Glycoconj J (2009) 26:647–661 DOI 10.1007/s10719-008-9219-4 Post-translational and transcriptional regulation of glycolipid glycosyltransferase genes in apoptotic breast carcinoma cells: VII. Studied by DNA-microarray after treatment with L-PPMP Rui Ma & N. Matthew Decker & Vesta Anilus & Joseph R. Moskal & Joseph Burgdorf & James R. Johnson & Manju Basu & Sipra Banerjee & Subhash Basu Received: 15 July 2008 /Revised: 4 November 2008 /Accepted: 25 November 2008 / Published online: 29 January 2009 # Springer Science + Business Media, LLC 2009 Abstract Functions of glycosphingolipids on the eukary- GLTs were investigated by enzymatic assay and DNA otic cell membranes during the onset of oncogenic microarray analyses. These GLTs are involved in biosyn- processes and cell death are not well understood. thesis of LeX and sialosyl-LeX (neolactosyl-ceramide series) Inhibitors of glycosphingolipid biosynthesis were recently such as GalT-4 (UDP-Gal: LcOse3cer β−galactosyltransferase, found to trigger apoptosis in many carcinoma including GalT-5 (UDP-Gal: nLcOse4Cer α1, 3galactosyltransferase, breast cancer SKBR-3, MCF-7, and MDA-468 cells FucT-3 (GDP-Fucose: LM1 α1, 4fucosyltransferase). A similar through either intrinsic or extrinsic apoptotic pathways as effect was observed with the GLTs involved in the biosyntheses we previously reported. These anti-cancer inhibitors could of Gg-series gangliosides, such as SAT-4 (CMP-NeuAc: increase ceramide concentration by blocking functions of GgOse4Cer α2, 3sialyltransferase, and SAT-2 (CMP-NeuAc: glycolipid glycosyltransferases (GLTs). In this study, using GM3 α2, 8sialyltransferase). The glycol-related gene DNA- a novel fluorescent dye (ASK-0) revealed the damage of cell microarrays also suggested the transcriptional regulation of organelle membranes by an inhibitor of glucosylceramide several GLTs involved in the biosynthesis of neolactosylcer- biosynthesis (L-PPMP). A highly drug- and cell-dependent amide containing cell-surface antigens in these apoptotic breast regulation of MAPKs was also found by cis-platin and L- carcinomacells.Intheearlyapoptoticstages(2to6hafterL- PPMP when inducing apoptosis in SKBR-3, MCF-7, and PPMP treatment) in addition to GlcT-1 gene, several genes MDA-468 cells. A dose and time-dependent regulation of (βGalTs and βGlcNAcTs) in the SA-Lea pathway were stimulated. R. Ma : N. Matthew Decker : V. Anilus : J. R. Johnson : M. Basu : S. Basu (*) Keywords Apoptosis . AKS-0 . Breast carcinoma cells . Department of Chemistry and Biochemistry, Carpases . cis-platin . DNA-microarray. GSL . The University of Notre Dame, Gangliosides . Glycosyltransferases . FucT. GlcT. GD3 . Notre Dame, IN 46556, USA . X . e-mail: [email protected] GD1b D-PDMP L-PPMP LM1 Le MAPKinases SA-Lea . SA-LeX . PSS-380 . Phosphatidylserine J. R. Moskal : J. Burgdorf The Falk Center for Molecular Therapeutics, Abbreviation Department of Biomedical Engineering, McCormick School of Engineering and Applied Sciences, GSLs Glycosphingolipids Northwestern University, GLTs glycolipid glycosyltransferases Evanston, IL 60201, USA GalT galactosyltransferases SAT sialyltransferases S. Banerjee Department of Cancer Biology, Cleveland Clinic Foundation, FucT fucosyltransferases Cleveland, OH 44129, USA GlcT glycosyltransferase 648 Glycoconj J (2009) 26:647–661 L-PPMP L-threo-1-phenyl-2-palmitoylamino- death because GlcT has at least two distinct functions, one 3-morpholino-1-propanol of which is a negative regulatory factor for intracellular D-PDMP D-threo-1-phenyl-2-decanoylamino- ceramide content through ceramide glycosylation. The 3-morpholino-1-propanol other is a positive regulatory role of the glycolipid X SA-Le sialyl-fucosyl-neolactotetraosyl- biosynthesis. The action of ceramide-like substances (D- ceramide and L-PDMP) on sphingolipid glycosyltransferases, purified cis-Platin cis-diamino-dichloro platinum lactosylceramide synthase, has also been studied [14]. Using the GlcT-specific inhibitor L-threo-PPMP [11-13]in human breast carcinoma cells [14-19], it is suggested that a systematic study of different stereo isomers of these Introduction inhibitors of GSL biosynthesis is needed to establish a relation of these GSL biosynthesis inhibitors to the Studies on initiation and regulation of the apoptotic apoptotic processes in the level of other glycosyltransferase cascades are of great interest in oncogenic drug discovery. activities [20, 21]. Such studies would be important in the Glycosphingolipids (GSLs) comprise an important outer- search for chemotherapeutic drugs for treatment of breast leaflet structural and signaling lipid in the higher eukaryotic and other highly metastatic cancer cancers. cells. Importantly, the glycolipid-enriched membrane Abnormal expression of any particular glycosyltransferase domain (Rafts) also includes proteins related to signal may lead to a distorted sequence and may give rise to aberrant transduction including the apoptotic signal. GSLs are oligosaccharides in tumor and metastatic cancer cells [14, 22]. synthesized from ceramide in the Golgi bodies [1–4], and Many tumor-associated antigens are characterized as glyco- subsequently distributed to different areas of the cell. The lipids and glycoproteins [23–25]. Comparative studies glycosphingolipids are derived from the sphingolipid between normal and tumor cells showed changes in glycan ceramide by the action of a glucosyltransferase, GlcT structures during oncogenic transformation. Complete bio- [1–5], to generate glucosylceramide, a precursor of other synthetic steps of these GSLs have been established [6–8]. long chain glycolipids [6–9] (Fig. 1). However, ceramide is SA-Lex (SAα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,3 widely agreed to be a pro-apoptotic messenger in eukary- Galβ1,4Glc-Ceramide) and SA-Lea(SAα2,3Galβ1,3 otic cells [10]. If the GSLs biosynthesis is blocked [11–13] (Fucα1,4)GlcNAcβ1,3Galβ1,4 Glc-Ceramide) have been in the first committed step of glucosyltransferase (GlcT), suggested as markers for metastatic breast carcinoma cells the ceramide will accumulate in the cells, which may be the [26]. Exact abnormalities of these oligosaccharide-antigens cause for apoptosis. At present it is not known why ablation (glycolipid or glycoproteins) and the regulation of these of the GlcT gene causes the enhancement of apoptotic cell GSLs during apoptosis in tumor or cancer cells is not known Ceramide D-/L-PDMP GlcT D-/L-PPMP Galα1- 3Galβ1- 4GlcNAcβ1-3Lc2 Glc-Cer GalT-2 nLc5 GlcNAcT- 1 Galβ1-4Glc-Cer SAT-1 Galβ1-4Glc-Cer GlcNAcβ1-3Lc2 α2-3 GalT-5 GM3 Lc2 Lc3 SA GalT-4 SAT- 2 GalT-5’ Galβ1-4Glc-Cer GalNAcT-1 GD3 Galβ1- 4GlcNAcβ1-3Lc2 SA Galα1-4Lc2 nLc4 GalNAcβ1-4Gal-Glc-Cer α2-8 Gb3 GalNAcT-1 GalNAcT-2 SAT-3 SA GM2 GlcNAcT-2 SA Galβ1- 4GlcNAcβ1-3Lc2 GalT-3 β GalNAc 1-4Gal-Glc-Cer α 2-3 LM1 β GD2 GalNAcβ1-3Gb3 SA GlcNAc 1-3nLc4 Galβ1- 3GalNAc-Gal-Glc-Cer SA FucT-3 GalT-3 GbOsc4Cer inLc5 SAT-4 SA GM1 SA Galβ1- 4GlcNAcβ1-3Lc2 GlcNAcT-3 Galβ1-3GalNAc-Gal-Glc-Cer α1-3 Gal-GalNAc-Gal-Glc-Cer GD1b SA Fuc GlcNAcβ1-3 i α2-3 SA X nLc4 GD1a SA-Le SA SA GlcNAcβ1-6 SA I Ii Fig. 1 Proposed pathways for biosynthesis of sialosyl glycosphingolipids in animal cells Glycoconj J (2009) 26:647–661 649 or has been poorly understood until now. Our present goal is MAb (20G11), rabbit anti-human-phos-SAPK/JNK MAb to correlate the changes in the expression of cancer cell (98F2) were from Cell Signaling Technology, Inc. (Danvers, surface glycosphingolipids (Glyco-biomarkers) and the MA); Rabbit anti-human-actin, Goat anti-mouse-IgG-ALP signal transduction with apoptosis induced by anti-cancer conjugate, Goat anti-rabbit-IgG-ALP conjugate were from drugs, inhibitors of glycosphingolipid biosynthesis such as Sigma Chemical Co. (St. Louis, MO). L-PPMP. Microarray-based assays are rapidly becoming one of the Staining agents Trypan blue, Propidium iodide were from powerful technologies in high-throughput-gene-expression Sigma Chemical Co. (St. Louis, MO); PSS-380 and analyses. In order to focus on intrinsically complex Squaraine-derived Rotaxane AKS-0, and MitoTracker Red regulation of synthesis and degradation of the specific 580 were kind gifts of Dr. Smith and Dr. Goodson glycoconjugates that are essential for breast cancer, a total (Department of Chemistry and Biochemistry, University of 359 genes comprising the human glycobiology micro- of Notre Dame, IN). array are complied from currently available NCBI/EMBL/ TIGR human sequence database and the Consortium for Substrates for glycosyltransferases: donors UDP-[3H]- 3 Functional Glycomics-CAZY databases [27]. A unique Galactose, [ H]-L-fucose were from American Radiolabeled 3 sense 45-mer oligonucleotides corresponding to mRNAs Chemicals, Inc. (St. Louis, MO). GDP-[ H]-L-Fucose was of each gene are individually synthesized, purified, and prepared in our laboratory [21] and all other glycosphingo- immobilized via a 5′ amino linker onto aldehyde-coated lipids were the stock prepared and identified in our microarrays. The labeled apoptotic cell aRNA from L- laboratory previously [28, 29]. PPMP-treated cells were hybridized with the array to study the change of GSL-GLT expression. DNA microarray agents RNeasy Mini Kit was purchased from Qiagen (Germany); Amino Allyl MessageAmp™ II aRNA Amplification Kit, Ambion (Austin, TX); Micro Material and methods Bio-Spin Chromatography Columns were purchased from Bio-Rad Laboratories (Hercules, CA), Glycobiology micro- Cells MCF-7, MDA-468, and SKBR-3 human breast array [30] containing all human 359 glyco-related genes adenocarcinoma cell lines were the kind gifts of