Indian Journal of Experimental Biology Vol. 37, August 1999, pp. 737-749

Review Article

Gangliosides : Potential diagnostic as well as therapeutic target for cancer

Sandip Saha

Department of Metabolic Regulation, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road. Calcutta 700 026, India

A large number of studies have been performed and are still being carried out to understand the basis and genetic control mechanism of cancer cells. Unfortunately, knowledge derived from such studies is still fragmentary and is far from allowing us any significant practical application. However, essentially all cancer cells are characterized by aberration in their cell surface glycosylations which are reflected in aberrant functional activity of the cancer cells. Aberrant glycosylation per se may be the basis of inappropriate cell-cell and cell-matrix interactions, which are reflected in the abnormal behaviour of tumor cells, such as uncontrolled cell growth, invasiveness and metastatic potential.Such aberrant structures on the cancer cell surface may be effective targets for prevention, diagnosis and treatment of cancers. With the invention of monoclonal antibody (MAb) technique, analysis of such structures on the cell surface has become realistic. Perhaps the earliest chemical evidence suggesting the occurrence of aberrant glycosylation in human cancer was the changes detected in A, B blood group antigens. A large quantity of several fucose-containing glycosphingolipids were found to be accumulated in various types of human cancers, identified as Lewisa

(Lea), Lewisb (Leb), and Lewisx (LeX) antigens, related

to blood group antigens ABH. Later with the application of MAb technique several other sialic acid containing Lewis antigens, sialyl- Le", sialyl-Leb

and sialyl-LeX were detected on human cancer cell surface. When glycosylation pattern of normal and oncogenically transformed cells were compared ill vitro with the application of cell culture and oncogenic transformation of cultured cells ill vitro by DNA and RNA viruses, transformation-dependent changes of gangliosides and other glycolipids on transformed cell surface were clearly demonstrated by many investigators. In many experimental animal tumor model s neutral glycosphingolipids as well as

acidic glycosphingolipids like gangliosides have been identified as tumor-specific and/or tumor-associated antigens, which are immunogenic in tumor host. Moreover, tumor-gangliosides play important role in regulation of humoral as well as cellular immune factors of tumor hosts. So, these antigens, spec iall y gangliosides may be potential targets for immunological manipulation to get therapeut ic benefit to tumor-host.

Biomolecular characteristics and metabolism of gangliosides

Gangliosides comprise a family of acidic glycolipids, characterised by the presence of sia li c acid (SA:N-acetyl neuraminic acid : NeuAc : NANA) and are present in the plasma membrane of most vertebrate cells . They are m'ost abundant in brain ganglion tissues, and are found in other ti ssues as well'. Since they were first iso lated from brain ganglion tissues, they got the name gangliosides. They are located asymmetrically in the outer membrane of cells with their negat ively charged hydrophilic oligosaccharide chains oriented towards the extracellular environment and is assumed to play an important role in recognition phenomena, while the hydrophobic lipid part is inserted into the membrane and is suggested to participate in the process of signal transduction2

.J. The oligosaccharide part of gangliosides is made up of sialic acid , hexoses, and N-acetylated hexosamines . The hydrophobic part, called ceramide is made up of a long chain fatty acid linked through an amide bond to an amino alcohol sphingosine. The oligosaccharides are linked through a glycosidic bond to C I of the sphingosine. The structure of one such common ganglioside called GM, is shown on nex t page.

The important physiological role of gangliosides was suggested as involvement in the synapti c

738 INDIAN J EXP SIOL, AUGUST 1999

N-acetyl-galactosamine Galactose Glucose Sphingosine

rp-CH20HO 0

, O-CH, o OH I

HCNH

I OH HCOH

Galactose

Hvf-. CH:lOHOO~ 0

~ OH

Sialic acici

tranSmISSIOn of nerve impulses, although the exact mechanism of action is still not clear. Very small amount of gangliosides is also present in intracellular structure, possibly in vesicles involved in transport of gangliosides from the site of biosynthesis and degradation to and from the plasma membrane of the cell. Tissues and individual cell types, possess a well defined ganglioside pattern, represented by a mixture of different molecular species at fixed ratios. The pattern is constant for a given animal and an established functional status of the cell, suggesting that the biosynthesis of gangliosides in the cell follows a well defined established paradigm, a.nd is under a strict control. An overall picture of ganglioside metabolism in a cell is presented in Fig. I (ref. 4), which consists of de novo biosynthesis, biosynthesi s by recycling of catabolic products, biosynthesi s by direct glycosylation and lysosomal degradation.

The de novo biosynthesis of gangliosides begins with the formation of ceramide, catalysed by a fatty acyl transferase in the presence of fatty acyl-CoA and a long chain base, sphingosine5. The different saccharide units are sequentially added to the ceramide by the action of glycosyltransferases in the presence of the corresponding donar sugar­nucleotides . The sequence of glycosylation follows the order which is dictated by the substrate specificity of the involved enzymes6

. Recruitment of substrates occurs in the cytosol, with the only exception of CMP-sialic acid, which is mainly produced in the nucleus7. Formation of ceramide and glucosyl­ceramide probably takes place in the endoplasmic reticulum (or cis-Golgi), while further glycosylations in the Golgi (or trans-Golgi) apparatus8

. Lactosyl­ceramide is the common precursor for all the

Fatty acid

< .. ...... Ceramide ....... ;>

gangliosides of the ganglio-series. Each ganglioside is biosynthesized by a separate multi -glycosy l­transferase system9 located in the Golgi apparatus membrane and orienting its catalytic sites on the luminal side 10. The final products of gangli os ide biosynthesi s leave the Golgi apparatus and reach the plasma membrane, possibly through a vesic le­mediated process l l

. The sequential biosynthesis of ganglio-series gangliosides occurs mainly through two pathways, namely , Basu-Kaufman-Roseman pathway' 2- '5 and Svennerholm-Holmgren pathway '6.'7

(Fig. 2t There is another asialo-pathway called Handa-Burton pathway IS, starting wi th conversion of Lac-Cer to Gg3(as ia lo-GM2) followed by the formation of GM2 (Fig. 2) . Gg3 could also be converted to Gg4(asialo-GM I) and GM I b, a lthough thi s route has not been demonstrated in normal human tissue. GM I or asialo-GM I can be converted to fucosyl-GM 1 or fucosyl-asialo-GM I as a mlllor pathway in normal ti ssue. So the main neutral glycolipid backbone structures of the gangli o-series gangliosides are as follows!.'9 .

Galp 1-+4Glc P 1-+ 1 Cerami de GalNAcp 1-+4Galp 1-+4Glc P 1-+ 1 Ccramidc Galp 1-+-3GaINAcp 1-+4Galp 1--+4Glc P 1-+ 1 Ccramidc Fucal-+2Galp 1-+3Ga1NAcp 1-+4Galp 1-+4Glc p 1-+ 1 CcrallliLic

To these backbone one or more sia lic acid residues are linked mainly to galactose to form mono-, di-, tri-, or higher sialic acid containing gangli os ides as shown in Fig. 2.

Aberrant glycosylation in human ca ncer was initially suggested by the discovery of an accumulation of unusual glycosphingolipids in a large variety of human cancers, which chemically and immunochemically were identified as Lewisa (Lea),

SAHA: GANGLIOSIDES: POTENTIAL DIAGNOSTIC & THERAPEUTIC TARGET 739

Glycoproteins Gangliosides

Exogenous GangliOside

Man NAc

Gte

Fatty Acid Serine LYSOSOME

Fig. I-Schematic representation of the main processes of ganglioside metabolism: de /I0VO biosynthesis , biosynthesis by recycling of catabolic byproducts, biosynthesis by direct glycosylation, lysosomal degradation.

Lewisb(Leb) and LewisX(LeX), analogs of blood group antigens ABH, having the following structures20.21 :

Gal~ I ~3GlcNAc~ I ~3Gal~ I ~4Glc~ I ~ I Cerami de (Lea) 4 i laFuc

Gal~ I ~3GIcNAc~ I ~3Gal~ I ~4GIc~ I ~ I Ceramide (Leh)

2 4 i i laFuc laFuc Gal~ I ~4GlcNAc~ I ~3Gal~ I ~-4G\c~ I ~ I Cerami de (leX)

3 i laFuc

A decade later when the monoclonal antibody (MAb) approach was ihtroduced in analysis of human tumor antigens it became clear that many of the MAbs selected on the basis of preferential reactivity with tumor cells over normal cells were identified as being directed to other analogs of Lewis antigens, such as sialyl-Lea

, sialyl-Leb and sialyl-Lex. Sialyl­

Lewis antigens have been suggested to play an important role in adhesion of cancer cells to endothelium and homing of metastatic cancer cells22

.

Increased expression of sialyl-Lea and sialyl-LeX

antigens have been observed in liver metastasis of human colorectal carcinoma23 . These antigens are also expressed in primary tumor and metastatic lymph

d . h . N no es In uman gastrIc cancers .

Oncofetal pathway of gangliosides The major ganglio-series antigens accumulating in

human cancer and defined by MAbs which are present only in trace amounts in normal ti ssues, are the precursors in synthesis of ganglio-series gangliosides, e.g., G03 accumulated in melanoma and defined by various MAbs25-27, G02 in melanoma and neuroblastoma28.29 and GM1 in melanoma30.3I. Accumulation of GM3 in various types of melanomas was confirmed by an antibody directed to GM/ 2

Gg3(asialo-GM2), which accumulates in mouse sarcoma KIMSV and lymphoma L517833

.34

, is also present in human Hodgkin"s lymphoma' 5. Presence of asialo-GM2(Gg3) in various but limited types of human cancer could be due to blocked asialo-core synthesis, initiated by the Handa-Burton pathway. Various murine and human hybridomas have been established which secrete antibodies showing preferential reactivity with human me lanoma and neuroblastoma; many of these antibodies were found to be directed to GM/0.3I ) 6 and G02 ganglio­sides28.29.37. Accumulation of G03 and GO] in

association with deletion of higher gangliosides suggests a blocking of the Basu-Kaufman-Roseman pathway. However, recent studies indicate that synthesis of precursor gangliosides such as GO) and G02 is also greatly enhanced3s. It is therefore assumed that accumulation of precursor gangliosides results from both blocked synthesis of higher

740 INDIAN J EXP SIOL, AUGUST 1999

gangliosides and enhanced synthesis of the precursors. It is not yet known whether the asialo­ganglio pathway initiated by the Handa-Burton synthesis route is enhanced in certain human cancers. Gg4(asialo-GM 1) was claimed to be a marker of human lymphoblastic leukemia39

. A MAb prepared against small cell lung carcinoma was found to be directed to fucosyl-GM 140. Fuentes et . af. reported significant level of GDJ and 9-0-acetyl -GDJ

41 M' expression on small cell lung cancers .ouse lymphoma L5178 and its variant cell lines showing different metastatic potential contain GM 1b and its new analog GD 1a 42. Rat ascites hepatoma also contains GD 1a

43• The presence of GD 1a in human

cancer remains to be studied .

Functional changes as related to ganglioside changes in cancer

The concept that membrane gangliosides may regulate cell growth has developed slowly during the last two decades, based initially on changes observed in ganglioside synthesis associated with oncogenic transformation, cell cycle and contact inhibition of cell growth. An aberrant profi le of cell surface glycolipids, which occurs as a consequence of blocked synthesis or neosynthes is, may cause a failure of cellular interaction through a carbohydrate­recognizing protein (cognin) at the surface of counter­part cells, although cognin is unchanged on transformation. The cognin cou ld be a glycosy!

44 4~ . I I . 46 0 transferase , hydrolase - or al1lma ect1l1 . ne

Basu-Kaufman-Roseman Pathway

Svennerholm-Holmgren Pathway

NeuAc Tranferase V

NeuAc Transferase IV

Gal Transferase

GalNAc Transferase

NeuAc Transferase I

r GTla

~l GDla

~r GMt

t ~l

~f GM2

~ GM3

, NeuAc A

~t Transferase II

~ OGle

f GQlb 'f GPlc

t GTlb ~ GQlc

t GDlb

~ GTlc

~ GD2 ~ GT2

i GD3 , GT3

)

A NeuAc Transferase III

~i Cor.mido

o _G~L e GalNAc • NeuAc

Fig. 2-Lines of de /l OVO biosynthesis of gangli osides as the resul t of substrate specificity of the in volved sialyl-transferase

SAHA: GANGLIOSIDES: POTENTIAL DIAGNOSTIC & THERAPEUTIC TARGET 741

phase of cell recognItIOn involves cell attachment through adhesion matrix47 , adhesion plaque48, or detergent insoluble matrix49. Although gangliosides are not considered as functional components of adhesion plaque, thay could play important role in cell adhesion since gangliosides are present in substrate attachment matrix as well as in detergent­insoluble matrixS0.51 and gangliosides can inhibit cell

adhesion on fibronectin-coated substrate52 as well as h b 'f' II 45 H on ot er su strates nonspec l Ica y. owever,

certain gangliosides such as GM 3, which is ubiquitously di stributed in a large variety of cells and which is deleted or reduced greatly on transformation , may have a basic function in regulation of cell growth. Recent studies on cell growth regulation in

chemically defined medium revealed that GM3 may regulate the function of the receptor for fibroblast

growth factor5' . Similarly, GM I in other types of cells

may regulate receptor function for platele t-de rived growth factor and perhaps other growth factor receptors as we1l 54. Involvement of gangliosides in two basic cellular functions: i) ce ll adhesion and recognition and ii) regulation and maintenance of growth factor receptor function suggest that defect ive synthesis of these gangliosides may cause loss of growth control and anchorage-dependent cell proliferation, which are the most common denominators of oncogenesis. Gangliosides have been shown to inhibit POGF-induced signal transduction events in human glioma cellsss . Many of the growth factor receptors, e .g., epidermal growth factor, insulin, platele t-der ived growth factor, insulin-like growth factor and colony stimulating factor-I have been shown to be protein tyrosine kinase, a key enzyme that transduces their prolife ration signal via tyrosine phosphorylationS6-s8

. Numerolls studies have

been focused on the role of genes coding for prote in kinases in controlling protein tyros ine phosphorylation . Recently , considerable attention has been given to prote in tyros ine phosphatase enzymes (PTPs) that may oppose the action of protein tyrosine kinases, by dephosphorylating tyrosyl residues in response to changes in cellular milieu . Thus, genes for PTPs may play the role of cancer suppressor genes or antioncogenes by regulating the level and patterns of prote in tyrosine phosphorylation and, consequently cell proliferation. Loss of function of these genes may then result in deregulated cell growth. In fact, PTPy gene at 3p21 has been shown to be a candidate suppressor gene and loss of this gene

has correlation with development of human lung and kidney cancers59

. Now, gangliosides have been shown to regulate activity of many of these prote in tyros ine kinases which are in many cases growth factor

56-58 S I f I" d . I receptors . 0, ro e 0 gang IOSI es III t 1e interaction between protein tyros ine kinases and PTPs III relation to antioncogenes deserves further

explorations. . GangJiosides have amphipathic property, that is

they are capable of being incorporated into cellul ar membrane when exogenous ly added, thu s, the ce lls di splay new antigenic property and growth be hav iour. This approach has been ex tensive ly used to ide ntify the function of specific gangliosides . Enri ch ment of certain gangliosides found abundantl y III

differentiated cells induces te rminal diffe renti at ion.

For example, HL60 cell s, incubated with GM, a monosi aloganglioside, differentiate into monocytes/

macrophages and the cell surface contained hi gh GM3 whereas the same cells, incubated with s ial opara­globoside (S PG), diffe rentiate into granul ocytes which had high SPG. Similarly , phenotype of

neuronal cells can be induced by gang li os ide GQ lb, a tetrasialogangl ioside . Thus enri chment of spec i fie gangliosides triggers and defines the direction o f terminal diffe rentiation and enhanced ga ngli oside

levels in membrane are the result as we ll as the cause of di fferentiation6061 .

Moreove r, the inducti on and maintenance of malignant transformati on61

-65 and exp ress ion o f

f· .. 66.f,7 . . . (,RJ,')

trans ormlng genes III vit ro , tumorI ge nI c It y d . . 1ft' d II 70-7' I an metastatIc potentIa 0 tran s orme ce s . 1a ve

been observed to have close re lati on with the change in composition and concentration of gangli os ides of transformed cells. Oncogenes, which tri gge r a series of cascade reactions leading to ex press ion of a number of tumor-assoc iated phenotypes , were found originally in retroviruses, subsequently in norma l cells, and are activated and amplified in some human cancers. A poss ible corre latio n between oncogene activation and aberrant sy nthes is of g lyco lipids had been found previously , e.g., GM, synthesis as re lated

. . 67 r"' h · I d to src activatIon , ug, synt eSls as re ate to ras transfection in NIH 3T3 cells from human tumors 74,

and GO, and other ganglioside_chilnges as re lated to . 7'; .. .

adenOVIrus oncogenes · . In cells transf·onned by retroviral infection, oncogene activation is we ll correlated with aberrant glycosylation 67

. Aberrant glycosylation was detectable in essentially all human cancers by various MAb probes in combination . In

742 INDIAN J EXP BIOL, AUGUST 1999

contrast, the estimated incidence of activated oncogenes in human cancers detected by previously known oncogene probes was approximately 20-30%76-78.

Ganglioside changes in cell surface have been suggested to have role in defining tumorigenicity and metastatic property. The low tumorigenic hybrids resulting from a fu sion between high- and low­tumorigenic cells in which tumorigenicity was suppressed68 showed a higher content of GM1 and long-chain neutral glycolipids but low levels of GD or GT gangliosides69

. The high-tumorigenic hybrids res~lting from a fusion between two high-tumorigenic varIants showed, in general , a low GM1 content and a hi gh level of GD or GT gangliosides6

.i. Interestingly ,

a lo~ GM3 content was al so correlated with a highly malIgnant "free form" ascites hepatoma, whereas a high GM3 content was found in the less malignant adhesive form ("island form") hepatoma79

. The tumorigenicity of NIL cell variants has been correlated with the absence of cell density-dependent response of GM.1 synthesis with, however, a few

. 62 exceptIOn . Yogeeswaran and his associates7o•71

claimed that metastatic property of B 16 melanoma and 3T3 sarcoma cells induced by KiMuSV showed a correlation with ganglioside composition and organi zation . The highly metastatic melanoma showed a loss of GM3 crypticity and an enhanced level of GT or GD gangliosides70

, and a highly metastatic KiMuSV -induced sarcoma showed a high level of and greater exposure of Gg3 and high level of GT or GD ganglioside71

. Thus, with the exception of malignant hepatoma, the degree of sialylation in gangliosides and in glycoproteins were claimed to be closely related to the degrees of metastatic property72. Thomas et ai.reported that surface expression of GD, in human melanoma was correlated to both metastati~ potential in vivo and radiosensitivity in vitro71

.

Diagnostic implications of ganglioside changes associated with cancer

Whatever the function of ganglios ide changes in tumor cells may be, they can be applied in the diagnosis of human cancers. A sensitive radio­immunoassay was introduced by Koscielak and hi s associates80 using rad iolabeled lactosyl sphingosine­BSA conjugate. The method was designed to determine the sel1lm antibody level directed to lac losyl -sphingosine, and the level was claimed to be

well correlated with development of cancer. This sensitive technique can also be used in case of gangliosides. Enhanced levels of 2---3sialyl-Lewis" antigen in sera of patients with various types of gastrointestinal cancers were observed by inhibition of MAb-N-J9-9 binding to colonic tumor ce ll sRI and b " d ' h" 81 Y san WIC assay -. Elevated leve l of thi s anti cren b

was observed in sera of patients with vari ous types of gastrointestinal tumors83

. Of particular interest is the high incidence of thi s antigen elevat ion leve l in the sera of patients with pancreati c cancer, which is otherwise difficult to diagnose in its earl y stages84

.85

.

However this antigen is absent in sera of Le(a-b-) patients . Another antigen with a similar stl1lcture sialyJ-Lewisx, difined by antibody CSLEX- I ~6 wa~ also found to be elevated in sera of several types of human cancers (including lung and breast) and showed a pattern of high elevation complementary to that of sialyl-Lewi s". Sialyl-Lewisx antigen has an advantage that it is expressed in tu mors in Le(a-b-) patients83

. The antigen with a si milar epitope, defined by the antibody FH6, having a long chain sialyl lactosamine with internal fucosy lation (s ialyl-LeX-1 or sialyl dimeric LeX) was al so found to be elevated in the sera of patients with vari ous types of cancerX7

The antibody showed a high incidence of pos iti vity in sera of patients with lung adenocarcinoma (86%) and pancreatic cancer (85 %), but relati vely low positi ve incidence in other human cancers (55%). Nevertheless, the antibody may have di agnostic va lue since it rarely gave fal se pos iti ve resultsRx

. A hi crh incidence of positi ve cases was found with ano th~r antibody, CA50, which defines the 2---3s ialyl type 1 chain . The antibody was able to detec t monosialoganglioside in 60-90% of co lorectal or pancreatic adenocarcinomas or their metastases, but was essentiall y unreacti ve with normal co lonic mucosa89

. The antibody CA50 was able to detect enhanced level of antigens in sera of patient s with various types of cancers, particularl y colorectal cancer (50-75 %), uterine cancer (75 %) and prostate cancer (90%)90. A MAb FH-7 was establi shed which was capable of detecting a new sialylatecl type I chain , disialyl Lea antigen(2---3, 2---6 sial yl Lea) in the sera of pancreatic, colonic and bladder cancers')I . An antibody directed to gastric cance r, NCC-ST-439, establ ished by Hi rohashi et al.,)l, was capable of detecting high levels of a sia lylated Le type anti gen in the sera of patients with pancreati c. co lorectal and b t

. 97 I . reas carcll10ma -. n pati ents wih co lorec tal

SAHA: GANGLIOSIDES: POTENTIAL DIAGNOSTIC & THERAPEUTIC TARGET 743

carcinoma, the antigen levels decreased sharply after surgical intervention93

.

We observed in our investigation with Ehrlich Ascites carcinoma model that gangliosides in the plasma of Ehrlich ascites tumor-bearing mice gradually increased with progress of tumor growth, thus suggesting diagnostic significance of gangliosides94

. We also observed that gangliosides obtained from tumor-plasma enhanced tumor growth when adoptively transferred with tumor cell s to normal mice94

. However, individual gangliosides acted differently. Out of six gangliosides separated from tumor-plasma two components, each enhanced tumor growth, whereas, the remaining four components, each inhibited tumor growth. Neither of the two enhancing components in various combinations with the four inhibitory components could restore the enhancing effect. The enhancing effect was restored only when both the enhancing components were mixed with the remaining four ganglioside components. The two enhancing gangliosides were found to be of tumor cell origin, one a trisialoganglioside and the other a disialoganglioside95

. Moreover, we observed that tumor growth enhancing or inhibitory effect of tumor growth modifiers like vitamin A and vitamin C were reflected in the plasma gangliosides profile. Three specific plasma gangliosides changed in concentration and showed some definite correlation with tumor growth modifying effect of vitamin A and vitamjn C. Two of them were of tumor cell origin, as mentioned above and the third one of host origin96

.

All these information suggested that proper manipulation , modulation or elimination of these tumor enhancing and tumor inhibitory gangliosides of plasma may lead to therapeutic benefit to tumor­bearing host.

More recently , ganglioside was used for tumor imaging. Localisation of human osteosarcoma grown in nude mice was targeted by whole or F(abh fragments of anti-G02 ganglioside MAb 3F8. The targeting was highly efficient and specific for the osteosarcoma, but not for Ewing'S tumor, which did not express G02 antigen97

• Interestingly, the same

antigen G02 has been found in human neuroblastoma, melanoma, small cell lung carcinoma and certain brain tumors. The antibody 3F8, defining G02

antigen, is therefore the common imaging reagent for

these tumor types . Expression of G02 ganglioside in normal tissues is highly restricted in humans ; in mice,

it is found only in the thymus. The anti-G02 MAb 3F8 has also been used for imaging of small-ce ll lung cancers in human patients98

. Another study in vo lved • • • <)1)

GM3 ganglIosIde In melanoma as a target . There are not many reports on the diagnostic as

well as prognostic implication of gangli os ides. However, a good number of studi es indicating correlation between plasma leve ls of s ial yl-Lewis antigens, structural analogues of gangli os ides and various types of human cancers and the corre lati on of plasma gangliosides with progress of Ehrlich ascites tumor growth observed in our stud ies suggest that potentiality of gangliosides as diagnosti c as well as prognostic markers deserves further ex plorati on. Thi s is supported by our recent studies with human cancer patients. We have observed that plasma leve ls of gangliosides are e levated in va ri ous types of human cancers by 4 to 6 times the normal va lue . These elevated levels come down to almost normal levels after surgical removal of the primary tumors , indicating that plasma gangli os ide leve ls may possibly be used as an indicator of response of the cancer patients to treatment modalities (unpubli shed data).

Escape of tumor cells from immune-destruction of host as related to gangliosides

It is now a well established fact that when a tumor develops in a host, it is recogni sed as foreign by the immune machinery of the host due to presence of tumor- associated and/or tumor-spec ifi c antigens on its surface. The recognition is expressed by the generation of anti-tumor antibody' oo, anti-tumor

100 101 h 102. 103 d effector lymphocytes . , macrop ages an other cellular as well as humoral factors 10.1, which are supposed to kill the tumor. But it does not happen in reality. Tumors some how escape the immune destruction by the host and continue to grow. Although the humora l factors, i.e., antibody produced against the tumor and the cellular factors , i.e., effector cytotoxic lymphocytes, macrophages and other cellular factors produced against the tumor can kill the tumor cells under in vitro conditi on, but fail to do so in ViVOI05 .106 . Tumors induce both humoral loo.lo7.108

d II I 109 110 . . h' an ce u ar' ImmunosuppreSS Ive mec an Isms which impose restriction on the functional acti vity of anti-tumor immune components of the host. Among many theories the most recent one sugges ts that tumor cells continuously shed surface anti gens in the ­circulation of the host, which well in advance react

744 INDIAN J EXP BIOL, AUGUST 1999

with the humoral as well as cellular immune components of the host in the circulation, generated against the tumor and thus precipitate the anti-tumor immune factors of the host, before they can reach the t 108 III I .. II I h umor mass ' . mtla y t lese s ed surface antigens of the tumor cells were suggested to be protein or glycoprotein in nature"2

,((3. But recent studies have characterized these antigens in many tumors as glycolipid or rather as ganglioside in naturel14-1 18 . In fact, gangliosides shed by tumor cells in the circulation have been implicated as suppressor of host immune responses . Because, tumor-derived gangliosides and gangliosides shed by tumor cells have been observed to inhibit generation of antibody producing cells in vitro l14

, inhibit functional activity of effector cytotoxic T-lymphocytes" 5

."6,18, macro­

phages and NK-cells in vitro I 17,1 19,120. There are reports in which activation of lymphocytes by monoclonal antibodies to GO]121,122, mediation of AOCC reaction and complement dependent cytotoxicity of tumor cells by anti-G02 and anti .. GO) antibody in vitro i23

,124, lysis of human tumor cells of neuroectodermal origin by monoclonal anti-G02 and anti-GO) in vitro l25 and killing of neuroblastoma cells

. by neutrophils obtained from G-CSF-treated cancer patients and targeted by monoclonal anti-G02 anti Fc gamma RI antibody in vitro 126 have been demonstrated. Even immunization of mice with irradiated EL-4 tumor cells containing G02 as surface an tigens has been shown to produce specific cytotoxic T-Iymphocytes which protected the host from EL-4 lymphoma and the CTL response was observed to be specific for G02

127. This later report

indicated that immunization with ganglioside may produce not only tumor-specific anti-ganglioside antibody but also produce tumor-specific cellular factors in the host to counteract the tumor growth.

Therapeutic significance of gangliosides associated with cancer

Immunotherapy of human cancer is possible if the right antibodies directed to the right markers are chosen. Although GM2, G02 and GO) are present in normal brain and other tissues in minute quantities, they are overexpressed in tumors of neuroectodermal ongln, particularly In melanomas, sarcomas, neuroblastomas and astrocytomas and these gangliosides have bee;-J shown to be immunogenic in

,26,27 II ' h 28 11 . mIce as we as 111 umans ,- , thus suggestll1g thal acti ve speci fic immunotherapy with tumor

gangliosides may lead to therapeuic effect in host bearing animal tumors as well as in human cancer patients.

Active immunization with tumor antigens to suppress tumor growth has had little success in the past. However, since tumor spec ifi c andlor tumor­associated antigens have been weI! establi shed as glycolipid or as sialic acid containing glycolipids, the approach should be re-evaluated using these purified antigens. Wallack and associates developed melanoma vaccines by infecting human melanoma cells grown in vitro with Vacc inia vi rus . The virus shed in the supernatant was pelleted, and SOllg protein aliquots of the pellet ("Vacc inia melanoma oncolysate", VMO) were injected intradermall y in melanoma patients. The patients were divided into two groups, showing either IgM or IgG response, and the incidence of recurrence of the melanoma was correlated with the titre of an tibodies directed to gangliosides. The higher the antioanolioside loG b b b

antibody titre in the sera of patients, the lower the . 'd 1 28 , 12~- . II1CI ence of recurrence . Therel'ore, the essential epitope of YMO vaccine was suggested to be gangliosides. Morton et al. immunized 149 patients with stage II melanoma after surgery. Patients were divided into three groups: group I received onl y surgery: group 2 received surgery and adjuvant BCG; group 3 received surgery and adju vant BCG with melanoma tumor-cell vaccine containing GD], GMz and G02 . A significant number of group 3 (but not group I or 2) patients developed antibodies against GD2(1gM, two patients), GM2(IgM, ten patients) and GM2(IgG, two patients)' 30. Since gangliosides GM2,

G02 and GO) represent potential targets for immunotherapy of melanoma, a systematic study was carried out by Livingston et al. 131, WI ich attempted to elicit immune response against gangliosides . BCG coated with GM2 elicited anti-GM2 antibody response in patients with melanoma, part icularly those pretreated with cyclophosphamide. Anti-GM2

antibodies in vaccinated patients were of the IgM class, and were cytotoxic for melanoma cell s expressing GM2 in the presence of human complement132

. In a follow-up study, tumor size was reduced and length of survival was increased in patients showing anti-GM2 antibody response to vaccination, as compared with patient s who did not show anti-GM2 response iD

. The cOl11bina~ion of GM2

-KLH plus QS-I and GM2/BCG vaccine .c.:onsi stently

SAHA: GANGLIOSIDES: POTENTIAL DIAGNOSTIC & THERAPEUTIC TARGET 745 ; !

produced high titre IgM antidodies to this ganglioside , in most melanoma patients with associated improved I d' . f d' . d . 1112114115 S' '1 I Isease- ree con Ihon an survlva ... , '. Iml ar y,

GD3, another ganglioside, predominantly expressed on human melanoma cell surface has been used in its lactone form for specific active immunotherapy with success J36

. Since GD, is poorly immunogenic in mice and human, in one study several liposome formulations of GD, were used to increase the immunogenicity of GD, during active immunization 137. In a recent study with Ehrlich carcinoma tumor model we have shown that direct immunization with a trisialoganglioside of tumor cell origin can inhibit Ehrlich ascites as well as subcutaneous solid tumor growth significantly and the antibody produced in the sera was found to be of IgM type 138.

The IgG I muose MAb R24, defining the GD3 ganglioside that is highly expressed on human melanoma cell surfaces, activated human effector cell functions, causing lysis of melanoma cells through complement-antibody mediated cytotoxici ty l39.

Another IgG3 MAb directed to GD3, B3. 6, showed a clear antibody-mediated melanoma cell lysis27. Subsequently, a phase I clinical trial of R24 antibody in patients with malignant melanoma was carried out. Of 12 patients tested, 3 showed clear regression of tumors, and 4 patients showed a partial response l40

.

I 12. He Istrom et at. . reported three mouse monoclonal

IgG3 antibodies directed to GD3 All three antibodies mediated antibody-dependent cytotoxicity in vitro and inhibited outgrowth of human melanoma xenografts in nude mice. Melanomas, regardless of

species, all express high concentrations of GM3. Antihody M2590, claimed to be specific to

melanoma, was found to be directed to GM3. The

IgG3 version of anti -GM3 antibody (DH2), originally

directed to GM3 lactone, was growth inhibitory to mouse and human melanoma in vitro, displayed antibody-dependent cytotoxicity, and inhibited B 16 melanoma growth in syngeneic mice99

. Thus, the DH2 antibody was similar in function to R24, but displayed a more striking effect on melanoma growth in syngeneic mice. The importance of complement­mediated cytotoxicity was fully demonstrated by the effcet of human IgM MAb L 72, which was directed to GD2 ganglioside. Irie and Morton 141 injected thi s antibody into cutaneous metastasis of eight melanoma patients on a daily or weekly basi s. Regression was

seen in all tumors except those of two patient~ whose

tumors were shown to have low GD2 expression. One patient with melanoma satellitosis, treated w!th L 72 showed complete regression of tumor with no sign of recurrence. Furthermore, with the exception of mild erythema, no significant side effects were observed in any patient. Another MAb 14. 18 produced against

GD2 was shown to inhibit human neuroblastoma cell growth in vitro as well as in vivo in nude mice '42.

MAbs against fucosyl-GMI was used 'successfully by Brezieka et al. '43 to inhibit growth of small-cell lung carcinomas in vitro as well as in vivo in nude mi ce .

Moreover, MAbs to GM2/ GD2/GD3 has been used succesfully for passive immunotherapy of melanoma growth and its metastatic spread99.'40. In a phase I trial

murine MAb to GD2 has been used with success for the treatment of pediatric patients with neuroblastoma

and osteosarcomal 44. In another phase I trial anti-GD,

MAb R24 and recombinant human M-CSF combination has been used for the treatment of metastasis of human melanomas '45 . Zhang ef at. used MAb 3F8 against GD2 to eradicate successfully micrometastasis in syngeneic mice l46. Even a human

MAb to GD2 has been applied for the treatment of human melanomas 141. The MAbs used so far for therapy of human cancers were mostly derived from animal sourses, which had some practical problems. So, different approaches to generate human MAbs against tumor-specific and/or tumor-associated gangliosides have been carried out recently' 47.

Anti-idiotype MAb carrying the internal image of

ganglioside GM3 was used effectively by Yamamoto et at. 148 for specific immunotherapy of tumor. Foon el

al. used anti-idiotype antibody to GD1 for clinical trial of 12 melanoma patients with advanced disease. One patient showed complete clinical response and the remaining patients partial responses l49. We have shown that passive immunization with a polyclonal IgM antibody to a trisialogangli os ide of tumor ce ll origin raised in rabbit can inhibit Ehrli ch ascites as well as subcutaneous solid tumor growth in Vl VO

significantlylJ8. We have also shown thal immunization with anti-idiotype antibody to this trisialoganglioside raised in Sprague Dawley rats can inhibit Ehrlich ascites as we ll as subcutaneous solid tumor growth by 60% and 81 % respectively l3H The effect was better than that with direct immunization with the tri sialoganglioside or passive immuni zation with its polyclonal IgM antibody.

746 INDIAN J EXP BIOL, AUGUST 1999

Specific delivery of chemotherapeutic drugs by antibody-drug conjugates directed to glycolipid targets through biotinyl drug, biotinyl antibodies, and avidin was developed by Urdal and Hakomori l5o to kill tumors. Kiura et al. suggested on the basis of their studies on the alteration of ganglioside composition in cislpatin-resistant lung cancer cell line that the alteration of ganglioside composition may be actively involved 111 the acquisition of drug­resistance l 51

.

Conclusion

In conclusion it may be stated that proper manipulation of active and passive immunization with gangliosides of tumor cell origin may increase the tumor killing ability of anti-ganglioside antibodies and ganglioside-specific other immune factors of the host and thus may lead to potential therapeutic effect. Immunization with anti-idiotype antibody may play an important role in those cases where antigen is available in very low quantity or is antigenically very weak, specially complex carbohydrate antigens with weak antigenICity. Since an Jg(anti-idiotype) molecule carrying the internal image of a cancer antigen may produce immune response similar to that of cancer antigen and possibly a better response than the antigen itself, the anti-idiotype antibody may serve as a substitute for cancer antigen which in most cases are not available in quantities sufficient for immunization. As the tumor cell surface gangHosides are weak antigenically and they are not available in quantities sufficient for immunization, the anti­idiotype antibody approach may be more efficient for getting better therapeutic response in their case. Moreover, the reports in which tumor cell surface gangliosides have been successfully used for immunotherapy are mostly with human melanomas and few others like neuroblastomas and osteosarcomas, and there are very few reports on animal tumor models regarding therapeutic application of tumor gangliosides . So, there should be more studies with various types of animal tumor models in this respect to get stronger basis for the therapeutic applicability of tumor gangliosides to other human cancers. Identification of gangliosides with tumor growth inhibitory effect in various other types of human cancers may lead to development of therapeutic modalities for the treatment of those cancers . In addition, delivery of the combination of MAbs to proper gangliosides and specific anti-cancer

drugs to tumor targets may be a crucial approach in control of human cancers in the near future. So far there is no report on the role of gangliosides in the regulation of activity of protein tyros ine kinases and PTPs in relation to cancer. So, thi s area of investigation should be explored to give therapeuti c benefit to cancer patients.

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