OBJECTIVE: One of the models we study is the ovarian granulosa cell (GC), a somatic cell lineage that is critically important for maintenance of the female germ line and many endocrine functions of the ovaries. The objective of this study is to clarify the significance of ceramide and the role of ceramide metabolism in dictating the fate of cells exposed to stress. METHODS: We first treated GC with a C8-ceramide analog or an amine derivative of ceramide that cannot be metabolized by ceramidase (C8-ceramine). Northern blot analysis was performed to evaluate mRNA of acid ceramidease expression regualted by gonadotropin and in situ hybridization was done to identify the mRNA expression of acid ceramidase in ovaries. RESULTS: After 6 hours, C8-ceramide (50 micro M) triggered apoptosis in only 28 +/- 6% of the cells, whereas C8-ceramine (50 micro M) induced apoptosis in all cells (LD50=1 micro M). These data suggested that ceramidase activity is a critical determinant of GC survival. In situ hybridization showed that mRNA of acid ceramidase was highly expressed in GC in growing follicle. mRNA of acid ceramidase was expressed abundantly in granulosa cells and ovaries and its expression was significantly increased by gonadotropin in granulosa cells in in situ hybridization. Forty two hour after gonadotropin treatment, mRNA expression of acid ceramidase in granulosa cells was two fold increased cells comparing with no treatment control in northern blot analysis (P<0.05). In copora lutea, elevated mRNA expression of acid ceramidase was decreased. CONCLUSION: We concluded that GC possess inherently high levels of ceramidase activity, and that ceramidase has important for metabolizing ceramind to maintain GC survival in the ovary.
Acid Ceramidase ; Apoptosis ; Blotting, Northern ; Cell Lineage ; Ceramidases* ; Female ; Germ Cells ; Gonadotropins ; Granulosa Cells* ; Humans ; In Situ Hybridization ; Metabolism ; Ovary ; RNA, Messenger

BACKGROUND: Ceramides are the main lipid component of the stratum corneum and are a structurally heterogeneous and complex group of sphingolipids of which sphingoid bases are the basic structural constituents. Altered levels of sphingoid bases have been reported in skin conditions that involve dryness and barrier disruption, including atopic dermatitis. OBJECTIVE: The purpose of this study was to investigate the altered levels of sphingoid bases in psoriatic epidermis and their relationship with the clinical severity of the psoriasis. METHODS: Samples from the lesional and non-lesional epidermis were obtained from eight psoriasis patients. Levels of sphingosine and sphinganine were analyzed by high-performance liquid chromatography. The expression of ceramide synthase and ceramidase proteins, which are related to sphingosine and sphinganine metabolism, were measured using Western blot analysis. RESULTS: Levels of sphingosine and sphinganine in the lesional epidermis were significantly higher than those in the non-lesional epidermis. Although there was no altered ceramide synthase and ceramidase, there was a highly significant positive correlation between the % change of ceramidase, the degradative enzyme of ceramide into sphingosine, and the Psoriasis Area Severity Index (PASI) score. CONCLUSION: The levels of sphingosine and sphinganine were significantly increased in psoriatic epidermis and the % change of ceramidase was positively correlated with the clinical severity of psoriasis.
Blotting, Western ; Ceramidases ; Ceramides ; Chromatography, Liquid ; Epidermis ; Humans ; Oxidoreductases ; Proteins ; Psoriasis ; Skin ; Sphingolipids ; Sphingosine

BACKGROUND: Ceramides are the main lipid component of the stratum corneum and are a structurally heterogeneous and complex group of sphingolipids of which sphingoid bases are the basic structural constituents. Altered levels of sphingoid bases have been reported in skin conditions that involve dryness and barrier disruption, including atopic dermatitis. OBJECTIVE: The purpose of this study was to investigate the altered levels of sphingoid bases in psoriatic epidermis and their relationship with the clinical severity of the psoriasis. METHODS: Samples from the lesional and non-lesional epidermis were obtained from eight psoriasis patients. Levels of sphingosine and sphinganine were analyzed by high-performance liquid chromatography. The expression of ceramide synthase and ceramidase proteins, which are related to sphingosine and sphinganine metabolism, were measured using Western blot analysis. RESULTS: Levels of sphingosine and sphinganine in the lesional epidermis were significantly higher than those in the non-lesional epidermis. Although there was no altered ceramide synthase and ceramidase, there was a highly significant positive correlation between the % change of ceramidase, the degradative enzyme of ceramide into sphingosine, and the Psoriasis Area Severity Index (PASI) score. CONCLUSION: The levels of sphingosine and sphinganine were significantly increased in psoriatic epidermis and the % change of ceramidase was positively correlated with the clinical severity of psoriasis.
Blotting, Western ; Ceramidases ; Ceramides ; Chromatography, Liquid ; Epidermis ; Humans ; Oxidoreductases ; Proteins ; Psoriasis ; Skin ; Sphingolipids ; Sphingosine

UV-irradiation is a major factor of photo-aged skin, by which pigmentation, wrinkles and laxity are increased. In addition, the epidermal barrier is disrupted, ultimately causing dryness in photo-aged skin. As an effort to search dietary sources for improving the dryness of UV irradiated skin, the dietary effect of red ginseng based functional foods on the epidermal level of ceramides, a major lipid maintaining epidermal barrier, was determined in this study. Albino hairless mice were fed either a control diet [group UV (UV-irradiated control)] or diets with 0.5% (group M0.5) or 1% (group M1.0) of red ginseng extracts mixed with Torilis fructus and Corni fructus (66.7% red ginseng) in parallel with UV irradiation for 5 wks. A normal control group (group C) was fed a control diet without UV irradiation for 5 wks. The epidermal level of ceramides in group UV was significantly lower than that in group C, in which ceramidase, an enzyme involved in ceramide degradation, was highly expressed. In group M0.5, the epidermal level of ceramide was significantly increased to the level even higher than in group C. In addition, protein expression of serine palmitoyl transferase (SPT), a key enzyme involved in de novo ceramide synthesis, was increased in group M0.5. However the epidermal levels of ceramides as well as of ceramidase protein expression in group M1.0 did not differ from those in group UV. In conclusion, we demonstrate that dietary supplementation of red-ginseng extracts mixed with Torilis fructus and Corni fructus at a level of 0.5% level in diet increased the epidermal level of ceramides coupled with the elevated expression of SPT protein.
Animals ; Ceramidases ; Ceramides ; Cornus ; Diet ; Dietary Supplements ; Functional Food ; Mice ; Mice, Hairless ; Panax ; Pigmentation ; Proteins ; Serine ; Skin ; Transferases

B13 analogues are being considered as therapeutic agents for cancer cells, since B13 is a ceramide analogue and inhibits ceramidase to promote apoptosis in cancer cells. B13 sulfonamides are assumed to have biological activity similar to B13, since they are made by bioisosterically substituting the carboxyl moiety of B13 with sulfone group. Twenty B13 sulfonamides were evaluated for their in vitro cytotoxicities against human colon cancer HT-29 and lung cancer A549 cell lines using MTT assays. Replacement of the amide group with a sulfonamide group increased cytotoxicity in both cancer cell lines. The sulfonamides with long alkyl chains exhibited activities two to three times more potent than that of B13 and compound (15) had the most potent activity with IC50 values of 27 and 28.7microM for HT-29 and A549, respectively. The comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were used to carry out QSAR molecular modeling of these compounds. The predictive CoMSIA models for HT-29 and A549 gave cross-validated q2 values of 0.703 and 0.830, respectively. From graphical analysis of these models, we suppose that the stereochemistry of 1,3-propandiol is not important for activity and that introduction of a sulfonamide group and long alkyl chains into B13 can increase cytotoxicity.
Apoptosis ; Cell Line ; Ceramidases ; Colonic Neoplasms ; Humans ; Inhibitory Concentration 50 ; Lung Neoplasms ; Models, Molecular ; Quantitative Structure-Activity Relationship ; Sulfonamides

PURPOSE: Borage oil (BO) and safflower oil (SO) are efficacious in reversing epidermal hyperproliferation, which is caused by the disruption of epidermal barrier. In this study, we compared the antiproliferative effect of dietary BO and SO. Altered metabolism of ceramide (Cer), the major lipid of epidermal barrier, was further determined by measurement of epidermal levels of individual Cer, glucosylceramide (GlcCer), and sphingomyelin (SM) species, and protein expression of Cer metabolizing enzymes. METHODS: Epidermal hyperproliferation was induced in guinea pigs by a hydrogenated coconut diet (HCO) for 8 weeks. Subsequently, animals were fed diets of either BO (group HCO + BO) or SO (group HCO + SO) for 2 weeks. As controls, animals were fed BO (group BO) or HCO (group HCO) diets for 10 weeks. RESULTS: Epidermal hyperproliferation was reversed in groups HCO + BO (67.6% of group HCO) and HCO + SO (84.5% of group HCO). Epidermal levels of Cer1/2, GlcCer-A/B, and beta-glucocerebrosidase (GCase), an enzyme of GlcCer hydrolysis for Cer generation, were higher in group HCO + BO than in group HCO, and increased to levels similar to those of group BO. In addition, epidermal levels of SM1, serine palmitoyltransferase (SPT), and acidic sphingomyelinase (aSMase), enzymes of de novo Cer synthesis and SM hydrolysis for Cer generation, but not of Cer3-7, were higher in group HCO + BO than in group HCO. Despite an increase of SPT and aSMase in group HCO + SO to levels higher than in group HCO, epidermal levels of Cer1-7, GlcCer-A/B, and GCase were similar in these two groups. Notably, acidic ceramidase, an enzyme of Cer degradation, was highly expressed in group HCO + SO. Epidermal levels of GlcCer-C/D and SM-2/3 did not differ among groups. CONCLUSION: Dietary BO was more prominent for reversing epidermal hyperproliferation by enhancing Cer metabolism with increased levels of Cer1/2, GlcCer-A/B, and SM1 species, and of GCase proteins.
Animals ; Borago* ; Carthamus tinctorius* ; Ceramidases ; Cocos ; Diet ; Epidermis* ; Glucosylceramidase ; Guinea Pigs* ; Guinea* ; Hydrogen ; Hydrolysis ; Metabolism* ; Safflower Oil* ; Serine C-Palmitoyltransferase ; Sphingomyelin Phosphodiesterase

B13, a ceramide analogue, is a ceramidase inhibitor and induces apoptosis to give potent anticancer activity. A series of thiourea B13 analogues was evaluated for their in vitro cytotoxic activities against human renal cancer Caki-2 and leukemic cancer HL-60 in the MTT assay. Some compounds (12, 15, and 16) showed stronger cytotoxicity than B13 and C6-ceramide against both tumor cell lines, and compound (12) gave the most potent activity with IC50 values of 36 and 9 microM, respectively. Molecular modeling of thiourea B13 analogues was carried out by comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). We obtained highly reliable and predictive CoMSIA models with cross-validated q2 values of 0.707 and 0.753 and CoMSIA contour maps to show the structural requirements for potent activity. These data suggest that the amide group of B13 could be replaced by thiourea, that the stereochemistry of 1,3-propandiol may not be essential for activity and that long alkyl chains increase cytotoxicity.
Apoptosis ; Cell Line, Tumor ; Ceramidases ; Ceramides ; HL-60 Cells ; Humans ; Inhibitory Concentration 50 ; Kidney Neoplasms ; Models, Molecular ; Structure-Activity Relationship ; Thiourea

Recently, a mitochondrial ceramidase has been identified and cloned, whose mitochondrial localization strongly suggests the existence of an unexpected mitochondrial pathway of ceramide metabolism that may play a key role in mitochondrial functions, especially in the regulation of apoptosis. To explore the biological effect of mitochondrial ceramidase on cells, pcDNA 3.1/His-CDase plasmid, containing mitochondrial ceramidase cDNA sequence, was transducted into K562 cells mediated by liposome, and G418 was used to screen for positive colonies. A stable transfected K562 cell line was established and named as 'K562TC'. The difference between K562 and K562TC cells in chemotheraputic cytotoxicity response and serum-withdrawal resistance and Bcl-2 protein expression were evaluated by MTT assay, annexin V/PI test, flow cytometry or Western blotting, respectively. The results showed that although survival was comparable between K562 and K562TC cells after exposed to adriamycin, etoposide or arsenious acid, K562TC cells with elevated Bcl-2 protein expression level as identified by FCM or Western blotting revealed stronger resistance to apoptosis induced by serum withdrawal than their parental cells. Inhibition of mitochondrial ceramidase expression in K562TC cells by its specific antisense oligodeoxynucleotide was correlated with a decrease in Bcl-2 protein level. N, N-dimethylsphingosine, a sphingosine kinase inhibitor, depleted intracellular sphingosine-1-phosphate production, also abrogated Bcl-2 protein expression in K562TC cells, while Bcl-2 protein level in K562 cells was up-regulated by exogenous sphingosine-1-phosphate. It is concluded that mitochondrial ceramidase overexpression in K562 cells leads to markedly elevated level of Bcl-2 protein and results in more resistance to serum withdrawal. This effect is initiated not by sphingosine, the direct metabolite of mitochondrial ceramidase, but via sphingosine-1-phosphate, its phosphorylated form. This is the first evidence that mitochondrial ceramidase, through its sphingoid metabolite sphingosine-1-phosphate, up-regulates Bcl-2 protein expression in K562 cells.
Amidohydrolases ; physiology ; Apoptosis ; Arsenites ; pharmacology ; Ceramidases ; Doxorubicin ; pharmacology ; Etoposide ; pharmacology ; Humans ; K562 Cells ; Lysophospholipids ; physiology ; Mitochondria ; enzymology ; Oligonucleotides, Antisense ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Sphingosine ; analogs & derivatives ; physiology ; Up-Regulation

Ceramides are the main lipid component maintaining the lamellae structure of stratum corneum, as well as lipid second messengers for the regulation of cellular proliferation and/or apoptosis. In our previous study, psoriatic skin lesions showed marked decreased levels of ceramides and signaling molecules, specially protein kinase C-alpha (PKC-alpha) and c-jun N-terminal kinase (JNK) in proportion to the psoriasis area and severity index (PASI) scores, which suggested that the depletion of ceramide is responsible for epidermal hyperproliferation of psoriasis via downregulation of proapoptotic signal cascade such as PKC-alpha and JNK. In this study, we investigated the protein expression of serine palmitoyltransferase (SPT) and ceramidase, two major ceramide metabolizing enzymes, in both psoriatic epidermis and non-lesional epidermis. The expression of SPT, the ceramide generating enzyme in the de novo synthesis in psoriatic epidermis, was significantly less than that of the non-lesional epidermis, which was inversely correlated with PASI score. However, the expression of ceramidase, the degradative enzyme of ceramides, showed no significant difference between the lesional epidermis and the non-lesional epidermis of psoriatic patients. This might suggest that decreased expression of SPT protein is one of the important causative factors for decreased ceramide levels in psoriasis.
Adolescent ; Adult ; Amidohydrolases/*biosynthesis/metabolism ; Apoptosis ; Cell Proliferation ; Ceramidases ; Ceramides/chemistry ; Child ; Epidermis/metabolism ; Female ; Humans ; JNK Mitogen-Activated Protein Kinases/metabolism ; Male ; Models, Biological ; Protein Kinase C-alpha/metabolism ; Psoriasis/*blood/diagnosis ; Serine C-Palmitoyltransferase/*biosynthesis