Project description:Patients with peritoneal metastasis (PM) of colorectal cancer (CRC) have poorer overall survival outcomes than those without PM. Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment and mediate CRC progression and PM. It is imperative to identify and develop novel therapeutic targets for PM-CRC driven by CAFs. Using lipidomics, we reveal that the abundance of phosphatidylcholine (PC) with unsaturated acyl chains was increased in clinical PM-CRC specimens. Additionally, we found that CAFs were present at a higher relative abundance in primary PM-CRC tumors and that membrane fluidity in CRC cells was increased after incubation with CAF-conditioned medium (CM) through three independent methods: lipidomics, fluorescence recovery after photobleaching (FRAP), and generalized polarization. Then, we found that increased membrane fluidity can enhance glucose uptake and metabolism, as supported by real-time bioenergetics analysis and U-13C glucose labeling. Interestingly, stearoyl-CoA desaturase 1 (SCD), the rate-limiting enzyme in the biosynthesis of unsaturated fatty acids (uS-FAs), was expressed at low levels in PM and associated with poor prognosis in CRC patients. Importantly, by untargeted metabolomics analysis and fatty acid ([U-13C]-stearic acid) tracing analyses, we found that CRC cells take up lipids and lipid-like metabolites secreted from CAFs, which may compensate for low SCD expression. Both in vitro and in vivo experiments demonstrated that sodium palmitate (C16:0) treatment could decrease the CAF-induced change in cell membrane fluidity, limit glucose metabolism, suppress cell invasiveness, and impair tumor growth and intraperitoneal dissemination. An increased C16:0 concentration was shown to induce apoptosis linked to lipotoxicity. Furthermore, C16:0 effectively enhanced the antitumor activity of 5-fluorouracil (5-FU) in vitro and was well tolerated in vivo. Taken together, these findings suggest that adding the saturated fatty acid (S-FA) C16:0 to neoadjuvant chemotherapy may open new opportunities for treating PM-CRC in the future.

Project description:The aim of this work is to investigate whether A. thaliana senses low temperature by perceiving changes in membrane fluidity. To this end, we have performed an experiment to test whether mutant or transgenic plants with altered membrane lipid composition, regulate their gene expression in the same manner as wild type plants in response to cold. Previous work has demonstrated that a change in the expression levels of a number of genes is important in acquiring tolerance to low temperatures. Chemicals which rigidify cell membranes in such a way as to mimic the effects of cold have been shown to be able to induce the expression of such genes. However, because of the non-specific nature of such chemical treatments, it has not been possible to demonstrate unequivocally that the changes in gene expression observed were the result of changes in membrane fluidity. All of the mutants used in our experiment, fab1, fad2-2 and the fad3/fad7/fad8 mutant, have increased lipid saturation levels compared to wild type plants and are thought to have reduced membrane fluidity. The fab1 mutant is also known to be sensitive to chilling. In the fab1 mutant the elongation of 16:0 fatty acids to 18:0 is reduced. The fad2-2 mutant has reduced 18:1 desaturase activity and hence reduced amounts of polyunsaturated phospholipids. The fad3/fad7/fad8 triple mutant is deficient in 18:2 desaturase activity and consequently unable to synthesise trienoic fatty acids. The transgenic line used contained a 35S::FAD3 transgene and in contrast to the mutants tested, should have increased lipid desaturation and increased membrane fluidity. A. thaliana ecotype Col-0 was used as the wild type control for the fab1 and fad2-2 mutants, in addition to the 35S::FAD3 line. The fad3/fad7/fad8 mutant had previously been transformed with the 35S::apoaequorin transgene and a Columbia line expressing apoaequorin under the control of the same promoter, was included to control for the presence of aequorin. Nine day old seedlings grown in petri-dishes on MS were transferred from their growth room (20 oC, 16 h photoperiod, 100 E m-2 s-1) to a growth cabinet (20 oC, 16 h photoperiod,160 m-2 s-1) 24 hours before the experiment began. The next day, one petri-dish of seedlings from each line of plants used was transferred to a cabinet running at 5 oC (16 h photoperiod,160 E m-2 s-1). Control plates remained at 20 oC. Seedlings were harvested after three hours and frozen in liquid nitrogen.

Project description:The aim of this work is to investigate whether A. thaliana senses low temperature by perceiving changes in membrane fluidity. To this end, we have performed an experiment to test whether mutant or transgenic plants with altered membrane lipid composition, regulate their gene expression in the same manner as wild type plants in response to cold. Previous work has demonstrated that a change in the expression levels of a number of genes is important in acquiring tolerance to low temperatures. Chemicals which rigidify cell membranes in such a way as to mimic the effects of cold have been shown to be able to induce the expression of such genes. However, because of the non-specific nature of such chemical treatments, it has not been possible to demonstrate unequivocally that the changes in gene expression observed were the result of changes in membrane fluidity. All of the mutants used in our experiment, fab1, fad2-2 and the fad3/fad7/fad8 mutant, have increased lipid saturation levels compared to wild type plants and are thought to have reduced membrane fluidity. The fab1 mutant is also known to be sensitive to chilling. In the fab1 mutant the elongation of 16:0 fatty acids to 18:0 is reduced. The fad2-2 mutant has reduced 18:1 desaturase activity and hence reduced amounts of polyunsaturated phospholipids. The fad3/fad7/fad8 triple mutant is deficient in 18:2 desaturase activity and consequently unable to synthesise trienoic fatty acids. The transgenic line used contained a 35S::FAD3 transgene and in contrast to the mutants tested, should have increased lipid desaturation and increased membrane fluidity. A. thaliana ecotype Col-0 was used as the wild type control for the fab1 and fad2-2 mutants, in addition to the 35S::FAD3 line. The fad3/fad7/fad8 mutant had previously been transformed with the 35S::apoaequorin transgene and a Columbia line expressing apoaequorin under the control of the same promoter, was included to control for the presence of aequorin. Nine day old seedlings grown in petri-dishes on MS were transferred from their growth room (20 oC, 16 h photoperiod, 100 E m-2 s-1) to a growth cabinet (20 oC, 16 h photoperiod,160 m-2 s-1) 24 hours before the experiment began. The next day, one petri-dish of seedlings from each line of plants used was transferred to a cabinet running at 5 oC (16 h photoperiod,160 E m-2 s-1). Control plates remained at 20 oC. Seedlings were harvested after three hours and frozen in liquid nitrogen. 12 samples were used in this experiment

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The significance of membrane fluidity of feeder cell-derived substrates for maintenance of iPS cell stemness.

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The significance of membrane fluidity of feeder cell-derived substrates for maintenance of iPS cell stemness.

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