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Detail description of synthesis and supporting data

Figure S1. In vitro profiling of SCH900776 and its selected analogs. Figure S2. Analogs of MU380 with the N-trifluoromethylpyrazol pharmacophore. Figure S3. Dot plots of flow cytometric analysis using γH2AX and Annexin V staining related to Figure 2D. Figure S4. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentrations in combination with HU in the concentration range shown on the x-axis (related to Figure 3A). Figure S5. Dynamics of cytotoxic effects of CHK1 inhibitors alone and in combination with HU. Figure S6. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentration in combination with GEM in concentration range shown on the x-axis (related to Figure 3B). Figure S7. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentration in combination with irradiation in the dose range shown on the x-axis. Figure S8. Western blot analysis of total CHK1 and p-CHK1 (S296) levels in a panel of selected cell lines after indicated treatments. Figure S9. Drug retention analysis. Figure S10. MU380 induces CHK1 pS345 in mice hair follicles. Figure S11. Bioluminescent signal of SHO mice bearing MiaPaCa2 luc xenografts treated with GEM, SCH900776, or MU380 alone or in indicated combinations. Weight of MiaPaCa2 luc tumor-bearing animals. Representative images of γH2AX staining in MiaPaCa2 luc tumor sections.

Detail description of Material and Methods

Supplementary Legends for Supplemetary Figure S1 - S11

Sphingolipids (SLs), glycosphingolipids (GSLs), and eicosanoids are bioactive lipids, which play important roles in the etiology of various diseases, including cancer. However, their content and roles in cancer cells, and in particular in the exosomes derived from tumor cells, remain insufficiently characterized. In this study, we evaluated alterations of SL and GSL levels in transformed cells and their exosomes, using comparative HPLC-MS/MS analysis of parental human bronchial epithelial cells HBEC-12KT and their derivative, benzo[a]pyrene-transformed HBEC-12KT-B1 cells with the acquired mesenchymal phenotype. We examined in parallel SL/GSL contents in the exosomes released from both cell lines. We found significant alterations of the SL/GSL profile in the transformed cell line, which corresponded well with alterations of the SL/GSL profile in exosomes derived from these cells. This suggested that a majority of SLs and GSLs were transported by exosomes in the same relative pattern as in the cells of origin. The only exceptions included decreased contents of sphingosin, sphingosin-1-phosphate, and lactosylceramide in exosomes derived from the transformed cells, as compared with the exosomes derived from the parental cell line. Importantly, we found increased levels of ceramide phosphate, globoside Gb3, and ganglioside GD3 in the exosomes derived from the transformed cells. These positive modulators of epithelial–mesenchymal transition and other pro-carcinogenic processes might thus also contribute to cancer progression in recipient cells. In addition, the transformed HBEC-12KT-B1 cells also produced increased amounts of eicosanoids, in particular prostaglandin E2. Taken together, the exosomes derived from the transformed cells with specifically upregulated SL and GSL species, and increased levels of eicosanoids, might contribute to changes within the cancer microenvironment and in recipient cells, which could in turn participate in cancer development. Future studies should address specific roles of individual SL and GSL species identified in the present study.

The development of colon cancer, one of the most common malignancies, is accompanied with numerous lipid alterations. However, analyses of whole tumor samples may not always provide an accurate description of specific changes occurring directly in tumor epithelial cells. Here, we analyzed in detail the phospholipid (PL), lysophospholipid (lysoPL), and fatty acid (FA) profiles of purified EpCAM+ cells, isolated from tumor and adjacent non-tumor tissues of colon cancer patients. We found that a number of FAs increased significantly in isolated tumor cells, which also included a number of long polyunsaturated FAs. Higher levels of FAs were associated with increased expression of FA synthesis genes, as well as with altered expression of enzymes involved in FA elongation and desaturation, including particularly fatty acid synthase, stearoyl-CoA desaturase, fatty acid desaturase 2 and ELOVL5 fatty acid elongase 5 We identified significant changes in ratios of specific lysoPLs and corresponding PLs. A number of lysophosphatidylcholine and lysophosphatidylethanolamine species, containing long-chain and very-long chain FAs, often with high numbers of double bonds, were significantly upregulated in tumor cells. Increased de novo synthesis of very long-chain FAs, or, altered uptake or incorporation of these FAs into specific lysoPLs in tumor cells, may thus contribute to reprogramming of cellular phospholipidome and membrane alterations observed in colon cancer.

Aims The purpose of the study was to evaluate the occurrence of Campylobacter jejuni and Campylobacter coli in the aquatic environment based on the water origin, seasonality and physico-chemical properties. Methods and results The occurrence of Campylobacter jejuni and Campylobacter coli was determined in waste (29) or surface (56) waters in 4 different seasons. The air and water temperatures were measured during sampling and chemical analyses of water samples for ammonium, chloride, chlorine, nitrite, nitrate, phosphate, and iron were performed. The thermotolerant Campylobacter spp. were more frequently detected in wastewater (59%; 17 positive samples) compared to surface water (38%; 21 positive samples), with the highest rate in autumn (67% of samples positive) and with a higher Campylobacter coli occurrence than Campylobacter jejuni (31% vs. 26%). Ammonium (above 0.2 mg/L) and chloride ion concentrations (above 60 mg/L) favour Campylobacter jejuni. Similarly, Campylobacter coli occurrence in water was supported by ammonium (above 0.2 mg/L), chloride (above 60 mg/L) and in addition by phosphate ion concentrations (below 0.7 mg/L). Conclusions Campylobacter presence in water is influenced by physico-chemical parameters such as concentrations of ammonium and chloride ions. Significance and impact of study Water environment is an alternative source of Campylobacter. The concentration of ammonium and chloride ions can be used as a basis for successful prediction of the potential occurrence of C. jejuni and C. coli in wastewater and surface water in future.

Sphingolipids (SLs) are important signaling molecules and functional components of cellular membranes. Although SLs are known as crucial regulators of neural cell physiology and differentiation, modulations of SLs by environmental neurotoxicants in neural cells and their neuronal progeny have not yet been explored. In this study, we used in vitro models of differentiated neuron-like cells, which were repeatedly exposed during differentiation to model environmental toxicants, and we analyzed changes in sphingolipidome, cellular morphology and gene expression related to SL metabolism or neuronal differentiation. We compared these data with the results obtained in undifferentiated neural cells with progenitor-like features. As model polychlorinated organic pollutants, we used 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3′-dichlorobiphenyl (PCB11) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153). PCB153 revealed itself as the most prominent deregulator of SL metabolism and as potent toxicant during early phases of in vitro neurogenesis. TCDD exerted only minor changes in the levels of analysed lipid species, however, it significantly changed the rate of pro-neuronal differentiation and deregulated expression of neuronal markers during neurogenesis. PCB11 acted as a potent disruptor of in vitro neurogenesis, which induced significant alterations in SL metabolism and cellular morphology in both differentiated neuron-like models (differentiated NE4C and NG108-15 cells). We identified ceramide-1-phosphate, lactosylceramides and several glycosphingolipids to be the most sensitive SL species to exposure to polychlorinated pollutants. Additionally, we identified deregulation of several genes related to SL metabolism, which may be explored in future as potential markers of developmental neurotoxicity.

Identification of changes of phospholipid (PL) composition occurring during colorectal cancer (CRC) development may help us to better understand their roles in CRC cells. Here, we used LC-MS/MS-based PL profiling of cell lines derived from normal colon mucosa, or isolated at distinct stages of CRC development, in order to study alterations of PL species potentially linked with cell transformation. We found that a detailed evaluation of phosphatidylinositol (PI) and phosphatidylserine (PS) classes allowed us to cluster the studied epithelial cell lines according to their origin: i) cells originally derived from normal colon tissue (NCM460, FHC); ii) cell lines derived from colon adenoma or less advanced differentiating adenocarcinoma cells (AA/C1, HT-29); or, iii) cells obtained by in vitro transformation of adenoma cells and advanced colon adenocarcinoma cells (HCT-116, AA/C1/SB10, SW480, SW620). Although we tentatively identified several PS and PI species contributing to cell line clustering, full PI and PS profiles appeared to be a key to the successful cell line discrimination. In parallel, we compared PL composition of primary epithelial (EpCAM-positive) cells, isolated from tumor and adjacent non-tumor tissues of colon cancer patients, with PL profiles of cell lines derived from normal colon mucosa (NCM460) and from colon adenocarcinoma (HCT-116, SW480) cells, respectively. In general, higher total levels of all PL classes were observed in tumor cells. The overall PL profiles of the cell lines, when compared with the respective patient-derived cells, exhibited similarities. Nevertheless, there were also some notable differences in levels of individual PL species. This indicated that epithelial cell lines, derived either from normal colon tissue or from CRC cells, could be employed as models for functional lipidomic analyses of colon cells, albeit with some caution. The biological significance of the observed PL deregulation, or their potential links with specific CRC stages, deserve further investigation.

The natural environment and water are among the sources of Campylobacter jejuni and Campylobacter coli. A limited number of protocols exist for the isolation of campylobacters in poorly filterable water. Therefore, the goal of our work was to find a more efficient method of Campylobacter isolation and detection from wastewater and surface water than the ISO standard. In the novel rapid culture method presented here, samples are centrifuged at high speed, and the resuspended pellet is inoculated on a filter, which is placed on Campylobacter selective mCCDA agar. The motile bacteria pass through the filter pores, and mCCDA agar suppresses the growth of background microbiota on behalf of campylobacters. This culture-based method is more efficient for the detection and isolation of Campylobacter jejuni and Campylobacter coli from poorly filterable water than the ISO 17995 standard. It also is less time-consuming, taking only 72 h and comprising three steps, while the ISO standard method requires five or six steps and 144–192 h. This novel culture method, based on high-speed centrifugation, bacterial motility, and selective cultivation conditions, can be used for the detection and isolation of various bacteria from water samples.

Metabolic reprogramming leading to alterations in lipid metabolism and lipid-mediated signaling may contribute to colorectal cancer (CRC) development and progression. We hypothesized that a detailed description of changes in sphingolipidome of specific cellular subpopulation residing in colon cancer tissue may help to discriminate between normal and transformed colon epithelial cells. Using HPLC-mass spectrometry, we analyzed the EpCAM-positive cells isolated from tumor and adjacent non-tumor tissues of colon cancer patients, aiming to identify potential lipid biomarkers specific for CRC. We then employed RT-qPCR-based methodology in order to analyze expression of SL metabolism-related genes in the isolated EpCAM-positive tumor cells and/or in unseparated colon tumor tissues. We observed significant changes in sphingolipid (SL) species in the EpCAM-positive tumor cells, with the accumulation of lactosylceramide (LacCer) being the most prominent. B4GALT5 and B4GALT6 genes were identified as two potential gene candidates contributing to LacCer accumulation. We further identified additional genes of SL and fatty acid metabolism (e.g. SPHK1, GBA2, NEU3, GLA, FASN, PLA2G10), which were significantly altered in colon tumor tissue. The present results indicate that the EpCAM-positive cells are a major contributor to LacCer accumulation in CRC tissue, and may help to identify novel CRC-specific lipid/gene biomarkers.

// Eva Slabakova 1, 2 , Gvantsa Kharaishvili 3 , Monika Smějova 1, 4 , Zuzana Pernicova 1, 2 , Tereza Suchankova 1 , Jan Remsik 1, 2, 5 , Stanislav Lerch 1, 5 , Nicol Strakova 1, 2 , Jan Bouchal 3 , Milan Kral 6 , Zoran Culig 2, 7 , Alois Kozubik 1, 5 , Karel Soucek 1, 2, 5 1 Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Brno, Czech Republic 2 Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne’s University Hospital Brno, Brno, Czech Republic 3 Department of Clinical and Molecular Pathology and Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic 4 Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic 5 Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic 6 Department of Urology, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic 7 Division of Experimental Urology, Department of Urology, Medical University of Innsbruck, Austria Correspondence to: Karel Soucek, e-mail: ksoucek@ibp.cz Eva Slabakova, e-mail: slabakova@ibp.cz Keywords: epithelial-mesenchymal transition, MDM2/MDMX, SNAI2/SLUG, TWIST, prostate/breast cancer Received: July 28, 2015 Accepted: September 15, 2015 Published: September 25, 2015 ABSTRACT Plasticity of cancer cells, manifested by transitions between epithelial and mesenchymal phenotypes, represents a challenging issue in the treatment of neoplasias. Both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are implicated in the processes of metastasis formation and acquisition of stem cell-like properties. Mouse double minute (MDM) 2 and MDMX are important players in cancer progression, as they act as regulators of p53, but their function in EMT and metastasis may be contradictory. Here, we show that the EMT phenotype in multiple cellular models and in clinical prostate and breast cancer samples is associated with a decrease in MDM2 and increase in MDMX expression. Modulation of EMT-accompanying changes in MDM2 expression in benign and transformed prostate epithelial cells influences their migration capacity and sensitivity to docetaxel. Analysis of putative mechanisms of MDM2 expression control demonstrates that in the context of defective p53 function, MDM2 expression is regulated by EMT-inducing transcription factors Slug and Twist. These results provide an alternative context-specific role of MDM2 in EMT, cell migration, metastasis, and therapy resistance.

Docosahexaenoic acid (DHA) and sodium butyrate (NaBt) exhibit a number of interactive effects on colon cancer cell growth, differentiation, or apoptosis; however, the molecular mechanisms responsible for these interactions and their impact on cellular lipidome are still not fully clear. Here, we show that both dietary agents together induce dynamic alterations of lipid metabolism, specific cellular lipid classes, and fatty acid composition. In HT-29 cell line, a model of differentiating colon carcinoma cells, NaBt supported incorporation of free DHA into non-polar lipids and their accumulation in cytoplasmic lipid droplets. DHA itself was not incorporated into sphingolipids; however, it significantly altered representation of individual ceramide (Cer) classes, in particular in combination with NaBt (DHA/NaBt). We observed altered expression of enzymes involved in Cer metabolism in cells treated with NaBt or DHA/NaBt, and exogenous Cer 16:0 was found to promote induction of apoptosis in differentiating HT-29 cells. NaBt, together with DHA, increased n-3 fatty acid synthesis and attenuated metabolism of monounsaturated fatty acids. Finally, DHA and/or NaBt altered expression of proteins involved in synthesis of fatty acids, including elongase 5, stearoyl CoA desaturase 1, or fatty acid synthase, with NaBt increasing expression of caveolin-1 and CD36 transporter, which may further promote DHA incorporation and its impact on cellular lipidome. In conclusion, our results indicate that interactions of DHA and NaBt exert complex changes in cellular lipidome, which may contribute to the alterations of colon cancer cell differentiation/apoptotic responses. The present data extend our knowledge about the nature of interactive effects of dietary fatty acids.

The short-chain and n-3 polyunsaturated fatty acids exhibit anticancer properties, and they may mutually interact within the colon. However, the molecular mechanisms of their action in colon cancer cells are still not fully understood. Our study focused on the mechanisms responsible for the diverse effects of sodium butyrate (NaBt), in particular when interacting with docosahexaenoic acid (DHA), in distinct colon cancer cell types, in which NaBt either induces cell differentiation or activates programmed cell death involving mitochondrial pathway. NaBt activated autophagy both in HT-29 cells, which are sensitive to induction of differentiation, and in nondifferentiating HCT-116 cells. However, autophagy supported cell survival only in HT-29 cells. Combination of NaBt with DHA-promoted cell death, especially in HCT-116 cells and after longer time intervals. The inhibition of autophagy both attenuated differentiation and enhanced apoptosis in HT-29 cells treated with NaBt and DHA, but it had no effect in HCT-116 cells. NaBt, especially in combination with DHA, activated PPARγ in both cell types. PPARγ silencing decreased differentiation and increased apoptosis only in HT-29 cells, therefore we verified the role of caspases in apoptosis, differentiation and also PPARγ activity using a pan-caspase inhibitor. In summary, our data suggest that diverse responses of colon cancer cells to fatty acids may rely on their sensitivity to differentiation, which may in turn depend on distinct engagement of autophagy, caspases and PPARγ. These results contribute to understanding of mechanisms underlying differential effects of NaBt, when interacting with other dietary fatty acids, in colon cancer cells.

Although beneficial effects of the dietary n-3 docosahexaenoic acid (DHA) or butyrate in colon carcinogenesis have been implicated, the mechanisms of their action are not fully clear. Here, we investigated modulations of composition of individual phospholipid (PL) classes, with a particular emphasis on cardiolipins (CLs), in colon cells treated with DHA, sodium butyrate (NaBt), or their combination (DHA/NaBt), and we evaluated possible associations between lipid changes and cell fate after fatty acid treatment. In two distinct human colon cell models, foetal colon (FHC) and adenocarcinoma (HCT-116) cells, we compared patterns and composition of individual PL classes following the fatty acid treatment by HPLC-MS/MS. In parallel, we measured the parameters reflecting cell proliferation, differentiation and death. In FHC cells, NaBt induced primarily differentiation, while co-treatment with DHA shifted their response towards cell death. In contrast, NaBt induced apoptosis in HCT-116 cells, which was not further affected by DHA. DHA was incorporated in all main PL types, increasing their unsaturation, while NaBt did not additionally modulate these effects in either cell model. Nevertheless, we identified an unusually wide range of CL species to be highly increased by NaBt and particularly by DHA/NaBt, and these effects were more pronounced in HCT-116 cells. DHA and DHA/NaBt enhanced levels of high molecular weight and more unsaturated CL species, containing DHA, which was specific for either differentiation or apoptotic responses. We identified a wide range of CL species in the colon cells which composition was significantly modified after DHA and NaBt treatment. These specific CL modulations might contribute to distinct cellular differentiation or apoptotic responses.

The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples.