[Can Analysis of Cellular Lipidome Contribute to Discrimination of Tumour and Non-tumour Colon Cells?] / Muze analýza bunecného lipidomu prispet k rozlisení nádorových a nenádorových bunek tlustého streva?

BACKGROUNDS: Colon cancer development is often characterized by abnormalities in lipid synthesis and metabolism, which may influence energetic balance, structure and function of biological membranes, or production of specific mediators and cell signalling. The changes in lipid profile and metabolism (lipidome) may significantly affect cell behaviour and response to therapy. Permanent epithelial cell lines at various stages of cancer development are used for better understanding of this topic on cellular and molecular levels. In our study, we hypothesized that detailed analyses of colon cancer cell line lipidomes may help to identify major alterations in the amount and profile of specific lipid classes/species, which can contribute to their different response to various stimuli. MATERIAL AND METHODS: Cellular lipids were isolated from six human epithelial cell lines derived from tissues at various stages of tumour development. Liquid chromatography coupled with tandem mass spectometry analyses were performed in order to determine amount and mass profiles of all phospholipid (PL), lysophospholipid (lysoPL) and sphingolipid classes. The data was statistically evaluated (cluster and discrimination analyses) with respect to mutual comparison of cell lines and to significantly discriminating lipid types. RESULTS: The results of cluster analysis arranged cell lines in order corresponding to their level of transformation (normal cells, adenoma, carcinoma and lymph node metastasis). The results of discrimination analyses revealed the most discriminating lipid types and distinction in PL: lysoPL ratios. Particularly, significant correlation of the amount and profiles of both specific lysoPL and sphingolipid classes with cell transformation level were observed. Similar approaches are now applied to compare lipidomes of colon epithelial cells isolated from tumour vs. non-tumour samples of colon cancer patients. CONCLUSION: Our results indicate that a) selected cancer cell lines are suitable model for lipidomic studies that can serve as a basis for subsequent clinical research, b) cellular lipidome analyses may help to discriminate tumour and non-tumour cells in clinical samples, where specific types of lipids could serve as biomarkers.Key words: colon cancer - cell lines - liquid chromatography - mass spektrometry - phospholipids - sphingolipids - bioinformatics The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. This work was supported by Czech Health Research Council, grant No. AZV 15-30585A.Submitted: 19. 3. 2018Accepted: 18. 4. 2018.

Histopathological autoptic findings in 8 patients with pandemic influenza A (H1N1) pneumonia.

UNLABELLED: In the years 2009 and 2010 a novel influenza A (H1N1) caused the first influenza pandemic after 41 years. In the Czech Republic it culminated in November and December 2009 and there were 101 laboratory-confirmed deaths. Another few cases occurred later in the year 2010 and at the beginning of 2011. Here we report 8 autoptic cases of patients who died between 2009 and 2011 with confirmed H1N1 influenza and underwent a post mortem examination at the Institute of Pathology, General University Hospital in Prague, Czech Republic. This group differs from the others reported in literature by having a higher age as well as a higher percentage of patients with pre-existing severe comorbidities including malignant diseases. All 8 patients developed atypical pneumonia with subsequent respiratory failure. In this article we present these cases with related clinical data and findings in other organs, but we focus primarily on the findings in the respiratory tract which were shown to be approximately similar to those in the other studies and case reports. Nevertheless there were also some noteworthy variations. The most prominent feature observed was diffuse alveolar damage accompanied by intraalveolar haemorrhage and inflammatory infiltrate of variable extent. Less frequent features included cytopathic changes of pneumocytes and their desquamation, reactive changes of bronchial epithelium, intraalveolar fibrinous exudate, minor necroses, residual necrotizing bronchitis, focal granulation tissue and incipient fibrosis. In one case we found an extraordinary vascular change of uncertain origin. In conclusion, this group of patients is slightly atypical and differ in some features from those in other published studies and case reports concerning novel pandemic influenza. By reporting them we wish to extend the number of described cases, which may contribute to a better understanding of the pathogenesis of novel influenza infection. KEYWORDS: influenza A virus, subtype H1N1 - viral pneumonia - diffuse alveolar damage.

Tribology provides an in vitro tool that correlated to in vivo sensory data on the mouthfeel of coated tablets.

Tribology is an emerging technique in the pharmaceutical field for texture and mouthfeel studies. Due to its relevance to oral sensory perception, tribology supports the development of novel products in the food industry. This study explores tribology as a tool to optimise the mouthfeel and ease of swallowing of pharmaceutical coatings and coated tablets. We measured the lubricating properties of eight pharmaceutical coatings using two methods: surface tribology and thin film tribology. As food science is more advanced in texture and mouthfeel studies, methods were developed from this field with the intention to mimic tablet ingestion. Further, the link between tribological measurements and the sensory evaluation of the coated tablets obtained by a human panel was explored. We have demonstrated that discrimination of tablets with different coatings using tribology is feasible. The viscosity, solubility and composition of the coating formulations played an important factor in lubrication. For the first time, tribology was used to analyse the lubricating properties of conventional tablet coatings and a linear relationship between tribology and the oral sensory perception, i.e. slipperiness and stickiness, was demonstrated. Tribology has the potential to become a valuable formulation tool to characterise the lubricating behaviour of coated tablets in the context of oral sensory perception.

Sensory attributes of coated tablets: Developing a formal lexicon and sensory wheel.

The patient's sensory experience when taking an oral medicine is important in the assessment of its palatability, and acceptability. The aim of this study was to develop tools useful for standardisation of sensory assessment of coated tablets: a lexicon and a sensory wheel. Two randomised, double-blind sensory assessments were performed involving 83 and 52 heathy adult volunteers and two sets of coated tablets. By adapting the principles used by food sciences, a free-text description of conventional, bitter-tasting or tasteless, coated tablets was performed. In the first assessment, volunteers described the sensory attributes of the first set of tablets. The attributes collected were then validated using a second set of tablets in a separate study with different volunteers. The appropriateness and semantics of each sensory attribute was analysed. Twenty attributes most relevant for assessment of coated tablets were selected for the lexicon and associated with explicit definitions. A collection of all attributes that could possibly be triggered by coated tablets were organised in the form of a sensory wheel. This study provides a valuable insight into the sensory experience while taking a coated tablet and presents tools which can accelerate the development of palatable medicines.

Developing methodology to evaluate the oral sensory features of pharmaceutical tablet coatings.

Acceptability of medicines is critical for effective pharmacotherapy. The aim of this study was to investigate the oral sensory properties of tablet coatings to determine how mouthfeel can improve acceptability. A randomised double-blind study was performed in 84 adult volunteers (51% ≥55 years). Each participant received 4 placebo tablets (3 coated and 1 uncoated) to evaluate (i) ease of swallowing and (ii) palatability. Visual analogue scales (VAS) were used to capture sensory parameters. Acceptability was assessed using the following parameters: ease of swallowing; amount of water taken with the tablet; rank order of preference; roughness; adhesiveness and slipperiness. Ease of swallowing was determined to be the most sensitive measure of acceptance. The best coating was the one that was reported to be the most slippery and smooth. The presence of a coating improved ease of swallowing, mouthfeel and overall palatability. This study demonstrates that slippery coatings improve acceptability of tablets. The study also demonstrates the value of VAS to measure the sensory attributes of coated tablets.

The interaction of butyrate with TNF-alpha during differentiation and apoptosis of colon epithelial cells: role of NF-kappaB activation.

We demonstrated that TNF-alpha suppressed differentiation and potentiated cell death induced by butyrate (NaBt) in both adenocarcinoma HT-29 and fetal FHC human colon cells in vitro. Since TNF-alpha is a typical activator of NF-kappaB pathway, we studied the role of NF-kappaB activation in cell differentiation and death during the TNF-alpha and NaBt co-treatment. TNF-alpha induced rapid NF-kappaB activation in both HT-29 and FHC cell lines and this effect was differently modulated by NaBt in these two cell lines. In HT-29 cells, NaBt potentiated NF-kappaB activity induced by TNF-alpha after 4h treatment. However, this initial potentiation of NF-kappaB activity was not observed in FHC cells. During additional time of TNF-alpha and NaBt co-treatment, NaBt decreased the TNF-alpha-mediated NF-kappaB activity in both cell types. We also detected a different response of HT-29 and FHC cells after the pre-treatment with the NF-kappaB inhibitor parthenolide. Our results indicated that NaBt-mediated differentiation and apoptosis of colon epithelial cells can be modulated by TNF-alpha. Furthermore, we found significant differences in the mechanism of the NaBt and TNF-alpha co-treatment effects between cells of non-cancer and cancer origin, suggesting that the NF-kappaB pathway may be more effectively involved in these processes in cancer cells.

Non-dioxin-like polychlorinated biphenyls induce a release of arachidonic acid in liver epithelial cells: a partial role of cytosolic phospholipase A(2) and extracellular signal-regulated kinases 1/2 signalling.

Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) have been shown to act as tumor promoters in liver; however, the exact mechanisms of their action are still only partially understood. One of the interesting effects of NDL-PCBs is the acute inhibition of gap junctional intercellular communication (GJIC), an effect, which has been often found to be associated with tumor promotion. As previous studies have suggested that NDL-PCB-induced disruption of lipid signalling pathways might correspond with GJIC inhibition, we investigated effects of PCBs on the release of arachidonic acid (AA) in the rat liver epithelial WB-F344 cell line, a well-established model of liver progenitor cells. We found that both 2,2',4,4'-tetrachlorobiphenyl (PCB 47) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), but not the dioxin-like, non-ortho-substituted, 3,3',4,4',5-pentachlorobiphenyl (PCB 126), induce a massive release of AA. The AA release, induced by PCB 153, was partially inhibited by extracellular signal-regulated kinases 1/2 (ERK1/2) signalling inhibitor, U0126, and by cytosolic phospholipase A(2) (cPLA(2)) inhibitor, AACOCF(3). Although PCB 153 induced both ERK1/2 and p38 activation, the specific p38 kinase inhibitor, SB203580, had no effect on AA release. Inhibitors of other phospholipases, including phosphatidylcholine-specific phospholipase C or phosphatidylinositol-specific phospholipase C, were also without effect. Taken together, our findings suggest that the AA release, induced by non-dioxin-like PCBs in liver progenitor cell line, is partially mediated by cytosolic PLA(2) and regulated by ERK1/2 kinases. Our results suggest that more attention should be paid to cell signalling pathways regulated by AA or eicosanoids after PCB exposure, which might be involved in their toxic effects.

5-Lipoxygenase inhibitors potentiate effects of TGF-beta 1 on the differentiation of human leukemia HL-60 cells.

It was clearly demonstrated that two structurally different inhibitors of 5-lipoxygenase (5-LPO) metabolism (leukotriene synthesis), i.e. MK-886 and esculetin, when combined with transforming growth factor-beta 1 (TGF-beta 1), significantly enhanced the differentiation but did not change proliferation (i.e. cell number and cell cycle parameters) of human leukemia HL-60 cells in vitro. Although cell morphology and measurement of cell surface antigens (CD11b, CD14, and CD66b) after 48 h of combined treatment with MK-886 and TGF-beta 1 suggested a shift of the HL-60 cell population into more differentiated stages of myelopoiesis, cells with a fully mature phenotype were not observed. The effects on differentiation were better distinguished in the functional parameters of differentiation, i.e. oxidative burst of cells as detected by chemiluminescence and the nitroblue tetrazolium reduction test. Detailed statistical analysis of these data proved a significant synergistic interaction between TGF-beta 1 and inhibitors of 5-LPO metabolism. The differentiation effects of TGF-beta 1 alone and especially of its combination with MK-886 were most pronounced when the cells were pretreated with dimethyl sulfoxide or all-trans-retinoic acid. The results imply that either the lack of 5-LPO metabolites or a certain type of mis-balance in arachidonic acid metabolism can modulate the TGF-beta 1 effect on myeloid differentiation.

Modulation of death receptor-mediated apoptosis in differentiating human myeloid leukemia HL-60 cells.

Differentiating myeloid cells may become resistant to various apoptotic stimuli. In the present study, dimethyl sulfoxide (DMSO) and all-trans retinoic acid (ATRA) were found to modulate the sensitivity of HL-60 cells to death receptor-mediated apoptosis in a time-dependent manner. During the early stages of differentiation, DMSO treatment increased the response of HL-60 cells to tumor necrosis factor alpha; (TNF-alpha), but enhanced responsiveness was lost during later differentiation stages. In contrast, ATRA treatment induced resistance to TNF-alpha-induced apoptosis. HL-60 cells were resistant to Fas-mediated apoptosis but were sensitized by culturing in serum-free conditions. Similar to its effect on TNF-alpha sensitivity, DMSO pretreatment augmented the response to Fas-mediated signaling, which coincided with increased expression of Fas on DMSO-pretreated cells. However, during the later stages of DMSO-induced differentiation, sensitivity to anti-Fas antibody-induced apoptosis declined significantly, although Fas expression was still elevated. The reduced sensitivity to anti-Fas treatment partially correlated with increased Fas-associated phosphatase-1 mRNA expression. Thus, regardless of either Fas up-regulation or potentiation of TNF-alpha-mediated apoptosis during early DMSO-induced differentiation, a slow increase in resistance to apoptosis mediated through these death receptors occurs during DMSO-induced differentiation, which contrasts with the rapid induction of resistance following treatment with ATRA.

Retinoid X receptor suppresses transformation by the v-myb oncogene.

The v-myb oncogene of avian myeloblastosis virus causes acute monoblastic leukemia in vivo and transforms myelomonocytic cells in culture. Retinoids are potent regulators of proliferation and differentiation in various cell types, and they can initiate differentiation in certain types of leukemic cells. However, the BM2 v-myb-transformed chicken monoblastic cell line is resistant to retinoic acid treatment. We found that overexpression of the retinoid X receptor confers sensitivity of BM2 cells to retinoic acid, resulting in induction of growth arrest and terminal differentiation. In contrast, the frequency of apoptosis was not affected by the retinoid X receptor in this cell type. We also demonstrated that suppression of transformation by v-Myb results from the negative effect of retinoid X receptor on v-Myb transactivation function, similar to that previously described for the retinoic acid receptor. The retinoid X receptor-induced inhibition of transactivation by v-Myb seems to be enhanced by a cell type-specific factor(s), which is not required by retinoic acid receptor.

The effects of parenteral lipid emulsions on cancer and normal human colon epithelial cells in vitro.

Differences in lipid metabolism of tumor and normal tissues suggest a distinct response to available lipid compounds. In this study, the in vitro effects of five types of commercial parenteral lipid emulsions were investigated on human cell lines derived from normal fetal colon (FHC) or colon adenocarcinoma (HT-29). Changes of the cellular lipid fatty acid content, cell oxidative response, and the cell growth and death rates were evaluated after 48 h. No effects of any type of emulsions were detected on cell proliferation and viability. Compared to the controls, supplementation with lipid emulsions resulted in a multiple increase of linoleic and linolenic acids in total cell lipids, but the content of arachidonic, eicosapentaenoic, and docosahexaenoic acids decreased particularly in HT-29 cells. The concentration of emulsions which did not affected HT-29 cells increased the percentage of floating and subG0/G1 FHC cells probably due to their higher reactive oxygen species production and lipid peroxidation. Co-treatment of cells with antioxidant Trolox reduced the observed effects. Our results imply that lipid emulsions can differently affect the response of colon cells of distinct origin.

The effect of nordihydroguaiaretic acid, an inhibitor of prostaglandin and leukotriene biosynthesis, on hematopoiesis of gamma-irradiated mice.

The effects of the inhibition of the cyclooxygenase and lipoxygenase metabolic pathways of arachidonic acid on the postirradiation recovery of hematopoietic functions in mice were investigated. Nordihydroguaiaretic acid (NDGA), an inhibitor of prostaglandin (PG) and leukotriene (LT) production, was given to animals in single doses (0.015 to 0.75 mg/mouse) 1 hour before 5 Gy of total-body gamma-irradiation. Enhanced hematopoietic recovery in terms of exogenous and endogenous spleen colonies, femoral granulocyte-macrophage colony-forming cells and peripheral blood granulocyte levels was observed at higher doses of NDGA. The treatment used influenced neither lymphocyte nor erythrocyte postirradiational levels or hemoglobin concentration. A comparison of the effects induced by a high dose of NDGA (0.3 mg per mouse) with those observed after an isomolar dose of indomethacin (an inhibitor of PG production) indicated only slight differences between these two drugs. An isomolar dose of esculetin (an inhibitor of LT production) had no effect on the postirradiation behavior of hematopoiesis. The results suggest that the inhibition of PG production plays the main role in the mechanism of NDGA action. Inhibition of LT production seems to be of less importance for hematopoiesis in these in vivo conditions.

TNF-alpha modulates the differentiation induced by butyrate in the HT-29 human colon adenocarcinoma cell line.

The aim of this study was to determine whether and how tumour necrosis factor alpha (TNF-alpha) modulates butyrate effects. After the treatment of human colon adenocarcinoma HT-29 cells with sodium butyrate (NaBt), TNF-alpha or with their combinations we detected cell cycle (flow cytometry), cell proliferation (amidoblack and MTT assays), the amount of dead (floating) and apoptotic cells (flow cytometry and fluorescence microscopy), and the level of differentiation by alkaline phosphatase (ALP) activity (spectrophotometry), relative F-actin content (confocal laser scanning microscopy analysis) and E-cadherin expression (Western blot analysis). Both TNF-alpha and NaBt decreased cell growth in a dose-dependent manner. After combined treatment of the cells with both agents used, either none or additive effects were observed as compared with NaBt treatment alone. The level of dead and apoptotic cells was dose-dependently increased after this combined treatment. In contrast, TNF-alpha suppressed ALP activity and F-actin accumulation induced by NaBt. The results suggest that TNF-alpha does not influence significantly the antiproliferative effects of NaBt but, contrary to its potentiation of apoptosis, it markedly reduces NaBt-induced differentiation of HT-29 colon adenocarcinoma cells.

Eicosanoid inhibitors enhance synergistically the effect of transforming growth factor beta 1 on CCL 64 cell proliferation.

The interactions between drugs suppressing the production of arachidonic acid metabolites-eicosanoids and transforming growth factor beta 1 (TGF-beta 1) were investigated using CCL64 cells. These experiments, designed as complete factorial combination of treatments, demonstrated that both esculetin and eicosatetraynoic acid significantly potentiated the inhibitory effect of TGF-beta 1 on [3H]thymidine incorporation. The expression of overadditive effects depended both on the type and concentration of combined factors. These results corresponded with cell cycle analysis data (increased cell number in G1 and decreased cell number in S and G2/M phases) and with the results monitoring cell number following treatment with eicosatetraynoic acid, esculetin, 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-di methyl propanoic acid (MK-886) and indomethacin. Summarizing, the degree of significance of combined effects supports the hypothesis of synergistic potentiation of TGF-beta 1 effects caused by eicosanoid inhibitors. The results indicate that either the lack of some eicosanoids or a certain type of misbalance in the metabolism of arachidonic acid leading to its abundance might modulate TGF-beta 1 effects on the cell cycle and proliferation in CCL64 cells.

Inhibitors of lipoxygenase metabolism exert synergistic effects with retinoic acid on differentiation of human leukemia HL-60 cells.

The interaction between drugs suppressing the production of arachidonic acid metabolites and inducers of granulocytic differentiation, i.e., all-trans retinoic acid and dimethyl sulphoxide (DMSO) was investigated using the human myeloid leukemia HL-60 cell line. The experiments were designed as a complete factorial combination of treatments and used chemiluminescence as a marker of cell oxidative burst (level of differentiation). It was clearly demonstrated that two structurally different inhibitors of 5-lipoxygenase metabolism, i.e., 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-di methyl propanoic acid (MK-886) and esculetin, significantly potentiated the HL-60 cell differentiation induced by retinoic acid or DMSO. Detailed mathematical evaluation of the results revealed the synergistic character of the interaction. The most significant effects were achieved with a combination of 5-lipoxygenase inhibitors and low doses of retinoic acid. These results were confirmed by analysis of cell morphology and expression of cell surface antigen CD11b after treatment of the cells with selected concentrations of agents. In contrast to those on differentiation, no additional effects of MK-886 or esculetin on cell proliferation (cell number and cell cycle parameters) and apoptosis were observed. An inhibitor of cyclooxygenases, indomethacin, affected neither cell proliferation nor differentiation of cells. The results implied that either modulation of 5-lipoxygenase metabolism or a certain type of imbalance in arachidonic acid metabolism could modulate the effects of retinoic acid or DMSO on myeloid cell differentiation.

Inhibitors of arachidonic acid metabolism potentiate tumour necrosis factor-alpha-induced apoptosis in HL-60 cells.

We investigated whether and how could various modulators of arachidonic acid metabolism affect apoptosis induced by tumour necrosis factor-alpha (TNF-alpha) in human myeloid leukaemia HL-60 cells. These included arachinonyltrifluoromethyl ketone (AACOCF3; cytosolic phospholipase A2 inhibitor), indomethacin (cyclooxygenase inhibitor), MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethyl propanoic acid; 5-lipoxygenase-activating protein inhibitor), nordihydroguaiaretic acid (general lipoxygenase inhibitor), and arachidonic acid itself. Incubation of HL-60 cells with nordihydroguaiaretic acid resulted in apoptosis and it was characterised by mitochondria membrane depolarisation, release of cytochrome c from mitochondria into cytosol and activation of caspase-3. Indomethacin and nordihydroguaiaretic acid synergistically potentiated TNF-alpha-induced apoptosis, while arachidonic acid, AACOCF3 and MK-886 did not modulate its effects. Furthermore, indomethacin potentiated apoptosis in cells treated with a differentiating agent, all-trans retinoic acid, which induces resistance to TNF-alpha. However, the observed effects were probably not associated either with the cyclooxygenase- or lipoxygenase-dependent activities of indomethacin and nordihydroguaiaretic acid, respectively. Since indomethacin may reportedly activate peroxisome proliferator-activated receptors (PPARs), the effects of specific ligands of PPARs on apoptosis were studied as well. It was found that selective PPARs ligands had no effects on TNF-alpha-induced apoptosis. The findings suggest that arachidonic acid metabolism does not play a key role in regulation of apoptosis induced by TNF-alpha in the present model. Nevertheless, our data raise the possibility that indomethacin could potentially be used to improve the treatment of human myeloid leukaemia.

Effects of drugs inhibiting prostaglandin or leukotriene biosynthesis on postirradiation haematopoiesis in mouse.

Two non-steroidal anti-inflammatory drugs, i.e. indomethacin (INDO), an inhibitor of prostaglandin production, and esculetin (ESCUL), an inhibitor of leukotriene production, were tested for their ability to modify haematopoiesis in three experimental systems: (a) in vitro clonal proliferation of marrow GM-CFC from the irradiated mouse was found to be augmented by addition of INDO at a low concentration, and inhibited by ESCUL in a dose-dependent manner; (b) in the lethally irradiated and bone marrow-transplanted mice treated with the drugs in the postirradiation period, stimulatory effects of INDO on CFU-S and GM-CFC populations and an inhibitory effect of ESCUL on GM-CFC were observed; and (c) when the drugs were administered i.p. to mice 1 h before 5-Gy irradiation, INDO enhanced the postirradiation recovery of haematopoietic indices such the numbers of CFU-S, GM-CFC, peripheral blood granulocytes, and nucleated bone marrow cells, while ESCUL had no effect or even inhibited the recovery of these indices. Survival curves for CFU-S and GM-CFC showed that altered haematopoietic recovery in the INDO- and ESCUL-pretreated mice was not due to changes of intrinsic radiosensitivity of pluripotent (CFU-S) or committed (GM-CFC) stem cell populations. These results confirm earlier findings suggesting an inhibitory role of prostaglandins on haematopoiesis, and provide evidence that endogenous leukotrienes might play a positive role in the regulation of haematopoietic functions in an irradiated organism.

Lipoxygenase inhibitors suppress proliferation of G5:113 fibrosarcoma cells in vitro but they have no anticancer activity in vivo.

Nordihydroguaiaretic acid (NDGA) and esculetin, both nonspecific inhibitors of lipoxygenases (LOX), were found to suppress expressively the in vitro proliferation of fibrosarcoma cells G5:113 in concentrations ranging from 10 to 50 microM. Subsequent flow-cytometric analysis of the cell cycle showed that both these drugs significantly decreased the percentage proportion of cells in the G0/G1-phase and simultaneously increased significantly this proportion in the S-phase. No apoptosis was detected in the whole range of concentrations studied, from 2.5 to 50 mM. On the contrary, in experiments in vivo, neither NDGA nor esculetin had any curative effect if they were repeatedly injected intraperitoneally (i.p.) into mice bearing tumors growing from subcutaneously (s.c.) transplanted G5:113 cells. Pretreatment of the fibrosarcoma cells with NDGA or esculetin in vitro preceding their s.c. transplantation into mice did not result in suppression of the tumor growth, either. Finally, if G5:113 cells were injected intravenously and the mice were subsequently treated repeatedly with i.p. injections of NDGA, decreased survival and increased number of surface lung metastases were observed in the NDGA-treated group. Thus the suppressive action of inhibitors of LOX on the growth of fibrosarcoma cells in vitro was not reflected in their anti-tumor effects in vivo.

Lipoxygenase inhibitors induce arrest of tumor cells in S-phase of the cell cycle.

Inhibitors of the lipoxygenase pathway of arachidonic acid metabolism represent a potential anti-tumor drugs. These compounds have been found to inhibit the growth and induce the apoptosis of various tumor cells both in vitro and in vivo. In this study, the effects of the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid (NDGA) on the progression of the cell cycle were investigated in eight mammalian cell lines of different origin. Flow cytometric analyses of cell cycle distribution after staining of DNA with propidium iodide or 7-aminoactinomycin D and DNA synthesis using incorporation of 5-bromo-2'-deoxy-uridine showed that both esculetin and NDGA suppress cell growth by interrupting the progression of cells through S-phase that results in their accumulation in this phase of the cell cycle. The possible mechanisms of these effects and the significance of the findings for the improvement of anticancer therapy targeted on cell cycle is discussed.

Monitoring river sediments contaminated predominantly with polyaromatic hydrocarbons by chemical and in vitro bioassay techniques.

Extracts of sediment samples collected from the Morava River and its tributaries (Czech Republic) were examined for mutagenic, dioxin-like, and estrogenic activities. Moreover, the human leukemic HL-60 cell line was tested as a potential model for the detection of effects of environmental contaminants on cell proliferation and differentiation processes. Analytical data indicate that the sediments were contaminated predominantly with polycyclic aromatic hydrocarbons (PAHs) and phthalate esters. The sums of concentrations of 16 U.S. Environmental Protection Agency priority PAHs ranged from 0.8 to 13.2 micrograms/g and those of phthalates reached up to 3,000 ng/g, while only low levels of chlorinated hydrocarbons were found. The main goal of the present study was to determine effects of PAH prevalence on in vitro bioassays, with special emphasis on dioxin-like activity. The dioxin-like activity was tested using a reporter gene assay based on chemical-activated luciferase expression (the CALUX assay). Significant dioxin-like activity (2.6-40.1 micrograms/g benzo[a]pyrene equivalents and 5.9-48.2 ng/g 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents) was detected in all samples, and the results obtained with various exposure times or with both crude and PAH-deprived extracts indicate that the response was probably caused almost exclusively by the presence of high concentrations of PAHs. This corresponds with results of chemical analyses and indicates that various exposure times would allow a discrimination between dioxin-like activities of persistent compounds and easily metabolized aryl hydrocarbon (Ah) receptor inducers. Only sediment extracts containing the highest concentrations of PAHs were mutagenic, as determined by the umu assay. Estrogenic activity was found in several samples (4.75-22.61 pg/g estradiol equivalents) using cells stably transfected with an estrogen-responsive element linked to a luciferase promoter. Noncytotoxic doses of extracts had no effects on HL-60 cell proliferation, while two of the tested crude extracts significantly enhanced their all-trans retinoic acid-induced differentiation. These activities were not associated with phthalate esters and/or PAHs. Our results indicate that cellular and biochemical in vitro assays based on various specific modes of action may yield data complementary to results of mutagenicity tests and that they could be useful in environmental risk assessment. High levels of PAHs are apparently associated with dioxin-like and mutagenic activities rather than with estrogenic activity.