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.

• MeSH
• analýza hlavních komponent MeSH
• epitelové buňky metabolismus   patologie   MeSH
• fosfolipidy metabolismus   MeSH
• kolon patologie   MeSH
• lidé MeSH
• lipidomika * MeSH
• nádorové buněčné linie MeSH
• nádory tračníku metabolismus   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• antitumorózní látky farmakologie   MeSH
• apoptóza účinky léků   MeSH
• autofagie účinky léků   MeSH
• buněčná diferenciace účinky léků   MeSH
• buňky HT-29 MeSH
• butyráty farmakologie   MeSH
• HCT116 buňky MeSH
• kaspasa 3 genetika   metabolismus   MeSH
• kyselina máselná farmakologie   MeSH
• kyseliny dokosahexaenové farmakologie   MeSH
• lidé MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• nádory tračníku patologie   MeSH
• PPAR gama genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

• MeSH
• apoptóza účinky léků   MeSH
• buněčná diferenciace účinky léků   MeSH
• fosfolipidy chemie   MeSH
• HCT116 buňky MeSH
• kaspasa 3 genetika   metabolismus   MeSH
• kolon cytologie   účinky léků   MeSH
• kyselina máselná farmakologie   MeSH
• kyseliny dokosahexaenové farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• proliferace buněk účinky léků   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

To examine reciprocal or unilateral implications between two cell destruction processes, autophagy and apoptosis, in 5-Fluorouracil (5-FU)-treated tumor cells, a combination of chemical inhibitors, RNAi and genetic approaches were used. In contrast to cancer cells harboring obstructed apoptosis, either at the DISC or the mitochondrial level, p53-deficiency generated signs of autophagy deregulation upon chemotherapy. On the other, hand disruption of lysosomal function by chloroquine, caused a profound decrease in apoptotic markers appearing in response to 5-FU. DR5, which is essential for 5-FU-induced apoptosis, accumulated in lysosomes and autophagosomes upon chloroquine treatment. Since neither 3-MA, RNAi of critical autophagy regulators or inhibition of cathepsins reversed apoptosis in a similar manner, it is likely that not autophagy per se but rather correct receptor transport is an important factor for 5-FU cytotoxicity. We found that apoptosis generated by TRAIL, the cognate ligand for DR5, remained unchanged upon chloroquine lysosomal interference, indicating that 5-FU activates the receptor by a discrete mechanism. In support, depletion of membrane cholesterol or hampering cholesterol transport drastically reduced 5-FU cytotoxicity. We conclude that targeting of lysosomes by chloroquine deregulates DR5 trafficking and abrogates 5-FU- but not TRAIL-stimulated cell elimination, hence suggesting a novel mechanism for receptor activation.

• MeSH
• antimetabolity antitumorózní farmakologie   MeSH
• apoptóza MeSH
• autofagie * MeSH
• buněčná membrána metabolismus   MeSH
• chlorochin chemie   MeSH
• cholesterol chemie   MeSH
• fagozomy MeSH
• fluorouracil chemie   MeSH
• HCT116 buňky MeSH
• lidé MeSH
• ligandy MeSH
• lyzozomy metabolismus   MeSH
• makrolidy chemie   MeSH
• mitochondrie metabolismus   MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• protein TRAIL farmakologie   MeSH
• RNA interference MeSH
• signální transdukce účinky léků   MeSH
• TRAIL receptory metabolismus   MeSH
• transport proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Despite recent advances in targeted therapeutics, administration of 5-fluorouracil (5-FU) remains a common clinical strategy for post-surgical treatment of solid tumors. Although it has been proposed that RNA metabolism is disturbed by 5-FU treatment, the key cytotoxic response is believed to be enzymatic inhibition of thymidylate synthase resulting in nucleotide pool disproportions. An operating p53 tumor suppressor signaling network is in many cases essential for the efficiency of chemotherapy, and malfunctions within this system remain a clinical obstacle. Since the fate of chemotherapy-insensitive tumor cells is rarely described, we performed a comparative analysis of 5-FU toxicity in p53-deficient cells and conclude that p53 acts as a facilitator rather than a gatekeeper of cell death. Although p53 can act as a regulator of several cellular stress responses, no rerouting of cell death mode was observed in absence of the tumor suppressor. Thus, the final death outcome of 5-FU-treated p53-/- cells is demonstrated to be caspase-dependent, but due to a slow pace, accumulation of mitochondrial reactive oxygen species contributes to necrotic characteristics. The oligomerization status of the p53 target gene DR5 is determined as a significant limiting factor for the initiation of caspase activity in an intracellular TRAIL-dependent manner. Using several experimental approaches, we further conclude that RNA-rather than DNA-related stress follows by caspase activation irrespectively of p53 status. A distinct 5-FU-induced stress mechanism is thereby functionally connected to a successive and discrete cell death signaling pathway. Finally, we provide evidence that silencing of PARP-1 function may be an approach to specifically target p53-deficient cells in 5-FU combinatorial treatment strategies. Together, our results disclose details of impaired cell death signaling engaged as a consequence of 5-FU chemotherapy. Obtained data will contribute to the comprehension of factors restraining 5-FU efficiency, and by excluding DNA as the main stress target in some cell types they propose alternatives to currently used and suggested synergistic treatment regimens.

• MeSH
• antimetabolity antitumorózní farmakologie   MeSH
• apoptóza účinky léků   MeSH
• fluorescenční protilátková technika MeSH
• fluorouracil farmakologie   MeSH
• imunoblotting MeSH
• imunoprecipitace MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• polymerázová řetězová reakce MeSH
• protein TRAIL metabolismus   MeSH
• RNA účinky léků   MeSH
• signální transdukce účinky léků   fyziologie   MeSH
• transdukce genetická MeSH
• transmisní elektronová mikroskopie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Intestinal homeostasis is precisely regulated by a number of endogenous regulatory molecules but significantly influenced by dietary compounds. Malfunction of this system may result in chronic inflammation and cancer. Dietary essential n-3 polyunsaturated fatty acids (PUFAs) and short-chain fatty acid butyrate produced from fibre display anti-inflammatory and anticancer activities. Both compounds were shown to modulate the production and activities of TNF family cytokines. Cytokines from the TNF family (TNF- α, TRAIL, and FasL) have potent inflammatory activities and can also regulate apoptosis, which plays an important role in cancer development. The results of our own research showed enhancement of apoptosis in colon cancer cells by a combination of either docosahexaenoic acid (DHA) or butyrate with TNF family cytokines, especially by promotion of the mitochondrial apoptotic pathway and modulation of NF κ B activity. This review is focused mainly on the interaction of dietary PUFAs and butyrate with these cytokines during colon inflammation and cancer development. We summarised recent knowledge about the cellular and molecular mechanisms involved in such effects and outcomes for intestinal cell behaviour and pathologies. Finally, the possible application for the prevention and therapy of colon inflammation and cancer is also outlined.

• MeSH
• apoptóza MeSH
• butyráty metabolismus   MeSH
• cytokiny metabolismus   MeSH
• dieta MeSH
• kolon patologie   MeSH
• kyseliny dokosahexaenové metabolismus   MeSH
• lidé MeSH
• mitochondrie patologie   MeSH
• myši MeSH
• nádory metabolismus   MeSH
• nenasycené mastné kyseliny metabolismus   MeSH
• NF-kappa B metabolismus   MeSH
• střeva metabolismus   MeSH
• tumor nekrotizující faktory metabolismus   MeSH
• zánět metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

We show that the plant quaternary benzo[c]phenanthridine alkaloid sanguilutine (SL) is a strong inducer of caspase-independent non-apoptotic death in human melanoma cells. Necrostatin-1, a specific inhibitor of necroptosis, completely reversed the cytotoxic effect of SL, suggesting that necroptosis was a predominant type of cell death induced by SL in these cells. In addition, we showed that SL can trigger an autophagic response, as confirmed by GFP-LC3 puncta formation and LC3-II accumulation. Interestingly, we observed a significant decrease in the viability of melanoma cells treated with combination of autophagy inhibitors (3-methyladenine, bafilomycin-A1 and LY294002) and SL. Our results further indicated that autophagy may serve as a pro-survival mechanism, delaying the induction of necroptosis in melanoma cells. The ability of SL to induce caspase-independent non-apoptotic cell death (necroptosis) suggests its possible therapeutic potential in the treatment of apoptosis-resistant melanoma tumours. Furthermore, SL might serve as a useful tool for studying the mechanisms of necroptosis and autophagy induction and the interplay between these two processes.

• MeSH
• adenin analogy a deriváty   farmakologie   MeSH
• antitumorózní látky antagonisté a inhibitory   farmakologie   MeSH
• autofagie účinky léků   MeSH
• benzofenantridiny antagonisté a inhibitory   farmakologie   MeSH
• imidazoly farmakologie   MeSH
• indoly farmakologie   MeSH
• kaspasy metabolismus   MeSH
• lidé MeSH
• melanom patologie   MeSH
• nádorové buněčné linie MeSH
• nekróza chemicky indukované   MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• protoonkogenní proteiny c-bcl-2 metabolismus   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH