Ganglioside Biosynthesis in Developing Brains and Apoptotic Cancer Cells: X

Neurochem Res (2012) 37:1245–1255 DOI 10.1007/s11064-012-0762-9

OVERVIEW

Ganglioside Biosynthesis in Developing Brains and Apoptotic Cancer Cells: X. Regulation of Glyco-genes Involved in GD3 and Sialyl-Lex/a Syntheses

Subhash Basu • Rui Ma • Joseph R. Moskal • Manju Basu

Received: 11 November 2011 / Revised: 12 March 2012 / Accepted: 22 March 2012 / Published online: 10 April 2012 Ó Springer Science+Business Media, LLC 2012

Abstract Gangliosides, the acidic glycosphingolipids D-PDMP (inhibitor of glucosylceramide biosynthesis), (GSLs) containing N-acetylgalactosamine and sialic acid Betulinic Acid (a triterpinoid isolated from bark of certain are ubiquitous in the central nervous system. At least six trees and used for cancer treatment in China), Tamoxifen a DSL-glycosyltransferase activities (GLTs Gangliosides, drug in use in the west for treatment of early stages of the the acidic glycosphingolipids (GSLs) containing N-acety- disease in breast cancer patients), and cis-platin (an lgalactosamine and sialic acid (or NAc-Neuraminic acid) inhibitor of DNA biosynthesis used for testicular cancer are ubiquitous in the central nervous system. At least six patients) were used for induction of apoptosis in the above- GSL-glycosyltransferase activities (GLTs) of Basu-Rose- mentioned cell lines. Within 2–6 h, transcriptional modu- man pathway catalyzing the biosynthesis of these gan- lation of a number of glyco-genes was observed by DNA- gliosides have been characterized in developing chicken micro-array (containing over 300 glyco genes attached to brains. Most of these glyco-genes are expressed in the early the glass cover slips) studies. Under long incubation time stages (7–17 days) of brain development and lowered in (24–48 h) almost all of the glyco-genes were downregu- the adult stage, but the cause of reduction of enzymatic lated. The cause of these glyco-gene regulations during activities of these GLTs in the adult stages is not known. In apoptotic induction in metastatic carcinoma cells is order to study glyco-gene regulation we used four clonal unknown and needs future investigations for further metastatic cancer cells of colon and breast cancer tissue explanations. These apoptotic agents could be employed as origin (Colo-205, SKBR-3, MDA-468, and MCF-3). The a new generation of anti-cancer drugs after properly glyco-genes for synthesis of SA-LeX and SA-LeA (which delivered to the patients. contain N-acetylglucosamine, sialic acid and fucose) in these cells were modulated differently at different phases Keywords Apoptosis Á Breast cancer cells Á (between 2 and 48 h) of apoptotic inductions. L-PPMP, Ganglioside biosynthesis Á GD3 Á SA-Lex Á Glyco-gene regulation

Special Issue: In Honor of Bob Leeden. Introduction S. Basu (&) Á M. Basu Department of Chemistry and Biochemistry and Cancer Drug Delivery Research Foundation, University of Notre Dame, In the field of Glycosphingolipids (GSLs), the term POBox-752, Notre Dame, IN 46556, USA ‘‘Ganglioside’’ is meant for the GSLs containing sialic e-mail: [email protected] acids (NeuAc or NeuGc). These compounds were isolated R. Ma and purified from brain tissues [1–5] and peripheral ner- Global Assay Development, Siemens Healthcare Diagnostic vous systems [6–10]. Since discovery of GM2-ganglioside Div., Elkhart, IN 46515, USA from human Tay-Sachs brain by Klenk and his associates in 1939 [1] and GM3 from horse erythrocytes by Yama- J. R. Moskal The Falk Center for Molecular Therapeutics, Northwestern kawa and his co-workers in 1951 [11]. Structures of over University, Evanston, IL 60201, USA 180 gangliosides are documented in literature [12]. Kuhn 123 1246 Neurochem Res (2012) 37:1245–1255 and Wiegandt first established the chemical structures of acidic-GSLs were being established in different laborato- GM1, GD1a, and GD1b in 1963 [3]. Structures of all these ries around world, interest in searching the mechanism of naturally occurring gangliosides revealed they could be their synthesis in the normal tissues and cancer cells began classified in three families (Fig. 1) according to the third at almost the same time. The biosynthetic pathway-a for sugar present in the oligosaccharide chains next to cera- synthesis of GD1a ganglioside from ceramide was pro- mide: (1) Ganglio- (GalNAc-Gal-Glc-ceramide), (2) Lacto- posed by Basu and Roseman in 1975 [26–32] (Fig. 3) and (GlcNAc-Gal-Glc-ceramide), and (3) Globo- (Gal-Gal- was first established in embryonic chicken brains. All of Glc-ceramide). However, the simplest gangliosides G7 those glycosyltransferases were isolated, characterized, and (NeuAc2, 3Gal-Ceramide), GM3 (NeuAca2, 3Galb1, cloned in different laboratories. These days it is proved that 4Glc-ceramide), and GD3 (NeuAca2, 8-NeuAca2, 3Galb1, a different gene product catalyzes each enzymatic activity, 4Glc-ceramide) are free of hexosamines (Fig. 2). Sialic and isozymes are widely distributed in different animal acid-containing gangliosides were first characterized in systems. Expression of these genes in normal and diseased brain tissues; later on distribution of these acidic Glyco- stages could be studied by analysis of mRNAs, but very sphingolipids (GSLs) containing both NeuAc and NeuGc little is known about the regulation of these gene expres- has been characterized in blood and other tissues of higher sions during varied biological conditions. Recognition of eukaryotes except in marine animals. In recent years GM3, the exact regulation of these Glyco-genes would give us GD3, LD1, SA-Lex, and SA-Lea have been characterized in tools for disease control and cure in humans. many cancer cells [13–16] including breast and colon In this article an attempt is made to compare gene reg- carcinomas. In case of various neurological inherited dis- ulation of glycosyltransferases in developing brains eases, deposition of specific gangliosides in the tissues is with apoptotic cancer cells. All the enzymes of GD3 and recognized. GM2 is known to be the main ganglioside deposited in Tay-Sachs-diseased (TSD) brain, but how Abbreviated Names Structures excess deposition of this ganglioside hinders the normal Hexosamine Free α function in TSD brain [17, 18] remains unknown. Recently, G7 NeuAc 2-3Gal-Cer NeuAcα2-3Galβ1-4Glc-Cer Dwek and his associates indicated [19] using mouse GM3 GD3 NeuAca2-8NeuAca2-3Galb1-4Glc-Cer models of GM1 and GM2 gangliosidosis that inflammation may play an important role in the pathogenesis of the GalNAcβ1-4Gal-Glc-Cer GalNAc-containing α2-3 gangliosides. GD3 ganglioside has been found in excess in GM2 NeuAc Galβ1-3GalNAcβ1-4Gal-Glc-Cer human meningiomas [20], gliomas [21], and melanomas α2-3 [22]. However, the relationship of these gangliosides to the GM1 NeuAc Galβ1-3GalNAcβ1-4Gal-Glc-Cer exact physiological disorders is not yet known. Both GD1a α2-3 α2-3 x a NeuAc NeuAc SA-Le and SA-Le are present on the cell surfaces of β β GlcNAc-containing Gal 1-4GlcNAc 1-3Gal-Glc-Cer α α1-3 breast and colon cancers [23–25]. A direct role of these X 2-3 SA-Le NeuAc Fuc acidic GSLs to cancer metastasis has not been established. During the last five decades while the structures of these Fig. 2 Structure of gangliosides with and without NAc-hexosamines

Fig. 1 Broad classiﬁcation of (Glycose)n—O—Sphingosine glycosphingolipids (GSLs) | CERAMIDE n=1 to 10 glycoses NH R=C to C | Fatty acid 13 15 CO—R

ACIDIC - GSLs NEUTRAL- GSLs

Glucuronyl- Sialyl- Mono- Di- Oligo-glycosyl- Gal-cer Glc-cer Gal-Glc-cer ceramide Mono- Di-

Polysialo- Blood Groups Globosides Gangliosides- …. (….GlclNAc-Gal-Glc-cer) (….Gal-Gal-Glc-cer) (GalNAc-Gal-Glc-cer) (NeuAc or, NeuGc)

Series—[Ganglio-Gg] —[Lacto-Lc] —[Globo-Gb]

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Fig. 3 Basu-Roseman Ceramide D-/L-PDMP ganglioside pathway and cancer GlcT D-/L-PPMP Galα1- 3Galβ1- 4GlcNAcβ1-3Lc2 GSL pathways Inhibitors Glc-Cer GalT-2 nLc5 GlcNAcT- 1 Galβ1-4Glc-Cer SAT-1 Galβ1-4Glc-Cer GlcNAcβ1-3Lc2 α GalT-5 2-3 Lc2 Lc3 SA GM3 GalT-4 SAT- 2 Galβ1-4Glc-Cer GalNAcT-1 β β SA GD3 Gal 1- 4GlcNAc 1-3Lc2 α β 2-8 nLc4 GalNAc 1-4Gal-Glc-Cer SA GalNAcT-1 SAT-3 SA GM2 GlcNAcT-2 Galβ1- 4GlcNAcβ1-3Lc2 GalT-3 β GalNAc 1-4Gal-Glc-Cer α2-3 LM1 SA GlcNAcβ1-3nLc4 β SA GD2 FucT-3 Gal 1- 3GalNAc-Gal-Glc-Cer GalT-3 inLc5 SA SAT-4 SA GM1 Galβ1- 4GlcNAcβ1-3Lc2 GlcNAcT-3 Galβ1-3GalNAc-Gal-Glc-Cer α1-3 SA Fuc Gal-GalNAc-Gal-Glc-Cer GD1b GlcNAcβ1-3 i α SA 2-3 SA-LeX nLc4 SA SA GD1a GlcNAcβ1-6 SA GT1b I Carcino-Embryonic Ii

Basu-Roseman Pathway Pathway-b Antigen Ii Core GSLs (or Pathway-a) (Cancer Cells) Lymphoma Cells Embryonic Brain

Sialyl-LeX/A biosynthesis are expressed in both the sys- the GlcNAc-containing nLcOse4-ceramide (the intermedi- tems (embryonic brains and breast cancer cells). The age- ate of gangliosides SA-Lex) was established in rabbit bone dependent expression studies of those glycolipid glyco- marrow [50, 51], human neuroblastoma (IMR-32) cells syltransferases raised the question whether those enzymes [52], cultured TSD cells [53], mouse T- and B-lymphomas could be modulated also under induced apoptotic condi- [54, 55], bovine spleen [56, 57], embryonic chicken brains tions of cancer cells. Of course, the expression of glyco- [58–60], and carcinoma cells of colon origin [61–63]. sphingolipids on the surfaces of both apoptotic and Complete biosynthesis of SA-Lex from nLcOse4-ceramide nonapoptotic cells has not yet been revealed exhaustively. to SA-Lex (Fig. 3) has been achieved in colo-205 and However, our finding on the activity and DNA-microarrays embryonic chicken brains [60, 61]. Biosynthesis in studies suggested these could be different at different time vitro of Ii-core glycosphingolipids (GlcNAcbeta1,6(Glc- points (2 and 24 h) and with different concentrations of NAcbeta1,3)Galbeta1,4Glc-R) was achieved in mouse the apoptotic agents (L-PPMP; 2–16 lmol). This, in turn, T-lymphomas from nLcOse4-cer by two different N-acet- would reflect the biological behavior of cells after alter- ylglucosaminyltransferases (GlcNAcT-1 and GlcNAcT-2 ation of glycolipid expression. This can be a very powerful [55]. The GSL-GLTs of the Basu-Roseman Pathway tool to go toward the functional studies (by the future (Fig. 3) for biosynthesis of GD1a ganglioside from lacto- researchers) of cell surface properties of the Glyco- sylceramide (Fig. 3) were characterized in Golgi-bodies sphingolipids. Because of the limited scope of this mini- isolated from rat liver in the early seventies [64]. Biosyn- review we have quoted only the relevant references in this thesis of galactocerebroside from the hydroxyfattyacid- article. containing ceramide was reported in embryonic chicken brains (17-to 19-day old) [65], which is precursor of brain G7 ganglioside as revealed by Yu et al. in mouse brain Ganglioside Biosynthesis in Golgi-Membranes [66]. During the enzymatic studies of GM3 to GD1a bio- of Embryonic Chicken Brains synthesis in embryonic chicken brains, enzymatic synthesis of GD3 was achieved also [30]. Further elongation of chain The Basu and Roseman pathway for ganglioside was first length for polysialo-gangliosides (pathway-b; Fig. 3) from established in developing embryonic chicken brains GD3 was established in ER-Golgi membrane complex (Fig. 3). Characterization in vitro of each of those enzymes isolated from rat liver in mid-nineties [67]. The genes of was based on substrate specificity and chemical charac- most of the GSL-GLTs in both Basu-Roseman pathway terization of the corresponding products at the optimum and pathway-b have been cloned these days in many other age of the embryo. All the naturally occurring purified laboratories and not reviewed here. Regulations of these gangliosides were used as substrates, and products were genes are not yet well understood. However, the limited isolated and purified under optimum kinetic conditions gene expressions of the glycosyltransferases activities of [26–49]. The pathway starting from lactosylceramide up to these pathways could be an explanation for inherited

123 1248 Neurochem Res (2012) 37:1245–1255

diseases (e.g. Tay-Sachs disease) [53] in addition to the 100% Galβ1-3GalNAcβ1-4Gal-Glc-Cer Galβ1-3GalNAcβ1-4Gal-Glc-Cer alterations of GSL-glycosidases [67]. NeuAc (GM1b) 80%

Regulation of GSL-Glycosyltransferases in Developing Brains 60%

As mentioned above, the glycosyltransferase activities % Activities (GSL-GLTS) which catalyze synthesis of GD1a ganglio- 40% sides were first characterized in 9- to 15-day-old embryonic chicken brains. The GalT-1 (cerebroside synthase) activity 20% was barely detectable in young embryos (between 7 and 6 11 16 21 ECB Ages 15 days) but began to increase at 15 days (Fig. 4)[65]. Specificities of all these enzymatic activities were deter- Fig. 5 SAT-4 Activities in developing chicken brains mined under optimum conditions [38], which gave ratio- nale to arrange in the increasing order of chain length of the oligosaccharides [32, 44, 48, 49]. In the de novo situ- studied also [68]. Very little is known about the cause of ation sialylation of lactosylceramide first was concluded regulation of ganglioside and cerebroside biosynthesis in from the substrate competitive studies of GalNAcT-1 developing animal brains. It is important to mention that activity with lactosylceramide and GM3 gangliosides [27, galactocerebrosides are enriched in myelin and the factors 28, 32, 42, 58]. It was revealed that specific activities of initiate myelin synthesis may cause the increase in galac- membrane-bound GLTs (GalT-2, GalT-3 and GalT-1) were tocerebroside synthesis also. Whether transcriptional different with the progress of age of the embryos (Fig. 4) regulation of these GSL-GalTs occurs during brain devel- [65]. Both GalT-2 and GalT-3 appeared between 9 and opment has not been reported yet (Fig. 6). Recently, Yu 13 days and then showed marked decrease during the next and his collaborators have reported [69] histone acetyla- 4–5 days as the embryo developed before hatching. On the tion-mediated GSL-GLT gene regulation in mouse devel- other hand the GalT-1 activity was very low between 9 and oping brains. Chromatin immunoprecipitation assay of 13 days and started appearing on day 15 and reached peak both GalNAcT-1 (GM2 synthase) and SAT-2 (GD3 syn- activity before hatching (Fig. 4)[65]. It was proposed that thase) genes showed the histone H3 acetylation was highly the GalT-1 gene is expressed in developing brains before correlated to their mRNA expression levels during devel- myelination only. A similar age effect of SAT-1 [27, 28], opment of mouse brains. The correlation between histone- SAT-3 [60], and SAT-4 (Fig. 5) was also observed. Opti- H3 acetylation and expression of some GLTs involved in mum specific activities were observed between 9 and the GalNAc-containing gangliosides is the indirect begin- 12 days of embryonic age (Fig. 5). Similar increase of ning of brain development study. Using some clonal GSL-GLT activities in developing mouse brain had been mouse neuroblastoma cell lines (e.g. adrenergic clone (N1E - 115) and cholinergic clone (NS-20), we showed the difference of activities during induced differentiation

GalT-1 with (But)2-cAMP [70]. It was also identiﬁed that GalT-4 GalT-2 GalT-3 (nLcOse4cer synthase) was highly active in these cells 100 compared to other GSL-galactosyltransferases. However, uncontrolled differentiation of neuroblastoma cells in gene 80 regulation studies brought ambiguity in the conclusions regarding regulation of biosynthesis. 60 % Activities 40 Induction of Apoptosis in Colon and Breast Cancer

20 Cells by Novel Agents

Apoptosis is a naturally occurring process by which normal 7 9 11 13 15 17 19 21 Adult cells are directed to program death activating several sig- ECB Ages naling pathways by some inducer molecules from outside. Fig. 4 GalT-1, GalT-2, and GalT-3 activities in developing chicken It is a distinctive mode of cell death (through self- brains destruction) with characteristic changes in morphologic 123 Neurochem Res (2012) 37:1245–1255 1249 features, which is regulating the size of animal tissues. In (inhibitor of DNA biosynthesis), Betulinic acid (a trite- contrast, the process of necrosis in cancer cells is a pro- rpinoid used as an anti-cancer agent in China as an alter- gressive disintegration of cells, which cause nearby viable native medicine), Tamoxifen (a common anti-cancer agent cells to disintegrate. Viable cells discriminate apoptosis used for the treatment of breast cancers today), GD3, and necrosis targets via distinct cell surface receptors GD1b, and Melphalan (a scrambler for Golgi bodies) in the [71–74]. range of 2–16 lmol concentrations. Structures of these Unlike necrosis, apoptotic cell death is less damaging in apoptotic compounds are shown in Fig. 7. Inhibition of cell the patients carrying highly metastatic breast carcinomas. growth (IC-50) was different with three different metastatic Studies of induction (or initiation) and regulation of breast cancer cell lines when tested with a single apoptotic apoptotic cascades in breast and colon cancer cells are of agent (L-PPMP) for 24 h [75]. All these chemicals (Fig. 7) prime interest in anti-cancer drug discovery. If we search induced apoptosis by activating IMCAP (internal mito- this topic in the Pubmed, we will obtain at least 1,000 chondrial caspase activation pathway) (Fig. 8). On the reports regarding apoptosis in breast cancer cells. Induction other hand, cis-platin (used in the treatment of testicular of apoptosis can be classiﬁed in four categories: (1) cancers) induced apoptosis [77, 78] occurs in those cell Internal mitochondrial-caspase activation pathway (IM- lines at much higher concentrations (50–150 lM) by EB- CAP); (2) External Bad receptor activating pathway (EB- RAP (external Bad-receptor activation pathway) followed RAP); (3) NFkappaB activation pathway (NFKBP); and (4) by activation of Caspase-8 (Fig. 8). Several new chemicals Cascades of protein kinase activation Pathways (CPKAP) have been tested in recent years for induction of the (Fig. 8). apoptotic process by activating IMCAP, NFKBAP (NF- Using highly metastatic breast carcinoma cells (SKBR- kappaB activation pathway), EBRAP, or PKAP (protein 3, MDA-468, and MCF-7), we have reported in recent kinase activation pathway). We also tested activation of years [33, 75–78] apoptotic induction by D-/L-PPMP, apoptosis via all other pathways in the above-mentioned D-PDMP (inhibitors of GSL biosynthesis), cis-platin cell lines using those apoptotic agents [79–81]. Human

Fig. 6 Different GSL- Acceptors Linkage Catalyzed galactosyltransferase Gene GLT Products GalT-2 Glc-Ceramide Galβ1-4Glc-Cer

GlclNAcβ1-4Gal-Glc-Cer GalT-3 Galβ1-3GM2 NeuAc

GalT-4 LcOse3Cer (GlcNAcβ1-3Gal-Glc-Cer) Galβ1-4LcOse3Cer (Type-2 Core)

GalT-4’ GlcNAcβ1-3Gal-Glc-Cer Galβ1-3LcOse3Cer (Type-1 Core)

GalT-5 nLcOse4Cer Galα1-3nLcOse4Cer

L-/D-PPMP cis-Platin Betulinic Acid

Galβ1-4Glc-CERAMIDE Galβ1-3GalNAcβ1-4Galβ1-4Glc-CERAMIDE α2-3 α2-3 NeuAc NeuAc GD3 GD1b α2-8 α2-8 NeuAc NeuAc

Fig. 7 Structures of different apoptotic agents 123 1250 Neurochem Res (2012) 37:1245–1255

cis-Platin GSL (GD3/GD1b)

P-Choline Fas/TNF Glc-Gal (GD3) ? Sphase ? SA CGase SA Caspase-8 L-/D-PPMP FADD CERAMIDE Inhibitor PTP

IKKs Caspase-3 Mitochondrion GSL Cyto-C Sphingosine Apaf-1 p53 IkB Active Casp-3 (p17/12) Caspase-9 NF-kB Sph-1-P Ubi PKC NF-kB P DFF/ICAD/PARP etc.

DNA FLIP,cIAP2 BFL1,BCL-XL DNA Damage

Fig. 8 Apoptotic signal pathways by anti-cancer agents

MCF-7 breast cancer cells are resistant to pro-apoptotic Expression of sialosyl-LewisX/A is markedly enhanced in stimuli due to Caspase-3 inactivation. However, reconsti- solid tumors, but the molecular mechanisms underlying are tution of human MCF-7 breast cancer cells with the Cas- not well understood, until the glycosyltransferases and their pase-3 gene does not sensitize these cells to the inhibitory genes involved in the step-wise synthesis of sialyl-LewisX/ action of ROSC (roscovitine) and OLO (Olomycine). A determinant have been extensively been characterized Apoptotic mechanisms of MCF-7 cell by cis-platin or [50, 60, 61, 83–86]. Cancer cells are known to exhibit a L-PPMP is still unknown [74–80]. Quercetin (a natural metabolic shift from oxidative to elevated anaerobic gly- polyphenolic compound) induced apoptosis in many colysis (Warburg effect), which is correlated with the human cancer cells, including MCF-7 human breast cancer increased gene expression of sugar transporters and gly- cells [81]. The involvement of possible signaling pathways colytic enzymes induced by common cancer-speciﬁc gene and the roles of quercetin in the apoptosis of other cancer alterations. The increased sialyl-LewisX/A expression in cells remains undeﬁned. The recent results [81] suggest cancer could be a link in the chains of these events. Recent quercetin may induce apoptosis by direct activation of results by Kannagi [87, 88] indicated that these events Caspase cascade through IMCAP bypassing the Caspase-3 accompanying transcription induction of a set of genes activation process without involving Caspase-3. A direct closely related to its expression. Studies on the regulations relation of activation of Caspase-3 with the transcriptional of these GSLs would generate unambiguous proof of regulation of Glyco-genes in breast and colon cancer Glyco-gene regulation. As mentioned above the inhibitors apoptotic cells is the theme of our future research. of Glycosphingolipids (L-/D-PPMP) (Fig. 7) and DNA (cis-platin) biosynthesis, Betulinic acid (an herbal origin of a triterpinoid used for cancer treatment in China), Mel- phalan, and disialosylgangliosides (GD3 and GD1b) Regulation of Lacto-series GSLs Biosynthesis induced apoptosis [76] (intrinsic mitochondrial or extrinsic in Metastatic Clonal Cells During Apoptosis receptor mediated pathways) (Fig. 8) in human breast (SKBR-3, MCF-7, and MDA-468) and colon (Colo-205) Evidence indicates that blood group-related Lewis (Le) cell. These chemicals are suggested to be potential anti- antigens (glycosphingolipids: GSLs, and Glycoproteins: cancer drugs [77, 78]. Analysis of glycosphingolipid GPs) are tumor-associated cell surface molecules. The composition of MDA-MB-231 and MCF-7 human breast LeX, LeA, LeB, LeY, and their sialosyl-derivatives cancer cells (BCCs) showed abundant presence of GM3, (Fig. 2), all N-acetylglucosaminyl-containing glycoconju- GM2, GM1, and GD1a in both the cell lines [82]. The gate antigens, are over-expressed on the surfaces of breast, 18-fold increased amount of GM3 ganglioside suggests colon, and ovarian cancer cells during metastasis. some role for this simple ganglioside in the growth 123 Neurochem Res (2012) 37:1245–1255 1251 regulation in MDA-MB-231 BCCs [82]. However, inser- Post-translational Regulation of Glyco-genes During tion of GM3 ganglioside into the plasma membrane of Apoptosis of Breast Cancer Cells MCF-7 cells blocked the growth stimulatory effect of EGF. Biosynthesis of glycosphingolipids in both Ganglio (Gg)- A dose- and time-dependent down-regulation of GLTs was and Lacto (Lc)-series pathway before and during apoptosis investigated by GLT enzymatic assay (Table 2) after 48 h has been studied in recent years in our laboratory [33, 75– of apoptotic induction [75–80]. These GLTs are involved in 80]. Regulations of some of these Glyco-genes were biosynthesis of LeX and sialosyl-LeX (neolactosyl-ceramide observed in posttranslational and transcriptional levels as series) such as GalT-4 (UDP-Gal: LcOse3cer beta-galac- discussed in the following sections (Table 1) in the indi- tosyltransferase, GalT-5 (UDP-Gal: nLcOse4Cer alpha1, cated breast cancer cell lines. 3galactosyltransferase (Fig. 3), and FucT-3 (GDP-Fucose:

Table 1 Metastatic breast cancer clones (MCF-7, MDA-468, and SKBR-3) derived from pleural effusion of breast carcinoma Cells HER2 ER PgR p53 Caspase-3

MCF-7 Normal ?? Normal Mutant MDA-468 Normal -- Mutant Normal SKBR-3 High -- Mutant Normal THER2 (c-erbB-2/neu) protein: HER2 comes from a proto-oncogene encoding a transmembrane glycoprotein of 185 kDa (p185(HER2)) with intrinsic tyrosine kinase activity. HER2 gives the cells different responsiveness to anti-cancer drugs versus HER2 negative breast cancers cells Estrogen receptor (ER) and progesterone receptor (PgR)

Table 2 Posttranslational activities of GSL: glycosyltransferases in apoptotic breast cancer cells (after 48 h of induction) GSL-GLT Catalyzed reaction Enzymatic activity

GalT-4 Lc3 (GlcNAc-Gal-Glc-Cer) ? Galb-Lc3 Decrease (SKBR-3/cisP, L-PPMP and MDA-468/L-PPMP, 48 h) GalT-5 Lc4) (Gal-GlcNAc-Gal-Glc-Cer) ? Gala-Lc4 Decrease (SKBR-3/cisP and MDA-468/L-PPMP-48 h) SAT-2 GM3 ? GD3 Decrease (MCF-7/cisP,L-PPMP-48 h) SAT-4 GM1 ? GD1a Decrease (MCF-7/L-PPMP, SKBR-3/cisP and MDA-468/L- PPMP-48 h) SAT-4ı´ Gg4 (Gal-GalNAc-Gal-Glc-Cer) ? GM1b Decrease (SKBR-3/cisP and MDA-468/L-PPMP-48 h) FucT-3 LM1 ? SA-LeX Decrease (SKBR-3/L-PPMP-48 h)

Fig. 9 Signiﬁcant gene list with GLT Gene Cell Line Linkage Formed Fold Change 10 % false discovery rate from Name Glyco-microarray of human α breast cancer cell samples ST8SIA4 NeuAc 2-8NeuAc-R1 1.39 treated with 2 micromolar GlcNAcβ1-3 GCNT2 GlcNAcβ1-3nLc4 nLc4 1.39 L-PPMP for 2 h GlcNAcβ1-6 MCF-7 B3GALT2 Lc3 Galβ1-3Lc3 1.36 B3GALNT2 Gb3 GalNAcβ1-3Gb3 1.31 B3GALT5 Lc3 Galβ1-3Lc3 1.16 UGT8 Galβ1-1Cer -1.14 GlcNAcβ1-3 MDA-468 GCNT2 GlcNAcβ1-3nLc4 nLc4 -1.11 GlcNAcβ1-6

Galβ1-3 SKBR-3 GCNT1 Galβ1-3GalNAcα-R2 GalNAcα1-R2 -1.19 GlcNAcβ1-6

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Table 3 Signiﬁcance analysis of microarrays (SAM) [89] Gene Name MCF-7 MCF-7 Symbol Gene name 2h 24 h

NM_000188 1.20 0.67 HK1 Hexokinase 1 NM_000194 1.19 0.60 HPRT1 Hypoxanthine phosphoribosyltransferase 1 (Lesch-Nyhan Syndrome) NM_001069 1.45 0.38 TUBB2A Tubulin, beta2a NM_002629 1.11 1.20 PGAM1 Phosphoglycerate mutase 1 (brain) NM_005573 1.40 0.46 LMNB1 Lamin B1 NM_033170 1.19 1.16 B3GALT5 UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 5 (B3GALT5), transcript vai NM_170707 1.35 0.68 LMNA Lamin A/C

SKBR-3 SKBR-3 2h 24 h

NM_152932 1.35 0.87 GLT8D1 Glycosyltransferase 8 domain containing 1

MDA-468 MDA-468 2h 24 h

NM_006082 0.78 0.77 TUBA6 Tubulin, Alpha, Ubiquitous Results of Glyco microarray data of human breast cancer cell samples treated with 2 micromolar L-PPMP for 2 and 24 h as indicated

LM1 alpha1, 4fucosyltransferase) [33]. A similar effect was apoptotic carcinoma cells was supported by grants from NIH: NS- observed with the GLTs involved in the biosyntheses of Gg- 18005 (Jacob Javits Award) and from NCI: CA-14764. series gangliosides, such as SAT-4 (CMP-NeuAc: GgO- se4Cer alpha2, 3sialyltransferase), and SAT-2 (CMP-Neu- Ac: GM3 alpha2, 8sialyltransferase) (Fig. 3)[75, 78–80]. References

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