Vitamin E metabolite 13'-carboxychromanols inhibit pro-inflammatory enzymes, induce apoptosis and autophagy in human cancer cells by modulating sphingolipids and suppress colon tumor development in mice.
ABSTRACT: Vitamin E forms are substantially metabolized to various carboxychromanols including 13'-carboxychromanols (13'-COOHs) that are found at high levels in feces. However, there is limited knowledge about functions of these metabolites. Here we studied ?T-13'-COOH and ?TE-13'-COOH, which are metabolites of ?-tocopherol and ?-tocotrienol, respectively. ?TE-13'-COOH is also a natural constituent of a traditional medicine Garcinia Kola. Both 13'-COOHs are much stronger than tocopherols in inhibition of pro-inflammatory and cancer promoting cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), and in induction of apoptosis and autophagy in colon cancer cells. The anticancer effects by 13'-COOHs appeared to be partially independent of inhibition of COX-2/5-LOX. Using liquid chromatography tandem mass spectrometry, we found that 13'-COOHs increased intracellular dihydrosphingosine and dihydroceramides after short-time incubation in HCT-116 cells, and enhanced ceramides while decreased sphingomyelins during prolonged treatment. Modulation of sphingolipids by 13'-COOHs was observed prior to or coinciding with biochemical manifestation of cell death. Pharmaceutically blocking the increase of these sphingolipids partially counteracted 13'-COOH-induced cell death. Further, 13'-COOH inhibited dihydroceramide desaturase without affecting the protein expression. In agreement with these mechanistic findings, ?TE-13'-COOH significantly suppressed the growth and multiplicity of colon tumor in mice. Our study demonstrates that 13'-COOHs have anti-inflammatory and anticancer activities, may contribute to in vivo anticancer effect of vitamin E forms and are promising novel cancer prevention agents.
Project description:Vitamin E gamma-tocotrienol (?TE) is known to have anticancer effects, but mechanisms underlying these actions are not clear. Here using liquid chromatography tandem mass spectrometry, we show that ?TE induced marked changes of sphingolipids including rapid elevation of dihydrosphingosine and dihydroceramides (dhCers) in various types of cancer cells. The elevation of dihydrosphingolipids coincided with increased cellular stress, as indicated by JNK phosphorylation, and was prior to any sign of induction of apoptosis. Chemically blocking de novo synthesis of sphingolipids partially counteracted ?TE-induced apoptosis and autophagy. Experiments using 13C3, 15N-labeled l-serine together with enzyme assays indicate that ?TE inhibited cellular dihydroceramide desaturase (DEGS) activity without affecting its protein expression or de novo synthesis of sphingolipids. Unlike the effect on dhCers, ?TE decreased ceramides (Cers) after 8-h treatment but increased C18:0-Cer and C16:0-Cer after 16 and 24 h, respectively. The increase of Cers coincides with ?TE-induced apoptosis and autophagy. Since ?TE inhibits DEGS and decreases de novo Cer synthesis, elevation of Cers during prolonged ?TE treatment is likely caused by sphingomeylinase-mediated hydrolysis of sphingomyelin. This idea is supported by the observation that an acid sphingomeylinase inhibitor partially reversed ?TE-induced cell death. Our study demonstrates that ?TE altered sphingolipid metabolism by inhibiting DEGS activity and possibly by activating SM hydrolysis during prolonged treatment in cancer cells.
Project description:NF-?B plays a central role in pathogenesis of inflammation and cancer. Many phytochemicals, including ?-tocotrienol (?TE), a natural form of vitamin E, have been shown to inhibit NF-?B activation, but the underlying mechanism has not been identified. In this study, we show that ?TE inhibited cytokine-triggered activation of NF-?B and its upstream regulator TGF-?-activated kinase-1 in murine RAW 264.7 macrophages and primary bone marrow-derived macrophages. In these cells, ?TE induced upregulation of A20, an inhibitor of NF-?B. Knockout of A20 partially diminished ?TE's anti-NF-?B effect, but ?TE increased another NF-?B inhibitor, Cezanne, in A20(-/-) cells. In search of the reason for A20 upregulation, we found that ?TE treatment increased phosphorylation of translation initiation factor 2, I?B?, and JNK, indicating induction of endoplasmic reticulum stress. Liquid chromatography-tandem mass spectrometry analyses revealed that ?TE modulated sphingolipids, including enhancement of intracellular dihydroceramides, sphingoid bases in de novo synthesis of the sphingolipid pathway. Chemical inhibition of de novo sphingolipid synthesis partially reversed ?TE's induction of A20 and the anti-NF-?B effect. The importance of dihydroceramide increase is further supported by the observation that C8-dihydroceramide mimicked ?TE in upregulating A20, enhancing endoplasmic reticulum stress, and attenuating TNF-triggered NF-?B activation. Our study identifies a novel anti-NF-?B mechanism where A20 is induced by stress-induced adaptive response as a result of modulation of sphingolipids, and it demonstrates an immunomodulatory role of dihydrocermides.
Project description:Initial research on vitamin E and cancer has focused on ?-tocopherol (?T), but recent clinical studies on cancer-preventive effects of ?T supplementation have shown disappointing results, which has led to doubts about the role of vitamin E, including different vitamin E forms, in cancer prevention. However, accumulating mechanistic and preclinical animal studies show that other forms of vitamin E, such as ?-tocopherol (?T), ?-tocopherol (?T), ?-tocotrienol (?TE), and ?-tocotrienol (?TE), have far superior cancer-preventive activities than does ?T. These vitamin E forms are much stronger than ?T in inhibiting multiple cancer-promoting pathways, including cyclo-oxygenase (COX)- and 5-lipoxygenase (5-LOX)-catalyzed eicosanoids, and transcription factors such as nuclear transcription factor ?B (NF-?B) and signal transducer and activator of transcription factor 3 (STAT3). These vitamin E forms, but not ?T, cause pro-death or antiproliferation effects in cancer cells via modulating various signaling pathways, including sphingolipid metabolism. Unlike ?T, these vitamin E forms are quickly metabolized to various carboxychromanols including 13'-carboxychromanols, which have even stronger anti-inflammatory and anticancer effects than some vitamin precursors. Consistent with mechanistic findings, ?T, ?T, ?TE, and ?TE, but not ?T, have been shown to be effective for preventing the progression of various types of cancer in preclinical animal models. This review focuses on cancer-preventive effects and mechanisms of ?T, ?T, ?TE, and ?TE in cells and preclinical models and discusses current progress in clinical trials. The existing evidence strongly indicates that these lesser-known vitamin E forms are effective agents for cancer prevention or as adjuvants for improving prevention, therapy, and control of cancer.
Project description:The factors that contribute to the development of ulcerative colitis (UC), are still not fully identified. Disruption of the colon barrier is one of the first events leading to invasion of bacteria and activation of the immune system. The colon barrier is strongly influenced by sphingolipids. Sphingolipids impact cell-cell contacts and function as second messengers. We collected blood and colon tissue samples from UC patients and healthy controls and investigated the sphingolipids and other lipids by LC-MS/MS or LC-QTOFMS. The expression of enzymes of the sphingolipid pathway were determined by RT-PCR and immunohistochemistry. In inflamed colon tissue, the de novo-synthesis of sphingolipids is reduced, whereas lactosylceramides are increased. Reduction of dihydroceramides was due to posttranslational inhibition rather than altered serine palmitoyl transferase or ceramide synthase expression in inflamed colon tissue. Furthermore, in human plasma from UC-patients, several sphinglipids change significantly in comparison to healthy controls. Beside sphingolipids free fatty acids, lysophosphatidylcholines and triglycerides changed significantly in the blood of colitis patients dependent on the disease severity. Our data indicate that detraction of the sphingolipid de novo synthesis in colon tissue might be an important trigger for UC. Several lipids changed significantly in the blood, which might be used as biomarkers for disease control; however, diet-related variabilities need to be considered.
Project description:Leukodystrophies due to abnormal production of myelin cause extensive morbidity in early life; their genetic background is still largely unknown. We aimed at reaching a molecular diagnosis in Ashkenazi-Jewish patients who suffered from developmental regression at 6-13?months, leukodystrophy and peripheral neuropathy.Exome analysis, determination of alkaline ceramidase activity catalysing the conversion of C18:1-ceramide to sphingosine and D-ribo-C12-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-phytoceramide to NBD-C12-fatty acid using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and thin layer chromatography, respectively, and sphingolipid analysis in patients' blood by LC-MS/MS.The patients were homozygous for p.E33G in the ACER3, which encodes a C18:1-alkaline ceramidase and C20:1-alkaline ceramidase. The mutation abolished ACER3 catalytic activity in the patients' cells and failed to restore alkaline ceramidase activity in yeast mutant strain. The levels of ACER3 substrates, C18:1-ceramides and dihydroceramides and C20:1-ceramides and dihydroceramides and other long-chain ceramides and dihydroceramides were markedly increased in the patients' plasma, along with that of complex sphingolipids, including monohexosylceramides and lactosylceramides.Homozygosity for the p.E33G mutation in the ACER3 gene results in inactivation of ACER3, leading to the accumulation of various sphingolipids in blood and probably in brain, likely accounting for this new form of childhood leukodystrophy.
Project description:Bacteria alter the biophysical properties of their membrane lipids in response to environmental cues, such as shifts in pH or temperature. In essence, lipid composition determines membrane structure, which in turn influences many basic functions, such as transport, secretion, and signaling. Like other members of the phylum Bacteroidetes, the oral anaerobe Porphyromonas gingivalis possesses the ability to synthesize a variety of novel membrane lipids, including species of dihydroceramides that are distinct, yet similar in structure to sphingolipids produced by the human host. The role of dihydroceramides in the physiology and pathogenic potential of the human microbiota is only beginning to be explored; yet there is increasing data indicating that these lipids play a role in human diseases, such as periodontitis and multiple sclerosis. Here, we report on the identification of a gene (PG1780) in the chromosome of P. gingivalis strain W83 encoding a putative serine palmitoyltransferase, the enzyme that catalyzes the first step in sphingolipid biosynthesis. While we were able to detect dihydroceramides in whole lipid extracts of P. gingivalis cells as well as crude preparations of outer membrane vesicles, sphingolipids were absent in the PG1780 mutant strain. Moreover, we show that the synthesis of sphingolipids plays an essential role in the long-term survival of the organism as well as its resistance to oxidative stress. Further, a PG1780 mutant displayed much lower activity of cell-associated arginine and lysine gingipains, yet slightly higher activity in the corresponding culture supernates, which we hypothesize is due to altered membrane properties and anchoring of these proteases to the cell surface. In addition, we determined that sphingolipid production is critical to the presentation of surface polysaccharides, with the mutant strain displaying less K-antigen capsule and more anionic polysaccharide (APS). Overall, we have discovered that, in addition to their role in pathogenicity, the synthesis of sphingolipids is critical to the cellular homeostasis and persistence of this important dental pathogen.
Project description:Lipoxygenases (LOX) are key enzymes for the oxidative metabolism of polyunsaturated fatty acids into biologically active products. Clinical data on comparative levels of various LOX products in tumorigenesis are lacking. Therefore, we examined the profiles of several LOX products (5-LOX, 12-LOX, 15-LOX-1, and 15-LOX-2) by liquid chromatography/tandem mass spectrometry in the major steps of colorectal tumorigenesis (normal, polyp, and cancer) in a clinical study of 125 subjects (49 with normal colon, 36 with colorectal polyps, and 40 with colorectal cancer) who underwent prospective colorectal biopsies to control for various potential confounding factors (e.g., diet, medications). Mean 13-hydroxyoctadecadienoic acid (13-HODE) levels were significantly higher in normal colon [mean, 36.11 ng/mg protein; 95% confidence interval (95% CI), 31.56-40.67] than in paired colorectal cancer mucosa (mean, 27.01 ng/mg protein; 95% CI, 22.00-32.02; P = 0.0002), and in normal colon (mean, 37.15 ng/mg protein; 95% CI, 31.95-42.34) than in paired colorectal polyp mucosa (mean, 28.07 ng/mg protein; 95% CI, 23.66-32.48; P < 0.001). Mean 13-HODE levels, however, were similar between the left (mean, 37.15 ng/mg protein; 95% CI, 31.95-42.35) and the right normal colon (mean, 32.46 ng/mg protein; 95% CI, 27.95-36.98; P = 0.09). No significant differences with regard to 12- or 15-hydroxyeicosatetraenoic acid or leukotriene B(4) levels were detected between normal, polyp, and cancer mucosae. 15-LOX-1 inhibited interleukin-1beta expression. This study establishes that reduced 13-HODE levels are a specific alteration in the LOX product profile associated with human colorectal tumorigenesis.
Project description:Colorectal cancer (CRC) continues to be a major cause of morbidity and mortality. The arachidonic acid (AA) pathway and linoleic acid (LA) pathway have been implicated as important contributors to CRC development and growth. Human 15-lipoxygenase 1 (15-LOX-1) converts LA to anti-tumor 13-S-hydroxyoctadecadienoic acid (13-HODE)and 15-LOX-2 converts AA to 15-hydroxyeicosatetraenoic acid (15-HETE). In addition, human 12-LOX metabolizes AA to pro-tumor 12-HETE. In rodents, the function of 12-LOX and 15-LOX-1 and 15-LOX-2 is carried out by a single enzyme, 12/15-LOX. As a result, conflicting conclusions concerning the role of 12-LOX and 15-LOX have been obtained in animal studies. In the present studies, we determined that PD146176, a selective 15-LOX-1 inhibitor, markedly suppressed 13-HODE generation in human colon cancer HCA-7 cells and HCA-7 tumors, in association with increased tumor growth. In contrast, PD146176 treatment led to decreases in 12-HETE generation in mouse colon cancer MC38 cells and MC38 tumors, in association with tumor inhibition. Surprisingly, deletion of host 12/15-LOX alone led to increased MC38 tumor growth, in association with decreased tumor 13-HODE levels, possibly due to inhibition of 12/15-LOX activity in stroma. Therefore, the effect of 12/15-LOX on colorectal tumorigenesis in mouse models could be affected by tumor cell type (human or mouse), relative 12/15 LOX activity in tumor cells and stroma as well as the relative tumor 13-HODE and 12-HETE levels.
Project description:Hepatocellular carcinoma (HCC) shows a remarkable heterogeneity and is recognized as a chemoresistant tumor with dismal prognosis. In previous studies, we observed significant alterations in the serum sphingolipids of patients with HCC. This study aimed to investigate the in vitro effects of sorafenib, which is the most widely used systemic HCC medication, on the sphingolipid pathway as well as the effects of inhibiting the sphingolipid pathway in HCC. Huh7.5 and HepG2 cells were stimulated with sorafenib, and inhibitors of the sphingolipid pathway and cell proliferation, viability, and concentrations of bioactive metabolites were assessed. We observed a significant downregulation of cell proliferation and viability and a simultaneous upregulation of dihydroceramides upon sorafenib stimulation. Interestingly, fumonisin B1 (FB1) and the general sphingosine kinase inhibitor SKI II were able to inhibit cell proliferation more prominently in HepG2 and Huh7.5 cells, whereas there were no consistent effects on the formation of dihydroceramides, thus implying an involvement of distinct metabolic pathways. In conclusion, our study demonstrates a significant downregulation of HCC proliferation upon sorafenib, FB1, and SKI II treatment, whereas it seems they exert antiproliferative effects independently from sphingolipids. Certainly, further data would be required to elucidate the potential of FB1 and SKI II as putative novel therapeutic targets in HCC.
Project description:Systemic vitamin E metabolites have been proposed as signaling molecules, but their physiological role is unknown. Here we show, by library screening of potential human vitamin E metabolites, that long-chain ω-carboxylates are potent allosteric inhibitors of 5-lipoxygenase, a key enzyme in the biosynthesis of chemoattractant and vasoactive leukotrienes. 13-((2R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-2,6,10-trimethyltridecanoic acid (α-T-13'-COOH) can be synthesized from α-tocopherol in a human liver-on-chip, and is detected in human and mouse plasma at concentrations (8-49 nM) that inhibit 5-lipoxygenase in human leukocytes. α-T-13'-COOH accumulates in immune cells and inflamed murine exudates, selectively inhibits the biosynthesis of 5-lipoxygenase-derived lipid mediators in vitro and in vivo, and efficiently suppresses inflammation and bronchial hyper-reactivity in mouse models of peritonitis and asthma. Together, our data suggest that the immune regulatory and anti-inflammatory functions of α-tocopherol depend on its endogenous metabolite α-T-13'-COOH, potentially through inhibiting 5-lipoxygenase in immune cells.