Isolation of sphingoid bases of sea cucumber cerebrosides and their cytotoxicity against human colon cancer cells

Sea cucumber is a health-beneficial food, and contains a variety of physiologically active substances including glycosphingolipids. We show here the sphingoid base composition of cerebrosides prepared from sea cucumber and the cytotoxicity against human colon cancer cell lines. The composition of sphingoid bases prepared from sea cucumber was different from that of mammals, and the major constituents estimated from mass spectra had a branched C17-19 alkyl chain with 1-3 double bonds. The viability of DLD-1, WiDr and Caco-2 cells treated with sea cucumber sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. The sphingoid bases induced such a morphological change as condensed chromatin fragments and increased the caspase-3 activity, indicating that the sphingoid bases reduced the cell viability by causing apoptosis in these cells. Sphingolipids of sea cucumber might therefore serve as bioactive dietary components to suppress colon cancer.     

Apoptosis Induction by Wheat-flour Sphingoid Bases in DLD-1 Human Colon Cancer Cells

The apoptotic effects of plant sphingoid bases prepared from wheat-flour cerebroside on human colorectal cancer DLD-1 cells were examined. The viability of DLD-1 cells treated with such plant sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. Morphological changes such as condensed chromatin fragments were found, so those sphingoid bases reduced cell viability through causing apoptosis in these cells.     

Apoptosis induction by wheat-flour sphingoid bases in DLD-1 human colon cancer cells

The apoptotic effects of plant sphingoid bases prepared from wheat-flour cerebroside on human colorectal cancer DLD-1 cells were examined. The viability of DLD-1 cells treated with such plant sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. Morphological changes such as condensed chromatin fragments were found, so those sphingoid bases reduced cell viability through causing apoptosis in these cells.     

Sphingoid bases and ceramide induce apoptosis in HT-29 and HCT-116 human colon cancer cells

Complex dietary sphingolipids such as sphingomyelin and glycosphingolipids have been reported to inhibit development of colon cancer. This protective role may be the result of turnover to bioactive metabolites including sphingoid bases (sphingosine and sphinganine) and ceramide, which inhibit proliferation and stimulate apoptosis. The purpose of the present study was to investigate the effects of sphingoid bases and ceramides on the growth, death, and cell cycle of HT-29 and HCT-116 human colon cancer cells. The importance of the 4,5-trans double bond present in both sphingosine and C(2)-ceramide (a short chain analog of ceramide) was evaluated by comparing the effects of these lipids with those of sphinganine and C(2)-dihydroceramide (a short chain analog of dihydroceramide), which lack this structural feature. Sphingosine, sphinganine, and C(2)-ceramide inhibited growth and caused death of colon cancer cells in time- and concentration-dependent manners, whereas C(2)-dihydroceramide had no effect. These findings suggest that the 4,5-trans double bond is necessary for the inhibitory effects of C(2)-ceramide, but not for sphingoid bases. Evaluation of cellular morphology via fluorescence microscopy and quantitation of fragmented low-molecular weight DNA using the diphenylamine assay demonstrated that sphingoid bases and C(2)-ceramide cause chromatin and nuclear condensation as well as fragmentation of DNA, suggesting these lipids kill colon cancer cells by inducing apoptosis. Flow cytometric analyses confirmed that sphingoid bases and C(2)-ceramide increased the number of cells in the A(0) peak indicative of apoptosis and demonstrated that sphingoid bases arrest the cell cycle at G(2)/M phase and cause accumulation in the S phase. These findings establish that sphingoid bases and ceramide induce apoptosis in colon cancer cells and implicate them as potential mediators of the protective role of more complex dietary sphingolipids in colon carcinogenesis.     

Evaluation of sphinganine and sphingosine as human breast cancer chemotherapeutic and chemopreventive agents

No comparative study of the effects of sphingolipid metabolites on proliferation and differentiation in normal human breast epithelial cells versus stem cells and tumorigenic cells has been reported. The purpose of this study was to evaluate the chemotherapeutic and chemopreventive potential of sphingoid bases (sphingosine and sphinganine) using a novel cell culture system of normal human breast epithelial cells (HBEC) developed from breast tissues of healthy women obtained during reduction mammoplasty (Type I HBEC with stem cell characteristics and Type II HBEC with basal epithelial cell phenotypes) and transformed tumorigenic Type I HBEC. The results show that sphinganine inhibited the growth and induced apoptosis of transformed tumorigenic Type I HBEC more potently than sphingosine (IC(50) for sphinganine 4 microM; sphingosine 6.4 microM). Both sphinganine and sphingosine at high concentrations (8-10 lM) arrested the cell cycle at G(2)/M. Sphinganine inhibited the growth and caused death of Type I HBEC more strongly than sphingosine. In comparison, Type II HBEC (normal differentiated cells) were less sensitive to the growth-inhibitory effects of sphingoid bases than Type I HBEC (stem cells) or transformed tumorigenic Type I HBEC, suggesting that sphingoid bases may serve as chemotherapeutic agents. At concentrations (0.05, 0.1, and 0.5 microM) that are below the growth-inhibitory range, sphingoid bases induced differentiation of Type I HBEC to Type II HBEC, as detected morphologically and via expression of a tumor suppressor protein, maspin, which is a marker of Type II HBEC. Thus, sphingoid bases may function as chemotherapeutic as well as chemopreventive agents by preferentially inhibiting cancer cells and eliminating stem cells from which most breast cancer cells arise.     

海参多糖抑制人肾癌细胞A498的生长转移作用机制

肾细胞癌(RCC)是最常见的恶性癌之一,癌症转移是目前导致肾癌患者死亡的主要原因之一。MMP-9被发现在许多具有侵袭性和转移能力的人类癌症中过表达,其表达和分泌受到NF-κB调控;VEGF在维持原发性癌和转移瘤生长所需的血管生成中发挥重要作用,其表达也受到活化的NF-κB调节。海参的多种活性物质在抗氧化、抗菌和抗癌方面都有出色的作用,而抗癌的主要机制则包括诱导癌细胞凋亡、抑制癌细胞生长、减少癌细胞转移等。本研究通过利用不同浓度的海参多糖处理人肾癌细胞A498,采用MTT细胞增殖实验、粘附实验、迁移实验和小室侵袭实验,研究了海参多糖对A498细胞的生长转移的影响;采用蛋白印记法检测了海参多糖对A498细胞内MMP-9、NF-κBp65和VEGF表达水平的影响。结果表明,海参多糖能够显著抑制A498细胞的增殖活力、粘附能力、迁移能力和侵袭能力,并且全都表现出明显的剂量依赖性;中浓度(100μg/mL)和高浓度(200μg/mL)的海参多糖能够显著下调A498细胞内MMP-9、NF-κBp65和VEGF的表达。这些结果说明海参多糖能有效抑制人肾癌细胞A498的生长、转移和侵袭,可能的机制是通过抑制NF-κB信号通路下调MMP-9和VEGF的表达,从而发挥抗癌细胞转移的作用。     

Sphingomyelin in the suppression of colon tumors: prevention versus intervention

Intestinal cells are regularly exposed to sphingolipid metabolites, i.e., ceramide and sphingoid bases, after hydrolysis of complex sphingolipids from the diet. These metabolites are known regulators of cell growth, differentiation, and death. Non-pharmacological amounts in the diet have been shown to inhibit early stages of chemically induced colon cancer in mice. To distinguish between chemopreventive and chemotherapeutic effects of sphingomyelin supplements, mice were fed sphingomyelin before and after tumor initiation. Both applications drastically reduced tumor formation, without a significant difference among the groups, indicating that sphingolipids are as effective in the chemoprevention of tumors as in early intervention. The normalization of cell proliferation and rate of apoptosis, but not the induction of differentiation, seem to be key players in the suppression of tumor formation by dietary sphingomyelin. This may have implications for the development of a cancer prevention or treatment strategy with sphingolipids as an alternative to conventional drugs.     

Influence of p53 expression on sensitivity of cancer cells to bleomycin

In this study, we determined whether p53 expression affected the sensitivity of non–small cell lung cancer (NSCLC) and colon cancer cells to bleomycin (BLM). Two human NSCLC cell lines (A549 expressing wild‐type p53 and p53‐null H1299) and two colon cancer cell lines (HCT116 p53+/+ and its p53 deficient subline HCT116 p53?/?) were subjected to treatment with BLM. Cells were treated with various concentrations of BLM, and cellular viability was assessed by formazan assay. To investigate the role of p53 in BLM sensitivity, we transduced cells with adenovirus with wild‐type p53, dominant‐negative p53, and GFP control, and analyzed the effect on cellular viability. Cells expressing wild‐type p53 were more sensitive to BLM than p53‐null cells in both NSCLC and colon cancer cells. Sensitivity to BLM of the cells with wild‐type p53 was reduced by overexpression of dominant‐negative p53, while BLM sensitivity of p53‐null cells was increased by wild‐type p53 in both NSCLC cells and colon cancer cells. In conclusion, our data show that p53 sensitizes all four cancer cells lines tested to BLM toxicity and overexpression of p53 confers sensitivity to the cytotoxic activity of the anticancer agent in p53‐null cells. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:260–269, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20334     

Transcriptional analysis of VEGF-D and TGFβ genes in MCF7 cells exposed to saponin isolated from Holothuria leucospilota (sea cucumber)

Background:

Marine natural products contain a wide range of bioactive compounds with therapeutic properties that have revealed crucial properties in the treatment of some diseases. Some of these compounds have recently received considerable attention for drug discovery. In this study we examined the anti-angiogenic effect of saponin isolated from Holothuria leucospilota (sea cucumber) through evaluation of vascular endothelial growth factor D (VEGF-D) and transforming growth factor-β (TGFβ) expression in a breast cancer cell line.

Methods:

To investigate the effect of SCS on VEGF-D and TGF-β expression in breast cancer cells, the cells were treated with various concentrations of sample. After 48 h the viability of the cells was evaluated by trypan blue staining, and VEGF-D and TGFβ mRNA expression was were evaluated by real time-PCR.

Results:

Our results revealed that SCS can suppress cell viability and VEGF-D and TGFβ mRNA expression in breast cancer cells. Sea cucumber saponin at a concentration of 12 μg/ml inhibited VEGF-D and TGFβ expression more than 90% compared with controls.

Conclusion:

Findings suggest that SCS could inhibit tumor growth via inhibition of angiogenesis.Key Words: Sea cucumber, Saponin, Angiogenesis, Anticancer     

Effects of silk sericin on the proliferation and apoptosis of colon cancer cells

Sericin is a silk protein woven from silkworm cocoons (Bombyx mori). In animal model, sericin has been reported to have anti-tumoral action against colon cancer. The mechanisms underlying the activity of sericin against cancer cells are not fully understood. The present study investigated the effects of sericin on human colorectal cancer SW480 cells compared to normal colonic mucosal FHC cells. Since the size of the sericin protein may be important for its activity, two ranges of molecular weight were tested. Sericin was found to decrease SW480 and FHC cell viability. The small sericin had higher anti-proliferative effects than that of the large sericin in both cell types. Increased apoptosis of SW480 cells is associated with increased caspase-3 activity and decreased Bcl-2 expression. The anti-proliferative effect of sericin was accompanied by cell cycle arrest at the S phase. Thus, sericin reduced SW480 cell viability by inducing cell apoptosis via caspase-3 activation and down-regulation of Bcl-2 expression. The present study provides scientific data that support the protective effect of silk sericin against cancer cells of the colon and suggests that this protein may have significant health benefits and could potentially be developed as a dietary supplement for colon cancer prevention.     

Biodiversity of sphingoid bases ("sphingosines") and related amino alcohols

"Sphingosin" was first described by J. L. W. Thudichum in 1884 and structurally characterized as 2S,3R,4E-2-aminooctadec-4-ene-1,3-diol in 1947 by Herb Carter, who also proposed the designation of "lipides derived from sphingosine as sphingolipides." This category of amino alcohols is now known to encompass hundreds of compounds that are referred to as sphingoid bases and sphingoid base-like compounds, which vary in chain length, number, position, and stereochemistry of double bonds, hydroxyl groups, and other functionalities. Some have especially intriguing features, such as the tail-to-tail combination of two sphingoid bases in the alpha,omega-sphingoids produced by sponges. Most of these compounds participate in cell structure and regulation, and some (such as the fumonisins) disrupt normal sphingolipid metabolism and cause plant and animal disease. Many of the naturally occurring and synthetic sphingoid bases are cytotoxic for cancer cells and pathogenic microorganisms or have other potentially useful bioactivities; hence, they offer promise as pharmaceutical leads. This thematic review gives an overview of the biodiversity of the backbones of sphingolipids and the broader field of naturally occurring and synthetic sphingoid base-like compounds.     

Disruption of the FATB gene in Arabidopsis demonstrates an essential role of saturated fatty acids in plant growth

Acyl-acyl carrier protein thioesterases determine the amount and type of fatty acids that are exported from the plastids. To better understand the role of the FATB class of acyl-acyl carrier protein thioesterases, we identified an Arabidopsis mutant with a T-DNA insertion in the FATB gene. Palmitate (16:0) content of glycerolipids of the mutant was reduced by 42% in leaves, by 56% in flowers, by 48% in roots, and by 56% in seeds. In addition, stearate (18:0) was reduced by 50% in leaves and by 30% in seeds. The growth rate was reduced in the mutant, resulting in 50% less fresh weight at 4 weeks compared with wild-type plants. Furthermore, mutant plants produced seeds with low viability and altered morphology. Analysis of individual glycerolipids revealed that the fatty acid composition of prokaryotic plastid lipids was largely unaltered, whereas the impact on eukaryotic lipids varied but was particularly severe for phosphatidylcholine, with a >4-fold reduction of 16:0 and a 10-fold reduction of 18:0 levels. The total wax load of fatb-ko plants was reduced by 20% in leaves and by 50% in stems, implicating FATB in the supply of saturated fatty acids for wax biosynthesis. Analysis of C(18) sphingoid bases derived from 16:0 indicated that, despite a 50% reduction in exported 16:0, the mutant cells maintained wild-type levels of sphingoid bases, presumably at the expense of other cell components. The growth retardation caused by the fatb mutation was enhanced in a fatb-ko act1 double mutant in which saturated fatty acid content was reduced further. Together, these results demonstrate the in vivo role of FATB as a major determinant of saturated fatty acid synthesis and the essential role of saturates for the biosynthesis and/or regulation of cellular components critical for plant growth and seed development.     

The novel EpCAM-targeting monoclonal antibody 3–17I linked to saporin is highly cytotoxic after photochemical internalization in breast,pancreas and colon cancer cell lines

The epithelial cell adhesion molecule (EpCAM) is expressed by a wide range of human carcinomas, making it an attractive diagnostic and therapeutic target in oncology. Its recent identification on cancer stem cells has raised further interest in its use for tumor targeting and therapy. Here, we present the characterization and therapeutic potential of 3–17I, a novel human EpCAM-targeting monoclonal antibody. Strong reaction of 3–17I was observed in all lung, colon, and breast human tumor biopsies evaluated. By flow cytometry and confocal fluorescence microscopy, we demonstrate that 3–17I specifically targets EpCAM-positive cell lines. We also show evidence for mAb-sequestration in endo-/lysosomes, suggesting internalization of 3–17I by receptor-mediated endocytosis. The ribosomal-inactivating toxin saporin was linked to 3–17I, creating the per se non-toxic immunotoxin 3–17I-saporin, a promising candidate for the drug delivery technology photochemical internalization (PCI). PCI is based on a light-controlled destruction of endolysosomal membranes and subsequent cytosolic release of the sequestered payload upon light exposure. EpCAM-positive human cancer cell lines MCF7 (breast), BxPC-3 (pancreas), WiDr (colon), and the EpCAM-negative COLO320DM (colon), were treated with 3–17I-saporin in combination with the clinically relevant photosensitizer TPCS2a (Amphinex), followed by exposure to light. No cytotoxicity was observed after treatment with 3–17I-saporin without light exposure. However, cell viability, proliferation and colony-forming capacity was strongly reduced in a light-dependent manner after PCI of 3–17I. Our results show that 3–17I is an excellent candidate for diagnosis of EpCAM-positive tumors and for development of clinically relevant antibody-drug conjugates, using PCI for the treatment of localized tumors.     

Intestinal absorption of dietary maize glucosylceramide in lymphatic duct cannulated rats

Sphingolipids are ubiquitous in all eukaryotic organisms. Various physiological functions of dietary sphingolipids, such as preventing colon cancer and improving the skin barrier function, have been recently reported. One of the common sphingolipids used as a foodstuff is glucosylceramide from plant sources, which is composed of sphingoid bases distinct from those of mammals. However, the fate of dietary sphingolipids derived from plants is still not understood. In this study, we investigated the absorption of maize glucosylceramide in the rat intestine using a lipid absorption assay of lymph from the thoracic duct. The free and complex forms of trans-4,cis-8-sphingadienine, the predominant sphingoid base of maize glucosylceramide, were found in the lymph after administration of maize glucosylceramide. This plant type of sphingoid base was detected in the ceramide fraction and N-palmitoyl-4,8-sphingadienine (C16:0-d18:2) and N-tricosanoyl-4,8-sphingadienine (C23:0-d18:2) were identified by LC-MS/MS. The cumulative recovery of 4t,8c-sphingadienine in the lymph was very low. These results indicate that dietary glucosylceramide originating from higher plants is slightly absorbed in the intestine and is incorporated into ceramide structures in the intestinal cells. However, it appears that the intact form of sphingoid bases is not reutilized well in the tissues.     

Regulation of transplasmalemma electron transport in oat mesophyll cells by sphingoid bases and blue light

Long-chain sphingoid bases inhibit transplasmalemma electron transport in certain animal cells in part by inhibiting protein phosphorylation. As a first step in determining whether similar regulatory processes exist for cell surface redox activity in plants, peeled leaf segments of Avena sativa L. cv Garry were exposed to sphingoid bases and other long chain lipids. Sphingoid bases which are the most active inhibitors of protein kinase C in animal cells inhibit transplasmalemma electron transport by mesophyll cells in the dark as measured by reduction of exogenous ferricyanide. In white light, however, the same compounds markedly stimulate redox activity. The stimulation by sphingoid bases in the light is not eliminated by the inhibitor of photosynthesis, 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU). Redox activity remaining in the presence of DCMU and sphingoid bases can be observed in blue but not red light. A tentative hypothesis considering the involvement of two separate redox systems is presented in an attempt of explain the disparate action of sphingoid bases on electron transport across the plasmalemma.     

Sphingomyelinase action inhibits phorbol ester-induced differentiation of human promyelocytic leukemic (HL-60) cells

Prior studies showed that sphingomyelinase action and the free sphingoid bases inhibited protein kinase C (Kolesnick, R. N., and Clegg, S. (1988) J. Biol. Chem. 263, 6534-6537). The present studies investigated whether sphingomyelinase action also inhibited a biologic process mediated via protein kinase C, phorbol ester-induced differentiation of HL-60 promyelocytic cells into macrophages. The potent phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated time- and concentration-dependent conversion of HL-60 cells into macrophages, ED50 congruent to 5 x 10(-10) M. Differentiation involved growth inhibition, adherence of the suspended cells to tissue culture plastic, morphologic changes, and development of specific enzymatic markers. Sphingomyelinase, which increased the level of sphingoid bases and inactivated protein kinase C, prevented this event. In control incubations, cell number increased 2.10-fold over 24 h, and 2 +/- 1% of the cells were adherent. In incubations with TPA (0.5 nM), cell number increased only 1.75-fold, and 30% were adherent. Sphingomyelinase (3.8 x 10(-5) unit/ml) restored growth to incubations containing TPA to 2.02-fold and reduced adherence to 15%. Sphingomyelinase (3.8 x 10(-2) unit/ml) also restored growth partially and reduced adherence to a maximal concentration of TPA (3 nM). Similar results were obtained with the sphingoid base sphingosine (3-4.5 microM). Sphingomyelinase antagonized the morphologic changes associated with conversion to the macrophage phenotype. Untreated HL-60 cells presented typical promyelocytic morphology with large nuclei, little cytoplasm, and uniformity of nuclear and cell shape. TPA induced a larger cell population with abundant cytoplasm and unusual shape. Sphingomyelinase prevented these changes. Sphingomyelinase blocked TPA-induced increases in the macrophage marker enzymes, acid phosphatase and alpha-naphthyl acetate esterase. These studies indicate that the action of a sphingomyelinase, like the sphingoid bases, blocks phorbol ester-induced differentiation of HL-60 cells into macrophages and provides further support for the concept that sphingomyelinase action may be sufficient to comprise a physiologically relevant inhibitory pathway for protein kinase C.     

TheDPL1Gene Is Involved in Mediating the Response to Nutrient Deprivation inSaccharomyces cerevisiae

Sphingosine-1-phosphate is a sphingolipid metabolite involved in the regulation of cell proliferation in mammalian cells. The major route of sphingosine-1-phosphate degradation is through cleavage at the C_2–3bond by sphingosine phosphate lyase. The recent identification of the first dihydrosphingosine/sphingosine phosphate lyase gene inSaccharomyces cerevisiaeestablishes that phosphorylated sphingoid base metabolism is conserved throughout evolution. Thedpl1Δ deletion mutant, which accumulates endogenous phosphorylated sphingoid bases, exhibits unregulated proliferation upon approach to stationary phase. The increased proliferation rate during respiratory growth was associated with failure to appropriately recruit cells into the G₁phase of the cell cycle. Several genes were found to be overexpressed or prematurely expressed during nutrient deprivation in thedpl1Δ strain, including glucose-repressible genes and G₁cyclins. These studies implicate a role forDPL1and phosphorylated sphingoid bases in the regulation of global responses to nutrient deprivation in yeast.