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Glycolipid of human pancreatic cancer; the appearance of neolacto-series (type 2 chain) glycolipid and the presence of incompatible blood group antigen in tumor tissues
21
Biochimica et Biophysics Acta, 1125 (1992) 21-27 0 1992 Elsevier Science Publishers B.V. Ail rights reserved 00052760/92/$05.00
BBALIP 53868
Glycolipid of human pancreatic cancer; the appearance of ne~la~t~“seri~s (type 2 chain) glyc~li~id and the presence of incompatible blood group antigen in tumor tissues Hiroshi Hattori a, Kei-ichi Uemura b, Hideki Ishihara a and Hirayuki Ogata ’ a ~u~ra~o~ for CeEI 3~olo~~ Pharma Research Laboratories, Hoechsf Japan Limired, Kawagoe, Suirama {J&an), b department of Lipid B~ochern~st~~Institute of ~ardiouasc~iur Disease, ~hi~shu ~~~~ers~~ School of Medicine, Asahi, Matsumoto &zpanJ and ’ Blood transfusion seruice, Shinshu University Hospital, Asahi, Matsumolo (Japan]
(Received 22 August 1991)
Key words: Tumor associated carbohydrate antigen; ~~~cosphi~goli~id~Pancreatic cancer; Blwd group antigen; Neolacto-series
carbohydrate chain (type 2) Glycolipid isolated from normal and cancerous human pancreatic tissues were characterized chemically and immunologically. The major neutral glycolipids in both normal and cancerous tissues were composed of globo-series glycolipids and lacto-series glycolipids. The mole percentage of fucolipids in the total neutral glycolipids of normal tissues was XI-40%, and in general the fucofipids corresponded to blood group glycolipids related to the patient’s blood group, however, in cancerous tissues the amount of these fucolipids was decreased. fmmunostaining revealed that normal tissues contained only lacto-series (type 1 chain) glycolipids. In contrast, cancerous tissues contained the neolacto-series (type 2 chain) glycolipids as we11 as the lacto-series glycolipids. Incompatible blood group antigens, A active glycolipids in a blood type 0 patient and B active glycolipids in a blood type A patient, were also detectable in the neutral glycolipid fractions of the pancreatic cancer tissues.
Intmduction
Cancer associated aberrant giycosyiation is documented by the study of giycoiipid epitopes, although the same epitopes are also found in giycoprateins. The choice of giycotipids for study may be due to the fact that glycolipids can easily be purified to homogeneity, while the extremely heterogeneous oiigosaccharide chains present in giycoproteins make purification difficult [1,2]. Giycoiipid evaluation has a point of application in the analysis of pancreatic cancer. The incidence of this disease is now increasing steadily, however, its diagnosis is difficult. There are pancreatic cancer associated
carbohydrate antigens which have been characterized and are useful for clinical diagnosis [3]. These antigens have been detected by classical immunochemicai anaiysis as well as by a recentIy developed monodonal antibody [l]. Glycalipids of human normal pancreatic tissues have been previously analyzed, Breimer et al. [4,5] reported blood group active glycolipids of human normal pancreas. Wherret and Hakomori [63 observed the accumulation of B active fucoiipid in pancreas of Fabry’s disease patient. In this paper, chemical and immunoiogi~a~ characterization of glycoiipids from both human normal and malignant pancreatic tissues is described. Materials
TLC, thin layer e~roma~ograph~~GUI, gas liquid chromatography; PBS, phosphate-buffered saline; BSA, bovine serum
Abbreviations:
albumin. The glycolipid designations used, according to the recommendations of the Nomenclature Committee of the International Union of Pure and Applied Chemistry [291, are: Lc,, lactotetraosylceramide; nLc,, lactoneotetraosyiceramide; Lea; II14FucLc,; Leb, IV’Fuc, II14FucLc4; Le”, II13FucnLc,; LeY; IV2Fuc, IIi3FucnLc,. Correspondence: H. Hattori, Laboratory for Cell Biology, Pharma Research Laboratories, Hoechst Japan Limited, l-3-2, Minamidai, Kawagoe, Saitama, Japan 350.
and Methods
Giycolipid preparations were made from four sampies of human normal pancreatic tissue, two samples of human adenocarcinoma and 1 sample of bile duct adenocarcinoma. Age, sex, blood group (A, 13, 0) and pathology of each patient are shown in Table I. The isolation and purification of glycolipids were described in a previous paper [7]. Briefly, lipids were extracted from the tissue with 20 vol. each of ehioroform/ methanol (C/M) (2: 1, 1: 1, v/v) and C/M/water
22
(2 : 1: 0.5, v/v>, and the combined extracts were evaporated to dryness. The total mixture of lipids was applied to a DEAE-Sephadex A-25 column (Pharmacia Fine Chemical, Uppsala, Sweden). The neutral lipids were then eluted with chloroform/ methanol/ water (30:60:8, v/v> and the acidic lipids were eluted with chloroform/ methanol/O.8 M sodium acetate (30 : 60 : 8, v/v). The neutral glycolipids were separated from the neutral lipid fraction according to the method of Saito and Hakomori [S], then further purified by silica gel column chromatography (silica gel 60, Merck, Darmstadt, Germany). The final purification of individual neutral glycolipids was by preparative thin-layer chromatography (silica gel 60 plate, Merck) with chloroform/ methanol/water (65 : 2.5: 4) or (60 : 35 : 8) as the solvent system. Analytical procedures
TLC was carried out on a silica gel 60 plate (Merk) with chloroform/ methanol/ water (65 : 25 : 4 or 60 : 35 : 8) as the solvent system. The carbohydrate composition and content of the individuat glycolipids were determined by GLC analysis of the trimethylsilyl ether derivatives of the methylglycosides obtained after methanolysis at 95°C for 3 h with 3% HCl/methanol. GLC was performed with a glass column (3 mm i.d. X 2 m) packed with 2% OV-1 programmed at 2”C,/min from 140 to 210°C. Enzymatic hydrolysis of carbohydrate linkages in the glycolipids was carried out with cu-galactosidase, a-N-acetylgalactosaminidase, cy-fucosidase and neuraminidase [9].
blocked with 5% bovine serum albumin in phosphatebuffered saline and then incubated for 2 h with antibodies. The TLC plate was reincubated for 1 h with a 500-foid dilution of biotin conjugated anti-mouse immunoglobulin (DAKOPATTS). After washing, a peroxidase conjugated avidin-biotin complex solution (VECTASTAIN; Vector, CA) was added to the plate and allowed to react for 1 h at room temperature. After washing, for the final step, the chromatogram was incubated for 10-30 min with a substrate solution (KONICA IMMUNOSTAIN ’ HRP, Konica, Tokyo, Japan). Results GlycoZipid profile of normat and cancerous Mayan pancreatic tissues
The TLC separation of the neutral glycolipid fraction from normal human pancreatic tissues, pancreatic cancer tissues and bile duct cancer tissue is shown in Fig. I. Normal pancreatic tissues (samples l-4) contained glycolipids having a more complex carbohydrate structure as compared to the cancerous tissues. Quantitative and qualitative estimations of the neutral glycolipids from the various clinical samples are presented in Table II. The major neutral glycolipids found in normal pancreas tissues were glucosyIceramide, galactosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, lacto(neo)tetraosylceramide and fucolipids. Total content of neutral glycolipids from normal and cancerous tissues was 0.868-2.837 Fmol/g protein tissue and no differences were ob-
An t~bodies and lectins
Rabbit anti-Forssman antibody was obtained a previously described [lo]. Mouse monoclonal anti-A, anti-B and anti-H antibodies were purchased from Dakopatt (Glostrap, Denmark) and mouse monoclonal anti-Lea and L,eh antibodies were from Chem Biomed (Edmonton, Canada). UIex europaeus aggl~tinin I (UEA-I) and Grifforia simpficifolia II (GS-II) were purchased from E. Y. Laboratories (San Mateo, CA). Mouse monoclonal anti-LeX antibody (SW11 and anti-LeY antibody (AH6) were donated by Dr. Sen-itiroh Hakomori (Washington Universi~, Seattle, WA). Rabbit anti-nLc, was obtained by a published procedure [ll] and purified by affinity column. Peroxidase-conjugated UEA-I was purchased from E. Y. Laboratories.
The immunostaining procedure employed was a modification of the method described by Uemura et al. [12]. Approx. 50 pg of the total neutral glycolipid fraction was applied to a high performance aluminum sheet silica gei 60 TLC plate (Merck) and the plate was developed with a mixture of chlorofo~/methanol/ water (60: 35 : 8, v/v). Subsequently, the plate was
a b
C
d
01234567F 1.TLC
of the neutral glycolipids from normal and cancerous Fig. human pancreatic tissues. Lane 0, erythrocytes of OJx(a - b+ ); Lane 1-4, normal pancreas; Lane 5 and 6, pancreatic cancer; Lane 7, bile duct cancer; Lane F, Forssman glycolipid. a, Glc-Cer; b, Lac-Cer; c, GbOse,Cer; d, GbOse,Cer. Developing solvent, chloroform/ methano~/water (65 : 25: 4); ~sualization, cupric phosphoric acid reagent.
23 TABLE
1
Pancreas tissues examined in this study Sample no. (TLC No.)
Age
I 2 3 4 5 6 7
60 39 61 58 63 71 58
TABLE
Sex
Blood
Histology
group Male Male Female Female Male Male Female
B B B B A 0 B
Normal pancreas Normal pancreas Normal pancreas Normal pancreas Pancreatic cancer (ductal Pancreatic cancer (ductal Bile duct carcinoma
adenocarcinomal adenocarcinoma)
II
Neutral glycolipids of normal and cancerous human pancreatic tissues The contents tissue.
of each neutral
glycolipid
separated
Normal (sample
No.):
Gal-Cer Glc-Cer Gal-Glc-Cer Gal-Gal-Glc-Cer GalNAc-Gal-Gal-Glc-Cer Gal-GlcNAc-Gal-Glc-Cer in CMH in CDH in CTH Fuc-Gal-GlcNAc-Gal-Glc-Cer Fuc-Gal-GlcNAc-Gal-Glc-Cer F!ic Gal-Gal-GlcNAc-Gal-Glc-Cer F:c GalNAc-GBI-GlcNAc-Gal-Glc-Cer Fuc Fuc-Gal-GlcNAc-Gal-GlcNAc -Gal-Glc-Cer
by preparative
TLC was determined
pancreas
Pancreatic
are expressed
cancer
Bile duct cancer
1
2
3
4
5
6
7
0.063 0.055 0.474 0.156 0.208 0.076
0.202 0.452 0.601 0.583 0.196 0.073
0.222 0.316 0.326 0.180 0.091 0.045
0.164 0.090 0.198 0.244 0.142 0.061
0.262 0.242 0.385 0.700 0.221 0.203
0.068 0.315 0.586 0.244 0.344 0.268
_
trace trace 0.020
trace 0.010
0.141 0.021 _
_ _
0.183
_ _
_ 0.273
0.040 _ 0.172
0.198
_
0.518
_
0.288
_
0.096
_ 0.028
Total
0.868
1.129
2.837
1.488
1.005
2.386
1.825
III
Acidic glycolipids of normal and cancerous human pancreatic tissues
Normal Normal Normal Normal
pancreas pancreas pancreas pancreas
Pancreatic cancer Pancreatic cancer Bile duct cancer
as pmol/g
0.120 0.067 0.190 0.091 0.097 0.030
Fucolipid
TABLE
by GLC. The values
HSOs-Gal-Cer &moI/g protein)
Ganglioside lipid-bound sialic acid &mol/g protein)
(sample (sample (sample (sample
11 2) 3) 41
0.573 0.981 0.371 0.792
3.20 2.90 0.10 0.59
(sample (sample (sample
5) 6) 7)
1.542 0.850 1.780
2.21 1.81 6.92
protein
1, 2 and 4; the B active glycolipid and Le” active glycolipid in sample 3; the A active glycolipid in sample 5, and the H active glycolipid in sample 6. These blood group active glycolipids were also determined by enzymatic hydrolysis with exoglycosidase (data not shown). The quantitative estimation of acidic glycolipids (sulfatide and ganglioside) is shown in Table III. Acidic glycolipids were increased in the cancerous tissues as compared to normal tissues except for sample 2. TLC pattern of gangliosides were varied in each sample (Fig. 2). TLC-immunostaining of glycolipids from cancerous human pancreatic tissues
ab1234567 Fig. 2. TLC of gangliosides from normal and cancerous human pancreatic tissues. Lane 1-4, normal pancreas; Lane 5 and 6, pancreatic cancer; Lane 7, bile duct cancer; Lane a, standard GM, and CD,; Lane b, gangliosides from human brain. Developing solvent, chloroform/methanol/O.O2% CaCI, (60: 35: 8); visualization, resorcinol-HCI reagent.
served between the two. Normal pancreatic tissues also contain blood group active glycolipids, the fucolipids, which reflect the individual’s blood group. The mole percentage of blood group active fucolipids in the total neutral glycolipid was 20-40%. In contrast, cancerous tissue from blood group 0 or A patients showed decreased fucolipids. GLC analysis of the glycolipid from cancerous tissues revealed the presence of fucose in fractions of ceramide monosaccharide, ceramide disaccharide and ceramide trisaccharide (Table II>. The major fucolipids were the B active glycolipid in samples
C
normal and
To confirm the presence of blood group active fucolipids in both pancreatic cancer and normal tissues, TLC-immunostaining was performed utilizing affinity purified rabbit anti-paragloboside antibody, rabbit anti-Forssman antibody, mouse monoclonal anti-A, anti-B, anti-H antibodies, anti-Le”, anti-Let’, anti-Lex, anti-LeY antibodies, UEA-I lectin and GS-II lectin. The TLC immunostaining with anti-Le”, anti-Leh, antiA, anti-B and UEA-I is shown in Fig. 3. As expected, incompatible blood group antigens were detected only in pancreatic cancer tissues. Blood group A active glycolipids were observed in a pancreatic cancer tissue of the blood type 0 patient (Fig. 3E lane 6), and B active glycolipids were found in a cancerous tissue of the blood type A patient (Fig. 3D lane 5). The appearance of neolacto-series glycolipid in human pancreatic cancer tissues
Since the major changes in glycosylation detected in most human cancer are type 2 blood group chain, we investigated the reactivities of anti-Le”, anti-LeY, anti-
D
E
25 tion which had been treated with neuraminidase. Neolactotetraosylceramide was observed in the cancerous tissues (Fig. 41, and Le” and sialosyl Le” were also detected only in cancerous tissues (Fig. 5). Results of TLC-immunostaining with various antibodies in neutral glycolipid fractions of human pancreatic normal and cancerous tissues are listed in Table IV. Discussion
a1234567b Fig. 4. TLC-immunostaining of the neutral glycolipids from normal and cancerous human pancreatic tissues with anti-nlc, antibody. Lane 1-4, normal pancreas; Lane 5 and 6, pancreatic cancer; Lane 7, bile duct cancer; Lane a, isolated nLc,; lane b, neutral glycolipids from a human gastric cancer tissue.
nLc, antibodies in normal and cancerous pancreatic tissues. TLC immunostaining was performed with these anti-type 2 carbohydrate sequence antibodies on either the neutral glycolipid fractions or acidic glycolipid frac-
Alteration of carbohydrate composition in pancreatic cancer tissues is of practical importance since many carbohydrate containing tumor associated antigens observed in pancreatic carcinoma are applied as markers in clinical practice [31. This paper described the analysis of glycolipids from normal pancreatic tissues and pancreatic carcinoma tissues where marked differences were observed. Two types of aberrant glycosylations in tumor cells have been described: (A) incomplete synthesis with or without precursor accumulation; and (B) neosynthesis [13]. In this study both were observed. TLC and GLC analyses revealed a reduction of the more complex fucolipids in cancerous tissues. In transformed cells, precursor accumulation with incomplete synthesis has been previously observed and reported by many research groups [14]. The ganglioside precursor accumulates in human neuroecto-
A
12345678
1234567
Fig. 5. TLC-immunostaining of the glycolipids from normal and cancerous human pancreatic tissues with anti-Le” antibody. A: stained with anti&+ antib~y. B: stained with anti-Le’ antibody after treatment of the acidic glycolipid fraction with sialidase. Lane 1-4, normal pancreas; Lane 5 and 6, pancreatic cancers; Lane 7, bile duct cancer; Lane a, neutral glycolipids from a human gastric cancer tissue.
26 TABLE IV Results of TLC-immunosfoining of the glycolipids isolated from normal and cancerous human pancreatic tissues
Normal pancreas
Sample
Mood
Blood group substances
No.
smw
A
1
Le”
Leb
Le”
LeY
r&c,
sialo LeX
sialo Le”
type1
type 1
type:!
type2
type2
toe2
bwl
+ + + +
+ + -t +
_
-
-
_ -
f + i +
-
f -t
-
+ + * +
3
fi
type 1
type 1
type2
type2
type2
-
LJEAf (H) type1 Me2
4
B B B B
-
f + + +
Pancreatic cancer
5 6
A 0
+ _t
+ -
-
+ +
f +
“t -I-
+ -
-
+ +
Rife duct cancer
7
B
-
f
-
-
+
f
i
-
f
2 3
GSIl (amino CTH)
__
-
+
--
+
_“.
+
+ +
-
f
+
-
+, positively stained; +, faintly stained; - , negatively stained
dermaf tumors,I~pbomas and teukemia fl]. Lewis retated antigens (Lea, Leb) have been observed histochemically in human pancreatic normal and cancer tissues 1151, however, in our observations, changes in content of Le” and Leb glycolipids in pancreatic cancer tissues were not detected. We previously reported Le” active glycofipids accumulating in gastric cancer tissues in place of the non-fucosylated glycolipids f16]_ In a gastric cancer tissue sample from a small p blood type patient, the amount of fucolipids was reduced compared to uninvolved surrounding mucosae associated with cancerous tissues [7]. Cfycofipids of tissues from patients with colon cancer and renal cancer have also been investigated revealing the accumulation of lactosylceramide as a stem glycolipid [14]. Acidic glycolipids were not examined in detail in this study, however, sialylation and sulfation seem to be enhanced in pancreatic cancer tissues as is found in other human cancerous tissues [I]. The expression of incompatible histo-blood group antigens in tissue has been investigated 1171.The presence of incompatible histo-blood group antigens (glycolipids) was thought to result from ‘neosynthesis’ in cancerous tissues. The presence of incompatible blood group A antigens in blood group 0 or B patients has been reported by several groups [16,X8-211, and recently the presence of type 1 chain A antigen, either monofucosyl type 1 chain A or ALeb has been detected by monoclonal antibody [22]. In this study, blood group A active glycofipids were also detected in pancreatic cancer tissues of blood group 0 patients as well as in gastric cancer tissues [7,16]. Furthermore, blood group B glycolipid was found in the cancerous tissues of the blood group A patient. The occurrence of incompatible blood group I3 antigen in a glycofipid fraction has not been reported.
The type 2 chain is the major carrier for the blood group ABH determinants of human red blood ceils, but is virtually absent in normal human adult intestinal and gastric mucosae [23,24]. Le” and LeY antigens (type 2 chain) were immunohistochemically observed in human pancreatic cancer tissues [25] and Le’ antigens were expressed in pancreatic cancer tissues. in this study, type 2 carbohydrate structures were present in the pancreatic cancer tissues. This fact is supported by findings that many monoclonal antibodies directed to human cancerous tissues have been identified as being directed to type 2 chain based structures [1,26], GLC analysis revealed the presence of fucose in the ceramide monoglycoside, ceramide digiycoside and ceramide triglycoside fractions of the pancreatic cancer tissues. Fucolipids with short carbohydrate chain are known to be distributed in gastrointestinal tract tissues f27, 281 and fucosygceramide was described by Watanabe et al. This type of fucolipid might play an important role in malignant epithelial cells. Acknowledgements We wish to thank Dr. Sen-itiroh Hakomori for providing valuable antibodies and important advice, Dr. Julian N. Kanfer for helpful discussion and reviewing the manuscript.
1 Hakomori, S. (19891 Advances in cancer research (Wound G.E.V. and Klein, G., eds.), Vol. 52, pp. 257-331. Academic Press, Orlando. 2 Ranuvala, El, and Finne, J. (1979) FEBS. Lett. 97, l-8. 3 Chung, Y.S., No, J.J.L., Kim, Y.S., Tanaka, B., Nakata, B., Hiura, A., Motoyoshi, H., Stake, K. and Umeyama, K. (1987) Cancer (Phila.) 60. fh3h-1643.
27 4 Breimer, M.E. (1984) Arch. Biochem. Biophys. 228, 71-85. 5 Breimer, M.E., Karlsson, K.A. and Samuelsson, B.E. (1981) J. Biol. Chem. 256, 3810-3816. 6 Wherret, J.R. and Hakomori, S. (1973) J. Biol. Chem. 248, 3046-3051. 7 Hattori, H., Uemura, K., Ogata, H., Katsuyama, T., Taketomi, T. and Kanfer, J.N. (1987) Cancer Res. 47, 1968-1972. 8 Saito, T. and Hakomori, S. (1971) J. Lipid Res. 12, 257-259. 9 Spiro, R.G. (1972) Methods Enzymol. 28, 3-43. 10 Uemura, K., Yuzawa, M. and Taketomi, T. (1979) Jpn. J. Exp. Med. 49, l-8. 11 Uemura, K., Yuzawa, M. and Taketomi, T. (1978) J. Biochem. 83, 1191-1201. 12 Uemura, K., Macher, B.A., DeGregorio, M., Scudder, P., Buehler, J., Knapp, W. and Feizi, T. (1985) Biochim. Biophys. Acta 846, 26-36. 13 Hakomori, S. (1985) Cancer Res. 45, 2405-2414. 14 Hakomori, S. (1984) Annu. Rev. Immunol. 2, 103-126. 15 Ernst, C., Atkinson, B., Wysocka, M., Blaszczyk, M., Herlyn, M., Sears, H., Steplewski, Z. and Koprowski, H. (1984) Labo. Invest. 50, 394-401. 16 Hattori, H., Uemura, K. and Taketomi, T. (1981) Biochim. Biophys. Acta 666, 361-369. 17 Lloyd. K.O. (1986) Am. J. Clin. Pathol. 87, 129-139.
18 Hakkinen, I. (1970) J. Natl. Cancer Inst. 44, 1183-1193. 19 Breimer, M.E. (1980) Cancer Res. 40, 897-908. 20 Yokota, M., Warner, G. and Hakomori, S. (1981) Cancer Res. 41, 4185-4190. 21 Uemura, K., Hattori, H., Kitazawa, N. and Taketomi, T. (1982) J. Immunol. Methods 53, 221-232. 22 Clausen, H., Hakomori, S., Graem, N. and Dabelsteen, E. (1986) J. Immunol. 136, 326-330. 23 McKibbin, J.M., Spencer, W.A., Smith, E.L., Masson, J.E., Karlsson, K.A., Samuelsson, B.E., Li, Y.T., and Li, S.C. (1982) J. Biol. Chem. 257, 755-760. 24 Hattori, H., Uemura, K. and Taketomi, T. (1980) Jpn. J. Exp. Med. 50, 145-149. 25 Kim. Y.S., Itzkowitz, S.H., Yuan, M., Chung, Y.S. Satake, K., Umeyama, K. and Hakomori, S. (1988) Cancer Res. 48, 475-482. 26 Solter, D. and Knowles, B.B. (1978) Proc. Natl. Acad. Sci. USA 75, 5565-5569. 27 Watanabe, K., Matsubara, T. and Hakomori, S. (1976) J. Biol. Chem. 251, 2385-2387. 28 Yoshino, T., Watanabe, K. and Hakomori, S. (1982) Biochemistry 21, 928-934. 29 IUPAC-IUB Commission on Biochemical Nomenclature (1977) Lipids 12, 455-468.
Biochimica et Biophysics Acta, 1125 (1992) 21-27 0 1992 Elsevier Science Publishers B.V. Ail rights reserved 00052760/92/$05.00
BBALIP 53868
Glycolipid of human pancreatic cancer; the appearance of ne~la~t~“seri~s (type 2 chain) glyc~li~id and the presence of incompatible blood group antigen in tumor tissues Hiroshi Hattori a, Kei-ichi Uemura b, Hideki Ishihara a and Hirayuki Ogata ’ a ~u~ra~o~ for CeEI 3~olo~~ Pharma Research Laboratories, Hoechsf Japan Limired, Kawagoe, Suirama {J&an), b department of Lipid B~ochern~st~~Institute of ~ardiouasc~iur Disease, ~hi~shu ~~~~ers~~ School of Medicine, Asahi, Matsumoto &zpanJ and ’ Blood transfusion seruice, Shinshu University Hospital, Asahi, Matsumolo (Japan]
(Received 22 August 1991)
Key words: Tumor associated carbohydrate antigen; ~~~cosphi~goli~id~Pancreatic cancer; Blwd group antigen; Neolacto-series
carbohydrate chain (type 2) Glycolipid isolated from normal and cancerous human pancreatic tissues were characterized chemically and immunologically. The major neutral glycolipids in both normal and cancerous tissues were composed of globo-series glycolipids and lacto-series glycolipids. The mole percentage of fucolipids in the total neutral glycolipids of normal tissues was XI-40%, and in general the fucofipids corresponded to blood group glycolipids related to the patient’s blood group, however, in cancerous tissues the amount of these fucolipids was decreased. fmmunostaining revealed that normal tissues contained only lacto-series (type 1 chain) glycolipids. In contrast, cancerous tissues contained the neolacto-series (type 2 chain) glycolipids as we11 as the lacto-series glycolipids. Incompatible blood group antigens, A active glycolipids in a blood type 0 patient and B active glycolipids in a blood type A patient, were also detectable in the neutral glycolipid fractions of the pancreatic cancer tissues.
Intmduction
Cancer associated aberrant giycosyiation is documented by the study of giycoiipid epitopes, although the same epitopes are also found in giycoprateins. The choice of giycotipids for study may be due to the fact that glycolipids can easily be purified to homogeneity, while the extremely heterogeneous oiigosaccharide chains present in giycoproteins make purification difficult [1,2]. Giycoiipid evaluation has a point of application in the analysis of pancreatic cancer. The incidence of this disease is now increasing steadily, however, its diagnosis is difficult. There are pancreatic cancer associated
carbohydrate antigens which have been characterized and are useful for clinical diagnosis [3]. These antigens have been detected by classical immunochemicai anaiysis as well as by a recentIy developed monodonal antibody [l]. Glycalipids of human normal pancreatic tissues have been previously analyzed, Breimer et al. [4,5] reported blood group active glycolipids of human normal pancreas. Wherret and Hakomori [63 observed the accumulation of B active fucoiipid in pancreas of Fabry’s disease patient. In this paper, chemical and immunoiogi~a~ characterization of glycoiipids from both human normal and malignant pancreatic tissues is described. Materials
TLC, thin layer e~roma~ograph~~GUI, gas liquid chromatography; PBS, phosphate-buffered saline; BSA, bovine serum
Abbreviations:
albumin. The glycolipid designations used, according to the recommendations of the Nomenclature Committee of the International Union of Pure and Applied Chemistry [291, are: Lc,, lactotetraosylceramide; nLc,, lactoneotetraosyiceramide; Lea; II14FucLc,; Leb, IV’Fuc, II14FucLc4; Le”, II13FucnLc,; LeY; IV2Fuc, IIi3FucnLc,. Correspondence: H. Hattori, Laboratory for Cell Biology, Pharma Research Laboratories, Hoechst Japan Limited, l-3-2, Minamidai, Kawagoe, Saitama, Japan 350.
and Methods
Giycolipid preparations were made from four sampies of human normal pancreatic tissue, two samples of human adenocarcinoma and 1 sample of bile duct adenocarcinoma. Age, sex, blood group (A, 13, 0) and pathology of each patient are shown in Table I. The isolation and purification of glycolipids were described in a previous paper [7]. Briefly, lipids were extracted from the tissue with 20 vol. each of ehioroform/ methanol (C/M) (2: 1, 1: 1, v/v) and C/M/water
22
(2 : 1: 0.5, v/v>, and the combined extracts were evaporated to dryness. The total mixture of lipids was applied to a DEAE-Sephadex A-25 column (Pharmacia Fine Chemical, Uppsala, Sweden). The neutral lipids were then eluted with chloroform/ methanol/ water (30:60:8, v/v> and the acidic lipids were eluted with chloroform/ methanol/O.8 M sodium acetate (30 : 60 : 8, v/v). The neutral glycolipids were separated from the neutral lipid fraction according to the method of Saito and Hakomori [S], then further purified by silica gel column chromatography (silica gel 60, Merck, Darmstadt, Germany). The final purification of individual neutral glycolipids was by preparative thin-layer chromatography (silica gel 60 plate, Merck) with chloroform/ methanol/water (65 : 2.5: 4) or (60 : 35 : 8) as the solvent system. Analytical procedures
TLC was carried out on a silica gel 60 plate (Merk) with chloroform/ methanol/ water (65 : 25 : 4 or 60 : 35 : 8) as the solvent system. The carbohydrate composition and content of the individuat glycolipids were determined by GLC analysis of the trimethylsilyl ether derivatives of the methylglycosides obtained after methanolysis at 95°C for 3 h with 3% HCl/methanol. GLC was performed with a glass column (3 mm i.d. X 2 m) packed with 2% OV-1 programmed at 2”C,/min from 140 to 210°C. Enzymatic hydrolysis of carbohydrate linkages in the glycolipids was carried out with cu-galactosidase, a-N-acetylgalactosaminidase, cy-fucosidase and neuraminidase [9].
blocked with 5% bovine serum albumin in phosphatebuffered saline and then incubated for 2 h with antibodies. The TLC plate was reincubated for 1 h with a 500-foid dilution of biotin conjugated anti-mouse immunoglobulin (DAKOPATTS). After washing, a peroxidase conjugated avidin-biotin complex solution (VECTASTAIN; Vector, CA) was added to the plate and allowed to react for 1 h at room temperature. After washing, for the final step, the chromatogram was incubated for 10-30 min with a substrate solution (KONICA IMMUNOSTAIN ’ HRP, Konica, Tokyo, Japan). Results GlycoZipid profile of normat and cancerous Mayan pancreatic tissues
The TLC separation of the neutral glycolipid fraction from normal human pancreatic tissues, pancreatic cancer tissues and bile duct cancer tissue is shown in Fig. I. Normal pancreatic tissues (samples l-4) contained glycolipids having a more complex carbohydrate structure as compared to the cancerous tissues. Quantitative and qualitative estimations of the neutral glycolipids from the various clinical samples are presented in Table II. The major neutral glycolipids found in normal pancreas tissues were glucosyIceramide, galactosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, lacto(neo)tetraosylceramide and fucolipids. Total content of neutral glycolipids from normal and cancerous tissues was 0.868-2.837 Fmol/g protein tissue and no differences were ob-
An t~bodies and lectins
Rabbit anti-Forssman antibody was obtained a previously described [lo]. Mouse monoclonal anti-A, anti-B and anti-H antibodies were purchased from Dakopatt (Glostrap, Denmark) and mouse monoclonal anti-Lea and L,eh antibodies were from Chem Biomed (Edmonton, Canada). UIex europaeus aggl~tinin I (UEA-I) and Grifforia simpficifolia II (GS-II) were purchased from E. Y. Laboratories (San Mateo, CA). Mouse monoclonal anti-LeX antibody (SW11 and anti-LeY antibody (AH6) were donated by Dr. Sen-itiroh Hakomori (Washington Universi~, Seattle, WA). Rabbit anti-nLc, was obtained by a published procedure [ll] and purified by affinity column. Peroxidase-conjugated UEA-I was purchased from E. Y. Laboratories.
The immunostaining procedure employed was a modification of the method described by Uemura et al. [12]. Approx. 50 pg of the total neutral glycolipid fraction was applied to a high performance aluminum sheet silica gei 60 TLC plate (Merck) and the plate was developed with a mixture of chlorofo~/methanol/ water (60: 35 : 8, v/v). Subsequently, the plate was
a b
C
d
01234567F 1.TLC
of the neutral glycolipids from normal and cancerous Fig. human pancreatic tissues. Lane 0, erythrocytes of OJx(a - b+ ); Lane 1-4, normal pancreas; Lane 5 and 6, pancreatic cancer; Lane 7, bile duct cancer; Lane F, Forssman glycolipid. a, Glc-Cer; b, Lac-Cer; c, GbOse,Cer; d, GbOse,Cer. Developing solvent, chloroform/ methano~/water (65 : 25: 4); ~sualization, cupric phosphoric acid reagent.
23 TABLE
1
Pancreas tissues examined in this study Sample no. (TLC No.)
Age
I 2 3 4 5 6 7
60 39 61 58 63 71 58
TABLE
Sex
Blood
Histology
group Male Male Female Female Male Male Female
B B B B A 0 B
Normal pancreas Normal pancreas Normal pancreas Normal pancreas Pancreatic cancer (ductal Pancreatic cancer (ductal Bile duct carcinoma
adenocarcinomal adenocarcinoma)
II
Neutral glycolipids of normal and cancerous human pancreatic tissues The contents tissue.
of each neutral
glycolipid
separated
Normal (sample
No.):
Gal-Cer Glc-Cer Gal-Glc-Cer Gal-Gal-Glc-Cer GalNAc-Gal-Gal-Glc-Cer Gal-GlcNAc-Gal-Glc-Cer in CMH in CDH in CTH Fuc-Gal-GlcNAc-Gal-Glc-Cer Fuc-Gal-GlcNAc-Gal-Glc-Cer F!ic Gal-Gal-GlcNAc-Gal-Glc-Cer F:c GalNAc-GBI-GlcNAc-Gal-Glc-Cer Fuc Fuc-Gal-GlcNAc-Gal-GlcNAc -Gal-Glc-Cer
by preparative
TLC was determined
pancreas
Pancreatic
are expressed
cancer
Bile duct cancer
1
2
3
4
5
6
7
0.063 0.055 0.474 0.156 0.208 0.076
0.202 0.452 0.601 0.583 0.196 0.073
0.222 0.316 0.326 0.180 0.091 0.045
0.164 0.090 0.198 0.244 0.142 0.061
0.262 0.242 0.385 0.700 0.221 0.203
0.068 0.315 0.586 0.244 0.344 0.268
_
trace trace 0.020
trace 0.010
0.141 0.021 _
_ _
0.183
_ _
_ 0.273
0.040 _ 0.172
0.198
_
0.518
_
0.288
_
0.096
_ 0.028
Total
0.868
1.129
2.837
1.488
1.005
2.386
1.825
III
Acidic glycolipids of normal and cancerous human pancreatic tissues
Normal Normal Normal Normal
pancreas pancreas pancreas pancreas
Pancreatic cancer Pancreatic cancer Bile duct cancer
as pmol/g
0.120 0.067 0.190 0.091 0.097 0.030
Fucolipid
TABLE
by GLC. The values
HSOs-Gal-Cer &moI/g protein)
Ganglioside lipid-bound sialic acid &mol/g protein)
(sample (sample (sample (sample
11 2) 3) 41
0.573 0.981 0.371 0.792
3.20 2.90 0.10 0.59
(sample (sample (sample
5) 6) 7)
1.542 0.850 1.780
2.21 1.81 6.92
protein
1, 2 and 4; the B active glycolipid and Le” active glycolipid in sample 3; the A active glycolipid in sample 5, and the H active glycolipid in sample 6. These blood group active glycolipids were also determined by enzymatic hydrolysis with exoglycosidase (data not shown). The quantitative estimation of acidic glycolipids (sulfatide and ganglioside) is shown in Table III. Acidic glycolipids were increased in the cancerous tissues as compared to normal tissues except for sample 2. TLC pattern of gangliosides were varied in each sample (Fig. 2). TLC-immunostaining of glycolipids from cancerous human pancreatic tissues
ab1234567 Fig. 2. TLC of gangliosides from normal and cancerous human pancreatic tissues. Lane 1-4, normal pancreas; Lane 5 and 6, pancreatic cancer; Lane 7, bile duct cancer; Lane a, standard GM, and CD,; Lane b, gangliosides from human brain. Developing solvent, chloroform/methanol/O.O2% CaCI, (60: 35: 8); visualization, resorcinol-HCI reagent.
served between the two. Normal pancreatic tissues also contain blood group active glycolipids, the fucolipids, which reflect the individual’s blood group. The mole percentage of blood group active fucolipids in the total neutral glycolipid was 20-40%. In contrast, cancerous tissue from blood group 0 or A patients showed decreased fucolipids. GLC analysis of the glycolipid from cancerous tissues revealed the presence of fucose in fractions of ceramide monosaccharide, ceramide disaccharide and ceramide trisaccharide (Table II>. The major fucolipids were the B active glycolipid in samples
C
normal and
To confirm the presence of blood group active fucolipids in both pancreatic cancer and normal tissues, TLC-immunostaining was performed utilizing affinity purified rabbit anti-paragloboside antibody, rabbit anti-Forssman antibody, mouse monoclonal anti-A, anti-B, anti-H antibodies, anti-Le”, anti-Let’, anti-Lex, anti-LeY antibodies, UEA-I lectin and GS-II lectin. The TLC immunostaining with anti-Le”, anti-Leh, antiA, anti-B and UEA-I is shown in Fig. 3. As expected, incompatible blood group antigens were detected only in pancreatic cancer tissues. Blood group A active glycolipids were observed in a pancreatic cancer tissue of the blood type 0 patient (Fig. 3E lane 6), and B active glycolipids were found in a cancerous tissue of the blood type A patient (Fig. 3D lane 5). The appearance of neolacto-series glycolipid in human pancreatic cancer tissues
Since the major changes in glycosylation detected in most human cancer are type 2 blood group chain, we investigated the reactivities of anti-Le”, anti-LeY, anti-
D
E
25 tion which had been treated with neuraminidase. Neolactotetraosylceramide was observed in the cancerous tissues (Fig. 41, and Le” and sialosyl Le” were also detected only in cancerous tissues (Fig. 5). Results of TLC-immunostaining with various antibodies in neutral glycolipid fractions of human pancreatic normal and cancerous tissues are listed in Table IV. Discussion
a1234567b Fig. 4. TLC-immunostaining of the neutral glycolipids from normal and cancerous human pancreatic tissues with anti-nlc, antibody. Lane 1-4, normal pancreas; Lane 5 and 6, pancreatic cancer; Lane 7, bile duct cancer; Lane a, isolated nLc,; lane b, neutral glycolipids from a human gastric cancer tissue.
nLc, antibodies in normal and cancerous pancreatic tissues. TLC immunostaining was performed with these anti-type 2 carbohydrate sequence antibodies on either the neutral glycolipid fractions or acidic glycolipid frac-
Alteration of carbohydrate composition in pancreatic cancer tissues is of practical importance since many carbohydrate containing tumor associated antigens observed in pancreatic carcinoma are applied as markers in clinical practice [31. This paper described the analysis of glycolipids from normal pancreatic tissues and pancreatic carcinoma tissues where marked differences were observed. Two types of aberrant glycosylations in tumor cells have been described: (A) incomplete synthesis with or without precursor accumulation; and (B) neosynthesis [13]. In this study both were observed. TLC and GLC analyses revealed a reduction of the more complex fucolipids in cancerous tissues. In transformed cells, precursor accumulation with incomplete synthesis has been previously observed and reported by many research groups [14]. The ganglioside precursor accumulates in human neuroecto-
A
12345678
1234567
Fig. 5. TLC-immunostaining of the glycolipids from normal and cancerous human pancreatic tissues with anti-Le” antibody. A: stained with anti&+ antib~y. B: stained with anti-Le’ antibody after treatment of the acidic glycolipid fraction with sialidase. Lane 1-4, normal pancreas; Lane 5 and 6, pancreatic cancers; Lane 7, bile duct cancer; Lane a, neutral glycolipids from a human gastric cancer tissue.
26 TABLE IV Results of TLC-immunosfoining of the glycolipids isolated from normal and cancerous human pancreatic tissues
Normal pancreas
Sample
Mood
Blood group substances
No.
smw
A
1
Le”
Leb
Le”
LeY
r&c,
sialo LeX
sialo Le”
type1
type 1
type:!
type2
type2
toe2
bwl
+ + + +
+ + -t +
_
-
-
_ -
f + i +
-
f -t
-
+ + * +
3
fi
type 1
type 1
type2
type2
type2
-
LJEAf (H) type1 Me2
4
B B B B
-
f + + +
Pancreatic cancer
5 6
A 0
+ _t
+ -
-
+ +
f +
“t -I-
+ -
-
+ +
Rife duct cancer
7
B
-
f
-
-
+
f
i
-
f
2 3
GSIl (amino CTH)
__
-
+
--
+
_“.
+
+ +
-
f
+
-
+, positively stained; +, faintly stained; - , negatively stained
dermaf tumors,I~pbomas and teukemia fl]. Lewis retated antigens (Lea, Leb) have been observed histochemically in human pancreatic normal and cancer tissues 1151, however, in our observations, changes in content of Le” and Leb glycolipids in pancreatic cancer tissues were not detected. We previously reported Le” active glycofipids accumulating in gastric cancer tissues in place of the non-fucosylated glycolipids f16]_ In a gastric cancer tissue sample from a small p blood type patient, the amount of fucolipids was reduced compared to uninvolved surrounding mucosae associated with cancerous tissues [7]. Cfycofipids of tissues from patients with colon cancer and renal cancer have also been investigated revealing the accumulation of lactosylceramide as a stem glycolipid [14]. Acidic glycolipids were not examined in detail in this study, however, sialylation and sulfation seem to be enhanced in pancreatic cancer tissues as is found in other human cancerous tissues [I]. The expression of incompatible histo-blood group antigens in tissue has been investigated 1171.The presence of incompatible histo-blood group antigens (glycolipids) was thought to result from ‘neosynthesis’ in cancerous tissues. The presence of incompatible blood group A antigens in blood group 0 or B patients has been reported by several groups [16,X8-211, and recently the presence of type 1 chain A antigen, either monofucosyl type 1 chain A or ALeb has been detected by monoclonal antibody [22]. In this study, blood group A active glycofipids were also detected in pancreatic cancer tissues of blood group 0 patients as well as in gastric cancer tissues [7,16]. Furthermore, blood group B glycolipid was found in the cancerous tissues of the blood group A patient. The occurrence of incompatible blood group I3 antigen in a glycofipid fraction has not been reported.
The type 2 chain is the major carrier for the blood group ABH determinants of human red blood ceils, but is virtually absent in normal human adult intestinal and gastric mucosae [23,24]. Le” and LeY antigens (type 2 chain) were immunohistochemically observed in human pancreatic cancer tissues [25] and Le’ antigens were expressed in pancreatic cancer tissues. in this study, type 2 carbohydrate structures were present in the pancreatic cancer tissues. This fact is supported by findings that many monoclonal antibodies directed to human cancerous tissues have been identified as being directed to type 2 chain based structures [1,26], GLC analysis revealed the presence of fucose in the ceramide monoglycoside, ceramide digiycoside and ceramide triglycoside fractions of the pancreatic cancer tissues. Fucolipids with short carbohydrate chain are known to be distributed in gastrointestinal tract tissues f27, 281 and fucosygceramide was described by Watanabe et al. This type of fucolipid might play an important role in malignant epithelial cells. Acknowledgements We wish to thank Dr. Sen-itiroh Hakomori for providing valuable antibodies and important advice, Dr. Julian N. Kanfer for helpful discussion and reviewing the manuscript.
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