorescence signal and the mTHPC dose in the range from 0.075 to 0.5 mg/kg at times between 8 and 96 h after injection. No significant selectivity in mTHPC fluorescence between the tumoral and normal mucosa of the hamsters was found at any of the applied conditions. The sensitivity of the normal and tumoral hamster cheek pouch mucosa to mTHPC photodynamic therapy as a function of the light dose was determined by light irradiation at 650 nm and 150 mW/cm2, 4 days after the injection of a drug dose of 0.15 mg/kg. These results were compared with irradiations of the normal oral and normal and tumoral bronchial mucosa of 37 patients under the same conditions. The reaction to PDT of both types of human mucosae was considerably stronger than that of the hamster cheek pouch mucosa. The sensitivity to PDT became comparable between hamster and human mucosa when the drug dose for the hamster was increased to 0.5 mg/kg. A significant therapeutic selectivity between the normal and neoplastic hamster cheek pouch was observed. Less selectivity was found following irradiations of normal mucosa and early carcinomas in the human bronchi. The pharmacodynamic behavior of mTHPC was determined by test irradiations of the normal mucosa of hamsters and patients between 6 h and 8 days after injection of 0.5 and 0.15 mg/kg in the hamsters and the patients, respectively. The normal hamster cheek pouch showed a maximum response to irradiation 6 h after injection and then decreased continuously to no observable reaction at 8 days after injection. The reaction of the normal human oral mucosa, however, showed an increasing sensitivity to the applied light between 6 h and 4 days after mTHPC injection and then decreased again at 8 days. The hamster model with the chemically induced early squamous cell cancer in the cheek pouch thus showed some similarity to the early squamous cell cancer of the human oral mucosa considering the PK. However, a quantitative difference in fluorescence signal for identical mTHPC doses as well as a significant difference in pharmacodynamic behavior were also observed. The suitability of this animal model for the optimization of PDT parameters in the clinic is therefore limited. Hence great care must be taken in screening new dyes for PDT of early squamous cell cancer of the upper aerodigestive tract based upon observables in the hamster cheek pouch model.     

A proteomic approach to mimic fibrosis disease evolvement by an in vitro cell line.

Subepithelial fibrosis in asthma involves an increase in the thickening of the lamina reticularis and is due to increased deposition of collagen I, III and V, and fibronectin. The cause of the thickening of the reticular layer is not known in detail, however, it is proposed to be caused by bronchial myofibroblasts. The transformation of fibroblasts to myofibroblasts may be contributed by inflammatory cytokines. In this paper we have studied and compared in vivo tissue material with a human fibroblast target cell. A normal primary fetal fibroblast cell line and HFL-1 (human fibroblast lurg cells) were used as a comparison between fibroblasts from human central biopsies regarding morphology and cell proliferation. Both cell morphology and cell proliferation rate was similar between the different set of cell cultures. Furthermore, it could be concluded that fibroblasts cultures from patients with asthma were surrounded by more extracellular matrix molecules compared to the primary cell line HFL-1, which may mimic the in vivo situation during formation of fibrosis. We wanted to investigate if differential protein display by two-dimensional (2-D) gel electrophoresis and subsequent protein identification by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry could reveal proteins induced by cytokine stimulation that can be correlated to the transformation of normal human fetal lungs cells into a more myofibroblast like phenotype. After stimulation with transforming growth factor-beta (TGF-beta) several myofibroblast markers were found to be regulated. Especially cytoskeletal and cytoskeletal-associated proteins like actin isoforms and tropomyosin, proteins that are responsible for contraction as well as transportation of extra cellular matrix proteins, which are overproduced in the formation of fibrosis. These results indicate that TGF-beta, which is increased in a fibrotic process, participates in the transformation of fibroblasts to myofibroblasts.     

TNF-alpha-induced up-regulation of intercellular adhesion molecule-1 is regulated by a Rac-ROS-dependent cascade in human airway epithelial cells

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the lung airway epithelium is associated with the epithelium-leukocyte interaction, critical for the pathogenesis of various lung airway inflammatory diseases such as asthma. However, little is known about how ICAM-1 is up-regulated in human airway epithelial cells. In this study, we show that tumor TNF-alpha induces monocyte adhesion to A549 human lung airway epithelium and also up-regulation of ICAM-1 expression. These effects were significantly diminished by pre-treatment with diphenyliodonium (DPI), an inhibitor of NADPH oxidase-like flavoenzyme. In addition, the level of reactive oxygen species (ROS) was increased in response to TNF-alpha in A549 cells, suggesting a potential role of ROS in the TNF-alpha-induced signaling to ICAM-1 expression and monocyte adhesion to airway epithelium. Further, we found out that expression of RacN17, a dominant negative mutant of Rac1, suppressed TNF-alpha-induced ROS generation, ICAM-1 expression, and monocyte adhesion to airway epithelium. These findings suggest that Rac1 lies upstream of ROS generation in the TNF-alpha-induced signaling to ICAM-1 expression in airway epithelium. Finally, pretreatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, reduced TNF-alpha-induced ICAM-1 expression and both DPI and RacN17 significantly diminished NF-kappaB activation in response to TNF-alpha. Together, we propose that Rac1-ROS-linked cascade mediate TNF-alpha-induced ICAM-1 up-regulation in the airway epithelium via NF-kappaB-dependent manner.     

Cell-type specific protoporphyrin IX metabolism in human bladder cancer in vitro

5-Aminolevulinic acid (ALA)-supported fluorescence endoscopy of the urinary bladder results in a detection rate of bladder cancer superior to that of white light endoscopy. The different accumulation of the metabolite protoporphyrin IX (PPIX) in tumor cells after ALA instillation is poorly understood; however, it is crucial to optimize diagnosis and potential phototherapy. For systematic analysis of cell-type specific PPIX accumulation and metabolism two human bladder carcinoma cell lines (RT4 and J82), a normal urothelial cell line (UROtsa), and a fibroblast cell line (N1) were chosen, and grown in two different growth states to model important tissue components of the urinary bladder, i.e. tumor, normal epithelium and stroma. To quantitate PPIX content, fluorescence intensities measured by flow cytometry were matched with cellular PPIX extraction values, and related to relative ferrochelatase activity, cellular iron content, number of transferrin receptors per cell and porphobilinogen deaminase (PBGD) activity. For in vitro experiments, the initial correlation of relative flow cytometric and spectrometric measurements of PPIX provides a calibration curve for consequent flow cytometric PPIX quantification. Lower fluorescence of normal cells could be explained by significant differences of ferrochelatase activity and iron content in comparison to tumor cells. However, the content of iron was not related to transferrin receptor content. PBGD activity seemed to play a minor role for the differential accumulation of PPIX in urothelial cells. In conclusion, the in vitro culture of urothelial cells and fibroblasts indicates that the most important metabolic step for PPIX accumulation in the urinary bladder is the transition from PPIX to heme. Further investigation of PPIX metabolism does support the validation of photodynamic diagnosis, and might also lead the way to a highly specific tumor related molecule.