Protection of p53 wild type cells from taxol by nutlin-3 in the combined lung cancer treatment.

BACKGROUND: Mutations within the tumor suppressor TP53 gene are one of the most common genetic alterations present at high frequency in human tumors and have been shown to be associated with resistance to radio-chemotherapy. The lack of the wild type TP53 gene in cancer cells could be exploited for therapeutic advantage using a sequence of two antagonistic drugs. The aim of this study was to selectively kill p53 deficient cells (FaDu and H1299) by taxol and to protect p53 wild type cells (A549) by the prior administration of nutlin-3 in comparison to certain known anticancer drugs (5-fluorouracil, camptothecin, roscovitine). METHODS: Cytotoxic and cytostatic properties of 5-fluorouracil, camptothecin, roscovitine and nutlin-3 administrating alone or in combination with taxol were investigated in vitro by flow cytometry. RESULTS: It was found that nutlin-3 induced growth arrest and protected A549 cells from taxol. FaDu and H1299 cells responded to the same treatments with mitotic arrest and massive apoptosis. Other compounds (5-fluorouracil, camptothecin and roscovitine) revealed weaker selectivity and elevated toxicity in comparison to nutlin-3. CONCLUSIONS: We propose a therapeutic strategy protecting normal cells from taxol while increasing apoptosis selectively in p53-deficient cells using nutlin-3.

Improvement of radiation-mediated immunosuppression of human NSCLC tumour xenografts in a nude rat model.

Human tumour xenografts in a nude rat model have consistently been used as an essential part of preclinical studies for anticancer drugs activity in human. Commonly, these animals receive whole body irradiation to assure immunosuppression. But whole body dose delivery might be inhomogeneous and the resulting incomplete bone marrow depletion may modify tumour behaviour. To improve irradiation-mediated immunosuppression of human non-small cell lung cancer (NSCLC) xenografts in a nude rat model irradiation (2 + 2 Gy) from opposite sides of animals has been performed using a conventional X-ray tube. The described modification of whole body irradiation improves growth properties of human NSCLC xenografts in a nude rat model. The design of the whole body irradiation mediated immunosuppression described here for NSCLC xenografts may be useful for research applications involving other types of human tumours.

Protection of p53 wild type cells from taxol by genistein in the combined treatment of lung cancer.

This study specifies the basic principles to selectively kill p53-deficient cells (H1299, FaDu) by taxol and to protect p53 wild type cells (A549) by the prior administration of structurally related flavonoids (apigenin, genistein, and quercetin). Cytotoxic and cytostatic properties of flavonoids were investigated in vitro by flow cytometry and were compared to known anticancer drugs (cisplatin, doxorubicin, etoposide). It was confirmed that doxorubicin induced growth arrest and protected A549 cells from taxol while simultaneously killing or blocking H1299 and FaDu cancer cells. It was found that doxorubicin could be successfully substituted in this way by the isoflavone genistein used at physiologically relevant concentrations. The other compounds analyzed revealed less selectivity (apigenin, cisplatin) or demonstrated higher toxicity (cisplatin, etoposide, and quercetin). We concluded that genistein-based therapy may have antagonistic effects when combined with mitotic poisons. The proposed therapeutic strategy allows protection of p53 wild type cells from taxol and selectively increases apoptosis in p53-deficient cells. This strategy exploits the naturally occurring compound that can be used without significant toxicity in rather high concentrations as present in common diets.

Preliminary assessment of dynamic contrast-enhanced CT implementation in pretreatment FDG-PET/CT for outcome prediction in head and neck tumors.

BACKGROUND: Recently published data show some controversy concerning the impact of [18F]-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) in predicting head and neck tumors (HNT) outcome. Assessment of tumor blood supply parameters using dynamic contrast-enhanced CT (DCE-CT) may deliver additional information concerning this important question. PURPOSE: To evaluate the contribution of DCE-CT implemented in pretherapeutic FDG-PET/CT protocol for prognosis prediction in patients with HNT. MATERIAL AND METHODS: Ten consecutive patients (median age 50 years, range 47-74 years) with histologically proven HNT underwent FDG-PET/CT with DCE-CT before treatment. FDG uptake was measured by maximum standardized uptake value (SUV(max)). Relative tumor blood volume (rTBV) was determined from DCE-CT using Patlak analysis. Intratumoral heterogeneity was assessed by means of lacunarity analysis. Obtained values were compared with time-to-progression and overall survival. PET and DCE-CT images were compared on a pixel-by-pixel basis using Pearson coefficient of correlation. RESULTS: Three patients with lower FDG uptake (SUV(max): 8+/-1) and five patients with higher FDG uptake (SUV(max): 15+/-4, P=0.004) were free of local recurrence for 24 months. Two groups of patients with significantly differing lower (group A: 0.37+/-0.02, n=6) and higher (group B: 0.52+/-0.01, n=4; P<0.01), tumor heterogeneity (lacunarity) were identified. Corresponding mean rTBV was higher in group A (9.6+/-1.8 ml/100 ml) than in group B (6.2+/-0.6 ml/100 ml). All six patients with homogeneous tumor blood supply (lower lacunarity) and higher rTBV were free of local recurrence during 24 months, while two of four patients with heterogeneous tumor blood supply (higher lacunarity) and lower rTBV died during follow-up due to tumor relapse. A weak correlation between FDG-PET and DCE-CT rTBV was observed (R(2)=0.1). CONCLUSION: FDG-PET/CT and DCT-CT are complementary methods for surveillance assessment in patients with HNT. Implementation of DCE-CT in the pretreatment FDG-PET/CT protocol may improve tumor outcome prediction.

Noninvasive assessment and quantification of tumor vascularization using [18F]FDG-PET/CT and CE-CT in a tumor model with modifiable angiogenesis-an animal experimental prospective cohort study.

BACKGROUND: This study investigated the noninvasive assessment of tumor vascularization with clinical F-18-fluorodeoxyglucose positron emission tomography/computed tomography and contrast-enhanced computed tomography ([18F]FDG-PET/CT and CE-CT) in experimental human xenograft tumors with modifiable vascularization and compared results to histology. Tumor xenografts with modifiable vascularization were established in 71 athymic nude rats by subcutaneous transplantation of human non-small-cell lung cancer (NSCLC) cells. Four different groups were transplanted with two different tumor cell lines (either A549 or H1299) alone or tumors co-transplanted with rat glomerular endothelial (RGE) cells, the latter to increase vascularization. Tumors were assessed noninvasively by [18F]FDG PET/CT and contrast-enhanced CT (CE-CT) using clinical scanners. This was followed by histological examinations evaluating tumor vasculature (CD-31 and intravascular fluorescent beads). RESULTS: In both tumor lines (A549 and H1299), co-transplantation of RGE cells resulted in faster growth rates [maximal tumor diameter of 20 mm after 22 (± 1.2) as compared to 45 (± 1.8) days, p < 0.001], higher microvessel density (MVD) determined histologically after CD-31 staining [171.4 (± 18.9) as compared to 110.8 (± 11) vessels per mm2, p = 0.002], and higher perfusion as indicated by the number of beads [1.3 (± 0.1) as compared to 1.1 (± 0.04) beads per field of view, p = 0.001]. In [18F]FDG-PET/CT, co-transplanted tumors revealed significantly higher standardized uptake values [SUVmax, 2.8 (± 0.2) as compared to 1.1 (± 0.1), p < 0.001] and larger metabolic active volumes [2.4 (± 0.2) as compared to 0.4 (± 0.2) cm3, p < 0.001] than non-co-transplanted tumors. There were significant correlations for vascularization parameters derived from histology and [18F]FDG PET/CT [beads and SUVmax, r = 0.353, p = 0.005; CD-31 and SUVmax, r = 0.294, p = 0.036] as well as between CE-CT and [18F]FDG PET/CT [contrast enhancement and SUVmax, r = 0.63, p < 0.001; vital CT tumor volume and metabolic PET tumor volume, r = 0.919, p < 0.001]. CONCLUSIONS: In this study, a human xenograft tumor model with modifiable vascularization implementable for imaging, pharmacological, and radiation therapy studies was successfully established. Both [18F]FDG-PET/CT and CE-CT are capable to detect parameters closely connected to the degree of tumor vascularization, thus they can help to evaluate vascularization in tumors noninvasively. [18F]FDG-PET may be considered for characterization of tumors beyond pure glucose metabolism and have much greater contribution to diagnostics in oncology.

Transcriptome 3'end organization by PCF11 links alternative polyadenylation to formation and neuronal differentiation of neuroblastoma.

Diversification at the transcriptome 3'end is an important and evolutionarily conserved layer of gene regulation associated with differentiation and dedifferentiation processes. Here, we identify extensive transcriptome 3'end-alterations in neuroblastoma, a tumour entity with a paucity of recurrent somatic mutations and an unusually high frequency of spontaneous regression. Utilising extensive RNAi-screening we reveal the landscape and drivers of transcriptome 3'end-diversification, discovering PCF11 as critical regulator, directing alternative polyadenylation (APA) of hundreds of transcripts including a differentiation RNA-operon. PCF11 shapes inputs converging on WNT-signalling, and governs cell cycle, proliferation, apoptosis and neurodifferentiation. Postnatal PCF11 down-regulation induces a neurodifferentiation program, and low-level PCF11 in neuroblastoma associates with favourable outcome and spontaneous tumour regression. Our findings document a critical role for APA in tumorigenesis and describe a novel mechanism for cell fate reprogramming in neuroblastoma with potentially important clinical implications. We provide an interactive data repository of transcriptome-wide APA covering > 170 RNAis, and an APA-network map with regulatory hubs.

Age-related changes in the frequency of mesenchymal stem cells in the bone marrow of rats.

Mesenchymal stem cells (MSCs) are pluripotent cells that can differentiate into endothelial, osteogenic, adipogenic, and other lineages. In spite of the broad interest, little is known about the variation of MSC number in relation to the age of the donor. The aim of this study was to investigate the age-associated variations of bone marrow (BM) MSCs using a rat model. Cell populations were characterized by flow cytometry using light-scattering parameters, DNA content and a set of monoclonal antibodies and detected by magnetic resonance imaging (MRI). Single-cell analysis was performed by conventional fluorescent microscopy. BM mononucleated cells (MNCs) were isolated, in vitro culture of MSCs was established, and endothelial cells differentiation and intracellular magnetic labeling was shown. The amount of BM tissue obtainable from femurs and tibiae increased with age and reached a maximum in 8- to 12-week-old rats. At the same time, the proportional number of MNCs containing MSCs decreased. As a result, after 2 weeks of culture, the maximum yield of MSC number was registered from the youngest age group (4 weeks). MSCs were differentiated into endothelial cells by administration of vascular endothelial growth factor (VEGF) and subsequently revealed immunocytochemical and morphological characteristics of endothelial cells. The results of our study are the basis for further experiments with MSCs and their endothelial descendants, which may be labeled with different agents for cell tracking and detection experiments, but age-related changes in MSCs number should be taken into account whenever these cells are considered for practical applications.

A number of bone marrow mesenchymal stem cells but neither phenotype nor differentiation capacities changes with age of rats.

Bone marrow (BM) derived mesenchymal stem cells (MSC) are pluripotent cells which can differentiate into osteogenic, adipogenic and other lineages. In spite of the broad interest, the information about the changes in BM cell composition, in particularly about the variation of MSC number and their properties in relation to the age of the donor is still controversial. The aim of this study was to investigate the age associated changes in variations of BM cell composition, phenotype and differentiation capacities of MSC using a rat model. Cell populations were characterized by flow cytometry using light scattering parameters, DNA content and a set of monoclonal antibodies. Single cell analysis was performed by conventional fluorescent microscopy. In vitro culture of MSC was established and their phenotype and capability for in vitro differentiation into osteogenic and adipogenic cells was shown. Age related changes in tibiae and femurs, amount of BM tissue, BM cell composition, proportions of separated MSC and yield of MSC in 2 weeks of in vitro culture were found. At the same time, neither change in phenotype no in differentiation capacities of MSC was registered. Age-related changes of the number of MSC should be taken into account whenever MSC are intended to be used for investigations.

Glucagon-like peptide-1 receptor signalling reduces microvascular thrombosis, nitro-oxidative stress and platelet activation in endotoxaemic mice.

BACKGROUND AND PURPOSE: Excessive inflammation in sepsis causes microvascular thrombosis and thrombocytopenia associated with organ dysfunction and high mortality. The present studies aimed to investigate whether inhibition of dipeptidyl peptidase-4 (DPP-4) and supplementation with glucagon-like peptide-1 (GLP-1) receptor agonists improved endotoxaemia-associated microvascular thrombosis via immunomodulatory effects. EXPERIMENTAL APPROACH: Endotoxaemia was induced in C57BL/6J mice by a single injection of LPS (17.5 mg kg-1 for survival and 10 mg kg-1 for all other studies). For survival studies, treatment was started 6 h after LPS injection. For all other studies, drugs were injected 48 h before LPS treatment. KEY RESULTS: Mice treated with LPS alone showed severe thrombocytopenia, microvascular thrombosis in the pulmonary circulation (fluorescence imaging), increased LDH activity, endothelial dysfunction and increased markers of inflammation in aorta and whole blood (leukocyte-dependent oxidative burst, nitrosyl-iron haemoglobin, a marker of nitrosative stress, and expression of inducible NOS). Treatment with the DPP-4 inhibitor linagliptin or the GLP-1 receptor agonist liraglutide, as well as genetic deletion of DPP-4 (DPP4-/- mice) improved all these parameters. In GLP-1 receptor-deficient mice, both linagliptin and liraglutide lost their beneficial effects and improvement of prognosis. Incubation of platelets and cultured monocytes (containing GLP-1 receptor protein) with GLP-1 receptor agonists inhibited the monocytic oxidative burst and platelet activation, with a GLP-1 receptor-dependent elevation of cAMP levels and PKA activation. CONCLUSIONS AND IMPLICATIONS: GLP-1 receptor activation in platelets by linagliptin and liraglutide strongly attenuated endotoxaemia-induced microvascular thrombosis and mortality by a cAMP/PKA-dependent mechanism, preventing systemic inflammation, vascular dysfunction and end organ damage. LINKED ARTICLES: This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc.

The heat shock-induced cell cycle arrest is attenuated by weak electromagnetic fields.

Stress-induced effects in human acute leukaemia cells (HL-60) were studied by flow cytometry using the fluorescent dye carboxyfluorescein succinimidyl ester which allows the analysis of several successive cell generations for up to 10 days. Asynchronously cycling cells subjected to heat shock (30 min at 41 degrees C) responded in two distinct ways: while one fraction of the cell population (about 15%) re-entered the cell cycle after a short delay, other cells became arrested at different phases of the cell cycle and remained arrested for up to several days and finally underwent apoptosis. Weak electromagnetic fields (60 micro T, 50 Hz) alleviated the heat-induced block and the fraction of arrested cells was significantly smaller.

Chemical and biological characterization of cinnamic acid derivatives from cell cultures of lavender (Lavandula officinalis) induced by stress and jasmonic acid.

Cell cultures of lavender (Lavandula officinalis) were analyzed for the metabolite profile under normal growth conditions and under stress as well as after jasmonic acid treatment. The main compound synthesized was rosmarinic acid, which was also secreted into the culture medium. Different solvent extraction methods at different pH values altered the profile slightly. Anoxic stress induced the synthesis of a cinnamic acid derivative, which was identified as caffeic acid by gas chromatography-mass spectrometry. Caffeic acid was also induced after treatment of the cell cultures with jasmonic acid. Although the antioxidative activity of both compounds, rosmarinic acid and caffeic acid, was confirmed in an assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH), it was demonstrated that both substances have a low cytotoxic potential in vitro using acute myeloid leukemia (HL-60) cells. The potential of the system for finding new bioactive compounds is discussed.

Biological effects of epicuticular flavonoids from Primula denticulata on human leukemia cells.

The biological effects of epicuticular substances in farinose exudates accumulated on inflorescence shafts and calyces of Primula denticulata on human acute myeloid leukemia cells (HL-60) were analyzed. The crude material possessed little antioxidative capacity but strong cytostatic properties. Some of its known components (5-hydroxyflavone, 2'-hydroxyflavone, 5,2'-dihydroxyflavone, and 5,8-dihydroxyflavone) were further tested to identify the biologically active compounds. The effects of these flavones on cell cycle progression, mitochondrial membrane potential, and reactive oxygen species have been investigated by flow cytometry. The flavonol quercetin was included in the study as reference compound because of its known cytostatic properties and its activity as radical scavenger. Compared to quercetin the flavones induced little apoptosis (up to 40 microM), but despite their low toxicity, the Primula flavonoids possessed strong cytostatic properties even at low concentrations. The cell cycle distribution showed a characteristic time-dependent shift, giving evidence of a generally short-lived effect of the test compounds in the exposed cells. The antioxidative properties quantified according to two different methods correlated with the number of hydroxyl groups. Whereas quercetin strongly affected the mitochondrial membrane potential, none of the Primula flavones showed a comparable effect.

Radiosensitization of p53-deficient lung cancer cells by pre-treatment with cytostatic compounds.

BACKGROUND/AIM: This study specifies a strategy to improve radiotherapy by partial synchronization of p53-deficient cancer cells (FaDu and H1299) in mitosis using taxol, with protecting p53 wild-type cells (A549) by the prior administration of cytostatic compounds. Cytotoxic and cytostatic effects of ionizing radiation, cisplatin, doxorubicin and taxol, administrated alone or in combination were investigated in vitro by flow cytometry. RESULTS: A protective effect of doxorubicin but not cisplatin was found after administration of triple sequence with ionizing radiation and taxol. It was found that preliminary administration of doxorubicin induced growth arrest and protected A549 cells from the taxol/radiation treatment, while simultaneously killing FaDu and H1299 cells. CONCLUSION: The proposed therapeutic strategy allows protection of p53 wild-type cells and selectively increases radiosensitivity of p53-deficient cancer cells.

Usefulness of dynamic contrast enhanced computed tomography in patients with non-small-cell lung cancer scheduled for radiation therapy.

OBJECTIVE: The goal of this study was to investigate the local tumor blood supply parameters relative tumor blood volume (rTBV) and transfer coefficient (K(trans)) measurable with dynamic contrast enhanced computed tomography (DCE-CT) in patients with non-small-cell lung cancer (NSCLC) scheduled for radiation therapy (RT). MATERIALS AND METHODS: rTBV and K(trans) were measured before RT in 31 patients with clinically inoperable NSCLC (Stages I-III), which received (n=19) or did not receive (n=12) induction chemotherapy (IChT). Possible links between rTBV and K(trans) and time-to-progression (TTP), overall survival (OS) and maximum standardized uptake value (SUV(max)) from fluorodeoxyglucose positron emission tomography as well as histology were analyzed. RESULTS: NSCLC showed a wide range of rTBV and K(trans) values as estimated by DCE-CT (6.4±0.6ml/100ml and 18.2±1.5ml/100ml/min correspondingly). A significant difference in rTBV values in patients with IChT (4.6±0.6ml/100ml) and without IChT (7.5±0.9ml/100ml; p=0.023), depending on the number of cycles of the IChT and the clinical stage was found. A negative correlation between rTBV and TTP was revealed only in RT patients up-staged by FDG-PET/CT from stage III to stage IV (n=7, r=-0.96, p=0.0006). An inverse correlation between K(trans) and TTP (n=24, r=-0.53, p=0.008) was observed in all RT patients. No relevant correlation was detected between rTBV, K(trans) and SUV(max) or histologic subtypes and grading. CONCLUSIONS: Tumor blood supply parameters derived from DCE-CT are useful to characterize tumor vascularization before radiotherapy in patients with NSCLC and data on outcome prediction are supplemented.

Fusion of images in multistep analysis of neovascularisation.

PURPOSE: The aim of our study was to develop a method for the fusion of images received after repeated staining of the same sample taking into account spatial differences between the images. MATERIAL AND METHODS: A method of objective fusion performance was investigated on the images receiving during multistep staining of the xenograft tumour cross-sections. RESULTS: It was shown that several images receiving from different steps of staining procedures may be successfully fused by fluorescent marking of slide position with Trout red blood cells before analysis. CONCLUSIONS: Proposed technique provides an accurate rigid fusion of light and fluorescent images receiving during multistep image analysis under microscope and may be applied for study of neovascularisation.

Is pre-therapeutical FDG-PET/CT capable to detect high risk tumor subvolumes responsible for local failure in non-small cell lung cancer?

BACKGROUND AND PURPOSE: Local failure is a significant issue following radiotherapy (RT) for patients with non-small cell lung cancer (NSCLC). The aim of this study was to find out whether FDG-PET/CT is capable to predict tumor relapse location in patients with NSCLC, in particular to determine high risk tumors' subvolumes responsible for local failure. MATERIAL AND METHODS: Ten patients with locoregional relapse of NSCLC underwent FDG-PET/CT before, during, and in the 4-12 months following curative chemoradiotherapy (ChRT, 66 Gy) using a combined PET/CT scanner. Morphologic and metabolic tumor volumetry and an evaluation of FDG-uptake dynamics were performed. RESULTS: CT showed partial reduction of tumor volume after RT in all patients. PET-revealed partial in eight patients and complete metabolic response in two patients during RT. Six to nine months after RT, local failure was diagnosed in all patients with both methods. Tumor recurrences were localized mostly in the most active ones of pre-therapeutically metabolic regions of the primary tumor. CONCLUSIONS: Local failure in NSCLC appears most common at the primary site and within the irradiated target volume with the highest FDG uptake. This observation may be useful for further optimization of radiotherapy of NSCLC, for example, by the application of additional radiation dose to subvolumes of primary tumors with higher FDG uptake.

Lectin-binding pattern as tool to identify and enrich specific primary testis cells of the tilapia (Oreochromis niloticus) and medaka (Oryzias latipes).

Cell type-specific lectin binding is a useful tool for the analysis of developing systems. We describe the binding pattern of 21 different fluorescein isothiocyanate (FITC)-labelled lectins to the testis of two model teleost species, the medaka (Oryzias latipes) and the tilapia (Oreochromis niloticus). The analysis of the binding pattern was carried out on tissue sections (medaka and tilapia) and using primary culture cells (only tilapia). Lectin binding was studied by confocal microscopy and for histological analysis some sections were, in addition, stained with bodipy to gain additional information concerning the cytological organization of the cystic mode of spermatogenesis in fish. The observed differences in lectin staining of different cell types in primary cultures were quantified by flow cytometry. Only few lectins bound specifically to haploid cells while the reaction to diploid or tetraploid cells was generally stronger. However, the extracellular material around the haploid spermatids and spermatozoa in spermatocysts showed a strong staining reaction with several lectins (e.g., Phaseolus vulgaris Erythro agglutinin). The apparent differences in the cellular lectin-binding pattern can be used to identify particular cell types, to monitor their differentiation in vitro or to enrich particular cell types from heterogeneous cultures using magnetic beads coated with anti-FITC antibodies. Using the latter approach, we show that it is possible to enrich for gonial cells and at the same time deplete the preparation for haploid cells and Sertoli cells.

Quercetin metabolism in vital and apoptotic human leukaemia cells.

The metabolism of the flavonol quercetin in human leukaemia (HL-60) cells was investigated. The fluorescence that is elicited by quercetin upon binding to a target protein was quickly attenuated in vital cells, while apoptotic cells showed persistent fluorescence. The dynamics of induction and loss of fluorescence in the cells were quantified by flow cytometry. Several potential metabolites of quercetin, apart from isorhamnetin, had weak or no fluorogenic properties with test proteins. HPLC analysis showed that quercetin was metabolised to several substances, among them glycosylated metabolites. The loss of fluorescence in vital cells offers the unique opportunity to directly observe the metabolic conversion of quercetin in human cells.