TGF-β protects osteosarcoma cells from chemotherapeutic cytotoxicity in a SDH/HIF1α dependent manner

Background In the widespread adoption of chemotherapy, drug resistance has been the major obstacle to tumor elimination in cancer patients. Our aim was to explore the role of TGF-β in osteosarcoma-associated chemoresistance. Methods We performed a cytotoxicity analysis of methotrexate (MTX) and cisplatin (CIS) in TGF-β-treated osteosarcoma cells. Then, the metabolite profile of the core metabolic energy pathways in Saos-2 and MG-63 cell extracts was analyzed by 1H-NMR. We detected the expression of succinate dehydrogenase (SDH), STAT1, and hypoxia-inducible factor 1α (HIF1α) in TGF-β-treated osteosarcoma cells and further tested the effects of these molecules on the cytotoxicity induced by chemotherapeutic agents. Using in vivo experiments, we examined the tumor growth and survival time of Saos-2-bearing mice treated with a combination of chemotherapeutic agents and a HIF1α inhibitor. Results The metabolic analysis revealed enhanced succinate production in osteosarcoma cells after TGF-β treatment. We further found a decrease in SDH expression and an increase in HIF1α expression in TGF-β-treated osteosarcoma cells. Consistently, blockade of SDH efficiently enhanced the resistance of Saos-2 and MG-63 cells to MTX and CIS. Additionally, a HIF1α inhibitor significantly strengthened the anticancer efficacy of the chemotherapeutic drugs in mice with osteosarcoma cancer. Conclusion Our study demonstrated that TGF-β attenuated the expression of SDH by reducing the transcription factor STAT1. The reduction in SDH then caused the upregulation of HIF1α, thereby rerouting glucose metabolism and aggravating chemoresistance in osteosarcoma cells. Linking tumor cell metabolism to the formation of chemotherapy resistance, our study may guide the development of additional treatments for osteosarcoma.

Comparative analysis of L-fucose utilization and its impact on growth and survival of Campylobacter isolates

Campylobacter jejuni and Campylobacter coli were previously considered asaccharolytic, but are now known to possess specific saccharides metabolization pathways, including L-fucose. To investigate the influence of the L-fucose utilization cluster on Campylobacter growth, survival and metabolism, we performed comparative genotyping and phenotyping of the C. jejuni reference isolate NCTC11168 (human isolate), C. jejuni Ca1352 (chicken meat isolate), C. jejuni Ca2426 (sheep isolate), and C. coli Ca0121 (pig manure isolate), that all possess the L-fucose utilization cluster.All isolates showed enhanced survival and prolonged spiral cell morphology in aging cultures up to day seven in L-fucose-enriched MEMα medium (MEMαF) compared to MEMα. HPLC analysis indicated L-fucose utilization linked to acetate, lactate, pyruvate and succinate production, confirming the activation of the L-fucose pathway in these isolates. Highest consumption of L-fucose by C. coli Ca0121, is conceivably linked to its enhanced growth performance up to day 7, reaching 9.3 log CFU/ml compared to approximately 8.3 log CFU/ml for the C. jejuni isolates. Genetic analysis of their respective L-fucose clusters revealed several differences, including a 1 bp deletion in the Cj0489 gene of C. jejuni NCTC11168, causing a frameshift in this isolate resulting in two separate genes, Cj0489 and Cj0490, while no apparent phenotype could be linked to the presumed frameshift in the NCTC11168 isolate. Additionally, we found that the L-fucose cluster of C. coli Ca0121 was most distant from C. jejuni NCTC11168, but confirmation of links to L-fucose metabolism associated phenotypic traits in C. coli versus C. jejuni isolates requires further studies.ImportanceCampylobacter is the leading cause of gastroenteritis in humans worldwide, with increasing incidence and prevalence in recent years. The most prevalent species are Campylobacter jejuni and C. coli with 83% and 10% of all Campylobacter cases, respectively. Previously it was found that the majority of Campylobacter isolates are able to metabolize L-fucose (fuc+ isolates), a sugar that is widely present in the human gut. Putative roles for L-fucose in fuc+ C. jejuni isolates were found in growth, biofilm formation and virulence. Despite this, relatively little is known about L-fucose metabolism and the impact on growth and survival in fuc+ Campylobacter isolates. The results from our comparative genotyping and phenotyping study demonstrate that L-fucose, in both C. jejuni and C. coli fuc+ isolates, is involved in enhanced survival, prolonged spiral cell morphology and changes in the general metabolism. Possible links between phenotypes and differences in respective L-fucose gene clusters are discussed.

An Escherichia coli FdrA Variant Derived from Syntrophic Coculture with a Methanogen Increases Succinate Production Due to Changes in Allantoin Degradation

Here, we demonstrate the ability of E. coli to perform glycerol fermentation in coculture with the methanogen M. formicicum to produce succinate. We found that the production of succinate by E. coli significantly increased during successive cocultivation.

A sodium-translocating module linking succinate production to formation of a membrane potential in Prevotella bryantii

Ruminants such as cattle and sheep depend on the breakdown of carbohydrates from plant-based feedstuff which is accomplished by the microbial community in the rumen. Roughly 40% of the rumen microbiota belong to the family of Prevotellaceae which ferment sugars to organic acids such as acetate, propionate as well as succinate. These substrates are important nutrients for the ruminant. In a metaproteome analysis of the rumen of cattle, proteins that are homologous to the Na + -translocating NADH:quinone oxidoreductase (NQR) and the quinone:fumarate reductase (QFR) were identified in different Prevotella species. Here we show that fumarate reduction to succinate in anaerobically growing Prevotella bryantii is coupled to chemiosmotic energy conservation by a supercomplex composed of NQR and QFR. This S odium-translocating N ADH: F umarate oxido R eductase (SNFR) supercomplex was enriched by BN-PAGE and characterized by in-gel enzyme activity staining and mass spectrometry. High NADH oxidation (850 nmol min -1 mg -1 ), quinone reduction (490 nmol min -1 mg -1 ) and fumarate reduction (1200 nmol min -1 mg -1 ) activities, together with high expression levels, demonstrate that SNFR represents a charge-separating unit in P. bryantii . Absorption spectroscopy of SNFR exposed to different substrates revealed intramolecular electron transfer from the FAD cofactor in NQR to heme b cofactors in QFR. SNFR catalyzed the stoichiometric conversion of NADH and fumarate to NAD + and succinate. We propose that the regeneration of NAD + in P. bryantii is intimately linked to the build-up of an electrochemical gradient which powers ATP synthesis by electron transport phosphorylation. Importance Feeding strategies for ruminants are designed to optimize nutrient efficiency for animals and to prevent energy losses like enhanced methane production. Key to this are the fermentative reactions of the rumen microbiota, dominated by Prevotella sp. We show that succinate formation by P. bryantii is coupled to NADH oxidation and sodium-gradient formation by a newly described supercomplex consisting of Na + -translocating NADH:quinone oxidoreductase (NQR) and fumarate reductase (QFR), representing the S odium-translocating N ADH: F umarate oxido R eductase (SNFR) supercomplex. SNFR is the major charge-separating module, generating an electrochemical sodium gradient in P. bryantii . Our findings offer clues to the observation that use of fumarate as feed additive does not significantly increase succinate production, or decrease methanogenesis, by the microbial community in the rumen.

Isolation and Biochemical Characterization of Six Anaerobic Fungal Strains from Zoo Animal Feces

Anaerobic fungi are prime candidates for the conversion of agricultural waste products to biofuels. Despite the increasing interest in these organisms, their growth requirements and metabolism remain largely unknown. The isolation of five strains of anaerobic fungi and their identification as Neocallimastix cameroonii, Caecomyces spec., Orpinomyces joyonii, Pecoramyces ruminantium, and Khoyollomyces ramosus, is described. The phylogeny supports the reassignment of Neocallimastix californiae and Neocallimastix lanati to Neocallimastix cameroonii and points towards the redesignation of Cyllamyces as a species of Caecomyces. All isolated strains including strain A252, which was described previously as Aestipascuomyces dubliciliberans, were further grown on different carbon sources and the produced metabolites were analyzed; hydrogen, acetate, formate, lactate, and succinate were the main products. Orpinomyces joyonii was lacking succinate production and Khoyollomyces ramosus was not able to produce lactate under the studied conditions. The results further suggested a sequential production of metabolites with a preference for hydrogen, acetate, and formate. By comparing fungal growth on monosaccharides or on the straw, a higher hydrogen production was noticed on the latter. Possible reactions to elevated sugar concentrations by anaerobic fungi are discussed.