Effect of the TAT-RasGAP(317-326) peptide on apoptosis of human malignant mesothelioma cells and fibroblasts exposed to meso-tetra-hydroxyphenyl-chlorin and light

BACKGROUND: 5,10,15,20-Tetrakis(m-hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy (PDT) has shown insufficient tumor selectivity for the treatment of pleural mesothelioma. Tumor selectivity of mTHPC-PDT may be enhanced in the presence of the TAT-RasGAP(317-326) peptide which has the potential to specifically sensitize tumor cells to cytostatic agents. MATERIALS AND METHODS: H-meso-1 and human fibroblast cell cultures, respectively, were exposed to two different mTHPC doses followed by light delivery with and without TAT-RasGAP(317-326) administration. mTHPC was added to the cultures at a concentration of 0.04microg/ml and 0.10microg/ml, respectively, 24h before laser light illumination at 652nm (3J/cm(2), 40mW/cm(2)). TAT-RasGAP(317-326) was added to the cultures immediately after light delivery at a concentration of 20microM. The apoptosis rate was determined by scoring the cells displaying pycnotic nuclei. Cell viability was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: Light delivery associated with 0.04microg/ml mTHPC resulted in a significantly higher apoptosis rate in the presence of TAT-RasGAP(317-326) than without in H-meso-1 cells (p<0.05) but not in fibroblasts. In contrast, 1.0microg/ml mTHPC and light resulted in a significantly higher apoptosis rate in both H-meso-1 cells and fibroblasts as compared to controls (p<0.05) but the addition of TAT-RasGAP(317-326) did not lead to a further significant increase of the apoptosis rate of both H-meso-1 cells and fibroblasts as compared to mTHPC and light delivery alone. CONCLUSION: TAT-RasGAP(317-326) selectively enhanced the effect of mTHPC and light delivery on H-meso-1 cells but not on fibroblasts. However, this effect was mTHPC dose-dependent and occurred only at a low sensitizer dose.     

Characterization of the interaction between fibroblasts and tumor cells on a microfluidic co‐culture device

Fibroblasts and tumor cells have been involved in the process of cancer development, progression and therapy. Here, we present a simple microfluidic device which enables to study the interaction between fibroblasts and tumor cells by indirect contact co‐culture. The device is composed of multiple cell culture chambers which are connected by a parallel of cell migration regions, and it enables to realize different types of cells to communicate each other on the single device. In this work, human embryonic lung fibroblasts cells were observed to exhibit obvious migration towards tumor cells instead of normal epithelial cells on the co‐culture device. Moreover, transdifferentiation of human embryonic lung fibroblast cells was recognized by the specific expression of α‐smooth musle actin, indicating the effect of tumor cells on the behavior of fibroblasts. Furthermore, multiple types of cell co‐culture can be demonstrated on the single device which enables to mimic the complicated microenviroment in vivo. The device is simple and easy to operate, which enables to realize real‐time observation of cell migration after external stimulus. This microfluidic device allows for the characterization of various cellular events on a single device sequentially, faciliating the better understanding of interaction between heterotypic cells in a more complex microenvironment.     

Altered mitochondrial structure and motion dynamics in living cells with energy metabolism defects revealed by real time microscope imaging.

Using the real time microscope (RTM), a system applying new developments in light microscopy, we documented the spatial and temporal dynamics of mitochondrial behavior in human cultured skin fibroblasts. Without the use of stains or probes, we resolved fibroblast mitochondria as dark slender filaments of approximately 0.2 m wide and up to 10 m long, as well as a few smaller ovoid forms. In the living cell, the three most common mitochondrial movements were: (1) small oscillatory movements; (2) larger movements including filament extension, retraction, and branching as well as combinations of these actions; and (3) whole transit movements of single mitochondrial filaments. Skin fibroblasts from patients with mitochondrial complex I deficiency and normal fibroblasts during incubation with rotenone, or antimycin A, contained higher proportions of mitochondria in the swollen filamentous forms, nodal filaments, and ovoid forms rather than the slender filamentous forms in normal cells. Interestingly, decreased motility was observed with the more ovoid mitochondrial forms compared to the filamentous forms. We conclude that mitochondrial morphology and dynamic motion are strongly associated with changes in mitochondrial energy metabolism. Images documenting our observations are presented both at single time points and as QuickTime videos.     

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.     

The HGF-met signaling axis: emerging themes and targets of inhibition

The Met tyrosine kinase receptor is the only known receptor for hepatocyte growth factor (HGF). Downstream Met signaling is essential for embryonic development; however, aberrant Met signaling promotes tumor progression by facilitating cell proliferation, survival, migration, invasion, and metastasis. Tumor cell invasion is considered an important step in distant metastatic foci formation. Several recent reviews have focused on the pleiotropic effects of Met signaling in both tumor cells and in the surrounding stromal cells. This review will summarize the currently described mechanisms driving Met induced tumor cell progression and invasion, the role played by cells in the tumor stroma, and therapeutic approaches to block receptor activity. In addition, this review will also highlight two new areas of development: 1) attenuation of Met signaling via multiple mechanisms of action targeting tumor cells and cells in the surrounding stroma using plant-derived polyphenols and 2) the induction by HGF of atypical lysosome trafficking, leading to increased protease secretion and tumor cell invasion. These new areas of research will help to uncover novel therapeutic targets to block the HGF/Met signaling axis to slow cancer progression.     

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.     

Spectroscopic characterization of human and mouse primary cells,cell lines and malignant cells

Fourier transform infrared (FTIR) spectroscopy is currently being developed as a new optical approach to the diagnosis and characterization of cell or tissue pathology. The advantage of FTIR microspectroscopy over conventional FTIR spectroscopy in the diagnosis of malignancies is that it facilitates inspection of restricted regions of the cell culture or tissue. In this study, we set out to evaluate FTIR microspectroscopy as a diagnostic tool for identifying retrovirus-induced malignancies. Our study showed significant and consistent differences between cultures of different types of cells of both mouse and human origin, i.e. primary fibroblast cells (one to two passages in cell culture), fibroblast cell lines and malignant cells transformed by murine sarcoma virus. An impressive decrease in the levels of phosphate and other metabolites was seen in malignant cells compared with primary cells. The levels of these metabolites in the cell lines were significantly lower than in the primary cells but higher than in the malignant cells. In addition, the peak attributed to the PO2- symmetric stretching mode at 1082 cm(-1) in primary cells shifted significantly to 1085 cm(-1) for the cell line and to 1087 cm(-1) for the malignant cells. These differences taken together with differences in the shapes of various bands throughout the spectrum strongly support the possibility of developing FTIR microspectroscopy for the detection and study of malignant--and possibly premalignant--cells.     

Comparative sensitivity of microvascular endothelial cells, fibroblasts and tumor cells after in vitro photodynamic therapy with meso-tetra-hydroxyphenyl-chlorin

The phototoxic effect of meso-tetra-hydroxyphenyl-chlorin (mTHPC)-mediated photodynamic therapy (PDT) on human microvascular endothelial cells (hMVEC) was compared with that on human fibroblasts (BCT-27) and two human tumor cell lines (HMESO-1 and HNXOE). To examine the relationship between intrinsic phototoxicity and intracellular mTHPC content, we expressed cell survival as a function of cellular fluorescence. On the basis of total cell fluorescence, HNXOE tumor cells were the most sensitive and BCT-27 fibroblasts the most resistant, but these differences disappeared after correcting for cell volume. Endothelial cells were not intrinsically more sensitive to mTHPC-PDT than tumor cells or fibroblasts. Uptake of mTHPC in hMVEC increased linearly to at least 48 h, whereas drug uptake in the other cell lines reached a maximum by 24 h. No difference in drug uptake was seen between the cell lines during the first 24 h, but by 48 h hMVEC had a 1.8- to 2.8-fold higher uptake than other cell lines. Endothelial cells showed a rapid apoptotic response after mTHPC-mediated PDT, whereas similar protocols gave a delayed apoptotic or necrotic like response in HNXOE. We conclude that endothelial cells are not intrinsically more sensitive than other cell types to mTHPC-mediated PDT but that continued drug uptake beyond 24 h may lead to higher intracellular drug levels and increased photosensitivity under certain conditions.     

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.     

On-chip measurements of cell compressibility via acoustic radiation

Measurements of mechanical properties of biological cells are of great importance because changes in these properties can be strongly associated with the progression of cell differentiation and cell diseases. Although state of the art methods, such as atomic force microscopy, optical tweezers and micropipette aspiration, have been widely used to measure the mechanical properties of biological cells, all these methods involve direct contact with the cell and the measurements could be affected by the contact or any local deformation. In addition, all these methods typically deduced the Young's modulus of the cells based on their measurements. Herein, we report a new method for fast and direct measurement of the compressibility or bulk modulus of various cell lines on a microchip. In this method, the whole cell is exposed to acoustic radiation force without any direct contact. The method exploits the formation of an acoustic standing wave within a straight microchannel. When the polystyrene beads and cells are introduced into the channel, the acoustic radiation force moves them to the acoustic pressure node and the movement speed is dependent on the compressibility. By fitting the experimental and theoretical trajectories of the beads and the cells, the compressibility of the cells can be obtained. We find that the compressibility of various cancer cells (MCF-7: 4.22 ± 0.19 × 10(-10) Pa(-1), HEPG2: 4.28 ± 0.12 × 10(-10) Pa(-1), HT-29: 4.04 ± 0.16 × 10(-10) Pa(-1)) is higher than that of normal breast cells (3.77 ± 0.09 × 10(-10) Pa(-1)) and fibroblast cells (3.78 ± 0.17 × 10(-10) Pa(-1)). This work demonstrates a novel acoustic-based method for on-chip measurements of cell compressibility, complementing existing methods for measuring the mechanical properties of biological cells.     

Age-dependent DNA repair and cell cycle distribution of human skin fibroblasts in response to UVA irradiation

Ageing process in cells is associated with oxidative stress. Ultraviolet A produces reactive oxygen species responsible for accumulation of DNA and cellular damage. After the evaluation of antioxidant enzyme activities and oxidative stress markers at the basal state, we have studied the responses to UVA stress of coetaneous fibroblasts, isolated from different male donors (2-88 years, n=23) in terms of cytotoxicity, genotoxicity and DNA repair capacities. For this purpose, we have determined level of DNA damage using the comet assay (single strand breaks and alkali-labile sites) and the cell cycle distribution after a 5 J/cm2 irradiation. No differences with age were observed for antioxidant enzyme activities and oxidative stress markers. DNA strand breaks after UVA irradiation (5-20 J/cm2), was found to be age-dependent. DNA repair was slow and also significantly affected by ageing. The cell cycle distribution analysis showed that high repair correlated with high proliferative capacities at basal level. Twenty-four hours after the stress, fraction of young fibroblasts blocked in G1 phase was significantly increased whereas significant modifications concerned the G2-M phase for adult and older fibroblasts. These results indicate an age-dependent decline in the DNA repair capacities correlated with modifications of the cell cycle parameters.     

THE EFFECT OF LASER IRRADIATION ON THE RELEASE OF bFGF FROM 3T3 FIBROBLASTS

Studies have shown that low-level laser irradiation increases the proliferation of fibroblasts in cell culture. The mechanism of action is unknown. Basic fibroblast growth factor (bFGF) is a multifunctional polypeptide that has been detected in most tissues and which supports cell proliferation and differentiation. The purpose of this study was to determine whether laser irradiation (660 nm) can stimulate production of bFGF from fibroblast cells in cell culture. Our study showed that fibroblasts irradiated with laser energy at 2.16 J/cm² demonstrated increased cell proliferation and enhanced production of bFGF, whereas fibroblasts irradiated with laser energy at 3.24 J/cm² neither demonstrated increased cell proliferation or an enhanced release of bFGF as compared to the control group. These results provide direct evidence that the proliferation of fibroblasts as a result of stimulation by low level laser irradiation may be associated with the autocrine production of bFGF from fibroblasts.     

The role of the p53 tumor suppressor in the response of human cells to photofrin-mediated photodynamic therapy

Although there is evidence that the p53 tumor suppressor plays a role in the response of some human cells to chemotherapy and radiation therapy, its role in the response of human cells to photodynamic therapy (PDT) is less clear. In order to examine the role of p53 in cellular sensitivity to PDT, we have examined the clonogenic survival of normal human fibroblasts that express wild-type p53 and immortalized Li-Fraumeni syndrome (LFS) cells that express only mutant p53, following Photofrin-mediated PDT. The LFS cells were found to be more resistant to PDT compared to normal human fibroblasts. The D37 (LFS cells)/D37 (normal human fibroblasts) was 2.8 +/- 0.3 for seven independent experiments. Although the uptake of Photofrin per cell was 1.6 +/- 0.1-fold greater in normal human fibroblast cells compared to that in LFS cells over the range of Photofrin concentrations employed, PDT treatment at equivalent cellular Photofrin levels also demonstrated an increased resistance for LFS cells compared to normal human fibroblasts. Furthermore, adenovirus-mediated transfer and expression of wild-type p53 in LFS cells resulted in an increased sensitivity to PDT but no change in the uptake of Photofrin per cell. These results suggest a role for p53 in the response of human cells to PDT. Although normal human fibroblasts displayed increased levels of p53 following PDT, we did not detect apoptosis or any marked alteration in the cell cycle of GM38 cells, despite a marked loss of cell viability. In contrast, LFS cells exhibited a prolonged accumulation of cells in G2 phase and underwent apoptosis following PDT at equivalent Photofrin levels. The number of apoptotic LFS cells increased with time after PDT and correlated with the loss of cell viability. A p53-independent induction of apoptosis appears to be an important mechanism contributing to loss of clonogenic survival after PDT in LFS cells, whereas the induction of apoptosis does not appear to be an important mechanism leading to loss of cell survival in the more sensitive normal human fibroblasts following PDT at equivalent cellular Photofrin levels.     

Matrix metal loproteinases and the development of cancer

Proteolytic remodeling of the extracellular matrix is an important aspect of the creation and progression of cancer. Matrix metalloproteinases are important at several points during multistage neoplastic progression in tumor cells and responding blood vessels, inflammatory cells and stroma.     

Dual-label autoradiographic analysis of human skin fibroblast and myoblast proteins by two-dimensional polyacrylamide gel electrophoresis using immobilised pH gradients in the first dimension

Horizontal two-dimensional polyacrylamide gel electrophoresis with immobilised pH gradients in the first dimension has been applied to the analysis of human skin fibroblast and muscle myoblast total cell proteins. Excellent two-dimensional separations of skin fibroblast proteins were obtained using pH 4-10 immobilised pH gradient gels with a long interelectrode distance (16 cm), but resolution was degraded, particularly of the more acidic proteins, by the use of shorter (10 cm) gels. Improved resolution of acidic and basic proteins was obtained using separate pH 4-7 and pH 7-10 immobilised pH gradient gels respectively in the first dimension. Two-dimensional protein maps of skin fibroblast proteins were visualised both by silver staining and by autoradiography of samples labelled synthetically with [35S]methionine. Horizontal two-dimensional electrophoresis, using pH 4-7 and pH 7-10 immobilised pH gradient gels in the first dimension, was applied to the analysis of protein samples from skin fibroblasts and muscle myoblasts dual-labelled synthetically with [35S]methionine and [75Se]selenomethionine in an attempt to identify sets of proteins specific to each cell type. In addition, two-dimensional maps or protein samples derived from normal individuals and patients with Duchenne muscular dystrophy were compared to search for protein changes associated with the disease state. Although sets of qualitative protein spot differences were observed by visual inspection of the two-dimensional gels, more rigorous qualitative and quantitative analysis of the patterns using a computerised analysis system will be required to obtain the maximum amount of information from these data.     

Photoprotective properties of a hydrophilic extract of the fern Polypodium leucotomos on human skin cells

The effect of a hydrophilic extract of the fern Polypodium leucotomos (PLE) has been investigated in terms of photoprotection against UV-induced cell damage. PLE efficiently preserved human fibroblast survival and restored their proliferative capability when the cells were exposed to UVA light. This effect was specific and dose-dependent. Photoprotection was not restricted to fibroblasts, as demonstrated by its effect on survival and proliferation of the human keratinocyte cell line HaCat. Finally, treatment of the cells with PLE prevented UV-induced morphological changes in human fibroblasts, namely disorganisation of F-actin-based cytoskeletal structures, coalescence of the tubulin cytoskeleton and mislocalization of adhesion molecules such as cadherins and integrins. Our in vitro results demonstrate the photoprotective effect of PLE on human cells and support its use in the preventive treatment of sunburning and skin pathologies associated with UV-mediated damage.