Cellular effects of deoxynojirimycin analogues: uptake, retention and inhibition of glycosphingolipid biosynthesis

Deoxynojirimycin (DNJ) analogues are inhibitors of ceramide glucosyltransferase (CGT), which catalyses the first step in the glucosphingolipid (GSL) biosynthetic pathway. We have synthesized a series of DNJ analogues to study the contribution of N-alk(en)yl side chains (C4, C9 or C18) to the behaviour of these analogues in cultured HL60 cells. When cells were treated for 16 h at non-cytotoxic concentrations of inhibitor, a 40–50% decrease in GSL levels was measured by HPLC analysis of GSL-derived oligosaccharides following ceramide glycanase digestion of GSL and 2-aminobenzamide labelling of the released oligosaccharides. Using a novel technique for short-term [14C]galactose labelling of cellular GSL, we used compound inhibition of GSL biosynthesis as a marker for compound uptake into cells. Surprisingly, the uptake of all three of the DNJ analogues was extremely rapid and was not dependent upon the length of the N-alk(en)yl moiety. Compound uptake occurred in less than 1 min, as shown by the complete inhibition of GSL labelling in cells treated with all the DNJ analogues. Greatly increased cellular retention of N-cis-13-octadecenyl-DNJ was observed relative to the shorter-chain compounds, N-butyl-DNJ and N-nonyl-DNJ, as indicated by complete inhibition of CGT 24 h after removal of inhibitor from the culture medium. The present study further characterizes the properties of N-alk(en)ylated DNJs, and demonstrates that increasing the length of the side chain is a simple way of improving imino sugar retention and therefore inhibitory efficacy for CGT in cultured cells.

Ceramide Glycanase Activities in Human Cancer Cells

Ceramide glycanase (CGase) activities have been detected in different human tumor cells (colon, carcinoma Colo-205; neuroblastoma, IMR-32; breast cancer lines, SKBr3 and MCF7). However, the level of enzymatic activity is lower in these cells compared to that present in other mammalian tissues reported before (Basu, M., Kelly, P., Girzadas, M. A., Li, Z., and Basu, S. Methods Enzymol. (in press)). The majority of CGase activity was found in the 100,000g soluble supernatant fraction isolated from all these cell lines and tissues. Using the soluble enzyme, the requirement for optimum CGase activity was found to be consistent with previous observations found for rat and rabbit tissues (Basu, M., Dastgheib, S., Girzadas, M. A., O'Donnell, P. H., Westervelt, C. W., Li, Z., Inokuchi, J. I., and Basu, S. (1998) Acta Pol. Biochim. 42:327). The CGase activities from both Colo-205 and IMR-32 cells are optimum at a protein to detergent ratio of one. All the mammalian CGases, including human cancer cells, show an optimum pH between 5.5 and 5.8 in sodium acetate buffer. The CGase activities from cancer cells are found to be cation-independent; however, mercury, zinc, and copper ions seem to inhibit the enzyme activity substantially in both tumor cells lines. The mercury ion inhibition of CGase activities from all different sources indicates a possible structural homology in the CGase proteins. Radiolabeled substrates, labeled at the sphingosine double bond or at the 3-position of sphingosine without modifying double bond of sphingosine were used in this investigation. Both were active substrates with all enzyme preparations isolated from different cancer cells (apparent Km, 500 μM for nLcOse5[3H-DT]Cer and 350 μM for GgOse4[sph-3-3H]Cer with Colo-205 enzyme). Structural analogues of ceramide and sphingosine (L-PPMP, L-PDMP, alkylamines, and Tamoxifen) inhibited cancer cell CGase activities in vitro.

Hydrophobic nature of mammalian ceramide glycanases: purified from rabbit and rat mammary tissues.

The ceramide glycanase (CGase) activities, which cleave the intact oligosaccharide chain and the ceramide moiety of a glycosphingolipid, have been characterized from two mammalian sources. The enzymatic activities are almost comparable in rabbit and rat mammary tissues. The majority of the activities has been concentrated in the soluble fraction which could be partially purified using hydrophobic columns. The rabbit mammary ceramide glycanase activity has been purified up to 1438-fold using ion exchange and hydrophobic columns in tandem. The purified protein exhibited a molecular mass of 54 kDa which could be immunostained on the Western blot with clam anti-CGase polyclonal antibody. In addition, a 98 kDa protein also exhibited positive immunostain in a successive purified fraction with that antibody and is under investigation. The requirement for the optimal enzymatic activities are similar for both rabbit and rat CGase activities. The CGase activity requires the presence of detergent for optimal activity but is not dependent on the presence of any divalent cations. However, Hg2+, Zn2+, and Cu2+ are inhibitory to the enzymatic activities. It has been observed that rat as well as rabbit CGases are inhibited by both D- and L-PDMP (1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCl) and its higher analogue PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol.HCl). Alkyl amines containing C12 and higher chains are also found to inhibit both rat and rabbit CGase activities. Substantial levels of CGase activities have also been observed in various human tumor cells as well as in developing avian brains. These observations are significant in view of the recent findings that ceramide, which is one of the enzymatic reaction products of CGase activity, is mediating different cellular events like signal transduction and apoptosis. The role of this enzyme in development, metastasis and cellular regulation are anticipated.